Cruciferous vegetables are shown to be an important source of a variety of bioactive food components that may be useful for cancer prevention. A high dietary intake of cruciferous vegetables has been associated with a reduction in the incidence of a number of cancers including breast and prostate cancer. The anti-carcinogenic effects of cruciferous vegetables are mainly attributed to their high abundance of glucosinolates such as glucobrassicin, glucoraphanin, and progoitrin. Glucobrassicin, for example, can be hydrolyzed to produce a cancer preventive food component, indole-3-carbinol (I3C), that has been found in numerous epidemiological and pre-clinical studies to possess mammary cancer preventive properties. I3C spontaneously forms its derivatives including 3,3'-diindolymethane (DIM) after interaction with the acidity of the stomach. Mounting preclinical evidence in cell cultures and animal models suggests that both I3C and DIM have promising effects on mammary cancer prevention. However, it remains unclear how these dietary indoles interact with specific targets involved with breast cancer.In our previous cDNA micro-array studies, we demonstrated that I3C down-regulates the mRNA of ER which triggers many of estrogen's tumorigenic consequences. Since I3C is recognized to have a relatively low affinity for ER, the depression is likely not a direct effect. When MCF-7 cells were treated with AhR ligands including α-NF (10 μM), luteolin (10 uM), and DIM (as low as 1 uM) for 72 hours, there was a significant decrease in ER mRNA levels compared to controls suggesting the AhR may be involved with the suppressive effects of I3C on the ER. To test this hypothesis, we treated MCF7 cells with AhR siRNA to block the expression and function of this receptor. The results reveal that the suppressive effects of I3C and DIM on the ER may be independent of the AhR expression. Thus, the proposed USDA-NCI collaboration is designed to further investigate the mechanism that can explain how dietary indoles decrease ER expression and thus interfere with estrogen metabolism. The goal of this agreement is to 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 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. 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 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. Analyses will be performed in the laboratory of Dr. Tom Wang at the United States Department of Agriculture (USDA). The NCI will cover part of laboratory costs associated with these analyses as indicated below.In vitro studies with MCF-7 human breast cancer cells will be principally used for these investigations. Dr. Wang and I will examine a number of potential molecular targets for dietary indoles in the process of ER mediated cell signaling. These include PKA and MAPK that modulate the expression levels of ER. 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.
