2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The central objective of this proposal (investigating anti-cancer properties as well as cellular and molecular functions of phenolic phytochemicals in human cells) fits well with component #7 "Identifying the heath promoting properties of plant and animal foods" of National Program 107 - Human Nutrition. Numerous epidemiological studies have suggested that diets rich in fruits and vegetables and containing high levels of phytochemicals can lower the risk for chronic diseases such as cancer and cardiovascular conditions. Phenolics and their conjugates are phytochemicals commonly found in plant-derived foods that are part of the typical U.S. diet, and are reported to have beneficial health effects. In plants, phenolics are found as themselves or conjugated with other classes of phytochemicals. Phenolic conjugates are various groups of phytochemicals, including phenylpropenic acid amides. Phenylpropenic acid amides are found in plants, including bell peppers and cocoa, and may be consumed via plant-derived foods and products. However, potential biological effects of these phytochemicals have not been investigated and their long-term health effects are unknown. It is hypothesized that the phenolic conjugates can modulate cellular and molecular processes related to human health, thereby helping treat and/or prevent chronic diseases such as heart disease, obesity, diabetes, and cancer. However, without adequate information about cellular absorption, as well as cellular and molecular actions of the conjugate, it is not possible to accurately propose and/or assess their beneficial effects on human health, relative to nutrition. Therefore, a research project with the objectives of:. 1)absorption; and. 2)the cellular and molecular actions of phenolic conjugates, is proposed to acquire information on the cellular absorption and molecular functions of phytochemicals. This may lead to an understanding of their cellular and molecular mechanisms and to elucidation of their beneficial effects on human chronic diseases. Heart disease, diabetes, and cancer are serious chronic diseases affecting millions of Americans. These diseases are attributed to multiple non-biological factors. However, inflammation and inflammation-related cellular events are currently believed to play important roles in initiating those diseases. Compounds suppressing these cellular events have been explored and developed in major pharmaceutical companies. In a nutritional sense, if preventive and/or therapeutic effects of phytochemicals can be achieved through the daily diet, it would be a natural and effective means for providing reasonable solutions to modern health crises related to the referenced diseases. Traditionally, plants have the potential to prevent and/or treat human diseases because of their exposure to preventive and/or therapeutic phytochemicals. This is in contrast to medicines which provide therapeutic treatment on a short-term basis. However, there is still a huge gap in our knowledge of health effects from plants and their phytochemicals. Research is needed to study new phytochemicals. In this project, phenylpropenic acid amides found in bell peppers and cocoa have been studied and were found to have great potential to modulate cellular and molecular events related to heart disease and cancer (the primary and secondary causes of mortality in the U.S.). In this study, a mechanism-based approach is used to identify the cellular and molecular events critical for chronic diseases. Cellular and molecular processes associated with mitigating disease are being investigated, including signal transduction pathways, cell-cell interactions, cell activation, cell proliferation, and cell death (apoptosis). Techniques and methods used in this study include cell culture, reverse transcription-polymerase chain reaction (RT-PCR), flow-cytometry, several biochemical assays, and animal models. These tools are used for identifying specific cellular pathways and the molecular mechanisms responsible for purported or newly-proposed benefits of phenylpropenic acid amides on human health. 2.List the milestones (indicators of progress) from your Project Plan. FY 2005 - Synthesize and/or purify phenolic conjugates (clovamide-type phenylpropenic acid amides; N-caffeoyltryamine and analogues). - Develop the method to measure the conjugates in cell culture medium and biological samples. - Investigate anti-proliferation and other biological activities of the conjugates, using cell culture model. FY 2006 - Complete a manuscript of the conjugate (clovamide-type phenylopropenic acid amides; N-caffeoyltyramine and analogues) syntheses. - Complete a manuscript of cell study. - Initiate and continue cell studies investigating health effects of the conjugates related to human chronic diseases such as heart disease and cancer (apoptosis study). FY 2007 - Complete the assays for the conjugates in biological samples. - Complete cell studies related to biological activities. - Initiate and continue and continue cell signal studies [e.g. phosphorylartion, kinases, epidermal growth factor receptor (EGFR)]. - Start animal study. FY 2008 - Complete cell signal studies. - Initiate and continue cell-cell interaction study. - Continue animal study. FY 2009 - Complete cell-cell interaction study and animal study EGFR study. 4a.What was the single most significant accomplishment this past year? Inflammation is an outcome of cellular and molecular events responsive to extrinsic and intrinsic biological causes, believed to be involved in initiating human diseases, such as heart disease and cancer. Integrins are adhesion receptors connecting cells to components of the extracellular matrix or to counter receptors on other cells. In heart disease and cancer, numerous integrins are involved in mediating stable adhesion for cell migration, and modulating signal transduction cascades regulating cell survival, proliferation, and the expression of disease-related genes. Our laboratory has synthesized and characterized several clovamide-type phenylpropenic acid amides as anti-proliferative compounds. In particular, N-caffeoyltyramine was found to arrest the growth of several tumor cells via suppressing protein tyrosine kinase activities (e.g., epidermal growth factor receptor (EGFR)), possibly connected to the expression of numerous cancer-related gene products, including adhesion molecules. N-caffoeyldopamine and its analogues found in green peppers and cocoa can inhibit platelet activation via suppressing P-selectin expression (an integrin protein on platelets). The clovamide-type phenylpropenic acid amides currently being investigated are involved in suppressing the expression of integrin-member proteins, thereby modulating signal transduction cascades and inhibiting cell growth. For the first time, N-caffoeyldopamine and its analogues were synthesized and it was demonstrated that the compounds can inhibit P-selectin expression, a major cause of human cardiovascular disease. 4b.List other significant accomplishments, if any. Cellular redox imbalance induces radical oxygen species (ROS), implicated in many pathophysiological conditions in chronic diseases, such as heart disease and cancer. Due to its potential importance, the redox status of thiol groups is well balanced by biological reducing molecules and proteins. Among redox proteins, glutaredoxin is a protein that regenerates S-thiolated cysteines in proteins that result from oxidative stress. Thiol groups in proteins may act as redox sensitive switches, and are considered to be a key element in maintaining cellular redox balance. A messenger ribonucleic acid (mRNA) isoform of the human glutaredoxin (Grx1as) has been isolated. The nucleotide sequence, Northern blots and reverse transcription polymerase chain reaction (RT-PCR), genomic cloning, and chromosomal localization of Grx1as shows it has an extra 3'-untranslated region sequence originated from Grx1 via an alternative splicing. It has been demonstrated that the 3’- untranslated regions are critical to its mRNA stability These studies are of interest to molecular biologists as well as applied nutritionists. 4c.List any significant activities that support special target populations. None. 4d.Progress report. In collaboration with the Nutrient Requirements and Functions Laboratory (NRFL), it was demonstrated in the Phytonutrients Laboratory (PL), that N-caffeoyltyramine and its analogues suppressed growth of tumor cells. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Demonstrated that N-caffeoyltyramine and its analogues can have beneficial effects on cancer and heart disease. In particular, a patent application was filed for N-caffeoyldopamine and its analogues as potent compounds suppressing P-selectin on platelets, which can provide beneficial effects on cardiovascular diseases. Synthesized and characterized N-caffeoyldopamine and its analogues as potent compounds inhibiting platelet-leukocyte interactions via suppressing P-selectin expression on platelets. Updated phytochemical databases for facilitating phytochemical research which more than 1500 users visit daily to obtain information regarding phytochemicals, medicinal plants, and medicines. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Filed for a patent application "Phenylpropenic acid amides as potent beta-adrenoceptor agonists"(2005). 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None. Review Publications Park, J.B. 2005. N-coumaroyldopamine and n-caffeoyldepamine increase camp via beta 2-adrenoceptors in myelocytic U937 cells. Federation of American Societies for Experimental Biology Journal. 19:497-502. Park, J.B., Schoene, N.W. 2005. Phenylpropenic acid amide, n-caffeoyltyramine, increases camp via beta 2-adrenoceptors in human monocytic u937 cells; its effects on monocyte activation. [Abstract]. Experimental Biology 830(5):830. Park, J.B. 2005. N-coumaroyldopamine and its analogues found in cocoa (theobroma cacao l.) are potent beta-adrenoceptor agonists suppressing platelet activition via increasing camp production. [Abstract]. Experimental Biology 566(2): 566. Park, J.B. 2005. Quercetin. In: Coates, P., Blackman, M.R., Cragg, G., Levine, M., Moss, J., White, J., editors. Encyclopedia of Dietary Supplements. New York, NY. Marcell Dekker Press. p. 577-585. Park, J.B., Levin, M. 2005. Cloning, sequencing, and characterization of alternatively spliced glutaredoxin cDNA and its genomic gene. Journal of Biological Chemistry. 280:10427-10434.