Rit Vatsyayan

Anti-cancer effects of novel flavonoid vicenin-2 as a single agent and in synergistic combination with docetaxel in prostate cancer.

The present study was conducted to determine the efficacy of novel flavonoid vicenin-2 (VCN-2), an active constituent of the medicinal herb Ocimum Sanctum Linn or Tulsi, as a single agent and in combination with docetaxel (DTL) in carcinoma of prostate (CaP). VCN-2 effectively induced anti-proliferative, anti-angiogenic and pro-apoptotic effect in CaP cells (PC-3, DU-145 and LNCaP) irrespective of their androgen responsiveness or p53 status. VCN-2 inhibited EGFR/Akt/mTOR/p70S6K pathway along with decreasing c-Myc, cyclin D1, cyclin B1, CDK4, PCNA and hTERT in vitro. VCN-2 reached a level of 2.6±0.3μmol/l in serum after oral administration in mice which reflected that VCN-2 is orally absorbed. The i.v. administration of docetaxel (DTL), current drug of choice in androgen-independent CaP, is associated with dose-limiting toxicities like febrile neutropenia which has lead to characterization of alternate routes of administration and potential combinatorial regimens. In this regard, VCN-2 in combination with DTL synergistically inhibited the growth of prostate tumors in vivo with a greater decrease in the levels of AR, pIGF1R, pAkt, PCNA, cyclin D1, Ki67, CD31, and increase in E-cadherin. VCN-2 has been investigated for radioprotection and anti-inflammatory properties. This is the first study on the anti-cancer effects of VCN-2. In conclusion, our investigations collectively provide strong evidence that VCN-2 is effective against CaP progression along with indicating that VCN-2 and DTL co-administration is more effective than either of the single agents in androgen-independent prostate cancer.

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.

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.

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.

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 versatile transporter and an emerging target for cancer therapy

In the last few years, extensive research has been made to elucidate the functional significance of RLIP76. The resulting novel breakthroughs have helped us understand its transport and signaling functions. RLIP76 is a ubiquitously expressed, key stress-defensive, anti-apoptotic, multi-functional protein that transports glutathione-conjugates of electrophilic compounds, thus controlling the intracellular concentration of pro-apoptotic oxidized lipid byproducts and other xenobiotics such as chemotherapeutic agents. These properties place RLIP76 at a very important position in the hierarchy of the stress defense mechanism adopted by the cell. Selective over-expression of RLIP76 in malignant cells of diverse origin is one of the possible mechanisms by which these cells overcome chemotherapy and radiation induced oxidative damage. RLIP76 has also been shown to be an effective transporter of many conventional chemotherapeutic drugs. Such transport, if inhibited, can lead to increased cellular accumulation of drugs which in turn translates to enhanced drug sensitivity. Recent studies have shown that inhibition and/or depletion of RLIP76 by antibodies, siRNA, or antisense can lead to drastic and sustained regression of lung, kidney, melanoma, colon, and prostate cancer xenografts with no observed recurrence of tumors. All these findings converge on the fact that such inhibition/depletion of RLIP76 can be used clinically to terminate cancer growth and progression. In the present review, we will discuss the role of RLIP76 as a multi-drug transporter, its involvement in cancer, and the prospects of using RLIP76 inhibition as an emerging treatment for cancer.

Glutathione-Conjugate Transport by RLIP76 Is Required for Clathrin-Dependent Endocytosis and Chemical Carcinogenesis

Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76−/− MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis—RLIP76−/− mice will be deficient in chemical neoplasia—was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76−/−. PMA-induced skin carcinogenesis in RLIP76+/+ mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76−/− mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76−/− mice. In RLIP76−/− mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis. Mol Cancer Ther; 10(1); 16–28. ©2011 AACR.

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, 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.

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.