Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis

J Biol Chem. 1997 Aug 22;272(34):21388-95. doi: 10.1074/jbc.272.34.21388.


The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 microM C6-ceramide induced significant H2O2 production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect. Furthermore, no H2O2 production was observed in C6-ceramide-treated U937 rho degrees cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-kappaB and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H2O2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor). H2O2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramide-induced oxidative stress. Finally, C6-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antioxidants / pharmacology
  • Apoptosis / drug effects*
  • Calcium / metabolism
  • Ceramides / pharmacology*
  • DNA Fragmentation / drug effects
  • Humans
  • Hydrogen Peroxide / metabolism*
  • Membrane Potentials
  • Mitochondria / metabolism*
  • Mitochondria / physiology
  • NF-kappa B / metabolism
  • Reactive Oxygen Species / metabolism
  • Transcription Factor AP-1 / metabolism
  • Tumor Cells, Cultured


  • Antioxidants
  • Ceramides
  • NF-kappa B
  • Reactive Oxygen Species
  • Transcription Factor AP-1
  • Hydrogen Peroxide
  • Calcium