676. A Role for Ceramide in AdGFPFasLTET Gene Therapy

Abstract

Top of pageAbstract The application of gene therapy to cancer has many options in the choice of therapeutic genes but delivery of a corrective signal to every cell in the cancer is impossible. Thus, studies on improving the delivery of therapeutic genes or how to amplify the therapeutic response to gene therapy are urgently needed. In the DU145 model of prostate cancer, resistance to induction of apoptosis via Fas receptor signaling is due to overexpression of apoptotic resistance genes including cFLIPs [Hyer et al., Can Biol. and Ther. 1(4): 405–410, 2002]. We have also determined that expression of a FasL-GFP fusion gene overcomes resistance in infected cells [Hyer et al., Mol. Ther. 2(4):348–358, 2000] and kills the cell apoptotically. During this process apoptotic vesicles that signal through Fas to induce apoptosis are also produced and can be demonstrated to exert bystander activity [Hyer et al., Can. Gene Ther. 10(4):330–339, 2003]. DU145 cells overexpress apoptotic resistance genes and acid ceramidase (i.e. lowers ceramide levels), which we have shown (unpublished) produces a FasL resistant phenotype and reduces the bystander effects. This led us to examine the role of ceramide elevation in the bystander process by using small molecule ceramidase inhibitors. We have now demonstrated that acid ceramidase inhibitors, are highly efficient at activating cell death in DU145 cells in vitro using nontoxic doses in combination with AdGFPFasL virus at MOIs achievable in vivo. Administration of inhibitors activates proteasome function and transiently elevates ceramide. Mitochondrial depolarization also is observed. In vivo experiments using 75mg/kg of acid ceramidase inhibitor and AdGFPFasL provides real support for this combined gene therapy/small molecule therapy approach. We will present selected data at the meeting that explain, in part, the mechanisms of this combined therapy.