Glutathione S-transferases as antioxidant enzymes: small cell lung cancer (H69) cells transfected with hGSTA1 resist doxorubicin-induced apoptosis.

It has been suggested that the alpha-class glutathione S-transferases (GSTs) protect various cell types from oxidative stress and lipid peroxidation (LPO). In order to examine the protective role of alpha-class GST isozyme hGSTA1-1 against doxorubicin (DOX)-induced lipid peroxidation, cytotoxicity, and apoptosis, human small cell lung cancer (SCLC) H69 cells were stably transfected with hGSTA1. Immunological and biochemical characterization of hGSTA1-transfected cells revealed the expression of functionally active hGSTA1-1 localized near the cellular plasma membranes. hGSTA1-transfected cells acquired significantly increased resistance to the DOX-induced cytotoxicity by suppressing lipid peroxidation levels in these cells. Overexpression of hGSTA1-1 in cells inhibited DOX-mediated depletion of GSH and higher GSH levels were found in DOX-treated hGSTA1-transfected cells as compared with empty vector-transfected controls. hGSTA1-1 overexpression also provided protection to cells from DOX-induced apoptosis by inhibiting phosphorylation of c-Jun-N-terminal kinases (JNK), caspase-3 activation, and by preserving the levels of anti-apoptotic protein Bcl-2. These results are consistent with the idea that the alpha-class GSTs provide protection against oxidative stress by attenuating lipid peroxidation and these enzymes can modulate signaling for apoptosis.

Regulation of CD95 (Fas) expression and Fas-mediated apoptotic signaling in HLE B-3 cells by 4-hydroxynonenal.

The Fas (apo/CD95) receptor which belongs to the TNF-alpha family is a transmembrane protein involved in the signaling for apoptosis through the extrinsic pathway. During this study, we have examined a correlation between intracellular levels of 4-HNE and expression of Fas in human lens epithelial (HLE B-3) cells. Our results show that in HLE B-3 cells, Fas is induced by 4-HNE in a concentration- and time-dependent manner, and it is accompanied by the activation of JNK, caspase 3, and the onset of apoptosis. Fas induction and activation of JNK are also observed in various tissues of mGsta4 null mice which have elevated levels of 4-HNE. Conversely, when 4-HNE is depleted in HLE B-3 cells by a transient transfection with hGSTA4, Fas expression is suppressed. However, upon the cessation of hGSTA4 expression in these transiently transfected cells, Fas and 4-HNE return to their basal levels. Fas-deficient transformed HLE B-3 cells stably transfected with hGSTA4 show remarkable resistance to apoptosis. Also, the wild-type HLE B-3 cells in which Fas is partially depleted by siRNA acquire resistance to 4-HNE-induced apoptosis, suggesting an at least partial role of Fas in 4-HNE-induced apoptosis in HLE B-3 cells. We also demonstrate that during 4-HNE-induced apoptosis of HLE B-3 cells, Daxx is induced and it binds to Fas. Together, these results show an important role of 4-HNE in regulation of the expression and functions of Fas.

Depletion of 4-hydroxynonenal in hGSTA4-transfected HLE B-3 cells results in profound changes in gene expression.

Previously, we have shown that overexpression of 4-hydroxy-2-nonenal (HNE)-detoxifying enzyme glutathione S-transferase A4-4 (hGSTA4-4) in human lens epithelial cells (HLE B-3) leads to pro-carcinogenic phenotypic transformation of these cells [R. Sharma, et al. Eur. J. Biochem. 271 (2004) 1960-1701]. We now demonstrate that hGSTA4-transfection also causes a profound change in the expression of genes involved in cell adhesion, cell cycle control, proliferation, cell growth, and apoptosis, which is consistent with phenotypic changes of the transformed cells. The expression of p53, p21, p16, fibronectin 1, laminin gamma1, connexin 43, Fas, integrin alpha6, TGFalpha, and c-jun was down-regulated, while the expression of protein kinase C beta II (PKCbetaII), c-myc, cyclin-dependent kinase 2 (CDK2), and TGFbeta was up-regulated in transfected cells. These results demonstrate that HNE serves as a crucial signaling molecule and, by modulating the expression of genes, can influence cellular functions.