The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inf lammatory, anti-platelet, and anticarcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (Ϸ3-10 M) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.Resveratrol (trans-3, 4Ј, 5-trihydroxystilbene) occurs naturally in grapes and a variety of medicinal plants. In plants, resveratrol functions as a phytoalexin that protects against fungal infections (1). Because of its high concentration in grape skin, significant amounts of resveratrol are present in wine (2, 3), and it has been proposed to explain, at least in part, the apparent ability of moderate consumption of red wine to reduce the risk of cardiovascular disease (4 -7). Resveratrol also has been reported to have cancer chemopreventive activity (8). The similarity in structure between resveratrol and the synthetic estrogen diethylstilbestrol (DES; 4, 4Ј-dihydroxy-trans-␣, ␤-diethylstilbene) prompted us to investigate whether resveratrol might exhibit estrogenic activity, a property that is known to produce a cardioprotective benefit (9, 10).Estrogens, including phytoestrogens, act via the estrogen receptor, a member of the nuclear receptor superfamily. Estrogen binding to the receptor activates the transcription of estrogen-responsive target genes. We report here that resveratrol binds to and activates transcription by the estrogen receptor at concentrations that are comparable to those required for its other biological effects. EXPERIMENTAL PROCEDURESCell Culture. MCF-7 cells, subclone WS8 (estrogen receptor-positive), MDA-MB-231 cells, subclone 10A (estrogen receptor-negative), and T47D cells, subclone A18 (estrogen receptor-positive) are derived from human breast adenocarcinomas and were provided by V. Craig Jordan (Northwestern University Medical Sch...

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) a and fi binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

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We demonstrated previously that glucocorticoids differentially affect the levels of the two pituitary gonadotropins, LH and FSH, both in vivo and in vitro. In vivo, the effect of glucocorticoids is GnRH independent, indicating a direct action on the gonadotrope, and it leads to selective up-regulation of the pituitary content of FSH and FSH beta-subunit messenger RNA (mRNA). The objective of the present study was to confirm the direct action of corticosterone (B) on FSH beta-subunit mRNA in primary anterior pituitary cell culture and to assess whether the selective B-induced rise in FSH beta mRNA is mediated through altered stability of the FSH beta transcript. Anterior pituitary glands collected from randomly cycling female rats were dissociated with trypsin. Cells were incubated at 37 C for 48 h and subsequently exposed to vehicle or B (1.7 microM) for an additional 42 h. At the end of the incubation, media were sampled for FSH and LH, cells were lysed, and total RNA was isolated for Northern blot analysis. Exposure to B for 42 h caused direct and selective upregulation of FSH release, FSH content, and FSH beta mRNA; decreased alpha-subunit mRNA; and had no significant effect on LH release, LH content, or LH beta mRNA. To evaluate the mRNA stability of the three subunits, cells were exposed to the transcription blocker actinomycin D (act D; 5 micrograms/ml) for an additional 6 h. The combined 6-h treatment with B and act D slightly, but significantly, suppressed alpha-subunit mRNA and did not change LH beta mRNA, confirming a long half-life of the two gonadotropin subunit mRNAs. In contrast, FSH beta mRNA was significantly suppressed by act D to the same level in vehicle- and B-treated cells. The posttranscriptional decay rate was examined by sampling at 0, 1, 2, 3, and 6 h during the 6-h act D treatment period. Decay curves for FSH beta mRNA were parallel in vehicle- and B-treated cells, indicating that B did not alter FSH beta mRNA stability. We conclude that the selective B-induced rise in FSH beta mRNA is mediated at the level of transcription rather than mRNA stabilization.

Cortisol treatment for 6 or 12 days had no effect on serum FSH in intact males and animals castrated for 1 or 7 days, but pituitary FSH was increased by the steroid in both intact and castrate groups. In contrast, cortisol inhibited serum LH in both intact and castrated animals while only increasing pituitary levels of LH in 7 day castrates. Cortisol also increased the mRNA for FSH beta without affecting alpha or LH beta mRNAs. These data suggest that the selective increase in pituitary content of FSH may be due to a selective increase in FSH beta mRNA following exposure to cortisol.