Rlip depletion prevents spontaneous neoplasia in TP53 null mice.

TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53-/-) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53-/- mice, Rlip-/- mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53-/- mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53-/- mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53-/- mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/- mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.

Regulatory roles of glutathione-S-transferases and 4-hydroxynonenal in stress-mediated signaling and toxicity.

Glutathione-S-Transferases (GSTs) have primarily been thought to be xenobiotic metabolizing enzymes that protect cells from toxic drugs and environmental electrophiles. However, in last three decades, these enzymes have emerged as the regulators of oxidative stress-induced signaling and toxicity. 4-Hydroxy-trans 2-nonenal (HNE) an end-product of lipid peroxidation, has been shown to be a major determinant of oxidative stress-induced signaling and toxicity. HNE is involved in signaling pathways, including apoptosis, proliferation, modulation of gene expression, activation of transcription factors/repressors, cell cycle arrest, and differentiation. In this article, available evidence for a major role of GSTs in the regulation of HNE-mediated cell signaling processes through modulation of the intracellular levels of HNE is discussed.

Lens specific RLIP76 transgenic mice show a phenotype similar to microphthalmia.

RALBP1/RLIP76 is a ubiquitously expressed protein, involved in promotion and regulation of functions initiated by Ral and R-Ras small GTPases. Presence of multiple domains in its structure enables RLIP76 to be involved in a number of physiological processes such as endocytosis, exocytosis, mitochondrial fission, actin cytoskeleton remodeling, and transport of exogenous and endogenous toxicants. Previously, we have established that RLIP76 provides protection to ocular tissues against oxidative stress by transporting the glutathione-conjugates of the toxic, electrophilic products of lipid peroxidation generated during oxidative stress. Therefore, we developed lens specific RLIP76 transgenic mice (lensRLIP76 Tg) to elucidate the role of RLIP76 in protection against oxidative stress, but these transgenic mice showed impaired lens development and a phenotype with small eyes similar to that observed in microphthalmia. These findings prompted us to investigate the mechanisms via which RLIP76 affects lens and eye development. In the present study, we report engineering of lensRLIP76 Tg mice, characterization of the associated phenotype, and the possible molecular mechanisms that lead to the impaired development of eye and lens in these mice. The results of microarray array analysis indicate that the genes involved in pathways for G-Protein signaling, actin cytoskeleton reorganization, endocytosis, and apoptosis are affected in these transgenic mice. The expression of transcription factors, Pax6, Hsf1, and Hsf4b known to be involved in lens development is down regulated in the lens of these Tg mice. However, the expression of heat shock proteins (Hsps), the downstream targets of Hsfs, is differentially affected in the lens showing down regulation of Hsp27, Hsp40, up regulation of Hsp60, and no effect on Hsp70 and Hsp90 expression. The disruption in the organization of actin cytoskeleton of these Tg mice was associated with the inhibition of the activation of Cdc42 and down regulation of cofilin phosphorylation. These mice may provide useful animal model for elucidating the mechanisms of lens development, and etiology of microphthalmia.

Gsta4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity.

The alpha class glutathione s-transferase (GST) isozyme GSTA4-4 (EC2.5.1.18) exhibits high catalytic efficiency to-wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4-4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4-4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, activation of stress-related kinases in 4-HNE(Ac)3 treated Gsta4 null MEF were inversely correlated with the catalytic efficiency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4-4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE.

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.

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.

Role of SMC1 in overcoming drug resistance in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the hardest subtypes of breast cancer to treat due to the heterogeneity of the disease and absence of well-defined molecular targets. Emerging evidence has shown the role of cohesin in the formation and progression of various cancers including colon and lung cancer but the role of cohesin in breast cancer remains elusive. Our data showed that structural maintenance of chromosome 1 (SMC1), a subunit of the cohesin protein complex, is differentially overexpressed both at RNA and protein level in a panel of TNBC cell lines as compared to normal epithelial or luminal breast cancer cells, suggesting that the amplified product of this normal gene may play role in tumorigenesis in TNBC. In addition, our results show that induced overexpression of SMC1 through transient transfection enhanced cell migration and anchorage independent growth while its suppression with targeted small interfering RNA (siRNA) reduced the migration ability of TNBC cells. Increased expression of SMC1 also lead to increase in the mesenchymal marker vimentin and decrease in the normal epithelial marker, E-cadherin. Immunocytochemical studies along with flow cytometry and cell fractionation showed the localization of SMC1 in the nucleus, cytoplasm and also in the plasma membrane. The knockdown of SMC1 by siRNA sensitized the TNBC cells towards a PARP inhibitor (ABT-888) and IC50 was approximately three fold less than ABT-888 alone. The cytotoxic effect of combination of SMC1 suppression and ABT-888 was also confirmed by the colony propagation assay. Taken together, these studies report for the first time that SMC1 is overexpressed in TNBC cells where it plays a role in cell migration and drug sensitivity, and thus provides a potential therapeutic target for this highly invasive breast cancer subtype.

P300 regulates the human RLIP76 promoter activity and gene expression.

A 76-kDa Ral-interacting protein (RLIP76) has been implicated in the pathogenesis of cancer and diabetes. It is often over expressed in human malignant cell lines and human tumor samples and has been associated with metastasis and chemoresistance. RLIP76 homozygous knockout mice exhibit increased insulin sensitivity, hypoglycemia, and hypolipidemia, and resist cancer development. Little is known about the mechanism by which the expression of RLIP76 is regulated. In the present study, we functionally characterized the RLIP76 promoter using deletion mapping and mutational analysis to investigate the regulation of RLIP76 transcription. We have identified the promoter regions important for RLIP76 transcription, including a strong cis-activating element in the proximal promoter containing overlapping consensus cMYB and cETS binding sites. Transcription factor cMYB and the coactivator p300 associated with RLIP76 gene promoter as shown by CHIP assay. Knockdown of p300 in HEK293 cells reduced the activity of the promoter fragment containing wild type cMYB/cETS binding site in comparison to that with deleted or mutated cMYB/cETS binding site. Knockdown of p300 also decreased the RLIP76 expression as indicated by immunoblotting, immunocytochemistry and flow cytometry analysis. Thus, we report for the first time that p300 associates with the RLIP76 promoter via an overlapping cMYB and cETS binding site and regulates RLIP76 promoter activity and its expression.

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-3ß 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.

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.

The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4.

It is well established that 4-hydroxynonenal (HNE) plays a major role in oxidative stress-induced signaling and the toxicity of oxidants. Surprisingly our recent studies also demonstrate that low levels of HNE generated during oxidative stress promote cell survival mechanisms and proliferation. Since the expression and secretion of VEGF is known to be affected by Oxidative stress, during present studies, we have examined dose dependent effect of HNE on VEGF expression and secretion in a model of retinal pigment epithelial (RPE) cells in culture. Results of these studies showed that while inclusion of 0.1 µM HNE in the medium caused increased secretion of VEGF, its secretion and expression was significantly suppressed in the presence of >5 µM HNE in the media. These concentration dependent hormetic effects of HNE on VEGF secretion could be blocked by the over expression of GSTA4-4 indicating that these effects were specifically attributed to HNE and regulated by GSTA4-4. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVEC in matrigel when grown in media from RPE cells treated with 1 µM HNE. The corresponding media from GSTA4-4 over expressing RPE cells had no effect on migration and tube formation of HUVEC in matrigel. These results are consistent with earlier studies showing that at low concentrations, HNE promotes proliferative mechanisms and suggest that HNE induces VEGF secretion from RPE cells that acts in a paracrine fashion to induce angiogenic signaling mechanism in the endothelial cells. These findings may suggest a role of HNE and GSTA4-4 in oxidative stress induced proliferative retinopathies.

Inhibition of mercapturic acid pathway-mediated disposal of 4-hydroxynonenal causes complete and sustained remission of human cancer xenografts in nude mice.

Environmental electrophilic chemical carcinogens are detoxified via mercapturic acid pathway to be excreted as mercapturic acid derivatives. Mercapturic acid pathway is also involved in the metabolism of pro-apoptotic and toxic endogenous electrophiles such as 4-hydroxynonenal (HNE). HNE is a common denominator in stress induced signaling and is a pro-apoptotic second messenger that affects cell cycle signaling in a concentration dependent manner. It can regulate signaling for apoptosis, differentiation, and gene expression by interacting with the transcriptional factors, transcriptional repressors, membrane receptors and other proteins. First two rate limiting enzymes of the mercapturic acid pathway, GSTs that conjugate HNE to glutathione (GSH), and RLIP76 that excludes GHS-HNE conjugate from cells, regulate the intracellular concentration of HNE. Thus GSTs and RLIP76 can have a profound effect on cell cycle signaling. Our studies have established that increased HNE levels in cells promote apoptotic signaling while at decreased levels below its basal constituted levels HNE promote proliferation. A major outcome of these findings is that by blocking the mercapturic acid pathway mediated detoxification of HNE through the inhibition of RLIP76 catalyzed transport of GS-HNE, a complete remission of many human cancer xenografts in mice can be achieved.

Glutathione-conjugate transport by RLIP76 is required for clathrin-dependent endocytosis and chemical carcinogenesis.

Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76(-/-) MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis-RLIP76(-/-) mice will be deficient in chemical neoplasia-was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76(-/-). PMA-induced skin carcinogenesis in RLIP76(+/+) mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76(-/-) mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76(-/-) mice. In RLIP76(-/-) mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis.

Role of 4-hydroxynonenal in epidermal growth factor receptor-mediated signaling in retinal pigment epithelial cells.

Lipid peroxidation (LPO) end-product 4-hydroxynonenal (4-HNE) has been implicated in the mechanism of retinopathy. Lately it has been shown that besides being cytotoxic, 4-HNE plays an important role in oxidative stress-induced signaling. In this study, we have investigated the effect of 4-HNE on epidermal growth factor receptor (EGFR)-mediated signaling, its potential functional consequences, and the regulatory role of the 4-HNE metabolizing isozymes, glutathione S-transferase A4-4 (GSTA4-4) on this signaling in retinal pigment epithelial (RPE) cells. Our results showed that consistent with its known toxicity at relatively higher concentrations, 4-HNE induced cell death in RPE. However, at lower concentrations (as low as 0.1 µM) 4-HNE triggered phosphorylation of EGFR and activation of its down stream signaling components ERK1/2 and Akt that are known to be involved in cell proliferation. These effects of 4-HNE on EGFR could be attenuated by the over expression of GSTA4-4 that reduces intracellular levels of 4-HNE. Our results also indicated that 4-HNE-induced activation of EGFR is a protective mechanism against oxidative stress because EGFR, MEK, and PI3K inhibitors potentiated the toxicity of 4-HNE and also inhibited wound healing in a RPE cell model. These studies suggest that as an initial response to oxidative stress, 4-HNE induces protective mechanism(s) in RPE cells through EGFR-mediated signaling.

Evidence that Gsta4 modifies susceptibility to skin tumor development in mice and humans.

BACKGROUND: The incidence of nonmelanoma skin cancer (NMSC) is equivalent to that of all other cancers combined. Previously, we mapped the 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion susceptibility locus, Psl1, to distal chromosome 9 in crosses of sensitive DBA/2 mice with relatively resistant C57BL/6 mice. Here, we used the mouse two-stage skin carcinogenesis model to identify the gene(s) responsible for the effects of Psl1. METHODS: Interval-specific congenic mouse strains (n ≥ 59 mice per strain) were used to more precisely map the Psl1 locus. Having identified glutathione S-transferase α4 (Gsta4) as a candidate tumor promotion susceptibility gene that mapped within the delimited region, we analyzed Gsta4-deficient mice (n = 62) for susceptibility to skin tumor promotion by TPA. We used quantitative polymerase chain reaction, western blotting, and immunohistochemistry to verify induction of Gsta4 in mouse epidermis following TPA treatment and biochemical assays to associate Gsta4 activity with tumor promotion susceptibility. In addition, single-nucleotide polymorphisms (SNPs) in GSTA4 were analyzed in a case-control study of 414 NMSC patients and 450 control subjects to examine their association with human NMSC. Statistical analyses of tumor studies in mice were one-sided, whereas all other statistical analyses were two-sided. RESULTS: Analyses of congenic mice indicated that at least two loci, Psl1.1 and Psl1.2, map to distal chromosome 9 and confer susceptibility to skin tumor promotion by TPA. Gsta4 maps to Psl1.2 and was highly induced (mRNA and protein) in the epidermis of resistant C57BL/6 mice compared with that of sensitive DBA/2 mice following treatment with TPA. Gsta4 activity levels were also higher in the epidermis of C57BL/6 mice following treatment with TPA. Gsta4-deficient mice (C57BL/6.Gsta4(-/-) mice) were more sensitive to TPA skin tumor promotion (0.8 tumors per mouse vs 0.4 tumors per mouse in wild-type controls; difference = 0.4 tumors per mouse; 95% confidence interval = 0.1 to 0.7, P = .007). Furthermore, inheritance of polymorphisms in GSTA4 was associated with risk of human NMSC. Three SNPs were found to be independent predictors of NMSC risk. Two of these were associated with increased risk of NMSC (odds ratios [ORs] = 1.60 to 3.42), while the third was associated with decreased risk of NMSC (OR = 0.63). In addition, a fourth SNP was associated with decreased risk of basal cell carcinoma only (OR = 0.44). CONCLUSIONS: Gsta4/GSTA4 is a novel susceptibility gene for NMSC that affects risk in both mice and humans.

Mechanisms of 4-hydroxy-2-nonenal induced pro- and anti-apoptotic signaling.

In recent years, 4-hydroxy-2-nonenal (4-HNE) has emerged as an important second messenger in cell cycle signaling. Here, we demonstrate that 4-HNE induces signaling for apoptosis via both the Fas-mediated extrinsic and the p53-mediated intrinsic pathways in HepG2 cells. 4-HNE induces a Fas-mediated DISC independent apoptosis pathway by activating ASK1, JNK, and caspase-3. Parallel treatment of 4-HNE to HepG2 cells also induces apoptosis by the p53 pathway through activation of Bax, p21, JNK, and caspase-3. Exposure of HepG2 cells to 4-HNE leads to the activation of both Fas and Daxx, promotes the export of Daxx from the nucleus to cytoplasm, and facilitates Fas-Daxx binding. Depletion of Daxx by siRNA results in the potentiation of apoptosis, indicating that Fas-Daxx binding in fact is inhibitory to Fas-mediated apoptosis in cells. 4-HNE-induced translocation of Daxx is also accompanied by the activation and nuclear accumulation of HSF1 and up-regulation of heat shock protein Hsp70. All these effects of 4-HNE in cells can be attenuated by ectopic expression of hGSTA4-4, the isozyme of glutathione S-transferase with high activity for 4-HNE. Through immunoprecipitation and liquid chromatography-tandem mass spectrometry, we have demonstrated the covalent binding of 4-HNE to Daxx. We also demonstrate that 4-HNE modification induces phosphorylation of Daxx at Ser668 and Ser671 to facilitate its cytoplasmic export. These results indicate that while 4-HNE exhibits toxicity through several mechanisms, in parallel it evokes signaling for defense mechanisms to self-regulate its toxicity and can simultaneously affect multiple signaling pathways through its interactions with membrane receptors and transcription factors/repressors.

Role of lipid peroxidation in cellular responses to D,L-sulforaphane, a promising cancer chemopreventive agent.

D,L-sulforaphane (SFN), a synthetic analogue of the broccoli-derived l-isomer, is a highly promising cancer chemopreventive agent substantiated by inhibition of chemically induced cancer in rodents and prevention of cancer development and distant site metastasis in transgenic mouse models of cancer. SFN is also known to inhibit growth of human cancer cells in association with cell cycle arrest and reactive oxygen species-dependent apoptosis, but the mechanism of these cellular responses to SFN exposure is not fully understood. Because 4-hydroxynonenal (4-HNE), a product of lipid peroxidation (LPO), the formation of which is regulated by hGSTA1-1, assumes a pivotal role in oxidative stress-induced signal transduction, we investigated its contribution in growth arrest and apoptosis induction by SFN using HL60 and K562 human leukemic cell lines as a model. The SFN-induced formation of 4-HNE was suppressed in hGSTA1-1-overexpressing cells, which also acquired resistance to SFN-induced cytotoxicity, cell cycle arrest, and apoptosis. While resistance to SFN-induced cell cycle arrest by ectopic expression of hGSTA1-1 was associated with changes in levels of G2/M regulatory proteins, resistance to apoptosis correlated with an increased Bcl-xL/Bax ratio, inhibition of nuclear translocation of AIF, and attenuated cytochrome c release in cytosol. The hGSTA1-1-overexpressing cells exhibited enhanced cytoplasmic export of Daxx, nuclear accumulation of transcription factors Nrf2 and HSF1, and upregulation of their respective client proteins, gamma-GCS and HSP70. These findings not only reveal a central role of 4-HNE in cellular responses to SFN but also reaffirm that 4-HNE contributes to oxidative stress-mediated signaling.

Adenovirus-mediated overexpression of glutathione-s-transferase mitigates transplant arteriosclerosis in rabbit carotid allografts.

BACKGROUND: Cardiac transplant arteriosclerosis or cardiac allograft vasculopathy remains the leading cause of graft failure and patient death in heart transplant recipients. Endothelial cell injury is crucial in the development of human atherosclerosis and may play a role in allograft vasculopathy. Glutathione-S-transferase (GST) is known to protect endothelial cells from damage by oxidants and toxins. However, the contribution of human GST A4-4 (hGSTA4-4) to vascular cell injury and consequent transplant arteriosclerosis is unknown. METHODS: A recombinant adenoviral vector containing hGSTA4-4 gene was constructed and delivered to vascular endothelial cells in an in vivo rabbit carotid artery transplant model. Forty-five days after transplantation, allografts were harvested (n=28). Blood flow was measured by ultrasonography. In addition, grafts were analyzed by histology, morphometry, immunostaining, and western blot. RESULTS: The severity of arteriosclerosis in hGSTA4-4 transduced allografts was compared with control by measuring degree of stenosis by neointima. Decrease in blood flow in hGSTA4-4 transduced allografts was significantly less than control allografts, which also developed greater intimal thickening and stenosis than hGSTA4-4 transduced allografts in the proximal and distal regions of the graft. Leukocyte and macrophage infiltration was reduced in hGSTA4-4 transduced carotid arteries. CONCLUSION: Our data indicate that hGSTA4-4 overexpression protects the integrity of vessel wall from oxidative injury, and attenuates transplant arteriosclerosis.

A central role of RLIP76 in regulation of glycemic control.

OBJECTIVE: Pathology associated with oxidative stress frequently results in insulin resistance. Glutathione (GSH) and GSH-linked metabolism is a primary defense against oxidative stress. Electrophilic lipid alkenals, such as 4-hydroxy-t-2-nonenal (4HNE), generated during oxidative stress are metabolized primarily to glutathione electrophile (GS-E) conjugates. Recent studies show that RLIP76 is the primary GS-E conjugate transporter in cells, and a regulator of oxidative-stress response. Because RLIP76(-/-) mice are hypoglycemic, we studied the role of RLIP76 in insulin resistance. RESEARCH DESIGN AND METHODS: Blood glucose, insulin, lipid measurements, and hyperinsulinemic-euglycemic and hyperglycemic clamp experiments were performed in RLIP76(+/+) and RLIP76(-/-) C57B mice, using Institutional Animal Care and Use Committee-approved protocols. Time-resolved three-dimensional confocal fluorescence microscopy was used to study insulin endocytosis. RESULTS: The plasma insulin/glucose ratio was ordered RLIP76(-/-) < RLIP76(+/-) < RLIP76(+/+); administration of purified RLIP76 in proteoliposomes to RLIP76(+/+) animals further increased this ratio. RLIP76 was induced by oxidative or hyperglycemic stress; the concomitant increase in insulin endocytosis was completely abrogated by inhibiting the transport activity of RLIP76. Hydrocortisone could transiently correct hypoglycemia in RLIP76(-/-) animals, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fructose 1,6-bisphosphatase, in RLIP76(-/-). CONCLUSIONS: The GS-E conjugate transport activity of RLIP76 mediates insulin resistance by enhancing the rate of clathrin-dependent endocytosis of insulin. Because RLIP76 is induced by oxidative stress, it could play a role in insulin resistance seen in pathological conditions characterized by increased oxidative stress.

Role of RLIP76 in doxorubicin resistance in lung cancer.

Lung cancer is still a major cause of cancer deaths in spite of considerable efforts in its systemic therapy. Chemotherapy, along with local irradiation is frequently employed but as a palliative therapy. Inherent and acquired resistance in NSCLC and SCLC towards chemotherapeutic agents further makes chemotherapy an incommodious problem. The resistance mechanisms responsible for inherent DOX-resistance of NSCLC and acquired DOX-resistance in SCLC have been the subject of numerous investigations. This review will focus on the recent studies done for understanding the mechanism(s) of inherent and acquired resistance in NSCLC and SCLC and how these can be exploited for the future development of more effective novel biologic agents for the treatment of lung cancer.

RLIP76: a target for kidney cancer therapy.

RLIP76 is a multifunctional transporter protein that serves as an energy-dependent efflux mechanism for endogenously generated toxic metabolites as well as exogenous toxins, including chemotherapy drugs. Our recent studies in cultured cells, syngeneic animal tumor model, and in xenograft model have shown that RLIP76 serves a major cancer-specific antiapoptotic role in a wide variety of histologic types of cancer, including leukemia, melanoma, colon, lung, prostate, and ovarian cancer. Results of present studies in cell culture and xenograft model of Caki-2 cells show that RLIP76 is an important anticancer for kidney cancer because inhibition of RLIP76 function by antibody or its depletion by small interfering RNA or antisense DNA caused marked and sustained regression of established human kidney xenografts of Caki-2 cells in nude mouse.