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 nec rosis of several cell types, suggesting a critical role for reactive oxygen species (ROS) in cell death. However, how the cellular defense against oxi dative stress is connected with other cell death mediators remains unclear. We selectively investigated the interaction of seleno-glutathione peroxida se-1 (GPx-1), the major enzyme responsible for peroxide detoxification in m ammalian cells, with the cytotoxic response of T47D human breast cancer cel ls to doxorubicin, an anticancer drug known to promote production of ROS an d 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/GP x2 cells were significantly more resistant than T47D/H3 cells to doxorubici n (1 muM). The glutathione precursor, N-acetylcysteine also partially prote cted T47D/H3 cells from the lethal effect of doxorubicin, whereas L-buthion ine-(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, s phingomyelin 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 indu ced by exogenous cell-permeant ceramide. Moreover, the basal activity of ne utral sphingomyelinase was considerably lower in T47D/GPx2. Taken together, these results indicate that GPx-1 can regulate doxorubicin-induced cell de ath signaling at least in part by interfering with the activation of the sp hingomyelin-ceramide pathway.