Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species

Abstract

Ceramide is a sphingolipid that is generated in the signaling of inflammatory cytokines such as tumor necrosis factor (TNF), which exerts many functional roles depending on the cell type where it is produced. Since TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in generation of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C 2 -ceramide) to mitochondria led to an increase of fluorescence of dihydrorhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 M and maximal at 1-5 M C 2 , decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosine at the same concentration range, whereas the immediate precursor of C 2 , C 2 -dihydroceramide increased hydrogen peroxide at 1-5 M. Sphingosine generated hydrogen peroxide at concentrations 10 M, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C 2 was not triggered by mitochondrial permeability transition as C 2 did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C 2 ; however, interruption of electron flow at complex III by antimycin A potentiated the inductive effect of C 2 . Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of hydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted mitochondria. Mitochondria isolated from TNF-treated cells showed an increase (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a target of ceramide produced in the signaling of TNF whose effect on mitochondrial electron transport chain leads to overproduction of hydrogen peroxide and consequently this phenomena may account for the generation of reactive oxygen species during TNF cytotoxicity.

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

“…As DNR plus NOE treatment favors ceramide accumulation, it is conceivable that this lipid targets mitochondria eliciting a burst of ROS that precede mitochondrial depolarization and caspase activation. Indeed, the accumulation of ceramide in mitochondria has been reported to mediate tumor necrosis factor (TNF), Fas or radiation-induced cell death (Garcia-Ruiz et al, 1997;Matsko et al, 2001). In addition, the mitochondrial generation of ceramide upon forced expression of neutral sphingomyelinase in mitochondria leads to apoptotic cell death in MCF7 breast cancer cells (Birbes et al, 2001).…”

“…Combination of DNR plus NOE or AC siRNA targets mitochondria We next determined whether DNR plus NOE induced apoptosis via mitochondria (Garcia-Ruiz et al, 1997;Quillet-Mary et al, 1997;Ghafourifar et al, 1999). The morphological appearance of mitochondria from control HepG2 cells was oval shaped, in association with the endoplasmic reticulum and with organized cristae, and DNR treatment induced minor changes in these organelles (Figure 6a).…”

“…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).…”

Implication of mitochondria-derived reactive oxygen species, cytochrome C and caspase-3 in N-(4-Hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells

“…In the presence of respiratory substrates and oxygen, components`upstream' or downstream' of an inhibitor are reduced or oxidized, respectively and O 2 7 is produced by autoxidation of the reduced components of the respiratory chain (Richter and Schweizer, 1997). Thus, by using various inhibitors, the target sites of ROS generation in mitochondria have been characterized in several systems (Dawson et al, 1993;Garcia-Ruiz et al, 1997;Schulze-Ostho et al, 1992;Taylor et al, 1995;Turrens et al, 1985).…”

Ceramide: A common pathway for atherosclerosis?

“…Some studies showed that ROS participate in TNF-α-mediated ceramide formation, suggesting that the ROS-sensitive pathway may be located upstream of ceramide formation [29]. While other studies demonstrated that ceramide plays a role as an intermediate in TNF-α-mediated ROS production [30].…”