Rachel N Winter1, Juong G Rhee, Natasha Kyprianou. 1. Department of Biochemistry and Molecular Biology and Division of Urology, University of Maryland School of Medicine, Baltimore, MD, USA.
Abstract
BACKGROUND: The significance of caspase-1 in prostate cancer has recently been documented (Cancer Res 61: 1227-1232, 2001). In this study, we investigated the role of caspase-1 in radiation-induced apoptosis in order to identify the significance of this apoptotic initiator in radiation resistance. MATERIALS AND METHODS: Caspase-1 was over-expressed in DU-145 prostate cancer cells (which have weak endogenous expression of caspase-1), via transfection-mediated gene transfer. Stable transfectants were cloned and expression of caspase-1 was established at the mRNA and protein levels by RT-PCR and Western blot, respectively. Caspase-1 overexpressing clones were characterized for their apoptotic response to ionizing irradiation (0-9 Gy) on the basis of cell viability and Hoechst staining assays and profiling of expression of key apoptosis regulators, such as caspase -3 and -9. RESULTS: Caspase-1 transfectants exhibited a greater sensitivity in response to ionizing radiation than the neomycin control transfectants, as demonstrated by a dramatic loss in cell viability, that temporally correlated with apoptosis induction. Furthermore, caspase-1 overexpression resulted in a significant decrease in clonogenic survival following treatment with ionizing radiation, while the caspase-1 inhibitor, Z-YVAD.fmk, suppressed apoptosis induction in caspase-1 transfectants (p<0.008). The apoptotic effect was associated with increased expression of the pro-enzyme form of caspase-3 in both the caspase-1 transfectants and neo controls cells, with the activated caspase-3 being detected in caspase-1 transfectants only. While this activation of caspase-3 was paralleled by an elevated caspase-9 expression at 9 h post-irradiation, there was no major induction in Apaf-1 or cytochrome c release. CONCLUSION: The present study provides an initial mechanistic insight into the functional involvement of caspase-1 in changing the apoptotic threshold of prostate cancer cells to radiation. These findings will enhance the understanding of the molecular basis of prostate tumor radioresistance and may have significant clinical relevance in improving the therapeutic index of radiotherapy in prostate cancer patients.
BACKGROUND: The significance of caspase-1 in prostate cancer has recently been documented (Cancer Res 61: 1227-1232, 2001). In this study, we investigated the role of caspase-1 in radiation-induced apoptosis in order to identify the significance of this apoptotic initiator in radiation resistance. MATERIALS AND METHODS:Caspase-1 was over-expressed in DU-145 prostate cancer cells (which have weak endogenous expression of caspase-1), via transfection-mediated gene transfer. Stable transfectants were cloned and expression of caspase-1 was established at the mRNA and protein levels by RT-PCR and Western blot, respectively. Caspase-1 overexpressing clones were characterized for their apoptotic response to ionizing irradiation (0-9 Gy) on the basis of cell viability and Hoechst staining assays and profiling of expression of key apoptosis regulators, such as caspase -3 and -9. RESULTS:Caspase-1 transfectants exhibited a greater sensitivity in response to ionizing radiation than the neomycin control transfectants, as demonstrated by a dramatic loss in cell viability, that temporally correlated with apoptosis induction. Furthermore, caspase-1 overexpression resulted in a significant decrease in clonogenic survival following treatment with ionizing radiation, while the caspase-1 inhibitor, Z-YVAD.fmk, suppressed apoptosis induction in caspase-1 transfectants (p<0.008). The apoptotic effect was associated with increased expression of the pro-enzyme form of caspase-3 in both the caspase-1 transfectants and neo controls cells, with the activated caspase-3 being detected in caspase-1 transfectants only. While this activation of caspase-3 was paralleled by an elevated caspase-9 expression at 9 h post-irradiation, there was no major induction in Apaf-1 or cytochrome c release. CONCLUSION: The present study provides an initial mechanistic insight into the functional involvement of caspase-1 in changing the apoptotic threshold of prostate cancer cells to radiation. These findings will enhance the understanding of the molecular basis of prostate tumor radioresistance and may have significant clinical relevance in improving the therapeutic index of radiotherapy in prostate cancerpatients.
Authors: Jill M Miller; Joyce K Thompson; Maximilian B MacPherson; Stacie L Beuschel; Catherine M Westbom; Mutlay Sayan; Arti Shukla Journal: Cancer Prev Res (Phila) Date: 2014-01-15