Abstract
Abstract Background: Ovarian cancer is one of the most lethal gynecological malignancies. With existing treatment options, recurrence is very common with only 30% five year survival rate. The poor survival rate is associated mainly with acquired resistance to chemotherapy drugs, such as cisplatin. Therefore, identification of novel treatment options is highly desirable. Herein, we investigated the anti-cancer potential of ormeloxifene in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Materials and Methods: Ovarian cancer cells SKOV-3 (semi-resistant to cisplatin), A2780-CP (cisplatin-resistant), and A2780 (cisplatin-sensitive) were treated with ormeloxifene (1-27 µM) and the effect on cell growth was determined by MTS and colony formation assays. Apoptotic cells were identified using TUNEL and propidium iodide (PI) staining. The effect of ormeloxifene treatment on the expression of apoptosis and cell survival related proteins was determined by Western blotting and Flow Cytometry. Tetramethylrhodamine (TMRE, a stain sequestered by mitochondria) staining was performed to determine the effect of ormeloxifene on mitochondrial membrane potential. For that, cells were treated with ormeloxifene (10-20 µM) at three different time points (6, 12, and 18 hours), incubated with TMRE (100 nM), and were analyzed by flow cytometry. For in vivo tumor xenograft study, athymic nude (nu/nu) mice were injected with A2780CP (5 million) cells into the peritoneum. These mice were treated with ormeloxifene (50 or 100 µg/mice/week, for 3 subsequent weeks). On day 35 after cell injection, tumor burden was recorded. Statistical significance was determined using a two-tailed student t-test with equal variance. Results: In all three cell lines, ormeloxifene effectively inhibited cell growth in a dose-dependent manner in proliferation and colony forming assays. Further analysis using TUNEL and PI staining revealed a dose-dependent increase in percent of apoptotic cells. Immunoblotting assays showed a dose-dependent decrease in full-length caspase-9 and caspase-3 (indicating increased caspase activity), and a marked increase in cleaved Poly (ADP-ribose) polymerase (PARP). The TMRE assay also revealed a dose-and time-dependent decrease in mitochondrial membrane potential which is an early sign of the intrinsic apoptotic pathway. Interestingly, ormeloxifene treatment effectively blocked ovarian tumor growth of A2780CP ovarian cancer cells in xenograft mouse model. Conclusions: Ormeloxifene treatment effectively inhibits growth of cisplatin sensitive and resistant ovarian cancer cell via induction of caspase-mediated apoptosis. Our data suggest that ormeloxifene induces an intrinsic apoptotic pathway by altering mitochondrial membrane potential followed by PARP cleavage. Therefore, ormeloxifene may be a potential treatment for cisplatin-resistant and cisplatin-sensitive ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4521. doi:10.1158/1538-7445.AM2011-4521
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