Vitamin D(3) cryosensitization increases prostate cancer susceptibility to cryoablation via mitochondrial-mediated apoptosis and necrosis.

OBJECTIVES: To investigate the effect and molecular mechanisms of action of Vitamin D(3) (VD(3) ) 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 VD(3) 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 VD(3) cryosensitization. RESULTS: VD(3) 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 VD(3) 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 VD(3) as a cryosensitizer increases cryoablation efficacy through the increased activity of apoptosis as well as through necrosis. The data show that through VD(3) treatment the overall level of AI CaP cell tolerance to freezing is reduced to a level similar to that of AS CaP. VD(3) pre-treatment in conjunction with cryoablation may increase treatment efficacy and reduce disease recurrence for CaP patients.

Changing paradigms in biopreservation.

The field of cryopreservation has a long and successful history of in-depth study and progress. Advances in our knowledge base and our ability to cryopreserve cells have been consequential and have led to its widespread integration into academic, clinical, and agricultural settings. While many cell systems are successfully cryopreserved today, there remains significant cell loss associated with cryopreservation. Moreover, even today some cell systems remain uncryopreservable from a practical perspective. This is due to the diversity of post-freeze responses of individual cells to the various stressors experienced during the freeze-thaw process. In 1998, several independent groups reported on the direct involvement of apoptotic and necrotic cell death following cryopreservation (Baust, et al., 1998 and Borderie, et al., 1998). In addition to those reports, a substantial literature base describing the modulation of cell death through the use of various protease inhibitors, free radical scavengers, media formulations, and other novel compounds exist. These studies have identified diverse molecular-based, cellular responses to cryopreservation and have further demonstrated the significant improvements in cell survival through the modulation of molecular events. Numerous studies have reported on the molecular-based phenomena of cryopreservation-induced delayed onset cell death, yet our understanding of the pathway activation, progression, control, and the downstream effect on cell function remains in its infancy. To this end, modulation studies, such as targeted apoptotic control (TAC), have shown promise in furthering our understanding of the activation pathways and are proving to be a critical next step in the evolution of the cryopreservation sciences. This review provides an overview of the current literature on the mechanisms of cell death associated with cryopreservation failure.

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.

Cryoablative response of prostate cancer cells is influenced by androgen receptor expression.

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 degrees 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.

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems.

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (-5 to -40 degrees C), and cells were thawed followed by return to 37 degrees C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At -15 degrees C, PC-3 yielded approximately 55% viability versus approximately 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze-thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching -40 degrees C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze-thaw cycles, and passive thawing to provide maximum cell destruction.

Cryoablation of renal cancer: variables involved in freezing-induced cell death.

The detection of renal tumors has increased significantly over recent years resulting in a greater demand for novel, minimally invasive techniques. Cryoablation has emerged as a valuable treatment modality for the management of renal cancer. In an effort to detail the effects of freezing in renal cancer, the human renal cancer (RCC) cell line, 786-O, was evaluated in vitro. 786-O cells were exposed to a range of freezing temperatures from -5 to -40 degrees C and compared to non-frozen controls. The data show that freezing to -5 degrees C did not affect 786-O cell viability, while -10 degrees C, -15 degrees C, and -20 degrees C results in a significant loss of viability (23, 70, and 91%, respectively). A complete loss of cell viability was evident at temperatures of -25 degrees C and colder. Following this analysis, variables involved in the success of cryoablation were investigated. For each of the temperatures tested, extended freeze hold times and passive thawing rates resulted in more extensive cell damage. Additionally, a double freeze-thaw cycle significantly increased cell death compared to a single cycle (62% vs. 22% at -10 degrees C; 89% vs. 63% at -15 degrees C, respectively). While these variables play an important part in the effective application of cryoablation, a molecular understanding of the cell death involved is critical to improving efficacy. Apoptotic inhibition afforded 12% (-10 degrees C), 25% (-15 degrees C), and 11% (-20 degrees C) protection following freezing. Using fluorescence microscopy analysis, the results demonstrated that apoptosis peaked at six hours post-thaw. Next, apoptotic initiating agents including 5-FU and resveratrol (RVT) applied prior to freezing exposure resulted in a significant increase in cell death compared to either application alone. Importantly, the combination of RVT and freezing was noticeably less effective when applied to normal renal cells. The results herein demonstrate the efficacy of freezing and describe a novel therapeutic model for the treatment of renal cancer that may distinguish between cancer and normal cells.