coumestrol and Breast-Neoplasms

The growth of neoplasms originating from steroid hormone target tissues may be hormone-dependent. It has been clearly demonstrated that the oestrogen and/or progestagen receptor status predicts the response to endocrine treatment and the prognosis of breast cancer. However steroid receptor determination in tumour cytosols has several limitations, which can partly be resolved by (immuno) histochemical detection of steroid hormone-responsive tumour cells. A variety of histochemical techniques including autoradiography of tritiated steroids, immunohistochemistry with antibodies to steroid hormones, histochemistry with fluorescent ligands of low molecular weight and fluorochrome- or enzyme-labelled macromolecular probes are discussed. It is concluded that there is as yet no substantial evidence that these methods visualize steroid receptors or detect steroid hormone-responsiveness of tumour cells. On the other hand, immunohistochemical demonstration of oestrogen receptors with monospecific antibodies seems validated and indications have been obtained that this information is clinically relevant. Recent developments suggest that immunocytochemistry of receptors and specific hormone-induced proteins, flow cytometric analysis and probably hybridocytochemistry in the future will help to improve management of steroid hormone-dependent tumours.

Topics: Animals; Antibodies; Binding Sites; Breast Neoplasms; Coumestrol; Cytosol; Female; Fluorescent Dyes; Humans; Immunoenzyme Techniques; Ligands; Molecular Weight; Neoplasms; Receptors, Estrogen; Receptors, Steroid; Steroids

The propensity of breast cancer to preferentially metastasize to the skeleton is well known. Once established in bone metastatic breast cancers have a poor prognosis due to their ability to promote extensive bone loss which augments tumor burden. Unfortunately, current anti-resorptive therapies for skeletal metastasis are typically prescribed after secondary tumors have formed and are palliative in nature. One group of compounds with the potential to reduce both tumor burden and osteolysis are phytoestrogens (PE), but the mechanisms mediating a beneficial effect are unclear. Therefore, the current study examined the effect of genistein and coumestrol alone or in combination on breast cancer cell number, expression of mediators of preferential skeletal metastasis, bone matrix attachment and tumor-induced osteoclast formation. Results showed that genistein and coumestrol significantly reduced viable cell number in an estrogen receptor dependent manner (p < 0.05), whereas combinations of PE had no effect. In addition, genistein and coumestrol significantly reduced expression of genes driving epithelial to mesenchymal transition (snail), bone attachment (CXCR4 and integrin αV) and osteolysis (PTHrP and TNF-α). In keeping with this genistein and coumestrol significantly suppressed attachment of breast cancer cells to bone matrix and inhibited tumor and RANKL-induced osteoclast formation. Our data suggests that phytoestrogens not only decrease breast cancer cell viability but also antagonize essential tumor bone interactions that establish and drive the progression of skeletal metastasis.

Topics: Bone Matrix; Bone Neoplasms; Breast Neoplasms; Cell Survival; Coumestrol; Epithelial-Mesenchymal Transition; Female; Genistein; Humans; MCF-7 Cells; Osteogenesis; Osteolysis; Phytoestrogens

Tamoxifen users sometimes seek complementary and alternative medicine advice for treatment of a variety of illness and co-administer with phytoestrogen-containing herbs, resulting in an increasing concern of its influence in subsequent endometrial cancer risk. Our study aims to determine the prevalence of Chinese herbal products containing coumestrol, genistein, or daidzein and their association with subsequent endometrial cancer risk among tamoxifen-treated breast cancer survivors in Taiwan.. We selected all patients who were newly diagnosed with invasive breast cancer and received tamoxifen treatment between January 1, 1998, and December 31, 2008, from the National Health Insurance Research Database. Among the 26,656 tamoxifen-treated breast cancer survivors, we evaluated the usage, frequency of service, and prescription of Chinese herbal products containing coumestrol, genistein, or daidzein. The logistic regression method was employed to calculate the odds ratios for utilization of those herbal products. Cox proportional hazard regression was set to calculate the hazard ratios of endometrial cancer associated with such usage.. Of the patients surveyed, 36.2% (n=9652) of the tamoxifen-treated breast cancer survivors examined in the study had consumed Chinese herbal products containing coumestrol, genistein, or daidzein during the study period. Exposure to Ge Gen(Puerariae Radix) specifically was the most extensive. For it, the population consumed an average cumulative dose of above 180g. Compared to those who had never used Chinese herbal products, breast cancer survivors who had taken Chinese herbal products containing coumestrol, genistein, or daidzein concurrently with tamoxifen treatment did not have a higher hazard ratio for subsequent development of endometrial cancer.. Among those tamoxifen-treated female breast cancer survivors in Taiwan, consumption of Chinese herbal products containing coumestrol, genistein, or daidzein is negatively correlated with subsequent endometrial cancer risk.

Topics: Adult; Aged; Breast Neoplasms; Case-Control Studies; Coumestrol; Drugs, Chinese Herbal; Endometrial Neoplasms; Female; Genistein; Humans; Isoflavones; Middle Aged; Survivors; Taiwan; Tamoxifen; Young Adult

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC50 of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent Ki value of 7.67 μM. Coumestrol at 50μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21(Cip1/WAF1) pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22(phox) siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.

Topics: Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Colonic Neoplasms; Coumestrol; Down-Regulation; Female; Glycine max; HCT116 Cells; Humans; Kinetics; Male; Plant Extracts; Reactive Oxygen Species

An iron-based cross-dehydrogenative coupling (CDC) approach was applied for the diversity-oriented synthesis of coumestrol-based selective estrogen receptor modulators (SERMs), representing the first application of CDC chemistry in natural product synthesis. The first stage of the two-step synthesis of coumestrol involved a modified aerobic oxidative cross-coupling between ethyl 2-(2,4-dimethoxybenzoyl)acetate and 3-methoxyphenol, with FeCl3 (10 mol%) as the catalyst. The benzofuran coupling product was then subjected to sequential deprotection and lactonization steps, affording the natural product in 59% overall yield. Based on this new methodology other coumestrol analogues were prepared, and their effects on the proliferation of the estrogen receptor (ER)-dependent MCF-7 and of the ER-independent MDA-MB-231 breast cancer cells were tested. As a result, new types of estrogen receptor ligands having an acetamide group instead of the 9-hydroxyl group of coumestrol were discovered. Both 9-acetamido-coumestrol and 8-acetamidocoumestrol were found more active than the natural product against estrogen-dependent MCF-7 breast cancer cells, with IC50 values of 30 and 9 nM, respectively.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Coumestrol; Estrogen Receptor alpha; Estrogen Receptor Modulators; Female; Humans; Inhibitory Concentration 50; Iron; Molecular Structure; Receptors, Estrogen; Selective Estrogen Receptor Modulators

Some forms of estrogen are associated with breast cancer risk as well as with mammographic density (MD), a strong marker of breast cancer risk. Whether phytoestrogen intake affects breast density, however, remains unclear. We evaluated the association between serum levels of phytoestrogens and MD in postmenopausal women. We enrolled 269 women, ages 55-70 yr, who received a screening mammogram and had no history of postmenopausal hormone use. Subjects completed a survey on diet and factors related to MD and provided a blood sample for analysis of 3 phytoestrogens: genistein, daidzein, and coumestrol. We examined whether mean percent MD was related to serum level of phytoestrogens, adjusting for age and body mass index. Genistein and daidzein levels correlated with self-reported soy consumption. Mean percent MD did not differ across women with different phytoestrogen levels. For example, women with nondetectable genistein levels had mean density of 11.0% [95% confidence intervals (CI) = 9.9-12.4], compared to 10.5% (95% CI = 8.0-13.7) and 11.2% (95% CI = 8.7-14.6) for < and ≥ median detectable levels, respectively. In a population with relatively low soy intake, serum phytoestrogens were not associated with mammographic density. Additional studies are needed to determine effects of higher levels, particularly given patterns of increasing phytoestrogen intake.

Topics: Aged; Body Mass Index; Breast Neoplasms; Coumestrol; Diet; Feeding Behavior; Female; Genistein; Glycine max; Humans; Isoflavones; Mammography; Middle Aged; Postmenopause

Phytoestrogens have attracted attention as being safer alternatives to hormone replacement therapy (HRT) and as chemopreventive reagents for breast cancer because dietary soy isoflavone intake has been correlated with reduction in risk. To identify safe and effective phytoestrogen candidates for HRT and breast cancer prevention, we investigated the effects of daidzein, genistein, coumestrol, resveratrol and glycitein on cell growth, cell cycle, cyclin D1 expression, apoptosis, Bcl-2/Bax expression ratio and p53-dependent or NF-kappaB-dependent transcriptional activity in MCF-7 breast cancer cells. Phytoestrogens, except for glycitein, significantly enhanced estrogen-response-element-dependent transcriptional activity up to a level similar to that of 17beta-estradiol (E(2)). E(2) increased cell growth significantly, coumestrol increased cell growth moderately, and resveratrol and glycitein reduced cell growth. Phytoestrogens, except for glycitein, stimulated the promotion of cells to G(1)/S transition in cell cycle analysis, similar to E(2). This stimulation was accompanied by transient up-regulation of cyclin D1. While genistein, resveratrol and glycitein all increased apoptosis and reduced the Bcl-2/Bax ratio, resveratrol reduced this ratio more than either genistein or glycitein. Moreover, resveratrol significantly enhanced p53-dependent transcriptional activity, but slightly reduced NF-kappaB-dependent transcriptional activity. On knockdown analysis, genistein, resveratrol and glycitein all reduced the Bcl-2/Bax ratio in the presence of apoptosis-inducing stimuli, and estrogen receptor (ER) alpha silencing had no effect on these reductions. In contrast, in the absence of apoptosis-inducing stimuli, only resveratrol reduced the ratio, and ERalpha silencing abolished this reduction. Thus, resveratrol might be the most promising candidate for HRT and chemoprevention of breast cancer due to its estrogenic activity and high antitumor activity.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Coumestrol; Estradiol; Estrogen Receptor alpha; Female; Genistein; Humans; Isoflavones; Phytoestrogens; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Stilbenes

Results from epidemiological and experimental studies indicate that phytoestrogens may protect against breast cancer. Because one of the biological effects of phytoestrogens is probably estrogenic, it's possible that the preventive effect on breast cancer differs by estrogen receptor (ER) or progesterone receptor (PR) status of the tumor. We evaluated the associations between dietary phytoestrogen (isoflavonoids, lignans, and coumestrol) intake and risk of breast cancer and whether the ER/PR statuses of the tumor influence this relationship. In 1991-2 a prospective population-based cohort study among Swedish pre- and postmenopausal women was performed, making questionnaire data available for 45,448 women. A total of 1014 invasive breast cancers were diagnosed until December 2004. Cox proportional hazards models were performed to estimate multivariate risk ratios, 95% CI for associations with risk of breast cancer. Intakes of lignan, isoflavonoid, or coumestrol were not associated with breast cancer risk overall or before or after 50 y of age. The effects of lignans or isoflavonoids were independent of receptor status. However, intake of coumestrol was associated with decreased risk of receptor negative tumors (ER-PR-) but not positive tumors. The risk of ER-PR- tumors was significantly lower (50%) in women with intermediate coumestrol intake compared with those who did not consume any. In conclusion, we found no association between intake of isoflavonoids or lignans and breast cancer risk. Our results of a decreased risk of ER-PR- tumors in women with intermediate intake of coumestrol could be due to chance because of the low intake. The results should be confirmed in other studies.

Topics: Adult; Breast Neoplasms; Cohort Studies; Coumestrol; Diet; Dietary Fiber; Female; Flavonoids; Humans; Lignans; Middle Aged; Phytoestrogens; Prospective Studies; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; Surveys and Questionnaires; Sweden

Several estrogen mimics (xenoestrogens) inappropriately activate the estrogen receptor (ER) in the absence of endogenous ligand. Given the importance of the ER in breast cancer growth and regulation, delineating the impact of these agents under conditions related to tumor treatment is of significant importance. We examined the effect of two prevalent xenoestrogens (bisphenol A and coumestrol) on ER activation and ER-dependent mitogenesis in breast cancer cells. We show that the ability of these agents to induce mitogenesis was restricted to conditions of estrogen depletion, and that these agents failed to cooperate with estradiol to induce MCF-7 breast cancer cell growth. These observations are consistent with the impact of each agent specifically on exogenous ER activation as monitored in HeLa cells, wherein the xenoestrogens activated the receptor in the absence of estradiol but failed to cooperate with estrogen. Tamoxifen blocked bisphenol A and coumestrol-mediated ER activation, indicating that exposure to these agents is unlikely to disrupt such therapeutic intervention. The response of tumor-derived ER alleles to these xenoestrogens was also examined. Although the xenoestrogens failed to alter ER-Y537S function, the ER-D351Y mutant demonstrated an enhanced response to bisphenol A. Moreover, tamoxifen enhanced the agonistic effects of xenoestrogens on ER-D351Y. Lastly, we examined the impact of ER co-activator overexpression on xenoestrogen response. Bisphenol A and coumestrol exhibited differential responses to co-activators with regard to ER activation. However, when using mitogenesis as an endpoint, these co-activators were insufficient to provide a significant growth advantage. Combined, these data demonstrate that bisphenol A and coumestrol can impact ER activity and ER-dependent proliferation in breast cancer cells, but the influence of these agents is restricted to conditions of estrogen depletion, selective mutation of the ER, and expression of specific co-activators.

Topics: Benzhydryl Compounds; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Coumestrol; Estradiol; Female; Humans; Phenols; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Tamoxifen; Transcription, Genetic

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

Phytoestrogens have been described to be weak estrogens, SERMs or exhibit antiestrogenic properties. However, information about their activity in presence of estrogens is limited. Therefore, we have analysed the dose dependent combinatory activity of the phytoestrogens genistein (Gen), daidzein (Dai) and coumestrol (Cou), and 17beta-estradiol (E2) on cell proliferation and apoptosis induction in human MCF-7 breast cancer cells. Neither additive nor antagonistic effects on proliferation could be observed, but in contrast all phytoestrogens possessed the ability to inhibit apoptosis in the presence of 17beta-estradiol. In summary, our in vitro results demonstrate that Gen does not exhibit any antiestrogenic properties. The additive growth stimulatory effects of Gen, Dai and Cou in the presence of E2 are not the result of a stimulation of proliferation; these phytoestrogens, at least in MCF-7 cells, could be characterised as inhibitors of apoptosis.

Topics: Apoptosis; Breast Neoplasms; Cell Proliferation; Coumestrol; Drug Interactions; Estradiol; Estrogen Antagonists; Female; Genistein; Humans; Isoflavones; Phytoestrogens

In the presented study, we have analysed effects of the environmental estrogens bisphenol A (BPA), p-tert-octylphenol (OCT), o,p'-DDT (DDT) and coumestrol (COU) on cell proliferation, apoptosis induction, progesterone receptor (PR) and androgen receptor (AR) mRNA expression and ER alpha protein expression in comparison to estradiol (E2) and the selective ER modulator (SERM) raloxifene (RAL) and the pure antiestrogen faslodex (ICI 182780) in the human breast cancer cell line MCF-7. A dose dependent analysis of the cell cycle distribution of MCF-7 cells after administration of OCT, DDT and COU revealed a significant induction of cell proliferation and reduced rate of apoptosis. Maximum induction of cell proliferation and the lowest rate of apoptosis could be observed at a dose of 10(-6)M. Interestingly, administration of BPA reduces the rate of apoptosis, but does not enhance proliferation at any dose analysed. PR mRNA expression in MCF-7 cells was up regulated after administration of COU and DDT, whereas treatment with BPA and OCT did not effect PR mRNA expression. AR mRNA expression was down regulated by COU, but not effected by BPA, DDT and OCT. The expression of ER alpha protein in the breast cancer cells was slightly down regulated by COU and DDT, but unaffected by BPA and OCT. In summary and in comparison to the effects observed after administration of E2, RAL and ICI our data indicate that none of the analysed compounds exhibit properties comparable to RAL and ICI. COU and DDT exhibit properties which are very similar to E2. Administration of BPA and OCT did not effect any of the estrogen sensitive molecular parameters analysed. Nevertheless OCT is a very potent stimulator of cell proliferation in MCF-7 cells. Surprisingly, BPA is not able to induce the proliferation of MCF-7 breast cancer cells, but turns out to be a very potent inhibitor of apoptosis. For this reason and in agreement to the effects of BPA on the molecular parameters analysed, we conclude that BPA does not act in a classical estrogen like manner in MCF-7 breast cancer cells.

Topics: Apoptosis; Benzhydryl Compounds; Breast Neoplasms; Cell Division; Coumestrol; DDT; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens, Non-Steroidal; Female; Fulvestrant; Humans; Molecular Structure; Phenols; Raloxifene Hydrochloride; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Selective Estrogen Receptor Modulators; Surface-Active Agents; Tumor Cells, Cultured

A series of eight halogenated 2,4-diaryl-4H,5H-pyrano[3,2-c]benzopyran-5-ones have been synthesized, characterized and their stereochemistry determined. In a second stage of our work, the reported molecules were tested for their antiproliferative activity on MCF-7 breast carcinoma cells. Pharmacological results were compared with those of diethylstilbestrol (DES), an estrogen, as well as ICI 182,780, a pure antiestrogen. Then, these derivatives were evaluated for their capacity to activate the transcription of a reporter gene and for their affinity for human recombinant estrogen receptors alpha (hER alpha). These results were compared with those of coumestrol, a phytoestrogen structurally close to 2,4-diaryl-4H,5H-pyrano[3,2-c]benzopyran-5-ones, and with RU 58668, a pure antiestrogen. Although these derivatives exhibit a significant antiproliferative activity higher than that of ICI 182,780, neither of them displayed a significant estrogenicity or an affinity for hER alpha. Such results may suggest that their antiproliferative activity is not dependent of an antiestrogenic response.

Topics: Antineoplastic Agents, Phytogenic; Benzopyrans; Breast Neoplasms; Cell Division; Cell Survival; Chemical Phenomena; Chemistry, Physical; Chromatography, Thin Layer; Coumestrol; Drug Screening Assays, Antitumor; Estradiol Congeners; Estrogen Receptor alpha; Female; Humans; Magnetic Resonance Spectroscopy; Receptors, Estrogen; Spectrophotometry, Infrared; Tumor Cells, Cultured

The 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD 5) is involved in estrogen and androgen metabolism. In our study we tested the influence of environmental hormones, such as phytoestrogens (flavonoids, coumarins, coumestans), on reductive and oxidative 17beta-HSD activity of the human 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD 5). These dietary substances were shown to be potent inhibitors of aromatase, different 17beta-HSDs and seem to play an important role in delay of development of hormone dependent cancers. Our studies show that reductive and oxidative activity of the enzyme are inhibited by many dietary compounds, especially zearalenone, coumestrol, quercetin and biochanin A. Among the group of flavones inhibitor potency is growing with increasing number of hydroxylations. We suggest that these substances are bound to the hydrophilic cofactor-binding pocket of the enzyme. An interesting inhibition pattern is observed for 18beta-glycyrrhetinic acid, which has no influence on the oxidative but only on the reductive reaction. This indicates that this substrate binds to pH- and cofactor-depending sites at the active center of the enzyme.

Topics: 17-Hydroxysteroid Dehydrogenases; Aromatase Inhibitors; Binding Sites; Breast Neoplasms; Coumestrol; Diet; Enzyme Inhibitors; Estrogens; Estrogens, Non-Steroidal; Female; Gene Expression; Genistein; Glycine max; Glycyrrhetinic Acid; Humans; Hydrogen-Ion Concentration; Hydroxylation; Isoenzymes; Isoflavones; Male; Models, Molecular; Oxidation-Reduction; Phytoestrogens; Plant Preparations; Prostatic Neoplasms; Quercetin; Recombinant Proteins; Testosterone; Zearalenone

Phytoestrogen effects on estrogen action and tyrosine kinase activity have been proposed to contribute to cancer prevention. To study these mechanisms, a number of phytoestrogens and related compounds were evaluated for their effects on DNA synthesis (estimated by thymidine incorporation analysis) in estrogen-dependent MCF-7 cells in the presence of estradiol (E2), tamoxifen, insulin, or epidermal growth factor. We observed that 1) at 0.01-10 microM, genistein and coumestrol enhanced E2-induced DNA synthesis, as did 10 microM enterolactone. Chrysin at 1.0-10 microM and 10 microM luteolin or apigenin inhibited E2-induced DNA synthesis, as did all compounds at > 10 microM, 2) tamoxifen enhanced genistein-induced DNA synthesis but inhibited DNA synthesis induced by all other compounds, and 3) genistein enhanced insulin- and epidermal growth factor-induced DNA synthesis at 0.1-1.0 and 0.1-10 microM, respectively. At higher concentrations, inhibition was observed. Similar effects were seen with coumestrol. In conclusion, the effects of phytoestrogens in the presence of E2 or growth factors are concentration dependent and variable. At low concentrations, genistein and coumestrol significantly enhanced E2-induced and tyrosine kinase-mediated DNA synthesis; at high concentrations, inhibition was observed. Differing effects were observed with the other compounds. The variable effects of phytoestrogens on DNA synthesis must be considered when their roles in cancer prevention or treatment are evaluated.

Topics: 4-Butyrolactone; Anticarcinogenic Agents; Antineoplastic Agents, Hormonal; Breast Neoplasms; Chamomile; Coumestrol; DNA, Neoplasm; Epidermal Growth Factor; Estradiol; Estrogens, Non-Steroidal; Female; Flavonoids; Genistein; Humans; Insulin; Isoflavones; Lignans; Luteolin; Oils, Volatile; Phytoestrogens; Plant Preparations; Plants, Medicinal; Tamoxifen; Tumor Cells, Cultured

Thirteen isoflavonoids, flavonoids, and lignans, including some known phytoestrogens, were evaluated for their effects on DNA synthesis in estrogen-dependent (MCF-7) and -independent (MDA-MB-231) human breast cancer cells. Treatment for 24 hours with most of the compounds at 20-80 microM sharply inhibited DNA synthesis in MDA-MB-231 cells. In MCF-7 cells, on the other hand, biphasic effects were seen. At 0.1-10 microM, coumestrol, genistein, biochanin A, apigenin, luteolin, kaempferol, and enterolactone induced DNA synthesis 150-235% and, at 20-90 microM, inhibited DNA synthesis by 50%. Treatment of MCF-7 cells for 10 days with genistein or coumestrol showed continuous stimulation of DNA synthesis at low concentrations. Time-course experiments with genistein in MCF-7 cells showed effects to be reversed by 48-hour withdrawal of genistein at most concentrations. Induction of DNA synthesis in MCF-7 cells, but not in MDA-MB-231 cells, is consistent with an estrogenic effect of these compounds. Inhibition of estrogen-dependent and -independent breast cancer cells at high concentrations suggests additional mechanisms independent of the estrogen receptor. The current focus on the role of phytoestrogens in cancer prevention must take into account the biphasic effects observed in this study, showing inhibition of DNA synthesis at high concentrations but induction at concentrations close to probable levels in humans.

Topics: Anticarcinogenic Agents; Breast Neoplasms; Coumestrol; DNA; Estradiol; Estrogens, Non-Steroidal; Flavonoids; Genistein; Humans; Isoflavones; Kinetics; Lignans; Phytoestrogens; Plant Preparations; Tumor Cells, Cultured

Representative non-steroidal estrogens, from common environmental sources such as plants, pesticides, surfactants, plastics, and animal health products, demonstrated an ability to lower serum cholesterol and prevent bone loss. Specifically, select environmental estrogens (coumestrol, genistein, methoxychlor, bisphenol A, and zeranol) effectively lowered total serum cholesterol in an estrogen-dependent animal model, the ovariectomized rat. Of these entities, coumestrol, methoxychlor, and zeranol prevented ovariectomy-induced bone loss. In an in vitro environment, these compounds competed with 17beta-estradiol for estrogen receptor binding and stimulated cell proliferation in a human breast cancer cell line (MCF-7). In addition to their well-documented effects on reproductive tissue, various environmental estrogens can dramatically affect non-reproductive parameters such as cholesterol lowering and bone metabolism.

Topics: Adenocarcinoma; Animals; Anticholesteremic Agents; Benzhydryl Compounds; Binding, Competitive; Bone Density; Breast Neoplasms; Cell Line; Cholesterol; Coumestrol; Environmental Pollutants; Estradiol; Estrogens, Non-Steroidal; Female; Genistein; Humans; Isoflavones; Kinetics; Methoxychlor; Ovariectomy; Phenols; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Uterus; Zeranol

Estrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence-E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension. Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein-labeled estrogen probes. Base hydrolysis of the ester linkage in fluorescein-E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 X 10(8) M-1 for the ER and is a single component as determined by HPLC. Specific fluorescent uptake of coumestrol was performed on ER+ and ER- viable cell suspensions. When these coumestrol-cell suspensions were excited at 350-360 nm and the blue emission was measured using flow cytometry, the result was a fluorescence uptake that was not highly displaceable by excess nonfluorescence E2 probes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Breast Neoplasms; Cell Line; Coumestrol; Estradiol; Estrogens; Female; Flow Cytometry; Fluorescence; Fluorescent Dyes; Humans; Rats; Receptors, Estrogen; Structure-Activity Relationship; Uterus

Histochemical analyses estrogen (ER) and progesterone (PgR) receptors in breast cancer were statistically correlated with results of dextran-coated charcoal (DDC) and sucrose gradient assays. Correlated for ER was 91% of 363 cases, and for PgR 88% of 255 specimens. Breast cancer ER/PgR positivity by histochemistry correlated with a favorable clinical response to endocrine therapies in 72% of 25 cases, while ER/PgR negativity correlated with a lack of response in 96% of 22 cases with Stage IV disease. Nuclear ER/PgR correlated with a poor response to therapy in 8 of 12 patients. An in vitro technique to detect nuclear translocation of ER revealed two groups of ER positive cases, with 11 of 17 exhibiting translocation and 6 not displaying translocation. In prostatic carcinoma, 72% of 65 men were positive for ER and/or androgen receptor. Comparison of specimens obtained without and with electrocautery revealed a preponderance of nuclear binding in the latter, suggesting heat-induced nuclear translocation of receptor. coumestrol, a naturally fluorescent, entirely unaltered estrogen was also used for histochemical detection of ER. Results correlated with ER by DCC in 87% of 61 breast cancers. Coumestrol was additionally used to visually observe receptor and nuclear translocation of ER in intact whole cells in culture.

Topics: Breast Neoplasms; Carcinoma; Coumestrol; Estrogens; Female; Histocytochemistry; Humans; Male; Progesterone; Prostatic Neoplasms; Receptors, Androgen; Receptors, Estrogen; Receptors, Steroid

The interactions of phytoestrogens with estrogen receptors were studied in the human breast cancer cell line, MCF-7. The compounds tested were coumestrol, genistein, and formononetin and the mycotoxins, zearalenone and its reduced derivative, zearalenol. All but formononetin compete for binding of [3H]-estradiol to unfilled cytoplasmic estrogen receptor or unfilled nuclear estrogen receptor sites. Relative binding affinities are zearalenol HMP (high melting point isomer) greater than zearalenol LMP (low melting point isomer) greater than zearalenone = coumestrol greater than genistein greater than formononetin. Dissociation constants estimated from competition curves show that binding affinities are high. In contrast to estradiol, phytoestrogens bind only weakly to sex steroid-binding globulin; they also do not bind to corticosteroid-binding globulin. These compounds translocate the cytoplasmic estrogen receptor and bind to unfilled nuclear estrogen receptors in whole cells. Bound nuclear receptors are then processed in a manner similar to estradiol in a step which rapidly decreases total cellular estrogen receptors. The phytoestrogens are also biologically active; they can markedly enhance tumor cell proliferation. In sum, phytoestrogens interact with the estrogen receptors of human breast cancer cells in culture and, therefore, may affect estrogen-mediated events in these cells.

Topics: Breast Neoplasms; Cell Line; Coumarins; Coumestrol; Flavonoids; Humans; Isoflavones; Mycotoxins; Receptors, Estrogen; Sex Hormone-Binding Globulin; Zearalenone; Zeranol