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Objectives To investigate the effect and molecular mechanisms of action of Vitamin D3 (VD3) as a neo-adjunctive agent before cryosurgery in an effort to increase treatment efficacy for prostate cancer (CaP). To eliminate the potential for disease recurrence that exists at the periphery of the freeze lesion, where temperatures may be insufficient to destroy both androgen-sensitive (AS) and androgen-insensitive (AI) CaP. Methods Human CaP cells, LNCaP, were each genetically altered to express the AS and AI phenotypes and subjected to VD3 treatment and freezing in an in vitro and tissue-engineered model. Cell viability, caspase inhibitor and western blot studies were used to determine the basis of the different responses of AI and AS cells to VD3 cryosensitization. Results VD3 was found to be a highly effective cryosensitizer, resulting in a >50% overall increase in cell death after -15°C freezing. Fluorescence microscopy, western blot analysis and caspase protease assays confirmed that the increased activation of apoptosis was modulated through a mitochondrial-mediated pathway. Caspase inhibition studies showed that apoptosis played an integral role in cell death, with VD3 cryosensitivation-induced apoptotic events responsible for > 30% of the overall cell death after -15°C freezing. Conclusions The present study suggests that the use of VD3 as a cryosensitizer increases cryoablation efficacy through the increased activity of apoptosis as well as through necrosis. The data show that through VD3 treatment the overall level of AI CaP cell tolerance to freezing is reduced to a level similar to that of AS CaP. VD3 pre-treatment in conjunction with cryoablation may increase treatment efficacy and reduce disease recurrence for CaP patients.

The techniques of present-day cryosurgery performed with multiprobe freezing apparatus and advanced imaging techniques yield predictable and encouraging results in the treatment of prostatic and renal cancers. Nevertheless, and not unique to cryosurgical treatment, the rates of persistent disease demonstrate the need for improvement in technique and emphasize the need for proper management of the therapeutic margin. The causes of persistent disease often relate to a range of factors including selection of patients, understanding of the extent of the tumor, limitations of the imaging techniques, and failure to freeze the tumor periphery in an efficacious manner. Of these diverse factors, the one most readily managed, but subject to therapeutic error, is the technique of freezing the tumor and appropriate margin to a lethal temperature [Baust, J. G., Gage, A. A. The Molecular Basis of Cryosurgery. BJU Int 95, 1187-1191 (2005)]. This article describes the recent experiments that examine the molecular basis of cryosurgery, clarifies the actions of the components of the freeze-thaw cycle, and defines the resultant effect on the cryogenic lesion from a clinical perspective. Further, this review addresses the important issue of management of the margin of the tumor through adjunctive therapy. Accordingly, a goal of this review is to identify the technical and future adjunctive therapeutic practices that should improve the efficacy of cryoablative techniques for the treatment of malignant lesions.

OBJECTIVE To investigate in prostate cancer cells the consequences of androgen‐insensitivity (AI) development on the cellular and molecular responses to freezing, as a challenge in prostate cancer treatment occurs when the androgen‐sensitive (AS) phenotype switches to an AI phenotype, the latter of which is often refractory to many therapies. MATERIALS AND METHODS PC‐3 (AI) and LNCaP (AS) were each genetically altered to express the opposite phenotype and subjected to an in vitro freezing model. Viability, caspase inhibitor and Western blot studies were used to determine the basis of the differential responses of AI and AS cells. RESULTS LNCaP high‐passage cells, formed by repeated passage of LNCaP (AS) cells, were AI and showed a phenotypic shift to freeze resistance matching the freeze response of PC‐3 cells (AI). While stably transfected androgen receptor (AR)‐transfected cells (PC‐3 AR) had a freezing sensitivity similar to that of the LNCaP (AS) cell line. Importantly, AI cell lines survived and recovered from freezing exposure to temperatures as low as −40 °C whereas AS cell lines did not. Caspase inhibition studies and related fluorescent probes showed an elevated level of apoptotic involvement in both AS cell lines after freezing compared with their AI counterparts. Western blot analysis showed that AR expression was modified after exposure to freezing. CONCLUSION This study suggests that AS cancers may be far more sensitive to a freezing insult and this might be linked to elevated apoptosis and caspase activity. As such, cryoablation may prove most effective in cancer cells that have not yet progressed to a more resistant AI phenotype, but both generic variants can be fully ablated at sufficiently low temperatures.

Cryoablation has emerged as a primary therapy to treat prostate cancer. While effective, the assumption that freezing serves as a ubiquitous lethal stress is challenged by clinical experience and experimental evidence demonstrating time-temperature related cell death dependence. The age-related transformation from an androgen-sensitive (AS) to an androgen-insensitive (AI) phenotype is a major challenge in the management of prostate cancer. AI cells exhibit morphological changes and treatment resistance to many therapies. Since this resistance has been linked with α6β4 integrin overexpression as a result of androgen receptor (AR) loss, we investigated whether α6β4 integrin expression, as a result AR loss, contributes to the reported increased freeze tolerance of AI prostate cancer. A series of studies using AS (LNCaP LP and PC-3 AR) and AI (LNCaP HP and PC-3) cell lines were designed to investigate the cellular mechanisms contributing to variations in freezing response. Investigation into α6β4 integrin expression revealed that AI cell lines overexpressed this protein, thereby altering morphological characteristics and increasing adhesion characteristics. Molecular investigations revealed a significant decrease in caspase 8, 9, and 3 levels AI cells following freezing. Inhibition of α6β4 integrin resulted in increased caspase activity following freezing (similar to AS cells) and enhanced cell death. These data demonstrate that AI cells show an increase in post-freeze susceptibility following inhibition of α6β4 integrin function. Further understanding the role of androgen-receptor related α6β4 integrin expression in prostate cancer cells responses to freezing might lead to novel options for neo-adjunctive treatments targeting the AR signaling pathway.

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