Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.
Nordin Noraziah,Majid Nazia Abdul,Hashim Najihah Mohd,Rahman Mashitoh Abd,Hassan Zalila,Ali Hapipah Mohd
Drug design, development and therapy
Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.
Downregulation of DNMT3a expression increases miR-182-induced apoptosis of ovarian cancer through caspase-3 and caspase-9-mediated apoptosis and DNA damage response.
Lu Wei,Lu Tanmin,Wei Xin
In the present study, DNA (cytosine-5)-methyltransferase 3α (DNMT3a) is explored as an anticancer molecule in ovarian cancer treatment, and also the mechanistic link between DNMT3a and its regulatory signaling pathway in Caov-3 cells is provided. Firstly, DNMT3a protein expression in 12 freshly resected ovarian cancer patient tissues and tisssues from 8 ovariectomized patients was assessed. In the ovarian cancer tissues, DNMT3a expression was upregulated and miR-182 expression was downregulated. DNMT3a overexpression inhibited miR-182 expression and caspase-3 and -9 activity and suppressed p53 and c-Myc protein expression in Caov-3 cells. Secondly, miR-182 overexpression increased Caov-3 cell apoptosis, which however was reduced by DNMT3a (DNMT3 plasmid) overexpression. Downregulation of DNMT3a expression activated miR-182 expression and caspase-3 and -9 activity, and promoted p53 and c-Myc protein expression in Caov-3 cells. Collectively, a valuable anticancer mechanism of ovarian cancer was elucidated, by which downregulation of DNMT3a regulated miR-182 via caspase-3 and -9-mediated apoptosis and DNA damage response, which suggests that DNMT3a may be used as a potential strategy for therapeutic intervention in ovarian cancer.
Cleistopholine isolated from Enicosanthellum pulchrum exhibits apoptogenic properties in human ovarian cancer cells.
Nordin Noraziah,Majid Nazia Abdul,Mohan Syam,Dehghan Firouzeh,Karimian Hamed,Rahman Mashitoh Abdul,Ali Hapipah Mohd,Hashim Najihah Mohd
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:Cleistopholine is a natural alkaloid present in plants with numerous biological activities. However, cleistopholine has yet to be isolated using modern techniques and the mechanism by which this alkaloid induces apoptosis in cancer cells remains to be elucidated. HYPOTHESIS/PURPOSE:This study aims to isolate cleistopholine from the roots of Enicosanthellum pulchrum by using preparative-HPLC technique and explore the mechanism by which this alkaloid induces apoptosis in human ovarian cancer (CAOV-3) cells in vitro from 24 to 72 h. This compound may be developed as an anticancer agent that induces apoptosis in ovarian cancer cells. STUDY DESIGN/METHODS:Cytotoxicity was assessed via the cell viability assay and changes in cell morphology were observed via the acridine orange/propidium iodide (AO/PI) assay. The involvement of apoptotic pathways was evaluated through caspase analysis and multiple cytotoxicity assays. Meanwhile, early and late apoptotic events via the Annexin V-FITC and DNA laddering assays, respectively. The mechanism of apoptosis was explored at the molecular level by evaluating the expression of specific genes and proteins. In addition, the proliferation of CAOV-3-cells treated with cleistopholine was analysed using the cell cycle arrest assay. RESULTS:The IC50 of cleistopholine (61.4 µM) was comparable with that of the positive control cisplatin (62.8 µM) at 24 h of treatment. Apoptos is was evidenced by cell membrane blebbing, chromatin compression and formation of apoptotic bodies. The initial phase of apoptosis was detected at 24 h by the increase in Annexin V-FITC binding to cell membranes. A DNA ladder was formed at 48 h, indicating DNA fragmentation in the final phase of apoptosis. The mitochondria participated in the process by stimulating the intrinsic pathway via caspase 9 with a reduction in mitochondrial membrane potential (MMP) and an increase in cytochrome c release. Cell death was further validated through the mRNA and protein overexpression of Bax, caspase 3 and caspase 9 in the treated cells compared with the untreated cells. In contrast, Bcl-2, Hsp70 and survivin decreased in expression upon cleistopholine treatment. Cell cycle was arrested at the G0/G1 phase and cell population percentage significantly increased to 43.5%, 45.4% and 54.3% in time-dependent manner in the cleistopholine-treated CAOV-3 cells compared with the untreated cells at 24, 48 and 72 h respectively. CONCLUSION:The current study indicated that cleistopholine can be a potential candidate as a new drug to treat ovarian cancer disease.
Knockdown of BRCA2 enhances cisplatin and cisplatin-induced autophagy in ovarian cancer cells.
Wan Biao,Dai Leheyi,Wang Li,Zhang Ying,Huang Hong,Qian Guanhua,Yu Tinghe
Clinical implications of the BRCA2 expression level on treatments of ovarian cancer are controversial. Here, we demonstrated that platinum-resistant cancer had a higher percentage of high BRCA2 level (87.5% vs 43.6%, = 0.001), and that patients with a low BRCA2 level in cancer tissues had longer progression-free survival (with a median time of 28.0 vs 12.0 months, < 0.001) and platinum-free duration (with a median time of 19.0 vs 5.0 months, < 0.001) compared with those with a high BRCA2 level. In human ovarian cancer cell lines CAOV-3 and ES-2, cisplatin induced an upregulation of the RAD51 protein, which was inhibited after silencing ; silencing enhanced the action of cisplatin and Knockdown of BRCA2 promoted cisplatin-induced autophagy. Interestingly, the autophagy blocker chloroquine enhanced cisplatin in BRCA2-silenced cells accompanied by an increase in apoptotic cells, which did not occur in BRCA2-intact cells; chloroquine enhanced the efficacy of cisplatin against BRCA2-silenced CAOV-3 tumors , with an increase in LC3-II level in tumor tissues. Sensitization of cisplatin was also observed in BRCA2-silenced CAOV-3 cells after inhibiting ATG7, confirming that chloroquine modulated the sensitivity via the autophagy pathway. These data suggest that a low BRCA2 level can predict better platinum sensitivity and prognosis, and that the modulation of autophagy can be a chemosensitizer for certain cancers.
Influence of carboplatin on the proliferation and apoptosis of ovarian cancer cells through mTOR/p70s6k signaling pathway.
Zhou Honghui,Zhao Haibo,Liu Hui,Xu Xiang,Dong Xiaoli,Zhao Enfeng
Journal of B.U.ON. : official journal of the Balkan Union of Oncology
PURPOSE:To investigate the influence of carboplatin on the proliferation and apoptosis of ovarian cancer cells through mTOR/P70S6K signaling pathway. METHODS:The mRNA and protein expressions were detected via Western blotting and RT-PCR to study whether the mTOR/p70S6K signaling pathway was activated in OVCAR-3 and Caov-3 ovarian cancer cell lines. After cells were treated with different concentrations of carboplatin, the mRNA and protein expressions of mTOR, p70S6K and 4E-BP1 were detected via RT-PCR and Western blotting. OVCAR-3 cells were treated with 20 and 50 μM carboplatin for 4 hrs, and then apoptosis was analyzed and assessed. OVCAR-3 cells were treated with different concentrations of carboplatin (20, 50, 100, 150 and 200 μM) for 24 and 48 hrs, respectively. RESULTS:The mTOR signaling pathway was activated in OVCAR-3 and Caov-3 ovarian cancer cell lines. The mRNA level of mTOR in Caov-3 cells was higher, but that of p70S6K was lower. Carboplatin significantly reduced the mRNA expression of mTOR (p<0.01), whereas the mRNA expressions of p70S6K and 4E-BP1 in carboplatin-treated cells were increased in a dose-dependent manner (p<0.01). Carboplatin inhibited the mTOR protein expression in a dose-dependent manner (p<0.01). The proliferation of OVCAR-3 cells exposed to carboplatin was reduced compared with that of untreated cells (p<0.01), and the inhibitory effect of carboplatin on the proliferation of OVCAR-3 cells was time- and dose-dependent. CONCLUSION:The mTOR/p70S6K pathway was activated in ovarian cancer. Carboplatin could rapidly inhibit the expression of mTOR, and the phosphorylation of its major downstream effectors p70S6K and 4E-binding protein 1 (4E-BP1) arrested cells in G0/G1 phase and induced ovarian cancer cell apoptosis.
Plagioneurin B, a potent isolated compound induces apoptotic signalling pathways and cell cycle arrest in ovarian cancer cells.
Nordin Noraziah,Majid Nazia Abdul,Othman Rozana,Omer Fatima Abdelmutaal Ahmed,Nasharuddin Muhammad Nazil Afiq,Hashim Najihah Mohd
Apoptosis : an international journal on programmed cell death
Plagioneurin B belongs to acetogenin group has well-established class of compounds. Acetogenin group has attracted worldwide attention in the past few years due their biological abilities as inhibitors for several types of tumour cells. Plagioneurin B was isolated via conventional chromatography and tested for thorough mechanistic apoptosis activity on human ovarian cancer cells (CAOV-3). Its structure was also docked at several possible targets using Autodock tools software. Our findings showed that plagioneurin B successfully inhibits the growth of CAOV-3 cells at IC of 0.62 µM. The existence of apoptotic bodies, cell membrane blebbing and chromatin condensation indicated the hallmark of apoptosis. Increase of Annexin V-FITC bound to phosphatidylserine confirmed the apoptosis induction in the cells. The apoptosis event was triggered through the extrinsic and intrinsic pathways via activation of caspases 8 and 9, respectively. Stimulation of caspase 3 and the presence of DNA ladder suggested downstream apoptotic signalling were initiated. Further confirmation of apoptosis was conducted at the molecular levels where up-regulation in Bax, as well as down-regulation of Bcl-2, Hsp-70 and survivin were observed. Plagioneurin B was also seen to arrest CAOV-3 cells cycle at the G2/M phase. Docking simulation of plagioneurin B with CD95 demonstrated that the high binding affinity and hydrogen bonds formation may explain the capability of plagioneurin B to trigger apoptosis. This study is therefore importance in finding the effective compound that may offer an alternative drug for ovarian cancer treatment.
Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells.
Jeong Kang Jin,Park Soon Young,Seo Ji Hye,Lee Kyung Bok,Choi Wahn Soo,Han Jeung Whan,Kang Jae Ku,Park Chang Gyo,Kim Yong Kee,Lee Hoi Young
Experimental & molecular medicine
Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.