Cryosurgical modeling: Cytotoxic agent addition enhances freezing-induced cell death in a prostate cancer cell model

Cryosurgery is a treatment option that utilizes the destructive characteristics of low temperature and freezing to destroy unwanted tissue, such as cancer. The application of sub-zero temperatures results in the formation of a freeze zone that encompasses the desired tissue. The resultant isothermal characteristic of the "ice-ball" provides the lowest temperatures near the source (cryoprobe) and highest temperatures around the periphery. The formation of this cryogenic lesion has been the focus of many experiments and much debate. One debate revolves around the critical temperature necessary for complete cell and tissue destruction. A number of studies have shown that this critical temperature varies depending on the cell or tissue type, with some cells requiring only -15°C while others, including that of the prostate, require much lower temperatures (-80° to -100°C) for complete destruction. With temperatures ranging from -20°C to 37°C in and adjacent to the ice-ball periphery, the uncertainty of complete tissue destruction is apparent. This uncertainty is of great importance when the focus of the treatment is ablation of cancerous tissue.; Early improvements of the cryosurgical technique focused on the development of improved cryosurgical devices. Now, however, more attention is being focused at the cell and tissue level. The recent discovery that programmed cell death (apoptosis) plays a significant role in freezing-induced cell death, especially at those temperatures found around the periphery of the ice-ball, has been the major driving force of the research detailed in this dissertation. Since apoptotic cell death can be regulated to some extent, we hypothesized that manipulation of apoptosis could be accomplished by the addition of adjunctive methods, i.e., chemotherapeutic agents. We further hypothesized that the combined approach would lead to an enhancement of cell death at those temperatures associated with the periphery of the ice-ball, and therefore a more effective treatment option might be developed.; We report that: (1) The addition of a chemotherapeutic agent in combination with sub-zero temperature application results in enhanced cell death. (2) The most effective combination results from the addition of the chemotherapeutic agent prior to the freezing insult. (3) The chemocryo combination is not restricted to a single agent or a single cell type. (4) Apoptosis does occur following both freezing and the combination treatment. (5) The chemocryo combination reduced the necessary critical temperature to achieve complete cell destruction in a prostate cancer cell model from -100°C (freezing alone) to -40°C (combination). (6) Protein levels of an anti-apoptotic Bcl-2 family member (Bcl-2) are up-regulated following freezing in a human prostate cancer (PC-3) cell model. (7) The addition of chemotherapeutic agents resulted in an increased expression of a pro-apoptotic Bcl-2 family member (Bax). (8) The resultant cell death following the combination treatment occurred within the first 1--2 hours post-thaw. (9) End-stage cell death is primarily pathological i.e., necrotic cell death.; We conclude that (1) the combined treatment of chemotherapy and freezing can result in improved cryosurgical efficacy, (2) the incidence of recurrence in a prostate cancer model can be significantly reduced following the combined treatment, (3) a shift in the pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) ratio may be one cause of the enhanced cell death common to the combination treatment, and (4) a further understanding of the mechanisms of freezing injury may lead to improvement in the adjunctive measures and ultimately to improved cryosurgical ablation of tissues such as prostate cancer.