Intracellular glutathione depletion and reactive oxygen species generation are important in alpha-hederin-induced apoptosis of P388 cells.
alpha-Hederin, a pentacyclic triterpene saponin isolated from the seeds of Nigella sativa, was recently reported to have potent in vivo antitumor activity against LL/2 (Lewis Lung carcinoma) in BDF1 mice. In this study we observed that alpha-hederin caused a dose- and time-dependent increase in apoptosis of murine leukemia P388 cells. In order to evaluate the possible mechanisms for apoptosis, the effects of alpha-hederin on intracellular thiol concentration, including reduced glutathione (GSH), and protein thiols, and the effects of pretreatment with N-acetlycysteine (NAC), a precursor of intracellular GSH synthesis, or buthionine sulfoxime (BSO), a specific inhibitor of intracellular GSH synthesis, on alpha-hederin-induced apoptosis were investigated. It was found that alpha-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated alpha-hederin-induced apoptosis, while BSO augmented alpha-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after alpha-hederin treatment caused disruption of mitochondrial membrane potential (deltapsi(m)) and subsequently increased the production of reactive oxygen species (ROS) in P388 cells at an early time point. Bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, and cyclosporin (CsA) attenuated the alpha-hederin-induced loss of deltapsi(m), and ROS production. Thus, oxidative stress after alpha-hederin treatment is an important event in alpha-hederin-induced apoptosis. As observed in this study, permeability transition of mitochondrial membrane occurs after depletion of GSH and precedes a state of reactive oxygen species (ROS) generation. Further, we observed that alpha-hederin caused the release of cytochrome c from the mitochondria to cytosol, leading to caspase-3 activation. Our findings thus demonstrate that changes in intracellular thiols and redox status leading to perturbance of mitochondrial functions are important components in the mechanism of alpha-hederin-induced cell death.
Swamy
S Muthu Kumara
SM
Department of Pharmacology, Faculty of Medicine, National University of Singapore, Singapore.
Huat
Benny Tan Kwong
BT
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects; D007941:Leukemia P388 / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects; D031881:Nigella sativa / Q000737:chemistry; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D010936:Plant Extracts / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012503:Saponins / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D012503:Saponins; C010177:beta-hederin; D009828:Oleanolic Acid; D045304:Cytochromes c; D005978:Glutathione