Intracellular glutathione depletion and reactive oxygen species generation are important in α-hederin-induced apoptosis of P388 cells

Abstract

α-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 α-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 α-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 α-hederin-induced apoptosis were investigated. It was found that α-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated α-hederin-induced apoptosis, while BSO augmented α-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after α-hederin treatment caused disruption of mitochondrial membrane potential (ΔΨm) 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 α-hederin-induced loss of ΔΨm, and ROS production. Thus, oxidative stress after α-hederin treatment is an important event in α-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 α-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 α-hederin-induced cell death.