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

Ionizing radiation caused induction NFkappaB activity and Bcl-2 protein expression in the radioresistant p53 null human prostate cancer cell line, PC-3. Exposure of PC-3 cells to Ads-IkappaB super-repressor inhibited radiation-induced Bcl-2 expression indicating that radiation-induced NFkappaB activity is required for the induction of 1362 protein. PAR-4, a novel pro-apoptotic protein is a potent down-modulator of NFkappaB activity and bcl-2 protein expression. This study was undertaken to investigate the impact of PAR-4 expression on radiation-induced NFkappaB 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 box 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: box ratio in PC-3/PAR-4 cells and this led enhanced radiosensitivity. PAR-4 was found to inhibit the radiation-induced NFkappaB activity and NFkappaB transcriptional activity is essential for bcl-2 upregulation. In PC-3/Vector cells, radiation caused an increase in NFkB activity leading to upregulation of bcl-2 protein. However, in PC-3/PAR-4 cells, the radiation-induced NFkappaB 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 IkappaBalpha, which led to the loss of NFkappaB 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 NFkappaB activity and bcl-2 function respectively.