Flavonoids represent a family of phytochemicals found in many human food items. Among them, genistein (Gn) is found especially at high concentration in soybeans, an important element of Oriental diet. Epidemiological studies, in vivo animal studies and in vitro cell studies all suggested that Gn could play a role in the prevention of cancer. More work at the molecular level is needed to define the mechanism. This proposal addresses one potential mechanism: the ability of Gn to increase the expression of an antioxidant protein, metallothionein (MT). We have found that Gn increased the levels of MT protein and mRNA in human intestinal cells, Caco-2. The induction was synergistic with the stimulatory effect of zinc. Based on these observations, the proposal is designed to determine the physiological significance of MT induction; and the mode of Gn-MT gene interaction. We will compare the level of oxidative byproduct after tert- butylhydroperoxide challenge in cells with and without Gn treatment. In addition, animal study will be conducted to show that Gn feeding leads to an increase in the organ MT level and a decrease in the organ oxidative byproduct level. The mode of Gn-MT gene interaction will be investigated indirectly through the combination treatment of cells with Gn and other inducers of MT expression like copper, cadmium or cytokines. We will also perform nuclear run-on experiment and mRNA stability analysis in cells to directly confirm the effect of Gn at the transcriptional level. Reporter gene assays will then be conducted to determine the site of Gn-MT gene interaction on the cloned sequence of human MTIIA promoter. The results from the proposed studies will help to assess the essentiality of Gn in the human diet for reduction of cancer risk. These studies may also lead to the identification of a previous unknown pathway for Gn to regulate mammalian genes.
