Vimal Pandey

Diet-induced obesity increases melanoma progression: Involvement of Cav-1 and FASN

Recent population‐based epidemiological studies strongly hint towards a link between obesity and its occurrence as well as progression of several cancers including melanoma. Although effects of obesity on breast, colon and liver cancers have been extensively investigated, the links between obesity and melanoma remain largely unexplored. Present study aimed to understand the effect of high fat diet‐induced weight gain on susceptibility of C57BL/6J mice to melanoma. For this, mice routinely were fed on high fat diet for 6 months (HFD mice). Subsequently, mouse melanoma cells were injected subcutaneously in control as well as HFD mice and followed for tumor initiation and progression. We provide strong evidence that diet‐induced obesity leads to increased melanoma progression in male C57BL/6J mice. We observed that increased melanoma progression is associated with enhanced Cav‐1 and FASN expression in tumors from HFD mice. Cav‐1 and FASN are co‐ordinately regulated and Cav‐1 interacts with FASN in melanoma cells. Enhanced levels of Cav‐1, FASN and pAkt control melanoma cell proliferation. Our study establishes a causative relationship between diet‐induced obesity and melanoma progression as well as demonstrates that obesity affects important tumorigenic pathways in melanoma.

Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

Backgroundp53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV) E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5) is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment.ResultsTo determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well.ConclusionPresent study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

Hyperglycemia regulates MDR‐1, drug accumulation and ROS levels causing increased toxicity of carboplatin and 5‐fluorouracil in MCF‐7 cells

There is constant increase in number of diabetic cases thereby giving it status of a serious epidemic. Diabetes increases the risk of occurrence of several cancers including breast cancer and may also have a serious impact on the outcome of cancer treatment. In the present study we investigated effect of hyperglycemia on cytotoxic efficacy of carboplatin and 5‐fluorouracil in MCF‐7 cells. MCF‐7 cells were grown in 5.5 or 25 mM glucose chronically. We show that hyperglycemia favors proliferation of MCF‐7 cells and increases expression of cell cycle regulatory proteins cyclin E and cdk‐2. Hyperglycemia enhances cytotoxicity of carboplatin and 5‐fluorouracil in MCF‐7 cells by approximately 30% and decreases their IC50 by 1.5‐ and 1.3‐folds, respectively. Hyperglycemia reduces expression of P‐glycoprotein and promotes cell killing by increasing drug accumulation. Treatment with 40 µM verapamil, an inhibitor of P‐gp activity specifically increases killing of MCF‐7 cells cultured in 5.5 mM glucose. Further, this effect is synergized by elevated reactive oxygen species and treatment with N‐Acetylcysteine, an inhibitor of ROS, increases survival by 30 and 18% in carboplatin‐ and 5‐fluorouracil‐treated cells cultured in high glucose, respectively. Cytotoxicity of these drugs is associated with reduced activation of Akt and decreased transcriptional activation of NF‐κB. In conclusion, hyperglycemia potentiates cytotoxicity of drugs by reducing P‐gp expression and, increased ROS levels may partially or completely contribute to enhanced toxicity. J. Cell. Biochem. 112: 2942–2952, 2011.

Obesity induced rapid melanoma progression is reversed by orlistat treatment and dietary intervention: Role of adipokines

Obesity, owing to adiposity, is associated with increased risk and development of various cancers, and linked to their rapid growth as well as progression. Although a few studies have attempted to understand the relationship between obesity and melanoma, the consequences of controlling body weight by reducing adiposity on cancer progression is not well understood. By employing animal models of obesity, we report that controlling obesity either by orlistat treatment or by restricting caloric intake significantly slows down melanoma progression. The diminished tumor progression was correlated with decreased fat mass (adiposity) in obese mice. Obesity associated factors contributing to tumor progression were decreased in the experimental groups compared to respective controls. In tumors, protein levels of fatty acid synthase (FASN), caveolin (Cav)‐1 and pAkt, which are tumor promoting molecules implicated in melanoma growth under obese state, were decreased. In addition, increased necrosis and reduction in angiogenesis as well as proliferative markers PCNA and cyclin D1 were observed in tumors of the orlistat treated and/or calorically restricted obese mice. We observed that growth of melanoma cells cultured in conditioned medium (CM) from orlistat‐treated adipocytes was reduced. Adipokines (leptin and resistin), via activating Akt and modulation of FASN as well as Cav‐1 respectively, enhanced melanoma cell growth and proliferation. Together, we demonstrate that controlling body weight reduces adipose mass thereby diminishing melanoma progression. Therefore, strategic means of controlling obesity by reduced caloric diet or with antiobesity drugs treatment may render obesity‐promoted tumor progression in check and prolong survival of patients.

Glucose induced activation of canonical Wnt signaling pathway in hepatocellular carcinoma is regulated by DKK4

Elevated glycemic index, an important feature of diabetes is implicated in an increased risk of hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of this association are relatively less explored. Present study investigates the effect of hyperglycemia over HCC proliferation. We observed that high glucose culture condition (HG) specifically activates canonical Wnt signaling in HCC cells, which is mediated by suppression of DKK4 (a Wnt antagonist) expression and enhanced β-catenin level. Functional assays demonstrated that a normoglycemic culture condition (NG) maintains constitutive expression of DKK4, which controls HCC proliferation rate by suppressing canonical Wnt signaling pathway. HG diminishes DKK4 expression leading to loss of check at G0/G1/S phases of the cell cycle thereby enhancing HCC proliferation, in a β-catenin dependent manner. Interestingly, in NOD/SCID mice supplemented with high glucose, HepG2 xenografted tumors grew rapidly in which elevated levels of β-catenin, c-Myc and decreased levels of DKK4 were detected. Knockdown of DKK4 by shRNA promotes proliferation of HCC cells in NG, which is suppressed by treating cells exogenously with recombinant DKK4 protein. Our in vitro and in vivo results indicate an important functional role of DKK4 in glucose facilitated HCC proliferation.

Atomic force microscopy, biochemical analysis of 3T3-L1 cells differentiated in the absence and presence of insulin.

BACKGROUND There are ample evidences to demonstrate that differentiation of preadipocytes is associated with deposition of fat in cells. Still, it is unclear whether the differentiation process also alters membrane topology as well as cholesterol levels and whether insulin contributes to it. METHODS Membrane scanning of differentiated cells, along with freshly plated and 11 day preadipocytes, was performed using Atomic Force Microscopy (AFM) to gain qualitative information about cell surface properties as well as roughness. Moreover, glucose uptake, lipid analysis, expression profiling of transcription factors and signaling molecules involved in the process of differentiation was also performed. RESULTS We report (i) differentiation in the presence of 500 microM isobutylmethylxanthine (IBMX), 0.25 microM dexamethasone (DEX) with or without 0.1 microM (0.57 microg/ml) insulin directly alters membrane topology. (ii) At nano-levels, addition of insulin maintains plasma membrane roughness during differentiation in comparison with IBMX and DEX only. (iii) At macro levels, decreased fat accumulation in preadipocytes exposed to insulin during the initial stages of differentiation is a result of reduced expression and nuclear localization of sterol regulatory element binding protein (SREBP)-1. GENERAL SIGNIFICANCE This study reports a significant reduction of membrane cholesterol and total cholesterol (p<0.01) in cells differentiated in the presence of insulin.

Hepatocellular carcinoma-associated hypercholesterolemia: involvement of proprotein-convertase-subtilisin-kexin type-9 (PCSK9)

BackgroundPCSK9 regulates low-density lipoprotein cholesterol (LDLc) level and has been implicated in hypercholesterolemia. Aberrant plasma lipid profile is often associated with various cancers. Clinically, the relationship between altered serum lipid level and hepatocellular carcinoma (HCC) has been documented; however, the underlying cause and implications of such dyslipidemia remain unclear.MethodsThe present study includes the use of HepG2 tumor xenograft model to study the potential role of glucose (by providing 15% glucose via drinking water) in regulating PCSK9 expression and associated hypercholesterolemia. To support in vivo findings, in vitro approaches were used by incubating HCC cells in culture medium with different glucose concentrations or treating the cells with glucose uptake inhibitors. Impact of hypercholesterolemia on chemotherapy was demonstrated by exogenously providing LDLc followed by appropriate in vitro assays.ResultsWe observed that serum and hepatic PCSK9 level is decreased in mice which were provided with glucose containing water. Interestingly, serum and tumor PCSK9 level was upregulated in HepG2-tumor-bearing mice having access to water containing glucose. Additionally, elevated LDLc is detected in sera of these mice. In vitro studies indicated that PCSK9 expression was increased by high glucose availability with potential involvement of reactive oxygen species (ROS) and sterol regulatory element binding protein-1 (SREBP-1). Furthermore, it is also demonstrated that pre-treatment of cells with LDLc diminishes cytotoxicity of sorafenib in HCC cells.ConclusionTaken together, these results suggest a regulation of PCSK9 by high glucose which could contribute, at least partly, towards understanding the cause of hypercholesterolemia in HCC and its accompanied upshots in terms of altered response of HCC cells towards cancer therapy.