2002 Annual Report 1.What major problem or issue is being resolved and how are you resolving it? Numerous epidemiological studies have correlated human consumption of diets rich in fruits and vegetables containing high levels of polyphenols to lower risk for specific chronic diseases such as cancer and cardiovascular conditions. Flavonoids and phenolics are phytochemicals that are widely distributed in plant-derived foods of the typical U.S. diet. These compounds have been reported to contribute to the purported beneficial health effects of fruits and vegetables. However, understanding the specific biological mechanisms involved, relative to human nutrition, presents a challenging and researchable problem. Without adequate definitive information on the mechanisms of absorption as well as cellular and molecular functions of flavonoids, it is not possible to accurately assess their beneficial effects on human health. Moreover, optimal but safe intakes of flavonoids have yet to be determined; these data must be generated before any dietary recommendations can be made. Therefore, a research project having two objectives (absorption of flavonoids and phenolics, and cellular and molecular actions of flavonoids and phenolics) is proposed to gain information on the cellular absorption and molecular functions of the phytochemicals, which might lead us to a better understanding of cellular and molecular mechanisms and to elucidating the beneficial effects of the phytochemicals. We hypothesized that cellular processes that affect human health can be altered by phytochemicals, thereby preventing chronic diseases such as cancer. The phytochemicals, including flavonoids and phenolics, in combination with nutrients, may act in synergy to regulate cellular and molecular pathways and thereby lead to prevention of chronic diseases in humans. A mechanism-based approach is used to identify those basic biological processes that are critical for preventing chronic diseases. We focus on processes within cellular pathways that are associated with mitigation of disease states; these include cell cycle pathways and apoptosis pathways. Techniques/methods to monitor effects of phytochemicals on these processes include: employment of cell culture and an animal model, utilizing RT-PCR and DNA micro-array technology to monitor regulation of gene expression and other conventional biochemical assays. We are applying these tools toward identifying specific biological mechanisms and pathways responsible for purported benefits of phytochemicals on human health. 2.How serious is the problem? Why does it matter? Historically, phytochemicals have been accredited with providing beneficial effects on human health. Recently, public recognition of their beneficial effects has led to increased consumption of phytochemicals by a progressive change of daily dietary patterns and a health-oriented intake of supplements. Although the intake of phytochemicals has increased rapidly, their biological effects have not yet been fully assessed. Nevertheless, dietary intake of phytochemicals is now increasing, and seems to reduce risks for development of a variety of common chronic diseases, including cancer. The cost for medical treatment and care for the diseases exceeds $200 billion per year. The American Cancer Society estimated in 1996 that one-third of the 500,000 cancer deaths annually in the U.S. are due to a variety of dietary factors. The annual economic impact of cancer in the U.S. exceeds $104 billion. The staggering cost of nutrition-related diseases clearly points toward a need to develop effective strategies, based on research that elucidates the molecular mechanisms of action by diet-derived compounds, to lower the cost of health care by prevention of diseases. 3.How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The health promoting properties of plant and animal foods is a key component of ARS national programs for human nutrition. Also, the mission of the Nutrition Program (# 107) is to conduct research to identify optimal diet and nutrient intake, determine the nutritional constituents of foods and diets that sustain and promote health throughout the life cycle, and identify biomarkers and bioassays of nutritional relevance; to make improved human nutritional outcomes the ultimate goal of all aspects of food related agricultural research and production. The current research projects in the program pursue these goals, and the central objective in this proposal (cellular and molecular functions of flavonoids in human cells) fits well with this component of the ARS national program. 4.What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment During FY 2002: Components from plants, phytochemicals, may be useful in preventing or treating cancer due to their anti-proliferative effects. Dr. Park, Phytonutrients Laboratory, in collaboration with Dr. Schoene, Nutrient Requirements and Functions Laboratory, synthesized and characterized a phytochemical, N-coumaroyltyramine, and conducted cell cycle analysis using this compound. It was demonstrated that this phytochemical suppresses the growth of human tumor cells, including U937 and Jurkat cells, by inhibiting protein tyrosine kinases, thus arresting the cell cycle. This study indicates that N-coumaroyltyramine may be useful as an anti-proliferation agent because it inhibits key enzymes required for the growth of cancer cells. B. Other Significant Accomplishment(s), if any: Prostate cancer is a major cause of death in the United States; thus, an understanding of the mechanisms of cancer-preventive phytochemicals could lead to the development of improved strategies for cancer prevention. Dr. Wang, Phytonutrients Laboratory, in collaboration with Dr. Steve Hursting, National Cancer Institute, conducted cell culture studies using prostate cancer cells to assess the ability of a vitamin A derivative, N-4-hydrozyphenyl-retinamine (4-HPR), to induce death in these cancer cells. They demonstrated that 4-HPR can induce the death of prostate cancer cells in vitro by a process known as apoptosis. This work identified a potential mechanism for the prevention of prostate cancer and suggested better strategies for cancer prevention. C. Significant Accomplishments/Activities that Support Special Target Populations: None. D. Progress Report: We continued to search for phytochemicals with anti-proliferation activity, and elucidate their molecular mechanisms. N-coumaroyltyramine and other phytochemicals have been investigated as potent compounds to suppress growth of some types of cancer cells. Due to the strong potency of N-coumaroyltyramine, a confidential agreement with DTP (Developmental Therapeutics Program, NCI, NIH) was created to investigate the phytochemical further (NSC number assigned as #724607). 5.Describe your major accomplishments over the life of the project, including their predicted or actual impact? Identified phytochemicals with anti-proliferation activity and elucidated molecular mechanisms behind the activity using various techniques including cDNA microarray technique. Identified phytochemicals with anti-proliferation activity for developing potent compounds to prevent and/or treat cancer. Demonstrated that N-coumaroyltyramine suppress growth of cancer cells. Demonstrated that N-4-Hydroxyphenyl retinamide, a derivative of vitamin A, can induce cancer cell death. Demonstrated that beta-naphthoflavone, a synthetic flavone, can interfere with gene expression analysis using luciferase-based reporter gene. 6.What do you expect to accomplish, year by year, over the next 3 years? Year 1: Determine effects of flavonoids and phenolics on cell cycle, cell proliferation and apoptosis. Use cDNA microarray technology to perform gene expression profiling of cells treated with cancer preventive phytochemicals. Year 2: Continue to elucidate molecular, cellular effects of flavonoids and phenolics. Based on gene expression profiling results, studies will be designed to elucidate potential mechanisms involved. Year 3: Correlate in vitro observation with animal and/or human studies. 7.What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Advised and provided Dagmar Kollar (Garuda International, INC.) phytochemical database information about Octacosanol (2002). Advised and shared information with Dr. Leslie Taylor ( Raintree Nutrition, Inc) on phytochemical database (2002). Material transfer agreement (pJDL) between Dr. Park and Dr. Eck (NCI, NIH), and advised and transferred technology to Dr. Eck (NCI, NIH) pJDL reporter vector (2002). Advised and shared information with Dr. Leo Fedor (University at Buffalo) (2002) on utilizing phytochemical database. Advised and transferred technology to Dr. Yoshiaki (Okayama U., Japan) including pJDL reporter vector, and established a Material Transfer Agreement (pJDL) between Dr. Park and Dr. Yoshiaki (2002). Confidential agreement with Developmental Therapeutics Program Branch at NCI (2002) Applied for a patent application "N-caffeoyltyramine and its analoques as anti-proliferative agents against human cancer cells"(2002) 8.List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) None. Review Publications Park, J.B. N-coumaroyltyramine is a potent phytochemical to arrest the growth of human tumor cells. Biochemical and Biophysical Research Communications. 2002. v.292. p.1104-1110. Song, J., Daruwala, R., Eck, P., Park, J.B., Levine, M. Flavonoid inhibition of the intestinal vitamin C transporter SVCT(h). Journal of the Biological Chemistry. 2002. v.277. p.15252-15260. Hursting, D.S., Shen, J.C., Sun, S.Y., Wang, T.T., Phang, M.J., Perkins, S.N. Modulation of cyclophilin gene expression by N-4-(hydroxyphenyl) retinamide: Association with reactive oxygen species generation and apoptosis. Molecular Carcinogenesis. 2002. v.33(1). p.16-24. Wang, T.T. Beta-naphthoflavone, an inducer of xenobiotic metabolizing enzymes, inhibit firefly luciferase activity. Analytical Biochemistry. 2002. v.304(1). p.122-126.