RESUMO
Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme with tumor suppressor activity; however, the molecular mechanisms of MnSOD antitumor effects remain unclear. We hypothesized that MnSOD activity in cancer cells might cause downstream changes in the expression of other tumor suppressor genes. To determine whether maspin, a tumor suppressor gene that inhibits breast cancer cell invasion and metastasis, might be a target of MnSOD, we forced MnSOD expression in several human breast and prostate cancer cell lines by adenovirus-mediated gene transfer and measured maspin mRNA expression. Forced expression of MnSOD caused maspin mRNA to accumulate in a dose-dependent manner in both human breast and prostate cancer cells. Normal p53 was not necessary to mediate the effect of MnSOD because MnSOD up-regulated maspin in cells that harbor wild-type p53 and in cells that harbor mutant p53. Moreover, the effects of MnSOD on maspin were not due to demethylation of the maspin promoter. Analyses of maspin promoter activity, transcriptional run-on, and mRNA stability showed that maspin mRNA stability was the major mechanism for maspin up-regulation by MnSOD. Our findings identify a mechanism underlying MnSOD antitumor effects and provide evidence to support MnSOD as a genetic therapy in the treatment of human breast and prostate cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas/genética , Serpinas/genética , Superóxido Dismutase/fisiologia , Adenoviridae/genética , Sequência de Bases , Northern Blotting , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA , Dactinomicina/farmacologia , Feminino , Genes Supressores de Tumor , Vetores Genéticos/genética , Humanos , Cinética , Masculino , Dados de Sequência Molecular , Mutação/genética , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serpinas/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transcrição Gênica/genética , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
The ras proto-oncogene is frequently mutated in human tumors and functions to constitutively stimulate signal transduction cascades, resulting in unchecked proliferation and malignant transformation. In certain cells, superoxide functions as a signal-transduction messenger, mediating the downstream effects of ras and rac. We demonstrated previously that v-Ha-ras-transfected rat kidney epithelial cells (RECs) overproduced superoxide anion and that this superoxide production was mediated by ras. In the present study, we further demonstrated that v-Ha-ras overexpression transformed immortal nonmalignant RECs into malignant cancer cells; v-Ha-ras-transfected cells formed clones in soft agar, had high plating efficiency, and formed tumors in nude mice. Our data suggest that superoxide radical plays a role in ras-induced transformation; modulation of intracellular superoxide level by overexpression of manganese-containing superoxide dismutase or copper- and zinc-containing superoxide dismutase inhibited ras-induced transformation, as evidenced by in vitro studies of plating efficiency and by in vivo studies of tumor formation in nude mice. Overexpression of catalase (CAT) alone was found to have little effect on tumor cell growth, but overexpression of glutathione peroxidase 1 (GPx1) completely suppressed tumor cell growth in nude mice. This finding suggests that peroxides removed by GPx1, but not by CAT, are also involved in ras-induced transformation.