Abstract
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 μM). The glutathione precursor,N-acetylcysteine also partially protected T47D/H3 cells from the lethal effect of doxorubicin, whereasl-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.
Footnotes
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This work was supported by grants from INSERM, UniversitéP. Sabatier, and from the NCI of Canada (to M.-E.M.). V.G. is a recipient of the Association pour la Recherche contre le Cancer and the Ligue Régionale Contre le Cancer fellowships.
- Abbreviations:
- ROS
- reactive oxygen species
- GSH
- glutathione
- BSO
- l-buthionine-(S,R)-sulfoximine
- TNF
- tumor necrosis factor
- GPx
- glutathione peroxidase
- SMase
- sphingomyelinase
- SM
- sphingomyelin
- NAC
- N-acetylcysteine
- DAPI
- 4′,6-diamidino-2-phenylindole
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- DCFH-DA
- 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- Received April 30, 2001.
- Accepted June 11, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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