RESUMO
The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis in leukemia cells by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We report here that silencing and functional inhibition of Rac1 block Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. Using confocal, electron microscopy and coimmunoprecipitation, as well as glutathione S-transferase-fusion proteins, we provide evidence for a colocalization and physical interaction between the 2 proteins. This interaction is blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia cells and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anticancer drug design.
Assuntos
Apoptose , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Superóxidos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Inativação Gênica , Células HeLa , Humanos , Células Jurkat , Camundongos , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neuropeptídeos/genética , Fragmentos de Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
The critical role of mitochondria in cell fate decisions has been well documented over the years. These observations have highlighted the way mitochondrial physiology controls cell survival and growth in the normal settings, the critical role of mitochondrial outer membrane permeabilization and altered mitoenergetics in cell death execution, and most importantly the association of altered mitochondrial metabolism with pathological states, in particular cancer. Reprogramming of cell metabolism, an invariable finding in cancer cells, is tightly linked to mitoenergetics as is evidenced by up-regulation of nutrient uptake and a pro-oxidant tilt in the intracellular milieu. The latter has also been demonstrated in oncogene-induced carcinogenesis models, notably as a functional outcome of Bcl-2 overexpression. Interestingly, even in that model, mitochondria appear to be the target as well. Thus the association of metabolic re-circuiting and altered mitoenergetics with the process of transformation has resulted in a paradigm shift in the way cancer development and progression is viewed today, which has tremendous implications for the development of novel and strategic therapeutic modalities.