Ceramide in apoptosis signaling: relationship with oxidative stress.

Ceramide is one of the major sphingosine-based lipid second messengers that is generated in response to various extracellular agents. However, while widespread attention has focused on ceramide as a second messenger involved in the induction of apoptosis, important issues with regard to the mechanisms of ceramide formation and mode of action remain to be addressed. Several lines of evidence suggest that ceramide and oxidative stress are intimately related in cell death induction. This review focuses on the putative relationships between oxidative stress and sphingolipid metabolism in the apoptotic process and discusses the potential mechanisms that connect and regulate the two phenomena.

Glutathione peroxidase-1 overexpression prevents ceramide production and partially inhibits apoptosis in doxorubicin-treated human breast carcinoma cells.

Reduced glutathione and N-acetylcysteine can inhibit both apoptosis and necrosis of several cell types, suggesting a critical role for reactive oxygen species (ROS) in cell death. However, how the cellular defense against oxidative stress is connected with other cell death mediators remains unclear. We selectively investigated the interaction of seleno-glutathione peroxidase-1 (GPx-1), the major enzyme responsible for peroxide detoxification in mammalian cells, with the cytotoxic response of T47D human breast cancer cells to doxorubicin, an anticancer drug known to promote production of ROS and apoptotic mediator ceramide. The sensitivity to doxorubicin-mediated cell death was compared in T47D/H3 containing low levels of endogenous GPx and T47D/GPx2 transfectant cells, which overexpress GPx-1. We show that T47D/GPx2 cells were significantly more resistant than T47D/H3 cells to doxorubicin (1 microM). The glutathione precursor, N-acetylcysteine also partially protected T47D/H3 cells from the lethal effect of doxorubicin, whereas L-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione biosynthesis, sensitized both GPx-1--deficient and -proficient cells. Interestingly, in addition to a decrease in ROS production, the activation of neutral sphingomyelinase, sphingomyelin hydrolysis, and ceramide generation in response to doxorubicin was impaired in T47D/GPx2 cells compared with control cells. In contrast, GPx overexpression did not protect breast cancer cells from cell death induced by exogenous cell-permeant ceramide. Moreover, the basal activity of neutral sphingomyelinase was considerably lower in T47D/GPx2. Taken together, these results indicate that GPx-1 can regulate doxorubicin-induced cell death signaling at least in part by interfering with the activation of the sphingomyelin-ceramide pathway.

Lysosomal sphingomyelinase is not solicited for apoptosis signaling.

Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells.

Effect of nicotine and cotinine on the susceptibility to in vitro oxidation of LDL in healthy non smokers and smokers.

Cigarette smoke of which the major component is nicotine plays an important role in the development of cardiovascular diseases. To study the effect of in vitro incubation of LDL with nicotine and its metabolite, cotinine on a copper-induced peroxidation, we monitored the formation of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances production. The LDL studied were taken from six non-smokers (aged 41.5 years) and six smokers who consumed at least ten cigarettes per day (40.7 years). LDL oxidation with CuSO4 showed that cigarette smoking promotes LDL susceptibility to peroxidative modification. During the peroxidation of LDL with nicotine (O to 5 mmol/1) and CuSO4 (5 micromol/l), the formation of hydroperoxides decreased when nicotine concentrations increased and the production of TBARS increased in a concomitant manner. The results showed that the presence of nicotine destabilized the production of hydroperoxides in LDL and increased the formation of secondary oxidation products. On the other hand, cotinine had no effect on LDL oxidative susceptibility in smokers and non-smokers.

Effect of aluminium ions on liposomal membranes as detected by Laurdan fluorescence.

We report here an investigation of the influence of aluminium on iron-induced peroxidation in brain model membranes. Laurdan fluorescence emission spectra and generalised polarisation measurements have been used to investigate how ferrous and aluminium ions can affect the phase components of phospholipid membranes. An increase in the generalised polarisation of oxidised liposomes with respect to controls has been observed, which reveals the presence of a less polar environment surrounding the probe that changes the properties of the bilayer. Aluminium has been shown to facilitate iron-mediated oxidation as detected from emission fluorescence spectra. However, no quantitative influence has been calculated relative to general polarisation and derived phase state determinations. The structural influence of aluminium on membranes may therefore be less significantly marked than initially expected.