Autophagy precedes caspase-independent cell death in chemo-resistant ovarian cancer cells

Abstract

3216

Introduction: The role of autophagy in cancer is controversial. Initially autophagy was thought to represent an alternative pathway for cell death. However, more recently it has been suggested to play a role in tumor survival. In this study we describe how the induction of autophagy by a novel isoflavone derivative, NV128, induce cell death 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 NV128 (0.1, 1, and 10 ug/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. Autophagic markers LC3-II and Beclin-1 was determined by Western blot analysis. Results: NV128 treatment decreased cell viability in all tested EOC cells lines in a dose-dependent manner with IC50 between 1 and 5 ug/ml. Flow cytometry analysis revealed DNA fragmentation, with Hoechst and Propidium iodide double-positive cells increasing 2-4 fold after NV128 treatment. However, cell death was caspase independent as evidenced by the lack of caspases- 3/7, -8, and -9 activity and the inability of the caspase inhibitor, Z-VAD-FMK, to prevent cell death. Western blot analysis of autophagic markers showed early involvement of LC3-II and Beclin-1. Conclusion: We demonstrate that induction of autophagy by NV128 in chemo-resistant ovarian cancer cells is able to promote caspase- independent cell death. Our results suggest that the autophagic pathway can be an alternative target in EOC cells, especially those that are highly resistant to apoptosis.