Activation of caspase-independent programmed cell death overcomes apoptosis resistance in ovarian cancer cells

A50 Introduction: Chemoresistance is a major burden in the treatment of patients with ovarian cancer and is associated with failure to induce apoptosis. In panA¢â‘¬â\#8364;œresistant cancer cells, the full induction of the apoptotic cascade is inhibited due to high levels of expression of anti-apoptotic proteins, which prevent caspase activation. The objective of this study was to evaluate whether the activation of a caspase-independent alternative pathway could promote cell death in chemoresistant ovarian cancer cells. We describe the characterization of a novel compound, NV-128, which promotes cell death in a caspase-independent manner in chemo-resistant ovarian cancer cells.
 Methods: Eight primary cultures and two established epithelial ovarian cancer (EOC) cell lines were treated with increasing concentrations of NV-128 (0.1, 1, and 10 Μg/ml) with or without the pan-caspase inhibitor, Z-VAD-FMK. Cell viability was determined after 24h using the Celltiter 96 assay. DNA fragmentation was analyzed by flow cytometry with Hoechst and Propidium iodide staining. Activity of caspases- 3/7, -8, and -9 was measured using Caspase-Glo assay. Protein expression was determined by Western blot analysis.
 Results: NV-128 treatment decreased cell viability in all tested EOC cells lines in a dose-dependent manner with IC50 between 1 and 5 Μg/ml. Flow cytometry analysis revealed DNA fragmentation, with >90% cells staining double-positive for Hoechst and Propidium iodide after 24h. Cell death was however, caspase-independent as evidenced by the lack of caspases- 3/7, -8, and -9 activity and the inability of the pan-caspase inhibitor, Z-VAD-FMK, to prevent cell death. DNA fragmentation was observed as the result of the activation of an intracellular pathway involving: down-regulation of pAKT, cleavage of LC3 to LC3-II, Beclin mitochondrial translocation leading to Bcl2 inhibition, and nuclear translocation of EndoG.
 Conclusion: We describe an alternative pathway leading to DNA fragmentation and cell death, which does not depend on caspase activation. Our findings demonstrate the possibility of using therapeutic drugs, such as NV-128, which could overcome resistance to the classical caspase-dependent apoptosis and therefore have beneficial effects in chemoresistantovarian cancer patients.