Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis*

Abstract

The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 μm 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 ρ° cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-κB 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.