Ethnobotanical approach versus random approach in the search for new bioactive compounds: support of a hypothesis.

CONTEXT: Whether natural product drug discovery programs should rely on wild plants collected "randomly" from the natural environment, or whether they should also include plants collected on the basis of use in traditional medicine remains an open question. OBJECTIVE: This study analyzes whether plants with ethnomedical uses from Vietnam and Laos have a higher hit rate in bioassay testing than plants collected from a national park in Vietnam with the goal of maximizing taxonomic diversity ("random" collection). MATERIALS AND METHODS: All plants were extracted and subjected to bioassay in the same laboratories. Results of assays of plant collections and plant parts (samples) were scored as active or inactive based on whether any extracts had a positive result in a bioassay. Contingency tables were analyzed using χ(2) statistics. RESULTS: Random collections had a higher hit rate than ethnomedical collections, but for samples, ethnomedical plants were more likely to be active. Ethnomedical collections and samples had higher hit rates for tuberculosis, while samples, but not collections, had a higher hit rate for malaria. Little evidence was found to support an advantage for ethnomedical plants in HIV, chemoprevention and cancer bioassays. Plants whose ethnomedical uses directly correlated to a bioassay did not have a significantly higher hit rate than random plants. DISCUSSION: Plants with ethnomedical uses generally had a higher rate of activity in some drug discovery bioassays, but the assays did not directly confirm specific uses. CONCLUSIONS: Ethnomedical uses may contribute to a higher rate of activity in drug discovery screening.

An ethnobotanical survey of medicinal plants of Laos toward the discovery of bioactive compounds as potential candidates for pharmaceutical development.

CONTEXT: An ethnobotany-based approach in the selection of raw plant materials to study was implemented. OBJECTIVE: To acquire raw plant materials using ethnobotanical field interviews as starting point to discover new bioactive compounds from medicinal plants of the Lao People's Democratic Republic. METHODS: Using semi-structured field interviews with healers in the Lao PDR, plant samples were collected, extracted, and bio-assayed to detect bioactivity against cancer, HIV/AIDS, TB, malaria. Plant species demonstrating activity were recollected and the extracts subjected to a bioassay-guided isolation protocol to isolate and identify the active compounds. RESULTS: Field interviews with 118 healers in 15 of 17 provinces of Lao PDR yielded 753 collections (573 species) with 955 plant samples. Of these 955, 50 extracts demonstrated activity in the anticancer, 10 in the anti-HIV, 30 in the anti-TB, and 52 in the antimalarial assay. Recollection of actives followed by bioassay-guided isolation processes yielded a series of new and known in vitro-active anticancer and antimalarial compounds from 5 species. DISCUSSION: Laos has a rich biodiversity, harboring an estimated 8000-11,000 species of plants. In a country highly dependent on traditional medicine for its primary health care, this rich plant diversity serves as a major source of their medication. CONCLUSIONS: Ethnobotanical survey has demonstrated the richness of plant-based traditional medicine of Lao PDR, taxonomically and therapeutically. Biological assays of extracts of half of the 955 samples followed by in-depth studies of a number of actives have yielded a series of new bioactive compounds against the diseases of cancer and malaria.

Effect of deguelin on UVB-induced skin carcinogenesis.

Nonmelanoma skin cancer afflicts more than one million people in the U.S. annually, highlighting the need for more effective preventive regimens. We have investigated the ability of deguelin, a plant-derived rotenoid with cancer chemopreventive activity, to inhibit UVB-induced skin carcinogenesis with the SKh-1 mouse model. Topically-applied deguelin significantly inhibited the multiplicity of UVB-induced skin tumors, indicating potential as a human skin cancer chemopreventive agent. Mechanistic studies to determine the potential of deguelin to block a number of established UVB-induced molecular events yielded negative results [including UVB-induced AP-1 DNA binding, c-fos and TNFalpha mRNA induction, arachidonic acid release and UVB-induced phosphorylation of mTOR (Ser2448), akt (Ser473) and erk (Thr202/Tyr204)]. These results are of interest as they contradict a major hypothesis for the mode of action of deguelin, i.e., a general down regulation of signal transduction based on inhibition of NADH dehydrogenase and depletion of ATP levels. In the current work, however, deguelin was found to activate 5' AMP-activated kinase (AMPK), a protein that acts as a cellular energy sensor. This is the first report of a chemopreventive agent having this effect and suggests a possible role for AMPK in cancer chemoprevention.

Resveratrol inhibits rhabdomyosarcoma cell proliferation.

Rhabdomysarcoma is the most common soft tissue tumour in children under the age of 15. Although the introduction of multimodal treatment programmes, including chemotherapy, radiation therapy and excision have increased the overall survival, the chemotherapeutic agents currently used for the treatment of rhabdomyosarcoma exhibit considerable toxicity. The aim of this study was to investigate the effects and possible mechanism(s) of action of resveratrol on human embryonal rhabdomyosarcoma (RD) cells. Resveratrol is a natural polyphenolic compound produced in a number of edible plants and has received considerable attention as a potential chemopreventive and/or chemotherapeutic agent against various types of cancers. In the present study, resveratrol was shown to inhibit cell proliferation of RD cells in a dose-dependent manner with an IC50 of 48.1 micromol/l and induce an arrest in the S/G2 phase of the cell cycle. As evident from immunocytochemical data, resveratrol treatment increased the size of the RD cells. Furthermore, resveratrol treatment resulted in a significant downregulation of cyclin B expression as demonstrated by western blot analyses. In conclusion, the present study shows that resveratrol exerts a strong inhibition of rhabdomyosarcoma cell proliferation in part by arresting cells in S/G2 phase of the cell cycle. These findings warrant further investigation to establish potential use of resveratrol as a relatively non-toxic chemotherapeutic agent for the treatment of rhabdomyosarcoma.

Ethnobotany/ethnopharmacology and mass bioprospecting: issues on intellectual property and benefit-sharing.

Ethnobotany/ethnopharmacology has contributed to the discovery of many important plant-derived drugs. Field explorations to seek and document indigenous/traditional medical knowledge (IMK/TMK), and/or the biodiversity with which the IMK/TMK is attached, and its conversion into a commercialized product is known as bioprospecting or biodiversity prospecting. When performed in a large-scale operation, the effort is referred to as mass bioprospecting. Experiences from the mass bioprospecting efforts undertaken by the United States National Cancer Institute, the National Cooperative Drug Discovery Groups (NCDDG) and the International Cooperative Biodiversity Groups (ICBG) programs demonstrate that mass bioprospecting is a complex process, involving expertise from diverse areas of human endeavors, but central to it is the Memorandum of Agreement (MOA) that recognizes issues on genetic access, prior informed consent, intellectual property and the sharing of benefits that may arise as a result of the effort. Future mass bioprospecting endeavors must take heed of the lessons learned from past and present experiences in the planning for a successful mass bioprospecting venture.

In vitro assessment of intestinal permeability and hepatic metabolism of 4'-bromoflavone, a promising cancer chemopreventive agent.

1. The intestinal permeability and hepatic metabolism of the investigational cancer chemoprevention agent 4'-bromoflavone were investigated in vitro using human intestinal Caco-2 cell monolayers, human liver microsomes and human hepatocytes. Liquid chromatography-mass spectrometry and tandem mass spectrometry were used for quantitative analysis in support of the Caco-2 cell studies and for the characterization of metabolites of 4'-bromoflavone. 2. The Caco-2 cell model indicated that 4'-bromoflavone would be absorbed by the intestine at a moderate rate by means of direction-independent, passive diffusion. There was no indication of active transport or efflux. 3. Three monohydroxylated metabolites and one monohydroxylated, hydrated metabolite of 4'-bromoflavone were detected at relatively low levels in the human liver microsomal and hepatocyte incubations. The structures of these metabolites were confirmed by comparison with synthetic standards. Hydroxylation occurred on the A-ring of 4'-bromoflavone but not on the B-ring, probably due to deactivation of the B-ring by bromine. No phase II metabolites were detected following incubation of 4'-bromoflavone in these in vitro systems. 4. In conclusion, these studies predict that 4'-bromoflavone should show moderate oral bioavailability, and that it would probably be excreted as unchanged compound and monohydroxylated metabolites. The results might be helpful in the design of clinical trials and in the interpretation of pharmacokinetic studies of 4'-bromoflavone.

Prostate intraepithelial neoplasia in Noble rats, a potential intermediate endpoint for chemoprevention studies.

In most prostate chemoprevention studies conducted with animal models, the incidence and multiplicity of tumours have been used as endpoints for efficacy. However, the latency of tumours is usually over 1 year, making these studies costly and time consuming. The main purpose of this study was to assess the utility of prostate intraepithelial neoplasia (PIN), induced in Noble rats by continuous testosterone + oestradiol (T + E) administration, as a potential intermediate endpoint biomarker of efficacy in chemoprevention studies. Noble rats at the age of 12 weeks were treated for 36 weeks with T + E given subcutaneously via Silastic capsules. The incidence and multiplicity of PIN were assessed in various prostate glands by serial sections generated at three separate tissue levels. The efficacy of dehydroepiandrosterone (DHEA) and DHEA 8354 (1000 and 2000 mg/kg diet), difluoromethylornithine (DFMO) (1000 and 2000 mg/kg diet) and oltipraz (125 and 250 mg/kg diet) to inhibit PIN was assessed in two independent sets of experiments. T + E induced multiple PIN in the dorsolateral prostate (DLP) of 80-100% of the animals. DHEA and DHEA 8354 did not affect the incidence but decreased the multiplicity of PIN in the DLP, from 3.2 +/- 1.0 in control group to 1.5 +/- 1.0 in the low-dose and to 1.6 +/- 0.6 in the high-dose group for DHEA (P<0.05 and P<0.02, respectively), and to 1.9 +/- 0.8 in the high-dose (P<0.05) DHEA 8354. Both agents did not affect PIN in anterior prostate, seminal vesicles or ventral prostate. In a second experiment, DFMO and oltipraz were found not effective in inhibiting PIN. In this study, we provide new evidence that PIN in Noble rats, induced by continuous T + E treatment, is a useful intermediate endpoint for determining the efficacy of DHEA and other potential chemopreventive agents. The hormonal pathogenesis, high multiplicity, short latency, preferential location in the DLP, similarity in morphology and biology to PIN of human prostate, and the sensitivity to agents that suppress prostate carcinogenesis, makes PIN in Noble rats a promising intermediate endpoint for chemoprevention studies.

The UIC ICBG (University of Illinois at Chicago International Cooperative Biodiversity Group) Memorandum of Agreement: a model of benefit-sharing arrangement in natural products drug discovery and development.

The Convention on Biodiversity mandates a new approach to the discovery of natural product drugs, one that incorporates concepts of national ownership of genetic resources, intellectual property rights in traditional knowledge, and sharing of economic benefits with countries that are the source of new natural products. The International Cooperative Biodiversity Group (ICBG) program was established to support experimentation in implementation of the Convention through development and execution of international agreements for bioprospecting. The agreement of one such ICBG program, between the University of Illinois at Chicago and institutions in Vietnam and Laos, is presented here. The core elements contained in the single, five-way Memorandum of Agreement are the arrangements for intellectual property rights, treatment of informed consent, and plans for benefit-sharing (including the sharing of short- and long-term royalty benefits, capacity building, and community reciprocity). Program participants were able to develop a practical and flexible agreement that satisfies the wishes of all institutions that are parties to it.

Isolation of linoleic acid as an estrogenic compound from the fruits of Vitex agnus-castus L. (chaste-berry).

A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERalpha) and beta (ERbeta). The extract at 46 +/- 3 microg/ml displaced 50% of estradiol from ERalpha and 64 +/- 4 microg/ml from ERbeta. Treatment of the ER+ hormone-dependent T47D:A18 breast cancer cell line with the extract induced up-regulation of ERbeta mRNA. Progesterone receptor (PR) mRNA was upregulated in the Ishikawa endometrial cancer cell line. However, chaste-tree berry extract did not induce estrogen-dependent alkaline phosphatase (AP) activity in Ishikawa cells. Bioassay-guided isolation, utilizing ER binding as a monitor, resulted in the isolation of linoleic acid as one possible estrogenic component of the extract. The use of pulsed ultrafiltration liquid chromatography-mass spectrometry, which is an affinity-based screening technique, also identified linoleic acid as an ER ligand based on its selective affinity, molecular weight, and retention time. Linoleic acid also stimulated mRNA ERbeta expression in T47D:A18 cells, PR expression in Ishikawa cells, but not AP activity in Ishikawa cells. These data suggest that linoleic acid from the fruits of Vitex agnus-castus can bind to estrogen receptors and induce certain estrogen inducible genes.

Studies on di- and triterpenoids from Salvia staminea with cytotoxic activity.

A new ursane-type triterpenoid, 3 beta,11 alpha,21 alpha-trihydroxyurs-12-ene, named salvistamineol (1), has been isolated from the methanol extract of Salvia staminea. In addition to 1, the methanol extract yielded four known compounds and the acetone extract yielded twelve known compounds consisting of two sesquiterpenes, six diterpenoids, a triterpenoid, two steroids and one flavone. DNA damaging properties of the extracts and some isolated diterpenes were investigated against three yeasts and only taxodione gave a positive response and also showed the highest cytotoxic activity against a panel of cell lines among the investigated compounds in this study.

Traditionally-used antimalarials from the Meliaceae.

A quantitative ethnobotanical approach to antimalarial drug discovery led to the identification of Lansium domesticum Corr. Ser. (Meliaceae) as an important antimalarial used by Kenyah Dyak healers in Indonesian Borneo. Triterpenoid lansiolides with antimalarial activity were isolated from the bark and shown to have activity in both in vitro bioassays with Plasmodium falciparum, and in mice infected with P. berghei. A survey of African and tropical American Meliaceae led to further development of the limonoid gedunin from the traditionally used medicinal plants, tropical cedar, Cedrela odorata L., and neem, Azadirachta indica A. Juss. Gedunin has significant in vitro activity but initially showed poor in vivo activity. In vivo activity was improved by (1) incorporation into an easy to absorb suspension, (2) preparation of a more stable compound, 7-methoxygedunin; and (3) synergism with dillapiol, a cytochrome P450 3A4 inhibitor. The results show the potential for both antimalarial drug and phytomedicine development from traditionally used plants.

Deguelin inhibits the growth of colon cancer cells through the induction of apoptosis and cell cycle arrest.

As previously demonstrated, deguelin [(7aS, BaS)-13, 13a-dihydro-9,10-dimethoxy-3,3-dimethyl-3H-bis[1]benzo-pyrano[3,4-b:6',5'-e]pyran-7(7aH)-one mediates anti-proliferative properties in a variety of cell types. In the present study, deguelin was found to suppress the growth of HT-29 colon cancer cells with an IC(50) of 4.32 x 10(-8) M. The cells were arrested in the G1-S-phase of the cycle. Investigations of G1/S regulatory proteins by Western blot analyses showed an upregulation of p27, and decreased expression levels of cyclin E and CDK4. Furthermore, by 24 h, exposure to deguelin resulted in an increase in the hypophosphorylated form of Rb. Since hypophosphorylated pRb binds to and inactivates E2F1, additional studies were performed and downregulation of E2F1 was observed after 24 h of treatment with deguelin. These results are consistent with the observation that deguelin arrested cells in the G1-S- phase. In addition, based on ethidium bromide/acridine orange staining, detection of digoxigenin-labelled genomic 3'-OH DNA ends, and DNA laddering, it was found that deguelin exerts its growth inhibitory effects via the induction of apoptosis. Based on these data, the potential of deguelin to serve as a cancer chemotherapeutic agent for colon cancer may be suggested.

Brusatol-mediated induction of leukemic cell differentiation and G(1) arrest is associated with down-regulation of c-myc.

Employing the natural product quassinoid brusatol, we currently report cellular and molecular events leading to cell death or terminal differentiation in a panel of leukemic cells. Brusatol and bruceantin exerted significant cytotoxic effects with several leukemic cell lines, but not with K562 or normal lymphocytic cells. Cell lines that were less sensitive to the cytotoxic effects of brusatol responded primarily through induction of terminal differentiation. The differentiated phenotype in cell lines derived from acute or chronic myeloid leukemias (HL-60, K562, Kasumi-1, NB4, U937, BV173) was characterized for producing superoxide and non-specific esterase, and some with up-regulation of CD13 (cluster of differentiation) and down-regulation of CD15. Chronic myeloid leukemic cell lines, K562 and BV173, and acute lymphoblastic cell lines, SUPB13 and RS4;11, were induced to differentiate along the erythrocytic pathway. Withdrawal studies showed that brusatol treatment for 48 h was sufficient to induce commitment towards terminal differentiation in HL-60, K562 and SUPB13. Reh cells did not undergo maturation. Analysis of c-MYC protein expression revealed that brusatol or bruceantin down-regulated expression to undetectable levels in cell lines that were most sensitive, based on cell death or terminal differentiation. Generally, c-myc RNA was reduced, but to a lower extent than c-MYC protein levels, indicating c-myc expression was regulated by quassinoids at the post-transcriptional level. Thus, regulation of c-myc expression may represent a critical event that leads to terminal differentiation. Since these responses are facilitated at clinically achievable concentrations, quassinoids may be of value for the management of hematological malignancies.

Inhibition of HIV-1 reverse transcriptase and HIV-1 replication by Calophyllum coumarins and xanthones.

As part of our continuing study on the Calophyllum species, a number of coumarins, xanthones and chromene acids from different Calophyllum species of Sri Lanka were tested for inhibitory activity against the HIV-1 and its virally-encoded reverse transcriptase (RT). These compounds were found to be inactive in both the HIV-1 RT and whole virus systems. In contrast, cordatolide A and B demonstrated IC(50) values of 19.3 and 11.7 microM, respectively, against HIV-1 replication in a novel green fluorescent protein (GFP)-based reporter cell assay (HOG.R5).

A preliminary RAPD-PCR analysis of Cimicifuga species and other botanicals used for women's health.

Traditional taxonomic methods of botanical identification that rely primarily on morphological observations cannot be used efficiently when only powdered plant materials are available. Thus, our objectives were to determine if we could apply a molecular approach to: a) produce unique DNA profiles that are characteristic of the species, and b) determine if the geographical area or time of collection influences these DNA profiles. Towards this end, random amplified polymorphic DNA (RAPD) analyses were performed on a number of botanicals currently used for women's health. The test materials included samples from three species each of the genera Cimicifuga (Actaea) and Trifolium, as well as samples of Vitex agnus-castus L., Glycyrrhiza glabra L., Gingko biloba L., Valeriana officinalis L., Angelica sinensis (Oliv.) Diels, Viburnum prunifolium L., Humulus lupulus L., Vaccinium macrocarpon Ait., Panax ginseng C.A. Mey. Cimicifuga racemosa (L.) Nutt. and Trifolium pratense L. are currently under clinical investigation in our basic research laboratories and medical clinic for the relief of post-menopausal symptoms. Characteristic profiles produced with the OPC-15 primer could distinguish the three Cimicifuga species: C. racemosa, C. americana and C. rubifolia. Similar results were obtained with the three Trifolium species: Trifolium pratense L., Trifolium incarnatum L., and Trifolium repens L. Accessions of cultivated T. pratense collected from the same field at different times, produced identical profiles. Accessions of Cimicifuga species collected from different geographical areas produced similar but not identical DNA profiles; however, species-specific DNA fragments were identified. These results demonstrate that RAPD analysis can be applied to distinguish species when only powdered material is available for testing. This methodology can be applied to identify species of commercial value regardless of collection time or geographic area.

Cycloartane saponins from Astragalus peregrinus as modulators of lymphocyte proliferation.

From Astragalus peregrinus, four cycloartane-type saponins have been isolated and their structures elucidated by spectral means as 20(R),24(S)-epoxy-9 beta,19-cyclolanostane-3 beta,6 alpha,16 beta,25-tetrol 3-O-beta-D-glucopyranoside (1), 20(R),24(S)-epoxy-9 beta,19-cyclolanostane-3 beta,6 alpha,16 beta,25-tetrol 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranoside (2), 20(R),24(S)-epoxy-9 beta,19-cyclolanostane-3 beta,6 alpha,16 beta,25-tetrol 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside (3) and 20(R),25-epoxy-9 beta,19-cyclolanostane-3 beta,6 alpha,16 beta,24(S)-tetrol (24-O-acetyl)- 3-O-alpha-L-rhamnopyranosyl-(1-->2)-(6'-O-acetyl)-beta-D-glucopyranoside (4). Compounds 2 and 3 showed to stimulate the proliferation of mouse splenocytes and were not significantly cytotoxic.

Macharistol, a new cytotoxic cinnamylphenol from the stems of Machaerium aristulatum.

A new cinnamylphenol, macharistol (1), along with a known pterocarpan, (+)-medicarpin (2), were isolated as cytotoxic constituents from the stems of Machaerium aristulatum. In addition, a known pterocarpan, (+)-maackiain (3), and a known isoflavone, formononetin (4), were identified as inactive constituents. Compound 1 was evaluated in the in vivo hollow fiber assay with KB, Col-2, and hTERT-RPE1 cells and found to be inactive at the highest dose (25 mg/kg body weight) tested.

Aromatase inhibitors from Broussonetia papyrifera.

Bioassay-guided fractionation of an ethyl acetate-soluble extract from the whole plants of Broussonetia papyrifera, using an in vitro aromatase inhibition assay, led to the isolation of five new active compounds, 5,7,2',4'-tetrahydroxy-3-geranylflavone (1), isogemichalcone C (8), 3'-[gamma-hydroxymethyl-(E)-gamma-methylallyl]-2,4,2',4'-tetrahydroxychalcone 11'-O-coumarate (9), demethylmoracin I (10), and (2S)-2',4'-dihydroxy-2' '-(1-hydroxy-1-methylethyl)dihydrofuro[2,3-h]flavanone (11), and 10 known (12-21) compounds which were also found to be active. Of these compounds, the most potent were 9 (IC(50) 0.5 microM), 11 (IC(50) 0.1 microM), isolicoflavonol (12, IC(50) 0.1 microM), and (2S)-abyssinone II (13, IC(50) 0.4 microM). Additionally, six new compounds, 5,7,3',4'-tetrahydroxy-6-geranylflavonol (2), 5,7,3',4'-tetrahydroxy-3-methoxy-6-geranylflavone (3), (2S)-7,4'-dihydroxy-3'-prenylflavan (4), 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)propane (5), 1-(2,4-dihydroxy-3-prenylphenyl)-3-(4-hydroxyphenyl)propane (6), and 1-(4-hydroxy-2-methoxyphenyl)-3-(4-hydroxy-3-prenylphenyl)propane (7), were isolated and characterized, but proved to be inactive as aromatase inhibitors, as were an additional 21 known compounds. The structures of the new compounds (1-11) were elucidated by spectroscopic methods. Structure-activity relationships in the aromatase assay were determined for the benzofurans, biphenylpropanoids, coumarins, and various types of flavonoids (chalcones, flavans, flavanones, and flavones) obtained among a total of 42 constituents of B. papyrifera.

Estrogenic and antiestrogenic properties of resveratrol in mammary tumor models.

Trans-3,4',5-trihydroxystilbene (resveratrol), a phytoalexin present in grapes and grape products such as wine, has been identified as a chemopreventive agent. Recent studies performed with MCF-7 human breast cancer cells have demonstrated superestrogenic effects with resveratrol. In contrast, studies performed using estrogen receptor-transfected cell lines have shown that resveratrol acts as a mixed agonist/antagonist. The major objective of this study was to characterize the estrogen-modulatory effects of resveratrol in a variety of in vitro and in vivo mammary models. Thus, the effect of resveratrol alone and in combination with 17beta-estradiol (E2) was assessed with MCF-7, T47D, LY2, and S30 mammary cancer cell lines. With cells transfected with reporter gene systems, the activation of estrogen response element-luciferase was studied, and using Western blot analysis, the expression of E2-responsive progesterone receptor (PR) and presnelin 2 protein was monitored. Furthermore, the effect of resveratrol on formation of preneoplastic lesions (induced by 7,12-dimethylbenz(a)anthracene) and PR expression (with or without E2) was evaluated with mammary glands of BALB/c mice placed in organ culture. Finally, the effect of p.o. administered resveratrol on N-methyl-N-nitrosourea-induced mammary tumors was studied in female Sprague Dawley rats. As a result, in transient transfection studies with MCF-7 cells, resveratrol showed a weak estrogenic response, but when resveratrol was combined with E2 (1 nM), a clear dose-dependent antagonism was observed. Similar mixed estrogenic/antiestrogenic effects were noted with S30 cells, whereas resveratrol functioned as a pure estrogen antagonist with T47D and LY2 cells. Furthermore, in MCF-7 cells, resveratrol induced PR protein expression, but when resveratrol was combined with E2, expression of PR was suppressed. With T47D cells, resveratrol significantly down-regulated steady-state and E2-induced protein levels of PR. With LY2 and S30 cells, resveratrol down-regulated presnelin 2 protein expression. Using the mouse mammary organ culture model, resveratrol induced PR when administered alone, but expression was suppressed in the presence of E2 (1 nM). Furthermore, resveratrol inhibited the formation of estrogen-dependent preneoplastic ductal lesions induced by 7,12-dimethylbenz(a)anthracene in these mammary glands (IC50 = 3.2 microM) and reduced N-methyl-N-nitrosourea-induced mammary tumorigenesis when administered to female Sprague Dawley rats by gavage. Therefore, in the absence of E2, resveratrol exerts mixed estrogen agonist/antagonist activities in some mammary cancer cell lines, but in the presence of E2, resveratrol functions as an antiestrogen. In rodent models, carcinogen-induced preneoplastic lesions and mammary tumors are inhibited. These data suggest that resveratrol may have beneficial effects if used as a chemopreventive agent for breast cancer.