Global Deletion of Glutathione S-Transferase A4 Exacerbates Developmental Nonalcoholic Steatohepatitis.

We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor α (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We used it to probe the importance of lipid peroxidation in progression of NASH beyond simple steatosis. Feeding Gsta4-/-/Ppara-/- double-knockout (dKO) mice liquid diets containing corn oil resulted in a percentage fat-dependent increase in steatosis and necroinflammatory injury (P < 0.05). Increasing fat to 70% from 35% resulted in increases in formation of 4-hydroxynonenal protein adducts accompanied by evidence of stellate cell activation, matrix remodeling, and fibrosis (P < 0.05). Comparison of dKO mice with wild-type (Wt) and single knockout mice revealed additive effects of Gsta4-/- and Ppara-/- silencing on steatosis, 4-hydroxynonenal adduct formation, oxidative stress, serum alanine amino transferase, expression of tumor necrosis factor alpha, Il6, interferon mRNA, and liver pathology (P < 0.05). Induction of Cyp2e1 protein by high-fat diet was suppressed in Gsta4-/- and dKO groups (P < 0.05). The dKO mice had similar levels of markers of stellate cell activation and matrix remodeling as Ppara-/- single KO mice. These data suggest that lipid peroxidation products play a role in progression of liver injury to steatohepatitis in NASH produced by high-fat feeding during development but appear less important in development of fibrosis.

Increased 4-hydroxynonenal protein adducts in male GSTA4-4/PPAR-α double knockout mice enhance injury during early stages of alcoholic liver disease.

To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male 129/SvJ mice (wild-type, WT) and glutathione S-transferase A4-4-null (GSTA4-/-) mice for 40 days. GSTA4-/- mice were crossed with peroxisome proliferator-activated receptor-α-null mice (PPAR-α-/-), and the effects of EtOH in the resulting double knockout (dKO) mice were compared with the other strains. EtOH increased lipid peroxidation in all except WT mice (P < 0.05). Increased steatosis and mRNA expression of the inflammatory markers CXCL2, tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA) were observed in EtOH GSTA4-/- compared with EtOH WT mice (P < 0.05). EtOH PPAR-α-/- mice had increased steatosis, serum alanine aminotransferase (ALT), and hepatic CD3+ T cell populations and elevated mRNA encoding CD14, CXCL2, TNF-α, IL-6, CD138, transforming growth factor-ß, platelet-derived growth factor receptor-ß (PDGFR-ß), matrix metalloproteinase (MMP)-9, MMP-13, α-SMA, and collagen type 1 compared with EtOH WT mice. EtOH-fed dKO mice displayed elevation of periportal hepatic 4-hydroxynonenal adducts and serum antibodies against malondialdehyde adducts compared with EtOH feeding of GSTA4-/-, PPAR-α-/-, and WT mice (P < 0.05). ALT was higher in EtOH dKO mice compared with all other groups (P < 0.001). EtOH-fed dKO mice displayed elevated mRNAs for TNF-α and CD14, histological evidence of fibrosis, and increased PDGFR, MMP-9, and MMP-13 mRNAs compared with the EtOH GSTA4-/- or EtOH PPAR-α-/- genotype (P < 0.05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling, and fibrosis.

Down-regulation of glutatione S-transferase α 4 (hGSTA4) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection.

Patients with severe burns are highly susceptible to bacterial infection. While immunosuppression facilitates infection, the contribution of soft tissues to infection beyond providing a portal for bacterial entry remains unclear. We showed previously that glutathione S-transferase S1 (gstS1), an enzyme with conjugating activity against the lipid peroxidation byproduct 4-hydroxynonenal (4HNE), is important for resistance against wound infection in Drosophila muscle. The importance of the mammalian functional counterpart of GstS1 in the context of wounds and infection has not been investigated. Here we demonstrate that the presence of a burn wound dramatically affects expression of both human (hGSTA4) and mouse (mGsta4) 4HNE scavengers. hGSTA4 is down-regulated significantly within 1 wk of thermal burn injury in the muscle and fat tissues of patients from the large-scale collaborative Inflammation and the Host Response to Injury multicentered study. Similarly, mGsta4, the murine GST with the highest catalytic efficiency for 4HNE, is down-regulated to approximately half of normal levels in mouse muscle immediately postburn. Consequently, 4HNE protein adducts are increased 4- to 5-fold in mouse muscle postburn. Using an open wound infection model, we show that deletion of mGsta4 renders mice more susceptible to infection with the prevalent wound pathogen Pseudomonas aeruginosa, while muscle hGSTA4 expression negatively correlates with burn wound infection episodes per patient. Our data suggest that hGSTA4 down-regulation and the concomitant increase in 4HNE adducts in human muscle are indicative of susceptibility to infection in individuals with severely thermal injuries.

The human hGSTA5 gene encodes an enzymatically active protein.

BACKGROUND: Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional. METHODS: Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity. RESULTS: Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. CONCLUSIONS: hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme. GENERAL SIGNIFICANCE: Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions.

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.

Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4-hydroxynon-2-enal.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an "aging effector" via the formation of 4-HNE-protein adducts, and a resulting change in protein function.

Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells.

Efficient handling of oxidative stress is critical for the survival of organisms. The orphan nuclear receptor pregnane X receptor (PXR) is important in xenobiotic detoxification through its regulation of phase I and phase II drug-metabolizing/detoxifying enzymes and transporters. In this study we unexpectedly found that the expression of an activated human PXR in transgenic female mice resulted in a heightened sensitivity to paraquat, an oxidative xenobiotic toxicant. Heightened paraquat sensitivity was also seen in wild-type mice treated with the mouse PXR agonist pregnenolone-16alpha-carbonitrile. The PXR-induced paraquat sensitivity was associated with decreased activities of superoxide dismutase and catalase, enzymes that scavenge superoxide and hydrogen peroxide, respectively. Paradoxically, the general expression and activity of glutathione S-transferases, a family of phase II enzymes that detoxify electrophilic and cytotoxic substrates, was also induced in the transgenic mice. PXR regulates glutathione S-transferase expression in an isozyme-, tissue-, and sex-specific manner, and this regulation is independent of the nuclear factor-erythroid 2 p45-related factor 2/Kelch-like Ech-associated protein 1 pathway. In cell cultures, expression of activated human PXR sensitizes the cancerous colon and liver cells to the cytotoxic effect of paraquat, which is associated with an increased production of the reactive oxygen species. The current study reveals a novel function of PXR in the mammalian oxidative stress response, and this regulatory pathway may be implicated in carcinogenesis by sensitizing normal and cancerous tissues to oxidative cellular damage.

Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2.

Electrophilic stress caused by lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and/or related compounds may contribute to aging. The major mode of 4-HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2-2, the product of the Caenorhabditis elegans gst-10 gene, has the ability to conjugate 4-HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2-2 correlates highly with lifespan in a series of hypomorphic daf-2 mutants of C. elegans. The overexpression of CeGSTP2-2 in daf-2 is abrogated in daf-16; daf-2 mutants, indicating that expression of the gst-10 gene is modulated by insulin-like growth factor signaling. To determine whether the relationship between CeGSTP2-2 and lifespan is causal, we used RNAi to knock down CeGSTP2-2. Treatment with gst-10-specific dsRNA decreased CeGSTP2-2 protein in wild-type N2 and in daf-2 strains to an approximately equal level. The ability to conjugate 4-HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf-2 over N2 is due largely to the overexpression of CeGSTP2-2. RNAi-mediated knock-down of CeGSTP2-2 led to an increased susceptibility to 4-HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf-2 strains. These results indicate that CeGSTP2-2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf-2 mutants concordantly with other longevity assurance genes, probably via insulin-like growth factor signaling.

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal.

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2-2. CeGSTP2-2 (the product of the gst-10 gene) has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4-HNE). Transgenic C. elegans strains were generated in which the 5'-flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with particularly high catalytic efficiency for 4-HNE. The localization of both transgenes was similar to that of native CeGSTP2-2. The 4-HNE-conjugating activity in worm lysates increased in the order: control

The course of CCl4 induced hepatotoxicity is altered in mGSTA4-4 null (-/-) mice.

Glutathione S-transferases (GSTs) play a key role in cellular detoxification of environmental toxicants through their conjugation to glutathione (GSH). Recent studies have shown that the alpha-class GSTs also provide protection against oxidative stress and lipid peroxidation (LPO). GSTA4-4 is a member of a sub group of the alpha-class GSTs. It has been shown to metabolize 4-hydroxynonenal (4-HNE) with high catalytic efficiency through its conjugation to glutathione (GSH) and has been suggested to be a major component of cellular defense against toxic electrophiles such as 4-HNE generated during LPO. Since the hepatotoxicity of carbon tetrachloride (CCl(4)) has been suggested to be due to the generation of free radicals leading to membrane LPO, the present studies were designed to compare hepatotoxicity of CCl(4) in GSTA4-4 null (-/-) and wild type (+/+) mice. The results show that administration of a single dose of CCl(4) (1 ml/kg i.p.) resulted in time dependent hepatotoxicity in both -/- and +/+ mice; the extent of cellular damage by serum enzymes suggests that progression was more rapid in -/- mice, although injury was similar by 24 h. Histopathologic examination showed similar degrees of centrilobular necrosis by 24 h but much greater surrounding degenerative change, including cellular swelling, disarray, and vacuolization, in the liver of -/- mice. As expected -/- mice did not show any expression of mGSTA4-4; after CCl(4) a compensatory increase in the activities of total GST activity was noted at 24 h. Major alterations in other antioxidant enzymes was not observed. 4-HNE levels in the liver of -/- mice were about four-fold higher than in +/+ mice, suggesting a positive correlation between 4-HNE levels and the altered course of CCl(4) hepatotoxicity. These studies suggest that GSTA4-4 is an important component during the early stages (1-6 h) of cellular defense against oxidative stress and LPO although, it is not effective in protecting against the ultimate degree of overall cell injury.

Transactivation of the epidermal growth factor receptor mediates cholinergic agonist-induced proliferation of H508 human colon cancer cells.

Some human colon cancer cell lines (e.g., H508 cells) express M3 subtype muscarinic receptors that are activated by cholinergic agonists. The objective of the present study was to determine the cellular mechanisms underlying M3 muscarinic receptor-mediated proliferation of H508 human colon cancer cells. In H508 cells, but not in SNU-C4 cells that do not express muscarinic receptors, acetylcholine stimulated calcium-dependent phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and p90 ribosomal S6 kinase and consequent cell proliferation. Atropine or inhibitors of MAPK phosphorylation blocked these effects. Conversely, the actions of epidermal growth factor (EGF) on H508 cells were neither calcium dependent nor mediated by cholinergic mechanisms. Both acetylcholine- and EGF-induced phosphorylation of p44/42 MAPK was abolished in the presence of EGF receptor (EGFR) inhibitors (AG1478 and PD168393). In Chinese hamster ovary cells transfected with the rat M3 muscarinic receptor, which lack EGFR, acetylcholine-induced MAPK phosphorylation was not altered in the presence of EGFR inhibitors. In H508 cells, protein kinase C inhibitors did not alter acetylcholine- or EGF-induced MAPK phosphorylation. Finally, inhibition of EGFR activation abolished acetylcholine-induced H508 cell proliferation. These data indicate that, in H508 human colon cancer cells, cholinergic ligand interaction with M3 muscarinic receptors results in transactivation of EGFR, thereby stimulating cellular proliferation.

Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice.

Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive electrophilic α/ß-unsaturated aldehydes that have the potential to modify proteins. A primary mechanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic conjugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4(-/-) mice is due to enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased hepatic protein carbonylation in GSTA4(-/-) mice as evidenced by increased 4-HNE and MDA immunostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4(-)(/-) mice compared to their respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from the mitochondrial fractions revealed an increased propensity for modification of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4(-/-) mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4(-/-) PF or EtOH fed mice. Among the peptides/proteins identified, ACSL, ACOX2, MTP, and THIKB contribute to regulation of fatty acid metabolism and ARG1, ARLY, and OAT, which regulate nitrogen and ammonia metabolism having direct relevance to ethanol-induced liver injury. These data define a role for GSTA4-4 in buffering hepatic oxidative stress associated with chronic alcohol consumption and that this GST isoform plays an important role in protecting against carbonylation of mitochondrial proteins.

Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line.

Lithocholic acid (LA) conjugates interact with M3 receptors, the muscarinic receptor subtype that modulates colon cancer cell proliferation. This observation prompted us to examine the action of bile acids on two human colon cancer cell lines: H508, which expresses M3 receptors, and SNU-C4, which does not. Cellular proliferation was determined using a colorimetric assay. Interaction with muscarinic receptors was determined by measuring inhibition of muscarinic radioligand binding and changes in cellular inositol phosphate (IP) formation. Lithocholyltaurine (LCT) caused a dose-dependent increase in H508 cell proliferation that was not observed in SNU-C4 cells. After a 6-day incubation with 300 microM LCT, H508 cell proliferation increased by 200% compared to control. Moreover, in H508 cells, LCT caused a dose-dependent inhibition of radioligand binding and an increase in IP formation. LCT did not alter the rate of apoptosis in H508 or SNU-C4 cells. These data indicate that, at concentrations achievable in the gut, LA derivatives interact with M3 muscarinic receptors on H508 human colon cancer cells, thereby causing an increase in IP formation and cell proliferation. This suggests a mechanism whereby alterations in intestinal bile acids may affect the growth of colon cancer cells.

Glutathione-S-transferase A4-4 modulates oxidative stress in endothelium: possible role in human atherosclerosis.

The role of alpha-class mammalian glutathione S-transferases (GSTs) in the protection of many cell types, including vascular smooth muscle cells, against oxidant damage has been demonstrated, but the role of GSTs in the endothelial cell is not well studied. In order to examine the role of GSTs in the endothelial cell, a stable transfection of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, mouse isozyme of GSTs with activity in vascular wall, was established. Transfected cells demonstrated significantly higher GSTs enzyme activity and expressed significantly increased resistance to the cytotoxicity of allylamine, acrolein, 4-hydroxy-2-nonenal (4-HNE), and H(2)O(2) (P < 0.05). A significantly higher rate of proliferation and lower baseline level of intracellular malondialdehyde (MDA) and 4-HNE were present when compared to wild-type or vector-transfected MS1 endothelial cells (P < 0.05). Transfection protected MS1 endothelial cells from 4-HNE and H(2)O(2) induced apoptosis by inhibiting phosphorylation of c-Jun N-terminal kinases (p-JNK) and consequent activation of p53 and Bax. In early human fibrous atherosclerotic plaques, immunohistochemical studies demonstrated marked induction of hGSTA4-4 in endothelial cells overlying plaque, and in proliferating plaque vascular smooth muscle cells. Our results indicate that endothelial cell mGSTA4-4 can play a key role in protecting blood vessels against oxidative stress and, thus, is likely to be a critical defense mechanism against oxidants that act as atherogens.

Multiple transport proteins involved in the detoxification of endo- and xenobiotics.

Transport mechanisms involved in the exclusion of xeno- and endobiotic toxins from the cellular environment play a crucial role in protecting cells from toxicity of these compounds. A transporter designated as dinitrophenyl S-glutathione ATPase (DNP-SG ATPase) present in human erythrocyte membrane has been characterized in our laboratory. The unique functional features of this transporter include its ability to mediate ATP-dependent transmembrane movement of organic anions such as glutathione conjugates, as well as weakly cationic amphiphilic compounds such as doxorubicin and other substrates of P-glycoprotein. The substrate specificity profile of DNP-SG ATPase overlaps with those of the drug efflux pumps, P-glycoprotein, multidrug resistance associated protein (MRP), and the multi-specific organic anion transporters (MOAT) despite its distinct structural properties from these transporters. Possible functional interrelationships among these transporters is discussed in this review and it is proposed that analogous to the phase I and phase II drug metabolizing enzymes the xeno- and endobiotic transporters may belong to several distinct gene families members of which share overlapping catalytic properties. Their functional diversity covering a wide range of substrate affinities provides protection from structurally diverse xeno- and endobiotic toxicants.

Design, synthesis, and structure-activity relationships of haloenol lactones: site-directed and isozyme-selective glutathione S-transferase inhibitors.

Overexpression of glutathione S-transferase (GST), particularly the GST-pi isozyme, has been proposed to be one of the biochemical mechanisms responsible for drug resistance in cancer chemotherapy, and inhibition of overexpressed GST has been suggested as an approach to combat GST-induced drug resistance. 3-Cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone (1a), a lead compound of site-directed GST-pi inactivator, has been shown to potentiate the cytotoxic effect of cisplatin on tumor cells. As an initial step to develop more potent and more selective haloenol lactone inactivators of GST-pi, we examined the relationship between the chemical structures of haloenol lactone derivatives and their GST inhibitory activity. A total of 16 haloenol lactone derivatives were synthesized to probe the effects of (1) halogen electronegativity, (2) electron density of aromatic rings, (3) molecular size and rigidity, (4) lipophilicity, and (5) aromaticity on the potency of GST-pi inactivation. The inhibitory potency of each compound was determined by time-dependent inhibition tests, and recombinant human GST-pi was used to determine their inhibitory activity. Our structure-activity relationship studies demonstrated that (1) reactivity of the halide leaving group plays a weak role in GST inactivation by the haloenol lactones, (2) aromatic electron density may have some influence on the potency of GST inactivation, (3) high rigidity likely disfavors enzyme inhibition, (4) lipophilicity is inversely proportional to enzyme inactivation, and (5) an unsaturated system may be important for enzyme inhibition. This work facilitated understanding of the interaction of GST-pi with haloenol lactone derivatives as site-directed and isozyme-selective inactivators, possibly potentiating cancer chemotherapy.

Role of RLIP76 in lung cancer doxorubicin resistance: II. Doxorubicin transport in lung cancer by RLIP76.

ATP-dependent transport of doxorubicin (DOX) by recombinant human RLIP76 has been demonstrated previously in reconstituted proteoliposomes. In the preceding communication, we demonstrated that the ATPase activity of RLIP76 was 2-fold higher in NSCLC as compared with SCLC, and it correlated with their inherent DOX resistance. Present studies were performed to determine whether greater ATPase activity of RLIP76 in NSCLC translated into greater RLIP76 mediated DOX transport, and to determine the overall contribution of RLIP76 toward total DOX transport. Consistent with the greater RLIP76 ATPase activity in NSCLC, DOX transport in artificial proteoliposomes reconstituted with purified RLIP76 from NSCLC was 1.8-fold greater than in SCLC. Anti-RLIP76 antibodies completely abrogated DOX transport in these RLIP76 proteoliposomes, whereas anti-MRP or anti-Pgp antibodies had no effect on transport. DOX transport studies in crude membrane vesicles from SCLC and NSCLC also showed a 2.1-fold higher rate of transport in NSCLC as compared with SCLC. Anti-RLIP76 IgG, which recognized only RLIP76 in crude extracts of both SCLC and NSCLC, inhibited 67+/-4% (n=12 cell lines) of total DOX transport in crude membrane vesicles from both SCLC and NSCLC. Inhibition of DOX transport by anti-MRP and anti-Pgp antibody was 35+/-7% (n=12) and 2+/-0.3% (n=12), respectively. The mixture of the three antibodies inhibited DOX transport by 95+/-3% (n=12). These studies demonstrate that DOX transport activity of RLIP76 is significantly greater in NSCLC as compared with SCLC, and that RLIP76 is the major DOX transporter in lung cancer cell lines.

Role of RLIP76 in lung cancer doxorubicin resistance: III. Anti-RLIP76 antibodies trigger apoptosis in lung cancer cells and synergistically increase doxorubicin cytotoxicity.

RLIP76 (ral-binding protein, RalBP1) is a non-ABC multi-specific transporter of amphiphilic chemotherapeutic drugs such as doxorubicin (DOX) and glutathione-electrophile conjugates. In the present studies, we used polyclonal rabbit anti-human RLIP76 IgG to inhibit RLIP76 function for determining the role of RLIP76 in DOX resistance of NSCLC cells. Western blot analyses and immunohistochemistry studies showed no recognition of other protein in crude NSCLC cell homogenates by anti-RLIP76, confirming the specificity of anti-RLIP76 IgG. In immunohistochemistry and flow cytometry studies, these antibodies recognized RLIP76 domain(s) on the cell surface. Cells coated with anti-RLIP76 IgG accumulated significantly greater DOX than cells coated with pre-immune IgG. Synergy was calculated using the Chou-Talalay median effect analysis. Herceptin was the positive control, and pre-immune IgG and Rituxan (anti-CD20) were negative controls. The interaction of anti-RLIP76 IgG and DOX were markedly synergistic (CI 0.36+/-0.27). Lesser synergy was observed between Herceptin and DOX (CI 0.75+/-0.49). Interaction between Herceptin and anti-RLIP76 was only additive (CI 1.12+/-0.5). Human IgG, Rituxan, and rabbit pre-immune IgG controls had no effect on DOX toxicity. DNA-laddering confirmed that DOX triggered apoptosis. Anti-RLIP76 IgG alone as well as Herceptin alone also triggered apoptosis in all 6 NSCLC cell lines. Anti-RLIP76 IgG and Herceptin were shown to increase DOX accumulation in NSCLC. These results demonstrated that specific inhibition of the transport function of RLIP by anti-RLIP76 IgG can trigger apoptosis and synergistically increase DOX cytotoxicity in NSCLC.

Role of RLIP76 in lung cancer doxorubicin resistance: I. The ATPase activity of RLIP76 correlates with doxorubicin and 4-hydroxynonenal resistance in lung cancer cells.

RLIP76 functions as an ATP-dependent transporter of amphiphilic chemotherapeutic drugs such as doxorubicin (DOX, adriamycin), as well as of glutathione-conjugates of endogenous electrophilic toxins such as 4-hydroxynonenal (4HNE). RLIP76 couples transport and ATP-hydrolysis with a 1:1 stoichiometry, making the ATPase activity of RLIP76 an excellent surrogate for its transport activity. Present studies were performed to determine the relationship of the RLIP76 ATPase activity with DOX and 4HNE resistance in a panel of 13 native human lung cancer cell lines. RLIP76 was purified from each cell line and homogeneity demonstrated by SDS-PAGE and amino acid composition analysis. Anti-RLIP76 antibodies were shown by Ouchterlony double immunodiffusion tests to be non-cross-reactive with any other proteins including P-glycoprotein (Pgp) or multidrug resistance associated protein (MRP). These antibodies completely immunoprecipitated ATPase activity of purified RLIP76 fractions, further confirming homogeneity of purified RLIP76. RLIP76 ATPase purified from NSCLC cell lines was about 2-fold more active than that from SCLC in the absence of the stimulator dinitrophenyl S-glutathione (206+/-47, n=7 vs. 94+/-22, n=6, nmol/min/mg protein, respectively), or in its presence (340+/-60, n=7 vs. 186+/-32, n=6, nmol/min/mg; p<0.01). Partial tryptic digest revealed a 44 kDa internal fragment of RLIP76 beginning at Thr-294 in NSCLC cell lines. This fragment was absent from all SCLC, suggesting the possibility that the activity of RLIP76 in SCLC and NSCLC is differentially regulated through post-translational modifications. Taken together, these findings suggest that RLIP76 activity is a general determinant of 4HNE and DOX resistance, and that its activity contributes to the drug-resistant phenotype of NSCLC.

Selective interaction of bile acids with muscarinic receptors: a case of molecular mimicry.

Bile acids alter regulatory pathways in several cell types. The molecular basis for these actions is not fully elucidated, but lithocholyltaurine interacts functionally with muscarinic receptors on gastric chief cells. In the present report, we demonstrate selective interaction of bile acids with Chinese hamster ovary (CHO) cells expressing each of the five muscarinic receptors. Lithocholyltaurine decreases binding of a radioligand to muscarinic M3 receptors, but not to other muscarinic receptors. Sulfated lithocholyltaurine, the major human metabolite, inhibits radioligand binding to muscarinic M1, but not to M2 or M3 receptors. Post-receptor actions of lithocholyltaurine include modulation of acetylcholine-induced increases in inositol phosphate formation and mitogen-activated protein (MAP) kinase phosphorylation. Molecular modeling suggests that the specific and functional interaction of lithocholyltaurine with muscarinic receptors is most likely due to similar shape and surface charge distribution of portions of acetylcholine and the bile acid. We propose that bile acids are signaling molecules whose effects may be mediated by interaction with muscarinic receptors.