Suresh Guruswamy

Myocyte enhancer factor 2 (MEF2)-binding site is required for GLUT4 gene expression in transgenic mice. Regulation of MEF2 DNA binding activity in insulin-deficient diabetes.

We have previously demonstrated that important regulatory elements responsible for regulated expression of the humanGLUT4 promoter are located between −1154 and −412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491–23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription rate of theGLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue.

Flexible heteroarotinoids (Flex-Hets) exhibit improved therapeutic ratios as anti-cancer agents over retinoic acid receptor agonists

The anti-cancer activities and toxicities of retinoic acid (RA) and synthetic retinoids are mediated through nuclear RA receptors (RARs) and retinoid X receptors (RXRs) that act as transcription factors. Heteroarotinoids (Hets), which contain a heteroatom in the cyclic ring of an arotinoid structure, exhibit similar anti-cancer activities, but reduced toxicity in vivo, in comparison to parent retinoids and RA. A new class of Flexible Hets (Flex-Hets), which contain 3-atom urea or thiourea linkers, regulate growth and differentiation similar to RA, but do not activate RARs or RXRs. In addition, Flex-Hets induce potent apoptosis in ovarian cancer and in head and neck cancer cell lines through the intrinsic mitochondrial pathway. In this study, 4 cervical cancer cell lines were growth inhibited by micromolar concentrations of Flex-Hets to greater extents than RAR/RXR active retinoids. The most potent Flex-Het (SHetA2) inhibited each cell line of the National Cancer Institute’s human tumor cell line panel at micromolar concentrations. Oral administration of Flex-Hets (SHetA2 and SHetA4) inhibited growth of OVCAR-3 ovarian cancer xenografts to similar extents as administration of a RAR/RXR-panagonist (SHet50) and Fenretinide (4-HPR) in vivo. None of these compounds induced evidence of skin, bone or liver toxicity, or increased levels of serum alanine aminotransferase (ALT) in the treated mice. Topical application of Flex-Hets did not induce skin irritation in vivo, whereas a RAR/RXR-panagonist (NHet17) and a RARγ-selective agonist (SHet65) induced similar irritancy as RA. In conclusion, Flex-Hets exhibit improved therapeutic ratios for multiple cancer types over RAR and/or RXR agonists.

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis.

S-adenosyl L-methionine inhibits azoxymethane-induced colonic aberrant crypt foci in F344 rats and suppresses human colon cancer Caco-2 cell growth in 3D culture

S‐Adenosyl L‐methionine (SAM) is a universal methyl group donor to various intermediary metabolites, hormones, proteins, neurotransmitters, phospholipids and nucleic acids. Deficiency of folate, which plays a role in the synthesis of SAM leads to increased risk for colon cancer. This study tested the effectiveness of SAM supplementation in protecting against azoxymethane (AOM)‐induced colon carcinogenesis in male F344 rats. We also tested the effect of SAM on cyclooxygenase‐2 (COX‐2) in a macrophage cell line. Further, we developed a 3‐D culture model using Caco‐2 cells to test the effect of SAM on tumor spheroid size and number. Groups of rats were given the experimental diet containing either 0‐, 400‐ or 800‐ppm SAM, 1 week before the first AOM injection and continued until 8 weeks. In the control group, AOM produced a substantial number of aberrant crypt foci (ACF) (96 ± 8). Dietary administration of SAM significantly reduced the number of total ACF (400 ppm SAM, 68 ± 7.3, p < 0.01 and 800 ppm SAM, 57 ± 7.1, p < 0.001). SAM significantly decreased AOM‐induced colonic multicrypt foci in a dose‐dependent manner. Suppression of Lipopolysaccharide (LPS) induced COX‐2 protein expression was observed in a RAW264.7 cell line. We established growth of Caco‐2 cells as spheroids, in a 3D matrix of collagen and matrigel. Treatment with SAM decreased both size and number of spheroids in a dose‐dependent manner (p < 0.0001). These observations demonstrate for the first time that SAM can reduce the occurrence of ACF in AOM treated male F344 rats and suppress formation of human tumor spheroids and expression of COX‐2.

Inhibition of azoxymethane-induced colorectal cancer by CP-31398, a TP53 modulator, alone or in combination with low doses of celecoxib in male F344 rats.

Tumor suppressor p53 plays a major role in colorectal cancer development. The present study explores the effects of p53-modulating agent CP-31398 alone and combined with celecoxib on azoxymethane-induced aberrant crypt foci (ACF) and colon adenocarcinomas in F344 rats. Maximum tolerated doses were 400 and 3,000 ppm for CP-31398 and celecoxib, respectively. ACF and tumor efficacy endpoints were carried out on azoxymethane-treated 7-week-old rats (48 per group) fed the control AIN-76A diet. Two weeks after carcinogen treatment, rats were fed the diets containing 0, 150, or 300 ppm CP-31398, 300 ppm celecoxib, or 150 ppm CP-31398 plus 300 ppm celecoxib. ACF and colon adenocarcinomas were determined at 8 and 48 weeks after azoxymethane treatment, respectively. Dietary CP-31398 was shown to suppress mean colonic total ACF by 43% and multicrypt ACF by 63%; dietary CP-31398 at 150 and 300 ppm suppressed adenocarcinoma incidence by 30.4% (P < 0.02) and 44% (P < 0.005), respectively, and adenocarcinoma multiplicity by 51% (P < 0.005) and 65% (P < 0.0001), respectively. Dietary celecoxib suppressed colon adenocarcinoma incidence (60%; P < 0.0003) and multiplicity (70%; P < 0.0001). Importantly, combination of low-dose CP-31398 and celecoxib suppressed colon adenocarcinoma incidence by 78% and multiplicity by 90%. Rats that were fed the high-dose CP-31398 or a combination of low-dose CP-31398 and celecoxib showed considerable enhancement of p53 and p21(WAF1/CIP) expression, apoptosis, and reduced tumor cell proliferation in colonic tumors. These observations show, for the first time, that CP-31398 possesses significant dose-dependent chemopreventive activity in a well-established colon cancer model and that a combination of low-dose CP-31398 and celecoxib significantly enhanced colon cancer chemopreventive efficacy.

Multi-Target Approaches in Colon Cancer Chemoprevention Based on Systems Biology of Tumor Cell-Signaling.

Colorectal cancer is the leading cause of cancer related deaths in the United States. Although it is preventable, thousands of lives are lost each year in the U.S. to colorectal cancer than to breast cancer and AIDS combined. In colon cancer, the formation and progression of precancerous lesions like aberrant crypt foci and polyps is associated with the up-regulation of cycloxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and hydroxy methyl glutaryl CoA reductase (HMG-CoA reductase). The current review will focus on the signaling pathway involving COX-2 and HMG-CoA reductase enzymes and their downstream effectors in signaling mechanism. Cancer cells need huge pools of both cholesterol and isoprenoids to sustain their unlimited growth potential. Cholesterol by modulating caveolae formation regulates several signaling molecules like AKT, IGFR, EGFR and Rho which are involved in cell growth and survival. Cholesterol is also essential for lipid body formation which serves as storage sites for COX-2, eicosanoids and caveolin-1. Experimental studies have identified important mechanisms showing that COX-2, caveolin-1, lipid bodies and prenylated proteins is involved in carcinogenesis. Therefore multi-target, multi-drug approach is the ideal choice for effective colon cancer chemoprevention. This review will give an overview of the two pathways, their signaling networks, and the interactions between the components of the two networks in the activation and regulation of cell signaling involving growth/survival and explain the rationale for colon cancer chemoprevention using COX-2 inhibitors and statins.

Chemoprevention of colon and small intestinal tumorigenesis in APC min/+ mice by SHetA2 (NSC721689) without toxicity

The occurrence of intestinal polyps in people at high risk for developing colorectal cancer provides an opportunity to test the efficacy of chemoprevention agents. In this situation of treating otherwise healthy people, the potential for toxicity must be minimal. The small-molecule flexible heteroarotinoid (Flex-Het), called SHetA2, has chemoprevention activity in organotypic cultures in vitro and lack of toxicity at doses capable of inhibiting xenograft tumor growth in vivo. The objective of this study was to evaluate SHetA2 chemoprevention activity and toxicity in the APCmin/+ murine model. Oral administration of SHetA2 at 30 and 60 mg/kg five days per week for 12 weeks significantly reduced development of intestinal polyps by 40% to 60% depending on the dose and sex of the treatment group. Immunohistochemical and Western blot analysis of polyps showed reduced levels of cyclin D1 and proliferating cell nuclear antigen in both SHetA2 treatment groups. Western blot analysis also showed SHetA2 induction of E-cadherin, Bax, and caspase-3 cleavage along with reduction in Bcl-2, COX-2, and VEGF, consistent with SHetA2 regulation of apoptosis, inflammation, and angiogenesis. Neither dose caused weight loss nor gross toxicity in APCmin/+ or wild-type littermates. Magnetic resonance imaging (MRI) of cardiac function showed no evidence of SHetA2 toxicity. SHetA2 did not alter left ventricular wall thickness. In summary, SHetA2 exerts chemoprevention activity without overt or cardiac toxicity in the APCmin/+ model. SHetA2 modulation of biomarkers in colon polyps identifies potential pharmacodynamic endpoints for SHetA2 clinical trials. Cancer Prev Res; 6(9); 908–16. ©2013 AACR.

Abstract A53: Chemoprevention of colon cancer by PBIT, an iNOS‐selective inhibitor, administered alone or in combination with low‐doses of celecoxib, a COX‐2 selective inhibitor

Clinical and preclinical studies suggest that NSAIDs and cyclooxygenase (COX)‐2 inhibitors such as celecoxib, reduce the risk of colorectal cancer. However high doses of NSAIDs causes gastrointestinal toxicity and increased cardiovascular risk. Inducible nitric oxide synthase (iNOS), like COX‐2, is overexpressed in colon tumors. Interestingly, nitric oxide stimulates COX‐2 activity and contributes to the tumor growth. The present study examines the effects of S,S ′‐1,4‐phenylenebis(1,2‐ethanediyl)bisisothiourea (PBIT), an iNOS‐selective inhibitor on 1) azoxymethane (AOM)‐induced colon adenocarcinomas in F344 rats 2) evaluates the combined effects of a low‐dose PBIT and celecoxib on chemopreventive efficacy in rats. Seven‐week old male F344 rats (36/group) were fed control AIN‐76A diet and one week later, AOM was administered s.c to induce colonic tumors. Four‐weeks after the AOM treatment, groups of rats were fed experimental diets containing either 0, 50, 100 ppm of PBIT or 250 and 500 ppm of celecoxib or a combination of 50 ppm PBIT plus 250 ppm celecoxib. Forty‐eight weeks after AOM treatment, rats were killed and the intestinal tumors were evaluated. Multiple samples of colonic tumors from each group were assayed for expression and activity of iNOS, COX‐2 and markers of apoptosis and cell proliferation. We found that 50 and 100 ppm PBIT in diet suppressed colon adenocarcinoma incidence by 34.6% (p WAF1/CIP expression in colonic tumors. Administration of 100 ppm PBIT, 250 ppm or 500 ppm celecoxib and a combination of 50 ppm PBIT plus 250 ppm celecoxib suppressed tumor cell proliferation (BrdU index by 22–53%, p Citation Information: Cancer Prev Res 2010;3(1 Suppl):A53.

Abstract 954: A selective iNOS inhibitor N 6 -iminoethyl-lysine tetrazoleamide (NILT), suppress invasive colonic cancers and improves preventive efficacy of low-dose COX-2 inhibitor, celecoxib in F344 rats

Clinical and preclinical studies suggest that NSAIDs and cyclooxygenase (COX)-2 inhibitors such as celecoxib, reduce the risk of colorectal cancer. However, at high doses celecoxib causes gastrointestinal toxicity and an increased cardiovascular risk. Inducible nitric oxide synthase (iNOS), similar to COX-2, is overexpressed in colon tumors, and nitric oxide is shown to stimulate COX-2 activity and contributes to the tumor invasiveness. Thus, in the present study we tested a novel iNOS-selective inhibitor NILT for colon cancer inhibition and invasiveness in F344 rats and evaluated the combined low dose effects of NILT and celecoxib, to improve the chemopreventive efficacy in rats. Seven week old male F344 rats (36/group) were fed control AIN-76A diet and colon cancer was induced by administering azoxymethane (AOM), s.c., a single dose at week 8 and 9. Four weeks after the AOM treatment, groups of rats were fed experimental diets containing either 0, 100, 200 ppm of NILT or 250 and 500 ppm of celecoxib or a combination of 100 ppm NILT and 250 ppm celecoxib. Forty eight weeks after AOM treatment, rats were killed and the colonic tumors were evaluated. Multiple samples of colonic tumors from each group were assayed for activity levels of iNOS, COX-2, and expression levels of cytokines, chemokine and markers of apoptosis and cell proliferation. We found that dietary administration of 200 ppm NILT suppressed both colon adenocarcinoma incidence by 40% (p 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 954.

Abstract A140: Chemoprevention of familial adenomatous polyposis by a flexible‐heteroarotinoid (Flex‐Het), SHetA2, in APCMin mice

Colorectal cancer is the second most common cancer in the western world with 1 million new cases worldwide each year. Flexible Heteroarotinoids (Flex‐Hets) are a promising new class of anticancer compounds that regulate cell cycle progression, differentiation and apoptosis, as well as xenograft tumor growth and angiogenesis in many cancer types. The lead Flex‐Het, Sulfur heteroarotinoid A2 (SHetA2), prevents DMBA‐induced transformation in endometrial organotypic cultures in vitro and is currently in preclinical testing in the NCI RAPID program. In this study, the potential in vivo chemopreventive effects of SHetA2 were evaluated in APCmin/+ mice. Six‐week old APCmin/+ mice were assigned to three different treatment groups with normalized distributions of males and females. Drug was dissolved in corn oil (0 ‐ placebo, 30 mg/kg SHetA2, 60 mg/kg SHetA2) and administered by gavage five days per week for 12 weeks. There was no statistically significant difference in body weight between any of the treatment groups and controls. All mice were killed and the total number and size of tumors along the entire length of the intestine and colon were visually counted using a magnified lens. SHetA2‐treated groups exhibited dramatic reductions in the total number of intestinal tumors at both doses (SHetA2 30 mg/kg ‐ 60%, SHetA2 60 mg/kg ‐ 54%, P 2 mm), medium (1–2 mm) and small ( Citation Information: Cancer Prev Res 2010;3(1 Suppl):A140.