Triapine (3-aminopyridine-2-carboxyaldehyde Thiosemicarbazone) Induces Apoptosis In Chemoresistant Ovarian Cancer Cells

Objectives'. The development of resistance to chemotherapeutic agents is one of the main problems in ovarian cancer treatment. The chemoresistance in cancer chemotherapy has motivated researchers to develop new drugs that are more effective and have less side effect. Triapine is a potent ribonucleotide reductase inhibitor, which exerts its anti-neoplastic acitivity by inhibiting DNA synthesis and repair. And it has been found with broad spectrum antitumor activity and is undergoing I clinical trial. The objective of this study was to determine whether Triapine can inhibit the proliferation of ovarian cancer cells and to characterize the apoptotic cascade induced in response to this agent.Methods: Three established epithelial ovarian cancer (EOC) cell lines and two EOC cell lines isolated from malignant ascites were treated with 10-fold dilutions of Triapine (0.1-100 μM) for 48 hours. Some of the cultures have been previously determined to be resistant to either paclitaxel (R182, R179, A2780, SKOV3) or carboplatin (CP70, SKOV3, R179 and R182). Cell viability was determined by CellTiter 96 and the morphological features of apoptosis were observed using Hoechst staining. To characterizethe cascade induced by Triapine, treatments were also done with or without the addition of either caspase -8 or -9 inhibitors. The activation of caspases was determined using the Caspase-Glo?assay and intra-cellular downstream components of the apoptotic cascade were determined by Western blot analysis.Results: (1)A11 EOC cell lines treated with Triapine showed decreased cell viability in a dose-dependent manner. The LD50 for the majority of cell lines was <10 4.M. (2)Hoechst staining revealed nuclear shrinkage and chromatin condensation and fragmentation, which correlate with the occurrence of apoptosis. (3)The activities of caspases -8, -9, and -3 increased at least 6-fold, 8-fold, and 5-fold, respectively after Triapine treatment. (4)Western blots showed the activation of Bid and Bax and the inactivation of the apoptotic inhibitor, XIAP. There was no change observed in Bcl-2 levels. (S)Triapine-induced apoptosis was dependent on caspase- 8 activation and amplified by the mitochondrial pathway via Bid and Bax.Conclusion: The present findings demonstrate that Triapine induces cell death through the induction of apoptosis. The activation of Bax , caspase-9 and inactivation of XIAP indicate the involvement of the mithochondrial pathway in the apoptotic cascade. The demonstration that Triapine is effective in chemoresistant ovarian carcinoma cells will initiate the development of new combination therapy for ovarian cancer.