Mugdha Sukhthankar

A Green Tea Component Suppresses Posttranslational Expression of Basic Fibroblast Growth Factor in Colorectal Cancer

BACKGROUND & AIMS Green tea catechins are known to have anticarcinogenic effects. Epigallocatechin-3-gallate (EGCG) accounts for almost 50% of the total catechin content in green tea extract and has very potent antioxidant effects. EGCG also inhibits angiogenesis, possibly through the inhibition of proangiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which in turn, inhibits tumor growth and metastasis. However, the exact molecular mechanism by which EGCG suppresses bFGF expression is not known. Our objective was to elucidate the molecular mechanisms by which EGCG inhibits bFGF expression in colorectal cancer. METHODS We examined posttranslational regulation of bFGF by EGCG in human colorectal cancer cells. We also examined bFGF in intestinal tumor formation of APC(Min/+) mice with and without catechin treatment. RESULTS The bFGF protein was quickly degraded in the presence of EGCG, but a proteasome inhibitor suppressed this degradation. EGCG was also found to increase ubiquitination of bFGF and trypsin-like activity of the 20S proteasome, thereby resulting in the degradation of bFGF protein. Furthermore, EGCG suppressed tumor formation in APC(Min/+) mice, compared with vehicle-treated mice, in association with reduced bFGF expression. CONCLUSIONS The ubiquitin-proteasome degradation pathway contributes significantly to down-regulation of bFGF expression by EGCG. Catechin compounds have fewer adverse effects than chemotherapeutic agents and hence can be used as proof-of-concept in cancer therapeutics to suppress growth and metastasis by targeting proteins such as bFGF.

Nonsteroidal Anti-inflammatory Drug-Activated Gene-1 Expression Inhibits Urethane-Induced Pulmonary Tumorigenesis in Transgenic Mice

The expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) inhibits gastrointestinal tumorigenesis in NAG-1 transgenic mice (C57/BL6 background). In the present study, we investigated whether the NAG-1 protein would alter urethane-induced pulmonary lesions in NAG-1 transgenic mice on an FVB background (NAG-1Tg+/FVB). NAG-1Tg+/FVB mice had both decreased number and size of urethane-induced tumors, compared with control littermates (NAG-1Tg+/FVB = 16 ± 4 per mouse versus control = 20 ± 7 per mouse, P < 0.05). Urethane-induced pulmonary adenomas and adenocarcinomas were observed in control mice; however, only pulmonary adenomas were observed in NAG-1Tg+/FVB mice. Urethane-induced tumors from control littermates and NAG-1Tg+/FVB mice highly expressed proteins in the arachidonic acid pathway (cyclooxygenases 1/2, prostaglandin E synthase, and prostaglandin E2 receptor) and highly activated several kinases (phospho-Raf-1 and phosphorylated extracellular signal-regulated kinase 1/2). However, only urethane-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation was decreased in NAG-1Tg+/FVB mice. Furthermore, significantly increased apoptosis in tumors of NAG-1Tg+/FVB mice compared with control mice was observed as assessed by caspase-3/7 activity. In addition, fewer inflammatory cells were observed in the lung tissue isolated from urethane-treated NAG-1Tg+/FVB mice compared with control mice. These results paralleled in vitro assays using human A549 pulmonary carcinoma cells. Less phosphorylated p38 MAPK was observed in cells overexpressing NAG-1 compared with control cells. Overall, our study revealed for the first time that the NAG-1 protein inhibits urethane-induced tumor formation, probably mediated by the p38 MAPK pathway, and is a possible new target for lung cancer chemoprevention.

Compounds containing 2-substituted imidazole ring for treatment against human African trypanosomiasis.

A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitroimidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2-yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good solubility. The presence of the NO(2) group at the 5-position of the imidazole ring in 2-substituted imidazoles is the crucial factor to inhibit T. brucei.

Synthesis and comparison of antimalarial activity of febrifugine derivatives including halofuginone.

Febrifugine and its derivatives including halofuginone which possess very high activity against malaria were prepared synthetically from easily available starting material, 3-hydroxy picoline, and using simple reaction conditions. Synthesis of 2-amino-5, 6-methylenedioxy benzoic acid, (which is an intermediate for the process) is described. The selectivity enhancement in nitration of 3, 4-methylenedioxybenzaldehyde towards 6-nitro isomer was done with the help of surfactant. The antimalarial activity of synthesized compounds was determined by using in vitro assays against chloroquine sensitive (D6), chloroquine resistant (W2) Plasmodium falciparum strains for susceptibility and two mammalian cell lines (neuronal cell line NG108 and macrophage cell line J774) for cytotoxicity. The IC(50)s of halofuginone was observed to be the best among the synthesized derivatives of febrifugine.

Cell adhesion property affected by cyclooxygenase and lipoxygenase: Opto-electric approach.

Expression of cyclooxygenases (COX) and lipoxygenases (LOX) has been linked to many pathophysiological phenotypes, including cell adhesion. However, many current approaches to measure cellular changes are performed only in a fixed-time point. Since cells dynamically move in conjunction with the cell matrix, there is a pressing need for dynamic or time-dependent methods for the investigation of cell properties. In the presented study, we used stable human colorectal cancer cell lines ectopically expressing COX-1, COX-2, and 15LOX-1, to investigate whether expression of COX-1, COX-2, or 15LOX-1 would affect cell adhesion using our opto-electric methodology. In a fixed-time point experiment, only COX-1- and COX-2-expressing cells enhanced phosphorylation of focal adhesion kinase, but all the transfected cells showed invasion activity. However, in a real-time experiment using opto-electric approaches, transmitted cellular morphology was much different with tight adhesion being shown in COX-2 expressing cells, as imaged by differential interference contrast microscopy (DICM) and interference reflection contrast microscopy (IRCM). Furthermore, micro-impedance measurements showed a continued increase in both resistance and reactance of COX- and LOX-transfected cells, consistent with the imaging data. Our data indicate that both COX- and LOX-expressing cells have strong cell-to-cell and cell-to-substrate adhesions, and that cell imaging analysis with cell impedance data generates fully reliable results on cell adhesion measurement.

BCL-2 family protein, BAD is down-regulated in breast cancer and inhibits cell invasion.

We have previously demonstrated that the anti-apoptotic protein BAD is expressed in normal human breast tissue and shown that BAD inhibits expression of cyclin D1 to delay cell-cycle progression in breast cancer cells. Herein, expression of proteins in breast tissues was studied by immunohistochemistry and results were analyzed statistically to obtain semi-quantitative data. Biochemical and functional changes in BAD-overexpressing MCF7 breast cancer cells were evaluated using PCR, reporter assays, western blotting, ELISA and extracellular matrix invasion assays. Compared to normal tissues, Grade II breast cancers expressed low total/phosphorylated forms of BAD in both cytoplasmic and nuclear compartments. BAD overexpression decreased the expression of β-catenin, Sp1, and phosphorylation of STATs. BAD inhibited Ras/MEK/ERK and JNK signaling pathways, without affecting the p38 signaling pathway. Expression of the metastasis-related proteins, MMP10, VEGF, SNAIL, CXCR4, E-cadherin and TlMP2 was regulated by BAD with concomitant inhibition of extracellular matrix invasion. Inhibition of BAD by siRNA increased invasion and Akt/p-Akt levels. Clinical data and the results herein suggest that in addition to the effect on apoptosis, BAD conveys anti-metastatic effects and is a valuable prognostic marker in breast cancer.

Abstract 4591: Estrogen receptor beta controls a wide variety of functions in breast cancer cells

The role of ER alpha as the mediator of estrogen action in breast cancer (BC) cells is well known, however, the role of ER beta is controversial. The majority of BC expressing ER alpha also express ER beta. Some clinical studies suggest that ER beta expression is a good prognosticator; however, in others, especially where expression levels of ER beta > ER alpha, ER beta may be associated with more advanced BC. Previously, we showed that ER beta may regulate a number of signaling pathways (AACR, 2008). Our recent work demonstrates that ER beta has even wider actions in MCF7 and T47D cells stably overexpressing ER beta. Thus, ER beta decreased phospho (p)/ total Stat1, p Stat3, p Stat5/total Stat5. Expression of the oncogenic miR cluster 17-92 was highly reduced (p These results suggest that ER beta has a wide variety of actions in BC cells, as predicted by the regulation of 62 miRs (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 4591.

Abstract 1046: BAD is a multifunctional protein in breast cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BAD is a multifunctional protein in breast cancer cells R. Fernando1, M. Cekanova2, J. Woraratphoka2, M. Sukhthankar2, J. Bahn2, S. Dharmawardhana3, N. Siriwardana2, N. Moustaid-Moussa2, A. Strom4, S. Baek2, Q. Wong5, M. Zou5, R. Donnell2, J. Wimalasena2 1NCI, Bethesda, MD; 2University of Tennessee, Knoxville, TN; 3University of Puerto Rico Medical Sciences, San Juan, PR; 4University of Houston, Houston, TX; 5University of Oklahoma Medical Center, Oklahoma City, OK. BAD is a typical BH3 protein and, like many other BCL-2 family proteins, regulates apoptosis. However, several recent studies suggest that BAD has non-apoptotic roles. Previously, we had shown that BAD inhibits the MCF7 cell cycle via abrogation of cyclin D1 synthesis (JBC 282, 28864, 2007). Using tetracycline-regulated stable BAD overexpression in MCF7 cells, we now report that BAD regulates a wide variety of functions: since BAD binds to c-Jun (JBC, 2007), we investigated the ability of BAD to regulate the AP1 reporter activity. BAD inhibited MEK and activated RAS (V12, S35) regulated AP1 activity, but not the increase due to AKT. BAD overexpression blocked ERK activation by MEK1, but not by active AKT. Wildtype BAD inhibited estrogen- or serum-induced activation of c-Jun, JNK, and ERK, but not p38 MAPK. However, the S112/S136 double mutant BAD had no inhibitory effects, suggesting a requirement for phosphorylation. We then explored if BAD can regulate signaling pathways and found that it can inhibit beta catenin mRNA and protein expression. Several signaling proteins, including total AKT, active GSK 3 beta, and apoptosis-related soluble FasL, TNF alpha, TRAIL, EGFR, and RBM5, which may regulate FasL were significantly (p<0.01) regulated by BAD. BAD decreased invasion of MCF7 cells through Matrigel (p<0.05) and decreased cellular VEGF (p50% (p<0.01). Surprisingly, BAD decreased protein expression of PPAR gamma, active AMPK, LKB1, and active acetylCoA carboxylase (all at p<0.01). We analyzed the expression of several markers in breast cancer specimens: phospho (p)-BAD, BAD and cyclin D1 using tissue microarrays containing normal and neoplastic tissue. In addition, we checked the expression levels of ERK-1, phospho (p)-ERK1, AKT, and phospho-AKT in surgical pathology blocks with normal and neoplastic breast epithelial cells. As expected, cytoplasmic p-BAD expression was significantly and positively correlated with nuclear BAD, nuclear p-BAD, as well as nuclear cyclin D1. Our data showed that cytoplasmic cyclin D1 was positively correlated with cytoplasmic ERK-1, and cytoplasmic cyclin D1 was negatively correlated with nuclear AKT expression. Nuclear BAD and AKT were negatively correlated with nuclear cyclin D1 and nuclear ER-beta. Cytoplasmic expression of p-ERK1 was negatively correlated with nuclear AKT and positively correlated with nuclear cyclin D1, and nuclear ER-beta. Taken together, these results suggest that BAD may be a multifunctional protein. 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 1046.

The antitumorgenic actions of estrogen receptor beta in breast cancer cells.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #3056 Recent studies have suggested that ERβ may oppose actions of ERα in breast cancer cells and clinical studies indicate that loss of ERβ expression in ERα positive tumors is associated with a poor prognosis and perhaps resistance to tamoxifen therapy. Clinical evidence indicates that in post menopausal women hormone replacement therapy significantly decreased incidence of colon cancer, suggesting that estrogens have a protective role . We have recently found that in SW 480 colon cancer cells, (ER negative) stable expression of ERβ decreased SKP2, c-myc but not p27kip1 mRNA measured by real time PCR. These results were confirmed for c-myc and SKP2 at the protein level. However, ERβ expression increased only p27 protein levels. Addition of E2 did not regulate effects of ERβ expression. Similar results have been observed with T47D (ER positive breast cancer cells) stably over expressing ERβ. In the latter cells and MCF7 (ER alpha positive) cells over expressing ERβ, E2 induced β catenin protein expression was blocked by ERβ. The latter also blocked AKT and ERK activation by E2 and induced apoptosis, in an E2 independent fashion. ERβ expression also blocked E2 induced growth in MCF 7 cells. Antibody array analysis showed that ERβ regulated >100 proteins at or above the 2 fold level in T47D breast cancer cells,where as 2D PAGE/MS showed that over 60 proteins changed significantly, both types of analysis and Western blots indicate that ERβ regulates Sp1,HSP70,β catenin, c-Jun, JNK,c-Jun ,BAD among other proteins. Antibody arrays and MS have therefore revealed unsuspected targets of ERβ action. Thus, ERβ appears to oppose several actions of ERα in breast cancer cells and block colon cancer cell growth, independently of E2. Design of selective ERβ activating agents or delivery of ERβ using gene therapy may be of substantial utility in the treatment of breast and colon cancer. Category27. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3056.