P13: Vitamin D3 therapy increases cryoablation efficacy: A novel strategy for the treatment of prostate cancer

Abstract

Introduction Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances prostate cancer cell death. Vitamin D 3 (VD 3 ) is a natural steroidal hormone that has been shown to exhibit cytotoxic properties preferentially inducing apoptosis in a variety of human cancer cells. Human prostate cancer cells are known to be resistant to many cytodestructive agents, including cryoablation and VD 3 . Here, we evaluated the efficacy of VD 3 combined with cryoablation on androgen insensitive human prostate cancer (PC-3 and LNCaP-HP) cell death. Methods Freezing and VD 3 exposure to PC-3 and LNCaP-HP cells were performed using both 2D and 3D culture systems and efficacy was determined by cell viability assays. Resultant cell death and specific signaling components were determined using apoptotic inhibitors, fluorescence microscopy, protease activity assays and immunoblotting. Results Exposure of LNCaP-HP cells to freezing (-15°C) or VD 3 (50 μM) results in minimal cell death (15% and 10% respectively 3 days post treatment), while a complete loss of viability was observed with the combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a decrease in pro-caspase-9 and -3 between 6 and 18 h post-exposure. Caspase activation assays confirmed the reduction in pro-caspase levels was a result of caspase activation. Protease inhibitors were incorporated into the combination protocol to determine the overall contribution of caspase activity in cell death. Inhibition of caspase-9 significantly blocked the combination induced cell death compared to cells that did not receive the inhibitor (55% vs. 21% viable, respectively). The addition of the caspase-8 inhibitor resulted in only minimal protection, indicating a specific mitochondrial-mediated event. Importantly, the combination was not effective when applied to normal prostate cells. Conclusions VD 3 sensitizes CaP cells to cryoablation. The significant increase in cell death was attributed to the activation of apoptosis, specifically through mitochondrial -mediated events. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.