A novel TOX3-WDR5-ABCG2 signaling axis regulates the progression of colorectal cancer by accelerating stem-like traits and chemoresistance.
PLoS biology
The eradication of cancer stem cells (CSCs) with drug resistance confers the probability of local tumor control after chemotherapy or targeted therapy. As the main drug resistance marker, ABCG2 is also critical for colorectal cancer (CRC) evolution, in particular cancer stem-like traits expansion. Hitherto, the knowledge about the expression regulation of ABCG2, in particular its upstream transcriptional regulatory mechanisms, remains limited in cancer, including CRC. Here, ABCG2 was found to be markedly up-regulated in CRC CSCs (cCSCs) expansion and chemo-resistant CRC tissues and closely associated with CRC recurrence. Mechanistically, TOX3 was identified as a specific transcriptional factor to drive ABCG2 expression and subsequent cCSCs expansion and chemoresistance by binding to -261 to -141 segments of the ABCG2 promoter region. Moreover, we found that TOX3 recruited WDR5 to promote tri-methylation of H3K4 at the ABCG2 promoter in cCSCs, which further confers stem-like traits and chemoresistance to CRC by co-regulating the transcription of ABCG2. In line with this observation, TOX3, WDR5, and ABCG2 showed abnormal activation in chemo-resistant tumor tissues of in situ CRC mouse model and clinical investigation further demonstrated the comprehensive assessment of TOX3, WDR5, and ABCG2 could be a more efficient strategy for survival prediction of CRC patients with recurrence or metastasis. Thus, our study found that TOX3-WDR5/ABCG2 signaling axis plays a critical role in regulating CRC stem-like traits and chemoresistance, and a combination of chemotherapy with WDR5 inhibitors may induce synthetic lethality in ABCG2-deregulated tumors.
10.1371/journal.pbio.3002256
ABCG2 harboring the Gly482 mutation confers high-level resistance to various hydrophilic antifolates.
Shafran Assaf,Ifergan Ilan,Bram Eran,Jansen Gerrit,Kathmann Ietje,Peters Godefridus J,Robey Robert W,Bates Susan E,Assaraf Yehuda G
Cancer research
ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482- and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482- and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 micromol/L [3H]methotrexate, in R482- and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di- and triglutamate species of [3H]methotrexate at 4 hours of incubation with methotrexate and in the tetra- and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-gamma-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates.
10.1158/0008-5472.CAN-04-4547
Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma.
Fujita Hirofumi,Nagakawa Keisuke,Kobuchi Hirotsugu,Ogino Tetsuya,Kondo Yoichi,Inoue Keiji,Shuin Taro,Utsumi Toshihiko,Utsumi Kozo,Sasaki Junzo,Ohuchi Hideyo
Cancer research
One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies. Cancer Res; 76(7); 1837-46. ©2016 AACR.
10.1158/0008-5472.CAN-15-1484
Inactivation of PTEN increases ABCG2 expression and the side population through the PI3K/Akt pathway in adult acute leukemia.
Huang Fang-Fang,Wu Deng-Shu,Zhang Li,Yu Yan-Hui,Yuan Xiao-Yu,Li Wen-Jin,Chen Xiao-Ping,Zhao Xie-Lan,Chen Fang-Ping,Zeng Hui
Cancer letters
Both the occurrence and recurrence of acute leukemia (AL) might suggest the presence of leukemia stem cells. Side population (SP) cells, exhibiting stem cell-like properties, express ABCG2 (breast cancer resistance protein [BCRP]). This study revealed that over-expression of ABCG2 in Jurkat and HL60 cells led to an increased SP fraction, up-regulated levels of phosphorylated-PI3K and phosphorylated-Akt, and enhanced drug resistance, all of which could be attenuated by treatment with either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin. ABCG2 expression and SP cell counts were further characterized in 222 adult AL patients at three disease stages: upon diagnosis, at remission and at refractory/relapse (R/R), while 10 healthy donors served as the normal controls. Only a small fraction of the ABCG2+population (0.05-12.3%) and SP cells (0.02-1.60%) were observed in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. In the normal control population, the SP cell fraction represented a statistically higher percentage of total cells compared to the fraction of SP cells upon diagnosis or relapse in both AML and ALL. In addition, we demonstrated that ABCG2 expression and SP cell ratios can be upregulated by the inactivation of phosphatase and tensin homolog (PTEN) protein, achieved in this study by removing inhibition of the PI3K/Akt pathway. Collectively, this study suggests that the PTEN/PI3K/Akt pathway up-regulates ABCG2 expression and the SP cell population and is a potential AL-specific treatment target worth investigating further.
10.1016/j.canlet.2013.04.006
A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity.
Barghout Samir H,Aman Ahmed,Nouri Kazem,Blatman Zachary,Arevalo Karen,Thomas Geethu E,MacLean Neil,Hurren Rose,Ketela Troy,Saini Mehakpreet,Abohawya Moustafa,Kiyota Taira,Al-Awar Rima,Schimmer Aaron D
JCI insight
TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.
10.1172/jci.insight.141518
Nucleotide binding is the critical regulator of ABCG2 conformational transitions.
eLife
ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.
10.7554/eLife.83976
Expression and role of Notch signalling in the regeneration of rat tracheal epithelium.
Ma X-B,Jia X-S,Liu Y-L,Wang L-L,Sun S-L,Song N,Wang E-H,Li F
Cell proliferation
OBJECTIVES:This study is to explore the role of Notch signalling during the regeneration of rat tracheal epithelium after injury induced by 5-fluorouracil (5-FU). MATERIALS AND METHODS:We developed an ex vivo model of rat tracheal epithelial regeneration using 5-FU to induce injury. Expression levels of members of the Notch signalling pathway, ABCG2, CK19, and proliferating cell nuclear antigen (PCNA) were examined by reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. One group of tracheas were cultured in the medium with a gamma-secretase inhibitor or Jag-1 peptide after 5-FU treatment and another group were pre-treated with the gamma-secretase inhibitor or Jag-1 peptide before 5-FU treatment. The expression changes of ABCG2, CK19, and PCNA were examined by Western blot or immunofluorescence and the morphologic changes were observed by haematoxylin and eosin stain during the recovery process. RESULTS:Expression levels of Notch3, Jagged1, and Hey1 were increased in rat tracheal epithelial cells after treatment with 5-FU. During injury recovery, disruption of Notch signalling by treatment with the gamma-secretase inhibitor reduced expression of ABCG2 and PCNA, but promoted expression of CK19, while persistent activation of Notch signalling promoted expression of ABCG2 and PCNA, but reduced expression of CK19. Under both conditions, recovery from injury was reduced. However, blocking Notch signalling prior to 5-FU treatment led to the complete blockage of recovery, while activating Notch signalling before 5-FU treatment promoted recovery. CONCLUSIONS:During tracheal epithelial regeneration, Notch signalling maintains an undifferentiated state and promotes proliferation among a population of tracheal epithelial cells.
10.1111/j.1365-2184.2008.00569.x
Tyrosine kinase inhibitors enhanced the efficacy of conventional chemotherapeutic agent in multidrug resistant cancer cells.
Wu Shaocong,Fu Liwu
Molecular cancer
Multidrug resistance (MDR) triggered by ATP binding cassette (ABC) transporter such as ABCB1, ABCC1, ABCG2 limited successful cancer chemotherapy. Unfortunately, no commercial available MDR modulator approved by FDA was used in clinic. Tyrosine kinase inhibitors (TKIs) have been administrated to fight against cancer for decades. Almost TKI was used alone in clinic. However, drug combinations acting synergistically to kill cancer cells have become increasingly important in cancer chemotherapy as an approach for the recurrent resistant disease. Here, we summarize the effect of TKIs on enhancing the efficacy of conventional chemotherapeutic drug in ABC transporter-mediated MDR cancer cells, which encourage to further discuss and study in clinic.
10.1186/s12943-018-0775-3
Dicer-mediated upregulation of BCRP confers tamoxifen resistance in human breast cancer cells.
Selever Jennifer,Gu Guowei,Lewis Michael T,Beyer Amanda,Herynk Matthew H,Covington Kyle R,Tsimelzon Anna,Dontu Gabriela,Provost Patrick,Di Pietro Attilio,Boumendjel Ahcène,Albain Kathy,Miele Lucio,Weiss Heidi,Barone Ines,Ando Sebastiano,Fuqua Suzanne A W
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α (ERα)-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer. EXPERIMENTAL DESIGN:We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells and observed a concomitant increase in expression of the breast cancer resistance protein (BCRP). We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation and evaluated intracellular Tam efflux. RESULTS:In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as five mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor-initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux. CONCLUSION:Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity.
10.1158/1078-0432.CCR-11-1403
Histone deacetylase inhibitors influence chemotherapy transport by modulating expression and trafficking of a common polymorphic variant of the ABCG2 efflux transporter.
Basseville Agnes,Tamaki Akina,Ierano Caterina,Trostel Shana,Ward Yvona,Robey Robert W,Hegde Ramanujan S,Bates Susan E
Cancer research
Histone deacetylase inhibitors (HDI) have exhibited some efficacy in clinical trials, but it is clear that their most effective applications have yet to be fully determined. In this study, we show that HDIs influence the expression of a common polymorphic variant of the chemotherapy drug efflux transporter ABCG2, which contributes to normal tissue protection. As one of the most frequent variants in human ABCG2, the polymorphism Q141K impairs expression, localization, and function, thereby reducing drug clearance and increasing chemotherapy toxicity. Mechanistic investigations revealed that the ABCG2 Q141K variant was fully processed but retained in the aggresome, a perinuclear structure, where misfolded proteins aggregate. In screening for compounds that could correct its expression, localization, and function, we found that the microtubule-disrupting agent colchicine could induce relocalization of the variant from the aggresome to the cell surface. More strikingly, we found that HDIs could produce a similar effect but also restore protein expression to wild-type levels, yielding a restoration of ABCG2-mediated specific drug efflux activity. Notably, HDIs did not modify aggresome structures but instead rescued newly synthesized protein and prevented aggresome targeting, suggesting that HDIs disturbed trafficking along microtubules by eliciting changes in motor protein expression. Together, these results showed how HDIs are able to restore wild-type functions of the common Q141K polymorphic isoform of ABCG2. More broadly, our findings expand the potential uses of HDIs in the clinic.
10.1158/0008-5472.CAN-11-2008
ABCG2: a perspective.
Advanced drug delivery reviews
ABCG2, or breast cancer resistance protein (BCRP), is an ABC transporter that has been the subject of intense study since its discovery a decade ago. With high normal tissue expression in the brain endothelium, gastrointestinal tract, and placenta, ABCG2 is believed to be important in the protection from xenobiotics, regulating oral bioavailability, forming part of the blood-brain barrier, the blood-testis barrier, and the maternal-fetal barrier. Notably, ABCG2 is often expressed in stem cell populations, where it likely plays a role in xenobiotic protection. However, clues to its epigenetic regulation in various cell populations are only beginning to emerge. While ABCG2 overexpression has been demonstrated in cancer cells after in vitro drug treatment, endogenous ABCG2 expression in certain cancers is likely a reflection of the differentiated phenotype of the cell of origin and likely contributes to intrinsic drug resistance. Notably, research into the transporter's role in cancer drug resistance and its development as a therapeutic target in cancer has lagged. Substrates and inhibitors of the transporter have been described, among them chemotherapy drugs, tyrosine kinase inhibitors, antivirals, HMG-CoA reductase inhibitors, carcinogens, and flavonoids. This broad range of substrates complements the efficiency of ABCG2 as a transporter in laboratory studies and suggests that, while there are redundant mechanisms of xenobiotic protection, the protein is important in normal physiology. Indeed, emerging studies in pharmacology and toxicology assessing polymorphic variants in man, in combination with murine knockout models have confirmed its dynamic role. Work in pharmacology may eventually lead us to a greater understanding of the physiologic role of ABCG2.
10.1016/j.addr.2008.11.003
Influence of germline variations in drug transporters ABCB1 and ABCG2 on intracerebral osimertinib efficacy in patients with non-small cell lung cancer.
EClinicalMedicine
Background:Central nervous system (CNS) metastases are present in approximately 40% of patients with metastatic epidermal growth factor receptor-mutated (m+) non-small cell lung cancer (NSCLC). The EGFR-tyrosine kinase inhibitor osimertinib is a substrate of transporters ABCB1 and ABCG2 and metabolized by CYP3A4. We investigated relationships between single nucleotide polymorphisms (SNPs) 3435C>T, 421C>A and 34G>A, and and CNS treatment efficacy of osimertinib in m+ NSCLC patients. Methods:Patients who started treatment with osimertinib for m+ NSCLC between November 2014 and June 2021 were included in this retrospective observational multicentre cohort study. For patients with baseline CNS metastases, the primary endpoint was CNS progression-free survival (CNS-PFS; time from osimertinib start until CNS disease progression or death). For patients with no or unknown baseline CNS metastases, the primary endpoint was CNS disease-free survival (CNS-DFS; time from osimertinib start until occurrence of new CNS metastases). Relationships between SNPs and baseline characteristics with CNS-PFS and CNS-DFS were studied with competing-risks survival analysis. Secondary endpoints were relationships between SNPs and PFS, overall survival, severe toxicity, and osimertinib pharmacokinetics. Findings:From 572 included patients, 201 had baseline CNS metastases. No SNP was associated with CNS-PFS. Genotype 34GA/AA and/or 3435CC --present in 35% of patients-- was significantly associated with decreased CNS-DFS (hazard ratio 0.28; 95% CI 0.11-0.73; p = 0.009) in the multivariate analysis. This remained significant after applying a Bonferroni correction and internal validation through bootstrapping. 421CA/AA was related to more severe toxicity (27.0% versus 16.5%; p = 0.010). Interpretation:ABCG2 34G>A and 3435C>T are predictors for developing new CNS metastases during osimertinib treatment, probably because of diminished drug levels in the CNS. 421C>A was significantly related with the incidence of severe toxicity. Pre-emptive genotyping for these SNPs could individualize osimertinib therapy. Addition of ABCG2 inhibitors for patients without 34G>A should be studied further, to prevent new CNS metastases during osimertinib treatment. Funding:No funding was received for this trial.
10.1016/j.eclinm.2023.101955
Breast Cancer Resistance Protein and P-Glycoprotein Influence In Vivo Disposition of 11C-Erlotinib.
Traxl Alexander,Wanek Thomas,Mairinger Severin,Stanek Johann,Filip Thomas,Sauberer Michael,Müller Markus,Kuntner Claudia,Langer Oliver
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
UNLABELLED:(11)C-erlotinib is a PET tracer to distinguish responders from nonresponders to epidermal growth factor receptor-targeted tyrosine kinase inhibitors and may also be of interest to predict distribution of erlotinib to tissues targeted for treatment. The aim of this study was to investigate if the known interaction of erlotinib with the multidrug efflux transporters breast cancer resistance protein (humans, ABCG2; rodents, Abcg2) and P-glycoprotein (humans, ABCB1; rodents, Abcb1a/b) affects tissue distribution and excretion of (11)C-erlotinib and has an influence on the ability of (11)C-erlotinib PET to predict erlotinib tissue distribution at therapeutic doses. METHODS:Wild-type and Abcb1a/b or Abcg2 knockout mice underwent (11)C-erlotinib PET/MR scans, with or without the coinjection of a pharmacologic dose of erlotinib (10 mg/kg) or after pretreatment with the ABCB1/ABCG2 inhibitor elacridar (10 mg/kg). Integration plot analysis was used to determine organ uptake (CLuptake) and biliary excretion (CLbile) clearances of radioactivity. RESULTS:(11)C-erlotinib distribution to the brain was restricted by Abcb1a/b and Abcg2, and CLuptake into the brain was only significantly increased when both Abcb1a/b and Abcg2 were absent (wild-type mice, 0.017 ± 0.004 mL/min/g of tissue; Abcb1a/b((-/-))Abcg2((-/-)) mice, 0.079 ± 0.013 mL/min/g of tissue; P < 0.001). The pretreatment of wild-type mice with elacridar increased CLuptake into the brain to levels comparable to Abcb1a/b((-/-))Abcg2((-/-)) mice (0.090 ± 0.007 mL/min/g of tissue, P < 0.001). The absence of Abcb1a/b and Abcg2 led to a 2.6-fold decrease in CLbile (wild-type mice, 0.025 ± 0.005 mL/min/g of tissue; Abcb1a/b((-/-))Abcg2((-/-)) mice, 0.0095 ± 0.001 mL/min/g of tissue; P < 0.001). There were pronounced differences in distribution of (11)C-erlotinib to the brain, liver, kidney, and lung and hepatobiliary excretion into intestine between animals injected with a microdose and pharmacologic dose of erlotinib. CONCLUSION:ABCG2, ABCB1, and possibly other transporters influence in vivo disposition of (11)C-erlotinib and thereby affect its distribution to normal and potentially also tumor tissue. Saturable transport of erlotinib leads to nonlinear pharmacokinetics, possibly compromising the prediction of erlotinib tissue distribution at therapeutic doses from PET with a microdose of (11)C-erlotinib. The inhibition of ABCB1 and ABCG2 is a promising approach to enhance brain distribution of erlotinib to increase its efficacy in the treatment of brain tumors.
10.2967/jnumed.115.161273
An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells.
Oncogene
The side population (SP) in human lung cancer cell lines and tumors is enriched with cancer stem cells. An endogenous inhibitor of angiogenesis known as tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), characterized for its ability to inhibit matrix metalloproteinases (MMPs), has been shown by several laboratories to impede tumor progression through MMP-dependent or -independent mechanisms. We recently reported that forced expression of TIMP-2, as well as the modified form Ala+TIMP-2 (that lacks MMP inhibitory activity) significantly blocks growth of A549 human lung cancer cells in vivo. However, the mechanisms underlying TIMP-2 antitumor effects are not fully characterized. Here, we examine the hypothesis that the TIMP-2 antitumor activity may involve regulation of the SP in human lung cancer cells. Indeed, using Hoechst dye efflux assay and flow cytometry, as well as quantitative reverse transcriptase-PCR analysis, we found that endogenous TIMP-2 mRNA levels showed a significant inverse correlation with SP fraction size in six non-small cell lung cancer cell lines. In A549 cells expressing increased levels of TIMP-2, a significant decrease in SP was observed, and this decrease was associated with lowered gene expression of ABCG2, ABCB1 and AKR1C1. Functional analysis of A549 cells showed that TIMP-2 overexpression increased chemosensitivity to cytotoxic drugs. The SP isolated from TIMP-2-overexpressing A549 cells also demonstrated impaired migratory capacity compared with the SP from empty vector control. More importantly, our data provide strong evidence that these TIMP-2 functions occur independent of MMP inhibition, as A549 cells overexpressing Ala+TIMP-2 exhibited identical behavior to those overexpressing TIMP-2 alone. Our findings provide the first indication that TIMP-2 modulates SP phenotype and function, and suggests that TIMP-2 may act as an endogenous suppressor of the SP in human lung cancer cells.
10.1038/onc.2013.61
Versatile inhibitory effects of the flavonoid-derived PI3K/Akt inhibitor, LY294002, on ATP-binding cassette transporters that characterize stem cells.
Imai Yasuo,Yamagishi Hidetsugu,Ono Yuko,Ueda Yoshihiko
Clinical and translational medicine
Stem cells are undifferentiated cells capable of proliferation, self-renewal, and production of a large number of differentiated progeny. Stem cells exist even in malignancies. They are called cancer stem cells, which may represent the origin of these tumors and may be one of the reasons of chemoresistance. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is important for the maintenance of pluripotency in stem cells. Flow cytometry assay for identifying stem cells defines a side population of cells that displays low fluorescent dye and is highly enriched for stem cells. The dye efflux is attributed to expression of ATP-binding cassette transporters such as P-glycoprotein and breast cancer resistance protein (BCRP)/ABCG2, which also transport a variety of anticancer drugs. The PI3K/Akt pathway can modulate functions of ABC transporters through various mechanisms. Reportedly, inhibition of the PI3K/Akt pathway caused BCRP translocation in hematopoietic stem cells and glioma stem-like cells. On the other hand, a PI3K inhibitor, LY294002, reversed multidrug resistance in cancer cells that overexpress BCRP not by affecting BCRP translocation but putatively as a competitive inhibitor. Other PI3K inhibitors, wortmannin and PI-103, did not reverse BCRP-mediated drug resistance. Since LY294002 is a derivative of quercetin that is a naturally occurring flavonoid, its chemical structure is similar to those of a group of flavonoids but those of wortmannin and PI-103 are quite different. It is known that many flavonoids are inhibitors of BCRP and PI3K. LY294002 has also been reported to exert inhibitory effects on multidrug resistance-associated protein 1 (MRP1) function via dual mechanisms, competitive block of substrate transport and modulation of expression. Furthermore, LY294002 has been found to antagonize transport activity of P-glycoprotein without influencing its expression. Taken together, LY294002 can inhibit all BCRP, P-glycoprotein, and MRP1, which are three major ABC transporters that are highly expressed in stem cells and cause multidrug resistance. Due to its versatile effects, LY294002 could be a lead compound for developing more effective and tolerable reagents for cancer treatment.
10.1186/2001-1326-1-24
In vitro and in vivo clinical pharmacology of dimethyl benzoylphenylurea, a novel oral tubulin-interactive agent.
Rudek Michelle A,Zhao Ming,Smith Nicola F,Robey Robert W,He Ping,Hallur Gurulingappa,Khan Saeed,Hidalgo Manuel,Jimeno Antonio,Colevas A Dimitrios,Messersmith Wells A,Wolff Antonio C,Baker Sharyn D
Clinical cancer research : an official journal of the American Association for Cancer Research
Dimethyl benzoylphenylurea (BPU) is a novel tubulin-interactive agent with poor and highly variable oral bioavailability. In a phase I clinical trial of BPU, higher plasma exposure to BPU and metabolites was observed in patients who experienced dose-limiting toxicity. The elucidation of the clinical pharmacology of BPU was sought. BPU, monomethylBPU, and aminoBPU were metabolized by human liver microsomes. Studies with cDNA-expressed human cytochrome P450 enzymes revealed that BPU was metabolized predominantly by CYP3A4 and CYP1A1 but was also a substrate for CYP2C8, CYP2D6, CYP3A5, and CYP3A7. BPU was not a substrate for the efflux transporter ABCG2. Using simultaneous high-performance liquid chromatography/diode array and tandem mass spectrometry detection, we identified six metabolites in human liver microsomes, plasma, or urine: monomethylBPU, aminoBPU, G280, G308, G322, and G373. In patient urine, aminoBPU, G280, G308, and G322 collectively represented <2% of the given BPU dose. G280, G308, G322, and G373 showed minimal cytotoxicity. When BPU was given p.o. to mice in the presence and absence of the CYP3A and ABCG2 inhibitor, ritonavir, there was an increase in BPU plasma exposure and decrease in metabolite exposure but no overall change in cumulative exposure to BPU and the cytotoxic metabolites. Thus, we conclude that (a) CYP3A4 and CYP1A1 are the predominant cytochrome P450 enzymes that catalyze BPU metabolism, (b) BPU is metabolized to two cytotoxic and four noncytotoxic metabolites, and (c) ritonavir inhibits BPU metabolism to improve the systemic exposure to BPU without altering cumulative exposure to BPU and the cytotoxic metabolites.
10.1158/1078-0432.CCR-05-1037
Substrate overlap between Mrp4 and Abcg2/Bcrp affects purine analogue drug cytotoxicity and tissue distribution.
Takenaka Kazumasa,Morgan Jessica A,Scheffer George L,Adachi Masashi,Stewart Clinton F,Sun Daxi,Leggas Markos,Ejendal Karin F K,Hrycyna Christine A,Schuetz John D
Cancer research
The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2'-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [(125)I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy.
10.1158/0008-5472.CAN-06-4720
Multidrug-resistant tumor cells remain sensitive to a recombinant interleukin-4-Pseudomonas exotoxin, except when overexpressing the multidrug resistance protein MRP1.
de Jong Mariska C,Scheffer George L,Broxterman Henk J,Hooijberg Jan H,Slootstra Jerry W,Meloen Rob H,Kreitman Robert J,Husain Syed R,Joshi Bharat H,Puri Raj K,Scheper Rik J
Clinical cancer research : an official journal of the American Association for Cancer Research
Tumor cells may become resistant to conventional anticancer drugs through the occurrence of transmembrane transporter proteins such as P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or members of the multidrug resistance-associated protein family (MRP1-MRP5; ABCC1-ABCC5). In this report, we studied whether tumor cells that are cytostatic drug resistant because of overexpression of one of the above mentioned proteins are sensitive to a new anticancer agent, interleukin-4 toxin (IL-4 toxin). IL-4 toxin is a fusion protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE) [IL-4(38-37)-PE38KDEL]. Ninety-six-h cytotoxicity assays and 10-day clonogenic assays showed that drug-selected multidrug resistant (MDR) tumor cells that overexpress P-glycoprotein or breast cancer resistance proteins are still sensitive to IL-4 toxin. Also, tumor cells transfected with cDNA for MRP2-5 showed no resistance, or marginal resistance, only to the toxin as compared with the parent cells. In contrast, MRP1-overexpressing cells, both drug selected and MRP1 transfected, are clearly resistant to IL-4 toxin with resistance factors of 4.3 to 8.4. MRP1-overexpressing cells were not resistant to PE itself. IL-4 toxin resistance in MRP1-overexpressing cells could be reversed by the MRP1 inhibitors probenecid or MK571 and were not affected by glutathione depletion by DL-buthionine-S,R-sulfoximine. In a transport assay using plasma membrane vesicles prepared from MRP1-overexpressing cells, IL-4 toxin and IL-4, but not PE, inhibited the translocation of the known MRP1 substrate 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). These data suggest that MRP1-overexpressing cells are resistant to IL-4 toxin because of extrusion of this agent by MRP1. Still, the results of this study demonstrate that IL-4 toxin effectively kills most MDR tumor cells and, therefore, represents a promising anticancer drug.
Purification of breast cancer resistance protein ABCG2 and role of arginine-482.
Cellular and molecular life sciences : CMLS
Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding.
10.1007/s00018-006-6159-7
Olmutinib (HM61713) reversed multidrug resistance by inhibiting the activity of ATP-binding cassette subfamily G member 2 and .
Zhang Zhiqiang,Guo Xiaoran,To Kenneth K W,Chen Zhen,Fang Xiaona,Luo Min,Ma Chunling,Xu Jianhua,Yan Shirong,Fu Liwu
Acta pharmaceutica Sinica. B
Overexpressing of ATP-binding cassette (ABC) transporters is the essential cause of multidrug resistance (MDR), which is a significant hurdle to the success of chemotherapy in many cancers. Therefore, inhibiting the activity of ABC transporters may be a logical approach to circumvent MDR. Olmutinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), which has been approved in South Korea for advanced EGFR T790M-positive non-small cell lung cancer (NSCLC). Here, we found that olmutinib significantly increased the sensitivity of chemotherapy drug in ABCG2-overexpressing cells. Furthermore, olmutinib could also increase the retention of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABC transporter subfamily G member 2 (ABCG2)-overexpressing cells. In addition, olmutinib was found to stimulate ATPase activity and inhibit photolabeling of ABCG2 with [I]-iodoarylazidoprazosin (IAAP). However, olmutinib neither altered ABCG2 expression at protein and mRNA levels nor blocked EGFR, Her-2 downstream signaling of AKT and ERK. Importantly, olmutinib enhanced the efficacy of topotecan on the inhibition of S1-MI-80 cell xenograft growth. All the results suggest that olmutinib reverses ABCG2-mediated MDR by binding to ATP bind site of ABCG2 and increasing intracellular chemotherapeutic drug accumulation. Our findings encouraged to further clinical investigation on combination therapy of olmutinib with conventional chemotherapeutic drugs in ABCG2-overexpressing cancer patients.
10.1016/j.apsb.2018.06.002
WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics.
Bisson Isabelle,Prowse David M
Cell research
Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear beta-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector beta-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.
10.1038/cr.2009.43
P-glycoprotein, CYP3A, and plasma carboxylesterase determine brain and blood disposition of the mTOR Inhibitor everolimus (Afinitor) in mice.
Tang Seng Chuan,Sparidans Rolf W,Cheung Ka Lei,Fukami Tatsuki,Durmus Selvi,Wagenaar Els,Yokoi Tsuyoshi,van Vlijmen Bart J M,Beijnen Jos H,Schinkel Alfred H
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. EXPERIMENTAL DESIGN:We used wild-type, Abcb1a/1b(-/-), Abcg2(-/-), Abcb1a/1b;Abcg2(-/-), and Cyp3a(-/-) mice to study everolimus oral bioavailability and brain accumulation. RESULTS:Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b(-/-)and Abcb1a/1b;Abcg2(-/-), but not Abcg2(-/-)mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (∼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a(-/-), but not Abcb1a/1b(-/-), Abcg2(-/-), or Abcb1a/1b;Abcg2(-/-)mice, displayed highly (>5-fold) increased oral availability of everolimus. CONCLUSIONS:Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug-drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies.
10.1158/1078-0432.CCR-13-1759
Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity.
Nagai Shinjiro,Takenaka Kazumasa,Nachagari Deepa,Rose Charles,Domoney Kali,Sun Daxi,Sparreboom Alex,Schuetz John D
Cancer research
Purine nucleoside antimetabolites, such as clofarabine, are effective antileukemic agents. However, their effectiveness depends on an initial activation step in which they are monophosphorylated by deoxycytidine kinase (dCK). Some purine nucleoside antimetabolites and their monophosphate derivatives are exported by the ABC transporter ABCG2. Because clofarabine is a dCK substrate, and we show substantial variation in dCK and ABCG2 in myeloid leukemia, we hypothesized that the activity of dCK may modulate ABCG2-mediated resistance to clofarabine by regulating the formation of clofarabine monophosphate. We show that ABCG2 influence on clofarabine cytotoxicity was markedly influenced by dCK activity. When dCK expression was reduced by siRNA, clofarabine cytotoxicity was strongly reduced by enhanced ABCG2-mediated efflux. Conversely, dCK overexpression blunted ABCG2-mediated efflux of clofarabine by increasing the formation of clofarabine nucleotides. The use of an ABCG2 inhibitor confirmed that ABCG2 export of clofarabine is maximal when dCK levels are minimal. Analysis of intracellular clofarabine metabolites suggested that ABCG2 exported clofarabine more readily than clofarabine monophosphate. That ABCG2 primarily effluxes clofarabine, but not chlorfarabine-monophosphate, was confirmed by HPLC analysis of drug exported from ABCG2-overexpressing cells. Because the level and function of dCK and ABCG2 vary substantially among other types of cancer, these findings have important implications not only for clofarabine therapy but for purine nucleoside therapy in general. Therefore, we propose that addition of ABCG2 inhibitors would effectively increase the antitumor efficacy of purine nucleosides by blocking drug efflux that may be a significant mode of resistance when dCK levels are low.
10.1158/0008-5472.CAN-10-1919
The net electrostatic potential and hydration of ABCG2 affect substrate transport.
Nature communications
ABCG2 is a medically important ATP-binding cassette transporter with crucial roles in the absorption and distribution of chemically-diverse toxins and drugs, reducing the cellular accumulation of chemotherapeutic drugs to facilitate multidrug resistance in cancer. ABCG2's capacity to transport both hydrophilic and hydrophobic compounds is not well understood. Here we assess the molecular basis for substrate discrimination by the binding pocket. Substitution of a phylogenetically-conserved polar residue, N436, to alanine in the binding pocket of human ABCG2 permits only hydrophobic substrate transport, revealing the unique role of N436 as a discriminator. Molecular dynamics simulations show that this alanine substitution alters the electrostatic potential of the binding pocket favoring hydration of the transport pore. This change affects the contact with substrates and inhibitors, abrogating hydrophilic compound transport while retaining the transport of hydrophobic compounds. The N436 residue is also required for optimal transport inhibition of ABCG2, as many inhibitors are functionally impaired by this ABCG2 mutation. Overall, these findings have biomedical implications, broadly extending our understanding of substrate and inhibitor interactions.
10.1038/s41467-023-40610-5
The role of multidrug resistance efflux transporters in antifolate resistance and folate homeostasis.
Assaraf Yehuda G
Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy
Members of the ATP-binding cassette (ABC) transporters including P-glycoprotein (Pgp/ABCB1), multidrug resistance proteins (MRPs/ABCC) as well as breast cancer resistance protein (BCRP/ABCG2) function as ATP-dependent drug efflux transporters, which form a unique defense network against multiple chemotherapeutic drugs and cellular toxins. Among antitumor agents is the important group of folic acid antimetabolites known as antifolates. Antifolates such as methotrexate (MTX), pemetrexed and raltitrexed exert their cytotoxic activity via potent inhibition of folate-dependent enzymes essential for purine and pyrimidine nucleotide biosynthesis and thereby block DNA replication. Overexpression of MRPs and BCRP confers resistance upon malignant cells to various hydrophilic and lipophilic antifolates. Apart from their central role in mediating resistance to antifolates and other anticancer drugs, MRPs and BCRP have been recently shown to transport naturally occurring reduced folates. This was inferred from various complementary systems as follows: (a) Cell-free systems including ATP-dependent uptake of radiolabeled folate/MTX into purified inside-out membrane vesicles from stable transfectants and/or cells overexpressing these transporters, (b) Decreased accumulation of radiolabeled folate/MTX in cultured tumor cells overexpressing these transporters, as well as (c) In vivo rodent models such as Eisi hyperbillirubinemic rats (EHBR) that hereditarily lack MRP2 in their canalicular membrane and thereby display a bile that is highly deficient in various reduced folate cofactors and MTX, when compared with wild type Sprague-Dawley (SD) rats. In all cases, these folate/antifolate transporters functioned as high capacity, low affinity ATP-driven exporters. While the mechanism of cellular retention of (anti)folates is mediated via (anti)folylpolyglutamylation, certain efflux transporters including MRP5 (ABCC5) and BCRP were shown to transport both mono-, di- as well as triglutamate derivatives of MTX and folic acid. Furthermore, overexpression of MRPs and BCRP has been shown to result in decreased cellular folate pools, whereas loss of ABC transporter expression brought about a significant expansion in the intracellular reduced folate pool. The latter finding has important implications to antifolate-based chemotherapy as an augmented cellular folate pool results in a significant level of resistance to certain antifolates. Hence, the aims of the present review are: (a) To summarize and discuss the cumulative evidence supporting a functional role for various multidrug resistance efflux transporters of the ABC superfamily which mediate resistance to hydrophilic and lipophilic antifolates, (b) To describe and evaluate the recent data suggesting a role for these efflux transporters in regulation of cellular folate homeostasis under folate replete and deplete conditions. Furthermore, novel developments and future perspectives regarding the identification of novel antifolate target proteins and mechanisms of action, as well as rationally designed emerging drug combinations containing antifolates along with receptor tyrosine kinase inhibitors are being discussed.
10.1016/j.drup.2006.09.001
MET inhibitor tepotinib antagonizes multidrug resistance mediated by ABCG2 transporter: and study.
Acta pharmaceutica Sinica. B
Overexpression of ABCG2 transporter in cancer cells has been linked to the development of multidrug resistance (MDR), an obstacle to cancer therapy. Our recent study uncovered that the MET inhibitor, tepotinib, is a potent reversal agent for ABCB1-mediated MDR. In the present study, we reported for the first time that the MET inhibitor tepotinib can also reverse ABCG2-mediated MDR and by directly binding to the drug-binding site of ABCG2 and reversibly inhibiting ABCG2 drug efflux activity, therefore enhancing the cytotoxicity of substrate drugs in drug-resistant cancer cells. Furthermore, the ABCB1/ABCG2 double-transfected cell model and gene knockout cell model demonstrated that tepotinib specifically inhibits the two MDR transporters. In mice bearing drug-resistant tumors, tepotinib increased the intratumoral accumulation of ABCG2 substrate drug topotecan and enhanced its antitumor effect. Therefore, our study provides a new potential of repositioning tepotinib as an ABCG2 inhibitor and combining tepotinib with substrate drugs to antagonize ABCG2-mediated MDR.
10.1016/j.apsb.2021.12.018
Octamer 4 (Oct4) mediates chemotherapeutic drug resistance in liver cancer cells through a potential Oct4-AKT-ATP-binding cassette G2 pathway.
Wang Xiao Qi,Ongkeko Weg M,Chen Lin,Yang Zhen Fan,Lu Ping,Chen Kwok Kin,Lopez Jay P,Poon Ronnie T P,Fan Sheung Tat
Hepatology (Baltimore, Md.)
UNLABELLED:Chemoresistance presents a major obstacle to the efficacy of chemotherapeutic treatment of cancers. Using chemotherapeutic drugs to select drug-resistant cancer cells in hepatocellular carcinoma (HCC) and several other cancer cell lines, we demonstrate that chemoresistant cells displayed cancer stem cell features, such as increased self-renewal ability, cell motility, multiple drug resistance, and tumorigenicity. Octamer 4 (Oct4) messenger RNA (mRNA) levels were dramatically increased in chemoresistant cancer cells due to DNA demethylation regulation of Oct4. By functional study, Oct4 overexpression enhanced whereas Oct4 knockdown reduced liver cancer cell resistance to chemotherapeutic drugs in vitro and in xenograft tumors. It is known that the Oct4-TCL1-AKT pathway acts on embryonic stem cells and cancer stem cells in cell proliferation through inhibition of apoptosis. We further demonstrate that Oct4 overexpression induced activation of TCL1, AKT, and ABCG2 to mediate chemoresistance, which can be overcome by addition of the PI3K/AKT inhibitor; therefore, a direct pathway of Oct4-TCL1-AKT-ABCG2 or a combination of Oct4-TCL1-AKT with the AKT-ABCG2 pathway could be a potential new mechanism involved in liver cancer cell chemoresistance. Moreover, the clinical significance of the Oct4-AKT-ABCG2 pathway can be demonstrated in HCC patients, with a strong correlation of expression patterns in human HCC tumors. The role of the Oct4-AKT-ABCG2 axis in cancer cell chemoresistant machinery suggests that AKT pathway inhibition (PI3K inhibitors) not only inhibits cancer cell proliferation, but may also enhance chemosensitivity by target potential chemoresistant cells. CONCLUSION:Oct4, a transcriptional factor of pluripotent cells, can mediate chemoresistance through a potential Oct4-AKT-ABCG2 pathway.
10.1002/hep.23692
Sensitivity and acquired resistance of BRCA1;p53-deficient mouse mammary tumors to the topoisomerase I inhibitor topotecan.
Zander Serge A L,Kersbergen Ariena,van der Burg Eline,de Water Niels,van Tellingen Olaf,Gunnarsdottir Sjöfn,Jaspers Janneke E,Pajic Marina,Nygren Anders O H,Jonkers Jos,Borst Piet,Rottenberg Sven
Cancer research
There is no tailored therapy yet for human basal-like mammary carcinomas. However, BRCA1 dysfunction is frequently present in these malignancies, compromising homology-directed DNA repair. This defect may serve as the tumor's Achilles heel and make the tumor hypersensitive to DNA breaks. We have evaluated this putative synthetic lethality in a genetically engineered mouse model for BRCA1-associated breast cancer, using the topoisomerase I (Top1) poison topotecan as monotherapy and in combination with poly(ADP-ribose) polymerase inhibition by olaparib. All 20 tumors tested were topotecan sensitive, but response heterogeneity was substantial. Although topotecan increased mouse survival, all tumors eventually acquired resistance. As mechanisms of in vivo resistance, we identified overexpression of Abcg2/Bcrp and markedly reduced protein levels of the drug target Top1 (without altered mRNA levels). Tumor-specific genetic ablation of Abcg2 significantly increased overall survival of topotecan-treated animals (P < 0.001), confirming the in vivo relevance of ABCG2 for topotecan resistance in a novel approach. Despite the lack of ABCG2, a putative tumor-initiating cell marker, none of the 11 Abcg2(-/-);Brca1(-/-);p53(-/-) tumors were eradicated, not even by the combination topotecan-olaparib. We find that olaparib substantially increases topotecan toxicity in this model, and we suggest that this might also happen in humans.
10.1158/0008-5472.CAN-09-3367
ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging.
Zhang Yimao,Bressler Joseph P,Neal Jeff,Lal Bachchu,Bhang Hyo-Eun C,Laterra John,Pomper Martin G
Cancer research
Bioluminescence imaging (BLI) is becoming indispensable to the study of transgene expression during development and, in many in vivo models of disease such as cancer, for high throughput drug screening in vitro. Because reaction of d-luciferin with firefly luciferase (fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now show that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP affects BLI signal output from the substrate d-luciferin. In vitro studies show that d-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ABCC1), or multidrug resistance protein 2 (MRP2/ABCC2). d-Luciferin uptake within cells is shown to be modulated by ABC transporter inhibitors, including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C. Images of xenografts engineered to express transgenic ABCG2/BCRP, as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally express ABCG2/BCRP, show that ABCG2/BCRP expression and function within regions of interest substantially influence d-luciferin-dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during d-luciferin-based BLI and suggest novel high throughput methods for identifying new ABCG2/BCRP inhibitors.
10.1158/0008-5472.CAN-07-0944
ABCG2 expression, function, and promoter methylation in human multiple myeloma.
Turner Joel G,Gump Jana L,Zhang Chunchun,Cook James M,Marchion Douglas,Hazlehurst Lori,Munster Pamela,Schell Michael J,Dalton William S,Sullivan Daniel M
Blood
We investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance.
10.1182/blood-2005-10-009084
Sildenafil is not a useful modulator of ABCB1 and ABCG2 mediated drug resistance in vivo.
Lin Fan,Hoogendijk Lisette,Buil Levi,Beijnen Jos H,van Tellingen Olaf
European journal of cancer (Oxford, England : 1990)
AIM:Recently, sildenafil was reported to be an inhibitor of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in vitro. We have now investigated the in vivo potency of sildenafil. METHODS:By using wild-type and Abcb1; Abcg2 knockout mice we have investigated the effect of sildenafil on the brain penetration of two substrate drugs (docetaxel and topotecan). Next we have investigated if sildenafil was able to improve the efficacy of doxorubicin against P-glycoprotein expressing CT26 colon cancer cells in syngeneic Balb/c mice. RESULTS:Sildenafil administered orally at a dose of 50mg/kg did not improve the brain penetration of docetaxel and topotecan, although the plasma level of sildenafil was already much higher than can be achieved in humans. On the other hand, sildenafil increased the plasma levels of the cytotoxic drugs, but not by inhibition of Abcb1 or Abcg2, since this effect was also seen in Abcb1;Abcg2 knockout mice. The brain penetration of sildenafil was more than 20-fold higher in Abcb1;Abcg2 mice versus wild-type mice, indicating that sildenafil is a good substrate of the two transporters. Sildenafil was also not able to improve the efficacy of doxorubicin against subcutaneous CT26 tumours. The doxorubicin level in tumour tissue did increase, but so did the concentration of doxorubicin in plasma and heart. CONCLUSION:These results demonstrate that the potency and specificity of sildenafil as an inhibitor of ABCB1 and ABCG2 is not sufficient to warrant further clinical testing of this agent in combination with anticancer drugs.
10.1016/j.ejca.2012.12.028
Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels.
Zhou Sheng,Zong Yang,Ney Paul A,Nair Geeta,Stewart Clinton F,Sorrentino Brian P
Blood
ABCG2/BCRP is a member of the adenosine triphosphate-binding cassette (ABC) transporter family and is expressed in intestine, kidney, and liver, where it modulates the absorption and excretion of xenobiotic compounds. ABCG2 is also expressed in hematopoietic stem cells and erythroid cells; however, little is known regarding its role in hematopoiesis. Abcg2 null mice have increased levels of protoporphyrin IX (PPIX) in erythroid cells, yet the mechanism for this remains uncertain. We have found that Abcg2 mRNA expression was up-regulated in differentiating erythroid cells, coinciding with increased expression of other erythroid-specific genes. This expression pattern was associated with significant amounts of ABCG2 protein on the membrane of mature peripheral blood erythrocytes. Erythroid cells engineered to express ABCG2 had significantly lower intracellular levels of PPIX, suggesting the modulation of PPIX level by ABCG2. This modulating activity was abrogated by treatment with a specific ABCG2 inhibitor, Ko143, implying that PPIX may be a direct substrate for the transporter. Taken together, our results demonstrate that ABCG2 plays a role in regulating PPIX levels during erythroid differentiation and suggest a potential role for ABCG2 as a genetic determinant in erythropoietic protoporphyria.
10.1182/blood-2004-04-1566
ABCG2/BCRP: Specific and Nonspecific Modulators.
Peña-Solórzano Diana,Stark Simone Alexandra,König Burkhard,Sierra Cesar Augusto,Ochoa-Puentes Cristian
Medicinal research reviews
Multidrug resistance (MDR) in cancer cells is the development of resistance to a variety of structurally and functionally nonrelated anticancer drugs. This phenomenon has become a major obstacle to cancer chemotherapy seriously affecting the clinical outcome. MDR is associated with increased drug efflux from cells mediated by an energy-dependent mechanism involving the ATP-binding cassette (ABC) transporters, mainly P-glycoprotein (ABCB1), the MDR-associated protein-1 (ABCC1), and the breast cancer resistance protein (ABCG2). The first two transporters have been widely studied already and reviews summarized the results. The ABCG2 protein has been a subject of intense study since its discovery as its overexpression has been detected in resistant cell lines in numerous types of human cancers. To date, a long list of modulators of ABCG2 exists and continues to increase. However, little is known about the clinical consequences of ABCG2 modulation. This makes the design of novel, potent, and nontoxic inhibitors of this efflux protein a major challenge to reverse MDR and thereby increase the success of chemotherapy. The aim of the present review is to describe and highlight specific and nonspecific modulators of ABCG2 reported to date based on the selectivity of the compounds, as many of them are effective against one or more ABC transport proteins.
10.1002/med.21428
and Evaluation of ABCG2 (BCRP) Inhibitors Derived from Ko143.
Journal of medicinal chemistry
Breast cancer resistance protein (BCRP, ABCG2) is an efflux transporter that plays a crucial role in multidrug resistance to antineoplastic drugs. Ko143, an analogue of the natural product fumitremorgin C, is a potent inhibitor of ABCG2 but is rapidly hydrolyzed to an inactive metabolite . To identify ABCG2 inhibitors with improved metabolic stability, we have assessed a series of Ko143 analogues for their ability to inhibit ABCG2-mediated transport in -transduced MDCK II cells and determined the stability of the most potent compounds in liver microsomes. The most promising analogues were evaluated by positron emission tomography. , three of the tested analogues were potent ABCG2 inhibitors and stable in microsomes. , they increased the distribution of the ABCG2/ABCB1 substrate [C]tariquidar to the brain both in wild-type (with Abcb1a/b transport blocked by tariquidar) and Abcb1a/b(-/-) mice. One analogue was more potent than Ko143 in both animal models.
10.1021/acs.jmedchem.3c00168
Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells and o.
Zeng Fanpu,Wang Fang,Zheng Zongheng,Chen Zhen,Wah To Kenneth Kin,Zhang Hong,Han Qian,Fu Liwu
Acta pharmaceutica Sinica. B
Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR), which severely limits the clinical efficacy of chemotherapy. Currently, there is no FDA-approved MDR modulator for clinical use. In this study, rociletinib (CO-1686), a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells and in MDR tumor xenografts in nude mice, without incurring additional toxicity. Mechanistic studies revealed that in ABCG2-overexpressing cancer cells, rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates. Moreover, rociletinib, inhibited the ATPase activity, and competed with [I] iodoarylazidoprazosin (IAAP) photolabeling of ABCG2. However, ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib. In addition, rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function, thus increasing the cellular accumulation of the transporter substrate anticancer drugs. The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.
10.1016/j.apsb.2020.01.008
Restoration of chemosensitivity for doxorubicin and cisplatin in chondrosarcoma in vitro: BCL-2 family members cause chemoresistance.
van Oosterwijk J G,Herpers B,Meijer D,Briaire-de Bruijn I H,Cleton-Jansen A M,Gelderblom H,van de Water B,Bovée J V M G
Annals of oncology : official journal of the European Society for Medical Oncology
BACKGROUND:Chondrosarcomas are malignant cartilage-forming tumors notorious for their resistance to conventional chemo- and radiotherapy. Postulated explanations describe the inaccessibility due to abundant hyaline cartilaginous matrix, presence of multidrug resistance (MDR) pumps, and expression of anti-apoptotic BCL-2 family members. MATERIALS AND METHODS:We studied the sensitivity of chondrosarcoma cell lines (SW1353, CH2879, JJ012, OUMS27) and two primary cultures for doxorubicin and cisplatin. We examined the role of extracellular matrix using three-dimensional (3D) pellet models and MDR pump activity using fluorescence-activated cell sorter analysis. The role of BCL-2 family members was investigated using the BH3 mimetic ABT-737. RESULTS:Chondrosarcoma cells showed highest resistance to cisplatin. 3D cell pellets, morphologically strongly resembling chondrosarcoma in vivo, confirmed nuclear incorporation of doxorubicin. MDR pump activity was heterogeneous among cultures. Chondrosarcoma cells responded to ABT-737 and combination with doxorubicin led to complete loss of cell viability and apoptosis with cytochrome C release. CONCLUSIONS:Despite MDR pump activity and abundance of hyaline cartilaginous matrix, doxorubicin is able to accumulate in the cell nuclei. By repairing the apoptotic machinery, we were able to sensitize chondrosarcoma cells to doxorubicin and cisplatin, indicating an important role for BCL-2 family members in chemoresistance and a promising new treatment strategy for inoperable chondrosarcoma.
10.1093/annonc/mdr512
Overexpression of ABCC1 and ABCG2 confers resistance to talazoparib, a poly (ADP-Ribose) polymerase inhibitor.
Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy
AIMS:The overexpression of ABC transporters on cancer cell membranes is one of the most common causes of multidrug resistance (MDR). This study investigates the impact of ABCC1 and ABCG2 on the resistance to talazoparib (BMN-673), a potent poly (ADP-ribose) polymerase (PARP) inhibitor, in ovarian cancer treatment. METHODS:The cell viability test was used to indicate the effect of talazoparib in different cell lines. Computational molecular docking analysis was conducted to simulate the interaction between talazoparib and ABCC1 or ABCG2. The mechanism of talazoparib resistance was investigated by constructing talazoparib-resistant subline A2780/T4 from A2780 through drug selection with gradually increasing talazoparib concentration. RESULTS:Talazoparib cytotoxicity decreased in drug-selected or gene-transfected cell lines overexpressing ABCC1 or ABCG2 but can be restored by ABCC1 or ABCG2 inhibitors. Talazoparib competitively inhibited substrate drug efflux activity of ABCC1 or ABCG2. Upregulated ABCC1 and ABCG2 protein expression on the plasma membrane of A2780/T4 cells enhances resistance to other substrate drugs, which could be overcome by the knockout of either gene. In vivo experiments confirmed the retention of drug-resistant characteristics in tumor xenograft mouse models. CONCLUSIONS:The therapeutic efficacy of talazoparib in cancer may be compromised by its susceptibility to MDR, which is attributed to its interactions with the ABCC1 or ABCG2 transporters. The overexpression of these transporters can potentially diminish the therapeutic impact of talazoparib in cancer treatment.
10.1016/j.drup.2023.101028
A novel 7-modified camptothecin analog overcomes breast cancer resistance protein-associated resistance in a mitoxantrone-selected colon carcinoma cell line.
Perego P,De Cesare M,De Isabella P,Carenini N,Beggiolin G,Pezzoni G,Palumbo M,Tartaglia L,Pratesi G,Pisano C,Carminati P,Scheffer G L,Zunino F
Cancer research
We selected a mitoxantrone-resistant HT29 colon carcinoma cell line (HT29/MIT) that exhibited a very high degree of resistance to the selecting agent and marked resistance to topotecan and SN38, but limited resistance to doxorubicin. The development of drug resistance was independent of expression of P-glycoprotein or multidrug resistance-associated protein but was associated with high up-regulation of the breast carcinoma resistance protein (BCRP) as shown by Western blot analysis. BCRP overexpression was associated with a reduced intracellular accumulation of topotecan, a known substrate for BCRP. Conversely, a lipophilic 7-modified camptothecin analogue (ST1481) displayed a complete lack of cross-resistance in HT29/MIT cells, suggesting that the drug was not a substrate for BCRP because no defects in intracellular accumulation were found. This conclusion is consistent with the antitumor efficacy of ST1481 against a BCRP-expressing tumor. These results may have therapeutic implications because the antitumor efficacy of ST1481 is in part related to a good bioavailability after oral administration, and the drug is currently under Phase I clinical evaluation.
Functional ABCG2 is overexpressed on primary CML CD34+ cells and is inhibited by imatinib mesylate.
Jordanides Niove E,Jorgensen Heather G,Holyoake Tessa L,Mountford Joanne C
Blood
Imatinib mesylate (IM) therapy for chronic myeloid leukemia (CML) has transformed the treatment of this disease. However, the vast majority of patients, despite major responses, still harbor Philadelphia chromosome-positive (Ph(+)) cells. We have described a population of primitive Ph(+) cells that are insensitive to IM and may be a source of IM resistance. Cell line studies have suggested that the drug transporter ABCG2 may be a mediator of IM resistance, however there is considerable debate about whether IM is an ABCG2 substrate or inhibitor. We demonstrate here that primitive CML CD34(+) cells aberrantly overexpress functional ABCG2 but that cotreatment with IM and an ABCG2 inhibitor does not potentiate the effect of IM. We definitively show that IM is an inhibitor of, but not a substrate for, ABCG2 and that, therefore, ABCG2 does not modulate intracellular concentrations of IM in this clinically relevant cell population.
10.1182/blood-2006-02-003145
Delivery of Anti-miRNA for Triple-Negative Breast Cancer Therapy Using RNA Nanoparticles Targeting Stem Cell Marker CD133.
Yin Hongran,Xiong Gaofeng,Guo Sijin,Xu Congcong,Xu Ren,Guo Peixuan,Shu Dan
Molecular therapy : the journal of the American Society of Gene Therapy
Triple-negative breast cancer (TNBC) is an aggressive disease with a short median time from relapse to death. The increased aggressiveness, drug resistance, disease relapse, and metastasis are associated with the presence of stem cells within tumors. Several stem cell markers, such as CD24, CD44, CD133, ALDH1, and ABCG2, have been reported, but their roles in breast cancer tumorigenesis remain unclear. Herein, we apply RNA nanotechnology to deliver anti-microRNA (miRNA) for TNBC therapy. The thermodynamically and chemically stable three-way junction (3WJ) motif was utilized as the scaffold to carry an RNA aptamer binding to CD133 receptor and a locked nuclei acid (LNA) sequence for miRNA21 inhibition. Binding assays revealed the specific uptake of the nanoparticles to breast cancer stem cells (BCSCs) and TNBC cells. Functional assays showed that cancer cell migration was reduced, miR21 expression was inhibited, and downstream tumor suppressor PTEN and PDCD4 expressions were upregulated. In vitro and in vivo studies revealed that these therapeutic RNA nanoparticles did not induce cytokine secretion. Systemic injection of these RNA nanoparticles in animal trial demonstrated high specificity in TNBC tumor targeting and high efficacy for tumor growth inhibition. These results revealed the clinical translation potential of these RNA nanoparticles for TNBC therapy.
10.1016/j.ymthe.2019.04.018
Squamous cell cancers contain a side population of stem-like cells that are made chemosensitive by ABC transporter blockade.
Loebinger M R,Giangreco A,Groot K R,Prichard L,Allen K,Simpson C,Bazley L,Navani N,Tibrewal S,Davies D,Janes S M
British journal of cancer
Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteristics including ATP-dependent drug efflux and elevated tumorigenic potential. To determine whether aerodigestive squamous cell carcinomas (SCCs) contain a subpopulation of cancer stem cell-like cells, we performed Hoechst dye efflux assays using four independent cell lines. Results revealed the presence of a rare, drug effluxing stem cell-like side population (SP) of cells within all cell lines tested (SCC-SP cells). These cells resembled previously characterised epithelial stem cells, and SCC-SP cell abundance was positively correlated with overall cellular density and individual cell quiescence. Serial SCC-SP fractionation and passaging increased their relative abundance within the total cell population. Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo. Despite this, SCC-SP cells remained chemotherapeutically sensitive upon ATP-dependent transporter inhibition. Overall, these findings suggest that the existence of ATP transporter-dependent cancer stem-like cells may be relatively common, particularly within established tumours. Future chemotherapeutic strategies should therefore consider coupling identification and targeting of this potential stem cell-like population with standard treatment methodologies.
10.1038/sj.bjc.6604185
A multiple-targets alkaloid nuciferine overcomes paclitaxel-induced drug resistance in vitro and in vivo.
Liu Rui-Ming,Xu Peng,Chen Qi,Feng Sen-Ling,Xie Ying
Phytomedicine : international journal of phytotherapy and phytopharmacology
OBJECTIVE:Multidrug resistance (MDR) is the major barrier to the successful treatment of chemotherapy. Compounds from nature products working as MDR sensitizers provided new treatment strategies for chemo-resistant cancers patients. METHODS:We investigated the reversal effects of nuciferine (NF), an alkaloid from Nelumbo nucifera and Nymphaea caerulea, on the paclitaxel (PTX) resistance ABCB1-overexpressing cancer in vitro and in vivo, and explored the underlying mechanism by evaluating drug sensitivity, cell cycle perturbations, intracellular accumulation, function and protein expression of efflux transporters as well as molecular signaling involved in governing transporters expression and development of MDR in cancer. RESULTS:NF overcomes the resistance of chemotherapeutic agents included PTX, doxorubicin (DOX), docetaxel, and daunorubicin to HCT-8/T and A549/T cancer cells. Notably, NF suppressed the colony formation of MDR cells in vitro and the tumor growth in A549/T xenograft mice in vivo, which demonstrated a very strong synergetic cytotoxic effect between NF and PTX as combination index (CI) (CI<0.1) indicated. Furthermore, NF increased the intracellular accumulation of P-gp substrates included DOX and Rho123 in the MDR cells and inhibited verapamil-stimulated ATPase activity. Mechanistically, inhibition of PI3K/AKT/ERK pathways by NF suppressed the activation of Nrf2 and HIF-1α, and further reduced the expression of P-gp and BCRP, contributing to the sensitizing effects of NF against MDR in cancer. CONCLUSION:This novel finding provides a promising treatment strategy for overcoming MDR and improving the efficiency of chemotherapy by using a multiple-targets MDR sensitizer NF.
10.1016/j.phymed.2020.153342
Systematic screening identifies ABCG2 as critical factor underlying synergy of kinase inhibitors with transcriptional CDK inhibitors.
Breast cancer research : BCR
BACKGROUND:Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited treatment options and poor clinical prognosis. Inhibitors of transcriptional CDKs are currently under thorough investigation for application in the treatment of multiple cancer types, including breast cancer. These studies have raised interest in combining these inhibitors, including CDK12/13 inhibitor THZ531, with a variety of other anti-cancer agents. However, the full scope of these potential synergistic interactions of transcriptional CDK inhibitors with kinase inhibitors has not been systematically investigated. Moreover, the mechanisms behind these previously described synergistic interactions remain largely elusive. METHODS:Kinase inhibitor combination screenings were performed to identify kinase inhibitors that synergize with CDK7 inhibitor THZ1 and CDK12/13 inhibitor THZ531 in TNBC cell lines. CRISPR-Cas9 knockout screening and transcriptomic evaluation of resistant versus sensitive cell lines were performed to identify genes critical for THZ531 resistance. RNA sequencing analysis after treatment with individual and combined synergistic treatments was performed to gain further insights into the mechanism of this synergy. Kinase inhibitor screening in combination with visualization of ABCG2-substrate pheophorbide A was used to identify kinase inhibitors that inhibit ABCG2. Multiple transcriptional CDK inhibitors were evaluated to extend the significance of the found mechanism to other transcriptional CDK inhibitors. RESULTS:We show that a very high number of tyrosine kinase inhibitors synergize with the CDK12/13 inhibitor THZ531. Yet, we identified the multidrug transporter ABCG2 as key determinant of THZ531 resistance in TNBC cells. Mechanistically, we demonstrate that most synergistic kinase inhibitors block ABCG2 function, thereby sensitizing cells to transcriptional CDK inhibitors, including THZ531. Accordingly, these kinase inhibitors potentiate the effects of THZ531, disrupting gene expression and increasing intronic polyadenylation. CONCLUSION:Overall, this study demonstrates the critical role of ABCG2 in limiting the efficacy of transcriptional CDK inhibitors and identifies multiple kinase inhibitors that disrupt ABCG2 transporter function and thereby synergize with these CDK inhibitors. These findings therefore further facilitate the development of new (combination) therapies targeting transcriptional CDKs and highlight the importance of evaluating the role of ABC transporters in synergistic drug-drug interactions in general.
10.1186/s13058-023-01648-x
Calcium channel α2δ1 subunit is a functional marker and therapeutic target for tumor-initiating cells in non-small cell lung cancer.
Cell death & disease
It is hypothesized that tumor-initiating cells (TICs) with stem cell-like properties constitute a sustaining force to drive tumor growth and renew fully established malignancy. However, the identification of such a population in non-small cell lung carcinoma (NSCLC) has been hindered by the lacking of reliable surface markers, and very few of the currently available surface markers are of functional significance. Here, we demonstrate that a subpopulation of TICs could be specifically defined by the voltage-gated calcium channel α2δ1 subunit from non-small cell lung carcinoma (NSCLC) cell lines and clinical specimens. The α2δ1 NSCLC TICs are refractory to conventional chemotherapy, and own stem cell-like properties such as self-renewal, and the ability to generate heterogeneous tumors in NOD/SCID mice. Moreover, α2δ1 NSCLC cells are more enriched for TICs than CD133, or CD166 cells. Interestingly, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca influx into cells, which subsequently activate Calcineurin/NFATc2 signaling that directly activates the expression of NOTCH3, ABCG2. Importantly, a specific antibody against α2δ1 has remarkably therapeutic effects on NSCLC xenografts by eradicating TICs. Hence, targeting α2δ1 to prevent calcium influx provides a novel strategy for targeted therapy against TICs of NSCLC.
10.1038/s41419-021-03522-0
Vimentin as a potential therapeutic target in sorafenib resistant HepG2, a HCC model cell line.
Makol Ankita,Kaur Harpreet,Sharma Sakshi,Kanthaje Shruthi,Kaur Ramanpreet,Chakraborti Anuradha
Clinical and molecular hepatology
BACKGROUND/AIMS:Hepatocellular carcinoma (HCC) is the most common liver cancer with high mortality rate in patients suffering from liver diseases. The drug of choice used in advanced-stage of HCC is sorafenib. However, adaptive resistance has been observed in HCC patients undergoing long-term sorafenib treatment, lowering its effectiveness. Hence, it is important to overcome drug resistance to improve overall management of HCC. Here, we have identified a candidate biomarker for sorafenib resistance in a HCC model cell line, HepG2. METHODS:Initially, comparative proteomic profiling of parental HepG2 [HepG2 (P)] and sorafenib-resistant HepG2 [HepG2 (R)] cells was performed via MALDI (matrix-assisted laser desorption/ionization) which revealed the deregulation of vimentin in HepG2 (R) cells. Gene and protein level expression of vimentin was also observed through quantitative real-time polymerase chain reaction (qRT PCR) and fluorescence-activated cell sorting (FACS), respectively. Furthermore, withaferin A was used to study regulation of vimentin expression and its significance in sorafenib resistance. RESULTS:Both gene and protein level of vimentin expression was found to be downregulated in HepG2 (R) in comparison to HepG2 (P). Interestingly, the study demonstrated that withaferin A further lowered the expression of vimentin in HepG2 (R) cells in a dose-dependent manner. Also, inhibition of vimentin lowered ABCG2 expression and decreased cell viability in parental as well as sorafenib resistant HepG2 cells. CONCLUSION:Hence, our study for the first time highlighted the probable therapeutic potential of vimentin in sorafenib resistant HepG2, a HCC model cell line.
10.3350/cmh.2019.0031
Breast cancer resistance protein-mediated efflux of androgen in putative benign and malignant prostate stem cells.
Huss Wendy J,Gray Danny R,Greenberg Norman M,Mohler James L,Smith Gary J
Cancer research
Malignantly transformed stem cells represent a potential common nidus for the primary cancer and the recurrent cancer that arises after treatment failure. Putative prostate stem cells and prostate tumor stem cells in benign and malignant human prostate tissue, in primary human prostate xenografts, and in the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, are defined by expression of breast cancer resistance protein (BCRP), a marker of pluripotent hematopoietic, muscle, and neural stem cells, and by an absence of androgen receptor (AR) protein. Inhibition of BCRP-mediated efflux of dihydrotestosterone by novobiocin or fumitremorgin C in a rat prostate progenitor cell line that expresses BCRP and AR mRNAs, but minimal AR protein, results in stabilization and nuclear translocation of AR protein, providing a mechanism for lack of AR protein in BCRP-expressing stem cells. In both benign and malignant human prostate tissue, the rare epithelial cells that express BCRP and lack AR protein are localized in the basal cell compartment, survive androgen deprivation, and maintain proliferative potential in the hypoxic, androgen-deprived prostate. Putative prostate tumor stem cells that express BCRP but not AR protein in TRAMP are the source of a BCRP-negative and AR-negative, Foxa2- and SV40Tag-expressing, transit amplifying compartment that progresses to the poorly differentiated carcinomas that arise rapidly after castration. Therefore, BCRP expression isolates prostate stem/tumor stem cells from the prostate tissue microenvironment through constitutive efflux of androgen, protecting the putative tumor stem cells from androgen deprivation, hypoxia, or adjuvant chemotherapy, and providing the nidus for recurrent prostate cancer.
10.1158/0008-5472.CAN-04-2548
Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies.
Jiang X,Zhao Y,Smith C,Gasparetto M,Turhan A,Eaves A,Eaves C
Leukemia
The leukemic stem cells in patients with chronic myeloid leukemia (CML) are well known to be clinically resistant to conventional chemotherapy and may also be relatively resistant to BCR-ABL-targeted drugs. Here we show that the lesser effect of imatinib mesylate (IM) on the 3-week output of cells produced in vitro from lin(-)CD34(+)CD38(-) CML (stem) cells compared with cultures initiated with the CD38(+) subset of lin(-)CD34(+) cells is markedly enhanced (>10-fold) when conditions of reduced growth factor stimulation are used. Quantitative analysis of genes expressed in these different CML subsets revealed a differentiation-associated decrease in IL-3 and G-CSF transcripts, a much more profound decrease in expression of BCR-ABL than predicted by changes in BCR expression, decreasing expression of ABCB1/MDR and ABCG2 and increasing expression of OCT1. p210(BCR-ABL) and kinase activity were also higher in the lin(-)CD34(+)CD38(-) cells and formal evidence that increasing BCR-ABL expression decreases IM sensitivity was obtained from experiments with a cell line model. Nevertheless, within the entire CD34(+) subset of CML cells, BCR-ABL expression was not strongly affected by changes in cell cycle status. Taken together, these results provide the first evidence of multiple mechanisms of innate IM resistance in primitive and quiescent CML cells.
10.1038/sj.leu.2404609
Consequences of bile duct obstruction on intestinal expression and function of multidrug resistance-associated protein 2.
Dietrich Christoph G,Geier Andreas,Salein Nina,Lammert Frank,Roeb Elke,Oude Elferink Ronald P J,Matern Siegfried,Gartung Carsten
Gastroenterology
BACKGROUND & AIMS:Multidrug resistance-associated protein 2 (MRP2), a transporter of organic anions in hepatocytes, renal epithelial cells, and enterocytes, is differentially regulated in liver and kidney during cholestasis, but little is known about its regulation in the intestine. METHODS:We investigated duodenal protein expression of MRP2 in male Sprague-Dawley rats with bile duct ligation (BDL) or biliary diversion as well as in 20 cholestatic patients with biliary obstruction. RESULTS:In biliary obstruction, but not biliary depletion, intestinal Mrp2 protein mass was reduced to 9.3% +/- 5.5% of controls and mRNA to 40.5% +/- 20.8% of controls after 7 days. Binding of RXR alpha:RAR alpha heterodimers to the Mrp2 promoter element was significantly reduced in BDL rats. Cytokine blockade identified IL-1 beta as the responsible inducer of Mrp2 down-regulation. In humans with obstructive cholestasis, intestinal MRP2 protein expression was reduced to 27.3% +/- 20.3% of control patients; this reduction correlated with the duration of cholestasis and was reversible after reconstitution of bile flow by stenting of the common bile duct. However, no significant differences in MRP2 mRNA levels were detected by RT-PCR in humans. Intestinal protein expression of P-glycoprotein, breast cancer resistance protein (BCRP), and MRP3 was unchanged. In BDL rats, oral bioavailability of the Mrp2 substrate and food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was elevated 2.5 times compared with sham-operated rats. CONCLUSIONS:Cholestasis promotes down-regulation of MRP2 expression in the duodenum of rats and humans. Selective down-regulation during cholestasis might be the consequence of species-specific transcriptional and posttranscriptional mechanisms and contributes to higher bioavailability of a food-derived carcinogen.
10.1053/j.gastro.2003.12.046
Metabolic regulation of cancer cell side population by glucose through activation of the Akt pathway.
Liu P-P,Liao J,Tang Z-J,Wu W-J,Yang J,Zeng Z-L,Hu Y,Wang P,Ju H-Q,Xu R-H,Huang P
Cell death and differentiation
Side population (SP) cells within tumors are a small fraction of cancer cells with stem-like properties that can be identified by flow cytometry analysis based on their high ability to export certain compounds such as Hoechst 33342 and chemotherapeutic agents. The existence of stem-like SP cells in tumors is considered as a key factor contributing to drug resistance, and presents a major challenge in cancer treatment. Although it has been recognized for some time that tumor tissue niches may significantly affect cancer stem cells (CSCs), the role of key nutrients such as glucose in the microenvironment in affecting stem-like cancer cells and their metabolism largely remains elusive. Here we report that SP cells isolated from human cancer cells exhibit higher glycolytic activity compared to non-SP cells. Glucose in the culture environment exerts a profound effect on SP cells as evidenced by its ability to induce a significant increase in the percentage of SP cells in the overall cancer cell population, and glucose starvation causes a rapid depletion of SP cells. Mechanistically, glucose upregulates the SP fraction through ATP-mediated suppression of AMPK and activation of the Akt pathway, leading to elevated expression of the ATP-dependent efflux pump ABCG2. Importantly, inhibition of glycolysis by 3-BrOP significantly reduces SP cells in vitro and impairs their ability to form tumors in vivo. Our data suggest that glucose is an essential regulator of SP cells mediated by the Akt pathway, and targeting glycolysis may eliminate the drug-resistant SP cells with potentially significant benefits in cancer treatment.
10.1038/cdd.2013.131
c-Met represents a potential therapeutic target for personalized treatment in hepatocellular carcinoma.
You Hanning,Ding Wei,Dang Hien,Jiang Yixing,Rountree C Bart
Hepatology (Baltimore, Md.)
UNLABELLED:c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97-L, and MHCC97-H were used in this study to investigate the effect of c-Met inhibition using the small molecule selective c-Met tyrosine kinase inhibitor PHA665752. MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c-Met-positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met-positive MHCC97-L and MHCC97-H tumor growth, and PHA665752-treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met-negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met-positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression. CONCLUSION:c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.
10.1002/hep.24450
Functionally overlapping roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2) in the elimination of methotrexate and its main toxic metabolite 7-hydroxymethotrexate in vivo.
Vlaming Maria L H,Pala Zeliha,van Esch Anita,Wagenaar Els,de Waart Dirk R,van de Wetering Koen,van der Kruijssen Cornelia M M,Oude Elferink Ronald P J,van Tellingen Olaf,Schinkel Alfred H
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:ABCC2 (MRP2) and ABCG2 (BCRP) transport various endogenous and exogenous compounds, including many anticancer drugs, into bile, feces, and urine. We investigated the possibly overlapping roles of Abcg2 and Abcc2 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX). EXPERIMENTAL DESIGN:We generated and characterized Abcc2;Abcg2(-/-) mice, and used these to determine the overlapping roles of Abcc2 and Abcg2 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. RESULTS:Compared with wild-type, the plasma areas under the curve (AUC) for MTX were 1.6-fold and 2.0-fold higher in Abcg2(-/-) and Abcc2(-/-) mice, respectively, and 3.3-fold increased in Abcc2;Abcg2(-/-) mice. The biliary excretion of MTX was 23-fold reduced in Abcc2;Abcg2(-/-) mice, and the MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-MTX were not significantly altered in Abcg2(-/-) mice, but the areas under the curve were 6.2-fold and even 12.4-fold increased in Abcc2(-/-) and Abcc2;Abcg2(-/-) mice, respectively. This indicates that Abcc2 compensates for Abcg2 deficiency but that Abcg2 can only partly compensate for Abcc2 absence. Furthermore, 21-fold decreased biliary 7OH-MTX excretion in Abcc2;Abcg2(-/-) mice and substantial 7OH-MTX accumulation in the liver and kidney were seen. We additionally found that in the absence of Abcc2, Abcg2 mediated substantial urinary excretion of MTX and 7OH-MTX. CONCLUSIONS:Abcc2 and Abcg2 together are major determinants of MTX and 7OH-MTX pharmacokinetics. Variations in ABCC2 and/or ABCG2 activity due to polymorphisms or coadministered inhibitors may therefore substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.
10.1158/1078-0432.CCR-08-2940
Population analysis of erlotinib in adults and children reveals pharmacokinetic characteristics as the main factor explaining tolerance particularities in children.
White-Koning Melanie,Civade Elodie,Geoerger Birgit,Thomas Fabienne,Le Deley Marie-Cécile,Hennebelle Isabelle,Delord Jean-Pierre,Chatelut Etienne,Vassal Gilles
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:The aim of this pharmacokinetic-pharmacodynamic (PK-PD) analysis was to evaluate the pharmacologic characteristics of erlotinib and its main metabolite (OSI-420) in pediatric patients compared with those in adult patients. EXPERIMENTAL DESIGN:Plasma concentrations of erlotinib and OSI-420 of 46 children with malignant brain tumors included in a phase I study and 42 adults with head and neck carcinoma were analyzed by a population-pharmacokinetic method (NONMEM). The effect of several covariates and single nucleotide polymorphisms (SNP) in ABCB1, ABCG2, and CYP3A5 on pharmacokinetic parameters was evaluated. PK/PD relationships between plasma drug exposure Area Under the Curve (AUC) at day 1 and skin toxicity were studied in children and compared with the relationship observed in adults. RESULTS:A significant difference in erlotinib clearance (P = 0.0001), when expressed in L·h(-1)·kg(-1), was observed between children and adults with mean values of 0.146 and 0.095, respectively (mean difference = 0.051 L·h(-1)·kg(-1), SD = 0.0594). However, a common covariate model was obtained describing erlotinib clearance according to body weight, alanine aminotransferase, ABCB1, and CYP3A5 polymorphisms (2677G > T/A and 6986G > A) for both children and adult patients. The PK-PD relationship was very consistent between the children and adult groups with risk of skin toxicity rising with increasing erlotinib AUC. CONCLUSIONS:The nonlinear population approach applied to pharmacokinetic data combined with a pharmacokinetic-pharmacodynamic analysis revealed that the higher recommended dose in children (125 mg/m(2)/day) compared with adults (90 mg/m(2)/day) is mainly due to pharmacokinetic rather than pharmacodynamic particularities.
10.1158/1078-0432.CCR-10-3278
Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2.
Bi Xiaoli,Yuan Zhongwen,Qu Biao,Zhou Hua,Liu Zhongqiu,Xie Ying
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:Although silybin serves as a well-known hepatoprotective agent with prominent anti-inflammatory, anti-oxidant and anti-fibrotic activities, its low bioavailability limits its application in the treatment of chronic liver diseases. However, novel formulation products with increased solubility were not sufficient to achieve pharmacologically meaningful concentrations of silybin in the clinical studies even used at high dosage. HYPOTHESIS/PURPOSE:We hypothesized that inhibiting efflux transporter(s) and/or glucuronidation by piperine might enhance the bioavailability and efficacy of silybin. METHODS:Pharmacokinetics of silybin given alone or in-combination with piperine was determined by a validated LC-MS method. A CCl induced rat model of liver injury was prepared and verified for comparing the effects of silybin and combination treatment. To investigate the underlying mechanism, the inhibition effects of piperine on transportation of silybin were performed in Caco-2 and transfected MDCKII cell lines as well as sandwich-cultured rat hepatocytes (SCH). Human liver microsomes incubation was used for exploring the modulation effects of piperine on the phase-2 metabolism of silybin. RESULTS:In the present study, we demonstrated for the first time that piperine as a bioenhancer increased the bioavailability of silybin (146%- 181%), contributing to a boosted therapeutic effect in CCl-induced acute liver-injury rat model. The underlying mechanisms involved that piperine enhanced the absorption of silybin by inhibiting the efflux transporters including MRP2 and BCRP but not MDR1 in Caco-2 and transfected MDCKII cell lines. Moreover, piperine could inhibit the biliary excretion of silybin and conjugated metabolites in sandwich-cultured rat hepatocytes. Notably, we found that piperine did not affect the phase-2 metabolism of silybin. CONCLUSION:Efflux transporters play an important role in the pharmacokinetic behavior of flavolignans, and modulating these transporters by bioenhancer such as piperine could enhance the in vivo absorption of silybin, leading to more effective treatments.
10.1016/j.phymed.2018.09.217
Epigallocatechin-3-gallate attenuates head and neck cancer stem cell traits through suppression of Notch pathway.
Lee Sang Hyuk,Nam Hyo Jung,Kang Hyun Jung,Kwon Hye Won,Lim Young Chang
European journal of cancer (Oxford, England : 1990)
Most solid cancers including head and neck squamous carcinoma (HNSC) are believed to be initiated from and maintained by cancer stem cells (CSCs) that are responsible for treatment resistance, resulting in tumour relapse. Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea, can potently inhibit cancer growth and induce apoptosis in various cancers, including HNSC. However, its effect on HNSC CSCs is not well elucidated. In this study, we examined the anti-tumour effect of EGCG on HNSC CSCs. We demonstrated that EGCG inhibits the self-renewal capacity of HNSC CSCs by suppressing their sphere forming capacity, and attenuates the expression of stem cell markers, such as Oct4, Sox2, Nanog and CD44. EGCG treatment augmented cisplatin-mediated chemosensitivity by suppressing ABCC2 and ABCG2 transporter genes, which are putative molecules of treatment resistance of CSC. In addition, the combination treatment of EGCG and cisplatin inhibited tumour formation and induced apoptosis in a xenograft model. As one of mechanisms of suppression of HNSC CSC traits, EGCG decreased the transcriptional level of Notch, resulting in the inhibition of Notch signalling. Collectively, our data suggest that EGCG in combination with cisplatin can be used for the management of HNSC CSCs.
10.1016/j.ejca.2013.06.025
Decreased affinity for efflux transporters increases brain penetrance and molecular targeting of a PI3K/mTOR inhibitor in a mouse model of glioblastoma.
Becker Chani M,Oberoi Rajneet K,McFarren Stephan J,Muldoon Daniel M,Pafundi Deanna H,Pokorny Jenny L,Brinkmann Debra H,Ohlfest John R,Sarkaria Jann N,Largaespada David A,Elmquist William F
Neuro-oncology
BACKGROUND:Targeting drug delivery to invasive glioma cells is a particularly difficult challenge because these cells lie behind an intact blood-brain barrier (BBB) that can be observed using multimodality imaging. BBB-associated efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) influence drug distribution to these cells and may negatively impact efficacy. To test the hypothesis that efflux transporters influence brain pharmacokinetics/pharmacodynamics of molecularly targeted agents in glioma treatment, we assessed region-specific penetrance and molecular-targeting capacity for a PI3K/mTOR kinase inhibitor that has high substrate affinity for efflux transporters (GDC-0980) and an analog (GNE-317) that was purposely designed to have reduced efflux. METHODS:Brain tumor penetrance of GDC-0980 and GNE-317 was compared between FVB/n wild-type mice and Mdr1a/b(-/-)Bcrp(-/-) triple-knockout mice lacking P-gp and BCRP. C57B6/J mice bearing intracranial GL261 tumors were treated with GDC-0980, GNE-317, or vehicle to assess the targeted pharmacokinetic/pharmacodynamic effects in a glioblastoma model. RESULTS:Animals treated with GNE-317 demonstrated 3-fold greater penetrance in tumor core, rim, and normal brain compared with animals dosed with GDC-0980. Increased brain penetrance correlated with decreased staining of activated p-Akt, p-S6, and p-4EBP1 effector proteins downstream of PI3K and mTOR. CONCLUSIONS:GDC-0980 is subject to active efflux by P-gp and BCRP at the BBB, while brain penetrance of GNE-317 is independent of efflux, which translates into enhanced inhibition of PI3K/mTOR signaling. These data show that BBB efflux by P-gp and BCRP is therefore an important determinant in both brain penetrance and molecular targeting efficacy in the treatment of invasive glioma cells.
10.1093/neuonc/nov081
Breast cancer resistance protein in drug resistance of primitive CD34+38- cells in acute myeloid leukemia.
Raaijmakers Marc H G P,de Grouw Elke P L M,Heuver Leonie H H,van der Reijden Bert A,Jansen Joop H,Scheper Rik J,Scheffer George L,de Witte Theo J M,Raymakers Reinier A P
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38- stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML. EXPERIMENTAL DESIGN:BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38- cells. RESULTS:BCRP was preferentially expressed in leukemic CD34+38- cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38- cells. CONCLUSIONS:These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.
10.1158/1078-0432.CCR-04-0212
P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.
de Graaf D,Sharma R C,Mechetner E B,Schimke R T,Roninson I B
Proceedings of the National Academy of Sciences of the United States of America
P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.
10.1073/pnas.93.3.1238
Parthenolide inhibits cancer stem-like side population of nasopharyngeal carcinoma cells via suppression of the NF-κB/COX-2 pathway.
Liao Kun,Xia Bin,Zhuang Qun-Ying,Hou Meng-Jun,Zhang Yu-Jing,Luo Bing,Qiu Yang,Gao Yan-Fang,Li Xiao-Jie,Chen Hui-Feng,Ling Wen-Hua,He Cheng-Yong,Huang Yi-Jun,Lin Yu-Chun,Lin Zhong-Ning
Theranostics
Cancer stem cells play a central role in the pathogenesis of nasopharyngeal carcinoma and contribute to both disease initiation and relapse. In this study, cyclooxygenase-2 (COX-2) was found to regulate cancer stem-like side population cells of nasopharyngeal carcinoma cells and enhance cancer stem-like cells' characteristics such as higher colony formation efficiency and overexpression of stemness-associated genes. The regulatory effect of COX-2 on cancer stem-like characteristics may be mediated by ABCG2. COX-2 overexpression by a gain-of-function experiment increased the proportion of side population cells and their cancer stemness properties. The present study also demonstrated that in contrast to the classical chemotherapy drug 5-fluorouracil, which increased the proportion of side population cells and upregulated the expression of COX-2, parthenolide, a naturally occurring small molecule, preferentially targeted the side population cells of nasopharyngeal carcinoma cells and downregulated COX-2. Moreover, we found that the cancer stem-like cells' phenotype was suppressed by using COX-2 inhibitors NS-398 and CAY10404 or knocking down COX-2 with siRNA and shRNA. These findings suggest that COX-2 inhibition is the mechanism by which parthenolide induces cell death in the cancer stem-like cells of nasopharyngeal carcinoma. In addition, parthenolide exhibited an inhibitory effect on nuclear factor-kappa B (NF-κB) nucler translocation by suppressing both the phosphorylation of IκB kinase complex and IκBα degradation. Taken together, these results suggest that parthenolide may exert its cancer stem cell-targeted chemotherapy through the NF-κB/COX-2 pathway.
10.7150/thno.8387
Chronic myeloid leukemia CD34+ cells have reduced uptake of imatinib due to low OCT-1 activity.
Engler J R,Frede A,Saunders V A,Zannettino A C W,Hughes T P,White D L
Leukemia
Active influx of imatinib in chronic myeloid leukemia (CML) cells is mediated by the organic cation transporter 1 (OCT-1). Functional activity of OCT-1 (OCT-1 Activity) in mononuclear cells is an excellent predictor of molecular response over the first 24 months of imatinib therapy for chronic phase patients. CML progenitor cells are less sensitive to imatinib-induced apoptosis and are likely contributors to disease persistence. We investigated whether alterations in the expression and function of OCT-1 have a role in imatinib resistance in progenitors. We found the intracellular uptake and retention (IUR) of imatinib, OCT-1 Activity and OCT-1 mRNA expression are all significantly lower in CML CD34+ cells compared with mature CD34- cells (P<0.001). However, no differences in IUR or OCT-1 Activity were observed between these subsets in healthy donors. In contrast to OCT-1, ABCB1 and ABCG2 seemed to have no functional role in the transport of imatinib in CML CD34+ cells. Consistent with the observation that nilotinib uptake is not OCT-1 dependent, the IUR of nilotinib did not differ between CML CD34+ and CD34- cells. These results indicate that low imatinib accumulation in primitive CML cells, mediated through reduced OCT-1 Activity may be a critical determinant of long-term disease persistence.
10.1038/leu.2010.16
Synthesis and small-animal positron emission tomography evaluation of [11C]-elacridar as a radiotracer to assess the distribution of P-glycoprotein at the blood-brain barrier.
Dörner Bernd,Kuntner Claudia,Bankstahl Jens P,Bankstahl Marion,Stanek Johann,Wanek Thomas,Stundner Gloria,Mairinger Severin,Löscher Wolfgang,Müller Markus,Langer Oliver,Erker Thomas
Journal of medicinal chemistry
With the aim to develop a positron emission tomography (PET) tracer to assess the distribution of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in vivo, the potent third-generation P-gp inhibitor elacridar (1) was labeled with (11)C by reaction of O-desmethyl 1 with [(11)C]-methyl triflate. In vitro autoradiography and small-animal PET imaging of [(11)C]-1 was performed in rats (n = 3), before and after administration of unlabeled 1, as well as in wild-type, Mdr1a/b((-/-)) and Bcrp1((-/-)) mice (n = 3). In PET experiments in rats, administration of unlabeled 1 increased brain activity uptake 5.4-fold, whereas blood activity levels remained unchanged. In Mdr1a/b((-/-)) mice, brain activity uptake was 2.5-fold higher compared to wild-type animals, whereas in Bcrp1((-/-)) mice, brain activity uptake was only 1.3-fold higher. In vitro autoradiography showed that 63% of [(11)C]-1 binding was displaceable by an excess of unlabeled 1. As the signal obtained with [(11)C]-1 appeared to be specific for P-gp at the BBB, its utility for the visualization of cerebral P-gp merits further investigation.
10.1021/jm900940f
Reversal of different drug-resistant phenotypes by an autocatalytic multitarget multiribozyme directed against the transcripts of the ABC transporters MDR1/P-gp, MRP2, and BCRP.
Kowalski Petra,Surowiak Pawel,Lage Hermann
Molecular therapy : the journal of the American Society of Gene Therapy
A "multitarget multiribozyme" (MTMR) was constructed. It consists of three trans-acting hammerhead ribozymes directed against the transcripts of the ABC transporters MDR1/P-gp, BCRP, and MRP2; three cis-acting MDR1/P-gp-specific ribozymes; and three MDR1/P-gp-homologous spacer sequences. The trans-acting hammerhead ribozymes are liberated from the MTMR through autocatalytic self-cleavage by the cis-acting ribozymes. The MTMR was characterized with regard to its kinetic parameters. Comparison of the MTMR-specific kinetic values with those of the corresponding monoribozymes demonstrated that MTMR fragments could cleave their specific substrates without loss of efficiency. The MTMR was applied to three cell models, each overexpressing another ABC transporter, i.e., the gastric carcinoma cell line EPG85-257RDB expresses MDR1/P-gp, the cell variant EPG85-257RNOV synthesizes BCRP, and the ovarian carcinoma line A2780RCIS produces MRP2. In all cellular systems, the MTMR could specifically decrease the expression of the respective ABC transporter at the mRNA level (97% decrease in the MDR1/P-gp mRNA, 80% decrease in the BCRP mRNA, 96% decrease in the MRP2 mRNA) and the protein level. Resistance against the selection drug was reversed completely (100% in EPG85-257RDB) or by 94 (EPG85-257RNOV) or 63% (A2780RCIS). Thus, the MTMR technology provides a novel tool for gene therapeutic applications to reverse different ABC-transporter-dependent drug-resistant phenotypes.
10.1016/j.ymthe.2004.11.016
CXCR4 activation maintains a stem cell population in tamoxifen-resistant breast cancer cells through AhR signalling.
Dubrovska A,Hartung A,Bouchez L C,Walker J R,Reddy V A,Cho C Y,Schultz P G
British journal of cancer
BACKGROUND:Tamoxifen is commonly used for breast cancer therapy. However, tamoxifen resistance is an important clinical problem. Continuous treatment with conventional therapy may contribute to cancer progression in recurring cancers through the accumulation of drug-resistant cancer progenitors. METHODS:To investigate signalling mechanisms important for the maintenance and viability of drug-resistant cancer progenitors, we used microarray analysis, PCR array for genes involved in cancer drug resistance and metabolism, flow cytometry, soft agar colony formation assay, in vivo tumourigenicity assay and immunohistochemical analysis using tamoxifen-sensitive and tamoxifen-resistant breast cancer MCF7 cells. RESULTS:Downregulation of CXCR4 signalling by small molecule antagonist AMD3100 specifically inhibits growth of progenitor cell population in MCF7(TAM-R) cells both in vitro and in vivo. Microarray analysis revealed aryl hydrocarbon receptor (AhR) signalling as one of the top networks that is differentially regulated in MCF7(TAM-R) and MCF7 xenograft tumours treated with AMD3100. Further, small molecule antagonists of AhR signalling specifically inhibit the progenitor population in MCF7(TAM-R) cells and growth of MCF7(TAM-R) xenografts in vivo. CONCLUSION:The chemokine receptor CXCR4 maintains a cancer progenitor population in tamoxifen-resistant MCF7 cells through AhR signalling and could be a putative target for the treatment of tamoxifen-resistant breast cancers.
10.1038/bjc.2012.105
The PI3K subunits, P110α and P110β are potential targets for overcoming P-gp and BCRP-mediated MDR in cancer.
Molecular cancer
BACKGROUND:PI3K/AKT is a vital signaling pathway in humans. Recently, several PI3K/AKT inhibitors were reported to have the ability to reverse cancer multidrug resistance (MDR); however, specific targets in the PI3K/AKT pathways and the mechanisms associated with MDR have not been found because many of the inhibitors have multiple targets within a large candidate protein pool. AKT activation is one presumed mechanism by which MDR develops during cancer treatment. METHODS:The effects of inhibiting PI3K 110α and 110β by BAY-1082439 treatment and CRISPR/Cas9 knockout were examined to determine the possible functions of BAY-1082439 and the roles of PI3K 110α and 110β in the reversal of MDR that is mediated by the downregulation of P-gp and BCRP. Inhibition of AKT with GSK-2110183 showed that the downregulation of P-gp and BCRP is independent of generalized AKT inactivation. Immunofluorescence, immunoprecipitation, MTT, flow cytometry and JC-1 staining analyses were conducted to study the reversal of MDR that is mediated by P-gp and BCRP in cancer cells. An ATPase assay and a structural analysis were also used to analyze the potential mechanisms by which BAY-1082439 specifically targets PI3K 110α and 110β and nonspecifically influences P-gp and BCRP. RESULTS:By inhibiting the activation of the PI3K 110α and 110β catalytic subunits through both the administration of BAY-1082439 and the CRISPR/Cas9 deletion of Pik3ca and Pik3cb, the ATP-binding cassette transporters P-gp/ABCB1 and BCRP/ABCG2 were downregulated, thereby reestablishing the drug sensitivity of human epidermoid carcinoma and non-small cell lung cancer (NSCLC) MDR cells. Inhibition of AKT did not reverse the MDR mediated by P-gp or BCRP. The ABC family proteins and AKT may play MDR-enhancing roles independently. CONCLUSIONS:The reversal of the dual functions of ABC-transporter-mediated and AKT-activation-enhanced MDR through the inhibition or knockout of PI3K 110α or 110β promises to improve current strategies based on combined drug treatments to overcome MDR challenges.
10.1186/s12943-019-1112-1
PK11195, a peripheral benzodiazepine receptor (pBR) ligand, broadly blocks drug efflux to chemosensitize leukemia and myeloma cells by a pBR-independent, direct transporter-modulating mechanism.
Walter Roland B,Pirga Jason L,Cronk Michelle R,Mayer Sasha,Appelbaum Frederick R,Banker Deborah E
Blood
The peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux to chemosensitize cancer cells at least as well or better than the Pgp modulator, cyclosporine A (CSA). We now show that PK11195 broadly inhibits adenosine triphosphate (ATP)-binding cassette (ABC) transporters in hematologic cancer cell lines and primary leukemia-cell samples, including multidrug resistance protein (MRP), breast cancer resistance protein (BCRP), and/or Pgp. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but showed that pBR expression is unnecessary to PK11195-mediated efflux inhibition. PK11195 binds plasma-membrane sites in Pgp-expressing cells, stimulates Pgp-associated adenosine triphosphatase (ATPase) activity, and causes conformational changes in Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bind noncompetitively in Pgp-expressing cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites. Importantly, PK11195 concentrations that were effective in these in vitro assays can be safely achieved in patients. Because PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism and broadly blocks drug efflux by an apparently pBR-independent, ABC transporter-dependent mechanism, PK11195 may be a useful clinical chemosensitizer in cancer patients.
10.1182/blood-2005-02-0711
High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor.
Talanta
Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The K of xanthotoxin, bergapten, and naringenin were 5.14 μM, 4.57 μM, and 3.72 μM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.
10.1016/j.talanta.2024.125987
Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.
Muñoz-Martínez Francisco,Lu Peihua,Cortés-Selva Fernando,Pérez-Victoria José María,Jiménez Ignacio A,Ravelo Angel G,Sharom Frances J,Gamarro Francisco,Castanys Santiago
Cancer research
Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.
10.1158/0008-5472.CAN-04-1005
Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity.
Robey R W,Honjo Y,Morisaki K,Nadjem T A,Runge S,Risbood M,Poruchynsky M S,Bates S E
British journal of cancer
Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.
10.1038/sj.bjc.6601370
Increased delivery of chemotherapy to the vitreous by inhibition of the blood-retinal barrier.
Pascual-Pasto Guillem,Olaciregui Nagore G,Opezzo Javier A W,Castillo-Ecija Helena,Cuadrado-Vilanova Maria,Paco Sonia,Rivero Ezequiel M,Vila-Ubach Monica,Restrepo-Perdomo Camilo A,Torrebadell Montserrat,Suñol Mariona,Schaiquevich Paula,Mora Jaume,Bramuglia Guillermo F,Chantada Guillermo L,Carcaboso Angel M
Journal of controlled release : official journal of the Controlled Release Society
Treatment of retinoblastoma -a pediatric cancer of the developing retina- might benefit from strategies to inhibit the blood-retinal barrier (BRB). The potent anticancer agent topotecan is a substrate of efflux transporters BCRP and P-gp, which are expressed at the BRB to restrict vitreous and retinal distribution of xenobiotics. In this work we have studied vitreous and retinal distribution, tumor accumulation and antitumor activity of topotecan, using pantoprazole as inhibitor of BCRP and P-gp. We used rabbit and mouse eyes as BRB models and patient-derived xenografts as retinoblastoma models. To validate the rabbit BRB model we stained BCRP and P-gp in the retinal vessels. Using intravitreous microdialysis we showed that the penetration of the rabbit vitreous by lactone topotecan increased significantly upon concomitant administration of pantoprazole (P=0.0285). Pantoprazole also increased topotecan penetration of the mouse vitreous, measured as the vitreous-to-plasma topotecan concentration ratio at the steady state (P=0.0246). Pantoprazole increased topotecan antitumor efficacy and intracellular penetration in retinoblastoma in vitro, but did not enhance intratumor drug distribution and survival in mice bearing the intraocular human tumor HSJD-RBT-2. Anatomical differences with the clinical setting likely limited our in vivo study, since xenografts were poorly vascularized masses that loaded most of the vitreous compartment. We conclude that pharmacological modulation of the BRB is feasible, enhances anticancer drug distribution into the vitreous and might have clinical implications in retinoblastoma. CHEMICAL COMPOUNDS INCLUDED IN THIS MANUSCRIPT:Topotecan (PubChem CID: 60700) Pantoprazole sodium (PubChem CID: 15008962).
10.1016/j.jconrel.2017.08.018
Peptide transport by the multidrug resistance protein MRP1.
de Jong M C,Slootstra J W,Scheffer G L,Schroeijers A B,Puijk W C,Dinkelberg R,Kool M,Broxterman H J,Meloen R H,Scheper R J
Cancer research
Small hydrophobic peptides were studied as possible substrates of the multidrug resistance protein (MRP)-1 (ABCC1) transmembrane transporter molecule. As observed earlier for P-glycoprotein- (Pgp; ABCB1) overexpressing cells, MRP1-overexpressing cells, including cells stably transfected with the MRP1 cDNA, showed distinct resistance to the cytotoxic peptide N-acetyl-Leu-Leu-norleucinal (ALLN). Resistance to this peptide and another toxic peptide derivative, which is based on a Thr-His-Thr-Nle-Glu-Gly backbone conjugated to butyl and benzyl groups (4A6), could be reversed by MRP1 inhibitors. The reduced toxicity of 4A6 in MRP1-overexpressing cells was found to be associated with lower accumulation of a fluorescein-labeled derivative of this peptide. Glutathione (GSH) depletion had a clear effect on resistance to ALLN but hardly affected 4A6 resistance. In a limited structure-activity study using peptides that are analogous to 4A6, MRP1-overexpressing cells were found to be resistant to these peptides as well. Remarkably, when selecting A2780 ovarian cancer cells for resistance to ALLN, even in the absence of Pgp blockers, resulting cell lines had up-regulated MRP1, rather than any of the other currently known multidrug resistance transporter molecules including Pgp, MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCCS), and the breast cancer resistance protein ABCG2. ALLN-resistant, MRP1-overexpressing cells were found to be cross-resistant to 4A6 and the classical multidrug resistance drugs doxorubicin, vincristine, and etoposide. This establishes MRP1 as a transporter for small hydrophobic peptides. More extensive structure-activity relationship studies should allow the identification of clinically useful peptide antagonists of MRP1.
Natural flavonol fisetin attenuated hyperuricemic nephropathy via inhibiting IL-6/JAK2/STAT3 and TGF-β/SMAD3 signaling.
Ren Qian,Tao Sibei,Guo Fan,Wang Bo,Yang Letian,Ma Liang,Fu Ping
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:The naturally occurring flavonol fisetin (3,3',4',7-tetrahydroxyflavone), widely dispersed in fruits, vegetables and nuts, has been reported to exert anti-inflammatory, antioxidant and anti-angiogenic effects. Our previous study indicated fisetin ameliorated inflammation and apoptosis in septic kidneys. However, the potential nephroprotective effect of fisetin in hyperuricemic mice remains unknown. PURPOSE:The current study was designed to investigate the effect of fisetin on hyperuricemic nephropathy (HN) and explore the underlying mechanisms. METHODS:The HN was induced in mice by mixing of potassium oxonate (2400 mg/kg) and adenine (160 mg/kg) in male C57BL/6J mice. Fisetin (50 or 100 mg/kg) was orally administrated either simultaneously with the establishment of HN or after HN was induced. As a positive control, allopurinol of 10 mg/kg was included. Uric acid levels in the serum and urine as well as renal function parameters were measured. Renal histological changes were measured by periodic acid-Schiff (PAS) and Masson's trichrome stainings. The expression of gene/protein in relation to inflammation, fibrosis, and uric acid excretion in the kidneys of HN mice or uric acid-treated mouse tubular epithelial (TCMK-1) cells were measured by RNA-seq, RT-PCR, western blot and immunohistochemical analysis. RESULTS:Treatment with fisetin, regardless of administration regimen, dose-dependently attenuated hyperuricemia-induced kidney injury as indicated by the improved renal function, preserved tissue architecture, and decreased urinary albumin-to-creatinine ratio. Additionally, fisetin lowered uricemia by modulating the expression of kidney urate transporters including urate transporter 1(URAT1), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) and ATP binding cassette subfamily G member 2 (ABCG2). Moreover, hyperuricemia-induced secretions of proinflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and monocyte chemoattractant protein-1(MCP-1) in HN mice and uric acid-stimulated TCMK-1 cells were mitigated by fisetin treatment. Meanwhile, fisetin attenuated kidney fibrosis in HN mice with restored expressions of alpha-smooth muscle actin (α-SMA), collagen I and fibronectin. Mechanistically, fisetin regulated the aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling and transforming growth factor-β (TGF-β) signaling in the HN kidneys and uric acid-stimulated TCMK-1 cells. CONCLUSION:Fisetin lowered uricemia, suppressed renal inflammatory response, and improved kidney fibrosis to protect against hyperuricemic nephropathy via modulation of STAT3 and TGF-β signaling pathways. The results highlighted that fisetin might represent a potential therapeutic strategy against hyperuricemic nephropathy.
10.1016/j.phymed.2021.153552
The linker region of breast cancer resistance protein ABCG2 is critical for coupling of ATP-dependent drug transport.
Cellular and molecular life sciences : CMLS
The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.
10.1007/s00018-015-2118-5
Tanshinone I, a new EZH2 inhibitor restricts normal and malignant hematopoiesis through upregulation of and .
Theranostics
Tanshinone, a type of diterpenes derived from , is a particularly promising herbal medicine compound for the treatment of cancers including acute myeloid leukemia (AML). However, the therapeutic function and the underlying mechanism of Tanshinone in AML are not clear, and the toxic effect of Tanshinone limits its clinical application. Our work utilizes human leukemia cell lines, zebrafish transgenics and xenograft models to study the cellular and molecular mechanisms of how Tanshinone affects normal and abnormal hematopoiesis. WISH, Sudan Black and O-Dianisidine Staining were used to determine the expression of hematopoietic genes on zebrafish embryos. RNA-seq analysis showed that differential expression genes and enrichment gene signature with Tan I treatment. The surface plasmon resonance (SPR) method was used with a BIAcore T200 (GE Healthcare) to measure the binding affinities of Tan I. methyltransferase assay was performed to verify Tan I inhibits the histone enzymatic activity of the PRC2 complex. ChIP-qPCR assay was used to determine the H3K27me3 level of EZH2 target genes. We found that Tanshinone I (Tan I), one of the Tanshinones, can inhibit the proliferation of human leukemia cells and in the xenograft zebrafish model, as well as the normal and malignant definitive hematopoiesis in zebrafish. Mechanistic studies illustrate that Tan I regulates normal and malignant hematopoiesis through direct binding to EZH2, a well-known histone H3K27 methyltransferase, and inhibiting PRC2 enzymatic activity. Furthermore, we identified and as two possible downstream genes of Tan I's effects on EZH2. Together, this study confirmed that Tan I is a novel EZH2 inhibitor and suggested and as two potential therapeutic targets for myeloid malignant diseases.
10.7150/thno.53170
Imaging the Impact of the P-Glycoprotein (ABCB1) Function on the Brain Kinetics of Metoclopramide.
Pottier Géraldine,Marie Solène,Goutal Sébastien,Auvity Sylvain,Peyronneau Marie-Anne,Stute Simon,Boisgard Raphaël,Dollé Frédéric,Buvat Irène,Caillé Fabien,Tournier Nicolas
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
UNLABELLED:The effects of metoclopramide on the central nervous system (CNS) in patients suggest substantial brain distribution. Previous data suggest that metoclopramide brain kinetics may nonetheless be controlled by ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier. We used (11)C-metoclopramide PET imaging to elucidate the kinetic impact of transporter function on metoclopramide exposure to the brain. METHODS:(11)C-metoclopramide transport by P-glycoprotein (P-gp; ABCB1) and the breast cancer resistance protein (BCRP; ABCG2) was tested using uptake assays in cells overexpressing P-gp and BCRP. (11)C-metoclopramide brain kinetics were compared using PET in rats (n = 4-5) in the absence and presence of a pharmacologic dose of metoclopramide (3 mg/kg), with or without P-gp inhibition using intravenous tariquidar (8 mg/kg). The (11)C-metoclopramide brain distribution (VT based on Logan plot analysis) and brain kinetics (2-tissue-compartment model) were characterized with either a measured or an imaged-derived input function. Plasma and brain radiometabolites were studied using radio-high-performance liquid chromatography analysis. RESULTS:(11)C-metoclopramide transport was selective for P-gp over BCRP. Pharmacologic dose did not affect baseline (11)C-metoclopramide brain kinetics (VT = 2.28 ± 0.32 and 2.04 ± 0.19 mL⋅cm(-3) using microdose and pharmacologic dose, respectively). Tariquidar significantly enhanced microdose (11)C-metoclopramide VT (7.80 ± 1.43 mL⋅cm(-3)) with a 4.4-fold increase in K1 (influx rate constant) and a 2.3-fold increase in binding potential (k3/k4) in the 2-tissue-compartment model. In the pharmacologic situation, P-gp inhibition significantly increased metoclopramide brain distribution (VT = 6.28 ± 0.48 mL⋅cm(-3)) with a 2.0-fold increase in K1 and a 2.2-fold decrease in k2 (efflux rate), with no significant impact on binding potential. In this situation, only parent (11)C-metoclopramide could be detected in the brains of P-gp-inhibited rats. CONCLUSION:(11)C-metoclopramide benefits from favorable pharmacokinetic properties that offer reliable quantification of P-gp function at the blood-brain barrier in a pharmacologic situation. Using metoclopramide as a model of CNS drug, we demonstrated that P-gp function not only reduces influx but also mediates the efflux from the brain back to the blood compartment, with additional impact on brain distribution. This PET-based strategy of P-gp function investigation may provide new insight on the contribution of P-gp to the variability of response to CNS drugs between patients.
10.2967/jnumed.115.164350
Estrogen-mediated post transcriptional down-regulation of breast cancer resistance protein/ABCG2.
Imai Yasuo,Ishikawa Etsuko,Asada Sakiyo,Sugimoto Yoshikazu
Cancer research
Breast cancer resistance protein (BCRP)/ABCG2 mediates concurrent resistance to chemotherapeutic agents, such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan, by pumping them out of cells. We previously reported that BCRP transports sulfated estrogens. In the present study, we show that at physiologic levels, estrogens markedly decrease endogenous BCRP expression in the estrogen-responsive and estrogen receptor alpha (ERalpha)-positive human breast cancer MCF-7 cells, but not in estrogen-nonresponsive human cancer cells. 17 beta-Estradiol (E(2)) also significantly reduces exogenous BCRP expression, driven by a constitutive promoter, in BCRP-transduced estrogen-responsive and ERalpha-positive MCF-7 (MCF-7/BCRP) and T-47D cells, but not in BCRP-transduced estrogen-nonresponsive MDA-MB-231 and SKOV-3 cells. E(2) potentiates the cytotoxicity of SN-38, but not vincristine, in MCF-7/BCRP cells significantly, and increases cellular topotecan uptake in MCF-7 and MCF-7/BCRP cells. Antiestrogen tamoxifen partially reverses E(2)-mediated BCRP down-regulation in MCF-7 and MCF-7/BCRP cells and treatment of MCF-7/BCRP cells with an ERalpha small interfering RNA abolished E(2)-mediated BCRP down-regulation, suggesting that interaction of E(2) and ERalpha is necessary for BCRP down-regulation. E(2) does not affect endogenous BCRP mRNA levels in MCF-7 cells or exogenous BCRP mRNA levels in MCF-7/BCRP cells. The results from pulse-chase labeling experiments with MCF-7/BCRP cells suggest that decreased protein biosynthesis and maturation, but not alterations in protein turnover, might underlie E(2)-mediated BCRP down-regulation. These data indicate that estrogen down-regulates BCRP expression by novel posttranscriptional mechanisms. This is the first report of small molecules that can affect BCRP protein expression in cells and may therefore assist in establishing new strategies for regulating BCRP expression.
Progranulin promotes Temozolomide resistance of glioblastoma by orchestrating DNA repair and tumor stemness.
Bandey I,Chiou S-H,Huang A-P,Tsai J-C,Tu P-h
Oncogene
Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with a dismal prognosis. Current therapy of surgical removal combined with Temozolomide (TMZ) and radiation therapy only slightly prolongs the survival of GBM patients. Thus, it is essential to elucidate mechanism underlying its highly malignant properties in order to develop efficacious therapeutic regimens. In this study, we showed that progranulin (PGRN) was overexpressed in most GBM cell lines and the majority of human tumor samples. PGRN overexpression conferred GBM cells with tumorigenic properties and TMZ resistance by upregulating DNA repair (PARP, ATM, BRCA1, Rad51, XRCC1 and so on) and cancer stemness (CD133, CD44, ABCG2) genes, in part via an AP-1 transcription factor, specifically cFos/JunB. Curcumin, an AP-1 inhibitor, was also found to regulate PGRN promoter activity and expression including its downstream effectors aforementioned. These data suggested a feedforward loop between PGRN signaling and AP-1. PGRN depletion significantly decreased unlimited self-renewal and multilineage differentiation and the malignant properties of GBMs cells S1R1, and enhanced their vulnerability to TMZ. In addition, S1R1 depleted of PGRN also lost the ability to form tumor in an orthotopic xenograft mouse model. In conclusion, PGRN had a critical role in the pathogenesis and chemoresistance of GBM and functioned at the top of the hierarchy of cellular machinery that modulates both DNA repair pathways and cancer stemness. Our data suggest that a new strategy combining current regimens with compounds targeting PGRN/AP-1 loop like curcumin may significantly improve the therapeutic outcome of GBM.
10.1038/onc.2014.92
Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer.
Kazi Armina A,Gilani Rabia A,Schech Amanda J,Chumsri Saranya,Sabnis Gauri,Shah Preeti,Goloubeva Olga,Kronsberg Shari,Brodie Angela H
Breast cancer research : BCR
INTRODUCTION:Although aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2. METHODS:In vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance. RESULTS:Basal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP. CONCLUSIONS:One of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.
10.1186/bcr3609
Synthesis and biological evaluation of (hetero)arylmethyloxy- and arylmethylamine-phenyl derivatives as potent P-glycoprotein modulating agents.
Colabufo Nicola Antonio,Berardi Francesco,Perrone Roberto,Rapposelli Simona,Digiacomo Maria,Vanni Michael,Balsamo Aldo
Journal of medicinal chemistry
Starting from lead compounds 12b and 28b, previously characterized as P-glycoprotein (P-gp) modulating agents, two series of new compounds were investigated. Compounds 14a, b and 15a, b displayed high P-gp modulating activity in the submicromolar range (EC 50 values from 0.25 to 0.80 microM). Moreover, amino derivatives 23- 27 showed EC 50 values ranging from 0.085 to 0.90 microM. In the pyridyl series, the best result has been obtained for 4-pyridyl derivative 17b (EC 50 = 0.85 microM). The best P-gp modulating agents 14a, b, 15a, b, and 23- 27 also have been studied for determining their breast cancer resistance protein (BCRP) inhibition activity. The results demonstrated that only the amino derivatives 23- 27 displayed moderate BCRP inhibition activity.
10.1021/jm701267q
Small molecule antibody targeting HLA class I inhibits myeloma cancer stem cells by repressing pluripotency-associated transcription factors.
Leukemia
Cancer stem cells have been proposed to be responsible for tumorigenesis and recurrence in various neoplastic diseases, including multiple myeloma (MM). We have previously reported that MM cells specifically express HLA class I at high levels and that single-chain Fv diabody against this molecule markedly induces MM cell death. Here we investigated the effect of a new diabody (C3B3) on cancer stem cell-like side population (SP) cells. SP fraction of MM cells highly expressed ABCG2 and exhibited resistance to chemotherapeutic agents; however, C3B3 induced cytotoxicity in both SP cells and main population (MP) cells to a similar extent. Moreover, C3B3 suppressed colony formation and tumorigenesis of SP cells in vitro and in vivo. Crosslinking of HLA class I by C3B3 mediated disruption of lipid rafts and actin aggregation, which led to inhibition of gene expression of β-catenin and pluripotency-associated transcription factors such as Sox2, Oct3/4 and Nanog. Conversely, knockdown of Sox2 and Oct3/4 mRNA reduced the proportion of SP cells, suggesting that these factors are essential in maintenance of SP fraction in MM cells. Thus, our findings reveal that immunotherapeutic approach by engineered antibodies can overcome drug resistance, and provide a new basis for development of cancer stem cell-targeted therapy.
10.1038/leu.2012.78
Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance.
Volk Erin L,Farley Kate M,Wu Yan,Li Fei,Robey Robert W,Schneider Erasmus
Cancer research
Previously, we have reported that a multidrug-resistant, mitoxantrone (MX)-selected cell line, MCF7/MX, is highly cross-resistant to the antifolate methotrexate (MTX), because of enhanced ATP-dependent drug efflux (E. L. Volk et al., Cancer Res., 60: 3514-3521, 2000). These cells overexpress the breast cancer resistance protein (BCRP), and resistance to MTX as well as to MX was reversible by the BCRP inhibitor, GF120918. These data indicated that BCRP causes the multidrug-resistance phenotype. To further examine the role of this transporter in MTX resistance, and in particular the role of amino acid 482, we analyzed a number of BCRP-overexpressing cell lines. MTX resistance correlated with BCRP expression in all of the cell lines expressing the wild-type transporter, which contains an Arg at position 482. In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1-M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Concomitantly, the greatest reduction in MTX accumulation was observed in the MCF7/MX cells (BCRP(Arg)) as compared with cells expressing the Thr and Gly BCRP variants. Furthermore, the reduction in drug accumulation was sensitive to BCRP inhibition by GF120918. In conclusion, we have demonstrated a novel role for BCRP as a mediator of MTX resistance and have provided further evidence for the importance of amino acid 482 in substrate specificity.
Physical blood-brain barrier disruption induced by focused ultrasound does not overcome the transporter-mediated efflux of erlotinib.
Goutal Sébastien,Gerstenmayer Matthieu,Auvity Sylvain,Caillé Fabien,Mériaux Sébastien,Buvat Irène,Larrat Benoit,Tournier Nicolas
Journal of controlled release : official journal of the Controlled Release Society
Overcoming the efflux mediated by ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB) remains a challenge for the delivery of small molecule tyrosine kinase inhibitors (TKIs) such as erlotinib to the brain. Inhibition of ABCB1 and ABCG2 at the mouse BBB improved the BBB permeation of erlotinib but could not be achieved in humans. BBB disruption induced by focused ultrasound (FUS) was investigated as a strategy to overcome the efflux transport of erlotinib in vivo. In rats, FUS combined with microbubbles allowed for a large and spatially controlled disruption of the BBB in the left hemisphere. ABCB1/ABCG2 inhibition was performed using elacridar (10 mg/kg i.v). The brain kinetics of erlotinib was studied using C-erlotinib Positron Emission Tomography (PET) imaging in 5 groups (n = 4-5 rats per group) including a baseline group, immediately after sonication (FUS), 48 h after FUS (FUS + 48 h), elacridar (ELA) and their combination (FUS + ELA). BBB integrity was assessed using the Evan's Blue (EB) extravasation test. Brain exposure to C-erlotinib was measured as the area under the curve (AUC) of the brain kinetics (% injected dose (%ID) versus time (min)) in volumes corresponding to the disrupted (left) and the intact (right) hemispheres, respectively. EB extravasation highlighted BBB disruption in the left hemisphere of animals of the FUS and FUS + ELA groups but not in the control and ELA groups. EB extravasation was not observed 48 h after FUS suggesting recovery of BBB integrity. Compared with the control group (AUC = 1.4 ± 0.5%ID.min), physical BBB disruption did not impact the brain kinetics of C-erlotinib in the left hemisphere (p > .05) either immediately (AUC = 1.2 ± 0.1%ID.min) or 48 h after FUS (AUC = 1.1 ± 0.3%ID.min). Elacridar similarly increased C-erlotinib brain exposure to the left hemisphere in the absence (AUC = 2.2 ± 0.5%ID.min, p < .001) and in the presence of BBB disruption (AUC = 2.1 ± 0.5%ID.min, p < .001). AUC was never significantly different from AUC (p > .05), in any of the tested conditions. BBB integrity is not the rate limiting step for erlotinib delivery to the brain which is mainly governed by ABC-mediated efflux. Efflux transport of erlotinib persisted despite BBB disruption.
10.1016/j.jconrel.2018.11.009
Interaction of the multikinase inhibitors sorafenib and sunitinib with solute carriers and ATP-binding cassette transporters.
Hu Shuiying,Chen Zhaoyuan,Franke Ryan,Orwick Shelley,Zhao Ming,Rudek Michelle A,Sparreboom Alex,Baker Sharyn D
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:To compare side-by-side the uptake of sorafenib and sunitinib in vitro by human uptake solute carriers of the SLC22A and SLCO families, the transport by and inhibition of efflux ATP-binding cassette (ABC) transporters, and the role of ABCB1 in the plasma pharmacokinetics and brain penetration of these agents. EXPERIMENTAL DESIGN:Uptake of [(3)H]sorafenib or [(3)H]sunitinib was assessed in Xenopus laevis oocytes or mammalian cells transfected with cDNAs coding for human OATP1A2, OATP1B1, OATP1B3, OCT1, OAT2, OAT3, OCTN1, or OCTN2. Efflux and inhibition experiments were conducted in cells transfected with human ABCB1, ABCG2, ABCC2, or ABCC4. In vivo pharmacokinetic studies were done in knockout mice lacking Abcb1-type transporters. RESULTS:Intracellular uptake was not appreciably affected by any of the studied solute carriers and was minute relative to the respective prototypical substrates. Sorafenib and sunitinib showed concentration-dependent (1 and 10 micromol/L), low to moderate affinity for ABCB1 but were not affected by the other ABC transporters. Both agents inhibited all tested ABC transporters. The absence of Abcb1 had no affect on plasma pharmacokinetics, but brain penetration was moderately increased by 1.9- and 2.9-fold for sorafenib and sunitinib, respectively, in knockout animals versus controls. CONCLUSIONS:Unlike other tyrosine kinase inhibitors, sorafenib and sunitinib do not appear to rely on active transport to enter the cell nor are they high-affinity substrates for ABC efflux transporters. Based on these characteristics, these two drugs may be less susceptible to transporter-mediated alterations in systemic exposure and transporter-related resistance mechanisms.
10.1158/1078-0432.CCR-09-0048
ABC transporters and drug resistance in leukemia: was P-gp nothing but the first head of the Hydra?
Steinbach D,Legrand O
Leukemia
More than 30 years ago it was discovered that permeability glycoprotein (P-gp) can cause drug resistance. Over the following decades numerous studies showed that high expression of P-gp is associated with poor prognosis in acute myeloid leukemia in adults and that it causes multidrug resistance via ATP-dependent drug efflux. It was hoped that an inhibition of P-gp could sensitize resistant leukemic cells to chemotherapy and thus improve treatment results. Today we know that the family of ATP-binding cassette transporters (ABC transporters) comprises 48 different proteins. Some of them seem to be able to cause drug resistance as well as P-gp. This review focuses on emerging data on the clinical relevance of other ABC transporters, such as BCRP, MRP3, and ABCA3. When Heracles fought the ancient Hydra, he had to fight all the heads at ones but only one head was vital for the beast. Can we block all the relevant ABC transporters at once? Is there one transporter that is more important than the others?
10.1038/sj.leu.2404692
The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux.
Erlichman C,Boerner S A,Hallgren C G,Spieker R,Wang X Y,James C D,Scheffer G L,Maliepaard M,Ross D D,Bible K C,Kaufmann S H
Cancer research
Because the activities of HER family members are elevated and/or aberrant in a variety of human neoplasms, these cell surface receptors are receiving increasing attention as potential therapeutic targets. In the present study, we examined the effect of combining the HER family tyrosine kinase inhibitor CI1033 (PD 183805) with the topoisomerase (topo) I poison 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, in a number of different cell lines. Colony-forming assays revealed that the antiproliferative effects of simultaneous treatment with CI1033 and SN-38 were synergistic in T98G glioblastoma cells and HCT8 colorectal carcinoma cells, whereas sequential treatments were additive at best. In additional studies examining the mechanistic basis for these findings in T98G cells, immunoblotting revealed that the inhibitory effects of CI1033 on epidermal growth factor receptor autophosphorylation were unaffected by SN-38. Likewise, CI1033 had no effect on topo I polypeptide levels, localization, or activity. Nonetheless, CI1033 markedly enhanced the number of covalent topo I-DNA complexes stabilized by SN-38 or the related agent topotecan (TPT). Analysis of intracellular SN-38 levels by high-performance liquid chromatography and intracellular TPT levels by flow microfluorometry revealed that CI1033 increased the steady-state accumulation of SN-38 and TPT by 9.4 +/- 1.9- and 1.8 +/- 0.2-fold, respectively. Further evaluation revealed that the initial rate of TPT uptake was unaffected by CI1033, whereas the rate of efflux was markedly diminished. Additional studies demonstrated that T98G and HCT8 cells express the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. Moreover, CI1033 enhanced the uptake and cytotoxicity of SN-38 and TPT in cells transfected with BCRP but not empty vector. Conversely, CI1033 accumulation was diminished in cells expressing BCRP, suggesting that CI1033 is a substrate for this efflux pump. These results indicate that CI1033 can modulate the accumulation and subsequent cytotoxicity of two widely used topo I poisons in cells that have no history of previous exposure to these agents.
Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan.
Jonker J W,Smit J W,Brinkhuis R F,Maliepaard M,Beijnen J H,Schellens J H,Schinkel A H
Journal of the National Cancer Institute
BACKGROUND AND METHODS:Breast cancer resistance protein (BCRP/MXR/ABCP) is a multidrug-resistance protein that is a member of the adenosine triphosphate-binding cassette family of drug transporters. BCRP can render tumor cells resistant to the anticancer drugs topotecan, mitoxantrone, doxorubicin, and daunorubicin. To investigate the physiologic role of BCRP, we used polarized mammalian cell lines to determine the direction of BCRP drug transport. We also used the BCRP inhibitor GF120918 to assess the role of BCRP in protecting mice against xenobiotic drugs. Bcrp1, the murine homologue of BCRP, was expressed in the polarized mammalian cell lines LLC-PK1 and MDCK-II, and the direction of Bcrp1-mediated transport of topotecan and mitoxantrone was determined. To avoid the confounding drug transport provided by P-glycoprotein (P-gp), the roles of Bcrp1 in the bioavailability of topotecan and the effect of GF120918 were studied in both wild-type and P-gp-deficient mice and their fetuses. RESULTS:Bcrp1 mediated apically directed transport of drugs in polarized cell lines. When both topotecan and GF120918 were administered orally, the bioavailability (i.e., the extent to which a drug becomes available to a target tissue after administration) of topotecan in plasma was dramatically increased in P-gp-deficient mice (greater than sixfold) and wild-type mice (greater than ninefold), compared with the control (i.e., vehicle-treated) mice. Furthermore, treatment with GF120918 decreased plasma clearance and hepatobiliary excretion of topotecan and increased (re-)uptake by the small intestine. In pregnant GF120918-treated, P-gp-deficient mice, relative fetal penetration of topotecan was twofold higher than that in pregnant vehicle-treated mice, suggesting a function for BCRP in the maternal-fetal barrier of the placenta. CONCLUSIONS:Bcrp1 mediates apically directed drug transport, appears to reduce drug bioavailability, and protects fetuses against drugs. We propose that strategic application of BCRP inhibitors may thus lead to more effective oral chemotherapy with topotecan or other BCRP substrate drugs.
10.1093/jnci/92.20.1651
Tyrosine kinase inhibitor gefitinib enhances topotecan penetration of gliomas.
Carcaboso Angel M,Elmeliegy Mohamed A,Shen Jun,Juel Stephen J,Zhang Ziwei M,Calabrese Christopher,Tracey Lorraine,Waters Christopher M,Stewart Clinton F
Cancer research
Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, increases brain parenchymal extracellular fluid (ECF) accumulation of topotecan, a substrate of the ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp/MDR-1) and breast cancer resistance protein (BCRP/ABCG2). The effect of modulating these transporters on topotecan penetration in gliomas has not been thoroughly studied. Thus, we performed intracerebral microdialysis on mice bearing orthotopic human gliomas (U87 and MT330) and assessed topotecan tumor ECF (tECF) penetration and the effect of gefitinib on topotecan tECF penetration and intratumor topotecan distribution. We found that topotecan penetration (P(tumor)) of U87 was 0.96 +/- 0.25 (n = 7) compared with that of contralateral brain (P(contralateral), 0.42 +/- 0.11, n = 5; P = 0.001). In MT330 tumors, P(tumor) (0.78 +/- 0.26, n = 6) and P(contralateral) (0.42 +/- 0.11, n = 5) also differed significantly (P = 0.013). Because both tumor models had disrupted blood-brain barriers and similar P(tumor) values, we used U87 and a steady-state drug administration approach to characterize the effect of gefitinib on topotecan P(tumor). At equivalent plasma topotecan exposures, we found that P(tumor) after gefitinib administration was lower. In a separate cohort of animals, we determined the volume of distribution of unbound topotecan in tumor (V(u,tumor)) and found that it was significantly higher in groups receiving gefitinib, implying that gefitinib administration leads to a greater proportion of intracellular topotecan. Our results provide crucial insights into the role that transporters play in central nervous system drug penetration and provide a better understanding of the effect of coadministration of transporter modulators on anticancer drug distribution within a tumor.
10.1158/0008-5472.CAN-09-4264
β-carotene reverses multidrug resistant cancer cells by selectively modulating human P-glycoprotein function.
Teng Yu-Ning,Sheu Ming-Jyh,Hsieh Yow-Wen,Wang Ruey-Yun,Chiang Yao-Chang,Hung Chin-Chuan
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:The issue of multidrug resistance (MDR) cancer is one of the major barriers to successful chemotherapy treatment. The ATP-binding cassette (ABC) efflux transporters play an important role in the chemotherapeutic failure. Several generations of ABC efflux transporter inhibitors have been developed, however, none of them could provide better clinical outcome due to systemic toxicities and significant drug-drug interactions. Therefore, the present study focused on identifying the effect of the natural carotenoid on ABC transporters and may provide a safer choice to defeat MDR cancer. PURPOSE:The aim of the present study was to evaluate the inhibitory potency of β-carotene on the ABC efflux transporters, as well as the reversal effect of β-carotene toward MDR cancers. The underlying molecular mechanisms and inhibitory kinetics of β-carotene on the major ABC efflux transporter, P-glycoprotein, were further investigated. METHODS:The human P-gp (ABCB1/Flp-In(TM)-293), MRP1 (ABCC1/Flp-In(TM)-293) and BCRP (ABCG2/Flp-In(TM)-293) stable expression cells were established by using the Flp-In(TM) system. The cytotoxicity of β-carotene was evaluated by MTT assay in the established cell lines, sensitive cancer cell lines (HeLaS3 and NCI-H460) and resistant cancer cell lines (KB-vin and NCI-H460/MX20). Surface protein detection assay and eFluxx-ID Green Dye assay were applied for confirmation of surface expression and function of the transporters. The transporter inhibition potency of β-carotene was evaluated by calcein-AM uptake assay and mitoxantrone accumulation assay. Further interaction kinetics between β-carotene and P-gp were analyzed by rhodamine123 and doxorubicin efflux assay. The influence of β-carotene on ATPase activity was evaluated by Pgp-Glo(TM) Assay System. RESULTS:Among the tested ABC efflux transporters, β-carotene significantly inhibited human P-gp efflux function without altering ABCB1 mRNA expression. Furthermore, β-carotene stimulated both P-gp basal ATPase activity and the verapamil-stimulated P-gp ATPase activity. In addition, β-carotene exerted partially inhibitory effect on BCRP efflux function. The combination of β-carotene and chemotherapeutic agents significantly potentiated their cytotoxicity in both cell stably expressed human P-gp (ABCB1/Flp-In(TM)-293) and MDR cancer cells (KB-vin and NCI-H460/MX20). CONCLUSION:The present study indicated that β-carotene may be considered as a chemo-sensitizer and regarded as an adjuvant therapy in MDR cancer treatment.
10.1016/j.phymed.2016.01.008
The deubiquitylase UCHL3 maintains cancer stem-like properties by stabilizing the aryl hydrocarbon receptor.
Ouyang Lianlian,Yan Bin,Liu Yating,Mao Chao,Wang Min,Liu Na,Wang Zuli,Liu Shouping,Shi Ying,Chen Ling,Wang Xiang,Cheng Yan,Cao Ya,Xiao Desheng,Zhang Lingqiang,Liu Shuang,Tao Yongguang
Signal transduction and targeted therapy
Cancer stem cells (CSCs) exhibit highly aggressive and metastatic features and resistance to chemotherapy and radiotherapy. Aryl hydrocarbon receptor (AhR) expression varies among non-small cell lung cancers (NSCLCs), and the mechanisms that support abnormal AhR expression in CSCs remain elusive. Here, we identified ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, as a bona fide deubiquitylase of the AhR in NSCLC. UCHL3 was shown to interact with, deubiquitylate, and stabilize AhR in a manner dependent on its deubiquitylation activity. Moreover, we showed that UCHL3 promotes the stem-like characteristics and potent tumorigenic capacity of NSCLC cells. UCHL3 increased AhR stability and the binding of AhR to the promoter regions of the "stemness" genes ATP-binding cassette subfamily G member 2 (ABCG2), KLF4, and c-Myc. Depletion of UCHL3 markedly downregulated the "stemness" genes ABCG2, KLF4, and c-Myc, leading to the loss of self-renewal and tumorigenesis in NSCLCs. Furthermore, the UCHL3 inhibitor TCID induced AhR degradation and exhibited significantly attenuated efficacy in NSCLC cells with stem cell-like properties. Additionally, UCHL3 was shown to indicate poor prognosis in patients with lung adenocarcinoma. In general, our results reveal that the UCHL3 deubiquitylase is pivotal for AhR protein stability and a potential target for NSCLC-targeted therapy.
10.1038/s41392-020-0181-3
Development of a cyclin-dependent kinase inhibitor devoid of ABC transporter-dependent drug resistance.
Kaliszczak M,Patel H,Kroll S H B,Carroll L,Smith G,Delaney S,Heathcote D A,Bondke A,Fuchter M J,Coombes R C,Barrett A G M,Ali S,Aboagye E O
British journal of cancer
BACKGROUND:Cyclin-dependent kinases (CDKs) control cell cycle progression, RNA transcription and apoptosis, making them attractive targets for anticancer drug development. Unfortunately, CDK inhibitors developed to date have demonstrated variable efficacy. METHODS:We generated drug-resistant cells by continuous low-dose exposure to a model pyrazolo[1,5-a]pyrimidine CDK inhibitor and investigated potential structural alterations for optimal efficacy. RESULTS:We identified induction of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, in resistant cells. Assessment of features involved in the ABC transporter substrate specificity from a compound library revealed high polar surface area (>100 Å(2)) as a key determinant of transporter interaction. We developed ICEC-0782 that preferentially inhibited CDK2, CDK7 and CDK9 in the nanomolar range. The compound inhibited phosphorylation of CDK substrates and downregulated the short-lived proteins, Mcl-1 and cyclin D1. ICEC-0782 induced G2/M arrest and apoptosis. The permeability and cytotoxicity of ICEC-0782 were unaffected by ABC transporter expression. Following daily oral dosing, the compound inhibited growth of human colon HCT-116 and human breast MCF7 tumour xenografts in vivo by 84% and 94%, respectively. CONCLUSION:We identified a promising pyrazolo[1,5-a]pyrimidine compound devoid of ABC transporter interaction, highly suitable for further preclinical and clinical evaluation for the treatment of cancer.
10.1038/bjc.2013.584
Piperine, a piperidine alkaloid from Piper nigrum re-sensitizes P-gp, MRP1 and BCRP dependent multidrug resistant cancer cells.
Li Sen,Lei Yu,Jia Yingjie,Li Na,Wink Michael,Ma Yonggang
Phytomedicine : international journal of phytotherapy and phytopharmacology
Over-expression of P-gp, MRP1 and BCRP in tumor cells is one of the important mechanisms leading to multidrug resistance (MDR), which impairs the efficacy of chemotherapy. P-gp, MRP1 and BCRP are ABC (ATP-Binding Cassette) transporters, which can expel a variety of lipophilic anti-cancer drugs and protect tumor cells. During a screening of MDR reversal agents among alkaloids of various structural types, a piperidine alkaloid, piperine (a main piperidine alkaloid in Piper nigurm) was identified as an inhibitor. Piperine can potentiate the cytotoxicity of anti-cancer drugs in resistant sublines, such as MCF-7/DOX and A-549/DDP, which were derived from MCF-7 and A-549 cell lines. At a concentration of 50 μM piperine could reverse the resistance to doxorubicin 32.16 and 14.14 folds, respectively. It also re-sensitized cells to mitoxantrone 6.98 folds. In addition, long-term treatment of cells by piperine inhibits transcription of the corresponding ABC transporter genes. These results suggest that piperine can reverse MDR by multiple mechanisms and it may be a promising lead compound for future studies.
10.1016/j.phymed.2011.06.031
ABC-transporter upregulation mediates resistance to the CDK7 inhibitors THZ1 and ICEC0942.
Oncogene
The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use.
10.1038/s41388-019-1008-y
Amine linked flavonoid dimers as modulators for P-glycoprotein-based multidrug resistance: structure-activity relationship and mechanism of modulation.
Chan Kin-Fai,Wong Iris L K,Kan Jason W Y,Yan Clare S W,Chow Larry M C,Chan Tak Hang
Journal of medicinal chemistry
Here we report a great improvement in reversal potency of cancer drug resistance when flavonoid dimers possess a functionally substituted aminopolyethylene glycol linker. The most potent compound, 18, contains a N-benzyl group at the linker. It has many advantages including (1) high potencies in reversing P-glycoprotein (P-gp) mediated resistance in LCC6MDR cells to various anticancer drugs with EC(50) in the nanomolar range, (2) low toxicity and high therapeutic index, and (3) preferential inhibition of P-gp over multidrug resistance protein 1 and breast cancer resistance protein. Compound 18 stimulates P-gp-ATPase activity by 2.7-fold and mediates a dose-dependent inhibition of doxorubicin (DOX) transport activity. Lineweaver-Burk and Dixon plots suggest that 18 is a competitive inhibitor to DOX in binding to P-gp with a K(i) of 0.28-0.34 μM and a Hill coefficient of 1.17. Moreover, the LCC6MDR cell displays about 2.1-fold lower intracellular accumulation of 18 compared to the wild type, suggesting that 18 is a P-gp substrate as well.
10.1021/jm201121b
Lusianthridin targeting of lung cancer stem cells via Src-STAT3 suppression.
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:Cancer stem cells (CSCs) are well-recognized as a majority cause of treatment failure and can give rise to relapse. The discovery of compounds attenuating CSCs' properties is crucial for enabling advances in novel therapeutics to limit recurrence. CSCs' features in lung cancer are regulated through a reduction in Src-STAT3-c-Myc, which drives cancer progression, drug resistance, and metastasis. METHODS:The effect of lusianthridin suppresses CSC-like phenotypes was determined by 3D culture and anchorage independent growth. The expression of CSC markers and associated proteins were determined by Western blot analyses. Protein ubiquitination and degradation were assessed using immunoprecipitation. RESULTS:Herein, we report that lusianthridin, a pure compound from Dendrobium venustum, dramatically suppressed CSCs in lung cancer cells as verified by several CSC phenotype assessments and CSC markers. The CSC phenotypes in lusianthridin-treated cells were suppressed through downregulation of Src-STAT3-c-Myc pathways. Ectopic Src introduced by the transfection augmented CSC phenotypes in lung cancer cells through STAT3 (increased active p-STAT3) and c-Myc signals, while the ShRNA-Src transfection or Src inhibitor dasatinib exhibited opposite results. Treatment of the Src-overexpressing cells with lusianthridin resulted in the reversal of active STAT3 (p-STAT3) and c-Myc as well as the CSC marker CD133. Importantly, we confirmed the CSC-targeted activity of lusianthridin in CSC-rich primary lung cancer cells. The compound dramatically inhibited the formation of tumor spheres of primary lung cancer cells. Finally, we demonstrated that after CSC-attenuation by lusianthridin, the lung cancer cells exhibited significantly higher susceptibility to chemotherapeutic drugs. Such a sensitizing effect caused by pro-survival suppression and pro-apoptotic induction together with the abolishment of stemness indicated by the decrease in CSC markers CD133, ABCG2, and ALDH1A1. CONCLUSION:These findings revealed a novel pharmacological action and the underlying mechanism of lusianthridin in negatively regulating CSC-like phenotypes and sensitizing resistant cancer cells to cemetery.
10.1016/j.phymed.2019.152932
Expression of multidrug transporters MRP1, MRP2, and BCRP shortly after status epilepticus, during the latent period, and in chronic epileptic rats.
van Vliet Erwin A,Redeker Sandra,Aronica Eleonora,Edelbroek Peter M,Gorter Jan A
Epilepsia
PURPOSE:Overexpression of multidrug transporters may play a role in the development of pharmacoresistance by decreasing extracellular drug levels in the brain. However, it is not known whether overexpression is due to an initial insult or evolves more gradually because of recurrent spontaneous seizures. In the present study, we investigated the expression of different multidrug transporters during epileptogenesis in the rat. In addition, we determined whether these transporters affected phenytoin (PHT) distribution in the brain. METHODS:Expression of multidrug resistance-associated proteins MRP1 and MRP2 and breast cancer-resistance protein (BCRP) was examined after electrically induced status epilepticus (SE) by immunocytochemistry and Western blot analysis. Brain/blood PHT levels were determined by high-performance liquid chromatography (HPLC) analysis in the presence and absence of the MRP inhibitor probenecid. RESULTS:Shortly after SE, MRP1, MRP2, and BCRP were upregulated in astrocytes within several limbic structures, including hippocampus. In chronic epileptic rats, these proteins were overexpressed in the parahippocampal cortex, specifically in blood vessels and astrocytes surrounding these vessels. Overexpression was related to the occurrence of SE and was present mainly in rats with a high seizure frequency. Brain PHT levels were significantly lower in epileptic rats compared with control rats, but pharmacologic inhibition of MRPs increased the PHT levels. CONCLUSIONS:Overexpression of MRP and BCRP was induced by SE as well as recurrent seizures. Moreover, overexpression was associated with lower PHT levels in the brain, which was reversed through inhibition of MRPs. These data suggest that administration of antiepileptic drugs in combination with specific inhibitors for multidrug transporters may be a promising therapeutic strategy in pharmacoresistant patients.
10.1111/j.1528-1167.2005.00250.x
ALK interacts with c-Myc and promotes cancer stem cell-like properties in sarcoma.
Xu Bu-Shu,Chen Huo-Ying,Que Yi,Xiao Wei,Zeng Mu-Sheng,Zhang Xing
Oncogene
Soft tissue sarcoma (STS) is a highly malignant tumor with limited targeted therapies. A novel anaplastic lymphoma kinase (ALK) transcript, ALK, was identified recently and could be targeted by ALK inhibitors in melanoma. However, the clinical and functional role of aberrant ALK expression in STS remains unknown. Here we demonstrate that as a new ALK transcript, ALK is frequently found in STS. ALK expression correlates with a lower probability of progression-free survival in STS patients. Compared with the other ALK isoforms, ALK expresses not only in the cytoplasm, but also in the nucleus of sarcoma cells. Functionally, overexpression of ALK promoted cancer stem cell (CSC)-like properties in sarcoma cells by promoting sphere formation and upregulating the expression of stem cell markers. Moreover, the ALK inhibitors not only suppressed the oncogenic functions of ALK but also attenuated ALK-induced CSC-like properties by reducing the expression of stem cell markers such as c-Myc, ABCG2, BMI1, and OCT4 both in vitro and in vivo. Furthermore, ALK interacted with c-Myc and increased the binding of c-Myc to the ABCG2 promoter, resulting in the induction of stem cell-like properties. Together, these findings indicate that ALK may be a potential prognostic marker and therapeutic target for STS patients harboring such ALK aberrations.
10.1038/s41388-019-0973-5
The importance of breast cancer resistance protein to the kidneys excretory function and chemotherapeutic resistance.
Caetano-Pinto Pedro,Jansen Jitske,Assaraf Yehuda G,Masereeuw Rosalinde
Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy
The relevance of membrane transporters gained momentum in recent years and it is now widely recognized that transporters are key players in drug disposition and chemoresistance. As such, the kidneys harbor a variety of drug transporters and are one of the main routes for xenobiotic excretion. The breast cancer resistance protein (BCRP/ABCG2) is widely accepted as a key mediator of anticancer drug resistance and is a prominent renal drug transporter. Here, we review the role of BCRP in both processes and present a multitude of variables that can influence its activity. An increasing number of renally cleared chemotherapeutics, including tyrosine kinase inhibitors, described as BCRP substrates can modulate its activity via transcription factors and cellular signaling pathways, such as the phosphoinositide 3-kinase (PI3K) pathway. In addition to pharmacological actions, genetic variations, as well as differences between species and gender can affect BCRP function, which are also discussed. Furthermore, the role of BCRP in light of cancer treatments and the implications for novel therapeutic interventions that take into account renal function are discussed.
10.1016/j.drup.2017.01.002
Mammalian drug efflux transporters of the ATP binding cassette (ABC) family in multidrug resistance: A review of the past decade.
Chen Zhaolin,Shi Tianlu,Zhang Lei,Zhu Pengli,Deng Mingying,Huang Cheng,Hu Tingting,Jiang Ling,Li Jun
Cancer letters
Multidrug resistance (MDR) is a serious phenomenon employed by cancer cells which hampers the success of cancer pharmacotherapy. One of the common mechanisms of MDR is the overexpression of ATP-binding cassette (ABC) efflux transporters in cancer cells such as P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) that limits the prolonged and effective use of chemotherapeutic drugs. Researchers have found that developing inhibitors of ABC efflux transporters as chemosensitizers could overcome MDR. But the clinical trials have shown that most of these chemosensitizers are merely toxic and only show limited or no benefits to cancer patients, thus new inhibitors are being explored. Recent findings also suggest that efflux pumps of the ABC transporter family are subject to epigenetic gene regulation. In this review, we summarize recent findings of the role of ABC efflux transporters in MDR.
10.1016/j.canlet.2015.10.010
Reversal of ABCB1-related multidrug resistance by ERK5-IN-1.
Wang Fang,Li Delan,Zheng ZongHeng,Kin Wah To Kenneth,Chen Zhen,Zhong Mengjun,Su Xiaodong,Chen Likun,Fu Liwu
Journal of experimental & clinical cancer research : CR
BACKGROUND:Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. METHODS:The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. RESULTS:Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. CONCLUSIONS:Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.
10.1186/s13046-020-1537-9
Selective elimination of human pluripotent stem cells by a marine natural product derivative.
Kuo Ting-Fang,Mao Di,Hirata Nao,Khambu Bilon,Kimura Yasuhisa,Kawase Eihachiro,Shimogawa Hiroki,Ojika Makoto,Nakatsuji Norio,Ueda Kazumitsu,Uesugi Motonari
Journal of the American Chemical Society
One of the current obstacles to stem cell therapy is the tumorigenic potential of residual undifferentiated stem cells. The present study reports rediscovery of a synthetic derivative of okadaic acid, a marine polyether toxin, as a reagent that selectively induces the death of human pluripotent stem cells. Cell-based screening of 333 cytotoxic compounds identified methyl 27-deoxy-27-oxookadaate (molecule 1) as a substrate of two ATP-binding cassette (ABC) transporters, ABCB1 (MDR1) and ABCG2 (BCRP), whose expression is repressed in human embryonic stem cells and induced pluripotent stem cells. The results demonstrate that selective elimination of human pluripotent stem cells can be achieved by designing cytotoxic small molecules with appropriate ABC-transporter selectivity.
10.1021/ja501795c
Mouse breast cancer resistance protein (Bcrp1/Abcg2) mediates etoposide resistance and transport, but etoposide oral availability is limited primarily by P-glycoprotein.
Allen John D,Van Dort Sonja C,Buitelaar Marije,van Tellingen Olaf,Schinkel Alfred H
Cancer research
The breast cancer resistance protein [BCRP (BCRP/ABCG2)] has not previously been directly identified as a source of resistance to epipodophyllotoxins.However, when P-glycoprotein (P-gp)- and Mrp1-deficient mouse fibroblast and kidney cell lines were selected for resistance to etoposide, amplification and overexpression of Bcrp1 emerged as the dominant resistance mechanism in five of five cases. Resistance was accompanied by reduced intracellular etoposide accumulation. Bcrp1 sequence in all of the resistant lines was wild-type in the region spanning the R482 mutation hot spot known to alter the substrate specificity of mouse Bcrp1 (mouse cognate of BCRP) and human BCRP. Transduced wild-type Bcrp1 cDNA mediated resistance to etoposide and teniposide in fibroblast lines and trans-epithelial etoposide transport in polarized Madin-Darby canine kidney II cells. Bcrp1-mediated etoposide resistance was reversed by two structurally different BCRP/Bcrp1 inhibitors, GF120918 and Ko143. BCRP/Bcrp1 (inhibition) might thus impact on the antitumor activity and pharmacokinetics of epipodophyllotoxins. However, treatment of P-gp-deficient mice with GF120918 did not improve etoposide oral uptake, suggesting that Bcrp1 activity is not a major limiting factor in this process. In contrast, use of GF120918 to inhibit P-gp in wild-type mice increased the plasma levels of etoposide after oral administration 4-5-fold. It may thus be worthwhile to test inhibition of P-gp in humans to improve the oral availability of etoposide.
C@PA: Computer-Aided Pattern Analysis to Predict Multitarget ABC Transporter Inhibitors.
Namasivayam Vigneshwaran,Silbermann Katja,Wiese Michael,Pahnke Jens,Stefan Sven Marcel
Journal of medicinal chemistry
Based on literature reports of the last two decades, a computer-aided pattern analysis (C@PA) was implemented for the discovery of novel multitarget ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP) inhibitors. C@PA included basic scaffold identification, substructure search and statistical distribution, as well as novel scaffold extraction to screen a large virtual compound library. Over 45,000 putative and novel broad-spectrum ABC transporter inhibitors were identified, from which 23 were purchased for biological evaluation. Our investigations revealed five novel lead molecules as triple ABCB1, ABCC1, and ABCG2 inhibitors. C@PA is the very first successful computational approach for the discovery of promiscuous ABC transporter inhibitors.
10.1021/acs.jmedchem.0c02199
From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance.
Litman T,Druley T E,Stein W D,Bates S E
Cellular and molecular life sciences : CMLS
The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.
10.1007/PL00000912
A mutation hot spot in the Bcrp1 (Abcg2) multidrug transporter in mouse cell lines selected for Doxorubicin resistance.
Allen John D,Jackson Sonja C,Schinkel Alfred H
Cancer research
The recent identification of mutations at arginine 482 (R482) in human Breast Cancer Resistance Protein (BCRP) in two drug-selected cell lines largely explains some discrepancies observed in the cross-resistance profiles of human cell lines overexpressing this multidrug transporter. We find that each of three mouse cell lines independently selected for resistance to the anthracycline doxorubicin also acquired mutations in the cognate mouse transporter Bcrp1 exclusively at R482. Although the mouse Bcrp1 amino acid substitutions (M or S) are distinct from those seen in the human cell lines (G or T), they all have similar consequences: (a) greater resistance to anthracyclines (and bisantrene); (b) relatively lower resistance to topotecan; (c) greatly enhanced efflux of the dye rhodamine 123. The ready selection of R482X mutations seen in vitro might also occur in tumors treated with anthracyclines. Thus, it is noteworthy that the efficacy of Bcrp1 inhibitors applicable in vivo was not markedly affected by the presence of the mutations. We found that the Bcrp1 mutations all occurred after previous amplification and overexpression of the wild-type gene under doxorubicin selection; wild-type Bcrp1 is evidently able to mediate substantial resistance to anthracyclines, and this was confirmed in Bcrp1-transduced cell lines. These observations emphasize the general importance of the arginine at amino acid 482 for substrate specificity of the transporter, while reminding us that unmutated Bcrp1 remains a potential source of resistance to anthracyclines and a potential factor in anthracycline pharmacokinetics. The same is most likely true of human BCRP, given its profound similarities to mouse Bcrp1.
Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2.
Dai Chun-ling,Tiwari Amit K,Wu Chung-Pu,Su Xiao-Dong,Wang Si-Rong,Liu Dong-geng,Ashby Charles R,Huang Yan,Robey Robert W,Liang Yong-ju,Chen Li-ming,Shi Cheng-Jun,Ambudkar Suresh V,Chen Zhe-Sheng,Fu Li-wu
Cancer research
Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E(2)17betaG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic.
10.1158/0008-5472.CAN-08-0499
Identification of 14-3-3sigma as a contributor to drug resistance in human breast cancer cells using functional proteomic analysis.
Liu Yang,Liu Hailan,Han Baoguang,Zhang Jian-Ting
Cancer research
Multidrug resistance (MDR) is a major obstacle to successful cancer treatment. To understand the mechanism of MDR, many cancer cell lines have been established, and various mechanisms of resistance, such as ATP-binding cassette (ABC) transporter-mediated drug efflux, have been discovered. Previously, a MDR cell line MCF7/AdVp3000 was selected from breast cancer cell line MCF7 against Adriamycin, and overexpression of ABCG2 was thought to cause MDR in this derivative cell line. However, ectopic overexpression of ABCG2 in MCF7 cells could not explain the extremely high drug resistance level of the selected MCF7/AdVp3000 cells. We hypothesized that MCF7/AdVp3000 cells must have other resistance mechanisms selected by Adriamycin. To test this hypothesis, we compared the global protein profiles between MCF7 and MCF7/AdVp3000 cells. Following two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, 17 protein spots with differential levels between the two cell lines were identified. Although 14-3-3sigma, keratin 18, keratin 19, ATP synthase beta, protein disulfide isomerase, heat shock protein 27, cathepsin D, triose-phosphate isomerase, peroxiredoxin 6, and electron transfer flavoprotein were increased, nm23/H1, peroxiredoxin 2, nucleophosmin 1/B23, and inorganic pyrophosphatase were decreased in MCF7/AdVp3000 cells. The differential levels of these proteins were validated using Western blot. Furthermore, functional validation showed that the elevated 14-3-3sigma expression contributes considerably to the observed drug resistance in MCF7/AdVp3000 cells. We, thus, conclude that these proteins likely contribute to the resistance selected in the MCF7/AdVp3000 cells, and their altered expression in tumors may cause clinical resistance to chemotherapy.
10.1158/0008-5472.CAN-05-3801
Pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinolines: Novel compounds that reverse ABCG2-mediated resistance in cancer cells.
Karthikeyan Chandrabose,Malla Ritu,Ashby Charles R,Amawi Haneen,Abbott Kodye L,Moore Joshua,Chen Joel,Balch Curt,Lee Crystal,Flannery Patrick C,Trivedi Piyush,Faridi Jesika S,Pondugula Satyanarayana R,Tiwari Amit K
Cancer letters
Overexpression of ATP-binding cassette transporter (ABC) subfamily G2 in cancer cells is known to elicit a MDR phenotype, ultimately resulting in cancer chemotherapy failure. Here, we report, for the first time, the effect of eight novel pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline (IND) derivatives that inhibit ABCG2 transporter restoring cancer cell chemosensitivity. IND -4, -5, -6, -7, and -8, at 10 µM, and nilotinib at 5 µM, significantly potentiated (8-10 fold) the cytotoxicity of the ABCG2 substrates mitoxantrone (MX) and doxorubicin in HEK293 cells overexpressing ABCG2 transporter, MX (~14 fold) in MX-resistant NCI-H460/MX-20 small cell lung cancer, and of topotecan (~7 fold) in S1-M1-80 colon cancer cells which all stably expressing ABCG2. In contrast, cytotoxicity of cisplatin, which is not an ABCG2 substrate, was not altered. IND-5,-6,-7, and -8 significantly increased the accumulation of rhodamine-123 in multidrug resistant NCI-H460/MX-20 cells overexpressing ABCG2. Both IND-7 and -8, the most potent ABCG2 inhibitors, had the highest affinities for the binding sites of ABCG2 in modeling studies. In conclusion, the beneficial actions of new class of agents warrant further development as potential MDR reversal agents for clinical anticancer agents that suffer from ABCG2-mediated MDR insensitivity.
10.1016/j.canlet.2016.03.030
IRE1α-targeting downregulates ABC transporters and overcomes drug resistance of colon cancer cells.
Gao Qiang,Li Xiu-Xiu,Xu Yi-Ming,Zhang Jin-Zhao,Rong Shi-di,Qin Yan-Qing,Fang Jing
Cancer letters
Drug resistance is a big problem in cancer treatment and one of the most prominent mechanisms underlain is overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1, ABCC1 and ABCG2. Inhibition of ABC transporters is an important approach to overcome drug resistance. The inositol-requiring enzyme 1α (IRE1α), an arm of unfolded protein response (UPR), splices XBP1 mRNA to generate an active transcription factor XBP1s. UPR is implicated in drug resistance. However, the underlying mechanism is unclear. We found that the anticancer drugs such as 5-fluorouracil (5-FU) activated the IRE1α-XBP1 pathway to induce the expression of ABCB1, ABCC1 and ABCG2 in colon cancer cells. Inhibition of IRE1α RNase activity with small molecule 4μ8c suppressed the drug-induced expression of these ABC transporters and sensitized 5-FU-resistant colon cancer cells to drug treatment. In vivo xenograft assay indicates that administration of 4μ8C substantially enhanced the efficacy of 5-FU chemotherapy on 5-FU-resistant colon cancer cells. These results suggest that IRE1α-targeting might be a strategy to cope with drug resistance of colon cancer.
10.1016/j.canlet.2020.02.007
DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.
Cellular & molecular biology letters
BACKGROUND:Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers. METHODS:The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined. RESULTS:DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 μM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%). CONCLUSIONS:We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.
10.1186/s11658-018-0096-y
Effect of excipients on breast cancer resistance protein substrate uptake activity.
Yamagata Tetsuo,Kusuhara Hiroyuki,Morishita Mariko,Takayama Kozo,Benameur Hassan,Sugiyama Yuichi
Journal of controlled release : official journal of the Controlled Release Society
Breast cancer resistance protein (BCRP/ABCG2) plays an important role in drug disposition. To examine whether some currently used excipients could inhibit its function, we measured the uptake of [(3)H]mitoxantrone in BCRP-, P-glycoprotein (P-gp)- or green fluorescent protein (GFP)-expressing cells, in the presence or absence of 15 kinds of currently used excipients. Of 15 excipients, five (Cremophor EL, Tween 20, Span 20, Pluronic P85 and Brij 30) increased the uptake of [(3)H]mitoxantrone in BCRP-expressing cells. On the other hand, ten (Cremophor EL, Cremophor RH40, Tween 20, Tween 80, Span 20, Pluronic P85, vitamin E TPGS, Brij 30, Myrj 52 and Gelucire 44/14) significantly increased uptake in P-gp-expressing cells. No significant effects on intracellular ATP levels were observed following treatments with the excipients that inhibited BCRP function. Taken together, this study demonstrated that some excipients might be potent BCRP inhibitors, and there may be differences in the effects of excipients on the functions of BCRP and P-gp.
10.1016/j.jconrel.2007.08.021
Pharmacogenomic and pharmacokinetic determinants of erlotinib toxicity.
Rudin Charles M,Liu Wanqing,Desai Apurva,Karrison Theodore,Jiang Xuemin,Janisch Linda,Das Soma,Ramirez Jacqueline,Poonkuzhali Balasubramanian,Schuetz Erin,Fackenthal Donna Lee,Chen Peixian,Armstrong Deborah K,Brahmer Julie R,Fleming Gini F,Vokes Everett E,Carducci Michael A,Ratain Mark J
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:To assess the pharmacogenomic and pharmacokinetic determinants of skin rash and diarrhea, the two primary dose-limiting toxicities of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib. PATIENTS AND METHODS:A prospective clinical study of 80 patients with non-small-cell lung cancer, head and neck cancer, and ovarian cancer was performed. Detailed pharmacokinetics and toxicity of erlotinib were assessed. Polymorphic loci in EGFR, ABCG2, CYP3A4, and CYP3A5 were genotyped, and their effects on pharmacokinetics and toxicities were evaluated. RESULTS:A novel diplotype of two polymorphic loci in the ABCG2 promoter involving -15622C/T and 1143C/T was identified, with alleles conferring lower ABCG2 levels associated with higher erlotinib pharmacokinetic parameters, including area under the curve (P = .019) and maximum concentration (P = .006). Variability in skin rash was best explained by a multivariate logistic regression model incorporating the trough erlotinib plasma concentration (P = .034) and the EGFR intron 1 polymorphism (P = .044). Variability in diarrhea was associated with the two linked polymorphisms in the EGFR promoter (P < .01), but not with erlotinib concentration. CONCLUSION:Although exploratory in nature, this combined pharmacogenomic and pharmacokinetic model helps to define and differentiate the primary determinants of skin and gastrointestinal toxicity of erlotinib. The findings may be of use both in designing trials targeting a particular severity of rash and in considering dose and schedule modifications in patients experiencing dose-limiting toxicities of erlotinib or similarly targeted agents. Further studies of the relationship between germline polymorphisms in EGFR and the toxicity and efficacy of EGFR inhibitors are warranted.
10.1200/JCO.2007.13.1128
Flavonoid Monomers as Potent, Nontoxic, and Selective Modulators of the Breast Cancer Resistance Protein (ABCG2).
Wong Iris L K,Zhu Xuezhen,Chan Kin-Fai,Liu Zhen,Chan Chin-Fung,Chow Tsun Sing,Chong Tsz Cheung,Law Man Chun,Cui Jiahua,Chow Larry M C,Chan Tak Hang
Journal of medicinal chemistry
We synthesize various substituted triazole-containing flavonoids and identify potent, nontoxic, and highly selective BCRP inhibitors. , , and possess -methoxycarbonylbenzyloxy substitution at C-3 of the flavone moiety and substituted triazole at C-4' of the B-ring. They show low toxicity (IC toward L929 > 100 μM), potent BCRP-inhibitory activity (EC = 1-15 nM), and high BCRP selectivity (BCRP selectivity over MRP1 and P-gp > 67-714). They inhibit the efflux activity of BCRP, elevate the intracellular drug accumulation, and restore the drug sensitivity of BCRP-overexpressing cells. Like Ko143, remarkably exhibits a 100% 5D3 shift, indicating that it can bind and cause a conformational change of BCRP. Moreover, it significantly reduces the abundance of functional BCRP dimers/oligomers by half to retain more mitoxantrone in the BCRP-overexpressing cell line and that may account for its inhibitory activity. They are promising candidates to be developed into combination therapy to overcome MDR cancers with BCRP overexpression.
10.1021/acs.jmedchem.1c00779
Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance.
Imai Yasuo,Tsukahara Satomi,Asada Sakiyo,Sugimoto Yoshikazu
Cancer research
Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan. We found previously that sulfated estrogens are physiologic substrates of BCRP. Flavonoids with weak estrogenic activities are called phytoestrogens. In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells. Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP. These flavonoids showed marginal effect on the drug sensitivity of K562 cells. Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance. Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells. K562/BCRP cells also accumulated less [(3)H]genistein than K562 cells. [(3)H]genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells. Fumitremorgin C abolished the increased transport of [(3)H]genistein in LLC/BCRP cells compared with parental cells. TLC analysis revealed that genistein was transported in its native form but not in its metabolized form. These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux. The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients.
10.1158/0008-5472.CAN-04-0078
Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling.
British journal of cancer
BACKGROUND:The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers. METHODS:The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERalpha, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs. RESULTS:The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r(2)=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab. INTERPRETATION:Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.
10.1038/sj.bjc.6605553
Impact of cellular folate status and epidermal growth factor receptor expression on BCRP/ABCG2-mediated resistance to gefitinib and erlotinib.
Lemos C,Kathmann I,Giovannetti E,Calhau C,Jansen G,Peters G J
British journal of cancer
The effect of folate status on breast cancer resistance protein (BCRP)-mediated drug resistance to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, was investigated in two human colon cancer cell lines, WiDr and Caco-2, of which the latter displayed greater sensitivity to these drugs due to high EGFR expression. Caco-2 LF/LV cells, growing under low-folate (LF) conditions, showed increased BCRP protein expression compared with the high-folate (HF) counterpart, which was associated with 1.8-fold resistance to gefitinib. Of note, the BCRP-specific inhibitor Ko143 completely reverted this phenotype. WiDr LF cells also showed slightly increased BCRP expression compared with the HF cells, but no differences in gefitinib sensitivity were observed. Both Caco-2 LF/LV and WiDr LF cells showed 2.4- and 2.3-fold resistance to erlotinib, respectively, compared with their HF counterparts, which mechanistically seemed BCRP unrelated, as Ko143 had no effect on erlotinib activity. In conclusion, our data suggest that in EGFR-expressing Caco-2 cells, BCRP is one of the determinants of gefitinib resistance but not of erlotinib resistance. Beyond this, folate depletion can provoke an additional decrease in gefitinib and erlotinib activity by mechanisms dependent or independent of BCRP modulation.
10.1038/sj.bjc.6604980
Structure of the human multidrug transporter ABCG2.
Taylor Nicholas M I,Manolaridis Ioannis,Jackson Scott M,Kowal Julia,Stahlberg Henning,Locher Kaspar P
Nature
ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.
10.1038/nature22345
Circumvention of breast cancer resistance protein (BCRP)-mediated resistance to camptothecins in vitro using non-substrate drugs or the BCRP inhibitor GF120918.
Maliepaard M,van Gastelen M A,Tohgo A,Hausheer F H,van Waardenburg R C,de Jong L A,Pluim D,Beijnen J H,Schellens J H
Clinical cancer research : an official journal of the American Association for Cancer Research
This study was aimed at characterizing the role of BCRP/MXR/ABCP (BCRP) in resistance of the human ovarian tumor cell lines T8 and MX3 to camptothecins more extensively and investigating whether resistance can be reversed by inhibiting BCRP by GF120918. Camptothecins studied were topotecan, CPT-11, and its active metabolite SN-38, 9-aminocamptothecin, and the novel experimental camptothecins NX211, DX8951f, and BNP1350. Notably, DX8951f and BNP1350 appeared to be very poor substrates for BCRP, with much lower resistance factors observed both in T8 and MX3 cells than observed for the other camptothecins tested. In the presence of a nontoxic dose level of GF120918, the intracellular accumulation of topotecan in the T8 and MX3 cells was completely restored to the intracellular levels observed in the sensitive IGROV1 parental cell line. This resulted in almost complete reversal of drug resistance to topotecan and to most of the other topoisomerase I drugs tested in the T8 cell line and to complete reversal in the MX3 cells. However, coincubation of DX8951f or BNP1350 with GF120918 did not affect the cytotoxicity of either of these drugs significantly. From the combined data, we conclude that the affinities of topoisomerase I drugs for BCRP are, in decreasing order: SN-38 > topotecan > 9-aminocamptothecin approximately CPT-11 > NX211 > DX8951f > BNP1350. Furthermore, GF120918 appears to be a potent reversal agent of BCRP-mediated resistance to camptothecins, with almost complete reversal noted at 100 nM. Potential BCRP-mediated resistance to topoisomerase I inhibitors can also be avoided by using the BCRP-insensitive drugs DX8951f or BNP1350. This observation may have important clinical implications for future development of novel camptothecins.
Aromatic 2-(thio)ureidocarboxylic acids as a new family of modulators of multidrug resistance-associated protein 1: synthesis, biological evaluation, and structure-activity relationships.
Häcker Hans-Georg,Leyers Stefan,Wiendlocha Jeanette,Gütschow Michael,Wiese Michael
Journal of medicinal chemistry
Four series of aromatic carboxylic acids were prepared with a urea or thiourea moiety at the neighboring position to the carboxyl group and benzene or thiophene as aromatic scaffold. Using a calcein AM assay, these compounds were evaluated as inhibitors of multidrug resistance-associated protein 1 (MRP1) and selected compounds were examined toward P-glycoprotein (P-gp) as well as breast cancer resistance protein (BCRP) to assess selectivity for MRP1. Two 2-thioureidobenzo[b]thiophene-3-carboxylic acids (48, 49) were identified as particularly potent inhibitors of MRP1, with IC50 values of around 1 microM. The structural features of this new family of nontoxic MRP1 inhibitors include a (thio)urea disubstituted with preferentially two alkyl groups at the terminal nitrogen and an additional fused aromatic ring.
10.1021/jm900688v
Bioluminescent imaging of drug efflux at the blood-brain barrier mediated by the transporter ABCG2.
Bakhsheshian Joshua,Wei Bih-Rong,Chang Ki-Eun,Shukla Suneet,Ambudkar Suresh V,Simpson R Mark,Gottesman Michael M,Hall Matthew D
Proceedings of the National Academy of Sciences of the United States of America
ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter.
10.1073/pnas.1312159110
Photodynamic Therapy Synergizes with Irinotecan to Overcome Compensatory Mechanisms and Improve Treatment Outcomes in Pancreatic Cancer.
Huang Huang-Chiao,Mallidi Srivalleesha,Liu Joyce,Chiang Chun-Te,Mai Zhiming,Goldschmidt Ruth,Ebrahim-Zadeh Neema,Rizvi Imran,Hasan Tayyaba
Cancer research
The ability of tumor cells to adapt to therapeutic regimens by activating alternative survival and growth pathways remains a major challenge in cancer therapy. Therefore, the most effective treatments will involve interactive strategies that target multiple nonoverlapping pathways while eliciting synergistic outcomes and minimizing systemic toxicities. Nanoliposomal irinotecan is approved by the FDA for gemcitabine-refractory metastatic pancreatic cancer. However, the full potential of irinotecan treatment is hindered by several cancer cell survival mechanisms, including ATP-binding cassette G2 (ABCG2) transporter-mediated irinotecan efflux from cells. Here, we demonstrate that benzoporphyrin derivative-based photodynamic therapy (PDT), a photochemical cytotoxic modality that activates the apoptotic pathway, reduced ABCG2 expression to increase intracellular irinotecan levels in pancreatic cancer. Moreover, we show that PDT inhibited survivin expression. Although PDT potentiated irinotecan treatment, we also demonstrate that irinotecan reduced the tumoral expression of monocarboxylate transporter 4, which was upregulated by PDT. Notably, using orthotopic xenograft models, we demonstrate that combination of single low-dose PDT and a subclinical dose of nanoliposomal irinotecan synergistically inhibited tumor growth by 70% for 3 weeks compared with 25% reduction after either monotherapies. Our findings offer new opportunities for the clinical translation of PDT and irinotecan combination therapy for effective pancreatic cancer treatment.
10.1158/0008-5472.CAN-15-0391
PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells.
Bleau Anne-Marie,Hambardzumyan Dolores,Ozawa Tatsuya,Fomchenko Elena I,Huse Jason T,Brennan Cameron W,Holland Eric C
Cell stem cell
In normal brain, the side population (SP) phenotype is generated by ABC transporter activity and identifies stem cell and endothelial cell subpopulations by dye exclusion. By drug efflux, the ABCG2 transporter provides chemoresistance in stem cells and contributes to the blood brain barrier (BBB) when active in endothelial cells. We investigated the SP phenotype of mouse and human gliomas. In glioma endothelial cells, ABC transporter function is impaired, corresponding to disruption of the BBB in these tumors. By contrast, the SP phenotype is increased in nonendothelial cells that form neurospheres and are highly tumorigenic. In this cell population, Akt, but not its downstream target mTOR, regulates ABCG2 activity, and loss of PTEN increases the SP. This Akt-induced ABCG2 activation results from its transport to the plasma membrane. Temozolomide, the standard treatment of gliomas, although not an ABCG2 substrate, increases the SP in glioma cells, especially in cells missing PTEN.
10.1016/j.stem.2009.01.007
Increased oral bioavailability of topotecan in combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918.
Kruijtzer C M F,Beijnen J H,Rosing H,ten Bokkel Huinink W W,Schot M,Jewell R C,Paul E M,Schellens J H M
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:We discovered that breast cancer resistance protein (BCRP), a recently identified adenosine triphosphate-binding cassette drug transporter, substantially limits the oral bioavailability of topotecan in mdr1a/1b(-/-) P-glycoprotein (P-gp) knockout and wild-type mice. GF120918 is a potent inhibitor of BCRP and P-gp. The aim was to increase the bioavailability of topotecan by GF120918. PATIENTS AND METHODS:In cohort A, eight patients received 1.0 mg/m(2) oral topotecan with or without coadministration of one single oral dose of 1,000 mg GF120918 (day 1 or day 8). In cohort B, eight other patients received 1.0 mg/m(2) intravenous topotecan with or without 1,000 mg oral GF120918 to study the effect of GF120918 on the systemic clearance of topotecan. RESULTS:After oral topotecan, the mean area under the plasma concentration-time curve (AUC) of total topotecan increased significantly from 32.4 +/- 9.6 microg.h/L without GF120918 to 78.7 +/- 20.6 microg.h/L when GF120918 was coadministered (P =.008). The mean maximum plasma concentration of total topotecan increased from 4.1 +/- 1.5 microg/L without GF120918 to 11.5 +/- 2.4 microg/L with GF120918 (P =.008). The apparent bioavailability in this cohort increased significantly from 40.0% (range, 32% to 47%) to 97.1% (range, 91% to 120%) (P =.008). Interpatient variability of the apparent bioavailability was 17% without and 11% with GF120918. After intravenous administration of topotecan, coadministration of oral GF120918 had a small but statistically significant effect on the AUC and systemic clearance of total topotecan but no statistically significant effect on maximum plasma concentration and terminal half-life of total topotecan. CONCLUSION:Coadministration of the BCRP and P-gp inhibitor GF120918 resulted in a significant increase of the systemic exposure of oral topotecan. The apparent oral bioavailability increased from 40.0% without to 97.1% with GF120918.
10.1200/JCO.2002.12.116
Multidrug resistance proteins in gastrointestinal stromal tumors: site-dependent expression and initial response to imatinib.
Théou Nathalie,Gil Sophie,Devocelle Anne,Julié Catherine,Lavergne-Slove Anne,Beauchet Alain,Callard Patrice,Farinotti Robert,Le Cesne Axel,Lemoine Antoinette,Faivre-Bonhomme Laurence,Emile Jean-François
Clinical cancer research : an official journal of the American Association for Cancer Research
Gastrointestinal stromal tumors (GIST) are the most frequent mesenchymal tumors of the digestive tract and respond poorly to chemotherapy. A tyrosine kinase inhibitor treatment, imatinib mesylate, was recently shown to have antitumor effects in metastatic patients. However, this drug is a substrate for multidrug resistance (MDR) proteins. Therefore, we investigated the expression of ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP) by Western blotting in 21 GISTs and 3 leiomyosarcomas. All the GISTs were positive for either ABCB1 (86% of cases) or ABCC1 expression (62%), but negative for ABCG2. ABCB1 was expressed in all gastric GISTs, but in only 67% of nongastric GISTs. By contrast, ABCC1 expression was more common in nongastric tumors (78% versus 42%). The levels of these MDR proteins in gastric GISTs were higher for ABCB1 (P = 0.007) and lower for ABCC1 (P = 0.004) compared with nongastric GISTs. We found no correlation between MDR protein expression and the risk assessment. None of the six patients treated with imatinib was resistant, although all were positive for at least one MDR protein. These results confirm that gastric and nongastric GISTs have different biological characteristics and suggest that MDR proteins do not impair the initial response of the tumor to imatinib.
10.1158/1078-0432.CCR-05-0710
Dasatinib targets chronic myeloid leukemia-CD34+ progenitors as effectively as it targets mature cells.
Hiwase Devendra K,Saunders Verity A,Nievergall Eva,Ross Douglas D,White Deborah L,Hughes Timothy P
Haematologica
Dasatinib is effective in most chronic phase chronic myeloid leukemia patients both in first-line therapy and following imatinib failure. While imatinib uptake into CD34(+) cells is low compared to mononuclear cells, few data evaluate how well dasatinib targets primitive CML cells. This study compares intracellular concentration of dasatinib and Bcr-Abl kinase inhibition in CML-CD34(+) progenitors and mononuclear cells induced by dasatinib. The intracellular concentrations of dasatinib were similar between CML-CD34(+) and mononuclear cells (P=0.8). Similarly, there was no significant difference in the degree of dasatinib-mediated Bcr-Abl kinase inhibition. ABCB1 (MDR1) and ABCG2 inhibitors neither increased dasatinib intracellular concentration nor enhanced dasatinib-mediated Bcr-Abl kinase inhibition. In contrast to nilotinib, we show that dasatinib is not an ABCB1 inhibitor. Thus, dasatinib targets CML-CD34(+) progenitors as effectively as it targets mononuclear cells. ABCB1 and ABCG2 efflux pumps do not appear to influence the intracellular dasatinib concentration in CML-CD34(+) progenitors.
10.3324/haematol.2012.070268
Synthesis and structure-activity relationship studies of O-biphenyl-3-yl carbamates as peripherally restricted fatty acid amide hydrolase inhibitors.
Moreno-Sanz Guillermo,Duranti Andrea,Melzig Laurin,Fiorelli Claudio,Ruda Gian Filippo,Colombano Giampiero,Mestichelli Paola,Sanchini Silvano,Tontini Andrea,Mor Marco,Bandiera Tiziano,Scarpelli Rita,Tarzia Giorgio,Piomelli Daniele
Journal of medicinal chemistry
The peripherally restricted fatty acid amide hydrolase (FAAH) inhibitor URB937 (3, cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester) is extruded from the brain and spinal cord by the Abcg2 efflux transporter. Despite its inability to enter the central nervous system (CNS), 3 exerts profound antinociceptive effects in mice and rats, which result from the inhibition of FAAH in peripheral tissues and the consequent enhancement of anandamide signaling at CB1 cannabinoid receptors localized on sensory nerve endings. In the present study, we examined the structure-activity relationships (SAR) for the biphenyl region of compound 3, focusing on the carbamoyl and hydroxyl groups in the distal and proximal phenyl rings. Our SAR studies generated a new series of peripherally restricted FAAH inhibitors and identified compound 35 (cyclohexylcarbamic acid 3'-carbamoyl-5-hydroxybiphenyl-3-yl ester) as the most potent brain-impermeant FAAH inhibitor disclosed to date.
10.1021/jm4007017
Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2).
Pedersen Jenny M,Matsson Pär,Bergström Christel A S,Norinder Ulf,Hoogstraate Janet,Artursson Per
Journal of medicinal chemistry
The chemical space of registered oral drugs was explored for inhibitors of the human multidrug-resistance associated protein 2 (MRP2; ABCC2), using a data set of 191 structurally diverse drugs and drug-like compounds. The data set included a new reference set of 75 compounds, for studies of hepatic drug interactions with transport proteins, CYP enzymes, and compounds associated with liver toxicity. The inhibition of MRP2-mediated transport of estradiol-17beta-D-glucuronide was studied in inverted membrane vesicles from Sf9 cells overexpressing human MRP2. A total of 27 previously unknown MRP2 inhibitors were identified, and the results indicate an overlapping but narrower inhibitor space for MRP2 compared with the two other major ABC efflux transporters P-gp (ABCB1) and BCRP (ABCG2). In addition, 13 compounds were shown to stimulate the transport of estradiol-17beta-D-glucuronide. The experimental results were used to develop a computational model able to discriminate inhibitors from noninhibitors according to their molecular structure, resulting in a predictive power of 86% for the training set and 72% for the test set. The inhibitors were in general larger and more lipophilic and presented a higher aromaticity than the noninhibitors. The developed computational model is applicable in an early stage of the drug discovery process and is proposed as a tool for prediction of MRP2-mediated hepatic drug interactions and toxicity.
10.1021/jm7015683
Mitochondrial expression and functional activity of breast cancer resistance protein in different multiple drug-resistant cell lines.
Solazzo Michela,Fantappiè Ornella,D'Amico Massimo,Sassoli Chiara,Tani Alessia,Cipriani Greta,Bogani Costanza,Formigli Lucia,Mazzanti Roberto
Cancer research
The multidrug resistance (MDR) phenotype is characterized by the overexpression of a few transport proteins at the plasma membrane level, one of which is the breast cancer resistance protein (BCRP). These proteins are expressed in excretory organs, in the placenta and blood-brain barrier, and are involved in the transport of drugs and endogenous compounds. Because some of these proteins are expressed in the mitochondria, this study was designed to determine whether BCRP is expressed at a mitochondrial level and to investigate its function in various MDR and parental drug-sensitive cell lines. By using Western blot analysis, immunofluorescence confocal and electron microscopy, flow cytometry analysis, and the BCRP (ABCG-2) small interfering RNA, these experiments showed that BCRP is expressed in the mitochondrial cristae, in which it is functionally active. Mitoxantrone accumulation was significantly reduced in mitochondria and in cells that overexpress BCRP, in comparison to parental drug-sensitive cells. The specific inhibitor of BCRP, fumitremorgin c, increased the accumulation of mitoxantrone significantly in comparison with basal conditions in both whole cells and in mitochondria of BCRP-overexpressing cell lines. In conclusion, this study shows that BCRP is overexpressed and functionally active in the mitochondria of MDR-positive cancer cell lines. However, its presence in the mitochondria of parental drug-sensitive cells suggests that BCRP can be involved in the physiology of cancer cells.
10.1158/0008-5472.CAN-08-4315
Gefitinib modulates the function of multiple ATP-binding cassette transporters in vivo.
Leggas Markos,Panetta John C,Zhuang Yanli,Schuetz John D,Johnston Brad,Bai Feng,Sorrentino Brian,Zhou Sheng,Houghton Peter J,Stewart Clinton F
Cancer research
The 4-anilinoquinazoline (4-AQ) derivative gefitinib (Iressa) is an oral epidermal growth factor receptor tyrosine kinase inhibitor. Oral administration of 4-AQ molecules, such as gefitinib, inhibits ATP-binding cassette (ABC) transporter-mediated drug efflux and strongly increases the apparent bioavailability of coadministered drug molecules that are transporter substrates. Based on in vitro studies investigating 4-AQ interactions with several transporters, these effects have primarily been attributed to the inhibition of breast cancer resistance protein (BCRP; ABCG2). Although 4-AQ shows in vitro inhibition of P-glycoprotein [multidrug resistance protein (MDR1); ABCB1], the in vivo effect on this and other transporters is not known. In our studies, pretreatment of Abcg2(-/-) and Mdr1(a/b)(-/-) mice with gefitinib increased oral absorption and decreased systemic clearance of topotecan, a model substrate, indicating that additional transporters were inhibited. These results were extended to human orthologues using engineered cell lines to show that gefitinib inhibited the efflux of BCRP and MDR1 substrates and restored vincristine sensitivity in MDR1-expressing cells. Although gefitinib inhibited BCRP more potently than MDR1 (10-fold), the inhibition of both transporters occurred at clinically relevant concentrations (e.g., 1-5 micromol/L). These studies illustrate the broad implications for the therapeutic combination of gefitinib or other 4-AQ molecules with agents that are BCRP and MDR1 substrates. 4-AQ molecules may offer a means to increase the low and variable oral drug absorption of transporter substrates while decreasing interpatient variability and reversing tumor drug resistance.
10.1158/0008-5472.CAN-05-2915
Symmetric bis-chalcones as a new type of breast cancer resistance protein inhibitors with a mechanism different from that of chromones.
Winter Evelyn,Devantier Neuenfeldt Patrícia,Chiaradia-Delatorre Louise Domeneghini,Gauthier Charlotte,Yunes Rosendo Augusto,Nunes Ricardo José,Creczynski-Pasa Tânia Beatriz,Di Pietro Attilio
Journal of medicinal chemistry
Potent ABCG2 inhibitors were recently identified as asymmetric chromones with different types of substituents. We here synthesized symmetric bis-chalcones that were differently substituted and screened for their ability to inhibit mitoxantrone efflux from ABCG2-transfected HEK293 cells. Potent bis-chalcone inhibitors were identified, the efficiency depending on both position of the central ketone groups and the number and positions of lateral methoxy substituents. The best derivative, namely, 1p, was selective for ABCG2 over P-glycoprotein and MRP1, appeared not to be transported by ABCG2, and was at least as active on various drug-selected cancer cells overexpressing ABCG2. Compound 1p stimulated the ABCG2 basal ATPase activity by contrast to a chromone lead that inhibited it, suggesting different mechanisms of interaction. Combination of both types of inhibitors produced synergistic effects, leading to complete inhibition at very low concentrations.
10.1021/jm401879z
9-Deazapurines as Broad-Spectrum Inhibitors of the ABC Transport Proteins P-Glycoprotein, Multidrug Resistance-Associated Protein 1, and Breast Cancer Resistance Protein.
Stefan Katja,Schmitt Sven Marcel,Wiese Michael
Journal of medicinal chemistry
P-Glycoprotein (P-gp, ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2) are the three major ABC transport proteins conferring resistance to many structurally diverse anticancer agents, leading to the phenomenon called multidrug resistance (MDR). Much effort has been put into the development of clinically useful compounds to reverse MDR. Broad-spectrum inhibitors of ABC transport proteins can be of great use in cancers that simultaneously coexpress two or three transporters. In this work, we continued our effort to generate new, potent, nontoxic, and multiply effective inhibitors of the three major ABC transporters. The best compound was active in a very low micromolar concentration range against all three transporters and restored sensitivity toward daunorubicin (P-gp and MRP1) and SN-38 (BCRP) in A2780/ADR (P-gp), H69AR (MRP1), and MDCK II BCRP (BCRP) cells. Additionally, the compound is a noncompetitive inhibitor of daunorubicin (MRP1), calcein AM (P-gp), and pheophorbide A (BCRP) transport.
10.1021/acs.jmedchem.7b00788
Quantitative and Mechanistic Understanding of AZD1775 Penetration across Human Blood-Brain Barrier in Glioblastoma Patients Using an IVIVE-PBPK Modeling Approach.
Li Jing,Wu Jianmei,Bao Xun,Honea Norissa,Xie Youming,Kim Seongho,Sparreboom Alex,Sanai Nader
Clinical cancer research : an official journal of the American Association for Cancer Research
AZD1775, a first-in-class, small-molecule inhibitor of the Wee1 tyrosine kinase, is under evaluation as a potential chemo- and radiosensitizer for treating glioblastoma. This study was to prospectively, quantitatively, and mechanistically investigate the penetration of AZD1775 across the human blood-brain barrier (BBB). AZD1775 plasma and tumor pharmacokinetics were evaluated in 20 patients with glioblastoma. The drug metabolism, transcellular passive permeability, and interactions with efflux and uptake transporters were determined using human derived systems. A whole-body physiologically based pharmacokinetic (PBPK) model integrated with a four-compartment permeability-limited brain model was developed for predicting the kinetics of AZD1775 BBB penetration and assessing the factors modulating this process. AZD1775 exhibited good tumor penetration in patients with glioblastoma, with the unbound tumor-to-plasma concentration ratio ranging from 1.3 to 24.4 (median, 3.2). It was a substrate for ABCB1, ABCG2, and OATP1A2, but not for OATP2B1 or OAT3. AZD1775 transcellular passive permeability and active efflux clearance across MDCKII-ABCB1 or MDCKII-ABCG2 cell monolayers were dependent on the basolateral pH. The PBPK model well predicted observed drug plasma and tumor concentrations in patients. The extent and rate of drug BBB penetration were influenced by BBB integrity, efflux and uptake active transporter activity, and drug binding to brain tissue. In the relatively acidic tumor microenvironment where ABCB1/ABCG2 transporter-mediated efflux clearance is reduced, OATP1A2-mediated active uptake becomes dominant, driving AZD1775 penetration into brain tumor. Variations in the brain tumor regional pH, transporter expression/activity, and BBB integrity collectively contribute to the heterogeneity of AZD1775 penetration into brain tumors. .
10.1158/1078-0432.CCR-17-0983
Nonprenylated rotenoids, a new class of potent breast cancer resistance protein inhibitors.
Ahmed-Belkacem Abdelhakim,Macalou Sira,Borrelli Francesca,Capasso Raffaele,Fattorusso Ernesto,Taglialatela-Scafati Orazio,Di Pietro Attilio
Journal of medicinal chemistry
Two rotenoids isolated from Boerhaavia diffusa (Nyctaginaceae), boeravinones G (1) and H (2), have been found to potently inhibit the drug efflux activity of breast cancer resistance protein (BCRP/ABCG2), a multidrug transporter responsible for cancer cell resistance to chemotherapy. The isolation of nine additional rotenoid derivatives (3-11), including the new boeravinones I (10) and J (11), from the extract of B. diffusa roots allowed us to establish structure-activity relationships toward inhibition of BCRP-mediated drug transport activity. The results show the positive roles of a methoxy group at position 6 of ring B and the absence of a substituent at position 10, and the requirement for a 6a/12a double bond between rings B and C. In contrast, both contraction of ring B, to give a coumaronochromone (11), and tetrasubstitution of ring D appeared to be detrimental for the inhibitory potency. The present study provides the first data on the BCRP-inhibiting activity of rotenoid derivatives, indicating boeravinones as a new class of interesting BCRP inhibitors.
10.1021/jm061450q
Investigation of chalcones as selective inhibitors of the breast cancer resistance protein: critical role of methoxylation in both inhibition potency and cytotoxicity.
Journal of medicinal chemistry
ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC(50) values below 0.5 μM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6'-hydroxyl-2',4'-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided.
10.1021/jm2016528
Pyrrolopyrimidine Derivatives as Novel Inhibitors of Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1).
Schmitt Sven Marcel,Stefan Katja,Wiese Michael
Journal of medicinal chemistry
Five series of pyrrolo[3,2-d]pyrimidines were synthesized and evaluated with respect to potency and selectivity toward multidrug resistance-associated protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome multidrug resistance in cancer patients. We investigated differently substituted pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation assay. New compounds with high potency and selectivity were identified. Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased activity. Aliphatic and aliphatic-aromatic variations at position 5 and 6 revealed compounds with IC50 values in high nanomolar range. All investigated compounds had low affinity toward P-glycoprotein (P-gp, ABCB1). Pyrrolopyrimidines with small substituents showed moderate inhibition against breast cancer resistance protein (BCRP, ABCG2).
10.1021/acs.jmedchem.5b01644
Gefitinib enhances the antitumor activity and oral bioavailability of irinotecan in mice.
Stewart Clinton F,Leggas Markos,Schuetz John D,Panetta John C,Cheshire Pamela J,Peterson Jennifer,Daw Najat,Jenkins Jesse J,Gilbertson Richard,Germain Glen S,Harwood Franklin C,Houghton Peter J
Cancer research
As a single agent the ERBB1 inhibitor, gefitinib (Iressa; ZD1839) showed minimal activity against a panel of 10 pediatric tumor xenografts that do not express the ERBB1 receptor. However, combined with irinotecan (CPT-11), significantly greater than additive activity was observed in four of eight models (P < 0.05), and the combination showed enhanced activity against three additional tumor lines. Breast cancer resistance protein (ABCG2), a transporter that confers resistance to SN-38 (the active metabolite of irinotecan), was readily detected in six of nine xenograft models examined by immunohistochemistry. In vitro gefitinib potently reversed resistance to SN-38 only in a cell line that overexpressed functional ABCG2. However, overexpression of ABCG2 did not decrease accumulation nor increase the rate of efflux of [(14)C]gefitinib. On the basis of these results and the distribution of Abcg2 in mouse tissues, we assessed the ability of gefitinib to modulate irinotecan pharmacokinetics. Oral gefitinib coadministration resulted in no change in clearance of intravenously administered irinotecan. However, gefitinib treatment dramatically increased the oral bioavailability of irinotecan after simultaneous oral administration. It is concluded that gefitinib may modulate SN-38 activity at the cellular level to reverse tumor resistance mediated by ABCG2 through inhibiting drug efflux and may be used potentially in humans to modulate the oral bioavailability of a poorly absorbed camptothecin such as irinotecan.
10.1158/0008-5472.CAN-04-0096
Substituted chromones as highly potent nontoxic inhibitors, specific for the breast cancer resistance protein.
Valdameri Glaucio,Genoux-Bastide Estelle,Peres Basile,Gauthier Charlotte,Guitton Jérôme,Terreux Raphaël,Winnischofer Sheila M B,Rocha Maria E M,Boumendjel Ahcène,Di Pietro Attilio
Journal of medicinal chemistry
A series of 13 disubstituted chromones was synthesized. Two types of substituents, on each side of the scaffold, contributed to both the potency of ABCG2 inhibition and the cytotoxicity. The best compound, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one (6g), displayed high-affinity inhibition and low cytotoxicity, giving a markedly high therapeutic index. The chromone derivative specifically inhibited ABCG2 versus other multidrug ABC transporters and was not transported. It constitutes a highly promising candidate for in vivo chemosensitization of ABCG2-expressing tumors.
10.1021/jm201404w
HM30181 Derivatives as Novel Potent and Selective Inhibitors of the Breast Cancer Resistance Protein (BCRP/ABCG2).
Köhler Sebastian C,Wiese Michael
Journal of medicinal chemistry
The breast cancer resistance protein (BCRP, ABCG2) belongs to the superfamily of ATP binding-cassette (ABC) proteins. In addition to other physiological functions, it transports potentially cell-damaging compounds out of the cell using the energy from ATP hydrolysis. Certain tumors overexpressing BCRP were found to become resistant against various anticancer drugs. In previous work, we found that tariquidar analogues lacking the tetrahydroisoquinoline moiety selectively inhibit BCRP. In the present study, we synthesized 21 derivatives of the third-generation P-gp inhibitor HM30181, which is structurally related to tariquidar. The compounds were tested for their inhibitory activities against BCRP and screened against P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) to confirm the selectivity toward BCRP. The most potent compounds are selective toward BCRP and 2-fold more potent than the reference Ko143. Qualitative structure-activity relationship (SAR) analysis revealed that the presence of a methoxy group in the ortho or para position of at least one phenyl ring is beneficial for inhibitory activity. Furthermore, the cytotoxicity and multidrug resistance (MDR)-reversal ability of selected compounds were investigated. It was shown that they have a low cytotoxicity and the ability to reverse the BCRP-mediated SN-38 resistance.
10.1021/acs.jmedchem.5b00188
Dasatinib cellular uptake and efflux in chronic myeloid leukemia cells: therapeutic implications.
Hiwase Devendra K,Saunders Verity,Hewett Duncan,Frede Amity,Zrim Stephanie,Dang Phuong,Eadie Laura,To L Bik,Melo Junia,Kumar Sharad,Hughes Timothy P,White Deborah L
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:The organic cation transporter OCT-1 mediates active transport of imatinib. We recently showed that low OCT-1 activity is a major contributor to suboptimal response in chronic myeloid leukemia (CML) patients treated with imatinib. The relevance of OCT-1 activity and efflux pumps in determining intracellular uptake and retention (IUR) of dasatinib was assessed. EXPERIMENTAL DESIGN:The effect of OCT inhibitors on [14C]dasatinib and [14C]imatinib IUR was compared using peripheral blood mononuclear cells from newly diagnosed CML patients. The role of efflux transporters was studied using ABCB1- and ABCG2-overexpressing cell lines and relevant inhibitors. RESULTS:Unlike imatinib, there was no significant difference in the dasatinib IUR at 37 degrees C and 4 degrees C (P = 0.8), and OCT-1 inhibitors including prazosin did not reduce dasatinib IUR significantly. In CML mononuclear cells, prazosin inhibitable IUR was significantly higher for imatinib than dasatinib (6.38 versus 1.48 ng/200,000 cells; P = 0.002; n = 11). Patients with high OCT-1 activity based on their imatinib uptake had IC50(dasatinib) values equivalent to patients with low OCT-1 activity. Dasatinib IUR was significantly lower in ABCB1-overexpressing cell lines compared with parental cell lines (P < 0.05). PSC833 (ABCB1 inhibitor) significantly increased the dasatinib IUR (P < 0.05) and reduced IC50(dasatinib) (from 100 to 8 nmol/L) in K562-DOX cell line. The ABCG2 inhibitor Ko143 significantly increased dasatinib IUR in ABCG2-overexpressing cell lines and reduced IC(50)(dasatinib). CONCLUSION:Unlike imatinib, dasatinib cellular uptake is not significantly affected by OCT-1 activity, so that expression and function of OCT-1 is unlikely to affect response to dasatinib. Dasatinib is a substrate of both efflux proteins, ABCB1 and ABCG2.
10.1158/1078-0432.CCR-07-5095
Converting potent indeno[1,2-b]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2.
Jabor Gozzi Gustavo,Bouaziz Zouhair,Winter Evelyn,Daflon-Yunes Nathalia,Aichele Dagmar,Nacereddine Abdelhamid,Marminon Christelle,Valdameri Glaucio,Zeinyeh Waël,Bollacke Andre,Guillon Jean,Lacoudre Aline,Pinaud Noël,Cadena Silvia M,Jose Joachim,Le Borgne Marc,Di Pietro Attilio
Journal of medicinal chemistry
A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.
10.1021/jm500943z
Afatinib enhances the efficacy of conventional chemotherapeutic agents by eradicating cancer stem-like cells.
Wang Xiao-kun,He Jie-hua,Xu Jing-hong,Ye Sheng,Wang Fang,Zhang Hui,Huang Zhen-cong,To Kenneth Kin Wah,Fu Li-wu
Cancer research
Cancer stem cells (CSC) have garnered significant attention as a therapeutic focus, based on evidence that they may represent an etiologic root of treatment-resistant cells. Indeed, expression of the multidrug resistance protein ATP-binding cassette subfamily G member 2 (ABCG2) confers chemoresistance to CSCs, where it serves as a potential biomarker and therapeutic target. Here, we show that afatinib, a small-molecule inhibitor of the tyrosine kinases EGFR, HER2, and HER4, preferentially eliminated side population cells with CSC character, in both cell lines and patient-derived leukemia cells, by decreasing ABCG2 expression. In these cells, afatinib also acted in parallel to suppress self-renewal capacity and tumorigenicity. Combining afatinib with the DNA-damaging drug topotecan enhanced the antitumor effect of topotecan in vitro and in vivo. Mechanistic investigations suggested that ABCG2 suppression by afatinib did not proceed by proteolysis through the ubiquitin-dependent proteosome, lysosome, or calpain. Instead, we found that afatinib increased DNA methyltransferase activity, thereby leading to methylation of the ABCG2 promoter and to a decrease in ABCG2 message level. Taken together, our results advocate the use of afatinib in combination with conventional chemotherapeutic drugs to improve efficacy by improving CSC eradication.
10.1158/0008-5472.CAN-13-3553
Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters.
Mi Yan-Jun,Liang Yong-Ju,Huang Hong-Bing,Zhao Hong-Yun,Wu Chung-Pu,Wang Fang,Tao Li-Yang,Zhang Chuan-Zhao,Dai Chun-Ling,Tiwari Amit K,Ma Xiao-Xu,To Kenneth Kin Wah,Ambudkar Suresh V,Chen Zhe-Sheng,Fu Li-Wu
Cancer research
Apatinib, a small-molecule multitargeted tyrosine kinase inhibitor, is in phase III clinical trial for the treatment of patients with non-small-cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr, and HEK293/ABCB1 cells overexpressing ABCB1 and in S1-M1-80, MCF-7/FLV1000, and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photoaffinity labeling of both ABCB1 and ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration that produced a reversal of MDR, did not significantly alter the ABCB1 or ABCG2 protein or mRNA expression levels or the phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1-resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking the AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs.
10.1158/0008-5472.CAN-10-0111
Synthesis and Biological Evaluation of 4-Anilino-quinazolines and -quinolines as Inhibitors of Breast Cancer Resistance Protein (ABCG2).
Krapf Michael K,Wiese Michael
Journal of medicinal chemistry
Chemotherapeutic treatment of cancer often fails due to overexpression of the ATP-binding cassette (ABC) transport proteins, like ABCG2, triggering active efflux of various structurally unrelated drugs. This so-called multidrug resistance (MDR) may be reversed by selective, potent, and nontoxic inhibitors of ABCG2. As only a few potent inhibitors are known, new compounds based on a 4-substituted-2-phenylquinazoline scaffold were investigated. Substitution with hydroxy, cyano, nitro, acetamido, and fluoro led to high inhibitory activities toward ABCG2. The ability to reverse MDR of the most active compounds was confirmed in a MTT efficacy assay. Moreover, a negligibly low intrinsic cytotoxicity was found resulting in a high therapeutic ratio. Investigations of the inhibitory activity toward ABCB1 and ABCC1 yielded a high selectivity toward ABCG2 for the quinazoline compounds. Quinoline-based analogues showed lower inhibitory activity and selectivity. The study yielded a variety of promising compounds, some with superior properties compared to those of the standard inhibitor Ko143.
10.1021/acs.jmedchem.6b00330
Isopetasin and S-isopetasin as novel P-glycoprotein inhibitors against multidrug-resistant cancer cells.
Abdelfatah Sara,Böckers Madeleine,Asensio Maitane,Kadioglu Onat,Klinger Anette,Fleischer Edmond,Efferth Thomas
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:A major problem of cancer treatment is the development of multidrug resistance (MDR) to chemotherapy. MDR is caused by different mechanisms such as the expression of the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). These transporters efflux xenobiotic toxins, including chemotherapeutics, and they were found to be overexpressed in different cancer types. PURPOSE:Identification of novel molecules that overcome MDR by targeting ABC-transporters. METHODS:Resazurin reduction assay was used for cytotoxicity test. AutoDock 4.2. was used for molecular docking. The function of P-gp and BCRP was tested using a doxorubicin uptake assay and an ATPase assay. ROS generation was detected using flow cytometry for the measurement of HDCFH-DA fluorescence. Annexin/PI staining was applied for the detection of apoptosis. Bioinformatic analyses were performed using LigandScout 3.12. software and DataWarrior software. RESULTS:In our search for new molecules that selectively act against resistant phenotypes, we identified isopetasin and S-isopetasin, which are bioactive natural products from Petasites formosanus. They exerted collateral sensitivity towards leukemia cells with high P-gp expression in CEM/ADR5000 cells, compared to sensitive wild-type CCRF-CEM leukemia cells. Also, they revealed considerable activity towards breast cancer cells overexpressing breast cancer resistance protein, MDA-MB-231-BCRP clone 23. This motivated us to investigate whether the function of P-gp was inhibited. In-silico results showed the compounds bound with high affinity and interacted with key amino acid residues in P-gp . Then, we found that the two compounds increased doxorubicin accumulation in P-gp overexpressing CEM/ADR5000 by three-fold compared to cells without inhibitor. P-gp-mediated drug efflux was ATP-dependent. Isopetasin and S-isopetasin increased the ATPase activity of human P-gp in a comparable fashion as verapamil used as control P-gp inhibitor. As isopetasin and S-isopetasin exerted dual roles, first as cytotoxic compounds and then as P-gp inhibitors, we suggested that their P-gp inhibition is part of a larger complex of mechanisms to induce cell death in cancer patients. P-gp dysfunction induces mitochondrial stress to generate ATP. Upon continuing stress by P-gp inhibition, the mitochondria generate reactive oxygen species (ROS). Initially established for verapamil, this theory was validated in the present study for isopetasin and S-isopetasin, as treatment with the two candidates increased ROS levels in CEM/ADR5000 cells followed by apoptosis. CONCLUSION:Our study highlights the importance of isopetasin and S-isopetasin as novel ROS-generating and apoptosis-inducing P-gp inhibitors.
10.1016/j.phymed.2020.153196
Lack of interactions between breast cancer resistance protein (bcrp/abcg2) and selected antiepileptic agents.
Cerveny Lukas,Pavek Petr,Malakova Jana,Staud Frantisek,Fendrich Zdenek
Epilepsia
PURPOSE:Recent studies have indicated constitutive expression of efflux transporter, breast cancer resistance protein (BCRP, ABCG2), in endothelial cells of the blood-brain barrier (BBB). In epileptogenic brain tumors such as ganglioma, astrocytoma, anaplastic astrocytomas, or glioma multiforme, strong expression of BCRP in the microvasculature of the BBB was observed. Therefore it was hypothesized that this phenomenon could critically influence the bioavailability of drugs in these tumors and potentially contribute to the failure of antiepileptic treatment. The aim of this study was to test whether some commonly used antiepileptic drugs (AEDs) are substrates transported by human BCRP. In particular, we focused on phenobarbital, phenytoin, ethosuximide, primidone, valproate, carbamazepine, clonazepam, and lamotrigine. Furthermore, the inhibitory potency of these AEDs to BCRP was examined. METHODS:To study substrate affinity of tested AEDs to BCRP, transport experiments were performed in epithelial BCRP-expressing MDCKII-BCRP and MDCKII-parent cell lines cultured on microporous membrane. For detection of inhibitory potency of AEDs to BCRP, accumulation assays were carried out in MEF3.8-BCRP cells with known BCRP substrates, BODIPY FL prazosin and mitoxantrone. RESULTS:No obvious interactions of tested AEDs with BCRP transporter were observed. Therefore these drugs in relevant therapeutic concentrations are neither substrates nor inhibitors of BCRP. CONCLUSIONS:Based on our in vitro data we can conclude that resistance to treatment with the tested AEDs probably is not caused by the overexpression of BCRP in the BBB of epileptogenic brain tumors.
10.1111/j.1528-1167.2006.00453.x
The effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics and brain penetration of imatinib mesylate (Gleevec): implications for the use of breast cancer resistance protein and P-glycoprotein inhibitors to enable the brain penetration of imatinib in patients.
Breedveld Pauline,Pluim Dick,Cipriani Greta,Wielinga Peter,van Tellingen Olaf,Schinkel Alfred H,Schellens Jan H M
Cancer research
Imatinib mesylate (signal transduction inhibitor 571, Gleevec) is a potent and selective tyrosine kinase inhibitor, which was shown to effectively inhibit platelet-derived growth factor-induced glioblastoma cell growth preclinically. However, in patients, a limited penetration of imatinib into the brain has been reported. Imatinib is transported in vitro and in vivo by P-glycoprotein (P-gp; ABCB1), which thereby limits its distribution into the brain in mice. Previously, imatinib was shown to potently inhibit human breast cancer resistance protein (BCRP; ABCG2). Here, we show that imatinib is efficiently transported by mouse Bcrp1 in transfected Madin-Darby canine kidney strain II (MDCKII) monolayers. Furthermore, we show that the clearance of i.v. imatinib is significantly decreased 1.6-fold in Bcrp1 knockout mice compared with wild-type mice. At t = 2 hours, the brain penetration of i.v. imatinib was significantly 2.5-fold increased in Bcrp1 knockout mice compared with control mice. We tested the hypothesis that P-gp and BCRP inhibitors, such as elacridar and pantoprazole, improve the brain penetration of imatinib. Firstly, we showed in vitro that pantoprazole and elacridar inhibit the Bcrp1-mediated transport of imatinib in MDCKII-Bcrp1 cells. Secondly, we showed that co-administration of pantoprazole or elacridar significantly reduced the clearance of i.v. imatinib in wild-type mice by respectively 1.7-fold and 1.5-fold. Finally, in wild-type mice treated with pantoprazole or elacridar, the brain penetration of i.v. imatinib significantly increased 1.8-fold and 4.2-fold, respectively. Moreover, the brain penetration of p.o. imatinib increased 5.2-fold when pantoprazole was co-administered in wild-type mice. Our results suggest that co-administration of BCRP and P-gp inhibitors may improve delivery of imatinib to malignant gliomas.
10.1158/0008-5472.CAN-04-2416
Piperazinobenzopyranones and phenalkylaminobenzopyranones: potent inhibitors of breast cancer resistance protein (ABCG2).
Boumendjel Ahcène,Nicolle Edwige,Moraux Thomas,Gerby Bastien,Blanc Madeleine,Ronot Xavier,Boutonnat Jean
Journal of medicinal chemistry
In continuing research that led us to identify chromanone derivatives (J. Med. Chem. 2003, 46, 2125) as P-glycoprotein inhibitors, we obtained analogues able to modulate multidrug resistance (MDR) mediated by the breast cancer resistance protein (BCRP). The linkage of 5-hydroxybenzopyran-4-one to piperazines or phenalkylamines affords highly potent inhibitors of BCRP. By using sensitive (HCT116) and resistant colon cancer cells expressing BCRP, we evaluated the effect of 14 benzopyranone (chromone) derivatives on the accumulation and the cytotoxic effect of the anticancer drug, mitoxantrone. At 10 microM, three compounds increased both intracellular accumulation and cytotoxicity of mitoxantrone in HCT116/R cells with a comparable rate as fumitremorgin C and Gleevec used as reference inhibitors. The most potent molecules 5b and 5c are still active at 1 microM, whereas FTC shows weak inhibition. These molecules do not induce cell death as shown by the cell cycle distribution study, which makes them potential candidates for in vivo studies.
10.1021/jm050705h
New Inhibitors of Breast Cancer Resistance Protein (ABCG2) Containing a 2,4-Disubstituted Pyridopyrimidine Scaffold.
Krapf Michael K,Gallus Jennifer,Vahdati Sahel,Wiese Michael
Journal of medicinal chemistry
Multidrug resistance (MDR) occurring during cancer chemotherapy is a major obstacle for effectiveness and response to therapy and is often caused by ATP-binding cassette (ABC) efflux transporters. Belonging to the family of ABC transporters, breast cancer resistance protein is getting more and more in the spotlight of research. As a strategy to overcome MDR, inhibitors of ABC transporters were synthesized, which could be applied in combination with cytostatic drugs. For this purpose, 2,4-disubstituted pyridopyrimidine derivatives were synthesized. The investigations confirmed three key characteristics of good inhibitors: a low intrinsic cytotoxicity and a high potency and selectivity toward ABCG2. For selected compounds the interaction with ABCG2 was elucidated and their effect on ATPase activity and conformation sensitive 5D3 antibody binding was investigated. Their ability to reverse MDR in coadministration with the active metabolite of irinotecan and mitoxantron was confirmed.
10.1021/acs.jmedchem.7b01012
Mithramycin represses basal and cigarette smoke-induced expression of ABCG2 and inhibits stem cell signaling in lung and esophageal cancer cells.
Zhang Mary,Mathur Aarti,Zhang Yuwei,Xi Sichuan,Atay Scott,Hong Julie A,Datrice Nicole,Upham Trevor,Kemp Clinton D,Ripley R Taylor,Wiegand Gordon,Avital Itzak,Fetsch Patricia,Mani Haresh,Zlott Daniel,Robey Robert,Bates Susan E,Li Xinmin,Rao Mahadev,Schrump David S
Cancer research
Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we observed that exposure of esophageal cancer cells to cigarette smoke condensate (CSC) led to upregulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas. Furthermore, CSC increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor, Sp1, and Nrf2 within the ABCG2 promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly downregulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Microarray analyses revealed that mithramycin targeted multiple stem cell-related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies.
10.1158/0008-5472.CAN-11-3983
PI3K-mTOR Pathway Inhibition Exhibits Efficacy Against High-grade Glioma in Clinically Relevant Mouse Models.
Lin Fan,de Gooijer Mark C,Hanekamp Diana,Chandrasekaran Gayathri,Buil Levi C M,Thota Nishita,Sparidans Rolf W,Beijnen Jos H,Würdinger Tom,van Tellingen Olaf
Clinical cancer research : an official journal of the American Association for Cancer Research
The PI3K-AKT-mTOR signaling pathway is frequently activated in glioblastoma and offers several druggable targets. However, clinical efficacy of PI3K/mTOR inhibitors in glioblastoma has not yet been demonstrated. Insufficient drug delivery may limit the efficacy of PI3K/mTOR inhibitors against glioblastoma. The presence of the efflux transporters ABCB1/Abcb1 (P-glycoprotein, MDR1) and ABCG2/Abcg2 (BCRP) at the blood-brain barrier (BBB) restricts the brain penetration of many drugs. We used drug transport assays and performed pharmacokinetic/pharmacodynamic studies in wild-type and ABC-transporter knockout mice. The efficacy of PI3K-mTOR inhibition was established using orthotopic allograft and genetically engineered spontaneous glioblastoma mouse models. The mTOR inhibitors rapamycin and AZD8055 are substrates of ABCB1, whereas the dual PI3K/mTOR inhibitor NVP-BEZ235 and the PI3K inhibitor ZSTK474 are not. Moreover, ABCG2 transports NVP-BEZ235 and AZD8055, but not ZSTK474 or rapamycin. Concordantly, mice revealed increased brain penetration of rapamycin (13-fold), AZD8055 (7.7-fold), and NVP-BEZ235 (4.5-fold), but not ZSTK474 relative to WT mice. Importantly, ABC transporters limited rapamycin brain penetration to subtherapeutic levels, while the reduction in NVP-BEZ235 brain penetration did not prevent target inhibition. NVP-BEZ235 and ZSTK474 demonstrated antitumor efficacy with improved survival against U87 orthotopic gliomas, although the effect of ZSTK474 was more pronounced. Finally, ZSTK474 prolonged overall survival in Cre-LoxP conditional transgenic mice, mainly by delaying tumor onset. PI3K/mTOR inhibitors with weak affinities for ABC transporters can achieve target inhibition in brain (tumors), but have modest single-agent efficacy and combinations with (BBB penetrable) inhibitors of other activated pathways may be required. .
10.1158/1078-0432.CCR-16-1276
Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells.
Robey R W,Medina-Pérez W Y,Nishiyama K,Lahusen T,Miyake K,Litman T,Senderowicz A M,Ross D D,Bates S E
Clinical cancer research : an official journal of the American Association for Cancer Research
We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.
Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression.
Nakanishi Takeo,Shiozawa Ken,Hassel Bret A,Ross Douglas D
Blood
Imatinib, a potent tyrosine kinase inhibitor, is effluxed from cells by the breast cancer resistance protein (BCRP/ABCG2), yet published studies to date fail to demonstrate resistance to imatinib cytotoxicity in BCRP-overexpressing cells in vitro. We investigated cellular resistance to imatinib in BCR-ABL-expressing cells transduced and selected to overexpress BCRP (K562/BCRP-MX10). These cells exhibited a 2- to 3-fold increase in resistance to imatinib (P < .05) and a 7- to 12-fold increase in resistance to mitoxantrone, a known BCRP substrate. Resistance to imatinib was completely abolished by the specific BCRP inhibitor fumitremorgin C. Studies of the mechanism of the diminished resistance to imatinib compared with mitoxantrone revealed that imatinib decreased the expression of BCRP in K562/BCRP-MX10 cells without affecting mRNA levels. BCRP levels in cells that do not express BCR-ABL were not affected by imatinib. Loss of BCRP expression was accompanied by imatinib-induced reduction of phosphorylated Akt in the BCRP-expressing K562 cells. The phosphoinositol-3 kinase (PI3K) inhibitor LY294002 also decreased BCRP levels in K562/BCRP-MX10 cells. These studies show that BCRP causes measurable imatinib resistance, but this effect is attenuated by imatinib-mediated inhibition of BCR-ABL, which in turn downregulates overall BCRP levels posttranscriptionally via the PI3K-Akt pathway.
10.1182/blood-2005-10-4020
Flavonoid structure-activity studies identify 6-prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer resistance protein ABCG2.
Ahmed-Belkacem Abdelhakim,Pozza Alexandre,Muñoz-Martínez Francisco,Bates Susan E,Castanys Santiago,Gamarro Francisco,Di Pietro Attilio,Pérez-Victoria José M
Cancer research
Overexpression of breast cancer resistance protein ABCG2 confers multidrug resistance in cancer cells. The GF120918-sensitive drug efflux activity of human wild-type (R482) ABCG2-transfected cells was used for rational screening of inhibitory flavonoids and establishment of structure-activity relationships. Flavones were found more efficient than flavonols, isoflavones, and flavanones. Differentially substituted flavone derivatives indicated positive OH effects at position 5, in contrast to positions 3 and 7. A methoxy at position 7 was slightly positive in tectochrysin, whereas a strong positive effect was produced by prenylation at position 6. The potency of 6-prenylchrysin was comparable with that of GF120918 (IC50 = 0.3 micromol/L). Both 6-prenylchrysin and tectochrysin seemed specific for ABCG2 because no interaction was detected with either P-glycoprotein or MRP1. The ABCG2 resistance profile in vitro is altered by mutation at amino acid 482. The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Whereas GF120918 strongly inhibited the ATPase activity of wild-type ABCG2, neither 6-prenylchrysin nor tectochrysin altered the activity. In contrast, all three inhibitors stimulated the ATPase activity of mutant ABCG2. 6-Prenylchrysin at 0.5 micromol/L efficiently sensitized the growth of wild-type ABCG2-transfected cells to mitoxantrone, whereas higher concentrations were required for the mutant ones. In contrast, 1 micromol/L tectochrysin was sufficient to fully sensitize mutant ABCG2-transfected cells, whereas higher concentrations were required for the wild-type ones. Both flavones exhibited a lower intrinsic cytotoxicity than GF120918 and were apparently not transported by ABCG2. 6-Prenylchrysin and tectochrysin therefore constitute new and promising inhibitors for the reversal of ABCG2-mediated drug transport.
10.1158/0008-5472.CAN-04-1817
Drug transporters: recent advances concerning BCRP and tyrosine kinase inhibitors.
Lemos C,Jansen G,Peters G J
British journal of cancer
Multidrug resistance is often associated with the (over)expression of drug efflux transporters of the ATP-binding cassette (ABC) protein family. This minireview discusses the role of one selected ABC-transporter family member, the breast cancer resistance protein (BCRP/ABCG2), in the (pre)clinical efficacy of novel experimental anticancer drugs, in particular tyrosine kinase inhibitors.
10.1038/sj.bjc.6604213
Triazole Bridged Flavonoid Dimers as Potent, Nontoxic, and Highly Selective Breast Cancer Resistance Protein (BCRP/ABCG2) Inhibitors.
Zhu Xuezhen,Wong Iris L K,Chan Kin-Fai,Cui Jiahua,Law Man Chun,Chong Tsz Cheung,Hu Xuesen,Chow Larry M C,Chan Tak Hang
Journal of medicinal chemistry
The present work describes the syntheses of diverse triazole bridged flavonoid dimers and identifies potent, nontoxic, and highly selective BCRP inhibitors. A homodimer, , with -methoxycarbonylbenzyloxy substitution at C-3 of the flavone moieties and a triazole-containing linker (21 atoms between the two flavones) showed low toxicity (IC toward L929, 3T3, and HFF-1 > 100 μM), potent BCRP-inhibitory activity (EC = 1-2 nM), and high BCRP selectivity (BCRP selectivity over MRP1 and P-gp > 455-909). inhibits BCRP-ATPase activity, blocks the drug efflux activity of BCRP, elevates the intracellular drug accumulation, and finally restores the drug sensitivity of BCRP-overexpressing cells. It does not down-regulate the surface BCRP protein expression to enhance the drug retention. Therefore, and similar flavonoid dimers appear to be promising candidates for further development into combination therapy to overcome MDR cancers with BCRP overexpression.
10.1021/acs.jmedchem.9b00963
Overcoming Resistance to the THZ Series of Covalent Transcriptional CDK Inhibitors.
Gao Yang,Zhang Tinghu,Terai Hideki,Ficarro Scott B,Kwiatkowski Nicholas,Hao Ming-Feng,Sharma Bandana,Christensen Camilla L,Chipumuro Edmond,Wong Kwok-Kin,Marto Jarrod A,Hammerman Peter S,Gray Nathanael S,George Rani E
Cell chemical biology
Irreversible inhibition of transcriptional cyclin-dependent kinases (CDKs) provides a therapeutic strategy for cancers that rely on aberrant transcription; however, lack of understanding of resistance mechanisms to these agents will likely impede their clinical evolution. Here, we demonstrate upregulation of multidrug transporters ABCB1 and ABCG2 as a major mode of resistance to THZ1, a covalent inhibitor of CDKs 7, 12, and 13 in neuroblastoma and lung cancer. To counter this obstacle, we developed a CDK inhibitor, E9, that is not a substrate for ABC transporters, and by selecting for resistance, determined that it exerts its cytotoxic effects through covalent modification of cysteine 1039 of CDK12. These results highlight the importance of considering this common mode of resistance in the development of clinical analogs of THZ1, identify a covalent CDK12 inhibitor that is not susceptible to ABC transporter-mediated drug efflux, and demonstrate that target deconvolution can be accomplished through selection for resistance.
10.1016/j.chembiol.2017.11.007
A Kinome-Wide Selective Radiolabeled TrkB/C Inhibitor for in Vitro and in Vivo Neuroimaging: Synthesis, Preclinical Evaluation, and First-in-Human.
Bernard-Gauthier Vadim,Bailey Justin J,Mossine Andrew V,Lindner Simon,Vomacka Lena,Aliaga Arturo,Shao Xia,Quesada Carole A,Sherman Phillip,Mahringer Anne,Kostikov Alexey,Grand'Maison Marilyn,Rosa-Neto Pedro,Soucy Jean-Paul,Thiel Alexander,Kaplan David R,Fricker Gert,Wängler Björn,Bartenstein Peter,Schirrmacher Ralf,Scott Peter J H
Journal of medicinal chemistry
The proto-oncogenes NTRK1/2/3 encode the tropomyosin receptor kinases TrkA/B/C which play pivotal roles in neurobiology and cancer. We describe herein the discovery of [C]-(R)-3 ([C]-(R)-IPMICF16), a first-in-class positron emission tomography (PET) TrkB/C-targeting radiolabeled kinase inhibitor lead. Relying on extensive human kinome vetting, we show that (R)-3 is the most potent and most selective TrkB/C inhibitor characterized to date. It is demonstrated that [C]-(R)-3 readily crosses the blood-brain barrier (BBB) in rodents and selectively binds to TrkB/C receptors in vivo, as evidenced by entrectinib blocking studies. Substantial TrkB/C-specific binding in human brain tissue is observed in vitro, with specific reduction in the hippocampus of Alzheimer's disease (AD) versus healthy brains. We additionally provide preliminary translational data regarding the brain disposition of [C]-(R)-3 in primates including first-in-human assessment. These results illustrate for the first time the use of a kinome-wide selective radioactive chemical probe for endogenous kinase PET neuroimaging in human.
10.1021/acs.jmedchem.7b00396
Structure-activity relationships of chromone derivatives toward the mechanism of interaction with and inhibition of breast cancer resistance protein ABCG2.
Winter Evelyn,Lecerf-Schmidt Florine,Gozzi Gustavo,Peres Basile,Lightbody Mark,Gauthier Charlotte,Ozvegy-Laczka Csilla,Szakacs Gergely,Sarkadi Balazs,Creczynski-Pasa Tânia B,Boumendjel Ahcène,Di Pietro Attilio
Journal of medicinal chemistry
We recently identified a chromone derivative, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one, named here as chromone 1, as a potent, selective, nontoxic, and nontransported inhibitor of ABCG2-mediated drug efflux (Valdameri et al. J. Med. Chem. 2012, 55, 966). We have now synthesized a series of 14 derivatives to study the structure-activity relationships controlling both drug efflux and ATPase activity of ABCG2 and to elucidate their molecular mechanism of interaction and inhibition. It was found that the 4-bromobenzyloxy substituent at position 5 and the methoxyindole are important for both inhibition of mitoxantrone efflux and inhibition of basal ATPase activity. Quite interestingly, methylation of the central amide nitrogen strongly altered the high affinity for ABCG2 and the complete inhibition of mitoxantrone efflux and coupled ATPase activity. These results allowed the identification of a critical central inhibitory moiety of chromones that has never been investigated previously in any series of inhibitors.
10.1021/jm401649j
Potent and selective inhibitors of breast cancer resistance protein (ABCG2) derived from the p-glycoprotein (ABCB1) modulator tariquidar.
Kühnle Matthias,Egger Michael,Müller Christine,Mahringer Anne,Bernhardt Günther,Fricker Gert,König Burkhard,Buschauer Armin
Journal of medicinal chemistry
The efflux pumps ABCB1 (p-gp, MDR1) and ABCG2 (BCRP) are expressed to a high extent by endothelial cells at the blood-brain barrier (BBB) and other barrier tissues and are involved in drug resistance of tumor (stem) cells. Whereas numerous ABCB1 inhibitors are known, only a few ABCG2 modulators with submicromolar activity have been published. Starting from tariquidar (4) analogues as ABCB1 modulators, minimal structural modifications resulted in a drastic shift in favor of ABCG2 inhibition. Highest potency was found when the 3,4-dimethoxy-2-(quinoline-3-carbonylamino)benzoyl moiety in 4 was replaced with a 4-methoxycarbonylbenzoyl moiety bearing a hetarylcarboxamido group in 3-position, e.g., quinoline-3-carboxamido (5, IC(50): 119 nM) or quinoline-2-carboxamido (6, IC(50): 60 nM, flow cytometric mitoxantrone efflux assay, topotecan-resistant MCF-7 breast cancer cells); the selectivity for ABCG2 over ABCB1 was about 100-500 fold and the compounds were inactive at ABCC2 (MRP2). Chemosensitivity assays against MCF-7/Topo cells revealed that the nontoxic inhibitor 6 completely reverted ABCG2-mediated topotecan resistance at concentrations >100 nM, whereas 5 showed ABCG2 independent cytotoxicity. ABCG2 inhibitors might be useful for cancer treatment with respect to reversal of multidrug resistance, overcoming the BBB and targeting of tumor stem cells.
10.1021/jm8013822
The role of breast cancer resistance protein in acute lymphoblastic leukemia.
Plasschaert Sabine L A,van der Kolk Dorina M,de Bont Eveline S J M,Kamps Willem A,Morisaki Kuniaki,Bates Susan E,Scheffer George L,Scheper Rik J,Vellenga Edo,de Vries Elisabeth G E
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Overexpression of the transporter ABCG2, also known as breast cancer resistance protein and mitoxantrone resistance protein, can confer resistance to a variety of cytostatic drugs, such as mitoxantrone, topotecan, doxorubicin, and daunorubicin. This study analyzes the ABCG2 expression and activity in 46 human de novo acute lymphoblastic leukemia B- and T-lineage (ALL) samples. EXPERIMENTAL DESIGN:ABCG2 expression was measured flow cytometrically with the BXP-34 monoclonal antibody. ABCG2 functional activity was determined flow cytometrically by measuring mitoxantrone accumulation in combination with the ABCG2 inhibitor fumitremorgin C (FTC). To determine a possible effect of the transporters P-glycoprotein and multidrug resistance-associated protein (MRP1 and MRP2) on mitoxantrone accumulation, the accumulation was investigated in the presence of the P-glycoprotein inhibitor PSC 833 and MRP inhibitor MK-571. The ABCG2 gene was sequenced to investigate the amino acid at position 482. RESULTS:In B-lineage ALL (n = 23), the median BXP-34:IgG1 ratio was higher, namely 2.4 (range, 1.7-3.7), than in T-lineage ALL (n = 23; 1.9; range, 1.2-6.6; P = 0.003). The addition of FTC to mitoxantrone treatment caused a median increase in mitoxantrone accumulation of 21% (range, 0-140%) in B-lineage ALL. In T-lineage ALL, this FTC effect was less pronounced (5%; range, 0-256%; P = 0.013). The influence of FTC on mitoxantrone accumulation correlated with ABCG2 protein expression (r = 0.52; P < 0.001; n = 43). The increase in mitoxantrone accumulation, when FTC was added to cells treated with both PSC 833 and MK-571, correlated with the ABCG2 expression in B-lineage ALL but not in T-lineage ALL. Sequencing the ABCG2 gene revealed no ABCG2 mutation at position 482 in patients who accumulated more rhodamine after FTC. CONCLUSIONS:This study shows that ABCG2 is expressed higher and functionally more active in B-lineage than in T-lineage ALL.
A c-Met inhibitor increases the chemosensitivity of cancer stem cells to the irinotecan in gastric carcinoma.
Yashiro M,Nishii T,Hasegawa T,Matsuzaki T,Morisaki T,Fukuoka T,Hirakawa K
British journal of cancer
BACKGROUND:Cancer stem cells (CSCs) may be postulated mediators of the chemoresistance. This study aimed to determine an effective signal inhibitor with effects on the proliferation of CSCs in combination with anticancer drugs. METHODS:We used three gastric cancer cell lines and three side population (SP)-enriched CSC cell lines. We examined the combined effects of inhibitors against stemness signals, including c-Met inhibitor SU11274, and five anticancer drugs on the CSC proliferation and mRNA expression of chemoresistance-associated genes. RESULTS:The IC50 of irinotecan in SP-enriched CSC was 10.5 times higher than parent OCUM-2M cells, whereas that of oxaliplatin, taxol, gemcitabine, and 5-fluorouracil was 2.0, 2.8, 2.0, and 1.2, respectively. The SP cell lines had higher expression levels of UGT1A1, ABCG2, and ABCB1 than their parent cell lines. There was a synergistic antiproliferative effect with a combination of SU11274 and SN38 in SP cells, but not other inhibitors. The SU11274 significantly decreased the expression of UGT1A1, but not ABCG2 and ABCB1. The SN38 plus SU11274 group more effectively suppressed in vivo tumour growth by OCUM-2M/SP cells than either group alone. CONCLUSION:Cancer stem cells have chemoresistance to irinotecan. The c-Met inhibitor may be a promising target molecule for irinotecan-based chemotherapy of gastric cancer.
10.1038/bjc.2013.638
Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.
Brendel C,Scharenberg C,Dohse M,Robey R W,Bates S E,Shukla S,Ambudkar S V,Wang Y,Wennemuth G,Burchert A,Boudriot U,Neubauer A
Leukemia
The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.
10.1038/sj.leu.2404638
The dopamine D receptor antagonists PG01037, NGB2904, SB277011A, and U99194 reverse ABCG2 transporter-mediated drug resistance in cancer cell lines.
Hussein Noor,Amawi Haneen,Karthikeyan Chandrabose,Hall F Scott,Mittal Roopali,Trivedi Piyush,Ashby Charles R,Tiwari Amit K
Cancer letters
The ATP - binding cassette (ABC) family G2 (ABCG2) transporters are known to produce multidrug resistance (MDR) in cancer, thereby limiting the clinical response to chemotherapy. Molecular modeling data indicated that certain dopamine (DA) D receptor antagonists had a significant binding affinity for ABCG2 transporter. Therefore, in this in vitro study, we determined the effect of the D receptor antagonists PG01037, NGB2904, SB277011A, and U99194 on MDR resulting from the overexpression of ABCG2 transporters. The D receptor antagonists, at concentrations >100 μM, did not significantly affect the viability of H460-MX20, S1M1-80, A549-MX10 or wild type ABCG2 overexpressing (HEK293-R2) cells. However, at concentrations ranging from 0.01 to 10 μM, the D receptor antagonists PG01037, NGB2904, SB-277011A, and U99194 significantly increased the efficacy of the anticancer drugs mitoxantrone and doxorubicin in ABCG2-overexpressing MDR cells. Efflux studies indicated that both PG01037 and NGB2904, at a concentration of 5 μM, significantly decreased the efflux of rhodamine 123 from H460-MX20 cells. Interestingly, 5 μM of PG01037 or NGB2904 significantly decreased the expression levels of the ABCG2 protein, suggesting that these compounds inhibit both the function and expression of ABCG2 transporters at non-toxic concentrations.
10.1016/j.canlet.2017.03.015
The function of breast cancer resistance protein in epithelial barriers, stem cells and milk secretion of drugs and xenotoxins.
van Herwaarden Antonius E,Schinkel Alfred H
Trends in pharmacological sciences
The breast cancer resistance protein [BCRP (also known as ABCG2)] belongs to the ATP binding cassette (ABC) family of transmembrane drug transporters. BCRP has a broad substrate specificity and actively extrudes a wide variety of drugs, carcinogens and dietary toxins from cells. Situated in the apical plasma membrane of epithelial cells of the small and large intestine and renal proximal tubules and in the bile canalicular membrane of hepatocytes, BCRP decreases the oral availability and systemic exposure of its substrates. In several blood-tissue barriers BCRP reduces tissue penetration of its substrates and it protects haematopoietic stem cells from cytotoxic substrates. Moreover, BCRP is expressed in mammary gland alveolar epithelial cells during pregnancy and lactation, where it actively secretes a variety of drugs, toxins and carcinogens into milk. In apparent contradiction with the detoxifying role of BCRP in mothers, this contamination of milk exposes suckling infants and dairy consumers to xenotoxins. BCRP thus affects many important aspects of pharmacology and toxicology.
10.1016/j.tips.2005.11.007
Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2.
Dai Chun-ling,Liang Yong-ju,Wang Yan-sheng,Tiwari Amit K,Yan Yan-yan,Wang Fang,Chen Zhe-sheng,Tong Xiu-zhen,Fu Li-wu
Cancer letters
Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we evaluated the possible interaction of sunitinib with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP) in vitro. Our results showed that sunitinib completely reverse drug resistance mediated by ABCG2 at a non-toxic concentration of 2.5muM and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1 overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1 overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine 123 and methotrexate by ABCG2 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [(3)H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Overall, we conclude that sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic.
10.1016/j.canlet.2009.01.027
The acridone derivative MBLI-87 sensitizes breast cancer resistance protein-expressing xenografts to irinotecan.
Arnaud O,Boumendjel A,Gèze A,Honorat M,Matera E L,Guitton J,Stein W D,Bates S E,Falson P,Dumontet C,Di Pietro A,Payen L
European journal of cancer (Oxford, England : 1990)
The breast cancer resistance protein ABCG2 confers cellular resistance to irinotecan (CPT-11) and its active metabolite SN-38. We utilised ABCG2-expressing xenografts as a model to evaluate the ability of a non-toxic ABCG2 inhibitor to increase intracellular drug accumulation. We assessed the activity of irinotecan in vivo in SCID mice: irinotecan completely inhibited the development of control pcDNA3.1 xenografts, whilst only delaying the growth of ABCG2-expressing xenografts. Addition of MBLI-87, an acridone derivative inhibitor, significantly increased the irinotecan effect against the growth of ABCG2-expressing xenografts. In vitro, MBLI-87 was as potent as GF120918 against ABCG2-mediated irinotecan efflux, and additionally was specific for ABCG2. A significant sensitisation to irinotecan was achieved despite the fact that doses remained well below the maximum tolerated dose (due to the rather limited solubility of MBLI-87). This suggested that MBLI-87 is an excellent candidate to prevent drug efflux by ABCG2, without altering plasma concentrations of irinotecan and SN-38 after IP (intra-peritoneal) injections. This could constitute a useful strategy to improve drug pharmacology, to facilitate drug penetration into normal tissue compartments protected by ABCG2, and potentially to reverse drug resistance in cancer cells.
10.1016/j.ejca.2010.11.019
4-Anilino-2-pyridylquinazolines and -pyrimidines as Highly Potent and Nontoxic Inhibitors of Breast Cancer Resistance Protein (ABCG2).
Krapf Michael K,Gallus Jennifer,Wiese Michael
Journal of medicinal chemistry
Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transport proteins remains a major problem in the chemotherapeutic treatment of cancer and might be overcome by inhibition of the transporter. Because of the lack of understanding, the complex mechanisms involved in the transport process, in particular for breast cancer resistance protein (BCRP/ABCG2), there is a persistent need for studies of inhibitors of ABCG2. In this study, we investigated a systematic series of 4-substituted-2-pyridylquinazolines in terms of their inhibitory potency as well as selectivity toward ABCG2. For comparison, the quinazoline scaffold was reduced to the significantly smaller 4-methylpyrimidine basic structure. Furthermore, the cytotoxicity and the ability to reverse MDR was tested with the chemotherapeutic agents SN-38 and mitoxantrone (MX). Interaction of the compounds with ABCG2 was investigated by a colorimetric ATPase assay. Enzyme kinetic studies were carried out with Hoechst 33342 as fluorescent dye and substrate of ABCG2 to elucidate the compounds binding modes.
10.1021/acs.jmedchem.7b00441
Pharmacogenetics of ABCG2 and adverse reactions to gefitinib.
Cusatis George,Gregorc Vanesa,Li Jing,Spreafico Anna,Ingersoll Roxann G,Verweij Jaap,Ludovini Vienna,Villa Eugenio,Hidalgo Manuel,Sparreboom Alex,Baker Sharyn D
Journal of the National Cancer Institute
Gefitinib is an inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase with activity in non-small-cell lung cancer. Diarrhea and skin toxicity are prominent gefitinib-related adverse events that potentially limit its use. Gefitinib is a substrate for ABCG2 (ABCP, BCRP, MXR), a polymorphic efflux transporter protein that is highly expressed in the intestines and liver. Here we investigated associations between allelic variants of EGFR, ABCG2, and the transporter protein ABCB1 with diarrhea and skin toxicity in gefitinib-treated patients. One variant, a common functional single-nucleotide polymorphism (SNP) in the ABCG2 gene, was associated with diarrhea in 124 patients treated with oral gefitinib 250 mg once daily; seven (44%) of 16 patients heterozygous for ABCG2 421C>A (Q141K) developed diarrhea, versus only 13 (12%) of 108 patients homozygous for the wild-type sequence (P = .0046). However, this SNP was not associated with skin toxicity (P = .99). The finding suggests that patients with reduced ABCG2 activity due to a common genetic variant are at increased risk for substrate drug-induced diarrhea, with implications for optimizing treatment with such agents.
10.1093/jnci/djj469
Synthesis and Investigation of Tetrahydro-β-carboline Derivatives as Inhibitors of the Breast Cancer Resistance Protein (ABCG2).
Spindler Anna,Stefan Katja,Wiese Michael
Journal of medicinal chemistry
The breast cancer resistance protein (ABCG2) transports chemotherapeutic drugs out of cells, which makes it a major player in mediating multidrug resistance (MDR) of cancer cells. To overcome this mechanism, inhibitors of ABCG2 can be used. Only a few potent and selective ABCG2 inhibitors have been discovered, i.e., fumitremorgin C (FTC), Ko143, and the alkaloid harmine, which contain a tetrahydro-β-carboline or β-carboline backbone, respectively. However, toxicity and or instability prevent their use in vivo. Therefore, there is a need for further potent inhibitors. We synthesized and pharmacologically investigated 37 tetrahydro-β-carboline derivatives. The inhibitory activity of two compounds (51, 52) is comparable to that of Ko143, and they are selective for ABCG2 over ABCB1. Furthermore, they are able to reverse the ABCG2-mediated resistance toward SN-38 and inhibit the ATPase activity. The cytotoxicity data show that their inhibitory effect is substantially higher than their toxicity.
10.1021/acs.jmedchem.6b00035
Transcriptomics and Transposon Mutagenesis Identify Multiple Mechanisms of Resistance to the FGFR Inhibitor AZD4547.
Kas Sjors M,de Ruiter Julian R,Schipper Koen,Schut Eva,Bombardelli Lorenzo,Wientjens Ellen,Drenth Anne Paulien,de Korte-Grimmerink Renske,Mahakena Sunny,Phillips Christopher,Smith Paul D,Klarenbeek Sjoerd,van de Wetering Koen,Berns Anton,Wessels Lodewyk F A,Jonkers Jos
Cancer research
In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified as a key driver of invasive lobular carcinoma (ILC) in an insertional mutagenesis screen using the transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK-ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies. These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. .
10.1158/0008-5472.CAN-18-0757
Oligomerization domain of the multidrug resistance-associated transporter ABCG2 and its dominant inhibitory activity.
Xu Junkang,Peng Hui,Chen Qun,Liu Yang,Dong Zizheng,Zhang Jian-Ting
Cancer research
Overexpression of human ATP-binding cassette transporter ABCG2 in cancer cells causes multidrug resistance by effluxing anticancer drugs. ABCG2 is considered as a half transporter and is thought to function as a homodimer. However, recent evidence suggests that it may exist as a higher form of oligomer consisting of 12 subunits. In this study, we mapped the oligomerization domain of human ABCG2 to its transmembrane domain consisting of TM5-loop-TM6. This oligomerization domain, when expressed alone in HEK293 cells, also forms a homododecamer. Furthermore, this domain has activity that inhibits drug efflux and resistance function of the full-length ABCG2 likely by disrupting the formation of the homo-oligomeric full-length ABCG2. These findings suggest that human ABCG2 may exist and work as a homo-oligomer by interactions located in TM5-loop-TM6, and that ABCG2 oligomerization may be used as a target for therapeutic development to circumvent ABCG2-mediated drug resistance in cancer treatment.
10.1158/0008-5472.CAN-06-3169
Strategies to Inhibit ABCB1- and ABCG2-Mediated Efflux Transport of Erlotinib at the Blood-Brain Barrier: A PET Study on Nonhuman Primates.
Tournier Nicolas,Goutal Sebastien,Auvity Sylvain,Traxl Alexander,Mairinger Severin,Wanek Thomas,Helal Ourkia-Badia,Buvat Irène,Soussan Michael,Caillé Fabien,Langer Oliver
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
The tyrosine kinase inhibitor erlotinib poorly penetrates the blood-brain barrier (BBB) because of efflux transport by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), thereby limiting its utility in the treatment of non-small cell lung cancer metastases in the brain. Pharmacologic strategies to inhibit ABCB1/ABCG2-mediated efflux transport at the BBB have been successfully developed in rodents, but it remains unclear whether these can be translated to humans given the pronounced species differences in ABCG2/ABCB1 expression ratios at the BBB. We assessed the efficacy of two different ABCB1/ABCG2 inhibitors to enhance brain distribution of C-erlotinib in nonhuman primates as a model of the human BBB. METHODS:Papio anubis baboons underwent PET scans of the brain after intravenous injection of C-erlotinib under baseline conditions (n = 4) and during intravenous infusion of high-dose erlotinib (10 mg/kg/h, n = 4) or elacridar (12 mg/kg/h, n = 3). RESULTS:Under baseline conditions, C-erlotinib distribution to the brain (total volume of distribution [V], 0.22 ± 0.015 mL/cm) was markedly lower than its distribution to muscle tissue surrounding the skull (V, 0.86 ± 0.10 mL/cm). Elacridar infusion resulted in a 3.5 ± 0.9-fold increase in C-erlotinib distribution to the brain (V, 0.81 ± 0.21 mL/cm, P < 0.01), reaching levels comparable to those in muscle tissue, without changing C-erlotinib plasma pharmacokinetics. During high-dose erlotinib infusion, C-erlotinib brain distribution was also significantly (1.7 ± 0.2-fold) increased (V, 0.38 ± 0.033 mL/cm, P < 0.05), with a concomitant increase in C-erlotinib plasma exposure. CONCLUSION:We successfully implemented ABCB1/ABCG2 inhibition protocols in nonhuman primates resulting in pronounced increases in brain distribution of C-erlotinib. For patients with brain tumors, such inhibition protocols may ultimately be applied to create more effective treatments using drugs that undergo efflux transport at the BBB.
10.2967/jnumed.116.178665
Gefitinib ("Iressa", ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ABCG2-mediated drug resistance.
Nakamura Yoichi,Oka Mikio,Soda Hiroshi,Shiozawa Ken,Yoshikawa Megumi,Itoh Akiko,Ikegami Yoji,Tsurutani Junji,Nakatomi Katsumi,Kitazaki Takeshi,Doi Seiji,Yoshida Hisahiro,Kohno Shigeru
Cancer research
Gefitinib ("Iressa", ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non-small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the K(i) value was 1.01 +/- 0.09 micromol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.
10.1158/0008-5472.CAN-03-2417
2,4,6-Substituted Quinazolines with Extraordinary Inhibitory Potency toward ABCG2.
Krapf Michael K,Gallus Jennifer,Namasivayam Vigneshwaran,Wiese Michael
Journal of medicinal chemistry
Several members of the ABC transporter superfamily play a decisive role in the development of multidrug resistance (MDR) in cancer. One of these MDR associated efflux transporters is ABCG2. One way to overcome this MDR is the coadministration of potent inhibitors of ABCG2. In this study, we identified novel inhibitors containing a 2,4,6-substituted quinazoline scaffold. Introduction of a 6-nitro function led to extraordinarily potent compounds that were highly selective for ABCG2 and also able to reverse the MDR toward the chemotherapeutic drugs SN-38 and mitoxantrone. The binding of substrate Hoechst 33342 and the two potent inhibitors 31 and 41 which differ in their mechanism of inhibition was rationalized using the recently published cryo-EM structures of ABCG2. For a better understanding of the interaction between the inhibitors and ABCG2, additional investigations regarding the ATPase activity, the interaction with Hoechst 33342, and with the conformational sensitive 5D3 antibody were carried out.
10.1021/acs.jmedchem.8b01011
FL118, a novel camptothecin derivative, is insensitive to ABCG2 expression and shows improved efficacy in comparison with irinotecan in colon and lung cancer models with ABCG2-induced resistance.
Westover David,Ling Xiang,Lam Hong,Welch Jacob,Jin Chunyang,Gongora Celine,Del Rio Maguy,Wani Mansukh,Li Fengzhi
Molecular cancer
BACKGROUND:Irinotecan is a camptothecin analogue currently used in clinical practice to treat advanced colorectal cancer. However, acquired resistance mediated by the drug efflux pump ABCG2 is a recognized problem. We reported on a novel camptothecin analogue, FL118, which shows anticancer activity superior to irinotecan. In this study, we sought to investigate the potency of FL118 versus irinotecan or its active metabolite, SN-38, in both in vitro and in vivo models of human cancer with high ABCG2 activity. We also sought to assess the potency and ABCG2 affinity of several FL118 analogues with B-ring substitutions. METHODS:Colon and lung cancer cells with and without ABCG2 overexpression were treated with FL118 in the presence and absence of Ko143, an ABCG2-selective inhibitor, or alternatively by genetically modulating ABCG2 expression. Using two distinct in vivo human tumor animal models, we further assessed whether FL118 could extend time to progression in comparison with irinotecan. Lastly, we investigated a series of FL118 analogues with B-ring substitutions for ABCG2 sensitivity. RESULTS:Both pharmacological inhibition and genetic modulation of ABCG2 demonstrated that, in contrast to SN-38, FL118 was able to bypass ABCG2-mediated drug resistance. FL118 also extended time to progression in both in vivo models by more than 50% compared with irinotecan. Lastly, we observed that FL118 analogues with polar substitutions had higher affinity for ABCG2, suggesting that the nonpolar nature of FL118 plays a role in bypassing ABCG2-mediated resistance. CONCLUSIONS:Our results suggest that in contrast to SN-38 and topotecan, FL118 is a poor substrate for ABCG2 and can effectively overcome ABCG2-mediated drug resistance. Our findings expand the uniqueness of FL118 and support continued development of FL118 as an attractive therapeutic option for patients with drug-refractory cancers resulting from high expression of ABCG2.
10.1186/s12943-015-0362-9
Brain accumulation of dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by elacridar treatment.
Lagas Jurjen S,van Waterschoot Robert A B,van Tilburg Vicky A C J,Hillebrand Michel J,Lankheet Nienke,Rosing Hilde,Beijnen Jos H,Schinkel Alfred H
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Imatinib, a BCR-ABL tyrosine kinase inhibitor, is a substrate of the efflux transporters P-glycoprotein (P-gp; ABCB1) and ABCG2 (breast cancer resistance protein), and its brain accumulation is restricted by both transporters. For dasatinib, an inhibitor of SCR/BCR-ABL kinases, in vivo interactions with P-gp and ABCG2 are not fully established yet. EXPERIMENTAL DESIGN:We used Abcb1a/1b(-/-), Abcg2(-/-), and Abcb1a/1b;Abcg2(-/-) mice to establish the roles of P-gp and ABCG2 in the pharmacokinetics and brain accumulation of dasatinib. RESULTS:We found that oral uptake of dasatinib is limited by P-gp. Furthermore, relative brain accumulation, 6 hours after administration, was not affected by Abcg2 deficiency, but absence of P-gp resulted in a 3.6-fold increase after oral and 4.8-fold higher accumulation after i.p. administration. Abcb1a/1b;Abcg2(-/-) mice had the most pronounced increase in relative brain accumulation, which was 13.2-fold higher after oral and 22.7-fold increased after i.p. administration. Moreover, coadministration to wild-type mice of dasatinib with the dual P-gp and ABCG2 inhibitor elacridar resulted in a similar dasatinib brain accumulation as observed for Abcb1a/1b;Abcg2(-/-) mice. CONCLUSIONS:Brain accumulation of dasatinib is primarily restricted by P-gp, but Abcg2 can partly take over this protective function at the blood-brain barrier. Consequently, when both transporters are absent or inhibited, brain uptake of dasatinib is highly increased. These findings might be clinically relevant for patients with central nervous system Philadelphia chromosome-positive leukemia, as coadministration of an inhibitor of P-gp and ABCG2 with dasatinib might result in better therapeutic responses in these patients.
10.1158/1078-0432.CCR-08-2253
Pheophorbide a is a specific probe for ABCG2 function and inhibition.
Robey Robert W,Steadman Kenneth,Polgar Orsolya,Morisaki Kuniaki,Blayney Margaret,Mistry Prakash,Bates Susan E
Cancer research
Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2(-/-) knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649-15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 micro M of the cyclin-dependent kinase inhibitor UCN-01 or 1 micro M of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 micro M tariquidar, and ABCG2-transfected cells were 6-7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.
10.1158/0008-5472.can-03-3298
Fasudil increases temozolomide sensitivity and suppresses temozolomide-resistant glioma growth via inhibiting ROCK2/ABCG2.
Zhang Xin,Liu Xiuting,Zhou Wei,Yang Mengdi,Ding Yang,Wang Qing,Hu Rong
Cell death & disease
Resistance to temozolomide (TMZ) is a major clinical challenge in glioma treatment, but the mechanisms of TMZ resistance are poorly understood. Here, we provided evidence that ROCK2 acted redundantly to maintain resistance of TMZ in TMZ-resistant gliomas, and as a ROCK2 phosphorylation inhibitor, fasudil significantly suppressed proliferation of TMZ-resistant gliomas in vivo and vitro via enhancing the chemosensitivity of TMZ. Additionally, the membrane translocation of ABCG2 was decreased with fasudil by ROCK2/moesin pathway. We also showed that fasudil suppressed the expression of ABCG2 via ROCK2/moesin/β-catenin pathway. Our results reveal an indispensable role for ROCK2 and provide strong evidence for the therapeutic use of fasudil in the clinical setting for TMZ-resistant gliomas.
10.1038/s41419-017-0251-9
Tyrosine kinase inhibitor resistance in cancer: role of ABC multidrug transporters.
Ozvegy-Laczka Csilla,Cserepes Judit,Elkind N Barry,Sarkadi Balázs
Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy
Recent antitumor drug research has seen the development of a large variety of tyrosine kinase inhibitors (TKIs) with increasing specificity and selectivity. These are highly promising agents for specific inhibition of malignant cell growth and metastasis. However, their therapeutic potential also depends on access to their intracellular targets, which may be significantly affected by certain ABC membrane transporters. It has been recently shown that several human multidrug transporter ABC proteins interact with specific TKIs, and the ABCG2 transporter has an especially high affinity for some of these kinase inhibitors. These results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the treatment of cancer patients; moreover, the extrusion of TKIs by multidrug transporters may result in tumor cell TKI resistance. Interaction with multidrug resistance ABC transporters may also significantly modify the pharmacokinetics and toxicity of TKIs in patients.
10.1016/j.drup.2005.02.002
Roles of sildenafil in enhancing drug sensitivity in cancer.
Shi Zhi,Tiwari Amit K,Patel Atish S,Fu Li-Wu,Chen Zhe-Sheng
Cancer research
The phenomenon of multidrug resistance (MDR) has decreased the hope for successful cancer chemotherapy. The ATP-binding cassette (ABC) transporter superfamily is the largest transmembrane family. The overexpression of ABC transporters is a major determinant of MDR in cancer cells both in vitro and in vivo. Unfortunately, until recently, most of the strategies used to surmount ABC-transporter-mediated MDR have had limited success. An ideal modulator of MDR would be one that has a low liability to induce toxicity and alter the pharmacokinetic profile of antineoplastic drugs. Sildenafil, an inhibitor of cGMP-specific phosphodiesterase type 5, was found to significantly reverse ABC-transporter-mediated MDR. Our results indicate that sildenafil has differential inhibitory effects on ABC transporters: It significantly decreases the efflux activity of ABCB1 and ABCG2, but has no significant effects on ABCC1. Emerging evidence indicates that sildenafil and other phosphodiesterase type 5 inhibitors may enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs.
10.1158/0008-5472.CAN-11-0375
A Proof-of-Concept Study to Inhibit ABCG2- and ABCB1-Mediated Efflux Transport at the Human Blood-Brain Barrier.
Bauer Martin,Karch Rudolf,Wulkersdorfer Beatrix,Philippe Cécile,Nics Lukas,Klebermass Eva-Maria,Weber Maria,Poschner Stefan,Haslacher Helmuth,Jäger Walter,Tournier Nicolas,Wadsak Wolfgang,Hacker Marcus,Zeitlinger Markus,Langer Oliver
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
The adenosine triphosphate-binding cassette transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are 2 efflux transporters at the blood-brain barrier (BBB) that effectively restrict brain distribution of dual ABCB1/ABCG2 substrate drugs, such as tyrosine kinase inhibitors. Pharmacologic inhibition of ABCB1/ABCG2 may improve the efficacy of dual-substrate drugs for treatment of brain tumors, but no marketed ABCB1/ABCG2 inhibitors are currently available. In the present study, we examined the potential of supratherapeutic-dose oral erlotinib to inhibit ABCB1/ABCG2 activity at the human BBB. : Healthy men underwent 2 consecutive PET scans with C-erlotinib: a baseline scan and a second scan either with concurrent intravenous infusion of the ABCB1 inhibitor tariquidar (3.75 mg/min, = 5) or after oral intake of single ascending doses of erlotinib (300 mg, = 7; 650 mg, = 8; or 1,000 mg, = 2). : Although tariquidar administration had no effect on C-erlotinib brain distribution, oral erlotinib led, at the 650-mg dose, to significant increases in volume of distribution (23% ± 13%, = 0.008), influx rate constant of radioactivity from plasma into brain (58% ± 26%, = 0.008), and area under the brain time-activity curve (78% ± 17%, = 0.008), presumably because of combined partial saturation of ABCG2 and ABCB1 activity. Inclusion of further subjects into the 1,000-mg dose group was precluded by adverse skin events (rash). : Supratherapeutic-dose erlotinib may be used to enhance brain delivery of ABCB1/ABCG2 substrate anticancer drugs, but its clinical applicability for continuous ABCB1/ABCG2 inhibition at the BBB may be limited by safety concerns.
10.2967/jnumed.118.216432
The tyrosine kinase inhibitor imatinib mesylate enhances the efficacy of photodynamic therapy by inhibiting ABCG2.
Liu Weiguo,Baer Maria R,Bowman Mary Jo,Pera Paula,Zheng Xiang,Morgan Janet,Pandey Ravindra A,Oseroff Allan R
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:The ATP-binding cassette protein ABCG2 (breast cancer resistance protein) effluxes some of the photosensitizers used in photodynamic therapy (PDT) and, thus, may confer resistance to this treatment modality. Tyrosine kinase inhibitors (TKI) can block the function of ABCG2. Therefore, we tested the effects of the TKI imatinib mesylate (Gleevec) on photosensitizer accumulation and in vitro and in vivo PDT efficacy. EXPERIMENTAL DESIGN:Energy-dependent photosensitizer efflux and imatinib mesylate's effects on intracellular accumulation of clinically used second- and first-generation photosensitizers were studied by flow cytometry in murine and human cells with and without ABCG2 expression. Effects of ABCG2 inhibition on PDT were examined in vitro using cell viability assays and in vivo measuring photosensitizer accumulation and time to regrowth in a RIF-1 tumor model. RESULTS:Energy-dependent efflux of 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide-a (HPPH, Photochlor), endogenous protoporphyrin IX (PpIX) synthesized from 5-aminolevulenic acid, and the benzoporphyrin derivative monoacid ring A (BPD-MA, Verteporfin) was shown in ABCG2+ cell lines, but the first-generation multimeric photosensitizer porfimer sodium (Photofrin) and a novel derivative of HPPH conjugated to galactose were minimally transported. Imatinib mesylate increased accumulation of HPPH, PpIX, and BPD-MA from 1.3- to 6-fold in ABCG2+ cells, but not in ABCG2- cells, and enhanced PDT efficacy both in vitro and in vivo. CONCLUSIONS:Second-generation clinical photosensitizers are transported out of cells by ABCG2, and this effect can be abrogated by coadministration of imatinib mesylate. By increasing intracellular photosensitizer levels in ABCG2+ tumors, imatinib mesylate or other ABCG2 transport inhibitors may enhance efficacy and selectivity of clinical PDT.
10.1158/1078-0432.CCR-06-1599
Dacomitinib potentiates the efficacy of conventional chemotherapeutic agents via inhibiting the drug efflux function of ABCG2 in vitro and in vivo.
Guo Xiaoran,To Kenneth K W,Chen Zhen,Wang Xiaokun,Zhang Jianye,Luo Min,Wang Fang,Yan Shirong,Fu Liwu
Journal of experimental & clinical cancer research : CR
BACKGROUND:ATP-binding cassette subfamily G member 2 (ABCG2), a member of the ABC transporter superfamily proteins, mediates multidrug resistance (MDR) by transporting substrate anticancer drugs out of cancer cells and decreasing their intracellular accumulation. MDR is a major hurdle to successful chemotherapy. A logical approach to overcome MDR is to inhibit the transporter. However, no safe and effective MDR inhibitor has been approved in the clinic. METHODS:The MTT assay was used to evaluate cell cytotoxicity and MDR reversal effect. Drug efflux and intracellular drug accumulation were measured by flow cytometry. The H460/MX20 cell xenograft model was established to evaluate the enhancement of anticancer efficacy of topotecan by dacomitinib in vivo. To ascertain the interaction of dacomitinib with the substrate binding sites of ABCG2, the competition of dacomitinib for photolabeling of ABCG2 with [I]- iodoarylazidoprazosin (IAAP) was performed. Vanadate-sensitive ATPase activity of ABCG2 was measured in the presence of a range of different concentrations of dacomitinib to evaluate the effect of dacomitinib on ATP hydrolysis as the energy source of the transporter. A flow cytometry-based assay and western blotting were employed to study whether dacomitininb could inhibit the expression level of ABCG2. The mRNA expression levels of ABCG2 were analyzed by real-time quantitative RT-PCR. The protein expression level of AKT, ERK and their phosphorylations were detected by Western blotting. RESULTS:Here, we found that dacomitinib, an irreversible pan-ErbB tyrosine kinase inhibitor (TKI) in phase III clinical trial, could enhance the efficacy of conventional chemotherapeutic agents specifically in ABCG2-overexpressing MDR cancer cells but not in the parental sensitive cells. Dacomitinib was found to significantly increase the accumulation of ABCG2 probe substrates [doxorubicin (DOX),Rhodamine 123 (Rho 123) and Hoechst 33342] by inhibiting the transporter efflux function. Moreover, dacomitinib stimulated ABCG2 ATPase activity and competed with [I]-IAAP photolabeling of ABCG2 in a concentration-dependent manner. However, dacomitinib did not alter ABCG2 expression at protein and mRNA levels or inhibit ErbB downstream signaling of AKT and ERK. Importantly, dacomitinib significantly enhanced the efficacy of topotecan in ABCG2-overexpressing H460/MX20 cell xenografts in nude mice without incurring additional toxicity. CONCLUSIONS:These results suggest that dacomitinib reverses ABCG2-mediated MDR by inhibiting ABCG2 efflux function and increasing intracellular accumulation of anticancer agents. Our findings advocate further clinical investigation of combinations of dacomitinib and conventional chemotherapy in cancer patients with ABCG2-overexpressing MDR tumors.
10.1186/s13046-018-0690-x
Dacomitinib antagonizes multidrug resistance (MDR) in cancer cells by inhibiting the efflux activity of ABCB1 and ABCG2 transporters.
Fan Ying-Fang,Zhang Wei,Zeng Leli,Lei Zi-Ning,Cai Chao-Yun,Gupta Pranav,Yang Dong-Hua,Cui Qingbin,Qin Zuo-Dong,Chen Zhe-Sheng,Trombetta Louis D
Cancer letters
The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Numerous mechanisms have been recognized that cause MDR, but one of the most important mechanisms is overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporters, through which the efflux of various anticancer drugs against their concentration gradients is powered by ATP. In recent years, small molecular tyrosine kinase inhibitors (TKIs) have been developed for treatment in various human cancers overexpressing epidermal growth factor receptor (EGFR). At the same time, some TKIs have been shown to be capable of inhibiting ABC transporter-mediated MDR. Dacomitinib (PF-00299804) is a second generation, irreversible TKI, which has shown positive anticancer activities in some preclinical and clinical trials. As many TKIs are substrates or inhibitors of ABC transporters, this study investigates whether dacomitinib could interact with ABC subfamily members that mediate MDR, including ABCB1 (P-gp), ABCG2 (BCRP) and ABCC1 (MRP1). The results showed that dacomitinib at 1.0 μM significantly reversed drug resistance mediated by ABCB1 and ABCG2, but not ABCC1, doing so by antagonizing the drug efflux function in ABCB1- and ABCG2-overexpressing cell lines. The reversal effect on ABCB1-overexpressing cells is more potent than that on ABCG2-overexpressing cells. In addition, dacomitinib at reversal concentration affected neither the protein expression level nor the localization of ABCB1 and ABCG2. Therefore, the mechanisms of this modulating effect are likely to be the following: first, as an inhibitor of ABCB1 or ABCG2 transporters, dacomitinib binds to drug-substrate site in transmembrane domains (TMD) stably in a noncompetitive manner; or second, dacomitinib inhibits ATPase activity and maintains the stability of TMD conformation in a concentration-dependent manner thereby inhibiting the drug efflux function of ABCB1 or ABCG2 transporter. This study provides a useful combinational therapeutic strategy with dacomitinib and substrates of ABCB1 and/or ABCG2 transporters in ABCB1- or ABCG2-overexpressing cancers.
10.1016/j.canlet.2018.01.021
Identification of Thienopyrimidine Scaffold as an Inhibitor of the ABC Transport Protein ABCC1 (MRP1) and Related Transporters Using a Combined Virtual Screening Approach.
Silbermann Katja,Stefan Sven Marcel,Elshawadfy Randa,Namasivayam Vigneshwaran,Wiese Michael
Journal of medicinal chemistry
A virtual screening protocol with combination of similarity search and pharmacophore modeling was applied to virtually screen a large compound library to gain new scaffolds regarding ABCC1 inhibition. Biological investigation of promising candidates revealed four compounds as ABCC1 inhibitors, three of them with scaffolds not associated with ABCC1 inhibition until now. The best hit molecule-a thienopyrimidine-was a moderately potent, competitive inhibitor of the ABCC1-mediated transport of calcein AM which also sensitized ABCC1-overexpressing cells toward daunorubicin. Further evaluation showed that it was a moderately potent, competitive inhibitor of the ABCB1-mediated transport of calcein AM, and noncompetitive inhibitor of the ABCG2-mediated pheophorbide A transport. In addition, the thienopyrimidine could also sensitize ABCB1- as well as ABCG2-overexpressing cells toward daunorubicin and SN-38, respectively, in concentration ranges that qualified it as one of the ten best triple ABCC1/ABCB1/ABCG2 inhibitors in the literature. Besides, three more new multitarget inhibitors were identified by this virtual screening approach.
10.1021/acs.jmedchem.8b01821
Molecular Imaging of ABCB1 and ABCG2 Inhibition at the Human Blood-Brain Barrier Using Elacridar and C-Erlotinib PET.
Verheijen Remy B,Yaqub Maqsood,Sawicki Emilia,van Tellingen Olaf,Lammertsma Adriaan A,Nuijen Bastiaan,Schellens Jan H M,Beijnen Jos H,Huitema Alwin D R,Hendrikse N Harry,Steeghs Neeltje
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
Transporters such as ABCB1 and ABCG2 limit the exposure of several anticancer drugs to the brain, leading to suboptimal treatment in the central nervous system. The purpose of this study was to investigate the effects of the ABCB1 and ABCG2 inhibitor elacridar on brain uptake using C-erlotinib PET. Elacridar and cold erlotinib were administered orally to wild-type (WT) and knockout mice. In addition, brain uptake was measured using C-erlotinib imaging and ex vivo scintillation counting in knockout and WT mice. Six patients with advanced solid tumors underwent C-erlotinib PET scans before and after a 1,000-mg dose of elacridar. C-erlotinib brain uptake was quantified by pharmacokinetic modeling using volume of distribution (V) as the outcome parameter. In addition, O-HO scans to measure cerebral blood flow were acquired before each C-erlotinib scan. Brain uptake of C-erlotinib was 2.6-fold higher in knockout mice than in WT mice, measured as percentage injected dose per gram of tissue ( = 0.01). In WT mice, the addition of elacridar (at systemic plasma concentrations of ≥200 ng/mL) resulted in an increased brain concentration of erlotinib, without affecting erlotinib plasma concentration. In patients, the V of C-erlotinib did not increase after intake of elacridar (0.213 ± 0.12 vs. 0.205 ± 0.07, = 0.91). O-HO PET showed no significant changes in cerebral blood flow. Elacridar exposure in patients was 401 ± 154 ng/mL. No increase in V with increased elacridar plasma exposure was found over the 271-619 ng/mL range. When Abcb1 and Abcg2 were disrupted in mice, brain uptake of C-erlotinib increased both at a tracer dose and at a pharmacologic dose. In patients, brain uptake of C-erlotinib was not higher after administration of elacridar. The more pronounced role that ABCG2 appears to play at the human blood-brain barrier and the lower potency of elacridar to inhibit ABCG2 may be an explanation of these interspecies differences.
10.2967/jnumed.117.195800
AST1306, a potent EGFR inhibitor, antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance.
Zhang Hui,Wang Yi-Jun,Zhang Yun-Kai,Wang De-Shen,Kathawala Rishil J,Patel Atish,Talele Tanaji T,Chen Zhe-Sheng,Fu Li-Wu
Cancer letters
AST1306, an inhibitor of EGFR and ErbB2, is currently in phase I of clinical trials. We evaluated the effect of AST306 on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters. We found that AST1306 significantly sensitized the ABC subfamily G member 2 (ABCG2)-overexpressing cells to ABCG2 substrate chemotherapeutics. AST1306 significantly increased intracellular accumulation of [(3)H]-mitoxantrone in ABCG2-overexpressing cells by blocking ABCG2 efflux function. Moreover, AST1306 stimulated the ATPase activity of ABCG2. Homology modeling predicted the binding conformation of AST1306 to be within the transmembrane region of ABCG2. In conclusion, AST1306 could notably reverse ABCG2-mediated MDR.
10.1016/j.canlet.2014.04.008
Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib).
Elkind N Barry,Szentpétery Zsófia,Apáti Agota,Ozvegy-Laczka Csilla,Várady György,Ujhelly Olga,Szabó Katalin,Homolya László,Váradi András,Buday László,Kéri György,Német Katalin,Sarkadi Balázs
Cancer research
Iressa (ZD1839, Gefitinib), used in clinics to treat non-small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signaling-dependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa.
10.1158/0008-5472.CAN-04-3303
Superior Pyrimidine Derivatives as Selective ABCG2 Inhibitors and Broad-Spectrum ABCB1, ABCC1, and ABCG2 Antagonists.
Silbermann Katja,Li Jiyang,Namasivayam Vigneshwaran,Baltes Fabian,Bendas Gerd,Stefan Sven Marcel,Wiese Michael
Journal of medicinal chemistry
In the search for highly effective modulators addressing ABCG2-mediated MDR, 23 pyrimidines were synthesized and biologically assessed. Seven derivatives with (a) nitrogen- and/or halogen-containing residue(s) had extraordinary potencies against ABCG2 (IC < 150 nM). The compounds competitively inhibited ABCG2-mediated Hoechst 33342 transport but were not substrates of ABCG2. The most potent MDR reverser, compound , concentration-dependently increased SN-38-mediated cancer cell death at 11 nM (EC), time-dependently doubled SN-38 toxicity in a period of 7 days at 10 nM, and half-maximally accelerated cell death combined with SN-38 at 17 nM. No induction of ABCG2 was observed. Furthermore, 11 pyrimidines were revealed as triple ABCB1/ABCC1/ABCG2 inhibitors. Five possessed IC values below 10 μM against each transporter, classifying them as some of the 50 most potent multitarget ABC transporter inhibitors. The most promising representative, compound , reversed ABCB1-, ABCC1-, and ABCG2-mediated MDR, making it one of the three most potent ABC transporter inhibitors and reversers of ABC transporters-mediated MDR.
10.1021/acs.jmedchem.0c00961
Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance.
Shi Zhi,Tiwari Amit K,Shukla Suneet,Robey Robert W,Singh Satyakam,Kim In-Wha,Bates Susan E,Peng Xingxiang,Abraham Ioana,Ambudkar Suresh V,Talele Tanaji T,Fu Li-Wu,Chen Zhe-Sheng
Cancer research
Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions.
10.1158/0008-5472.CAN-10-3820
Imidazo[1,2-]Pyridine Derivatives as Novel Dual-Target Inhibitors of ABCB1 and ABCG2 for Reversing Multidrug Resistance.
Journal of medicinal chemistry
ABCB1 and ABCG2 are the important ATP-binding cassette (ABC) transporters associated with multidrug resistance (MDR). Herein, we designed a series of imidazo[1,2-]pyridine derivatives as dual-target inhibitors of ABCB1 and ABCG2 through the scaffold hopping strategy. Compound displayed potential efflux function inhibitory toward both ABCB1 and ABCG2 (reversal fold: ABCB1 = 8.35 and ABCG2 = 2.71) without obvious cytotoxicity. also enhanced the potency of antiproliferative drugs in vitro. Mechanistic studies demonstrated that slightly suppressed ATPase activity but did not affect the protein expression of ABCB1 or ABCG2. Notably, exhibited negligible CYP3A4 inhibition and enhanced the antiproliferative activity of adriamycin in vivo by restoring the sensitivity of resistant cells. Thus, may be effective clinically in combination with common chemotherapy agents. In summary, is a potential dual-target inhibitor that reverses MDR by blocking the efflux function of ABCB1 and ABCG2.
10.1021/acs.jmedchem.2c01862
Identification of inhibitors of ABCG2 by a bioluminescence imaging-based high-throughput assay.
Zhang Yimao,Byun Youngjoo,Ren Yunzhao R,Liu Jun O,Laterra John,Pomper Martin G
Cancer research
ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)-based assay was developed to allow high-throughput compound screening. This assay exploits our finding that d-luciferin, the substrate of firefly luciferase (fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of d-luciferin and enhance bioluminescence signal by increasing intracellular d-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty-seven compounds showed BLI enhancement, a measure of anti-ABCG2 activity, of > or =5-fold, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g., mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from a FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing.
10.1158/0008-5472.CAN-08-4866
ABCG2 Transporter Expression Impacts Group 3 Medulloblastoma Response to Chemotherapy.
Morfouace Marie,Cheepala Satish,Jackson Sadhana,Fukuda Yu,Patel Yogesh T,Fatima Soghra,Kawauchi Daisuke,Shelat Anang A,Stewart Clinton F,Sorrentino Brian P,Schuetz John D,Roussel Martine F
Cancer research
While a small number of plasma membrane ABC transporters can export chemotherapeutic drugs and confer drug resistance, it is unknown whether these transporters are expressed or functional in less therapeutically tractable cancers such as Group 3 (G3) medulloblastoma. Herein we show that among this class of drug transporters, only ABCG2 was expressed at highly increased levels in human G3 medulloblastoma and a mouse model of this disease. In the mouse model, Abcg2 protein was expressed at the plasma membrane where it functioned as expected on the basis of export of prototypical substrates. By screening ABC substrates against mouse G3 medulloblastoma tumorspheres in vitro, we found that Abcg2 inhibition could potentiate responses to the clinically used drug topotecan, producing a more than 9-fold suppression of cell proliferation. Extended studies in vivo in this model confirmed that Abcg2 inhibition was sufficient to enhance antiproliferative responses to topotecan, producing a significant survival advantage compared with subjects treated with topotecan alone. Our findings offer a preclinical proof of concept for blockade of ABCG2 transporter activity as a strategy to empower chemotherapeutic responses in G3 medulloblastoma.
10.1158/0008-5472.CAN-15-0030
Ins and outs of the ABCG2 multidrug transporter: an update on in vitro functional assays.
Hegedus Csilla,Szakács Gergely,Homolya László,Orbán Tamás I,Telbisz Agnes,Jani Márton,Sarkadi Balázs
Advanced drug delivery reviews
The major aim of this chapter is to provide a critical overview of the in vitro methods available for studying the function of the ABCG2 multidrug transporter protein. When describing the most applicable assay systems, in each case we present a short overview relevant to ABC multidrug transporters in general, and then we concentrate on the tools applicable to analysis of substrate-drug interactions, the effects of potential activators and inhibitors, and the role of polymorphisms of the ABCG2 transporter. Throughout this chapter we focus on recently developed assay systems, which may provide new possibilities for analyzing the pharmacological aspects of this medically important protein.
10.1016/j.addr.2008.09.007
Structural basis of small-molecule inhibition of human multidrug transporter ABCG2.
Jackson Scott M,Manolaridis Ioannis,Kowal Julia,Zechner Melanie,Taylor Nicholas M I,Bause Manuel,Bauer Stefanie,Bartholomaeus Ruben,Bernhardt Guenther,Koenig Burkhard,Buschauer Armin,Stahlberg Henning,Altmann Karl-Heinz,Locher Kaspar P
Nature structural & molecular biology
ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.
10.1038/s41594-018-0049-1
Tyrphostin RG14620 selectively reverses ABCG2-mediated multidrug resistance in cancer cell lines.
Wu Chung-Pu,Hsiao Sung-Han,Murakami Megumi,Lu Ming-Jie,Li Yan-Qing,Hsieh Chia-Hung,Ambudkar Suresh V,Wu Yu-Shan
Cancer letters
The multidrug resistance (MDR) phenotype associated with the overexpression of ATP-binding cassette (ABC) drug transporters ABCB1, ABCC1 and ABCG2 is a major obstacle in cancer chemotherapy. Numerous epidermal growth factor receptor (EGFR) inhibitors have previously been shown capable of reversing MDR in ABCG2-overexpressing cancer cells. However, most of them are not transporter-specific due to the substantial overlapping substrate specificity among the transporters. In this study, we investigated the interaction between ABCG2 and tyrphostin RG14620, an EGFR inhibitor of the tyrphostin family, in multidrug-resistant cancer cell lines. We found that at nontoxic concentrations, tyrphostin RG14620 enhances drug-induced apoptosis and restores chemosensitivity to ABCG2-overexpressing multidrug-resistant cancer cells. More importantly, tyrphostin RG14620 is selective to ABCG2 relative to ABCB1 and ABCC1. Our findings were further supported by biochemical assays demonstrating that tyrphostin RG14620 stimulates ATP hydrolysis and inhibits photoaffinity labeling of ABCG2 with IAAP, and by a docking analysis of tyrphostin RG14620 in the drug-binding pocket of this transporter. Taken together, our findings indicate that tyrphostin RG14620 is a potent and selective modulator of ABCG2 that may be useful to overcome chemoresistance in patients with drug-resistant tumors.
10.1016/j.canlet.2017.08.035
Imatinib mesylate is a potent inhibitor of the ABCG2 (BCRP) transporter and reverses resistance to topotecan and SN-38 in vitro.
Houghton Peter J,Germain Glen S,Harwood Franklin C,Schuetz John D,Stewart Clinton F,Buchdunger Elisabeth,Traxler Peter
Cancer research
Imatinib mesylate (Gleevec, STI571) is a kinase inhibitor selective for Bcr-Abl, activated c-Kit kinases, and platelet-derived growth factor receptor tyrosine kinase. Imatinib mesylate, similar to many other tyrosine kinase inhibitors (TKIs), such as members of the 4-anilinoquinazoline class, competes for ATP binding. Previously, 4-anilinoquinazoline TKIs have been shown to inhibit the function of the breast cancer resistance-associated drug transporter (ABCG2), reversing resistance to camptothecin derivatives topotecan and SN-38. However, the potential to inhibit ABCG2 for the 2-phenylamino-pyrimidine class of TKIs, exemplified by imatinib mesylate, has not been examined. Here, we show that imatinib mesylate potently reverses ABCG2-mediated resistance to topotecan and SN-38 and significantly increases accumulation of topotecan only in cells expressing functional ABCG2. However, overexpression of ABCG2 does not confer resistance to imatinib mesylate. Furthermore, accumulation and efflux of [(14)C]imatinib mesylate are unaltered between ABCG2-expressing and non-ABCG2-expressing cells or by ATP depletion. These results suggest that imatinib mesylate inhibits the function of ABCG2 but is not a substrate for this transporter.
10.1158/0008-5472.can-03-3344
Influence of the dual ABCB1 and ABCG2 inhibitor tariquidar on the disposition of oral imatinib in mice.
Gardner Erin R,Smith Nicola F,Figg William D,Sparreboom Alex
Journal of experimental & clinical cancer research : CR
BACKGROUND:Imatinib, a tyrosine kinase inhibitor currently approved for treatment of several malignancies, has been shown to be a substrate for multiple efflux-transporter proteins, including ABCB1 (P-glycoprotein) and ABCG2 (BCRP). The effect of inhibiting these transporters on tissue exposure to imatinib remains unclear. OBJECTIVE:To assess the role of these transporters on drug disposition, 50 mg/kg imatinib was administered to Balb/C mice, 30 minutes after receiving tariquidar (10 mg/kg), an inhibitor of both ABCB1 and ABCG2, or vehicle, via oral gavage. METHODS:Quantitative determination of imatinib in mouse plasma, liver and brain was performed using a newly-developed and validated liquid-chromatography-mass spectrometric method. RESULTS:Exposure to imatinib was 2.2-fold higher in plasma, liver and brain in mice that received tariquidar, as compared to those that received the vehicle (P = 0.001). The peak plasma concentration did not increase substantially, suggesting that tariquidar is affecting the distribution, metabolism and/or excretion of imatinib, rather than absorption. Though tariquidar increased the absolute exposure of imatinib, the brain-to-plasma ratio of imatinib was unaffected. CONCLUSION:This study suggests that intentional inhibition of ABCB1 and ABCG2 function at the blood-brain barrier is unlikely to significantly improve clinical outcome of imatinib with currently used dosing regimens.
10.1186/1756-9966-28-99
Downregulated ABCG2 enhances sensitivity to topoisomerase I inhibitor in epidermal growth factor receptor tyrosine kinase inhibitor-resistant non-small cell lung cancer.
Ohtsuka Kouki,Ohnishi Hiroaki,Morii Takeshi,Fujiwara Masachika,Kishino Tomonori,Ogura Wataru,Chiba Misaki,Matsushima Satsuki,Goya Tomoyuki,Watanabe Takashi
Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer
INTRODUCTION:Understanding the mechanisms of drug resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) is essential to develop novel chemotherapies for non-small cell lung cancer (NSCLC). Therefore, we analyzed the expression and function of ATP-binding cassette (ABC) transporters in EGFR TKI-resistant NSCLC. METHODS:In three newly established AG1478-resistant NSCLC cell lines, we evaluated the expression profile of ABC transporters and genotyping of ABCG2 by real-time polymerase chain reaction and elucidated their function by Hoechst dye efflux analyses. The growth-inhibitory effect of the topoisomerase I inhibitor Hoechst 33342, which is extruded by ABCG2, was also investigated in these cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS:In AG1478-resistant cells, significantly less ABCG2 was expressed, and the ratios of the cells with a strong ability to extrude Hoechst dye were remarkably smaller than in the parent cells. Because of the ABCG2 downregulation and loss of function due to C421A/C421A homozygosity, PC-14AG50R was thus considered to be more sensitive to Hoechst 33342 than the parental cells. All AG1478-resistant cells were more sensitive to the combination of Hoechst 33342 and AG1478 than to single agent. CONCLUSIONS:Resistance to EGFR TKI in NSCLC is associated with the downregulation of ABCG2 expression. A topoisomerase I inhibitor alone or in combination with EGFR TKI might offer a promising strategy for treating NSCLC that is resistant to EGFR TKI.
10.1097/JTO.0b013e3181f0b6af
SIS3, a specific inhibitor of Smad3 reverses ABCB1- and ABCG2-mediated multidrug resistance in cancer cell lines.
Wu Chung-Pu,Murakami Megumi,Hsiao Sung-Han,Liu Te-Chun,Yeh Ni,Li Yan-Qing,Hung Tai-Ho,Wu Yu-Shan,Ambudkar Suresh V
Cancer letters
One of the major challenges in cancer chemotherapy is the development of multidrug resistance phenomenon attributed to the overexpression of ATP-binding cassette (ABC) transporter ABCB1 or ABCG2 in cancer cells. Therefore, re-sensitizing MDR cancer cells to chemotherapy by directly inhibiting the activity of ABC transporters has clinical relevance. Unfortunately, previous attempts of developing clinically applicable synthetic inhibitors have failed, mostly due to problems associated with toxicity and unforeseen drug-drug interactions. An alternative approach is by repositioning drugs with known pharmacological properties as modulators of ABCB1 and ABCG2. In this study, we discovered that the transport function of ABCB1 and ABCG2 is strongly inhibited by SIS3, a specific inhibitor of Smad3. More importantly, SIS3 enhances drug-induced apoptosis and resensitizes ABCB1- and ABCG2-overexpressing cancer cells to chemotherapeutic drugs at non-toxic concentrations. These findings are further supported by ATPase assays and by a docking analysis of SIS3 in the drug-binding pockets of ABCB1 and ABCG2. In summary, we revealed an additional action of SIS3 that re-sensitizes MDR cancer cells and a combination therapy with this drug and other chemotherapeutic agents may be beneficial for patients with MDR tumors.
10.1016/j.canlet.2018.07.004
Selonsertib (GS-4997), an ASK1 inhibitor, antagonizes multidrug resistance in ABCB1- and ABCG2-overexpressing cancer cells.
Ji Ning,Yang Yuqi,Cai Chao-Yun,Lei Zi-Ning,Wang Jing-Quan,Gupta Pranav,Shukla Suneet,Ambudkar Suresh V,Kong Dexin,Chen Zhe-Sheng
Cancer letters
Overexpression of ATP-binding cassette (ABC) transporters is one of the most important mechanisms responsible for the development of multidrug resistance (MDR). Selonsertib, a serine/threonine kinase inhibitor, targets apoptosis signal-regulating kinase 1 (ASK1) and is now in phase III clinical trial for the treatment of non-alcoholic steatohepatitis (NASH). In this study, we investigated whether selonsertib could reverse MDR-mediated by ABC transporters, including ABCB1, ABCG2, ABCC1 and ABCC10. The results showed that selonsertib significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR-mediated by ABCC1 or ABCC10. Mechanism studies indicated that the reversal effect of selonsertib was related to the attenuation of the efflux activity of ABCB1 and ABCG2 transporters, without the protein level decrease or change in the subcellular localization of ABCB1 or ABCG2. Selonsertib stimulated the ATPase activity of ABCB1 and ABCG2 in a concentration-dependent manner, and in silico docking study showed selonsertib could interact with the substrate-binding sites of both ABCB1 and ABCG2. This study provides a clue into a novel treatment strategy, which includes a combination of selonsertib with antineoplastic drugs to attenuate MDR-mediated by ABCB1 or ABCG2 in cancer cells overexpressing these transporters.
10.1016/j.canlet.2018.10.007