Botanical dietary supplements are used by consumers to prevent and to treat prostate cancer. Recently, an animal prostate cancer model has allowed the safety and efficacy of these supplements to be tested. We have established colonies of TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, estrogen receptor-deficient (ERaKO and ER(3K0) mice, and ER-deficient mice expressing the TRAMP transgene. Exciting data from our model clearly show that ERaKO mice are highly protected against PDC (poorty differentiated carcinoma) ofthe prostate and ERpKO mice are highly susceptible to PDC. In addition, we recently have found that phytoestrogens, phytosterols, and oxysterols can inhibit hedgehog signaling, a pathway important in prostate cancer. And surprisingly we find that oxysterols can bind ERp 10OX better than ERa with a Kd of ~20nM. Our overall hvpothesis is that differential regulation bv 5 botanicals and their antioxidant/phytoestrogen/phvtosterol components, whose actions are mediated bv 5 kev pathwavs, will be effective in prostate cancer prevention and treatment. The 5 kev pathwavs to be examined are the (anti)oxidant(Nrf2/Keap1 and NADPH oxidase) pathwavs and their interactions with the NF-kappaB-. estrogen-, and hedgehog-signaling pathwavs. Our goals are to characterize responses of key prostate tumor biomarkers to these botanicals and to provide novel molecular mechanisms for these responses useful in conducting later clinical trials. Aim 1: Determine in vitro in prostate cancer cell lines the effects ofthe 5 botanicals and selected pure compounds on the activities, protein concentrations, and mRNAs expressed in 5 key pathwavs: Aim 2: Perform in vivo cancer prevention trials in single and double transgenic TRAMP mice to test if the cancer protective activity of some pure compounds and botanicals will be dependent upon Keap1/Nrf2 and hedgehog pathway components;Aim 3: Test potential active ingredients and complex botanical extracts from Aims #1 or #2 with the same ingredients used in much faster in vivo human xenograft models of prostate cancer;and Aim 4: Identify responses associated with cell proliferation, differentiation, apoptosis, and the five key pathways in vivo in prostate tissues in response to botanicals, correlate these responses with tumorigenesis, and continue the correlations with the components ofthe oxidant, inflammatory, hedgehog, and/or estrogen pathways. These studies will provide fundamental insights into prostate tumor biology, and help provide biomarkers to assess the role of these botanicals in modifying the incidence and progression of prostate tumors in humans.