Identify and characterize the molecular mechanisms through which dietary indoles I3C/DIM may exert their cancer preventive effects in breast cancer cells. Since the possible involvement of AhR through the ligand-dependent mechanism has been eliminated as a result of the short interfering (Si) RNA studies, we plan to assess additional signaling molecules that can influence the ER&#61537;expression as well as can be modulated by dietary indoles. These include protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) that have been shown to modulate the expression of ER&#61537;. We propose to use H89 and PD98059, the inhibitors of PKA and MAPK respectively, to examine if these agents can alter the inhibitory effects of I3C/DIM on the ER&#61537;expression. The activities of PKA and MAPK in response to the dietary I3C/DIM supplementation will be analyzed as well in the presence and in the absence of estrogen. The indole induced changes in transcription factors and cofactors will also be examined using imaging techniques such as GFP, RFP, or luciferase. In vitro studies with MCF-7 human breast cancer cells will be principally used for these investigations. A number of potential molecular targets for dietary indoles in the process of ER&#61537;mediated cell signaling will be examined. These include PKA and MAPK that modulate the expression levels of ER&#61537;. Once we identify the molecular targets for the effects I3C/DIM, we will further characterize the right concentration and duration of dietary exposure, and the surrogate biomarkers for cancer endpoints such as cancer cell proliferation and/or growth. In addition, the real-time in vitro imaging techniques will be used to examine the altered localization of transcription factors and cofactors in response to the bioactive vegetable component I3C.