Rajendra Sharma

Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death.

Within the last two decades, 4-hydroxynonenal has emerged as an important second messenger involved in the regulation of various cellular processes. Our recent studies suggest that HNE can induce apoptosis in various cells through the death receptor Fas (CD95)-mediated extrinsic pathway as well as through the p53-dependent intrinsic pathway. Interestingly, through its interaction with the nuclear protein Daxx, HNE can self-limit its apoptotic role by translocating Daxx to cytoplasm where it binds to Fas and inhibits Fas-mediated apoptosis. In this paper, after briefly describing recent studies on various biological activities of HNE, based on its interactions with Fas, Daxx, and p53, we speculate on possible mechanisms through which HNE may affect a multitude of cellular processes and draw a parallel between signaling roles of H(2)O(2) and HNE.

4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (-/-) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.

RLIP76 in Defense of Radiation Poisoning

PURPOSE To determine the role of RLIP76 in providing protection from radiation and chemotherapy. In the present report, we used RLIP76 to refer to both the mouse (Ralbp1) and the human (RLIP76) 76-kDa splice variant proteins (RLIP76) for convenience and to avoid confusion. In other reports, Ralbp1 refers to the mouse enzyme (encoded by the Ralbp1 gene), which is structurally and functionally homologous to RLIP76, the human protein encoded by the human RALBP1 gene. METHODS AND MATERIALS Median lethal dose studies were performed in RLIP76(-/-) and RLIP76(+/+) C57B mice after treatment with a single dose of RLIP76 liposomes 14 h after whole body radiation. The radiosensitivity of the cultured mouse embryonic fibroblasts and the effects of buthionine sulfoximine (BSO), amifostine, c-jun N-terminal kinase (JNK), protein kinase B (Akt), and MAPK/ERK kinase (MEK) were determined by colony-forming assays. Glutathione-linked enzyme activities were measured by spectrophotometric assays, glutathione by dithiobis-2-nitrobenzoic acid (DTNB), lipid hydroperoxides by iodometric titration, and aldehydes and metabolites by thiobarbitauric acid reactive substances and liquid chromatography-mass spectrometry (LCMS). RESULTS RLIP76(-/-) mice were significantly more sensitive to radiation than were the wild-type, and RLIP76 liposomes prolonged survival in a dose-dependent manner in both genotypes. The levels of 4-hydroxynonenal and glutathione-conjugate of 4-hydroxynonenal were significantly increased in RLIP76(-/-) tissues compared with RLIP76(+/+). RLIP76(-/-) mouse embryonic fibroblasts were markedly more radiosensitive than RLIP76(+/+) mouse embryonic fibroblasts, despite increased glutathione levels in the former. RLIP76 augmentation had a remarkably greater protective effect compared with amifostine. The magnitude of effects of RLIP76 loss on radiation sensitivity was greater than those caused by perturbations of JNK, MEK, or Akt, and the effects of RLIP76 loss could not be completely compensated for by modulating the levels of these signaling proteins. CONCLUSION The results of our study have shown that RLIP76 plays a central role in radiation resistance.

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

Background: HNE is an important signaling molecule. Results: HNE induces G2/M cell cycle arrest and phosphorylation of H2A.X. ATR/Chk1-mediated regulation of Cdc25C and activation of p21 is the predominant mechanism of HNE-induced cell cycle arrest. GSTA4-4 overexpression inhibits HNE-induced cell arrest. Conclusion: HNE causes DNA damage and G2/M arrest. Significance: HNE and GSTA4-4 play a role in the maintenance of genomic integrity. 4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.

Physiological and Pharmacological Significance of Glutathione-Conjugate Transport

Transport of the glutathione conjugates (GS-E) of electrophilic compounds generated during biotransformation of drugs and environmental pollutants is central to the mechanisms of defense against oxidative/electrophilic stress. In recent years emphasis has been placed on ATP-binding cassette (ABC) transport proteins in the transport of GS-E and their involvement in the detoxification mechanisms, including drug resistance. Recent studies, however, suggested that the majority of GS-E transport in human and rodent cells is mediated by a non-ABC, multifunctional stress-response protein, RLIP76 or RalBP1 (ral-binding GTPase activating protein 1), which also functions as an effector in the Ral-Ras-Rho signaling pathway. In this review, after briefly describing the major discoveries in the field of glutathione (GSH)-conjugate transport, recent findings are presented on the role of RLIP76 in ATP-dependent transport of GS-E, and the relevance of this transport process to the mechanisms of toxicity of xenobiotics, radiation, and endogenous electrophilic toxicants is described. Furthermore, recent studies suggesting a link between RLIP76 mediated GS-E transport and cell cycle signaling are presented.

Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis

Glutathione transferase isozyme A4 (GSTA4) exhibits high catalytic efficiency to metabolize 4‐hydroxynonenal (4‐HNE), a highly reactive lipid peroxidation product that has been implicated in the pathogenesis of various chronic diseases. We investigated the role of 4‐HNE in the mechanisms of unilateral ureteral obstruction (UUO)‐induced fibrosis and its modulation by GSTA4‐4 in a mouse model. Our data indicate that after UUO, accumulation of 4‐HNE and its adducts were increased in renal tissues, with a concomitant decrease in the expression of GSTA4‐4 in mice. As compared to wild‐type (WT) mice, UUO caused an increased expression of fibroblast markers in the interstitium of GSTA4 KO mice. Additionally, increased autophagy and tubular cell damage were more severe in UUO‐treated GSTA4 KO mice than in WT mice. Furthermore, GSK‐3β phosphorylation and expression of Snail, a regulator of E‐cadherin and Occludin, was found to be significantly higher in UUO‐inflicted GSTA4 KO mice. GSTA4 over‐expression prevented 4‐HNE‐induced autophagy activation, tubular cell damage and Snail nuclear translocation in vitro. The effects of long‐term expression of GSTA4 in restoration of UUO‐induced damage in mice with the GSTA4 inducible transposon system indicated that release of obstruction after 3 days of UUO resulted in the attenuation of interstitial SMAα and collagen I expression. This transposon‐delivered GSTA4 expression also suppressed UUO‐induced loss of tubular cell junction markers and autophagy activation. Together, these results indicate that 4‐HNE significantly contributes to the mechanisms of tubule injury and fibrosis and that these effects can be inhibited by the enhanced expression of GSTA4‐4. Copyright

Role of 4-hydroxynonenal in chemopreventive activities of sulforaphane.

Chemoprevention of cancer via herbal and dietary supplements is a logical approach to combating cancer and currently it is an attractive area of research investigation. Over the years, isothiocyanates, such as sulforaphane (SFN) found in cruciferous vegetables, have been advocated as chemopreventive agents, and their efficacy has been demonstrated in cell lines and animal models. In vivo studies with SFN suggest that in addition to protecting normal healthy cells from environmental carcinogens, it also exhibits cytotoxicity and apoptotic effects against various cancer cell types. Among several mechanisms for the chemopreventive activity of SFN against chemical carcinogenesis, its effect on drug-metabolizing enzymes that cause activation/neutralization of carcinogenic metabolites is well established. Recent studies suggest that SFN exerts its selective cytotoxicity to cancer cells via reactive oxygen species-mediated generation of lipid peroxidation products, particularly 4-hydroxynonenal (HNE). Against the background of the known biochemical effects of SFN on normal and cancer cells, in this article we review the underlying molecular mechanisms responsible for the overall chemopreventive effects of SFN, focusing on the role of HNE in these mechanisms, which may also contribute to its selective cytotoxicity to cancer cells.

Role of alpha class glutathione transferases (GSTs) in chemoprevention: GSTA1 and A4 overexpressing human leukemia (HL60) cells resist sulforaphane and curcumin induced toxicity

Alpha‐class glutathione transferases (α‐GSTs) have been shown to protect cells from the harmful effects of reactive oxygen species (ROS) induced lipid peroxidation (LPO) during oxidative stress caused by various physico‐chemical agents. While GSTA1‐1/A2‐2 isozymes exhibit high activity towards lipid and fatty acid hydroperoxides through their selenium independent glutathione peroxidase (GPx) activity, the GSTA4‐4 isozyme efficiently metabolizes the LPO product 4‐hydroxynonenal (4‐HNE) by conjugating it with glutathione (GSH). Because of the fact that ROS generated by the chemopreventive agents, sulforaphane (SFN) and curcumin (Cur), are implicated in the mechanisms of cancer cell killing, the present studies were designed to investigate the contribution of ROS induced LPO in the cytotoxic effects of these agents and the role of α‐class GSTs in modulating their toxicity. Human erythroleukemic (HL60) cells were stably transfected with the cDNA encoding the hGSTA1‐1 and mGsta4‐4 isozymes. After analysing the expression and activities of the respective GST isozymes, the effects of SFN and Cur on the extent of LPO, cytotoxicity and apoptosis were compared in empty vector (VT), hGSTA1‐1 and mGsta4‐4 expressing HL60 cells. These studies demonstrate that when compared with SFN, Cur was relatively more cytotoxic to HL60 cells. The ectopic expression of hGSTA1‐1 and mGsta4‐4 isozymes provided resistance to SFN and Cur induced cytotoxicity and apoptosis through a significant suppression of LPO in these cells. Overall, the results suggest that the expression of α‐class GSTs in cancer cells can modulate the therapeutic efficacy of chemopreventive agents. Copyright

Mechanisms of Chemopreventive Activity of Sulforaphane

d, l-Sulforaphane (SFN) found in cruciferous vegetables is a highly promising anticancer and chemopreventive agent. SFN has been shown to exhibit cytostatic and cytotoxic activities against a number of cancer cell types in vitro and inhibit chemically induced carcinogenesis in rodent models in vivo. SFN also prevents metastasis in mouse models of different cancer types. Cytostatic and cytotoxic activities of SFN have been attributed to several mechanisms including the reactive oxygen species (ROS)-dependent cell cycle arrest and apoptosis. Recent studies discussed in this chapter strongly suggest that 4-hydroxynonenal (HNE), the most abundant end product of ROS-induced lipid peroxidation of ω-6 fatty acids, is a major contributor to the chemopreventive activity of SFN. The chemopreventive activity of SFN, and perhaps its analogs found in cruciferous plants, may be attributed to HNE-induced selective apoptosis in cancer cells and simultaneous protection of neighboring normal cells from carcinogenic insult through the induction of defense mechanisms such as the activation of Nrf2 and Hsf1.