924. D-MAPP Analogue, LCL-204 Sensitizes DU145 Prostate Cancer Cells (PCa) to FasL Gene Therapy through Down-Regulation of Survivin

Abstract

Introduction and Objectives: Current treatment for advanced PCa is mainly palliative. Recurrence after androgen ablation with more progressive disease typically occurs within 18 months of treatment. Gene therapy is an alternative therapy currently under investigation and is hampered by inefficient delivery to all cancer cells. Sensitizing cancer cells to gene therapy (GT) may augment its effect and increases the bystander effect. FasL mediates apoptosis through caspases and survivin is a downstream inhibitor. We have previously shown that FasL GT can induce apoptosis in prostate cancer cells and can generate a bystander effect. Furthermore, it is becoming increasingly evident that sphingolipids in general and ceramide in particular play a role in prostate cancer regulation. LCL-204, Ceramide analogue, is able to initiate apoptosis through lysosomes. Herein, we show that LCL-204 can sensitize DU145 PCa cells to FasL GT and that survivin may play an important role in this effect. Methods: MTS assay to asses cell viability. Lysotracker red to demonstrate Lysosomal pH changes and methyl dancyl cadaverine dye to show autophagic vacuoles. Proteasomal activity assessed by proteasomal assay. Western blot to show protein changes. Transfection and siRNA to show effect of survivin. Results: pretreatment with low doses of LCL-204 sensitized DU145 cells to AdGFPFasLTET . LCL-204 caused Lysosomal pH changes within few minutes after treatment as demonstrated by FACS and increased autophagic vacuoles. This is followed by increased proteasomal activity and down-regulation of anti-apoptotic protein survivin. We demonstrated increased proteasomal activity was partial responsible for survivin degradation. This was partially inhibited by proteasome inhibitors (lactacystin and MG132) as demonstrated by western blot. We also protected DU145 cells against apoptosis by AdGFPFasLTET by Transfecting DU145 cells with survivin-GFP DNA. Finally, we were able to sensitize DU145 cells to FasL induced apoptosis by Targeting survivin using siRNA. Conclusion: Survivin plays a role in protecting cells again Fas induced apoptosis. Down regulation of survivin using LCL-204 can augment the effect of Fas induced apoptosis. LCL-204 can be a potential drug used to augment the effect of AdGFPFasLTET in prostate cancer.