Par-4, A Pro-Apoptotic Gene, Inhibits Radiation-Induced NFκB Activity and Bcl-2 Expression Leading to Induction of Radiosensitivity in Human Prostate Cancer Cells PC-3

Abstract

Ionizing radiation caused induction NFκB activity and Bcl-2 protein expression in the radioresistant p53 null human prostate cancer cell line, PC-3. Exposure of PC-3 cells to Ad5-IkB super-repressor inhibited radiation-induced Bcl-2 expression indicating that radiation-induced NFκB activity is required for the induction of Bcl-2 protein. PAR-4, a novel pro-apoptotic protein is a potent down-modulator of NFκB activity and bcl-2 protein expression. This study was undertaken to investigate the impact of PAR-4 expression on radiation-induced NFκB activity and Bcl-2 expression and its resultant radiation response in PC-3 cells. Western blot analysis indicated that enforced expression of PAR-4 in PC-3 cells down regulated radiation-induced bcl-2 protein, whereas in vector transfected cells radiation caused an induction of bcl-2 protein. In both transfectant cell lines, the bax protein levels remained unaltered after radiation. When compared to PC-3/Vector cells, PC-3/PAR-4 cells showed significant sensitivity to radiation-induced clonogenic inhibition and apoptosis. Thus, the down-regulation of bcl-2 protein by ectopic PAR-4 expression altered bcl-2: bax ratio in PC-3/PAR-4 cells and this led enhanced radiosensitivity. PAR-4 was found to inhibit the radiation-induced NFκB activity and NFκB transcriptional activity is essential for bcl-2 upregulation. In PC-3/Vector cells, radiation caused an increase in NFκB activity leading to upregulation of bcl-2 protein. However, in PC-3/PAR-4 cells, the radiation-induced NFκB activity was inhibited resulting in the transrepression of bcl-2 promoter and down-modulation of bcl-2 protein. In addition, PAR-4 was found to directly inhibit the phosphorylation and degradation of IκBa, which led to the loss of NFkB activity causing repression of endogenous and radiation-induced Bcl-2 protein. Together, these mechanisms suggest that PAR-4 is functionally required to cause radiation-induced apoptosis by abrogating the survival and anti-apoptotic effects of NFκB activity and bcl-2 function respectively.