Involvement of cytochrome c, Smac/Diablo release from mitochondria and caspase ‐3 and ‐6 activation in fatty acid‐ induced apoptosis in human blood lymphocytes

Abstract

The present study was undertaken to evaluate if the effect of a mixture of fatty acids upon normal human lymphocytes involves cytochrome c and Smac/Diablo release from the mitochondria, activation of caspases-3, -6 and -8 and expression of the anti-apoptotic blc-2 and the pro-apoptotic bax proteins. Peripheral blood lymphocytes from healthy subjects were isolated and treated for 24 h with increasing concentrations (0.2 and 0.4 mM) of the fatty acid mixture. Afterwards, cells were harvested and the cytochrome c and Smac/Diablo release from mitochondrial intermembrane space into cytosol and expression of blc-2 and bax were investigated by Western blotting analysis. Activities of caspases-3, -6 and -8 were determined by using spectrofluorometric assays. Lymphocytes treated with the fatty acid mixture presented increased cytochrome c and Smac/Diablo release from mitochondria as compared with control lymphocytes without treatment. Caspase −3 and −6 activities were increased by 146% and 22%, respectively, by the treatment with 0.4 mM concentration of the fatty acid mixture for 24 h. The expression of bcl-2 protein was significantly reduced in lymphocytes treated with the fatty acid mixture, by 40% at 0.2 mM and by 80% at 0.4 mM fatty acid concentration, whereas bax protein was not affected by the treatment. In conclusion, fatty acids induce apoptosis in human lymphocytes through cytochrome c and Smac/Diablo release from the mitochondria, activation of the caspase cascade and by decrease of anti-apoptotic bcl-2 protein content. Financial Support: FAPESP, CNPq, CAPES