Resveratrol exhibits cytostatic and antiestrogenic properties with human endometrial adenocarcinoma (Ishikawa) cells.

Trans-3,4',5-trihydroxystilbene (resveratrol), a polyphenolic compound found in the human diet, was reported recently to serve as an estrogen agonist with cultured MCF-7 cells transfected with estrogen response element-luciferase reporter plasmids. As currently shown, treatment of cultured human endometrial adenocarcinoma (Ishikawa) cells with resveratrol (concentrations as high as 10 microM) did not significantly increase the levels of an estrogen-inducible marker enzyme, alkaline phosphatase. To the contrary, when alkaline phosphatase was induced by treatment with 1 nM of 17beta-estradiol (E(2)), resveratrol exhibited a dose-dependent decrease in activity (IC(50) = 2.3 microM). Furthermore, when Ishikawa cells were treated with resveratrol as a single agent, estrogen-inducible progesterone receptor (PR) was not enhanced, and PR expression induced by treatment with E(2) was inhibited by resveratrol in a dose-dependent fashion at both the mRNA and protein levels. In addition, resveratrol mediated suppression of a functional activity of PR as demonstrated by down-regulation of alpha(1)-integrin expression induced by E(2) plus progesterone. With transient transfection experiments conducted with Ishikawa cells, antiestrogenic effects were confirmed by dose-dependent inhibition of E(2)-induced estrogen response element-luciferase transcriptional activity. Because resveratrol antagonized estrogenic effects in Ishikawa cells, competitive binding analyses were performed to examine the potential of displacing [(3)H]E(2) from human estrogen receptor (ER). Resveratrol showed no discernable activity with ER-alpha, but with ER-beta, E(2) was displaced with an IC(50) of 125 microM. However, mRNA and protein expression of ER-alpha but not ER-beta were suppressed by resveratrol in Ishikawa cells, in the concentration range of 5-15 microM. In addition, in the presence or absence of E(2), resveratrol inhibited Ishikawa cell proliferation in a time-dependent manner with cells accumulating in the S phase of the cycle < or =48 h. This effect was reversible. Analysis of some critical cell cycle proteins revealed a specific increase in expression of cyclins A and E but a decrease in cyclin-dependent kinase 2. These data suggest resveratrol exerts an antiproliferative effect in Ishikawa cells, and the effect may be mediated by both estrogen-dependent and -independent mechanisms.

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.

Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.

Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

Natural modulators of estrogen biosynthesis and function as chemopreventive agents.

There is clearly a need for novel breast cancer chemopreventive agents with enhanced potency and specificity with little or no side effects. To this end, several new chemical moieties have been synthesized or isolated from natural sources. In this review, we have described some agents currently in use or under development for treatment or prevention of breast cancer, as well as our own strategies for the discovery of natural product modulators of estrogen biosynthesis and function. In particular, bioassay-guided fractionation of active plant extracts is a unique method for identifying agents with novel mechanisms of action, some of which should be useful for prevention of human cancer. Further, with the advent of combinatorial chemistry and high throughput screening, even greater progress may now be expected with natural product leads.

Tie2-mediated multidrug resistance in malignant gliomas is associated with upregulation of ABC transporters.

Resistance and relapse are still primary causes that result in poor effectiveness of chemotherapy in malignant gliomas. Therefore, development of new therapeutic strategies requires the identification of key molecular pathways regulating chemoresistance. We previously found that abnormal high expression of the Tie2 receptor in gliomas was associated with tumor malignancy. Here, we studied the role of Tie2 activation in drug resistance by testing the cytotoxicity of several chemotherapeutic drugs in a panel of human glioma cell lines and brain tumor stem cells and found that Tie2 activation was significantly related to chemoresistance. The essential role of Tie2 in this phenotype was illustrated by silencing Tie2 using specific siRNA, and the subsequent abrogation of the angiopoietin 1 (Ang1)-mediated chemoresistance. Using quantitative real-time PCR and functional drug efflux studies, we observed that Tie2 activation resulted in increased expression of ATP-binding cassette (ABC) transporters. Consistent with these results, downmodulation of ABCG2 or ABCC2 resulted in the inability of Tie2 activation to induce a chemoresistant phenotype. Our results indicate that Tie2 activation may be important in modifying the evolution of gliomas during conventional chemotherapy regimens, and open new avenues for the search of more effective therapies to avoid the inevitable brain tumor recurrence.

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.

Activity-guided isolation of constituents of Cerbera manghas with antiproliferative and antiestrogenic activities.

Two new cardenolides, (-)-14-hydroxy-3beta-(3-O-methyl-6-deoxy-alpha-L-rhamnosyl)-11a lpha, 12alpha-epoxy-(5beta,14beta,17betaH)-card-20 (22)-enolide (1), (-)-14-hydroxy-3beta-(3-O-methyl-6-deoxy-alpha-L-glucopyranosyl)-11al pha,12alpha-epoxy-(5beta,14beta,17betaH)-card -20(22)-enolide (2), and a known cardenolide, (-)-17beta-neriifolin (3), were isolated from the roots of Cerbera manghas as antiproliferative and antiestrogenic principles when evaluated against a human colon cancer cell line (Col2) and the Ishikawa cell line, respectively. Two known lignans, (-)-olivil (4) and (-)-cycloolivil (5), were also isolated but were inactive in the assay systems used.

Activity-guided isolation of steroidal alkaloid antiestrogen-binding site inhibitors from Pachysandra procumbens.

Four novel steroidal alkaloids, (+)-(20S)-20-(dimethylamino)-3-(3'alpha-isopropyl)-lactam-5alpha-+ ++preg n-2-en-4-one (1), (+)-(20S)-20-(dimethylamino)-16alpha-hydroxy-3-(3'alpha-isopropyl) -la ctam-5alpha-pregn-2-en-4-one (2), (+)-(20S)-3-(benzoylamino)-20-(dimethylamino)-5alpha-pregn-2-en-++ +4beta -yl acetate (3), and (+)-(20S)-2alpha-hydroxy-20-(dimethylamino)-3beta-phthalimido-5 alpha- pregnan-4beta-yl acetate (4), as well as five known compounds, (-)-pachyaximine A (5), (+)-spiropachysine (6), (+)-axillaridine A (7), (+)-epipachysamine D (8), and (+)-pachysamine B (9), were isolated from Pachysandra procumbens, using a bioassay-guided fractionation based on inhibition of 3H-tamoxifen binding at the antiestrogen binding site (AEBS). Compounds 1-7 and 9 demonstrated significant activity as AEBS-inhibitory agents, and compounds 3, 5 and 9 were found to potentiate significantly the antiestrogenic effect mediated by tamoxifen in cultured Ishikawa cells. The structure elucidation of compounds 1-4 was carried out by spectral data interpretation.

Evaluation of selected lichens from iceland for cancer chemopreventive and cytotoxic activity.

Cancer chemopreventive effects of organic extracts from 29 species of lichens collected in Iceland were evaluated using a panel of in vitro bioassays whereby extracts were tested for potential to induce quinone reductase (QR) and differentiation of human promyelocytic leukemia (HL-60) cells, inhibit cyclooxygenase-1 (COX-1), phorbol ester-induced ornithine decarboxylase (ODC), aromatase and sulfatase, as well as for antioxidant, estrogenic/anti-estrogenic and antiproliferative activity. In addition, the extracts were tested for cytotoxicity against 12 cancer cell lines. The most significant results were exhibited by extracts from Xanthoria elegans and Alectoria nigricans , which respectively, induced QR activity (concentration to double activity = 4.8 µg/ml) and inhibited phorbol ester-induced ODC activity with mouse 308 cells in culture (IC 50 = 2.6 µg/ml). Moderate inhibition of [ 3 H]thymidine incorporation with HL-60 cells was exhibited by the Peltigera leucophlebia extract. Several extracts prevented estrogen formation from estrogen precursors by inhibiting the enzymatic activities of aromatase ( Sphaerophorus globosus , Cetrariella delisei , Melanelia hepatizon ) and sulfatase ( Cladonia gracilis , Sphaerophorus fragilis , S. globosus ). None of the extracts demonstrated significant cytotoxic effects with selected cell lines.

Aromatase and sulfatase inhibitors from Lepiota americana.

From an edible mushroom Lepiota americana Pk., (Agaricaceae), 2-aminophenoxazin-3-one that inhibited aromatase at IC50 = 5.7 microM and 3 beta-hydroxy-5,8-epidioxyergosta-6,22-diene that inhibited sulfatase at IC50 = 0.9 microM were isolated. Neither 2-aminophenoxazin-3-one was active against sulfatase nor was 3 beta-hydroxy-5,8-epidioxyergosta-6,22-diene active against aromatase.