44. Rapid induction of apoptosis at ultra low temperatures enhances the efficacy of prostate cancer cryoablation

Abstract

Investigations into the molecular-based responses of prostate cancer following cold exposure have led to the discovery of delayed-onset, apoptotic cell death within the periphery of cryolesions. The apoptotic pathway typically attributed to this delayed death is the intrinsic/mitochondrial-mediated pathway characterized by a loss of mitochondrial potential, release of cytochrome c, and activation of caspase-9. Recent studies, however, have shown that at lower temperatures within the core of the cryogenic lesion (<−20 °C) a rapid programmed cell death response occurs. Using an engineered, 3-dimensional prostate tumor model, we investigated these events to determine the signaling pathway(s) responsible for the cell death as a means of developing improved molecular based approaches for the cryoablation of prostate cancer. Human prostate cancer cells (PC3) were cultured in the 3D matrices for 7 days prior to experimentation. The tumor models were then frozen to −30 or −15 degrC and analyzed at various times post-thaw using fluorescence microscopy, flow cytometry, and Western blots. Results demonstrated that the activation of apoptotic cell death occurred within 30 min of thawing at ultra low temperatures. At −30 °C, ∼25% of cells were apoptotic at 30 min and by 6 h levels had dropped near those of controls. At elevated temperatures (−15 °C), the activation and progression of apoptosis was considerably delayed, peaking at ∼20% by 6–24 h post-thaw. Additionally, it was determined that early onset apoptosis was regulated through a unique, caspase dependent process compared to that seen within the freeze margins. This induction was found to progress through a membrane mediated pathway associated with more severe thermal stressors as indicated by the activation of cas-pase-8 at low (−30 °C) but not mild (−15 °C) temperatures. These data suggest that an apoptotic continuum exists throughout the cryolesion whereby the more severe the cryogenic stress, the faster programmed cell death is manifested. The identification of this rapid-onset apoptosis within the core of the ablative zone represents a novel finding in a region previously thought to be only necrotic. Ultimately, it is our aim to decipher the signaling pathways involved in triggering rapid-onset apoptosis such that these events can be manipulated to enhance cell death, thus improving the overall efficacy of cryosurgical procedures.