Daidzein Induces Intrinsic Pathway of Apoptosis along with ER α/ß Ratio Alteration and ROS Production.

BACKGROUND: Low risk of breast cancer is observed among females consuming a moderate quantity of soy throughout their life. The present study was conducted to evaluate the anticancer potential of Daidzein, one of the major Isoflavones in soy using Human breast cancer cells MCF-7. METHODS: MCF-7 were subjected to various doses of Daidzein treatment to determine the IC50 value. Onset of apoptosis was ascertained by AnnexinV assay and caspase 3/7 activity post treatment. Expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 was also assessed to further confirm apoptotic mode of cell death. ROS production post treatment with Daidzein was assessed to ascertain the apoptosis via intrinsic pathway. Expression of ER α and ER ß was evaluated by western blot analysis. RESULTS: Human breast cancer cells MCF-7 were found to be sensitive to Daidzein treatment, with an IC50 value of 50µM. Increased percentage of treated cells stained with Annexin V confirmed apoptosis mediated cell death. Activity of Caspase 3/7 activity was found to be 1.4-fold higher in Daidzein treated cells than control cells, confirming apoptosis. Daidzein caused over expression of Bax and down-regulated expression of Bcl2. There has been an outburst of ROS in Daidzein treated cells indicating that Daidzein induces apoptosis via intrinsic pathway. A decrease in the expression of ER α and increase in levels of ER ß has been observed which are conducive indicator of apoptosis. CONCLUSIONS: In conclusion, the present study suggests that Daidzein induces apoptosis in MCF-7 cells by mitochondrial pathway along with lowering the ratio of ER α/ß and an outburst of Reactive Oxygen Species(ROS).

Sex hormone-like Effects of Icariin on T-cells immune modulation in spontaneously hypertensive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium brevicornu Maxim as a Chinese herb, is recommended for the treatment of menopausal women with hypertension for 50 years. Icariin, as the main hydrophilic ingredient of Epimedium brevicornu Maxim, has been proven to be a plant sex hormone and lower blood pressure down. Here, we hypothesized that Icariin can regulate T cells differentiation which leads to the blood pressure decrease in castrated SHR rats. AIM OF THE STUDY: The present study aimed to investigate the effects of the exogenous estrogen, androgen and Icariin on T-cell modulation in hypertension. MATERIALS AND METHODS: Two weeks after castration, both male and female SHR rats were given estradiol, testosterone, and Icariin intervention respectively. Body weight, blood pressure, and heart rate were tested weekly. After six weeks, proportion of T helper cells (Th), cytotoxic T cells (Tc), and regulatory T cells (Tregs) in both peripheral blood mononuclear cells (PBMCs) and splenocytes were tested by flowcytometry. Serum levels of estrogen, testosterone, AngII, TNF-α, IL-17 were tested by Elisa. Aortic arches were isolated for HE and Masson staining. The expressions of ERß and AR in aorta were tested by Western-blot. RESULTS: In both male and female SHR rats, we found that Icariin and estradiol lower blood pressure, but testosterone elevates blood pressure. Similar as testosterone, Icariin can attenuate Tc and Th proportions and elevate Tregs proportion in both peripheral blood and splenocyte in male SHR, which can be blunt by flutamide. Besides, Icariin performs similar function as estradiol that attenuates Tc proportions and elevates Tregs proportion in both peripheral blood and splenocytes in female SHR, which leads to the lower blood pressure and can be partly blunt by fulvestrant. Testosterone increases AngII and TNF-α levels in serum, leading to the higher blood pressure in both male and female SHR rats. CONCLUSION: These results verified that Icariin, as a plant sex hormone, can regulate T cells differentiation related to blood pressure decrease in SHR rats.

Calycosin induces apoptosis in osteosarcoma cell line via ERß­mediated PI3K/Akt signaling pathways.

Previous studies have shown that calycosin, a natural phytoestrogen which is structurally similar to estrogen, inhibits proliferation and induces apoptosis in estrogen­dependent cancer types via the estrogen receptor (ER)ß­induced inhibition of PI3K/Akt. Therefore, the aims of the present study were to investigate the effects of calycosin on human osteosarcoma (OS), and to examine the molecular mechanisms associated with ERß. Human OS MG­63 cells were treated with various concentrations of calycosin, and MTT and flow cytometry assays were used to assess the effects of calycosin on cellular proliferation and apoptosis. In addition, protein expression levels of ERß, phosphorylated (p)­PI3K, p­Akt, cleaved poly (ADP­ribose) polymerase 1 (PARP) and cleaved caspase­3 were evaluated by western blot analysis. The present results suggested that calycosin inhibited proliferation and induced apoptosis in MG­63 cells. Furthermore, increased ERß expression was detected in OS MG­63 cells treated with calycosin, and an ERß inhibitor (PHTPP) reversed calycosin­induced cytotoxicity and apoptosis. Moreover, phosphorylation levels of PI3K and Akt were significantly downregulated after calycosin treatment, whereas PHTPP reversed their phosphorylation. ERß­mediated PI3K/Akt downstream signaling pathways were found to influence the activity of poly (ADP­ribose) polymerase 1 and caspase­3. Thus, the present results indicated that calycosin inhibited proliferation and induced apoptosis in OS MG­63 cells, and that these effects were mediated by ERß­dependent inhibition of the PI3K/Akt pathways.

Effects of Paeonia lactiflora Extract on Estrogen Receptor ß, TPH2, and SERT in Rats with PMS Anxiety.

This study is to investigate the effect of Paeonia lactiflora extract on PMS anxiety and on expression of estrogen receptor ß (ERß), tryptophan hydroxylase-2 (TPH2), and serotonin transporter (SERT) in the premenstrual syndrome (PMS) anxiety model rats. The vaginal smear and open field test were used to screen rats in nonreception phase of estrus cycle with similar macroscopic behaviors and regular estrus cycle. PMS anxiety model rats were prepared by electrical stimulation. RT-PCR and immunofluorescence were used to measure the expression of ERß, TPH2, and SERT. Compared with normal rats, the total distance in the open field test of the model rats was significantly increased (P < 0.05). The model rats showed nervous alertness, irritability, and sensitivity to external stimuli. After treatment with the Paeonia lactiflora extract, the total distance of rats was significantly reduced (P < 0.05). In reception stage, there was no significant difference in the mRNA and protein expression of ERß, TPH2, and SERT. In nonreception stage, the expression of ERß and TPH2 in the model group was significantly decreased (P < 0.05) as compared with the control group, but not SERT. Abnormal changes of the above indicators were reversed after the administration of the Paeonia lactiflora extract. In conclusion, Paeonia lactiflora extract can increase the expression of ERß and TPH2 and decrease SERT in PMS model rats, which may be one of the mechanisms underlying the effect of Paeonia lactiflora extract on PMS.

Estrogenic phytochemical from Labisia pumila (Myrsinaceae) with selectivity towards estrogen receptor alpha and beta subtypes.

Labisia pumila var. alata (Myrsinaceae) or "Kacip fatimah" is a famous Malay traditional herb used for the maintenance of women's health. The extracts of L.pumila displayed estrogenic activity in rats. Nonetheless, the estrogenic bioactives were not identified. The aim of the study is to identify estrogenic compounds contributing to the established estrogenic activity. Bioactivity-guided-isolation method guided the isolation of pure bioactives. The hexane extract was subjected to a series of silica gel flash and open column chromatography with increasing amount of ethyl acetate in hexane or methanol in chloroform. Each fraction or pure compounds were evaluated on it's estrogen receptor (ER) binding activity with the fluorescence polarization competitive ERα and ERß binding assay kit. Cytotoxic assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method was used to establish the cytotoxic activity of the compounds. Four alkyl resorcinols and a dimeric 1,4-benzoquinone, namely belamcandol B (1), 5-pentadec-10'-(Z)-enyl resorcinol (2), 1,3-dihydroxy-5-pentadecylbenzene (3), 5-(heptadec-12'-(Z)-enyl) resorcinol (4) and demethylbelamcandaquinone B (5) were identified with selective binding affinities towards either ERα or ERß exhibiting selectivity ratio from 0.15-11.9. Alkyl resorcinols (2)-(4) exhibited cytotoxic activity towards HL60 cells with IC50 values from 19.5-22.0 µM. Structural differences between compounds influence the binding affinities to ER subtypes. Further study is needed to establish the agonist or antagonist effect of these compounds on various tissues and to identify if these compounds exert cytotoxic activity through the ERs. When consuming L.pumila as a complementary medicine, careful consideration regarding it's estrogenic compound content should be given due consideration.

Erythroidine alkaloids: a novel class of phytoestrogens.

Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, ß-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-ß-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and ß-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010% and 0.005 ± 0.010%, respectively, whereas only ß-erythroidine bound to ERß (0.006 ± 0.010%). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and ß-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of phytoestrogens.

Impact of lycopene on epididymal androgen and estrogen receptors' expression in polychlorinated biphenyls-exposed rat.

The aim of the study was to evaluate the androgen (AR) and estrogen receptors' (ER) expression in epididymis of polychlorinated biphenyls (PCBs)-exposed rats. The rats were assigned to groups. Group I controls were treated with corn oil 80 µL/d intraperitoneally (ip), group II were treated with 2 mg/kg/d of A1254 ip; and group III were treated with 2 mg/kg/d of A1254 ip along with simultaneous oral supplementation of 4 mg/kg/d lycopene . The treatment was given daily for 30 days. After 24 hours of treatment, the rats were killed, and the epididymal regions (caput, corpus, and cauda) were dissected out, weighed, and prepared to estimate the levels of sialic acid, glyceryl phosphoryl choline (GPC), hydrogen peroxide (H2O2), and lipid peroxidation (LPO). The messenger RNA (mRNA) expressions of AR, ERα, and ERß were analyzed by reverse transcriptase-polymerase chain reaction, and ERα and ERß protein expressions were analyzed by immunoblotting. The toxicity of PCBs was also confirmed by histology. There was a marked decrease in epididymal weight, sialic acid, and GPC levels, while oxidative stress markers H2O2 and LPO were increased in PCBs-treated rats. The mRNA and protein expression of AR, ERα, and ERß were decreased in PCBs-treated groups, and the histology confirms the cytoplasmic damage in the regions of caput, corpus, and cauda in PCBs-treated rats. Simultaneous supplementation of lycopene to PCBs-exposed rats resulted in significant decrease in the oxidative stress markers as that of control, while the AR, ERα, and ERß gene expressions were near to control. The results suggest that lycopene has ameliorative effect against PCBs-induced toxicity in epididymis.

The rat prepubertal uterine myometrium and not the luminal epithelium is predominantly affected by a chronic dietary genistein exposure.

Current knowledge about dietary soy isoflavone-induced hormonal effects and potential priming effects for the responsiveness of the organism to other estrogens is insufficient. The present study examined the effects of pre- and postnatal soy isoflavone exposure on estrogen responsiveness by estrogen receptor agonists in the uteri of prepubertal Wistar rats. To this end, offspring were generated from dams already maintained on three dietary groups, (1) a phytoestrogen-free diet, (2) a soy isoflavone-rich diet with 232 ppm daidzein and 240 ppm genistein or (3) a custom-made diet supplemented with 700 ppm genistein (GEN). Then, F1 females continuously exposed to isoflavones from GD1 to PND21 and non-exposed controls were subjected to an immature uterotrophic assay to compare physiological parameters and the response to subcutaneous treatment with 17ß-estradiol, GEN or an estrogen receptor subtype (ERα and ERß)-specific agonist. Uterine wet weight (UWW), luminal epithelial height (LEH) and myometrial thickness (MMT) were determined. In addition, isoflavone plasma levels and mRNA expression profiles of relevant steroid receptors and of molecular markers for proliferation and estrogenicity were assessed for all groups. The influence of dietary isoflavones on the sensitivity to various estrogenic stimuli in these prepubertal animals was minor. Yet, the uterus of immature rats with high chronic GEN exposure alone showed already an increase in UWW, LEH and MMT. The myometrial response to GEN was more pronounced than that of the luminal epithelium, which may be due to a non-uniform distribution of steroid receptors, in particular the progesterone receptor. In conclusion, although the impact of a continuous, prenatally initiated exposure to dietary isoflavones on the uterine physiology of juvenile rats is modest, the possible priming effects of this exposure for beneficial or adverse late-onset consequences in adults should not be neglected.

Selective estrogen receptor modulators regulate stromal proliferation in human benign prostatic hyperplasia by multiple beneficial mechanisms--action of two new agents.

The existing drugs for benign prostatic hyperplasia (BPH) are partially effective with undesirable side-effects; hence new agents acting by different mechanism(s) are required as supplements. Modulation of estrogen receptor signaling using selective estrogen receptor modulators (SERMs) offers an alternative approach for BPH management. Using human BPH-derived stromal cells and tissue explants in culture we evaluated two SERMs, DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2 H-1-benzopyran (BP) and Ormeloxifene (Orm) in comparison to Tamoxifen (Tam) and 4-hydroxytamoxifen (OHT). BP, OHT and Tam were more effective than Orm in reducing stromal cell proliferation of human BPH. BP was either equipotent or more effective than OHT and Tam in increasing estrogen receptor(ER)-ß, TGFß1, Fas and FasL, and in decreasing ER-α, AR, EGF-R and IGF-I expressions in BPH stromal cells. BP, Tam and Orm (1.0 mg/Kg) reduced rat prostate weights by almost same extent as Finasteride (Fin, 5.0 mg/Kg); however combination treatment (SERM+Fin) was more effective. BP was exceptionally efficient in reducing IGF-1 and cleaving PARP while combination treatments more effectively increased bax:bcl-2 ratio. Fin reduced acinar diameter and prostatic DHT level but increased testosterone, estradiol (E(2)) and E(2)/T+DHT ratio. SERMs, especially BP, reduced epithelial cell height drastically without significantly altering steroid hormone levels and E(2)/T+DHT ratio. Combination treatment reduced both acinar diameter and epithelial cell height with modest increase in E(2), T and E(2)/T+DHT. The study reveals the potential of SERMs per se for BPH management, and more effectively in combination with a 5α-reductase inhibitor. BP appears promising for further evaluation as a drug candidate for BPH and prostate cancer.

[Influence of standardized extract of Epilobium angustifolium on estrogen receptor alpha and beta expression in in vivo model]. / Wplyw standaryzowanego wyciqgu z Epilobium angustifolium na ekspresj mRNA receptorów estrogenowych alpha i beta w modelu in vivo.

OBJECTIVE: Evaluation of the influence of the standardized extract from the herb of Epilobium angustifolium on ERalpha and ERbeta mRNA expression in rat ventral prostate tissue and free serum estradiol level. MATERIALS AND METHODS: Rats were divided into 6 groups with 10 animals. ERalpha and ERbeta mRNA expression in rat ventral prostate tissue level was performed using real-time PCR method in Light Cycler system. Serum-free estradiol was evaluated using immunoenzymatic technique. RESULTS: In our experimental model there was an increase of ERa mRNA level by 9% and decrease by 36% of ERbeta mRNA level in ventral prostate tissue in rats administrated with testosterone and E. angustifolium extract, in comparison with testosterone alone administrated animals. CONCLUSIONS: E. angustifolium standardized extract influenced the expression of estrogen receptor alpha and beta mRNAs in differential manner which may suggest its potentially therapeutic properties or causing of adverse effects in pharmacotherapy of estrogen-related disorders. More complex studies should be undertaken to evaluate safety and to improve the efficacy of using this herbal extract.

[Combined estrogenic activity of soybean extract used in a dietary supplement and ethinyl estradiol].

We examined the combined estrogenic activity of soybean extract used in a dietary supplement and ethinyl estradiol (EE) contained in an oral contraceptive. Olive oil (control), soybean extract (0.0036 or 0.36 g/kg corresponding to doses of total isoflavone of 0.83 or 83 mg/kg respectively), EE (1 or 10 microg/kg), and soybean extract+EE were administered to ovariectomized CD-1 mice by oral gavage for 4 consecutive days. Soybean extract (0.0036 or 0.36 g/kg) and EE (1 microg/kg) did not increase the relative uterine weight. The relative uterine weight of the soybean extract (0.0036 or 0.36 g/kg)+EE (10 microg/kg) group was significantly higher than that of the control. The relative uterine weight of the soybean extract (0.36 g/kg)+EE (10 microg/kg) group was also significantly higher than that of the EE (10 microg/kg) group. Soybean extract showed estrogenic activity for human estrogen receptor (hER)-alpha and -beta. Coadministration of EE with soybean extract increased the estrogenic activity for hER-alpha and -beta.

Comparative effects of R- and S-equol and implication of transactivation functions (AF-1 and AF-2) in estrogen receptor-induced transcriptional activity.

Equol, one of the main metabolites of daidzein, is a chiral compound with pleiotropic effects on cellular signaling. This property may induce activation/inhibition of the estrogen receptors (ER) a or b, and therefore, explain the beneficial/deleterious effects of equol on estrogen-dependent diseases. With its asymmetric centre at position C-3, equol can exist in two enantiomeric forms (R- and S-equol). To elucidate the yet unclear mechanisms of ER activation/inhibition by equol, we performed a comprehensive analysis of ERa and ERb transactivation by racemic equol, as well as by enantiomerically pure forms. Racemic equol was prepared by catalytic hydrogenation from daidzein and separated into enantiomers by chiral HPLC. The configuration assignment was performed by optical rotatory power measurements. The ER-induced transactivation by R- and S-equol (0.1-10 µM) and 17b-estradiol (E2, 10 nM) was studied using transient transfections of ERα and ERß in CHO, HepG2 and HeLa cell lines. R- and S-equol induce ER transactivation in an opposite fashion according to the cellular context. R-equol and S-equol are more potent in inducing ERα in an AF-2 and AF-1 permissive cell line, respectively. Involvement of ERα transactivation functions (AF-1 and AF-2) in these effects has been examined. Both AF-1 and AF-2 are involved in racemic equol, R-equol and S-equol induced ERα transcriptional activity. These results could be of interest to find a specific ligand modulating ER transactivation and could contribute to explaining the diversity of equol actions in vivo.

Comparison of hormonal activity of isoflavone-containing supplements used to treat menopausal complaints.

OBJECTIVES: The isoflavones present in red clover and soy are used as an alternative treatment for menopausal complaints and are commercially available as high-dose food supplements. These preparations contain varying amounts of active ingredients, often without detailed specifications. Thus, it is difficult to derive a recommended daily dose, and the reliability of these products is rather low. METHODS: We quantified the isoflavone content of 19 different isoflavone-containing preparations and compared their binding and transactivational activities with regard to estrogen receptor alpha, estrogen receptor beta, androgen receptor, progesterone receptor, peroxisome-proliferator-activated receptor, and aryl hydrocarbon receptor. RESULTS: The food supplements that we tested bound to and transactivated both the estrogen receptors and the other receptors. After comparing the isoflavone content quantified by us with the isoflavone content specified on the package labels, we found that at least the specified isoflavone content or more could be detected in only 5 of the 19 food supplements that we tested. CONCLUSIONS: Preparations containing isoflavones should be standardized for the isoflavone aglycone content to facilitate the prediction of theoretical hormonal activity, facilitate the intake of a controlled amount of isoflavones, and ensure greater product reliability.

In vitro estrogenic activity of Asplenium trichomanes L. extracts and isolated compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Asplenium trichomanes was used as an expectorant, anti-cough remedy, laxative, emmenagogue, abortifacient and for irregular menses. AIM OF THE STUDY: To investigate the in vitro estrogenic activity of Asplenium trichomanes extracts and isolated compounds and their ability to activate ERalpha and ERbeta. MATERIALS AND METHODS: Leaves infusion (IF), decoction (DC) and methanol extract (ME) were prepared. MCF7/EREluc cell line which expresses endogenous ERalpha, and SK-NBE cells transiently transfected with the estrogen receptors (ERalpha and ERbeta) were used for the estrogenic activity assays. Phytochemical investigations were performed (CC, HPLC, etc.) and structure of isolated compounds were achieved on the basis of 1D and 2D NMR techniques and HR-MS spectrometry. RESULTS: IF and ME were active in our MCF7 model; selectivity for the ERbeta receptor was observed in the SK-NBE test. Two new phenol derivatives, 4-vinyl-phenol-1-O-[alpha-L-rhamno(1-->6)-beta-d-glucopyranosyde] (1) and kaempferol-3-O-alpha-[2'acetyl]-arabinofuranosyl-7-O-alpha-L-rhamnopyranoside (2) were isolated with six known compounds (3-8). Compounds 2-4, 7 and 8 showed selectivity for the activation of the ERbeta receptor although with a moderate activity compared with 17-beta-estradiol. CONCLUSION: Further investigations about the estrogenic effects of this plant are needed but our data can, at least in part, explain some of its traditional use as emmeagogue.

Down-regulation of epidermal growth factor receptor induced by estrogens and phytoestrogens promotes the differentiation of U2OS human osteosarcoma cells.

In previous studies on HeLa cells we demonstrated estrogen-responsiveness of the epidermal growth factor receptor (EGFR) gene, as 17beta-estradiol (E(2)) and selective estrogen receptor modulators (SERMs) genistein (G), daidzein (D), and 4-hydroxytamoxifen (4OH-T) modulated its transcription in a ligand- and estrogen receptor (ER) isoform-specific way. This study describes further investigations into the role of ERs in mediating the effects induced by E(2) and SERMs on EGFR expression, and the relationship between the actions of ERs and EGFR in U2OS osteosarcoma cells stably expressing ERalpha or ERbeta. Cell number and DNA content determination revealed that E(2), G, and D inhibited proliferation and cell cycle progression and promoted apoptosis in both cell lines. In parallel, changes in cell morphology typical of osteoblast maturation were observed via optical microscopy. Consistently, quantitative PCR and Western blot analysis showed an up-regulation of markers of osteoblast differentiation and bone repair, and a decrease in EGFR expression. The transfection of specific antisense (AS) oligonucleotides strengthened our hypothesis that EGFR reduction caused changes in the proliferation/differentiation pattern comparable to those induced by ER ligands. The link between the ER and EGFR pathways was confirmed by treatment with 4OH-T, which decreased the EGFR level and produced differentiation effects via ERalpha, but induced both EGFR expression and proliferation effects via ERbeta. In conclusion, we show that also in U2OS cells, E(2) and SERMs are able to modulate the expression of the EGFR gene and can affect events strictly controlled by its signaling pathway, such as the maturation of osteoblasts.

Effects of a diet rich in sesame ( Sesamum indicum) pericarp on the expression of oestrogen receptor alpha and oestrogen receptor beta in rat prostate and uterus.

The expression of oestrogen receptors (ERalpha and ERbeta) in the prostate and uterus tissues of Wistar rats supplied for 8 weeks with a diet rich in sesame (Sesamum indicum) pericarp (30 %) was monitored. Eight male rats, aged 6 weeks, were divided into a control group fed on a normal diet, and an experimental one, provided with the normal diet enriched with 30 % sesame pericarp. A similar experiment was performed with female rats. At the end of the experiment, the prostate and uterus tissues were surgically removed and kept at - 80 degrees C for up to 2 months. Western blotting and quantitative real-time PCR (qRT-PCR) methods were used in order to investigate the levels of receptor proteins and mRNA. Significant increase in the expression of ERbeta in prostate and uterus was evident in both methods, while the magnitude of the observed alteration depended on the applied method. No statistically significant change was observed in the expression of ERalpha in uterus. In prostate, although the increase was more evident when investigated by means of qRT-PCR, the difference in expression of ERalpha was not statistically significant. In both tissues, a shift of the ratio of ERalpha:ERbeta in favour of ERbeta was evident, indicating, according to existing literature, a beneficial effect of the diet provided upon the health status of the organisms. It is suggested that this effect is attributed to the lignans present in the pericarp which exert phyto-oestrogenic activity.

Improved synthesis of unique estradiol-linked platinum(II) complexes showing potent cytocidal activity and affinity for the estrogen receptor alpha and beta.

We have recently reported the synthesis of a platinum(II) complex, made of estradiol, the female sex hormone, and a cisplatin analog, an anticancer drug, linked together by an eleven carbon atoms chain. The novel estradiol-Pt(II) hybrid molecule was synthesized in nine chemical steps with 10% overall yield. This new compound has been tested in vitro on estrogen-dependent (MCF-7) and -independent (MDA-MD-231) (ER(+) and ER(-)) cell lines. Interestingly, the biological activity was quite significant, more potent than that of cisplatin, the compound currently used in chemotherapy. The estrogen receptor binding affinity (ERBA) of this compound was very similar to that of 17beta-estradiol (E(2)) on both estrogen receptors (ERs), alpha and beta. In order to further study this type of molecule, we have decided to synthesize several analogs with the same estrogenic scaffold but with various chain lengths separating the estradiol from the toxic part of the molecule. This was planned in order to study the effect of the length of the linking chain on the biological activity of the hybrids. Four E(2)-Pt(II) hybrid molecules having 6-14 carbon atoms linking chain have been synthesized using a new synthetic methodology. They are synthesized in only eight chemical steps with 21% overall yield. The 17beta-estradiol-linked platinum(II) complexes have been tested for their receptor binding affinity as well as for their cytocidal activity on several breast cancer cell lines. The synthesis and biological results are reported herein.

Detection of estrogenically active substances in diets for sows by an in vitro bioassay supported by HPLC analysis.

There is considerable evidence that exogenous estrogenic compounds can have adverse effects on fertility. The main reason cited in literature for hyperestrogenism in pigs is contamination of feedstuffs by the mycotoxin zearalenone (Boehm, 2000), but further estrogenically active substances might also be involved in cases of impaired fertility with symptoms like enlarged, red-coloured vulvae in piglets, irregular estrus cycles and anestrus of sows (Bennetts et al., 1946; Drane et al., 1981). It is well known that soy used in diets for pigs as a main protein source contains phytoestrogens. Amongst them, isoflavones like genistein and daidzein are of particular interest. Aim of this study was to optimize and use an established bioassay (Kluczka, 2003) to determine estrogenic activity in feedstuffs for pigs related to isoflavones and further substances with estrogenic potential. This bioassay is a reporter gene assay based on stably transfected human embryonal kidney cells (HEK 293) that contains either alpha or beta estrogen receptor (alpha- or beta-HEK). The estrogenic activity measured in the luciferase assay was expressed in estradiol-equivalents (EEQ) and the results were compared with the isoflavone content (genistein, daidzein) obtained by chemical analysis using high performance liquid chromatography-Ultraviolet (HPLC-UV). Mean estrogenic activity in diets fed to sows in herds with altered fertility was 275.8 microg EEQ/kg feed in alpha-HEK cells and 295.0 microg EEQ/kg feed in beta-HEK cells. Feedstuffs from herds without any altered fertility showed an average estrogenic activity of 204.9 microg EEQ/kg feed in alpha-HEK and 213.3 microg EEQ/kg feed in beta-HEK. The estrogenic activity was strongly related to the concentration of the isoflavones (alpha-HEK, r(2)=0.9488; beta-HEK, r(2)=0.9427). Clinically relevant zearalenone concentrations (>50-150 microg/kg feed) displayed estrogenic effects in the bioassay that did not differ significantly from those caused by high isoflavone concentration because of the use of soy as protein source.

Modulation of the genomic estrogen receptor pathway by water extracts of Cirsium japonicum.

We investigated the estrogenic activity of Cirsium japonicum water extracts, which have long been used to treat vascular-related diseases. The activity of the extracts was characterized in a transient transfection system, using estrogen receptor isoforms and estrogen-responsive luciferase plasmids in HEK 293 cells. The extract activated both and estrogen receptors. Activation was inhibited by the estrogen receptor antagonist ICI 182,780, indicating that the effects were mediated through the estrogen receptor isoforms. Treatment with the extracts increased expression of the progesterone receptor and pS2 genes and expression of estrogen receptor was decreased in MCF-7 cells. These results suggested that the Cirsium japonicum water extracts showed estrogenic effects and may be a potential clinical application for treatment of estrogen related vascular diseases.

Acute dilatation to phytoestrogens and estrogen receptor subtypes expression in small arteries from women with coronary heart disease.

We tested if endothelial function and estrogen receptor (ER) expression differs between resistance arteries in subcutaneous circulation from postmenopausal women with coronary heart disease (CHD, congruent with 1 year after myocardial infarction, n=12) and aged matched controls (n=14); and if acute effects of phytoestrogens (genistein, resveratrol) could be of relevance for vascular protection. We utilized ex vivo small artery ( congruent with 350 microm) bioassays and found no difference in bradykinin (BK)-mediated dilatation between the groups. One-hour incubation with phytoestrogens (natural ER beta agonists), propyl-pyrazole-triol-trisphenol (PPT-selective ER alpha agonist) and 17beta-estradiol (17beta-E(2)-ER alpha/beta agonist) at 0.01 microM/L had no effect on BK-induced responses. Concentration-response curves (0.01-30 microM/L) to investigated compounds were also obtained and compared in separate arteries. We found that dilatation to phytoestrogens was enhanced in CHD if compared to controls (p<0.05), while responses to 17beta-E(2) remained similar. The dilatation to phytoestrogens was also higher if compared to 17beta-E(2) (p<0.05) in CHD. In controls, only responses to PPT, but not to phytoestrogens, were enhanced in comparison to 17beta-E(2) (p<0.05). Inhibition of NO synthase had no effect on dilatation induced by increasing concentrations of investigated compounds. ER beta expression was enhanced in the vascular wall from CHD women, while ER alpha predominated in the controls (p<0.05). We suggest that diet supplementation by phytoestrogens may provide cardiovascular benefit for postmenopausal women with CHD. The selective targeting of one of the ER subtype may have implications for women's cardiovascular health.