Elsevier

Cryobiology

Volume 49, Issue 1, August 2004, Pages 45-61
Cryobiology

Addition of anticancer agents enhances freezing-induced prostate cancer cell death: implications of mitochondrial involvement

https://doi.org/10.1016/j.cryobiol.2004.05.003Get rights and content

Abstract

Recent evidence suggests that the successful treatment of prostate cancer may require adjuvant therapies. Accordingly, a better understanding of the molecular mechanisms involved in current treatments may lead to enhanced efficacy by providing a basis for adjuvant therapies. In this study, we demonstrate that the combination of sub-lethal concentrations of chemotherapeutic agents prior to freezing (−15 °C) in a prostate cancer cell (PC-3) model results in enhanced efficacy over either treatment alone. Morphological analysis revealed that necrosis appeared to be the prevalent mode of cell death following adjuvant (in vitro) modeling, yet molecular analysis indicated that freezing and chemotherapy differentially activated apoptotic cascades through modulating opposing members of the Bcl-2 protein family. Freezing results in a time-dependent increase of the antiapoptotic Bcl-2 protein, while chemotherapy results in an increase of the pro-apoptotic Bax protein. Anti-apoptotic Bcl-2 protein levels increase over 3-fold following exposure to freezing. 5-Fluorouracil (5-FU) causes pro-apoptotic Bax levels to increase 2-fold during the drug exposure. The increase in Bax was also apparent following the combination of 5-FU/freezing, while Bcl-2 levels were maintained at or below control levels. This led to a shift in the Bcl-2 to Bax ratio to a pro-death tendency. Other effective cryo/chemo combinations were also found to provide similar effects. The combination of cisplatin/freezing resulted in a 4-fold increase in the ratio of Bax to Bcl-2 when compared to controls, which represented a 2-fold increase over the 5-FU/freezing-combination model. This increase may contribute to the continued reduction in cell number observed during the 13-day recovery period. Additionally, the addition of an apoptotic caspase inhibitor was not able to protect cultures from cell death following combination treatment. In conclusion, the data suggest that both Bcl-2 and Bax may, not only, play an important role in the efficacy of the cryo/chemo combination, but also the balance between the two may determine the role and extent of system destruction.

Section snippets

Cell culture

The human prostate cancer cell line, PC-3, was obtained from the American Type Culture Collection (Rockville, MD). Stock cultures were maintained at 37C in 95% air/5% CO2 in Falcon T 75 cm2-flasks. PC-3 cell cultures were grown in RPMI-1640 (Gibco/BRL Laboratories, Grand Island, NY) medium, supplemented with 10% fetal calf serum (Atlanta Biological, Norcross, Georgia) and 1% penicillin/streptomycin (Gibco/BRL). Stock cultures were subcultured every 7–8 days at approximately 95% confluence, and

Pre-exposure of chemotherapy agents results in enhanced cell death when combined with freezing

Previous reports have indicated that the addition of a sub-toxic concentration of 5-fluorouracil (5-FU) prior to freezing (−15 °C) resulted in enhanced cell death when compared to either treatment alone [7], [8]. In an attempt to further improve the “cryosurgical outcome” in these in vitro models, additional chemotherapeutic agents were examined to determine if they would result in similar synergistic outcomes. Following a 2-day recovery period, cell cultures exposed to −15 °C experienced a 35%

Discussion

Hormone refractory prostate cancer remains a therapeutic challenge. The successful treatment of this disease will require a further understanding of the molecular mechanisms involved in both the disease and the effective nature of treatment options. The purpose of this study was to investigate the involvement, if any, of the mitochondria in the increased efficacy of the cryo/chemo combination model. In this study, it was demonstrated that the addition of sub-lethal concentrations of a range of

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    This study was supported in part by NIH Grant No. R43RR16014.

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