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 (SF(2)=0.84; D(0)=437cGy), the PC-3/vector cells (SF(2)=0.4; D(0)=235cGy) were significantly sensitive to ionizing radiation (p<0.001). Exposure of DX at 5 microM 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.
Inayat
Mohammed S
MS
Department of Biological Sciences, University of Westminster, London, UK.
Chendil
Damodaran
D
Mohiuddin
Mohammed
M
Elford
Howard L
HL
Gallicchio
Vincent S
VS
Ahmed
Mansoor M
MM
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000528:radiation effects; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D003131:Combined Modality Therapy; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D005822:Genetic Vectors; D006801:Humans; D006877:Hydroxamic Acids / Q000627:therapeutic use*; D008297:Male; D008938:Mitosis / Q000187:drug effects / Q000528:radiation effects; D016328:NF-kappa B / Q000187:drug effects / Q000528:radiation effects; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000532:radiotherapy*; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000528:radiation effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000528:radiation effects; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D014162:Transfection; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494826:BAX protein, human; D006877:Hydroxamic Acids; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C035419:3,4-dihydroxybenzohydroxamic acid