Didox (A novel ribonucleotide reductase inhibitor) overcomes bcl-2 mediated radiation resistance in prostate cancer cell line PC-3

In this study, we investigated the influence of bcl-2 overexpression on the radiosensitizing potential of Didox (DX; 3,,4-Dihydroxybenzohydroxamic acid), a novel ribonucleotide reductase inhibitor, in p53-null prostate cancer cell line PC-3. The PC-3 cells were transfected with vector alone or ectopically overexpressed with CMV-bcl-2 construct. The effect of radiation (IR) or DX alone and in combination (pre and post IR exposure of DX) on cell survival was determined by colony-forming assay. The impact of these two treatments on the cell cycle was determined by flow cytometry. To further understand the molecular mechanism of DX-mediated radiosensitization, induction of pro-survival and pro-apoptotic factors were determined by Western blot and gel-shift assays respectively. When compared to PC-3/bcl-2 cells (SF2=0.84; D-0=437cGy), the PC-3/vector cells (SF2=0.4; D-0=235cGy) were significantly sensitive to ionizing radiation (P<0.001). Exposure of DX at 5 muM concentration prior or post to radiation in both PC-3/vector and PC-3/bcl-2 transfectants caused an increase in radiation enhancement ratios. A significant reduction in G(2)M phase was observed in cells exposed to DX post IR when compared to cells exposed to IR alone. Exposure to DX after radiation in PC-3/vector significantly abrogated radiation-induced bcl-2 upregulation, with a concomitant induction of bax protein. In PC-3/bcl-2 transfectants, DX exposure after IR caused an induction of bax protein. Gel shift assays indicated that in PC-3/vector cells when exposed to IR caused an induction of NFkappa-B activity however, DX down regulated the NFkappa-B activity. Radiation-induced NFkappa-B activity was abrogated in pre and post DX exposure in combination with IR. These findings indicate that DX mediates a potent radiosensitizing effect in p53 null prostate cancer cells by overcoming radiation induced NFkappa-B activity and bcl-2 expression.