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

Mol Cell Biochem. 2003 Mar;245(1-2):127-39. doi: 10.1023/a:1022807207948.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Cytochromes c / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Free Radical Scavengers / pharmacology
  • Glutathione / metabolism*
  • Humans
  • Intracellular Membranes / drug effects
  • Leukemia P388 / metabolism*
  • Leukemia P388 / pathology*
  • Mice
  • Mitochondria / drug effects
  • Nigella sativa / chemistry
  • Oleanolic Acid / analogs & derivatives*
  • Oleanolic Acid / pharmacology*
  • Oxidative Stress / drug effects
  • Plant Extracts / chemistry
  • Reactive Oxygen Species / metabolism*
  • Saponins / pharmacology*
  • Tumor Cells, Cultured

Substances

  • Enzyme Inhibitors
  • Free Radical Scavengers
  • Plant Extracts
  • Reactive Oxygen Species
  • Saponins
  • alpha-hederin
  • Oleanolic Acid
  • Cytochromes c
  • Glutathione