Temperature-dependent activation of differential apoptotic pathways during cryoablation in a human prostate cancer model.

BACKGROUND: Critical to the continual improvement of cryoablation efficacy is deciphering the biochemical responses of cells to low-temperature exposure. The identification of delayed-onset cell death has allowed for the manipulation of cellular responses through the regulation of apoptosis. We hypothesized that in addition to delayed apoptotic events associated with mild subfreezing temperatures (10 to -25 °C), cells exposed to ultra-low temperatures (<-30 °C) may undergo rapid, early-onset apoptosis. METHODS: Human prostate cancer model and cells (PC-3) were exposed to temperatures of -60, -30 and -15 °C to simulate a cryoablative procedure. Using a combination of flow-cytometry, fluorescent microscopy and western blot analyses, samples were assessed at various times post thaw to identify the presence, levels and the pathways involved in cell death. RESULTS: Exposure to temperatures <-30 °C yielded a significant apoptotic population within 30 min of thawing, peaking at 90 min (~40%), and by 6 h, only necrosis was observed. In samples only reaching temperatures >-30 °C, apoptosis was not noted until 6-24 h post thaw, with the levels of apoptosis reaching ~10% (-15 °C) and ~25% (-30 °C) at 6 h post thaw. Further, it was found that early-onset apoptosis progressed through a membrane-mediated mechanism, whereas delayed apoptosis progressed through a mitochondrial path. CONCLUSIONS: These data demonstrate the impact of apoptotic continuum, whereby the more severe cryogenic stress activated the extrinsic, membrane-regulated pathway, whereas less severe freezing activated the intrinsic, mitochondrial-mediated path. The rapid induction and progression of apoptosis at ultra-low temperatures provides an explanation as to why such results have not previously been identified following freezing. Ultimately, an understanding of the events and signaling pathways involved in triggering apoptosis following freezing may provide a path for selective induction of the rapid-onset and delayed programmed cell death pathways in an effort to improve the overall cryoablation efficacy.

Targeted induction of apoptosis via TRAIL and cryoablation: a novel strategy for the treatment of prostate cancer.

Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances cell death. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Human prostate cancer cells (PC-3) are resistant to many cytodestructive agents, including cryoablation and TRAIL. Here, we evaluated the effects of TRAIL combined with cryoablation on PC-3 and normal prostate (RWPE-1) cell death. Exposure of PC-3 cells to freezing (-10 degrees C) or TRAIL (500 ng/ml) results in minimal cell death, whereas a complete loss of viability is observed with the simultaneous combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a significant level of caspase-8 and -3 cleavage between 12 and 24 h post-exposure. Caspase activation assays provided similar results and also indicated a role for caspase-9. Inhibitors to caspase-8 and -9 along with a pan-caspase inhibitor were incorporated to determine which pathway was necessary for the combined efficacy. Inhibition of caspase-8 significantly blocked the combination-induced cell death compared to cells that did not receive the inhibitor (63% compared to 10% viable). The addition of the caspase-9 inhibitor resulted in only a minimal protection. Importantly, the combination was not effective when applied to normal prostate cells. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.