Shrawan K. Mishra

S100A4 calcium-binding protein is key player in tumor progression and metastasis: preclinical and clinical evidence

The fatality of cancer is mainly bestowed to the property of otherwise benign tumor cells to become malignant and invade surrounding tissues by circumventing normal tissue barriers through a process called metastasis. S100A4 which is a member of the S100 family of calcium-binding proteins has been shown to be able to activate and integrate pathways both intracellular and extracellular to generate a phenotypic response characteristic of cancer metastasis. A large number of studies have shown an increased expression level of S100A4 in various types of cancers. However, its implications in cancer metastasis in terms of whether an increased expression of S100A4 is a causal factor for metastasis or just another after effect of several other physiological and molecular changes in the body resulting from metastasis are not clear. Here we describe the emerging preclinical and clinical evidences implicating S100A4 protein, in both its forms (intracellular and extracellular) in the process of tumorigenesis and metastasis in humans. Based on studies utilizing S100A4 as a metastasis biomarker and molecular target for therapies such as gene therapy, we suggest that S100A4 has emerged as a promising molecule to be tested for anticancer drugs. This review provides an insight in the (1) molecular mechanisms through which S100A4 drives the tumorigenesis and metastasis and (2) developments made in the direction of evaluating S100A4 as a cancer biomarker and drug target.

The Indian Genome Variation database (IGVdb): A project overview

Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.

Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated ‘sandwich’ topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

Lupeol, a Novel Androgen Receptor Inhibitor: Implications in Prostate Cancer Therapy

Purpose: Conventional therapies to treat prostate cancer (CaP) of androgen-dependent phenotype (ADPC) and castration-resistant phenotype (CRPC) are deficient in outcome which has necessitated a need to identify those agents that could target AR for both disease types. We provide mechanism-based evidence that lupeol (Lup-20(29)-en-3b-ol) is a potent inhibitor of androgen receptor (AR) in vitro and in vivo. Experimental Design: Normal prostate epithelial cell (RWPE-1), LAPC4 (wild functional AR/ADPC), LNCaP (mutant functional/AR/ADPC), and C4-2b (mutant functional/AR/CRPC) cells were used to test the anti-AR activity of lupeol. Cells grown under androgen-rich environment and treated with lupeol were tested for proliferation, AR transcriptional activity, AR competitive ligand binding, AR–DNA binding, and AR–ARE/target gene binding. Furthermore, in silico molecular modeling for lupeol–AR binding was done. Athymic mice bearing C4-2b and LNCaP cell–originated tumors were treated intraperitoneally with lupeol (40 mg/kg; 3 times/wk) and tumor growth and surrogate biomarkers were evaluated. To assess bioavailability, lupeol serum levels were measured. Results: Lupeol significantly inhibited R1881 (androgen analogue) induced (i) transcriptional activity of AR and (ii) expression of PSA. Lupeol (i) competed antagonistically with androgen for AR, (ii) blocked the binding of AR to AR-responsive genes including PSA, TIPARP, SGK, and IL-6, and (iii) inhibited the recruitment of RNA Pol II to target genes. Lupeol sensitized CRPC cells to antihormone therapy. High-performance liquid chromatography analysis showed that lupeol is bioavailable to mice. Lupeol inhibited the tumorigenicity of both ADPC and CRPC cells in animals. Serum and tumor tissues exhibited reduced PSA levels. Conclusion: Lupeol, an effective AR inhibitor, could be developed as a potential agent to treat human CaP. Clin Cancer Res; 17(16); 5379–91. ©2011 AACR.

Acute exposure of apigenin induces hepatotoxicity in Swiss mice.

Apigenin, a dietary flavonoid, is reported to have several therapeutic effects in different diseases including cancer. Toxicity of Apigenin is however, least explored, and reports are scanty in literature. This warrants dose-specific evaluation of toxicity in vivo. In the present study, Apigenin was administered intraperitoneally to Swiss mice at doses of 25, 50, 100 and 200 mg/kg. Serum levels of alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) were measured along with the examination of liver histology, reactive oxygen species (ROS) in blood, lipid peroxidation (LPO), glutathione level, superoxide dismutase activity, catalase activity, glutathione S-transferase activity and gene expression in liver tissue. Increase in ALT, AST, ALP, ROS, ratio of oxidized to reduced glutathione (GSSG/GSH) and LPO, altered enzyme activities along with damaged histoarchitecture in the liver of 100 or 200 mg/kg Apigenin treated animals were found. Microarray analysis revealed the differential expression of genes that correspond to different biologically relevant pathways including oxidative stress and apoptosis. In conclusion, these results suggested the oxidative stress induced liver damage which may be due to the regulation of multiple genes by Apigenin at higher doses in Swiss mice.

Epicatechin-rich cocoa polyphenol inhibits Kras-activated pancreatic ductal carcinoma cell growth in vitro and in a mouse model

Activated Kras gene coupled with activation of Akt and nuclear factor‐kappa B (NF‐κB) triggers the development of pancreatic intraepithelial neoplasia, the precursor lesion for pancreatic ductal adenocarcinoma (PDAC) in humans. Therefore, intervention at premalignant stage of disease is considered as an ideal strategy to delay the tumor development. Pancreatic malignant tumor cell lines are widely used; however, there are not relevant cell‐based models representing premalignant stages of PDAC to test intervention agents. By employing a novel Kras‐driven cell‐based model representing premalignant and malignant stages of PDAC, we investigated the efficacy of ACTICOA‐grade cocoa polyphenol (CP) as a potent chemopreventive agent under in vitro and in vivo conditions. It is noteworthy that several human intervention/clinical trials have successfully established the pharmacological benefits of cocoa‐based foods. The liquid chromatography (LC)–mass spectrometry (MS)/MS data confirmed epicatechin as the major polyphenol of CP. Normal, nontumorigenic and tumorigenic pancreatic ductal epithelial (PDE) cells (exhibiting varying Kras activity) were treated with CP and epicatechin. CP and epicatechin treatments induced no effect on normal PDE cells, however, caused a decrease in the (i) proliferation, (ii) guanosine triphosphate (GTP)‐bound Ras protein, (iii) Akt phosphorylation and (iv) NF‐κB transcriptional activity of premalignant and malignant Kras‐activated PDE cells. Further, oral administration of CP (25 mg/kg) inhibited the growth of Kras‐PDE cell‐originated tumors in a xenograft mouse model. LC–MS/MS analysis of the blood showed epicatechin to be bioavailable to mice after CP consumption. We suggest that (i) Kras‐driven cell‐based model is an excellent model for testing intervention agents and (ii) CP is a promising chemopreventive agent for inhibiting PDAC development.

ROBO1, a tumor suppressor and critical molecular barrier for localized tumor cells to acquire invasive phenotype: Study in African‐American and Caucasian prostate cancer models

High‐risk populations exhibit early transformation of localized prostate cancer (CaP) disease to metastasis which results in the mortality of such patients. The paucity of knowledge about the molecular mechanism involved in acquiring of metastatic behavior by primary tumor cells and non‐availability of reliable phenotype‐discriminating biomarkers are stumbling blocks in the management of CaP disease. Here, we determine the role and translational relevance of ROBO1 (an organogenesis‐associated gene) in human CaP. Employing CaP‐progression models and prostatic tissues of Caucasian and African‐American patients, we show that ROBO1 expression is localized to cell‐membrane and significantly lost in primary and metastatic tumors. While Caucasians exhibited similar ROBO1 levels in primary and metastatic phenotype, a significant difference was observed between tumor phenotypes in African‐Americans. Epigenetic assays identified promoter methylation of ROBO1 specific to African‐American metastatic CaP cells. Using African‐American CaP models for further studies, we show that ROBO1 negatively regulates motility and invasiveness of primary CaP cells, and its loss causes these cells to acquire invasive trait. To understand the underlying mechanism, we employed ROBO1‐expressing/ROBO1‐C2C3‐mutant constructs, immunoprecipitation, confocal‐microscopy and luciferase‐reporter techniques. We show that ROBO1 through its interaction with DOCK1 (at SH3‐SH2‐domain) controls the Rac‐activation. However, loss of ROBO1 results in Rac1‐activation which in turn causes E‐Cadherin/β‐catenin cytoskeleton destabilization and induction of cell migration. We suggest that ROBO1 is a predictive biomarker that has potential to discriminate among CaP types, and could be exploited as a molecular target to inhibit the progression of disease as well as treat metastasis in high‐risk populations such as African‐Americans.

Protective Effect of Quercetin on Chloroquine-Induced Oxidative Stress and Hepatotoxicity in Mice

The present study was aimed to find out the protective effect of quercetin on hepatotoxicity resulting by commonly used antimalarial drug chloroquine (CQ). Swiss albino mice were administered with different amounts of CQ ranging from human therapeutic equivalent of 360 mg/kg body wt. to as high as 2000 mg/kg body wt. We observed statistically significant generation of reactive oxygen species, liver toxicity, and oxidative stress. Our observation of alterations in biochemical parameters was strongly supported by real-time PCR measurement of mRNA expression of key biochemical enzymes involved in hepatic toxicity and oxidative stress. However, the observed hepatotoxicity and accompanying oxidative stress following CQ administration show dose specific pattern with little or apparently no effect at therapeutic dose while having severe effects at higher dosages. We further tested quercetin, an antioxidant flavanoid, against CQ-induced hepatoxicity and found encouraging results as quercetin was able to drastically reduce the oxidative stress and hepatotoxicity resulting at higher dosages of CQ administration. In conclusion, our study strongly suggests co administration of antioxidant flavonoid like quercetin along with CQ for antimalarial therapy. This is particularly important when CQ is administered as long-term prophylactic treatment for malaria as chronic exposure has shown to be resulting in higher dose level of drug in the body.

Phosphatidylserine stimulates ceramide 1-phosphate (C1P) intermembrane transfer by C1P transfer proteins

Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, “soluble” phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.

CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation†

ABSTRACT The macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.