2-Methoxyestradiol enhances TRAIL mediated apoptosis in ovarian cancer cells but not in normal cells in both a caspase-dependent and independent manner

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 1920 Ovarian cancer constitutes the main cause of death from gynecologic cancers. Despite using extensive cytoreductive surgery followed by intensive chemotherapy there is poor survival at advanced stages. Thus, finding new strategies of treatment constitutes a great challenge. We already demonstrated that TRAIL, a member of TNF family, induces apoptosis in ovarian cancer cells. In addition, we have found that 2-methoxyestradiol (2ME), an estrogen metabolite, kills cancer but not normal ovarian cells. Objectives: We investigated if TRAIL induces apoptosis in primary cultured ovarian cancer cells and if 2ME enhances TRAIL mediated apoptosis in this type of cancer. Furthermore, we investigated the mechanisms behind any interaction between them. Methods: Cell viability was studied by the MTS assay. Apoptosis was confirmed through DNA laddering, FACS, in vitro caspase activity assay, and W-B of different proteins involved in the apoptotic cascade. Results: TRAIL induces cell death in primary cultured ovarian cancer cell lines (established from advanced epithelial carcinomas) but not in normal cells. More interesting TRAIL did not affected cell cultures established from benign ovarian tumors or low malignant potential ovarian cancers. 2ME also induces cell death in primary cultures of advanced ovarian cancers but not in benign ovarian tumors or normal ovary. Pretreatment with 2ME enhances TRAIL mediated apoptosis in advanced ovarian carcinomas with no effect on low malignant potential tumors or benign tumors or normal ovary. The interaction was synergistic by fractional inhibition analysis. The mechanism behind this interaction involved increase in TRAIL-R2 (RT-PCR, W-B), increase in BID cleavage, increase in BCL2 phosphorylation, increase in caspase-8, -9, and -3 activity, and increase in cytochrome-c release. Interestingly, the use of a non-selective caspase inhibitor (ZVAD-fmk) or selective caspase inhibitors (caspase-8, -9 inhibitors) only partially reverted the effect. The incomplete reversion was explained by increased release of AIF from mitochondria suggesting triggering of a caspase independent apoptosis cascade. The stable overexpression of BClxL in these cancer cells resulted in almost complete reversion of the synergistic effect observed with the combination of 2ME + TRAIL. Conclusions: These data suggest 2ME enhances TRAIL mediated apoptosis by increasing both extrinsic and intrinsic apoptotic cascades. The apoptotic effect is due to activation of both caspase dependent and independent cascades. TRAIL and 2ME, might be considered in the treatment of advanced ovarian carcinoma. (FONDECYT 1050744)