Nitric-oxide-induced Bax integration into the mitochondrial membrane commits MDA-MB-468 cells to apoptosis: essential role of Akt.

We have previously reported that nitric oxide (NO) induces apoptosis in MDA-MB-468 cells through its action on the mitochondria and the release of cytochrome c. In this study, we investigated the critical events that must occur after which these cells are committed to apoptosis. We used the long-acting NO donor DETA-NONOate, which, at a concentration of 1 mM, releases NO in the range produced by activated macrophages. Depolarization of mitochondrial membrane potential (MMP) occurred at 4 h of DETA-NONOate treatment, which returned to control values and which was followed by another wave of depolarization at 24 h. There was a 2-fold increase of cytochrome c in the cytosol at 6 h, but it was not until 36 h that the level of cytochrome c was increased by 15-fold. Although the initial release of cytochrome c from the mitochondria could be inhibited by cyclosporin A or by bongkrekic acid, the later release continued even in its presence. We observed that the later release of cytochrome c at 36 h was independent of MMP depolarization but was dependent on Bax integration into the mitochondrial membrane, which committed the cells to apoptosis. We also observed a decline in the levels of cytosolic phospho-Akt at 16-24 h of DETA-NONOate treatment. We also conclude that decrease in phospho-Akt is an essential event upstream from Bax integration in MDA-MB-468 cells.