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 M C6-ceramide induced significant H 2 O 2 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 H 2 O 2 production was observed in C6-ceramide-treated U937 °c ells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-B and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H 2 O 2 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). H 2 O 2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramideinduced 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.

Influence of Bcl-2 overexpression on the ceramide pathway in daunorubicin-induced apoptosis of leukemic cells

“…We have previously demonstrated that DNR induces DNA fragmentation in the HL60 myeloid cell line (Quillet-Mary et al, 1996). As expected, control HL60/ Neo cells behaved as the parental cell line, with 82.6+9.3% DNA fragmentation after 6 h exposure to 1 mM DNR (Figure 3b, left).…”

“…The supernatant (detergent soluble low molecular weight DNA) and pellet (intact chromatin DNA or large chromatin fragments) radioactivity was determined by liquid scintillation counting. Results were expressed as the percentage of fragmented DNA, in which the spontaneous DNA fragmentation of untreated cells had been subtracted, as previously described (Quillet-Mary et al, 1996).…”

“…or ionizing radiations, hydrogen peroxyde) (Haimovitz-Friedman et al, 1994;Verheij et al, 1996). We recently demonstrated that daunorubicin (DNR), an anthracyclin widely used in the treatment of hematologic malignancies, triggers apoptosis in U937 and HL60 cells (Quillet-Mary et al, 1996). Like TNF-a, DNR also induces activation of a neutral sphingomyelinase (SMase) that hydrolyses SM to produce ceramide .…”

Susceptibility of HIV-1-TAT transfected cells to undergo apoptosis. Biochemical mechanisms

“…Although most of the apoptotic stimulus known so far lead ®rst to the Dc m dissipation followed by a hyperproduction of ROS , it has been shown that in other apoptotic systems, ROS production precedes Dc m (France-Lanord et al, 1997; Macho et al, 1998;Quillet-Mary et al, 1997). To study the role of the mitochondria membrane potential and ROS generation in both K562 and K562-Tat cells, we incubated the cells under low serum conditions (0.1% FCS) for di erent times.…”

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo

“…Ceramide has been described to activate various cell death mechanisms including mitochondrial recruitment (Kolesnick, 2002). For instance, through interaction with mitochondria, ceramide elicits a burst of reactive oxygen species (ROS) and release of mitochondrial apoptotic proteins, thus engaging the mitochondrial apoptosome (Garcia-Ruiz et al, 1997;Quillet-Mary et al, 1997;Ghafourifar et al, 1999). Other sphingolipids with death-promoting activity include gangliosides, such as ganglioside GD3, which has been shown to mediate apoptosis by a dual mechanism involving interaction with mitochondria and suppression of survival pathways (Scorrano et al, 1999;Garcia-Ruiz et al, 2000;Rippo et al, 2000;Colell et al, 2001;Paris et al, 2002a).…”