4-Hydroxy-2-nonenal enhances tissue factor activity in human monocytic cells via p38 mitogen-activated protein kinase activation-dependent phosphatidylserine exposure.

OBJECTIVE: 4-hydroxy-2-nonenal (HNE) is one of the major aldehydes formed during lipid peroxidation and is believed to play a role in the pathogenesis of atherosclerosis. The objective of the present study is to investigate the effect of HNE on tissue factor (TF) procoagulant activity expressed on cell surfaces. APPROACH AND RESULTS: TF activity and antigen levels on intact cells were measured using factor Xa generation and TF monoclonal antibody binding assays, respectively. Exposure of phosphatidylserine on the cell surface was analyzed using thrombin generation assay or by binding of a fluorescent dye-conjugated annexin V. 2',7'-dichlorodihydrofluorescein diacetate was used to detect the generation of reactive oxygen species. Our data showed that HNE increased the procoagulant activity of unperturbed THP-1 cells that express traces of TF antigen, but had no effect on unperturbed endothelial cells that express no measurable TF antigen. HNE increased TF procoagulant activity but not TF antigen of both activated monocytic and endothelial cells. HNE treatment generated reactive oxygen species, activated p38 mitogen-activated protein kinase, and increased the exposure of phosphatidylserine at the outer leaflet in THP-1 cells. Treatment of THP-1 cells with an antioxidant, N-acetyl cysteine, suppressed the above HNE-induced responses and negated the HNE-mediated increase in TF activity. Blockade of p38 mitogen-activated protein kinase activation inhibited HNE-induced phosphatidylserine exposure and increased TF activity. CONCLUSIONS: HNE increases TF coagulant activity in monocytic cells through a novel mechanism involving p38 mitogen-activated protein kinase activation that leads to enhanced phosphatidylserine exposure at the cell surface.

Nutlin-3 enhances sorafenib efficacy in renal cell carcinoma.

The renal cell carcinoma (RCC) is one of the top 10 cancers in USA. The renal tumors are highly angiogenic and are resistant to conventional interventions, particularly radiotherapy. The advent of multi-specific tyrosine kinase inhibitor sorafenib has improved the progression-free survival in RCC, but overall survival in recurrent and metastatic RCC is still a concern that has lead to characterization of combinatorial regimens. Hence, we studied the effect of combination of nutlin-3, an MDM2 inhibitor, which increases p53 levels, and sorafenib in RCC. Sorafenib along with nutlin-3 synergistically inhibited the cell survival and enhanced caspase-3 cleavage leading to apoptosis in RCC. Nutlin-3 and sorafenib were more effective in reducing the migration of RCC, in combination than as single agents. Sorafenib and nutlin-3 decreased the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) and ERK along with inducing p53 activity. The sorafenib and nutlin-3 co-treatment lead to enhanced levels of p53, p-p53, and increase in the levels of p53 pro-apoptotic effector PUMA, Bax, and decrease in the anti-apoptotic Bcl-2 levels. Importantly, our studies revealed that sorafenib alone can activate p53 in a concentration dependent manner. Thus, co-treatment of nutlin-3 with sorafenib leads to increased half-life of p53, which in turn can be activated by sorafenib, to induce downstream pro-apoptotic and anti-proliferative effects. This is the first report showing the synergistic effect of sorafenib and nutlin-3 while providing a strong clinical-translational rationale for further testing of sorafenib and nutlin-3 combinatorial regimen in human RCC.

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.

2'-hydroxyflavanone inhibits proliferation, tumor vascularization and promotes normal differentiation in VHL-mutant renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the top ten cancers prevalent in USA. Loss-of-function mutations in the von Hippel-Lindau (VHL) gene constitute an established risk factor contributing to 75% of total reported cases of RCC. Loss-of-VHL leads to a highly vascularized phenotype of renal tumors. Intake of citrus fruits has been proven to reduce the risk of RCC in multicenter international studies. Hence, we studied the effect of 2'-hydroxyflavanone (2HF), an active anticancer compound from oranges, in RCC. Our in vitro investigations revealed that 2HF suppresses VHL-mutant RCC to a significantly greater extent than VHL-wild-type RCC by inhibiting epidermal growth factor receptor signaling, which is increased due to VHL mutations in RCC. Our results also revealed for the first time, that 2HF inhibits glutathione S-transferase pi activity. 2HF reduced cyclin B1 and CDK4 levels and induced G2/M phase arrest in VHL-mutant RCC. Importantly, 2HF inhibited the angiogenesis in VHL-mutant RCC by decreasing vascular endothelial growth factor expression. Our in vivo studies in mice xenografts confirmed our in vitro results as evident by decreased levels of proliferation marker, Ki67 and angiogenic marker, CD31, in 2HF-treated mice xenografts of VHL-mutant RCC. 2HF also increased the expression of E-cadherin in VHL-mutant RCC, which would be of significance in restoring normal epithelial phenotype. Collectively, our in vitro and in vivo results revealed the potent antiproliferative, anti-angiogenic and prodifferentiation properties of 2HF in VHL-mutant RCC, sparing normal cells, which could have significant implications not only in the specific management of VHL-mutant RCC but also towards other VHL syndromes.

The sensors and regulators of cell-matrix surveillance in anoikis resistance of tumors.

Normal cells continuously monitor the nature of their respective cellular microenvironment. They are equipped with an inherent molecular defense to detect changes that can precipitate and trigger an oncogenic cascade in the internal and external environment of cells. The process called anoikis unleashes many a characteristic molecular change in the cells which eventually program to cell death in response to cell detachment and inappropriate cellular attachment, both of which can otherwise potentiate the ability of cells to preferentially pursue a malignant course due to the release of molecular discipline which conforms them to a benign structural and functional spectrum. The initiation and propagation of signaling that serves as a switch to cell survival or cell death mediated by surveillance of cell microenvironment is comprised of many heterogeneous sets of molecules interacting mainly at the interface of cell-extracellular matrix. Transforming cells continuously reprogram their signaling characteristics in sensing and modulating the stimuli from cell surface molecules like integrins, cadherins and immunoglobulin family of cell adhesion molecules at adhesion complexes, which enables them to resist anoikis and metastasize to different organs. Actin cytoskeleton binds BIM and Bcl2 modifying factor (BMF), which are regulated by the adhesion status and consequent conformation of cytoskeleton in the cells. This review aims at an integrated synopsis of fundamental mechanisms of the critical interactions of cell surface molecules to facilitate a focused analysis of the differential regulation of signaling processes at cell-ECM junctions that collectively rein the anoikis resistance, which in turn impacts metastatic aggressiveness and drug resistance of tumors originating from respective organs.

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.

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.

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.

Molecular probing of the Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C2-transfer activity.

Two families of sterol C24-methyltransferase (SMT) are responsible for the formation of the ergostane (C(1)-transfer activity; SMT1) and stigmastane (C(2)-transfer activity: SMT2) sterol side chains, respectively. The fungal Saccharomyces cerevisiae SMT1 (Erg6p) operates the first C(1)-transfer in concerted fashion to form a single product whereas the protozoan and plant SMTs are bifunctional capable of catalyzing two sequential, mechanistically distinct C-methylation activities in the conversion of a Delta(24)-sterol acceptor to diverse doubly alkylated products. Previous mutation of the amino acids of Erg6p at D79, Y81 and E82 afforded C(1) or C(2)-transfer activities typical of the protozoan and plant SMT. In this study, scanning mutagenesis experiments involving a leucine replacement of 52 amino acids in Erg6p followed by substitution of key residues with functionally or structurally similar amino acids indicated that 5 new residues at positions Y192, G217, G218, T219 and Y223 can switch the course of C(1)-transfer activity to include plant-like C(2)-transfer activity. The data support a model in which several conserved and non-conserved amino acids located in distinct regions of the Erg6p regulate the course of the C-methylation reaction toward product differences.

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.

Sterol 24-C-methyltransferase: an enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans.

Growth of Cryptococcus neoformans was inhibited by nine nitrogen and sulfur-containing sterols with a heteroatom positioned at C3, C7, C24, C25 or C32 in the lanostane frame. Analysis of the sterol composition of control and treated cells by GC-MS and (1)H NMR has proven that the C-methylation reaction catalyzed by the sterol 24-C-methyltransferase (24-SMT) is the crucial first step in a kinetically favored pathway that fails to include obtusifoliol or zymosterol as intermediates. Cultures fed [methyl-(2)H(3)]methionine led to two deuterium atoms into each of the newly biosynthesized sterols forming a route lanosterol, eburicol (24(28)-methylene-24,25-dihydrolanosterol), 32-noreburicol and ergost-7-enol to ergosterol. Examination of the substrate specificity of a soluble 24-SMT from C. neoformans showed lanosterol to be the optimal acceptor molecule. Incubation with the test compounds generated induced amounts of lanosterol, eburicol or 32-noreburicol concurrent with a decrease of ergosterol. Among them 24(R,S),25-epiminolanosterol (inhibitor of 24-SMT) showed the most potent in vitro antifungal activity comparable to those of itraconazole (inhibitor of the 14-demethylase). Taken together, these data indicate that treatment with substrate-based inhibitors of 24-SMT, a catalyst not found in humans, can disrupt ergosterol homeostasis involved with fungal growth and therefore these compounds can provide leads for rational drug design of opportunistic pathogens.

Physiological and pharmacological significance of glutathione-conjugate transport.

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

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.

Inactivation of factor VIIa by antithrombin in vitro, ex vivo and in vivo: role of tissue factor and endothelial cell protein C receptor.

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼ 1% expression of the mouse TF level) and high human TF mice (HTF, ∼ 100% of the mouse TF level) were injected with human rFVIIa (120 µg kg(-1) body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40-50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF's role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process.

Role of tissue factor in Mycobacterium tuberculosis-induced inflammation and disease pathogenesis.

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.

Proteomic analysis of signaling network regulation in renal cell carcinomas with differential hypoxia-inducible factor-2α expression.

BACKGROUND: The loss of von Hippel-Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α) instead of hypoxia-inducible factor-1α (HIF1α). METHODS AND PRINCIPAL FINDINGS: We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography-mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335. CONCLUSIONS AND SIGNIFICANCE: Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.

Didymin induces apoptosis by inhibiting N-Myc and upregulating RKIP in neuroblastoma.

Neuroblastomas arise from the neural crest cells and represent the most common solid tumors outside the nervous system in children. The amplification of N-Myc plays a primary role in the pathogenesis of neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases of neuroblastomas. In this regard, dietary compounds which can target N-Myc and exert anticancer effects independent of p53 status acquire significance in the management of neuroblastomas. Hence, we investigated the anticancer properties of the flavonoid didymin in neuroblastomas. Didymin effectively inhibited proliferation and induced apoptosis irrespective of p53 status in neuroblastomas. Didymin downregulated phosphoinositide 3-kinase, pAkt, Akt, vimentin, and upregulated RKIP levels. Didymin induced G(2)/M arrest along with decreasing the levels of cyclin D1, CDK4, and cyclin B1. Importantly, didymin inhibited N-Myc as confirmed at protein, mRNA, and transcriptional level by promoter-reporter assays. High-performance liquid chromatography analysis of didymin-treated (2 mg/kg b.w.) mice serum revealed effective oral absorption with free didymin concentration of 2.1 µmol/L. Further in vivo mice xenograft studies revealed that didymin-treated (2 mg/kg b.w.) animals had significant reductions in tumors size compared with controls. Didymin strongly inhibited the proliferation (Ki67) and angiogenesis (CD31) markers, as well as N-Myc expression, as revealed by the histopathologic examination of paraffin-embedded section of resected tumors. Collectively, our in vitro and in vivo studies elucidated the anticancer properties and mechanisms of action of a novel, orally active, and palatable flavonoid didymin, which makes it a potential new approach for neuroblastoma therapy (NANT) to target pediatric neuroblastomas.

RLIP76, a glutathione-conjugate transporter, plays a major role in the pathogenesis of metabolic syndrome.

PURPOSE: Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76⁻/⁻ mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76⁻/⁻ mice. RESEARCH DESIGN AND METHODS: Blood glucose (BG) and lipid measurements were performed in RLIP76⁺/⁺ and RLIP76⁻/⁻ mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining. RESULTS: The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76⁻/⁻ mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK. CONCLUSIONS/SIGNIFICANCE: All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76⁻/⁻ mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome.

Targeting p53-null neuroblastomas through RLIP76.

The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53-mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53-mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of proapoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione conjugate transporter, RLIP76. The RLIP76-p53 complex was showed by both immunoprecipitation analyses of purified proteins and immunofluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα-stimulated transport of glutathione conjugates of 4HNE (GSHNE) and doxorubicin. Drug resistance was significantly greater for p53-mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53-mutant neuroblastoma, based on a model of regulation of p53-induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in overexpression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53-mutant neuroblastoma.