Chinthalapally V. Rao - US grants

Chemoprevention of Azoxymthane - Induced Foci of Aberrant Crypts in Colons of Rats Treated with Chemopreventive Agents. The efficacy of a variety of chemopreventive agents to reduce or inhibit colon cancer using male Fisher 344 rats treated with a carcinogen + - chemopreventive agents is being assessed. Aberrant crypt formation four weeks after the first carcinogen exposure is being measured according to methods of Tudek, Bird, and Bruce (Cancer Research 49:1236-1240, 1989). All rats, approximately 8 weeks old, are treated with two injections of 15 mg of azoxymethane (AOM) per kg of body weight one week apart. An dose selection study is performed prior to the chemoprevention study for each agent at five doses. For Protocol A: Administer each agent at five doses to approximately seven-week-old rats. Feed the rats the test chemopreventive agent from one week prior to and four seeks following the first carcinogen treatment. Then kill the rats and score the aberrant crypts (see Bird, Cancer Lett.37:147-151, 1987). For Protocol B: Administration of the chemopreventive agents shall begin 4 weeks after the first carcinogen dose and continued for four weeks. At eight weeks following the first carcinogen treatment aberrant crypts will be scored. For both protocols, the number of aberrant crypt foci per colon and the number of aberrant crypts per foci from unsectioned methylene-blue stained colons are being determined. The two doses to be used in the aberrant crypt foci study are determined from data produced in the rangefinding assay and obtain approval from the Project Officer. Ten animals are being used per group. There are 20 agents in protocol A and 10 in protocol B.

Uptake and Absorption of Selected Chemopreventive Agents in Experimental Animals The objective is to develop and standardize assay protocols for selected chemopreventive agents which arf highly sensitive, quantifiable, and reproducible for serum (or plasma) and tissue measurements. Measure the concentration of selected chemopreventive agents in blood serum from rats and mice fed standardized diets containing various levels of chemoprevention agents. Serum levels of agents is being measured using tissues received from other sites from studies in progress. Protocols are being developed for the measurement of chemopreventive agents in serum or tissue extracts that are sensitive and quantitative. Methodology is being characterized for sensitivity, linear range, within and between day to day coefficient of variation. Animals (mice, rats, hamsters) are being fed agents at two levels in diets Blood samples are collected and serum extracted from 10 animals at baseline and after two weeks of feeding in each different dose group. The samples are being analyzed to determine agent concentration (muM) in serum extracted in early morning. The variance and standard deviation of the mean values are being determined. The absorption ratio is being determined by comparing the serum concentration versus the food concentration. At least two tissues for each agent are being determined and analyzed.

The overall objective of the proposal is to elucidate the inhibitory mechanisms in colon carcinogenesis by the naturally occurring agents curcumin and phenylethyl-3-methylcaffeate (PEMC). Colon cancer is the second most common malignancy in the Western world. In the USA lone, 56,600 annual deaths are attributed to colon cancer. Recent evidence suggests that non-steroidal anti-inflammatory agents (NSAIDs), use of the drugs for the prevention of colon cancer is limited due to severe gastrointestinal and renal toxicity. Our studies and those by others indicate that curcumin and PEMC, which are present in turmeric and honey respectively, inhibit the metabolism of arachidonic acid (AA) and also block the formation of azoxymethane (AOM)-induced aberrant crypt foci and adenocarcinomas in the colon of rats. Both these agents possess anti-inflammatory and anti-tumor activities, but, importantly, they do not have the undesirable toxic side effects of the NSAIDs. Thus, it is important to delineate the precise mechanisms that lead to the specific effects by curcumin and PEMC which have promise as safer anti-cancer agents for humans. Several studies indicate that specific activities of enzymes that affect AA metabolism and the modification of certain transduction pathways lead to suppression of tumorigenesis in the colon by these agents. The propose study is aimed at the elucidation of inhibitory processes by examining the selectivity of effects of curcumin and PEMC on enzymes involved in AA metabolism during several stages of AOM-induced colon carcinogenesis in the rat model. Specifically, we will examine the effects of natural curcumin and PEMC on both expression (RNA and protein levels) and activities of inducible nitric oxide synthases (iNOS), cyclooxygenase (COX) isoforms, and of lipoxygenase (LOX)-mediated metabolism and 12-LOX expression in colonic mucosa and in colon tumors of male F344 rats during defined stages of carcinogenesis. Tissue distribution and comparative metabolism studies of curcumin and PEMC will be studied with synthetic [3H] curcumin and [3H]-PEMC in vivo in male F-344 rats.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to determine the chemopreventive efficacy of a combination of lovastatin (3-hydroxy-3-methylglutaryl CoA reductase (HMG-R) inhibitor and celecoxib (COX-2-selective inhibitor) against colon cancer and to gain an understanding of the mechanism(s) of tumor inhibition by these agents. Colorectal cancer is one of the most common human malignancies in the United States, anticipated to account for 137,000 new cases and about 56,000 deaths in the year 2001. Developing chemopreventive agent(s) that aim to suppress tumor cell growth, but not normal cell growth, by targeting specific genes/factors that are responsible for tumor growth provides a rational approach. Our studies and those of others indicate that COX-2 and HMG-R activities were up-regulated several-fold in colon tumors compared to normal mucosa and, importantly, the metabolites/molecules derived from these enzymes play a pivotal role in modulation of apoptosis and proliferation. Recent evidence from clinical trials, and in vivo and in vitro laboratory studies suggest that application of a combination of HMG-R inhibitors (cholesterol-lowering drugs) and COX-inhibitors (nonsteroidal anti-inflammatory drugs) produces synergistic colon cancer-inhibiting effects. Thus, it is important to systematically develop HMG-R inhibitors and their combination with COX-2 inhibitors for colon cancer prevention and delineate the specific mechanisms that lead to modulation of apoptosis and proliferation by these agents. Specifically, we will examine 1) the chemopreventive efficacy of lovastatin on azoxymethane (AOM)-induced colon carcinogenesis in rats (maximum tolerated dose selection; dose-response effects; and effectiveness during promotion/progression stages, 2) study the synergistic effects of lovastatin and celecoxib on AOM-induced colon carcinogenesis and assess effectiveness of these agents in combination on the promotion/progression stages, and 3) elucidate mechanisms by determining the effect(s) of lovastatin with or without combination of celecoxib on HMG-CoA reductase, FPTase, GGPTase, p53, p21CIP/WAF1, caspase-3 &- 6, Bax, Bcl-2, Fas and lamin B, COX-2, PPAR-y, p53, and prostaglandins levels. Finally, we will study the effects of these agents on cell proliferation, and apoptosis during different stages of colon carcinogenesis.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop the use of inducible nitric oxide synthase (iNOS)-selective inhibitors in the chemoprevention of colorectal cancer, and to gain an understanding of the cellular and molecular mechanism(s) of tumor inhibition by these agents. In addition, we will design the strategies for improving the efficacy of colon cancer prevention and treatment by concurrent application of iNOS- and COX-2 selective inhibitors. Colorectal cancer is one of the most common human malignancies in the United States, anticipated to account for 140,000 new cases and about 56,000 deaths in the year 2003. Developing treatment strategies, aimed at a specific molecular target that facilitate(s) tumor cell growth, uncontrolled expansion and invasion, provide a rational approach. Nitric oxide, produced by isoforms of NOS, has been implicated in several pathophysiological conditions including colon carcinogenesis. Our studies and those of others indicate that iNOS activities were up-regulated several-fold in colon tumors compared to normal mucosa and, importantly, nitric oxide or its reactive molecules derived from these enzymes play a pivotal role in modulation of apoptosis and proliferation. Recent evidence from our laboratory suggests that iNOS-selective inhibitors suppress chemically-induced colon carcinogenesis and also tumor formation in transgenic APC min mice. Thus, it is important to systematically develop iNOS-selective inhibitors for colon cancer prevention/treatment and delineate the specific mechanisms that lead to inhibition of tumorigenesis by these agents. Specifically, we will 1) examine the chemopreventive efficacy of different iNOS-inhibitors [PBIT, NILT and BIPPA] on azoxymethane (AOM)-induced colon carcinogenesis in rats (maximum tolerated dose selection; dose-response effects; and effectiveness during promotion/progression stages of colon carcinogenesis; 2) establish strategies to improve efficacy of colon cancer prevention and treatment by a combination of COX-2- and iNOS-selective inhibitors and 3) assess the cellular and molecular biomarkers associated with iNOS inhibition/COX-2 inhibition (apoptotic and proliferation changes, nitric oxide, 3-nitrotyrosine, and expression and activities of isoforms of NOS and COX) and study the modulation of gene expression profiles to identify changes in functional groups of genes associated with apoptosis, cell-cycle regulation and iNOS and COX-2 mediated signals and 4) understand the mechanisms by which inhibitors of iNOS and COX-2 modulate colon tumor cell proliferation and apoptosis.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop the use of inducible nitric oxide synthase (iNOS)-selective inhibitors in the chemoprevention of colorectal cancer, and to gain an understanding of the cellular and molecular mechanism(s) of tumor inhibition by these agents. In addition, we will design the strategies for improving the efficacy of colon cancer prevention and treatment by concurrent application of iNOS- and COX-2 selective inhibitors. Colorectal cancer is one of the most common human malignancies in the United States, anticipated to account for 140,000 new cases and about 56,000 deaths in the year 2003. Developing treatment strategies, aimed at a specific molecular target that facilitate(s) tumor cell growth, uncontrolled expansion and invasion, provide a rational approach. Nitric oxide, produced by isoforms of NOS, has been implicated in several pathophysiological conditions including colon carcinogenesis. Our studies and those of others indicate that iNOS activities were up-regulated several-fold in colon tumors compared to normal mucosa and, importantly, nitric oxide or its reactive molecules derived from these enzymes play a pivotal role in modulation of apoptosis and proliferation. Recent evidence from our laboratory suggests that iNOS-selective inhibitors suppress chemically-induced colon carcinogenesis and also tumor formation in transgenic APC min mice. Thus, it is important to systematically develop iNOS-selective inhibitors for colon cancer prevention/treatment and delineate the specific mechanisms that lead to inhibition of tumorigenesis by these agents. Specifically, we will 1) examine the chemopreventive efficacy of different iNOS-inhibitors [PBIT, NILT and BIPPA] on azoxymethane (AOM)-induced colon carcinogenesis in rats (maximum tolerated dose selection; dose-response effects; and effectiveness during promotion/progression stages of colon carcinogenesis; 2) establish strategies to improve efficacy of colon cancer prevention and treatment by a combination of COX-2- and iNOS-selective inhibitors and 3) assess the cellular and molecular biomarkers associated with iNOS inhibition/COX-2 inhibition (apoptotic and proliferation changes, nitric oxide, 3-nitrotyrosine, and expression and activities of isoforms of NOS and COX) and study the modulation of gene expression profiles to identify changes in functional groups of genes associated with apoptosis, cell-cycle regulation and iNOS and COX-2 mediated signals and 4) understand the mechanisms by which inhibitors of iNOS and COX-2 modulate colon tumor cell proliferation and apoptosis.

Abstract The overall goal of this proposal is to develop effective chemopreventive strategies and understand the molecular mechanism of colon tumor inhibition by a combination of statins (3- hydroxy-3methyl glutaryl CoA reductase (HMG-R) inhibitors) and polyamine inhibitor, DFMO against colon cancer. Colorectal cancer is one of the most common malignancies in both men and women in the US. In developing translational strategies, a preferable approach is to target multiple signaling pathways that selectively contribute towards tumor growth, so that it provides a synergistic/additive efficacy. Colon tumor cells produce high levels of polyamines endogenously through the activation of ornithine decarboxylase (ODC). DFMO selectively inhibits ODC activity and thereby endogenous polyamine synthesis and colon tumor growth. New insights into the transport of polyamines in tumor cells by caveolin-1 (cav-1) and SLC3A2 mediated pathways suggest that new approaches are needed for effective polyamine regulation in tumor cells. In spite of the fact that DFMO inhibits endogenous polyamine synthesis, tumor cells can still uptake extracellular polyamines through a cav-1 dependent endocytic mechanism. Further, Cav-1 plays an important role in the generation of nitric oxide (NO) and activation of AKT and Rho-signaling, leading to enhanced tumor cell proliferation and invasion. Our preliminary results suggest, that a combination of rosuvastatin with low-dose DFMO suppresses azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF), and aberrant crypt cell proliferation. Our in vitro and in vivo results suggest that cav-1 plays an important role in colon cancer, and statins exhibit colon tumor inhibition in part by modulating cav-1. Therefore, we want to develop a combination of statin and DFMO for colon cancer prevention/treatment and understand the role of cav-1 in colon cancer and its possible regulation by miRNAs. Specific aims are 1) Determine the in vivo efficacy of Rosuvastatin (Crestor) in a F344 rat colon carcinogenesis model (maximum tolerated/optimal dose selection; dose- response effects; and effectiveness during promotion/progression stages (early/late interventions). 2) Determine the combinational efficacy of atorvastatin/rosuvastatin and DFMO on the progression of colonic ACF (post-initiation/early stage) or adenoma (progression/late stage) to colon adenocarcinoma formation in rats. We also want to determine the effect of statins and DFMO on the levels of cav-1, polyamines, ODC activity, NO (iNOS and eNOS activities), SLC3A2, AKT and Rho signaling proteins, miRNA modifications in colonic mucosa and tumor tissues and correlate these results with colon tumor inhibition. 3) Define the role of cav-1 in colon carcinogenesis using cav-1-/- knockout mouse model and determine the effect of statins with or without DFMO on carcinogen induced colon tumor formation in cav- 1-/- and cav-1+/+ mice. In addition, proposed research will elucidate the role of selective miRNAs on the regulation of cav-1 in colon cancer cells, and assess the possible modulatory role of statins and DFMO on cav-1 miRNA regulation.

The overall objective is to determine the potential clinical usefulness of aspirin or naproxen and omeprazole administered together for colorectal cancer (CRC) prevention in preclinical models. In spite of extensive preclinical data on the efficacy of aspirin, naproxen and, to certain extent, omeprazole on CRC chemoprevention, there are significant concerns in translating preclinical doses to human clinical trials. Another concern is that long-term NSAIDs use is associated with gastrointestinal (GI) side-effects, thus limiting the application in individuals at high-risk for CRC. The hypothesis is that combining aspirin or naproxen with omeprazole will provide a safer approach to eliminate the GI toxicity and improve the chemopreventive efficacy of aspirin or naproxen. Thus, it is imperative to develop clinically relevant doses of aspirin or naproxen in combination with omeprazole for CRC prevention in high-risk individuals.

The purpose of this Task Order is to hold an orientation/kickoff meeting to discuss Standard Operating Procedures (SOP) for all Contractors awarded an Indefinite Delivery, Indefinite Quantity (ID/IQ) contract for the PREVENT Preclinical Efficacy and Intermediate Endpoints Biomarkers acquisition.

Several Genetically Engineered Mouse (GEM) models are available which mimic some or all of the features for pancreatic ductal adenocarcinoma (PDAC) development. However, due to varied molecular features in different models, selecting the appropriate model is challenging and critical information on incidence rates, tumor multiplicity, and rate of progression for chemoprevention studies is needed. In humans, pancreatic precursor lesion development and progression occurs in the adult pancreas whereas a number of GEM models involve activation of mutated driver mutations during late embryonic development. Induction of precancerous and cancerous pancreatic lesions in these models usually occurs in the absence of pancreatitis. However, pancreatitis appears to be an important risk factor for PDAC in human patients. Hence, optimizing mouse models that represent high-risk cohorts of pancreatic cancer subjects who will participate in human chemoprevention trials will be important for translating preclinical chemopreventive observations to human clinical trials. The overall objective of this task order is to determine and characterize the temporal development of neoplastic pancreatic lesions in the genetically engineered mouse (GEM) Pdx1Cre/LSL-KrasG12D;Tif1?flox/flox model for IPMN formation and Ela-CreERT;CAG-lox-KrasG12v model for cystadenomas, pancreatitis and PanIN formation and their progression to pancreatic ductal adenocarcinoma (PDAC). These models will be optimized for use in preclinical chemoprevention studies.

Prostate cancer (PC) is the most common cancer and the second leading cause of cancer death in men in the United States. Two major phase III PC chemoprevention trials failed to yield results supportive of Food and Drug Administration (FDA) approval of the respective agents for prevention. Selenium and vitamin E, chosen based on promising secondary endpoints in non-PC prevention trials, both failed to show a benefit in PC reduction in the Selenium and Vitamin E Cancer Prevention Trial (SELECT), although an increase in PC was observed with vitamin E. The Prostate Cancer Prevention Trial (PCPT) demonstrated a decrease in PC risk with finasteride, but cancers that did occur tended to be of higher grade, discouraging approval of the agent for a risk-reducing indication. Thus, a need exists for an effective but non-toxic chemopreventive intervention for PC risk reduction. Aspirin has been shown in multiple observational studies and clinical trials to be associated with reduction in risk of a number of cancers, particularly malignancies of the gastrointestinal tract. This feature, together with its relative non-toxicity and beneficial cardiovascular effects, suggests aspirin as a promising chemopreventive agent. Although the benefits are less for PC than for GI cancers, several meta-analyses have shown a 10% reduction in risk of developing or dying from PC in association with aspirin use. Similarly, randomized trials have demonstrated a decrease in risk of death. Yet, while prevention of colon and mammary cancers with aspirin has been studied extensively in the preclinical setting, no similar studies have been carried out for PC. The mechanisms underlying the preventive activity of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) are complex. However, their anti-inflammatory activity, including inhibition of cyclo-oxygenases 1 and 2 (COX-1, 2), resulting in reduction of several prostaglandins (PGs) as well as thromboxane A, have held center stage as potential anti-cancer mechanisms. This may be particularly applicable to PC, which involves inflammation. Additional mechanisms to explain the anti-cancer activity of aspirin and other NSAIDs have been proposed. Among these is enhancement of the immune system, suggesting a potential immunological therapeutic effect by these agents. As one example, evidence suggests that NSAIDs limit carcinogenesis by preventing PGE2-induced maturation of monocytes into immunosuppressive myeloid derived suppressor cells (MDSCs). This additional domain of NSAID activity suggests that combining agents such as aspirin with immunological interventions holds promise in the area of prevention. Prior to combining aspirin/NSAIDs with immune strategies, however, appropriate preclinical studies of the former alone are required. Prior experience has shown that preclinical efficacy studies are critical predictors of outcomes in follow-up clinical trials in humans. A stark example is the negative preventive outcomes observed in the SELECT trial which were predicted by animal studies. A promising example of an animal model of PC is the TMPRSS2-ERG fusion mouse. A recurrent chromosomal rearrangement in PC involves juxtaposition of the 5? untranslated region of the TMPRSS2 gene and ETS family genes, which consist of oncogenic transcription factors such as ERG and ETS. The TMPRSS2 gene is a prostate-specific, androgen-responsive, transmembrane serine protease gene, whereas the ETS and ERG genes encode transcription factors that lead to cell proliferation. Fusions of the TMPRSS2 untranslated 5? exons to the ERG or ETS ?cancer genes? are observed in about 50% of localized prostate cancers and are generally associated with overexpression of the oncogene (?ERG?) component. When fused, upregulation of TMPRSS2 in response to androgen stimulation leads to activation of the ?ERG? gene component and hence to cell proliferation. In the genetically engineered TMPRSS2-ERG fusion mouse model, the TMPRSS2-ERG fusion construct is under the control of the ARR2-Probasin promoter, which has been used in previous models of prostate cancer and prostate intraepithelial neoplasia. However, the TMPRSS2-ERG fusion alone does not induce prostate intraepithelial neoplasia (PIN), a precursor to invasive PC. Rather, it requires concomitant activation of the PI3 kinase pathway, as for example, via inactivation of PTEN. Support for the testing of aspirin in a TMPRSS2-ERG fusion mouse model comes from a small population-based case-control study of PC in which a significant reduction in relative risk with aspirin use was observed for fusion-positive cases, but not fusion-negative cases.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States, and recent evidence indicate that the incidences of CRC has increased in adults <50 years of age. As such, new prevention strategies are urgently needed. ONC201 is a small molecule drug candidate with in vitro cytotoxicity associated with the induction of TNF-related apoptosis-inducing ligand (TRAIL), and subsequent TRAIL-dependent cell death. In vivo, ONC201 induces TRAIL and causes potent antitumor effects when administered as a single dose in mice with human CRC xenografts. A phase I clinical trial with ONC201 in patients with advanced solid tumors, showed some evidence of efficacy (stable disease) at doses that were well tolerated (no drug-related toxicities greater than grade 1). Micromolar plasma concentrations were achieved in these subjects after oral dosing, concentrations that were in the preclinical therapeutic range. ONC-201 is currently being tested in a phase II clinical trial in subjects with select advanced solid tumor cancers. The purpose of this Task Order is to determine whether ONC201 has chemopreventive activity in a mouse CRC model.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the US, with more than 1.35 million new cases diagnosed each year. Although non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and cyclooxygenase-2 (COX-2) inhibitors were shown to be effective in preventing CRC in animal models and in the clinic, their use is severely limited by gastrointestinal and cardiovascular toxicities. Thus, there is an unmet need for the identification of safer chemopreventative agents for CRC. The third-generation angiotensin receptor blocker (ARB) Olmesartin Medoxomil is the most commonly prescribed medication in the US for the treatment of hypertension. ARBs and angiotensin converting enzyme (ACE) inhibitors have been shown to reduce inflammation and inhibit cell growth and survival, and several retrospective case-control studies have revealed an association between the use of these drugs and a reduced risk for several different types of cancer, including CRC. In addition, the angiotensin signaling inhibitors, Captopril and Telmisartan, have been shown to suppress the formation of colonic aberrant crypt foci in animal models of CRC. The known safety profiles, cardiovascular protective properties, and pre-clinical efficacy of ARBs and ACE inhibitors make them attractive candidates for CRC chemopreventative agents. The purpose of this Task Order is to evaluate Olmesartin Medoxomil for CRC prevention.

Project Summary The major purpose of this proposed research is to develop mechanistically based, effective, and safer agents for colorectal cancer (CRC) chemoprevention. Every year, about 150,000 Americans are diagnosed with colorectal cancer (CRC), the second leading cause of cancer-related mortality in the US. About 1.35 million new CRC cases are diagnosed worldwide, highlighting that CRC is a major health problem. Evidence from our group and others suggests that NSAIDs and select COX-2 inhibitors show significant inhibitory effects in preclinical models and patients with CRC, but these inhibitors are also associated with gastrointestinal (GI) toxicity and cardiovascular (CV) risk. Reasons for these risks include increased 5-LOX metabolites and reduced PGI2 synthesis. Thus, selectively targeting microsomal PG Synthase-1 (mPGES-1) and 5-LOX would block the protumorigenic PGE2/prothrombotic LTs, but spare the PGI2. This approach is ideal for developing efficient and safer CRC chemopreventive agents. Toward this end, through high-throughput and enzyme kinetics assays and short- term in vivo efficacy studies, we have discovered a novel dual mPGES-1 /5-LOX inhibitor, CDPDPA. In this proposal, we seek to further develop CDPDPA for CRC chemoprevention. We have designed the research strategies to assess the pharmaco-dynamic dose-response efficacy, understand the role of mPGES-1/5- LOX, improve efficacy and safety through combinatorial approaches, and evaluate CV risk, if any, of CDPDPA compared with COX-2 inhibitor. We have assembled a team with expertise in CRC chemoprevention and cardivascular research to undertake following aims. 1). Determine whether targeting both mPGES-1 and 5-LOX with CDPDPA is efficacious in AOM-induced rat colon adenocarcinoma treatment; 2) Determine the source and relative contribution of mPGES-1 and 5-LOX to colon tumor developmen;t 3). Determine whether combinational targeting of mPGES-1/5-LOX with statin would improve the colon tumor inhibition efficacy and reduce cardiovascular side effects compared with celecoxib, and; 4). Determine the potential CV risk, if any, of long-term administration of CDPDPA compared with Celecoxib in LDLr-/-ApoB100/100 mice. The completion of this project will significantly improve the safety and efficacy of this novel drug for the prevention and treatment of CRC.