Dehydrocrotonin and its derivative, dimethylamide-crotonin induce apoptosis with lipid peroxidation and activation of caspases-2,-6 and-9 in human leukemic cells HL60

A variety Of Stimuli can induce cells to undergo apoptosis, with one of the most reproducible inducers being mild oxidative stress following exposure to anticancer agents. Apoptosis involves events mediated by cysteine proteases (caspases) that are classified as initiators (-8, -9 and -12) or executors (-2, -3, -6 and -7). In this Study, we examined the mechanisms of apoptosis induced by dehydrocrotonin (DHC), a diterpene lactone isolated from the Amazonian plant Croton cajucara, and its synthetic derivative. dimethylamide-crotonin (DCR), in human HL60 promyelocytic leukemia cells. Flow cytometric analysis of HL60 cells after treatment for 72 h showed that DCR- and DHC-induced apoptosis, with maximum cell death at a concentration of 250 muM for both compounds. DCR and DHC were effective in triggering the activation of caspases-2, -6 and -9. The level of reduced glutathione, (GSH) decreased, whereas there was an increase in thiobarbituric acid-reactive substance (TBARS) production and in mitochondrial swelling. These effects on mitochondrial swelling, GSH content and lipid peroxidation were abolished by cyclosporine A, an inhibitor of the membrane permeability transition. The cytotoxicity of DHC and DCR was prevented by a high concentration of GSH (15 mM) in the culture medium. These results indicate that DCR and DHC produced apoptosis partly by oxidative stress-induced lipid peroxidation, which triggered the caspase cascade, that lead to apoptotic cell death in HL60 cells. Based on the pattern of caspase activation, on the increase in mitochondrial swelling and on the inhibitory action of cyclosporine A, we conclude that DCR and DHC triggered apoptosis in HL60 cells probably through cytochrome c release and apoptosome, formation. (C) 2004 Elsevier Ireland Ltd. All rights reserved.2034169912313