Sharad S. Singhal

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H(2)O(2) and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.

Curcumin–glutathione interactions and the role of human glutathione S-transferase P1-1

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.

TRANSPORT OF GLUTATHIONE CONJUGATES AND CHEMOTHERAPEUTIC DRUGS BY RLIP76 (RALBP1): A NOVEL LINK BETWEEN G-PROTEIN AND TYROSINE KINASE SIGNALING AND DRUG RESISTANCE

Our studies have shown that RLIP76 (RALBP1), a 76 kDa Ral‐binding, Rho/Rac‐GAP and Ral effector protein, is a novel multispecific transporter of xenobiotics as well as GS‐Es. Like previously characterized ABC transporters, it mediates ATP‐dependent transport of structurally unrelated amphiphilic xenobiotics and displays inherent ATPase activity, which is stimulated by its substrate allocrites. It does not have significant sequence homology with ABC transporters and differs from the ABC transporters in several other important aspects, including (i) lack of any close homologs in humans, (ii) lack of a classical Walker domain, (iii) integral membrane association without clearly defined transmembrane domains and (iv) its role as a direct link to Ras/Ral/Rho and EGF‐R signaling through its multifunctional nature, including GAP activity, regulation of exocytosis as well as clathrin‐coated pit–mediated receptor endocytosis. Its multifunctional nature derives from the presence of multiple motifs, including a Rho/Rac GAP domain, a Ral effector domain binding motif, 2 distinct ATP‐binding domains, a H+‐ATPase domain, PKC and tyrosine kinase phosphorylation sites and the ability to undergo fragmentation into multiple smaller peptides which participate as components of macromolecular functional complexes. One of the physiologic functions of RLIP76 is regulation of intracellular concentration of the electrophilic intermediates of oxidative lipid metabolism by mediating efflux of GS‐E formed from oxidative degradation of arachidonic acid, including leukotrienes and the 4HNE‐GSH conjugate. RLIP76‐mediated transport of amphiphilic chemotherapeutic agents such as anthracyclines and vinca alkaloids as well as GS‐E produced during oxidative metabolism places this multifunctional protein in a central role as a resistance mechanism for preventing apoptosis caused by chemotherapeutic agents and a variety of external/internal stressors, including oxidative stress, heat shock and radiation.

Regression of Lung and Colon Cancer Xenografts by Depleting or Inhibiting RLIP76 (Ral-Binding Protein 1)

Ral-binding protein 1 (RALBP1) is a stress-responsive and stress-protective multispecific transporter of glutathione conjugates (GS-E) and xenobiotic toxins. It is frequently overexpressed in malignant cells and plays a prominent antiapoptotic role selectively in cancer cells through its ability to control cellular concentration of proapoptotic oxidized lipid byproducts. In the absence of chemotherapy, depletion or inhibition of RALBP1 causes regression of syngeneic mouse B16 melanoma. Because RALBP1 transports anthracycline and Vinca alkaloid drugs, as well as GS-E, and because it confers resistance to these drugs, we proposed that depletion or inhibition of RALBP1 should cause regression of human solid tumors that overexpress RALBP1 and augment chemotherapy efficacy. Non-small-cell lung cancer (NSCLC) H358 and H520 and colon SW480 cell lines were used. Cytotoxic synergy between anti-RALBP1 immunoglobulin G (IgG), cis-diammine-dichloroplatinum (II) [CDDP], and vinorelbine was examined in cell culture and xenografts of NSCLC cells. Effects of RALBP1 depletion by antisense were examined in xenografts of NSCLC H358, NSCLC H520, and colon SW480 cells. RALBP1 depletion by phosphorothioate antisense was confirmed and was associated with rapid, complete, and sustained remissions in established s.c. human lung and colon xenografts. RALBP1 inhibition by anti-RALBP1 IgG was equally as effective as antisense and enhanced CDDP-vinorelbine in lung cancer xenografts. These studies show that RALBP1 is a transporter that serves as a key effector function in cancer cell survival and is a valid target for cancer therapy, and confirm that inhibitory modulation of RALBP1 transport activity at the cell surface is sufficient for antitumor effects.

Aldose Reductase Regulates Growth Factor-Induced Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Human Colon Cancer Cells

Inhibition of prostaglandin E(2) (PGE(2)) and cyclooxygenase (COX)-2 by nonsteroidal anti-inflammatory drugs reduces the progression of colon cancer. Inhibition of aldose reductase (AR; EC. 1.1.1.21.) by sorbinil or by antisense ablation prevented fibroblast growth factor-induced and platelet-derived growth factor-induced up-regulation of PGE(2) synthesis in human colon cancer cells, Caco-2. AR besides reducing aldo-sugars efficiently reduces toxic lipid aldehydes and their conjugates with glutathione. Inhibition of AR prevented growth factor-induced COX-2 activity, protein, and mRNA and significantly decreased activation of nuclear factor-kappaB and protein kinase C (PKC) and phosphorylation of PKC-beta2 as well as progression of Caco-2 cell growth but had no effect on COX-1 activity. Cell cycle analysis suggests that inhibition of AR prevents growth factor-induced proliferation of Caco-2 cells at S phase. Treatment of Caco-2 cells with the most abundant and toxic lipid aldehyde 4-hydroxy-trans-2-nonenal (HNE) or its glutathione-conjugate [glutathionyl-HNE (GS-HNE)] or AR-catalyzed product of GS-HNE, glutathionyl-1,4-dihydroxynonane (GS-DHN), resulted in increased COX-2 expression and PGE(2) production. Inhibition of AR prevented HNE- or GS-HNE-induced but not GS-DHN-induced up-regulation of COX-2 and PGE(2). More importantly, in vivo studies showed that administration of AR-small interfering RNA (siRNA), but not control siRNA, to nude mice bearing SW480 human colon adenocarcinoma cells completely arrested tumor progression. Collectively, these observations suggest that AR is an obligatory mediator of growth factor-induced up-regulation of COX-2, PGE(2), and growth of Caco-2 cells, indicating that inhibition of AR may be a novel therapeutic approach in preventing the progression of colon cancer.

RLIP76 Is a Major Determinant of Radiation Sensitivity

RLIP76 (RALBP1) is a glutathione-conjugate transporter that is a critical component of clathrin-coated pit-mediated endocytosis, as well as in stress responses. In cultured cells, it provides protection from stressors including heat, oxidant chemicals, chemotherapeutic agents, UV irradiation, and X-irradiation. Here, we show marked reduction in glutathione conjugate transport capacity and stepwise increase in radiation sensitivity associated with heterozygous or homozygous loss of the RLIP76 gene in mice. Survival after radiation in homozygous knockout animals was significantly shorter than either the heterozygous knockouts or the wild type. Delivery of recombinant RLIP76 to mice lacking RLIP76 via a liposomal delivery system rescued radiation sensitivity. Furthermore, treatment of wild-type mice with RLIP76-containing liposomes conferred resistance to radiation. These findings suggest that inhibiting RLIP76 could be used for sensitization to radiation during cancer therapy and that RLIP76 liposomes could be radioprotective agents useful for treatment of iatrogenic or catastrophic radiation poisoning.

RLIP76, a non-ABC transporter, and drug resistance in epilepsy

BackgroundPermeability of the blood-brain barrier is one of the factors determining the bioavailability of therapeutic drugs and resistance to chemically different antiepileptic drugs is a consequence of decreased intracerebral accumulation. The ABC transporters, particularly P-glycoprotein, are known to play a role in antiepileptic drug extrusion, but are not by themselves sufficient to fully explain the phenomenon of drug-resistant epilepsy. Proteomic analyses of membrane protein differentially expressed in epileptic foci brain tissue revealed the frequently increased expression of RLIP76/RALBP1, a recently described non-ABC multi-specific transporter. Because of a significant overlap in substrates between P-glycoprotein and RLIP76, present studies were carried out to determine the potential role of RLIP76 in AED transport in the brain.ResultsRLIP76 was expressed in brain tissue, preferentially in the lumenal surface of endothelial cell membranes. The expression was most prominent in blood brain barrier tissue from excised epileptic foci. Saturable, energy-dependent, anti-gradient transport of both phenytoin and carbamazepine were demonstrated using recombinant RLIP76 reconstituted into artificial membrane liposomes. Immunotitration studies of transport activity in crude membrane vesicles prepared from whole-brain tissue endothelium showed that RLIP76 represented the dominant transport mechanism for both drugs. RLIP76-/- knockout mice exhibited dramatic toxicity upon phenytoin administration due to decreased drug extrusion mechanisms at the blood-brain barrier.ConclusionWe conclude that RLIP76 is the predominant transporter of AED in the blood brain barrier, and that it may be a transporter involved in mechanisms of drug-resistant epilepsy.

RLIP76 (RALBP1)-mediated transport of leukotriene C4 (LTC4) in cancer cells: implications in drug resistance.

Increased active transport of LTC4 observed frequently in multidrug‐resistant cancer cells have been attributed to ABC‐transporter proteins particularly, MRP1. We have demonstrated recently that a novel non‐ABC transporter, RLIP76 (RALBP1) can also mediate ATP‐dependent transport of GSH‐conjugates (GS‐E) as well as doxorubicin (DOX). We demonstrate RLIP76 reconstituted in artificial liposomes can catalyze ATP‐dependent transport of LTC4, which can be modulated by PKC‐α. The ATPase activity of E. coli expressed homogenous RLIP76 was stimulated in a saturable fashion by LTC4 with half maximal stimulation at 130 nM. Proteoliposomes reconstituted with RLIP76 catalyzed temperature and osmolar sensitive ATP‐dependent transport of LTC4 with Km values of 5.1 mM and 210 nM for ATP and LTC4, respectively. Vmax for transport was found to be 3.2 nmol/min/mg. Colchicine inhibited LTC4 transport to 50% at 5.8 μM. PKC‐α catalyzed phosphorylation of RLIP76 and increased its transport activity by 2–3‐fold. Membrane vesicles prepared from the small (SCLC) and non‐small (NSCLC) lung cancer cell lines as well as HL‐60 (leukemia) and U937 (lymphoma) cell lines exhibited ATP‐dependent transport of LTC4, which was inhibited by anti‐RLIP76 antibodies. The rate of transport of LTC4 in SCLC (H69, H378) was half of that observed in NSCLC cell lines but after transfection with RLIP76, the transport rate of LTC4 in H69 became comparable to that in NSCLC cell lines. Anti‐RLIP76 antibodies inhibited LTC4 transport by 67–81% in all 8 cell lines examined, whereas N‐19 anti‐MRP1 antibodies inhibited transport of LTC4 by only 11–26%. These results suggest that RLIP76 is the major LTC4 transporter in cancer cells and that its transport activity is regulated by PKC‐α‐mediated phosphorylation.

Linking stress-signaling, glutathione metabolism, signaling pathways and xenobiotic transporters

Multi-specific drug-transport mechanisms are intricately involved in mediating a pleiotropic drug-resistance in cancer cells by mediating drug-accumulation defects in cells in which they are over-expressed. The existence and over-expression in drug-resistant neoplasms of transporter proteins belonging to ATP-binding cassette (ABC) family indicate that these myriad transporters contribute to the multidrug-resistance phenomena by removing or sequestering of toxins and metabolites. Another prominent mechanism of multispecific drug-resistance involves glutathione and glutathione linked enzymes, particularly those of the mercapturic acid pathway, which are involved in metabolism and excretion of both endogenous and exogenous electrophilic toxins. A key step in the mercapturic acid pathway, efflux of the glutathione-electrophile conjugate has recently been shown to be catalyzed largely by the stress-responsive protein RLIP76, a splice variant peptide endowed by the human gene RALBP1. The known involvement of RLIP76 in membrane signaling pathways and endocytosis has resulted in a new paradigm for transport and metabolism related drug-resistance in which RLIP76 plays a central role. Our recent studies demonstrating a key anti-apoptotic and stress-responsive role of RLIP76, and the demonstration of dramatic response in malignancies to RLIP76 depletion indicate that targeting this mercapturic acid pathway transporter may be a highly effective and multifaceted antineoplastic strategy.

The role of PKCα and RLIP76 in transport-mediated doxorubicin-resistance in lung cancer

In deletion mutant analyses of potential phosphorylation sites in RLIP76, we identified T297 and S509 as targets for phosphorylation by PKCα. Phosphorylation at T297 increased doxorubicin (DOX)‐transport activity ∼2‐fold for RLIP76 purified from recombinant source, or from three small (H69, H1417, H1618) and three non‐small cell, one each derived from H226 (squamous), H358 (bronchio alveolar), and H1395 (adenocarcinoma) lung cancer cell lines. T297 phosphorylation conferred sensitivity to tryptic digestion at R293. The specific activity for DOX‐transport by RLIP76 purified from non‐small cell, which was primarily in the phosphorylated form, was approximately twice that in small cell lung cancer cell lines. These finding offer a novel explanation for the observed intrinsic differences in sensitivity to DOX between non‐small cell and small cell lung cancer cell lines.