| It has been reported that MnSOD has a tumor suppressor effect in a wide variety of different cancer cell types and overexpressing MnSOD sensitizes cancer cells to certain anticancer chemotherapy regents, such as BCNU. The purpose of our studies are (1) to explore the mechanism of MnSOD overexpression induced cellular sensitization to BCNU; (2) to determine if increasing the endogenous superoxide production can enhance the antitumor effect of AdMnSOD plus BCNU; (3) to test if nitric oxide application can increase the antitumor effect of the combination of MnSOD overexpression plus BCNU.; BCNU treatment inhibited the glutathione reductase activity but not other major antioxidant enzymes, including MnSOD, CuZnSOD, and catalase, in breast cancer cell lines in a dose and time dependent manner. MnSOD overexpression sensitized cells to the cytotoxicity of BCNU and this sensitization could be reversed by pyruvate. AdMnSOD plus BCNU increased the GSSG percentage in cells and increased the DNA damage.; To test the effect of hydrogen peroxide accumulation on tumor killing, cells were pretreated with AdMnSOD plus BCNU, and then different superoxide-producing modalities (antimycin, TNF-alpha, adriamycin, photofrin(TM)-PDA, and ionizing radiation) were used. Cell viability was measured by the clonogenic assay or trypan blue dye exclusion assay and the production of hydrogen peroxide was detected with the DCFH probe. The results showed that when cells were treated with AdMnSOD plus BCNU and other superoxide radical-producing agents, there were significant increases in the cytotoxicity, the percentage of GSSG, and the prooxidant accumulation. In vivo experiment showed that the combination of AdMnSOD, BCNU, and adriamycin significantly decreased the xenograft volume and prolonged animal tumor-free survival.; To test if nitric oxide mediates the anti-tumor effect of MnSOD, iNOS cDNA was delivered by adenovirus-mediated transduction and different nitric oxide producers (SNP, SIN-1, and DETANONOate) were used. The production of nitric oxide and the cell survival were measured. The results showed that the combination of MnSOD, BCNU and nitric oxide producer significantly increase the cell killing induced by either alone. |
