A. Quilletmary et al., IMPLICATION OF MITOCHONDRIAL HYDROGEN-PEROXIDE GENERATION IN CERAMIDE-INDUCED APOPTOSIS, The Journal of biological chemistry, 272(34), 1997, pp. 21388-21395
The key events implicated in ceramide-triggered apoptosis remain unkno
wn. In this study we show that 25 mu M CG-ceramide induced significant
H2O2, production within 60 min, which increased up to 180 min in huma
n myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide h
ad no effect, Furthermore, no H2O2 production was observed in CG-ceram
ide-treated U937 rho degrees cells, which are mitochondrial respiratio
n-deficient. We also present evidence that ceramide-induced activation
of the transcription factors NF-kappa B and AP-I is mediated by mitoc
hondrial derived reactive oxygen species, Both H2O2 production, transc
ription factor activation as well as apoptosis could be inhibited by r
otenone and thenoyltrifluoroacetone (specific mitochondrial complexes
I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidin
e dithiocarbamate. These effects could be potentiated by antimycin A (
specific complex III mitochondrial inhibitor); H2O2 production was als
o inhibitable by ruthenium red, suggesting a role of mitochondrial cal
cium homeostasis alterations in ceramide-induced oxidative stress, Fin
ally, CG-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 respir
atory chain, as an early major mediator in ceramide-induced apoptosis.