Mukesh Sahu

RLIP76 Transports Sunitinib and Sorafenib and Mediates Drug Resistance in Kidney Cancer

RLIP76 is a stress‐responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It represents the predominant glutathione‐conjugate (GS‐E) transporter in cells. We have shown that RLIP76 plays a crucial role in defending cancer cells from radiation and chemotherapeutic toxin‐mediated apoptosis, and that its inhibition by antibodies or depletion by siRNA or antisense causes apoptosis in a number of cancer cell types. We demonstrated for the first time that the striking anti‐neoplastic effects with no evident toxicity in terms of either weight loss or metabolic effects are also demonstrable for the antibody, antisense and siRNA in a renal cell xenografts model of Caki‐2 cells (Singhal et al., Cancer Res., 2009, 69: 4244). Present studies were performed to determine if RLIP76 targeting is more broadly applicable in other kidney cancer cell lines, to compare the signaling effects of RLIP76 antisense with kinase inhibitors used in treatment of renal cell carcinoma, and to determine whether kinase inhibitors were substrates for transport by RLIP76. Results of these studies show that sorafenib as well as sunitinib are substrates for transport by RLIP76 thus are competitive inhibitors of GS‐E transport. Furthermore, kinase inhibition in the ERK as well as PI3K pathways by RLIP76 depletion is more profound and consistent and is more widely apparent in a number of renal carcinoma cell lines. These studies offer strong support for our overall hypothesis that RLIP76 is an overarching anti‐apoptosis mechanism that, if inhibited, can be more broadly effective in the treatment of renal cell carcinoma.

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.

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.

Diminished drug transport and augmented radiation sensitivity caused by loss of RLIP76

This study was undertaken to characterize the consequences of Ral‐interacting protein (RLIP76)‐loss with respect to drug resistance, transport, radiation resistance, and alternative transport mechanisms in mouse embryonic fibroblasts (MEFs). MEFs were derived from RLIP76+/+, RLIP76+/− and RLIP76−/− mice. The transport of doxorubicin (DOX), colchicine (COL), leukotriene C4 and dinitrophenyl S‐glutathione (DNP‐SG) was analyzed in inside‐out vesicles (IOVs) prepared from MEFs. We used immuno‐titration of transport activity to determine the contribution of RLIP76, MRP1, and p‐glycoprotein (Pgp) towards total transport activity. Loss of RLIP76 alleles resulted in significant sensitization to radiation, DOX, cisplatin, and vinorelbine (VRL). In IOVs prepared from MEFs, we observed a stepwise loss of transport activity. Loss of RLIP76 confers sensitivity to xenobiotics and radiation due to the loss of a common transport mechanism for glutathione–electrophile conjugates and xenobiotics.

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.

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.

Abstract 1525: RLIP76 transports sunitinib and sorafenib and mediates drug resistance in kidney cancer

RLIP76 is a stress-responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It functions as the predominant glutathione-electrophile conjugate (GS-E) transporter in cells. We have shown that RLIP76 plays a crucial role in defending cancer cells from radiation and chemotherapeutic toxin-mediated apoptosis, and its inhibition by antibodies or depletion by siRNA or antisense causes apoptosis in a number of cancer cell types. Recently, we have demonstrated for the first time the striking anti-neoplastic effects with no evident toxicity in terms of either weight loss or metabolic effects for the antibody, antisense and siRNA in a renal cell xenografts model of Caki-2 cells (Singhal et al., Cancer Res., 2009, 69: 4244). Present studies were performed to determine if RLIP76 targeting is more broadly applicable in other kidney cancer cell lines, to compare the signaling effects of RLIP76 antisense with kinase inhibitors used in treatment of renal cell carcinoma, and to determine whether kinase inhibitors were substrates for transport by RLIP76. Results of these studies show that sorafenib as well as sunitinib are substrates for transport by RLIP76 thus are competitive inhibitors of GS-E transport. Furthermore, kinase inhibition in the ERK as well as PI3K pathways by RLIP76 depletion is more profound and consistent and is more widely apparent in a number of renal carcinoma cell lines. These studies support the validity of RLIP76 as a target in kidney cancer therapy, and the functional model in which RLIP76 provides protection from chemical and radiant stress through its transport activity. Results of these studies revealed for the first time that sorafenib as well as sunitinib, receptor tyrosine kinase inhibitors (RTKIs), are substrates for transport by RLIP76. These studies offer strong support for our overall hypothesis that RLIP76 is an overarching anti-apoptosis mechanism that, if inhibited, can be more broadly effective in the treatment of renal cell carcinoma. (Supported in part by NIH Grants CA 77495 and CA 104661 (to SA), Cancer Research Foundation of North Texas (to SSS & SY), Institute for Cancer Research and the Joe & Jessie Crump Fund for Medical Education (to SSS)) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1525.