Resveratrol and hormonal balance -
prism San Diego, CA. K i was estimated by the formula described in The solubility of resveratrol has not been published.
No additional increase in the absorbance was observed when the resveratrol concentration exceeded μ m , indicating that μ m is the limit of resveratrol solubility. Unincorporated nucleotides were removed by centrifugation through a Centri-Spin 20 column Princeton Separations, Adelphia, NJ.
Labeled EREc38 50, cpm was incubated for 2 h at 4 C with a nuclear extract of baculovirus-expressed rhERα or rrERβ. Binding reactions were performed in TDPEKG buffer 40 m m Tris-HCl pH 7.
An ERα-specific antibody H, generously provided by Abbott Laboratories Abbott Park, IL , was diluted in TE buffer 10 m m Tris-HCl pH 8. ERβ-specific antibodies PA1— Affinity BioReagents, Inc.
Gels were dried under vacuum and autoradiographed on Kodak X-Omat film with an intensifying screen Lightning Plus from DuPont Co.
The ratio of ER-bound to free DNA was determined using a Packard Instruments InstantImager and associated software, Packard Imager for Windows v 2. The cells were transfected with 0. The transient transfection was performed using Transfast Promega Corp. according to directions supplied by Promega Corp.
The cells were harvested 30 h later, and the luciferase andβ -galactosidase β-gal activities were assayed All data for transient transfections were normalized by β-gal to account for transfection efficiency.
Cell proliferation was determined using the Cell Proliferation Kit 1 MTT according to the directions provided by the supplier Roche Molecular Biochemicals, Indianapolis, IN. Mock transfected cells were incubated with Transfast without added plasmid DNA and then treated with hormones or vehicle as described above.
Cell proliferation experiments were conducted concomitantly with transient transfection experiments. Cells cotransfected with ERα or ERβ and treated with 0. The absorbance of solubilized crystals was measured at nm in a Molecular Devices SpectraMAX plate reader.
Resveratrol was shown to bind ER in MCF-7 10 and rat uterine cytosol extracts ERβ for this binding is unknown. Competition binding experiments were used to determine the relative binding affinity of resveratrol for ERα and ERβ.
Resveratrol binds to rhERα Fig. The relative binding affinities RBA of resveratrol for ERα and ERβ are not statistically different Table 1. The equilibrium dissociation constants K I are different, reflecting higher affinity of E 2 binding to ERα than ERβ Table 1.
This is the first demonstration of resveratrol interaction with ERβ. Resveratrol competes with [ 3 H]E2 for binding rhERα and rrERβ. Competition binding experiments were performed with baculovirus expressed rhERα A and rrERβ B as described in Materials and Methods.
Increasing concentrations of either E 2 closed squares or resveratrol closed diamonds were mixed with Data were graphed as percent of saturation of the specific[ 3 H]E 2 binding capacity vs.
competitor concentration. These data indicate that resveratrol binds ERα and ERβ with an affinity approximately , and , fold lower than E 2 , respectively.
ERα and ERβ ligand binding competition experiments were performed and analyzed as described in Materials and Methods. The values are the mean from two experiments in which individual samples were performed in triplicate.
are given. The correlation coefficient r b is indicated. se cannot be calculated because of antilogarithm transformation of the confidence intervals for log 10 IC The IC 50 values obtained from these experiments indicate that resveratrol binds both ERα and ERβ with lower affinity than E 2 Table 1.
The estimated IC 50 values for resveratrol of Together, these data indicate that resveratrol binds ERα and ERβ with comparable binding affinity. Resveratrol has been reported to inhibit the proliferation of ER positive and negative cultured human breast cancer cells 10 , To determine the effect of resveratrol on CHO-K1 cell proliferation in a receptor-isoform-dependent assay, cells were transfected with expression vectors for either ERα or ERβ.
The mock transfected cells showed no alteration in proliferation regardless of cell treatment, indicating that TransFast is not toxic to the cells data not shown. Cells transfected with ERα or ERβ showed no alteration in cell proliferation with or without treatment with E 2 or 4-OHT Fig.
However, cells transfected with ERα or ERβ and treated with μ m resveratrol showed decreased proliferation.
Untransfected or mock-transfected cells showed no decrease in proliferation with μ m resveratrol treatment, indicating the effect is dependent upon ER expression Fig.
Similarly, cells transfected with pCMV-β-gal and treated μ m resveratrol show no decrease in cell proliferation. Cells treated with E 2 showed no decrease in viability. These data indicate that μ m resveratrol decreases cell proliferation only when the CHO-K1 cells are transfected with ERα or ERβ.
Effect of E 2 or resveratrol on the proliferation of ERα or ERβ- expressing CHO-K1 cells. The effect of the indicated concentrations of E 2 or resveratrol on the proliferation of CHO-K1 cells transfected with ERα or ERβ vs. nontransfected treatment only cells was measured by the tetrazolium dye MTT assay as described in Materials and Methods.
Data are presented as the mean ± sem of three different experiments in which each treatment was performed in triplicate. To determine if resveratrol affects the interaction of ERα or ERβ with an ERE, ERα or ERβ were incubated with E 2 , 4-OHT, or resveratrol and ERE binding was measured by EMSA Fig.
ERα bound specifically to EREc38, as indicated by the supershift of the entire bound complex with ERα-specific antibody H Fig.
The multiple ERα bands are due to truncated ERα present in the NE. As anticipated from previous reports 29 — 31 , preincubation of ERα with E 2 accelerated the migration of the ERα-EREc38 complex, while addition of 4-OHT slowed the migration of the 4-OHT-ERα-EREc38 complex compare Fig.
Resveratrol had no apparent effect on the migration of the ERα-EREc38 complex. These data indicate differences in ERα conformation in the presence of these ligands. Likewise, resveratrol did not affect the binding of H to ERα-EREc38, generating a supershifted complex similar to that of the E 2 -ERα-EREc38 complexed with H Fig.
ERα and ERβ bind an ERE in the presence of resveratrol. A, A nuclear extract of rh ERα 6. The reactions in lanes 1 and 6 included H H antibody to ERα. A nuclear extract of rh ERβ 1. The reactions in lanes 2 and 7 included 1 μl of PA1— and in lane 3, 1μ l of Y anti-ERβ antibodies.
For the reaction in both gels, 50, cpm 3. Incubation and EMSA reaction conditions are described in Materials and Methods. Similar results were detected for ERβ. Preincubation of ERβ with E 2 accelerated the migration of the ERβ-EREc38 complex, while addition of 4-OHT slowed the migration of the 4-OHT-ERβ-EREc38 complex compare Fig.
Resveratrol had no apparent effect on the migration of the ERβ-EREc38 complex compare Fig. These data indicate differences in ERβ conformation in the presence of these ligands. Resveratrol did not affect the interaction of antibody PA1— with the resveratrol-ERβ-EREc38 complex Fig.
While the concentration of resveratrol added was expected to fully occupy ERα or ERβ, based on the data shown in Fig. This inhibition of ER-ERE binding by resveratrol can also be observed by noting the increased amount of free EREc38 at the bottom of the gel Fig.
Resveratrol had a more pronounced inhibitory effect on ERβ-EREc38 binding compared with ERα-EREc38 binding. Like resveratrol, E 2 had more of an inhibitory effect on ERβ-EREc38 binding compared with ERα-EREc38 binding.
This observation indicates that ERβ-EREc38 binding is more labile than ERα-EREc38 binding. Resveratrol inhibits ERα and ERβ-EREc38 binding in vitro. Nuclear extracts of rh ERα A and rr ERβ B fmol were incubated with the indicated volumes or concentrations of ethanol EtOH , as vehicle control, or resveratrol range p m — μ m final.
ERα antibody H and ERβ antibody Y were added to lane 1 in A and B, respectively. EMSA was performed as described in Fig. C, The total amount of ER-EREc38 complex all retarded ERα-ERE bands in A formed with increasing concentrations of resveratrol or E 2 was quantitated.
There was no significant difference in the amount of either ERα or ERβ bound to EREc38 when incubated with 0. The data are the average ± sem of 2—4 independent EMSAs. The fills and symbols are indicated at the top of panel C. We used cotransfection assays in CHO-K1 cells to compare the transcriptional activities and ligand responsiveness of ERα and ERβ.
This cell line was selected because it requires exogenous ERα or ERβ to activate ERE-driven reporter gene expression and thus allows evaluation of the transcriptional response of each ER isoform in isolation with each ERE.
First, we examined the effect of resveratrol on reporter gene activity driven by one copy of a consensus ERE called EREc38 sequence in Materials and Methods with ERα. Resveratrol stimulated luciferase activity in a concentration-dependent manner up to 50 μ m , but μ m resveratrol inhibited luciferase activity Fig.
Because results from the MTT assay indicate that μ m resveratrol decreased cell proliferation in ERα-expressing cells Fig. E 2 , at concentrations of 1 or 10 n m , has higher agonist activity with ERα than 50 μ m resveratrol. Resveratrol at concentrations of 1, 10, or 50 μ m had no effect on basal luciferase activity from the pGL3-pro-luciferase parental vector data not shown.
Addition of n m 4-OHT blocked resveratrol activity with ERα Fig. While the fold induction of E 2 -stimulated luciferase detected is low, we note that others have reported that single EREs show significantly lower levels of transcriptional activation in response to E 2 compared with commonly used multiple tandem EREs 24 , 33 , These results indicate that the estrogen agonist activity of resveratrol is mediated by ERα-ERE interaction in transiently transfected CHO-K1 cells.
Resveratrol acts as an ERα and ERβ agonist in transient transfection assay. A, CHO-K1 cells were cotransfected with pGL3—1 EREc38 luciferase, pCMV-βgal, and either pCMV-ERα or pCMV-ERβ, indicated as different filled boxes , and treated with EtOH, as the vehicle control, or the indicated concentrations of E 2 , resveratrol, or resveratrol and 4-OHT.
B, CHO-K1 cells were cotransfected with pGL3-luciferase reporter plasmid bearing the EREs from the human c- fos FOS, open bars , pS2 closed bars , and PR PR, hatched bars genes, pCMV-βgal, and pCMV-ERα and treated with EtOH, E 2 , or resveratrol at the indicated concentration.
C, CHO-K1 cells were cotransfected with pGL3-luciferase reporter plasmid bearing the EREs from the human c- fos FOS , pS2, and PR PR genes, pCMV-βgal, and pCMV-ERβ and treated with EtOH, E 2 , resveratrol or resveratrol and 4-OHT at the indicated concentrations. The cells were transiently transfected and treated as described in Materials and Methods.
Cells were harvested 30 h after starting treatment, and the cell extracts were assayed for luciferase and β-gal activities. In each panel, the fold induction of luciferase activity was normalized for β-gal and is expressed as the ratio of RLU between treatment groups and the ethanol control which was set to 1.
Data are the mean ± sem from three to five different experiments in which each treatment was performed in triplicate within the experiment. Genes containing imperfect EREs, i. EREs that differ in one or more nucleotides in the inverted repeat of the consensus ERE, usually exhibit lower responsiveness to E 2 in reporter gene assays in transfected cells, compared with a consensus ERE 24 , Furthermore, ERα binds these EREs with reduced affinity compared with binding to EREc38 Refs.
Therefore, to determine whether resveratrol activates ERα-mediated reporter gene activity from imperfect EREs, we performed transient transfection assays using luciferase reporter plasmids containing the imperfect EREs and their native flanking regions from the human c- fos , pS2, and PR genes Fig.
E 2 did not induce as much luciferase activity from the natural, imperfect EREs from the human pS2 and PR genes as from the consensus EREc38 compare Fig. However, the luciferase activity induced by E 2 from the imperfect ERE from the human c- fos gene was similar than that induced from EREc While there was a significant increase in luciferase activity from pS2 when cells were treated with 10 n m E 2 , no significant dose-response relationship was detected for induction from Fos or PR As seen for EREc38, resveratrol induced significantly lower luciferase expression from Fos and pS2 compared with E 2.
The luciferase activity induced by 50μ m resveratrol from PR was comparable to that induced by 1 n m E 2. The induction of luciferase activity from the natural EREs by resveratrol was blocked by cotreatment with 4-OHT, indicating that ERα is responsible for resveratrol-induced reporter activity.
As seen for EREc38, treatment of the CHO-K1 cells with μ m resveratrol inhibited luciferase activity from the pS2, FOS, and PR EREs data not shown. In conclusion, the data from these transient transfection assays indicate that resveratrol acts as an estrogen agonist with ERα.
These results are similar to those detected in transiently transfected, ERα-expressing COS-1 cells with either a vitellogenin ERE or LH-β promoter-luciferase reporter plasmid ERβ has been shown, in transient transfection assays using a single or multiple tandem copies of a consensus ERE, to have lower activity in response to E 2 than ERα However, in COS-1 cells, ERβ induced higher reporter activity from the vitellogenin ERE than ERα in response to concentrations of E 2 ranging from 0.
Here, we observed that cotransfection of CHO-K1 cells with ERβ and EREc38 generated lower luciferase expression in response to E 2 compared with ERα Fig. Comparable protein expression levels of ERα and ERβ were achieved in these cells Western blot data not shown.
With ERβ, resveratrol stimulated luciferase expression from EREc38 in a concentration-dependent manner up to 50 μ m, whereas μ m resveratrol inhibited luciferase activity. These results are similar to those reported for ERβ with a vitellogenin ERE reporter in transiently transfected COS-1 cells In CHO-K1 cells, resveratrol-stimulated activity was inhibited by cotreatment with 4-OHT Fig.
Interestingly, in contrast to the differences in luciferase activity induced by E 2 with ERα and ERβ, resveratrol induced nearly identical levels of luciferase activity from EREc38 with either ERα or ERβ Fig. This indicates that resveratrol-liganded ERβ has similar transcriptional activity to E 2 -ERβ at a single perfect, palindromic ERE.
These data differ from those for ERβ expression in COS-1 cells in which μ m resveratrol showed higher induction of reporter activity from two tandem copies of the vitellogenin ERE than any of the concentrations of E 2 0.
These findings indicate that cell-specific factors influence the agonist activity of resveratrol with ERβ. Next we examined the induction of luciferase activity from the EREs from the human c- fos , pS2, and PR genes with ERβ Fig.
Please note that the scale for fold-induction of luciferase activity for revised Fig. As seen for ERα, E 2 induced lower activity from each of the imperfect EREs than from EREc38 compare Fig.
Unlike ERα for which E 2 stimulated more activity from Fos than the other natural EREs, there was no difference in the luciferase activity induced by ERβ with 10 n m E 2 from the imperfect EREs.
These data indicate that ERα and ERβ transactivate reporter gene expression differentially in response to E 2 from natural imperfect EREs in CHO-K1 cells.
Resveratrol stimulated ERβ-driven reporter activity from each natural-occurring ERE in a concentration-dependent manner, although the response with PR was not statistically different between resveratrol concentrations.
For both ERα and ERβ, PR was least responsive to resveratrol. As anticipated, the luciferase activity from the three imperfect EREs was lower than that induced from EREc38 compare Fig.
However, for Fos and pS2, the luciferase activity induced by 50 μ m resveratrol was greater than that stimulated by 1 or 10 n m E 2. As seen with ERα, treatment of the ERβ-transfected CHO-K1 cells with μ m resveratrol inhibited luciferase activity from all EREs due to decreased CHO-K1 cell proliferation Fig.
The induction of luciferase activity from each of the natural EREs by resveratrol with ERβ was blocked by cotreatment with 4-OHT, indicating that ERβ is responsible for resveratrol-induced reporter activity.
At 50 μ m , resveratrol induced identical levels of reporter activity from EREc38, Fos, and pS2 with ERα and ERβ. However, E 2 induced higher reporter activity from all EREs with ERα than ERβ.
For PR, ERβ was less active than ERα with resveratrol and E 2. Taken together, these results imply that resveratrol-liganded ERα and ERβ are equivalently transcriptionally active with the Fos and pS2 EREs. In contrast, E 2 -liganded ERβ interacts with EREc38 and the imperfect EREs from the human c- fos , pS2, and PR genes less productively than ERα.
Finally, these data indicate that the ER agonist activity of resveratrol is not identical for ERα and ERβ and varies with ERE sequence. greater activity than with 0. This phenomenon is not understood because these two reports show different definitions of superagonist activity.
In the more recent report, the reporter activity detected in MCF-7 cells treated with 0. We evaluated how resveratrol impacted E 2 -stimulated luciferase activity from EREc38 or the natural EREs from the human c- fos , pS2, and PR genes in CHO-K1 cells expressing either ERα Fig.
No additive activity was detected with ERα and 0. Interestingly, 50 μ m resveratrol had E 2 antagonist activity for EREc38 and PR, but not Fos or pS2.
We note that the relatively low fold induction that we observed with single EREs is similar to that reported by other investigators 33 , Resveratrol exhibits little or no antagonist activity with ERα and additive agonist activity with ERβ at various EREs.
CHO-K1 cells were cotransfected with pGL3-luciferase reporter plasmid bearing 1 EREc38 or the EREs from the human c- fos FOS , pS2, and PR PR genes, pCMV-βgal, and pCMV-ERα A or pCMV-ERβ B.
In both panels, cells were treated with 0. The transient transfection, treatment, and assay conditions were as described in Materials and Methods and in Fig. The fold induction of luciferase activity was normalized for β-gal and is expressed as the ratio of RLU between treatment groups and the vehicle control which was set to 1.
Data are the mean ± sem from three different experiments in which each treatment was performed in triplicate within the experiment.
In contrast to ERα, with ERβ the luciferase activity stimulated by 1 n m E 2 from EREc38 and PR was not antagonized by cotreatment with 50 μ m resveratrol. As seen for ERα, resveratrol had no effect on E 2 -induced reporter activity from pS2.
The results for Fos were similar for ERα and ERβ: increased activity with E 2 plus resveratrol. In no case was the activity of E 2 and resveratrol additive.
The activity induced by E 2 plus resveratrol for each ERE with ERβ was blocked by 4-OHT, indicating that it is dependent on ERβ AF-2 data not shown. As seen with the single EREs Figs. Resveratrol showed weak agonist activity for both ERα and ERβ. Similar to the data for a single copy of EREc38, resveratrol suppressed E 2 -ERα-stimulated luciferase activity from 2 EREc38 in a concentration-dependent manner.
In contrast, resveratrol did not inhibit E 2 -ERβ activity. This result indicates that ERα and ERβ respond differently to resveratrol at the same ERE. Resveratrol, when combined with E 2 exhibits antagonist activity with ERα, but no antagonist activity with ERβ.
Combined E 2 and resveratrol have different effects on ERα and ERβ. CHO-K1 cells were cotransfected with pGL3-luciferase reporter plasmid bearing 2 EREc38 , pCMV-βgal, and pCMV-ERα black bars or pCMV-ERβ hatched bars.
Epidemiological evidence indicates that phytoestrogens have biological activities including inhibition of cancer initiation and growth, reduction of serum cholesterol levels, and benefits in treating osteoporosis 39 , This gives a concentration of trans- resveratrol of 0.
Given the daily recommended dose of wine is ml 41 , it is estimated that a person would ingest 0. Resveratrol has chemopreventive and chemotherapeutic activities 1 , 2 and has been classified as a phytoestrogen because it binds to ERα with low affinity This finding contrasts with data showing that several phytoestrogens bind ERβ with higher affinity than ERα One possible explanation for the lower affinity of resveratrol binding to ERβ compared with other phytoestrogens is its structural similarity with diethylstilbestrol DES 10 , which binds ERβ with lower affinity than ERα Whether resveratrol-occupied ERα has agonist or antagonist activity has been controversial 10 , 14 , higher reporter activity than E 2 , in ERα-expressing MCF-7 cells 9 , However, another report found no evidence of superagonism in COS cells with either ERα or ERβ Data from our transient transfection assays in CHO-K1 cells using a consensus ERE or the natural imperfect EREs from the human c- fos , pS2, or PR genes indicate that resveratrol acts as an estrogen agonist with ERα and ERβ.
However, resveratrol does not display superagonist activity either alone or in combination with E 2. These results are similar to those detected in COS-1 cells transfected with ERα and either a vitellogenin ERE or LH-β promoter-luciferase reporter plasmid Our results demonstrate that the agonist activity of resveratrol with ERβ is fundamentally different from E 2 agonist activity because, in contrast to E 2 which induces higher activity of ERα than ERβ, resveratrol activated equal reporter activity from EREc38 with both ERα and ERβ.
In contrast, μ m resveratrol was reported to induce higher ERE-driven reporter activity by ERβ than any concentration of E 2 0. Because both we and Ashby et al. Importantly, our data indicate that resveratrol-liganded ERβ has higher transcriptional activity than E 2 - liganded ERβ at a single palindromic ERE.
Biochemical 29 , 30 , 44 , 45 and crystal structure 46 , 47 studies indicate ligand-specific differences in ERα conformation that impact interaction with coactivators. We observed clear differences in the migration of ERE-bound ERα and ERβ either unoccupied or occupied by E 2 , 4-OHT, or resveratrol.
These data indicate differences in ER conformation in the presence of resveratrol compared with E 2 or 4-OHT. The concentrations at which resveratrol inhibited ERE binding by ERα and ERβ in vitro are concentrations at which resveratrol exhibited agonist activity in transiently transfected cells.
Further experiments, e. in vivo DNase I footprinting, are needed to determine in vivo effects of resveratrol on ER-ERE binding. The authors concluded that this positioning of the transactivational helix 12 was consistent with the partial agonist activity of genistein Given the pharmacological activities of resveratrol observed here, we predict that resveratrol-bound ERβ LBD may show a structure similar to that of the genistein-occupied ERβ.
Because alterations in LBD conformation impact the interaction of ERα and ERβ with coactivators, further experiments are needed to assess coactivator effects on resveratrol-liganded ERβ activity.
While resveratrol reportedly gave a dose-dependent increase in reporter activity with concentrations as high as μ m with both ERα and ERβ in transiently transfected COS cells 14 , we observed that μ m resveratrol inhibited ERE-driven reporter activity in CHO-K1 cells.
Moreover, we observed decreases cell proliferation in CHO-K1 cells expressing ERα or ERβ and treated with μ m resveratrol. There was no significant decrease in CHO-K1 cell proliferation in cells treated with μ m resveratrol and expressing β-galactosidase or in mock-transfected cells under the same conditions.
While others have reported that E 2 treatment of cells stably overexpressing ERα inhibits cell proliferation 49 — 53 , E 2 at concentrations that stimulate reporter activity in transiently transfected CHO-K1 cells had no effect on cell proliferation in the presence or absence of transfected ERα or ERβ.
Therefore, stable expression of ERα in ERα negative cells appears to be required for E 2 -induced inhibition of cell proliferation in response to E 2 , but not resveratrol. We conclude that expression of ERα or ERβ is involved in the decrease in CHO-K1 cell proliferation with μ m resveratrol.
Others reported that μ m resveratrol inhibited the growth of ERα-expressing MCF-7 cells However, the mechanism for inhibition of MCF-7 cell proliferation may not be ERα-mediated since resveratrol also inhibited the growth of ER-negative breast cancer cells In addition to its agonist activity, resveratrol exhibited antiestrogenic activity in MCF-7 cells Resveratrol decreased the levels of transcription of PR, insulin-like growth factor-receptor, and transforming growth factor-α genes and stimulated the expression of transforming growth factor-β2, results similar to those elicited by tamoxifen in these cells While the authors concluded that the most likely mechanism for the antiestrogenic effect of resveratrol is its direct competition with E 2 for ER binding, they also suggested that resveratrol might prevent ER binding to EREs Our data support both suggestions because we observed that resveratrol competes with E 2 for ERα and ERβ binding and inhibited ERα and ERβ binding to EREc38 in vitro.
We observed both ER isoform-specific and ERE-specific differences in the agonist activity induced by resveratrol in CHO-K1 cells. For example with ERα, the activity induced by E 2 was greater than that stimulated by any concentration of resveratrol for EREc38, Fos, and pS2, but not PR In contrast, resveratrol and E 2 were equally transcriptionally active with ERβ at all EREs tested.
With ERα and PR, although the induction levels are low, they are significantly above the ethanol control values, and resveratrol-induced luciferase activity was comparable to that induced by E 2.
In addition to estrogen agonist activity, we also report that resveratrol has estrogen antagonist activity in CHO-K1 cells. These data are reminiscent of the lack of 4-OHT agonist activity with ERβ We speculate that the antagonist activity of resveratrol may be mediated by AF-1, which appears to be absent in ERβ.
Interestingly, the antagonist activity of resveratrol was only observed with EREc38, whether as a single or two tandem copies, and PR These data indicate that the ERE alters the pharmacological properties of resveratrol mediated by ERα.
This result agrees with our postulate that the ERE sequence acts as an allosteric modulator of ER activity Further experiments are needed to define exactly what regions of ERα and ERβ are necessary for resveratrol agonist and antagonist activity and to define the exact ERE sequence requirements for resveratrol antagonist activity with ERα.
For example, resveratrol induces phosphorylation of the mitogen-activated protein MAP kinase family members, extracellular regulated kinase 1 ERK1 , and ERK2, in neuroblastoma SH-SY5Y cells 55 Activation of the MAP kinase pathway has been shown to activate unliganded ERα through phosphorylation of serine in AF-1 However, this ligand-independent pathway does not appear to be important in our experiments because 4-OHT blocked both E 2 and resveratrol activity, indicating that direct activation of ERα through the LBD was responsible for the activity reported here.
While the pharmacokinetics of resveratrol metabolism have not yet been examined in humans, results from rodent studies indicate that two servings of red wine may provide two-digit micromolar serum concentrations of resveratrol 1 , i.
concentrations identical to those at which the pharmacological activities of resveratrol were observed here as well as reported by others 10 , 14 , The cell-type and ERE-sequence dependence of the transcriptional activity of resveratrol with ERα and ERβ may be related to cell-specific differences in the activity of enzymes that modulate ER function, e.
protein kinases, and in the expression of coactivator or corepressor proteins. Continued analysis of ERα and ERβ interaction with estrogenic ligands, estrogen-regulated genes, and coregulator proteins is necessary to gain a better understanding of how these receptors regulate estrogenic activity at the cellular and molecular level and whether the anticancer and cardioprotective activities of resveratrol are mediated by ER.
Supported by NIH Grant RDK, the Cancer Research Foundation of America, and a University of Louisville School of Medicine Research Grant to C. We thank the following companies for supplying reagents used in this study: resveratrol: Pharma Science, Montréal, Québec, Canada; and H Abbott Laboratories Abbott Park, IL.
We thank Rosemary L. Sims for her assistance in some of the experiments reported here. We thank Drs. Barbara J. Clark and Peter C. Kulakosky for their contributions to the experimental design and for reviewing this manuscript.
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In one study, forty postmenopausal females were given 1 gram of resveratrol daily for twelve weeks. Also, reduce the risk of breast cancer in females by regulating estrogen metabolism. In rats, resveratrol inhibit the enzyme activity, which activates estrogen receptors. It binds to them and makes them weak; this action has a balancing effect on the body.
According to health experts, when estrogen level becomes low in females during menopause , resveratrol can increase its level. Another research has concluded that high doses of resveratrol almost mg per day for four months are effective in reducing the level of serum androgen levels.
But its effects on levels of testosterone and DHEAS are not clear. The health statements have not yet been officially established by the European authorities.
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