Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis.
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.
Quillet-Mary
A
A
CJF INSERM 9503, Centre Claudius Régaud, Toulouse Cedex, France.
Jaffrézou
J P
JP
Mansat
V
V
Bordier
C
C
Naval
J
J
Laurent
G
G
D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology; D016328:NF-kappa B / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D002518:Ceramides; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D018808:Transcription Factor AP-1; D006861:Hydrogen Peroxide; D002118:Calcium