afimoxifene and daidzein

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.

Topics: Apoptosis; Biomarkers; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Shape; Down-Regulation; ErbB Receptors; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Genistein; Humans; Isoflavones; Osteoblasts; Osteosarcoma; Phytoestrogens; RNA, Messenger; Selective Estrogen Receptor Modulators; Tamoxifen; Time Factors

Women approaching menopause increasingly investigate alternatives to hormone replacement therapy. Plant phytoestrogens are being promoted as "natural" alternatives but there is a lack of substantive data to advocate their safe use in breast cancer patients receiving tamoxifen (TAM), or in those who have relapsed. The aim of our study was to investigate the proliferative effects and mode of action of the phytoestrogens genistein, daidzein and coumestrol on TAM-sensitive (-s) and resistant (-r) breast cancer cells under in vitro conditions designed to mimic the hormonal environment of the pre- and post-menopausal breast. At physiological concentrations (<10 microM) and under reduced estrogen (E2) conditions, genistein was mitogenic to TAM-s cells with TAM-r cells generally refractory. Daidzein and coumestrol were growth stimulatory irrespective of TAM sensitivity. Transcriptional activity was ERE-mediated. Combining phytoestrogens with E2 (simulating the pre-menopausal breast environment) had no effect on growth of TAM-s or TAM-r cells. Addition of 4-HT mimicked the hormonal environment in post-menopausal breast cancer patients receiving TAM. The growth inhibitory effects of 4-HT were abrogated in TAM-s cells when combined with genistein and coumestrol, and to a lesser extent, daidzein, where significant growth stimulatory effects were observed. In TAM-r cells, proliferation did not exceed control values. At phytoestrogen concentrations above 10 microM, growth inhibitory effects were seen, irrespective of estrogenic environment or cell sensitivity to TAM. Our in vitro data suggests that phytoestrogens could have potentially adverse mitogenic effects on tumour cells and should probably be avoided by patients who remain sensitive to TAM or in those with pre-existing and possibly undiagnosed breast tumours.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Coumestrol; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Female; Gene Expression; Genistein; Humans; Isoflavones; Luciferases; Phytoestrogens; Receptors, Progesterone; Response Elements; Tamoxifen; Transfection

The mechanism of phytoestrogen action in gonadal cells of ganders has not been elucidated. The aim of the study was to investigate in Biłgoraj ganders the possibility of phytoestrogen action via estrogen or androgen receptors or via protein tyrosine kinase pathways in Leydig cells. Genistein and daidzein (5 and 50 microM) as well as equol (50 microM) inhibited testosterone (T) secretion by incubated Leydig cells (1x10(5)/ml; 20 h; 37 degrees C). The effects of hydroxytamoxifen (estrogen receptor inhibitor) and cyproterone acetate (androgen receptor antagonist) on phytoestrogen inhibition of T release by Leydig cells were not observed. Lavendustin A (protein tyrosine kinases inhibitor) did not change T production. The influence of phytoestrogens seems not to be conducted via estrogen and androgen receptors or protein tyrosine kinases system in these cells, but further studies are required to completely examine the mechanism of phytoestrogens action in testes of ganders.

Topics: Animals; Equol; Geese; Genistein; Isoflavones; Leydig Cells; Male; Phenols; Phytoestrogens; Poland; Protein-Tyrosine Kinases; Radioimmunoassay; Tamoxifen; Testosterone

Through the analysis of the transient expression of the luciferase reporter gene in HeLa cells, an evaluation has been made of the transcriptional activity of oestrogens and of selective oestrogen receptor (ER) modulators (SERMs), mediated by the alpha and beta isoforms of the ER, on the epidermal growth factor receptor gene promoter. Oestrogen-activated ERbeta presents a lower transcriptional activity compared with ERalpha, probably due to structural differences in the AF-1 regions of the receptors. Also SERMs induce different responses depending on the receptor isoform bound. Indeed, the phyto-oestrogens, genistein and daidzein, act as weak agonists of the oestrogenic activity via ERalpha, but as full agonists when bound to ERbeta. The synthetic SERM 4OH-tamoxifen, on the other hand, displays an opposite behaviour since it exerts a full agonist action through ERalpha, but acts as a full antagonist via ERbeta. As we have previously shown for ERalpha, an ERbeta/Sp1 functional synergism has also been highlighted, by means of gel mobility shift assays. Moreover, our results show that the sensitivity of target tissues to oestrogens and SERMs can be affected by coexpression of ERs, depending on the formation of appropriate levels of homo- and heterodimers, thus providing a useful approach to predict the effects of hormonal treatment.

Topics: Binding Sites; Dimerization; Electrophoretic Mobility Shift Assay; ErbB Receptors; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Gene Expression Regulation; Genes, Reporter; Genistein; HeLa Cells; Humans; Isoflavones; Luciferases; Promoter Regions, Genetic; Protein Isoforms; Receptors, Estrogen; Recombinant Fusion Proteins; Selective Estrogen Receptor Modulators; Signal Transduction; Sp1 Transcription Factor; Tamoxifen; Transcription, Genetic; Transfection

Some of the isoflavonoids present in human diet as well as in urine are expected to exert biologic effects as they have been reported to bind to estrogen receptors and to be estrogenic in other species. This report describes the in vitro assessment of estrogenic effects of isoflavonoids using human endometrial cells and tissue. The relative estrogenic potencies (EC50 values) of estradiol, 3 dietary isoflavonoids (coumestrol, genistein and daidzein) and one of their metabolites (equol), were estimated by using a recently developed multiwell plate in vitro bioassay based on the estrogen-specific enhancement of alkaline phosphatase (AlkP) activity in human endometrial adenocarcinoma cells of the Ishikawa-Var I line. The maximal AlkP activity elicited by the isoflavonoids tested was as high as that achieved with estradiol and their effects were suppressed by the antiestrogens 4-hydroxytamoxifen and ICI 164,384. These results indicate that estradiol and the isoflavonoids exert their effects on AlkP by similar interactions with the estrogen receptor, with potencies depending on binding affinities. The estrogenic effect of equol was confirmed by another in vitro bioassay, based on the estrogen-stimulated enhancement of prostaglandin F2 alpha output by fragments of human secretory endometrium.

Topics: Adenocarcinoma; Alkaline Phosphatase; Biological Assay; Chromans; Coumestrol; Dinoprost; Endometrial Neoplasms; Endometrium; Equol; Estradiol; Female; Genistein; Humans; Isoflavones; Polyunsaturated Alkamides; Tamoxifen; Tumor Cells, Cultured