Chung Shu Yang - US grants

Cytochrome P-450ac is an enzyme existing is human, rat and other animal species, and is inducible by fasting, diabetes, ketogenic diets, as well as chemicals such as acetone, alcohols, pyrazole, and isoniazid. It is a key enzyme in the metabolism of acetone, N-nitrosodimethylamine, ethers, alkanes, CCl4, enflurane, and other xenobiotics. In the past few years, our laboratory has made major contributions to the understanding of this enzyme. Our long term objective is to understand the detailed molecular mechanisms of the regulation of this important enzyme, the structural basis for its catalytic activity, and its roles in the metabolism of drugs and environmental chemicals. The specific aims of this project are as follows: 1. To elucidate the mechanisms of the regulation of P-450ac by examining the roles of acetone, other ketone bodies, and hormones in the induction of this enzyme in the rat liver. 2. To characterize the molecular events in the regulation of P-450ac by measuring transcriptional and translational activities as well as the stabilities of mRNA and protein. 3. To investigate the sex-related differences and hormonal regulation of P-450ac in the mouse kidney at the physiological, biochemical, and molecular biological levels. 4. To understand the active site dynamics and catalytic functions of P-450ac by examining the structural characteristics of its substrates, by deriving structural information from the amino acid sequence, by modifying specific amino acid residues in the active site with a site-directed mutagenesis approach, and by studying the metabolism and toxicity of environmental chemicals. The project is expected to provide important new information concerning the regulation of P-450ac as well as its roles in the metabolism of drugs and toxic chemicals. This basic information should be very useful in our understanding of environmental toxicology, chemical carcinogenesis, and drug-nutrient interaction.

Tea is one of the most popular beverages worldwide. Whereas epidemiological studies have so far yielded inconclusive results, laboratory studies by us and others clearly indicate that green and black tea extracts inhibited carcinogenesis in animal models. In this project, we plan to further investigate the inhibitory actions of green an black teas and their components on nitrosamine-induced carcinogenesis in animal models with the following specific aims: 1. To determine the effects of green and black tea extracts and selected tea components on lung, forestomach, and esophageal carcinogenesis induced by specific nitrosoamines in animal models. The dose-response of the active components given at different stages of carcinogenesis will be studied systematically. 2. To elucidate the mechanisms of the inhibition of carcinogenesis by tea and tea components in the above models. Parameters related to their antioxidative, nucleophilic, and other properties pertaining to the carcinogenic processes will be examined. 3. To understand the biological effect of (-)-epigallocatechin gallate and other tea components by studying their absorption, metabolism, bioavailability, and tissue levels. 4. To fractionate green and black tea components, characterize their properties, determine their activities in short-term assays and prepare selected compounds in large quantities for the above studies. The project is expected to provide information fundamental to the understanding of the relationship between tea consumption and carcinogenesis. It will develop theoretical basis and laboratory methods for future epidemiological and intervention studies on tea and human cancer.

A variety of organic sulfides have been found to inhibit the toxicity or carcinogenicity of environmental chemicals. The investigators' previous studies indicate that rat hepatic cytochrome P450IIE1, an enzyme that activates low molecular weight organic compounds, is inhibited and inactivated by diallyl sulfide (DAS), a dietary chemical, and disulfiram (DSF), a drug used in avoidance therapy for alcohol abuse. The long-term goal of this project is to understand the biological action of this type of organic sulfide and to explore their usefulness in the prevention of chemical toxicity and carcinogenesis. The present proposal seeks to: further characterize the effects of DAS and DSF administration, both chronic and acute, on P450s and other enzymes in the liver and other tissues by specific enzyme assays and immunoquantitation; elucidate the molecular mechanisms of inhibition and inactivation of P450IIE1 by DAS and DSF in vivo and in vitro; delineate the effects of organic sulfides on the metabolism and toxicity of selected xenobiotics; explore the possible use of organic sulfides for the prevention of chemical toxicity; characterize other biological effects of organic sulfides; and evaluate the relevance of dietary levels of organic sulfides to the protection against chemical toxicity. P450IIE1 plays a vital role in catalyzing the activation of many environmental toxicants and carcinogens. The proposed studies on the inactivation of this enzyme by organic sulfides may form the basis for the prevention of toxicity and carcinogenesis by this type of compound either derived from the diet or taken as prophylactically.

Cytochrome P450 2E1 is a key enzyme in the biotransformation of environmental chemicals. The long term objectives of this project are to understand the catalytic and physiological functions of this enzyme and its roles in environmental toxicology. Significant progress toward these goals was made during the previous grant period. The specific aims of this renewal grant are as follows: 1. To establish a heterologous expression system for 2E1 with the aims of obtaining purified 2E1 for catalytic studies and of conducting site- directed mutagenesis studies to elucidate structure-function relationships. 2. To define the structural features of substrates and inhibitors of 2E1 and to understand the active-site dynamics of this enzyme. These will be accomplished by studying the kinetic parameters (Km, Vmax, Ki) of 2E1- catalyzed reactions and by active-site modeling together with a site- directed mutagenesis approach. 3. To elucidate the normal physiological functions of 2E1 by overexpressing and inactivating (by a homologous recombination approach) the 2E1 gene in transgenic mice. The effects of these genetic manipulations on the survival, development, and physiological functions will be investigated. 4. To further characterize the physiological functions of 2E1 and its roles in the activation or detoxification of environmental chemicals. Both transgenic mice and other animals will be used to study the tissue distribution of 2E1, the physiological role of the 2E1-dependent gluconeogenesis from acetone, roles of 2E1 in the generation of peroxides and the metabolism of lipid hydroperoxides, and roles of 2E1 in the activation or detoxification of selected environmental chemicals. 5. To characterize the metabolic activation of acetaminophen by 2E1 and other enzymes, and to delineate its modulation by dietary chemicals such as flavonoids. Both in vitro and in vivo studies will be pursued in rodents and in humans. The proposed work is expected to contribute significantly to our understanding of the unique catalytic activities and physiological functions of 2E1 as well as its roles in the activation or detoxification of environmental chemicals and drugs.

DESCRIPTION Esophageal cancer is one of the most common cancers worldwide with a well-demonstrated variation in its incidence among different populations. Exposure to environmental carcinogens is believed to be a major causative factor for human esophageal cancer and cytochrome P450 plays a key role in the metabolic activation of most environmental carcinogens. In this proposal the investigators will investigate the relationship between CYP2E1 polymorphisms and esophageal cancer risk. First, they will look for an association between genotypes at the CYP2E1 locus and esophageal cancer using patients and their matched non-cancer controls. They will also look for an interaction between genotypes at CYP2E1 and the glutathione S-transferase M1 (GSTM1) locus on cancer risk. Second, they will look for correlations between the CYP2E1 genotypes and phenotypic expression in non-cancerous esophageal samples. The expression of CYP2E1 will be determined by immunohistochemical analysis.

p53 gene /protein; tumor suppressor proteins; esophagus neoplasm; carcinogenesis; squamous cell carcinoma; early diagnosis; neoplasm /cancer genetics; loss of heterozygosity; oxidative stress; cancer risk; biomarker; prognosis; gene mutation; neoplasm /cancer diagnosis; preneoplastic state; neoplasm /cancer epidemiology; longitudinal human study; serial analysis of gene expression; flow cytometry; China; clinical research; immunocytochemistry; biopsy; human subject; microarray technology;

The purpose of this proposal is to obtain funds to support an international symposium entitled "Dietary Factors in Cancer Prevention: Molecular Mechanisms and Applications." The symposium, jointly-sponsored by the Environmental and Occupational Health Sciences Institute and the Cancer Institute of New Jersey, will be held at the Busch Campus of Rutgers, The State University, in Piscataway, New Jersey, from June 12 to 14, 1997. Funds are requested to support the travel and lodging of some of the invited speakers. The objective of the symposium is to discuss key issues concerning the use of dietary factors in cancer prevention. This symposium, with the participation of many leading investigators and younger scientists from different disciplines, will help promote and strengthen this research area. Through scientific presentations, discussion sessions, and poster sessions, the symposium will l) discuss molecular mechanisms by which dietary constituents and nutritional status affect cell growth and cancer formation, 2) identify relevant areas and effective approaches for future research, and 3) discuss approaches for extending laboratory studies to practical applications in human cancer prevention. The symposium will foster interactions between laboratory scientists and epidemiologists as well as between laboratory and clinical scientists in the development of translational research. It will also encourage the use of new biochemical and molecular tools in cancer-prevention research. A special feature of this symposium will be the use of "discussion sessions" to examine key issues and identify effective approaches for cancer prevention through diet. Such sessions on "Translational research," "Dietary modifications," and "Contributions from the food industry" will help to develop practical approaches for cancer prevention to achieve the goals of "Healthy People 2000."

DESCRIPTION: (Applicant's Abstract) The long-term goal of this project is to elucidate the mechanisms of inhibition of carcinogenesis by tea (Camellia sinensis) and to assess its usefulness in the prevention of human cancer. Previous studies have demonstrated that tea preparations inhibit carcinogenesis in animal models and that tea polyphenols inhibit cell transformation, proliferation, and related signal transduction pathways. Plasma and tissue levels of tea polyphenols and their metabolites in animals and humans have been determined. In this project the applicant and colleagues plan to further elucidate the mechanisms of action and identify the active components involved with the following specific aims: 1. To elucidate the mechanisms of inhibition of carcinogenesis by tea in the NNK-induced lung carcinogenesis model in A/J mice and in relevant cell lines. The applicant and colleagues will study the inhibition of cell proliferation and tumor promotion by tea and tea constituents, and relate the activity to pertinent signal transduction pathways (such as MAP-kinases and AP-1 activation) in short and long term animal experiments. In-depth mechanistic studies in cell lines, on the inhibition of AP-1 and NFkB and the upstream of protein kinase cascade, will complement the animal studies and provide basic understanding of the action of tea polyphenols in general. 2. To determine the blood, urine, and tissue levels of tea polyphenols and their metabolites in rodents and humans under different experimental conditions and to understand the factors influencing these levels. Improved methods will be developed to include the analysis of many newly identified metabolites in pharmacokinetic studies. The blood and tissue levels of these compounds will serve as a reference for evaluating the mechanisms of anti-carcinogenesis and for comparing results in mice and humans. 3. To synthesize and determine the biological activities of the metabolites identified in Aim 2 in cell lines and animal models. The applicant and colleagues will address key issues concerning the bioavailability and bioactivities of O-methyl, glucuronide, and sulfate derivatives and two ring-fission metabolites of tea catechins.

DESCRIPTION: (adapted from the investigator's abstract) The long-term goal of this project is to provide a mechanistic basis for the prevention of esophageal adenocarcinoma (EAC), a disease with a rapidly increasing incidence rate and a poor prognosis. For this purpose, the investigators have modified an esophagoduodenal anastomosis (EDA) model by giving iron supplements to rats. This model mimics human esophageal pathogenesis by causing chronic gastroduodenal-esophageal reflux and producing Barrett's esophagus (BE) and EAC, without the use of any carcinogen. Their hypothesis is that oxidative cellular damage, which is caused by reflux-induced inflammation, is a major factor driving the EAC pathogenic process, and thus preventable with the use of antioxidants and anti-inflammatory agents. They will test this hypothesis in the rat model and develop prevention approaches with the following specific aims: 1) To improve the nutritional status of the animals in their model and to investigate the effect of iron nutrition on oxidative cellular damage and the formation of BE/EAC. Rats (after surgery) will be maintained on an enriched AIN93 diet and supplemented with iron by different routes. Parameters reflecting inflammation and oxidative cellular damage and the formation of BE/EAC will be studied. 2) To test the hypothesis that oxidative stress is a major causative factor for the pathogenesis of EAC by studying its inhibition by antioxidative nutrients (agents). The effects of vitamin E, selenium, and N-acetylcysteine supplementation on oxidative damage parameters and their relationship with esophageal pathogenesis will be investigated. 3) To investigate the possible inhibition of BE/EAC formation by nonsteroidal anti-inflammatory drugs (NSAIDs). Sulindac, sulindac sulfone, and selective cyclooxygenase 2 (COX-2) inhibitors will be used to examine the roles of inflammation and COX-2 in esophageal adenocarcinogenesis. 4) To develop a model for studying the progression of esophageal metaplasia to adenocarcinoma and to study its inhibition by antioxidants and NASIDs.

DESCRIPTION (provided by applicant) The inhibitory action of tea (Camellia sinenis) and tea components against carcinogenesis has been demonstrated in animal models. The overall goal of this program project is to elucidate the mechanisms of inhibition of carcinogenesis by tea and the active components involved. The common hypothesis is that tea polyphenols and caffeine, and their metabolites, inhibit carcinogenesis by suppressing cell proliferation and enhance apoptosis via modulating key signal transduction and metabolic pathways. The inhibition of AP-l related signaling pathways and arachidonic acid metabolism will receive major attention. A novel hypothesis to be tested is that tea consumption lowers body fat levels and this effect contributes to the inhibition of carcinogenesis. The themes of the three Projects are as follows: Project 1, "Effects of Tea on the Formation and Growth of Skin Tumors," will investigate the inhibition of skin carcinogenesis in UVB-induced high-risk mice by orally and topically applied tea, caffeine, and EGCG; the mechanisms of growth inhibition and apoptosis induction; and the role of "lowering body fat levels" in anticarcinogenesis. Project 2, "Inhibition of Lung Carcinogenesis and General Mechanistic Studies," will study the entailed mechanisms in vivo and in vitro (especially on the inhibition of signal transduction kinases), the biotransformation and tissue levels of tea constituents, and the biological activities of key constituents and metabolites. Project 3, "Modulation of Signal Transduction Pathways by Tea Components" will focus on mechanisms key? to the inhibition of cell transformation and apoptosis in cell lines by caffeine and polyphenols as well as their derivatives, using cell and molecular biology approaches. Facilitated by the "Tea Chemistry and Analysis Core" and "Administrative Core, "the three Projects are highly interactive and integrated. Jointly, we intend to make major contributions to the use of tea for the I chemoprevention of cancer.

DESCRIPTION (provided by applicant): Diet is known to play important roles in influencing the development of human cancer, but the mechanisms are not well understood. The objective of this planning grant is to organize a multidisciplinary collaborative program to investigate the molecular mechanisms by which certain dietary constituents inhibit carcinogenesis. Our collaborative program will consist of investigators with demonstrated expertise in nutrition, carcinogenesis, molecular genetics, and signal transduction. We plan to select catechins, caffeine, and curcumin as the main dietary chemicals for mechanistic investigations. These compounds occur widely in fruits, beverages, or spices, and their cancer preventive activities have been demonstrated in animal models. It would be important to elucidate in depth their mechanisms of action and to determine whether these mechanisms are relevant in human cancer prevention. We plan to focus our studies on skin and colon cancers. Well established animal models and new transgenic mouse models, as well as relevant cell lines will be used. Our hypothesis is that catechins, caffeine, and curcumin suppress carcinogenesis by one or more of the following mechanisms: inhibition of inflammation, cell proliferation, cell transformation, and angiogenesis, and induction of apoptosis. We will study the key signaling and metabolic pathways leading to these processes, such as MAP-kinase, AP-1, Nfr,Kappa B,p53, and arachidonic acid metabolism. We will use DNA microarray and proteomic approaches to study multiple genetic pathways. A key feature of our program is to integrate studies in animal models, cell lines, and humans. Cell line studies will generate hypotheses on possible mechanisms of chemoprevention, and the hypotheses will be tested in animal models and humans. It would be important to elucidate the chemopreventive mechanisms most relevant to humans. We plan to create a structure to facilitate effective communication and interactions among the collaborators. A web-site is being set up to pool and share information and research results. We plan to establish facility Cores to facilitate the research. Monthly meetings and workshops among the investigators will be scheduled to design strategies and make study plans for the U54 application.

DESCRIPTION (provided by applicant): Hypermethylation of promoter CpG islands is an important mechanism to silence the expression of many tumor suppressors, DNA repair, and other genes in cancer. The long-term goal of this project is to study the inhibition and reversal of this process by dietary polyphenols for the purpose of prevention and treatment of cancer. In preliminary studies, we observed that treatment of human esophageal cancer KYSE 510 cells with the tea polyphenol EGCG caused the reversal of hypermethylation of RARbeta, p16, MGMT, and hMLH1 and the regaining of gene expression. EGCG also inhibited DNMT activity in nuclear extracts. This property of EGCG and related compounds could be explored for cancer chemoprevention and therapy. Our hypothesis is that EGCG and some other dietary polyphenols can inhibit DNMT, prevent or reverse the gene silencing caused by hypermethylation, and contribute to the inhibition of carcinogenesis, In this proposal, we plan to test this hypothesis with the following Specific Aims: 1. To quantify the demethylation and reactivation of hypermethylation-silenced genes (MSGs) such as RARbeta, p16, MGMT, and hMLH1 by EGCG, characterize the pattern of CpG demethylation, and determine the sustainability of the reactivated gene expression. 2. To elucidate the mechanisms by which EGCG inhibits DNMT activity and DNA hypermethylation. The leading hypothesis is that inhibition of DNMT1 by EGCG causes demethylation. Other mechanisms such as those involving AdoMet levels and other methyltransferases, will also be examined. 3. To establish the above observed effects as general phenomena by determining the spectrum of MSGs that are reactivated by EGCG (using methylation microarrays), examining the effects of EGCG on other cell lines (oral, colon, and prostate cancers), and investigating the effects of other dietary polyphenols. 4. To determine the possible synergistic effects in the reactivation of MSGs when EGCG is used in combination with a HDAC inhibitor (butyrate or TSA) or retinoic acid. Possible effects on global hypomethylation will be analyzed. 5. To determine whether EGCG, alone or in combination with other agents, can prevent hypermethylation of genes and tumor development in the intestinal tumorigenesis model in the Min mice.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The objective of this project is to study the inhibition of lung carcinogenesis by green tea polyphenols and the synergistic action when used in combination with atorvastatin (ATST, trade name Lipitor). Tea is commonly consumed by humans, and Lipitor is a popular cholesterol-lowering drug. Based on our preliminary results, we hypothesize that green tea polyphenols, especially the major polyphenol (-)- epigallocatechin-3-gallate (EGCG), interact synergistically with ATST in the inhibition of lung carcinogenesis. The possible use of the combination of EGCG and ATST for lung cancer prevention is an attractive strategy that needs more investigation. To test our hypothesis, these agents will be studied in a 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis model in A/J mice and related cell lines. The inhibitory action and the mechanisms involved will be thoroughly investigated with specific aims as follows: 1. Determine the inhibitory actions of EGCG and its combination with ATST in an NNK-induced mouse lung carcinogenesis model at the post-initiation and progression stages. We will use different concentrations of EGCG (0.1, 0.2, & 0.4%) and ATST (0.01, 0.02, & 0.04%) administered in the diet to determine the dose-response relationship and the interactions of these two agents in the inhibition of lung carcinogenesis. The inhibitory action will be correlated with the levels of EGCG and ATST in lung tissues and plasma. 2. Elucidate the mechanisms of inhibition of lung carcinogenesis by EGCG and its combination with ATST in NNK-treated mice. Using samples from Aim 1, we will examine the effects of the different treatments on cell proliferation, apoptosis, angiogenesis, and related molecular changes (e.g., Erk1/2, Akt, JNK, VEGF, arachidonic acid metabolism, and oxidative stress parameters) and on membrane association of small G-proteins using immunohistochemical and biochemical analyses. Short-term animal experiments with tumor-bearing mice will be used as a direct approach to obtain mechanistic information in vivo. 3. Delineate detailed mechanisms of lung cancer prevention by EGCG and its combination with ATST in lung cancer cell lines. Mechanisms of interaction between EGCG and ATST will be investigated. The activity of a green tea polyphenol mixture (Polyphenon E) will also be studied as a comparison. We will integrate the results from studies in vitro and in vivo to gain a better understanding of lung cancer preventive activities of EGCG and its combination with ATST. PUBLIC HEALTH RELEVANCE: The objective of this project is to study the inhibition of lung carcinogenesis by green tea polyphenols and the synergistic action when used in combination with atorvastatin (ATST, trade name Lipitor). Tea is commonly consumed by humans and Lipitor is a popular cholesterol-lowering drug. The possible use of the combination of tea polyphenols and Lipitor for lung cancer prevention is an attractive strategy and could have a large impact in public health. [unreadable] [unreadable] [unreadable]