Silencing of augmenter of liver regeneration inhibited cell proliferation and triggered apoptosis in U266 human multiple myeloma cells.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas
Augmenter of liver regeneration (ALR) is a thermostable cytokine that was originally identified to promote the growth of hepatocytes. This study was conducted to explore the expression and function of ALR in multiple myeloma (MM), a common hematologic malignancy. Real-time PCR and western blot analysis were performed to detect the expression of ALR in U266 human MM cells and healthy peripheral blood mononuclear cells (PBMCs). U266 MM cells were exposed to 20 or 40 μg/mL of recombinant ALR and tested for cell proliferation. Small interfering RNA-mediated silencing of ALR was done to investigate the role of ALR in cell proliferation, apoptosis, and cytokine production. Compared to PBMCs, U266 MM cells exhibited significantly higher levels of ALR at both the mRNA and protein levels. The addition of recombinant ALR protein significantly promoted the proliferation of U266 cells. In contrast, knockdown of ALR led to a significant decline in the viability and proliferation of U266 cells. Annexin-V/PI staining analysis demonstrated that ALR downregulation increased apoptosis in U266 MM cells, compared to control cells (20.1±1.1 vs 9.1±0.3%, P<0.05). Moreover, ALR depletion reduced the Bcl-2 mRNA level by 40% and raised the Bax mRNA level by 2-fold. Additionally, conditioned medium from ALR-depleted U266 cells had significantly lower concentrations of interleukin-6 than control cells (P<0.05). Taken together, ALR contributed to the proliferation and survival of U266 MM cells, and targeting ALR may have therapeutic potential in the treatment of MM.
10.1590/1414-431X20176139
Blocking the short isoform of augmenter of liver regeneration inhibits proliferation of human multiple myeloma U266 cells via the MAPK/STAT3/cell cycle signaling pathway.
Huang Wenqi,Sun Hang,Hu Ting,Zhu Dongju,Long Xianli,Guo Hui,Liu Qi
Oncology letters
Multiple myeloma (MM) is the second most common haematological malignancy and remains an incurable disease, with most patients relapsing and requiring further treatment. Augmenter of liver regeneration (ALR) is a vital protein affecting fundamental processes such as energy transduction, cell survival and regeneration. Silencing ALR inhibits cell proliferation and triggers apoptosis in human MM U266 cells. However, little is known about the role of 15-kDa-ALR on MM. In the present study, the role of 15-kDa-ALR in human MM cells was investigated. Blocking extracellular 15-kDa-ALR with an anti-ALR monoclonal antibody (McAb) decreased the proliferation and viability of U266 cells. However, the results of flow cytometry revealed no changes in apoptosis, and the expression levels of Bax, Bcl-2, caspase-3 and cleaved caspase-3 were not affected. However, combined treatment with anti-ALR McAb and epirubicin increased the apoptosis of U266 cells. RNA sequencing results indicated that the ERK1/2, JNK-MAPK and STAT3 signaling pathways, as well as the cell cycle, were associated with the mechanism of action of the anti-ALR McAb, and PCR, western blotting and cell cycle analysis confirmed these results. The present findings suggested that blocking extracellular 15-kDa-ALR in U266 cells with an anti-ALR McAb decreased cell proliferation via the MAPK, STAT3 and cell cycle signaling pathways without increasing apoptosis. Thus, 15-kDa-ALR may be a new therapeutic target for myeloma.
10.3892/ol.2021.12458
[Apoptosis Effects of GM3 Ganglioside on U266 Cells and Its Possible Mechanism].
Zhao Hui-Ying,Ma Yan-Ping
Zhongguo shi yan xue ye xue za zhi
OBJECTIVE:To investigate the proliferation- inhibitory and apoptosis inducing effect of ganglioside GM3 on human multiple myeloma cell line U266 cells and its possible mechanisms. METHODS:MTT assay and flow cytometry were used to observe the effects of GM3 ganglioside on proliferation and apoptosis of human myeloma cell line U266. Effects of different concentration of ganglioside GM3 on the mRNA expression level of BCL-2 and BAX were detected by Real-time PCR. RESULTS:MTT assay and Flow Cytometry showed that ganglioside GM3 could induce the apoptosis and inhibit the proliferation of multiple myeloma U266 cell line, and both the effects were enhanced with the increase of GM3 ganglioside concentration. Compared with the control group, the relative expression of BAX mRNA with the increase of GM3 concentration in experimental group was enhanced gradually(r=0.968), while the relative mRNA expression of anti-apoptotic gene BCL-2 was decreased gradually（r=-0.727). CONCLUSION:GM3 ganglioside can induce apoptosis and inhibit the proliferation of U266 cell line in a concentration dependent manner. The mechanism may be related with up- regulation of BAX expression and down-regulation of BCL-2.
10.7534/j.issn.1009-2137.2018.04.013
[Mechanisms of Apoptosis Induced by FTY720 in Multiple Myeloma Cell Line U266].
Liao Ai-Jun,Li Shu-Chen,Wu Bin,Hu Rong,Li Ying-Chun,Yao Kun,Yang Wei,Liu Zhuo-Gang
Zhongguo shi yan xue ye xue za zhi
OBJECTIVE:To investigate the effects of FTY720 on apoptosis in multiple myeloma cell line U266 and to clarify the molecular mechanism of apoptosis induced by FTY720. METHODS:U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the apoptotic rates were tested by flow cytometry with Annexin-V-FITC/PI staining. Then U266 cells were treated with 20 µmol/L FTY720 for 0, 6, 16 and 24 hours, the apoptotic rates were tested. U266 cells were treated with DMSO and FTY720 separately and then were stained with DAPI for 5 min. Drop the cells to the slides and cover the slide with the glass. The cells were observed by fluorescence microscopy. U266 cells were treated with 5 µmol/L FTY720 or together with different doses of Z-VAD-fmk (12.5, 25, 50 µmol/L), a pancaspase inhibitor, for 24 hours, then the cell viability was tested by CCK-8. U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expression of cleaved caspase-3 was tested by Western blot. U266 cells were treated with 0, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expressions of MCL-1, survivin, BCL-2, BID, BAX, BAK, P-ERK were tested by Western blot. RESULTS:The apoptotic rate increased in U266 cells treated with FTY720 and showed the characteristic of time-dependent and dose-dependent manner. Karyopyknosis and nuclearfragmentation could be observed in U266 cells treated with FTY720 after being stained with DAPI under fluorescent microscope. The same effect was not observed in the cells treated with DMSO. Z-VAD-fmk could rescue the apoptosis in U266 cells treated with FTY720 in dose-dependent manner. The expression of MCL-1, survivin and BCL-2 decreased in U266 cells treated with FTY720. The cleavage of BID could be observed in U266 cells treated with FTY720. FTY720 had no effect on the expression of BAX, BAK and P-ERK. CONCLUSION:FTY720 can induce the apoptosis in U266 cells, the apoptosis was Caspase-3-depended. The apoptosis induced by FTY720 is due to the decrease of MCL-1, survivin and BCL-2, which are the inhibitors of apoptosis. Meanwhile, the apoptosis was also due to the activation of BID, which is pro-apoptotic protein.
10.7534/j.issn.1009-2137.2015.06.018
Brazilin induces apoptosis and G2/M arrest via inactivation of histone deacetylase in multiple myeloma U266 cells.
Kim Bonglee,Kim Sun-Hee,Jeong Soo-Jin,Sohn Eun Jung,Jung Ji Hoon,Lee Min Ho,Kim Sung-Hoon
Journal of agricultural and food chemistry
Although brazilin [7,11b-dihydrobenz(b)indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol] isolated from Caesalpinia sappan was known to have various biological activities, including anti-inflammation, antibacteria, and antiplatelet aggregation, there is no report yet on its anticancer activity. In the present study, the anticancer mechanism of brazilin was elucidated in human multiple myeloma U266 cells. We found that brazilin significantly inhibited the activity of histone deacetylases (HDACs), transcription factors involved in the regulation of apoptosis and cell cycle arrest in U266 cells. Consistently, brazilin enhanced acetylation of histone H3 at Lys 23, indicating activation of histone acetyltransferase (HAT), and also suppressed the expressions of HDAC1 and HDAC2 at both protein and mRNA levels. Additionally, brazilin significantly increased the number of sub-G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells undergoing apoptosis and also activated caspase-3 and regulated the expression of Bcl-2 family proteins, including Bax, Bcl-x(L), and Bcl-2 in U266 cells, indicating that brazilin induces apoptosis through the mitochondria-dependent pathway. Interestingly, cell cycle analysis revealed that brazilin induced G2/M phase arrest along with apoptosis induction. Consistently, brazilin attenuated the expression of cyclin-dependent kinases (CDKs), such as cyclin D1, cyclin B1, and cyclin E, and also activated p21 and p27 in U266 cells. Furthermore, HAT inhibitor anacardic acid reversed activation of acetyl-histone H3 and cleavage of PARP induced by brazilin, while pan-caspase inhibitor Z-VAD-FMK001 did not affect the expression of HDAC induced by brazilin that brazilin mediates apoptosis via inactivation of HDAC in U266 cells. Notably, brazilin significantly potentiated the cytotoxic effect of standard chemotherapeutic agents, such as bortezomib or doxorubicin, in U266 cells. When our findings are taken together, they suggest that brazilin has potential as a chemotherapeutic agent alone or in combination with an anticancer agent for multiple myeloma treatment.
10.1021/jf302527p
Knockdown of c-Met inhibits cell proliferation and invasion and increases chemosensitivity to doxorubicin in human multiple myeloma U266 cells in vitro.
Que Wenzhong,Chen Junmin
Molecular medicine reports
c-Met, a receptor tyrosine kinase and its ligand, hepatocyte growth factor, are critical in cellular proliferation, motility and invasion and confer resistance to specific chemotherapeutic drugs. However, little is known about the impact of c-Met knockdown on the biological functions of human multiple myeloma U266 cells. The present study was designed to determine the role of c-Met in the proliferation and invasion of U266 cells, using RNA interference technology in vitro. In our study, the c-Met short hairpin RNA (shRNA) was successfully transfected into U266 cells, which resulted in the significant inhibition of transcription and expression of c-Met. The down-regulation of c-Met inhibited the proliferation potential, adherence and invasiveness of U266 cells, and also increased chemosensitivity to doxorubicin. The c-Met shRNA in U266 cells induced apoptosis and increased the accumulation of cleavage PARP and cleavage caspase-3. However, the expression of Bcl-2 and Bax did not change following the c-Met knockdown. Taken together, our data reveal that the down-regulation of c-Met inhibits proliferation and invasion and increases the chemosensitivity of U266 cells. Thus, the targeting of c-Met could be an effective therapeutic approach against multiple myeloma.
10.3892/mmr.2011.426
MiR-657/ATF2 Signaling Pathway Has a Critical Role in Dunn Extract-Induced Apoptosis in U266 and U937 Cells.
Lim Hyun Ji,Park Moon Nyeo,Kim Changmin,Kang Beomku,Song Hyo-Sook,Lee Hyemin,Kim Sung-Hoon,Shim Bum-Sang,Kim Bonglee
Cancers
Though Dunn (SSD) has been reported to have anti-virus, anti-osteoclastogenesis, and anti-inflammation activities, its underlying anti-cancer mechanism has never been elucidated in association with the role of miR-657 in endoplasmic reticulum (ER) stress-related apoptosis to date. SSD treatment exerted cytotoxicity in U266 and U937 cells in a dose-dependent manner. Also, apoptosis-related proteins such as PARP, procaspase-3, and Bax were regulated by SSD treatment. Furthermore, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed that a number of apoptotic bodies were increased by SSD. Interestingly, the ER stress-related proteins such as p-ATF2 and CHOP were elevated by SSD. Interestingly, reactive oxygen species (ROS) generation and cytotoxicity by SSD treatment were significantly reduced by -Acetyl-L-cysteine (NAC). Among the microRNAs (miRNAs) regulated by SSD treatment, miR-657 was most significantly reduced by SSD treatment. However, an miR-657 mimic reversed SSD-induced apoptosis by the attenuation of the expression of p-ATF2, CHOP, and PARP cleavage. Overall, these findings provide scientific evidence that miR657 is an onco-miRNA targeting the ER stress signal pathway and SSD induces apoptosis via the inhibition of miR-657, ROS, and the activation of p-ATF2 and CHOP as a potent anti-cancer agent for myeloid-originated hematological cancer.
10.3390/cancers11020150
Synergistic apoptotic effect of arabinoxylan rice bran (MGN-3/Biobran) and curcumin (turmeric) on human multiple myeloma cell line U266 in vitro.
Ghoneum M,Gollapudi S
Neoplasma
The present study was carried out to investigate the synergistic apoptotic potential of arabinoxylan rice bran (MGN-3/Biobran) and curcumin (turmeric) on human multiple myeloma (MM) cell line U266 , in vitro. U266 cells were cultured with MGN-3 (50 or 100μg/ml) and curcumin (2.5-10μM) for 3 days. The effects of MGN-3 and curcumin on the growth and survival of the U266 cells were determined by trypan blue, MTT assay, flow cytometry analysis of cancer cell cycle, and apoptosis. Expression of proapoptotic Bax, and antiapoptotic Bcl2 was determined by Western blot analysis. Treatment with MGN-3 alone or curcumin alone caused a dose-dependent inhibition in the proliferation of U266 cells. However, a synergistic effect was noticed post-treatment with both agents that maximized at 100μg/ml MGN-3 plus 10μM curcumin. This synergy was characterized by an 87% decrease in cell number and a 2.6 fold increase in the percentage of apoptotic U266 cells. Cell cycle analysis showed a 53% decrease in the percentage of cells in the G0-G1 phase treated with MGN-3 and curcumin (from 36% to 17%). Analysis of the expression of the pro and antiapoptotic molecules Bax and Bcl-2 revealed synergistic effects of these agents, as the expression of Bcl-2 was decreased and Bax was increased. This resulted in a cellular microenvironment favorable for apoptosis. We conclude that MGN-3 and curcumin synergize in the induction of U266 cell apoptosis. This data may establish the foundation for in vivo studies that could have therapeutic implications.
10.4149/neo_2011_02_118
Dual targeting of BCL2 and MCL1 rescues myeloma cells resistant to BCL2 and MCL1 inhibitors associated with the formation of BAX/BAK hetero-complexes.
Seiller Carolane,Maiga Sophie,Touzeau Cyrille,Bellanger Céline,Kervoëlen Charlotte,Descamps Géraldine,Maillet Laurent,Moreau Philippe,Pellat-Deceunynck Catherine,Gomez-Bougie Patricia,Amiot Martine
Cell death & disease
Multiple myeloma is a plasma cell malignancy that escapes from apoptosis by heterogeneously over-expressing anti-apoptotic BCL2 proteins. Myeloma cells with a t(11;14) translocation present a particular vulnerability to BCL2 inhibition while a majority of myeloma cells relies on MCL1 for survival. The present study aimed to determine whether the combination of BCL2 and MCL1 inhibitors at low doses could be of benefit for myeloma cells beyond the single selective inhibition of BCL2 or MCL1. We identified that half of patients were not efficiently targeted neither by BCL2 inhibitor nor MCL1 inhibitor. Seventy percent of these myeloma samples, either from patients at diagnosis or relapse, presented a marked increase of apoptosis upon low dose combination of both inhibitors. Interestingly, primary cells from a patient in progression under venetoclax treatment were not sensitive ex vivo to neither venetoclax nor to MCL1 inhibitor, whereas the combination of both efficiently induced cell death. This finding suggests that the combination could overcome venetoclax resistance. The efficacy of the combination was also confirmed in U266 xenograft model resistant to BCL2 and MCL1 inhibitors. Mechanistically, we demonstrated that the combination of both inhibitors favors apoptosis in a BAX/BAK dependent manner. We showed that activated BAX was readily increased upon the inhibitor combination leading to the formation of BAK/BAX hetero-complexes. We found that BCLXL remains a major resistant factor of cell death induced by this combination. The present study supports a rational for the clinical use of venetoclax/S63845 combination in myeloma patients with the potential to elicit significant clinical activity when both single inhibitors would not be effective but also to overcome developed in vivo venetoclax resistance.
10.1038/s41419-020-2505-1
Activation of reactive oxygen species/AMP activated protein kinase signaling mediates fisetin-induced apoptosis in multiple myeloma U266 cells.
Jang Ki Young,Jeong Soo-Jin,Kim Sun-Hee,Jung Ji Hoon,Kim Ji-Hyun,Koh Wonil,Chen Chang-Yan,Kim Sung-Hoon
Cancer letters
We investigated the molecular mechanisms responsible for fisetin-induced apoptosis in U266 cells. Fisetin elicited the cytotoxicity in U266 cells, manifested as an increased fraction of the cells with sub-G1 content or stained positively with TUNEL labeling. Fisetin enhanced caspase-3 activation, downregulation of Bcl-2 and Mcl-1(L), and upregulation of Bax, Bim and Bad. Fisetin activated AMPK as well as its substrate acetyl-CoA carboxylase (ACC), along with a decreased phosphorylation of AKT and mTOR. Fisetin also stimulated generation of ROS in U266 cells. Conversely, compound C or N-acetyl-L-cystein blocked fisetin-induced apoptosis. Our data suggest that fisetin-induced apoptosis in U266 cells is through ROS and AMPK pathways.
10.1016/j.canlet.2012.01.008
[Metformin Induces Apoptosis of Human Multiple Myeloma Cell U266 through the Mitochondrial Apoptotic Pathway].
Tong Jia-Yin,Wang Chong,Liu Yan-Fang,Wang Wei-Qiong,Hao Qian-Qian,Ma Jie,Sun Ling,Sun Hui
Zhongguo shi yan xue ye xue za zhi
OBJECTIVE:Metformin (Met) can inhibit the proliferation of tumor cells in vitro, its effects on multiple myeloma and action mechanisms have been not yet understood. The purpose of this study was to investigate the effect and molecular mechanism of metformin on human myeloma cells U266. METHODS:U266 cells were treated with different concentration of Met, the MTT was used to detect cell proliferation, the PI staining was used to detect the cell cycle, and the protein expression of BCL-2 family and the release of cytochrome C were assessed by Western blot. RESULTS:Metformin could inhibit the proliferation of U266 cell in a time- and concentration- dependent manner. The U266 cells were arrested in G/G phase after metformin treatment for 48 h, as compared with non-treated U266 cells. The proteins expression of BCL-2 and BCL-XL was down-regulated and the protein expression of BAX was up-regulated. The released of cytochrome C from mitochondria to cytoplasm was increased, and protein splicing of PARP was also enhanced. CONCLUSION:Metformin can inhibit the cell proliferation and induce U266 cell apoptosis through the mitochondrial apoptotic pathway.
10.7534/j.issn.1009-2137.2018.02.031
[Atractylenolide I Can Induce Apoptosis of U266 Cells and Enhance Bortezomib Effect].
Mai Zi-Xin,Yu Tian-Qi,Fan Ting-Ting
Zhongguo shi yan xue ye xue za zhi
OBJECTIVE:To investigate the effect of atractylenolide I on proliferation and apoptosis of U266 cells, and anti-multiple myeloma effect of bortezomib. METHODS:Bortezomib, bortezomib combined atractylenolide I and atractylenolide I at different concentrations were added into U266 cells respectively, cellular proliferation toxicity was evaluated by CCK-8 assay, apoptosis and cell cycle were detected by using flow cytometry with Annexin V-FITC/PI staining. RT-PCR and Western blot analysis were used to detect the mRNA and protein levels of targeting gene Caspase-3,Caspase-9,BCL-2,BAX,JAK2,STAT3 and IL-6, respectively. RESULTS:The proliferation of U266 cells could inhibited by atractylenolide I, and the apoptosis of U266 cells could be promoted by atractylenolide I, also, which showed a dose-dependent manner(P<0.00; r=0.99). Moreover, the atractylenolide I could regulat the mitochondrial pathway(P<0.01). The combination of 2 drugs could strengther the inhibition of U266 cell proliferation significantly, and the expression level of IL-6,JAK2,STAT3 and BCL-2 mRNA and protein could be decreased by single drug and 2 drugs both(P<0.01). CONCLUSION:Atractylenolide I significantly inhibits the proliferation of U266 cells and promotes their apoptosis. At the same time, it acts synergistically with bortezomib, which may be related to mitochondrial pathway, and probably related to the regulating of IL-6, JAK2 and STAT3 gene expression in signal pathway of JAK2/STAT3.
10.19746/j.cnki.issn.1009-2137.2020.01.030