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1.  Vitamin D3 cryosensitization increases prostate cancer susceptibility to cryoablation via mitochondrial-mediated apoptosis and necrosis 
Bju International  2011;109(6):949-958.
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.
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.
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.
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.
PMCID: PMC3235271  PMID: 21883825
cryosurgery; apoptosis; prostate cancer; vitamin D; adjunctive; cryosensitization; cryoablation
2.  Integrin Involvement in Freeze Resistance of Androgen-Insensitive Prostate Cancer 
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.
PMCID: PMC2869388  PMID: 20066006
androgen receptor; integrin; cryosurgery; apoptosis; caspase
3.  In Vitro Assessment of Apoptosis and Necrosis Following Cold Storage in a Human Airway Cell Model 
As advances in medical technology improve the efficacy of cell and tissue transplantation, a void remains in our knowledge base as to the specific molecular responses of cells to low-temperature storage. While much focus has been given to solution formulation for tissue perfusion during storage, investigations into cold exposure-induced complex molecular changes remain limited. The intent of this study was to quantify the levels of cell death following hypothermic storage in a lung cell model, establishing a foundation for future in-depth molecular analysis. Normal human lung fibroblasts (IMR-90) were stored for 1 day or 2 days and small airway epithelial cells (SAEC) were stored for 5 days or 7 days at 4°C in complete media, ViaSpan, or ViaSpan + pan-caspase (VI) inhibitor. (Poststorage viability was assessed for 3 days using alamarBlue™.) Sample analysis revealed that IMR-90 cells stored in ViaSpan remained 80% (±9) viable after 1 day of storage and 21% (±7) viable after 2 days of storage. SAEC cells stored in ViaSpan remained 81% (±5) viable after 5 days and 28% (±7) after 7 days. Microfluidic flow cytometry analysis of the apoptotic and necrotic populations in the ViaSpan-stored samples revealed that in the IMR-90 cells stored for 2 days, 7% of the population was apoptotic at 4-h poststorage, while ∼70% was identified as necrotic. Analysis of the SAEC cell system following 7 days of ViaSpan storage revealed an apoptotic peak of 19% at 4-h poststorage and a corresponding necrotic peak of 19%. Caspase inhibition during hypothermic storage increased viability 33% for IMR-90 and 25% for SAEC. Data revealed a similar pattern of cell death, through both apoptosis and necrosis, once the onset of cold storage failure began, implying a potential conserved mechanism of cold-induced cell death. These data highlight the critical need for a more in-depth understanding of the molecular changes that occur as a result of cold exposure in cells and tissues.
PMCID: PMC3205736  PMID: 22087352

Results 1-3 (3)