The stimulation of cell proliferation by quercetin is mediated by the estrogen receptor

Quercetin causes biphasic modulation of the proliferation of specific colon and mammary cancer cells. In this study, the possible involvement of the estrogen receptor (ER) in the stimulation of cell proliferation by quercetin was investigated. For this purpose, the effect of quercetin on cell proliferation was tested in ER-positive MCF-7 and T47D cells, and in ER-negative HCC-38 and MDA-MB231 cells. Quercetin stimulated proliferation of ER-positive cells only, suggesting this effect to be ER-dependent. In support of these results, quercetin induced ER-ERE-mediated gene expression in a reporter gene assay using U2-OS cells transfected with either ERalpha or ERbeta, with 10(5)-10(6) times lower affinity than 17beta-estradiol (E2) and 10(2)-10(3 )times lower affinity than genistein. Quercetin activated the ERbeta to a 4.5-fold higher level than E2, whereas the maximum induction level of ERalpha by quercetin was only 1.7 fold that of E2. These results point at the relatively high capacity of quercetin to stimulate supposed 'beneficial' ERbeta responses as compared to the stimulation of ERalpha, the receptor possibly involved in adverse cell proliferative effects. Altogether, the results of this study reveal that physiologically relevant concentrations of quercetin can exert phytoestrogen-like activity similar to that observed for the isoflavonoid genistein.     

Oxidation products of stigmasterol interfere with the action of the female sex hormone 17beta-estradiol in cultured human breast and endometrium cell lines

Phytosterols are constituents of plant membranes and are thus contained in low concentrations in vegetable products as well as at high concentrations in functional food designed to reduce serum cholesterol levels. Similar to ChOL, phytosterols are oxidized chemically in food and by biotransformation in vivo. Although oxyphytosterols have been detected in the serum of healthy human subjects, little is known of their biological activity. Therefore, the estrogenic and antiestrogenic activities of a mixture of six oxidation products of stigmasterol (oxy-StOL) were determined at the following endpoints: (i) the affinity to isolated human estrogen receptors (ER), (ii) the basal and 17beta-estradiol (E2)-induced expression of the alkaline phosphatase (AlP) in human endometrial adenocarcinoma (Ishikawa) cells, and (iii) the basal and E2-induced proliferation of human breast adenocarcinoma (MCF-7) cells. Oxy-StOL was able to replace E2 from human ERalpha and ERbeta and induced a weak estrogenic response in MCF-7 cells. Moreover, the E2-induced activity of the AlP in Ishikawa cells as well as the E2-induced proliferation of MCF-7 cells were decreased at noncytotoxic concentrations (up to 10 microM), indicating that at least one component of oxy-StOL represents an estrogen-active compound which might interfere with endogenous estrogens.     

Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity

Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.     

Functional associations between two estrogen receptors, environmental estrogens, and sexual disruption in the roach (Rutilus rutilus)

Wild male roach (Rutilus rutilus) living in U.K. rivers contaminated with estrogenic effluents from wastewater treatment works show feminized responses and have a reduced reproductive capability, but the chemical causation of sexual disruption in the roach has not been established. Feminized responses were induced in male roach exposed to environmentally relevant concentrations of the pharmaceutical estrogen 17alpha-ethinylestradiol, EE2 (up to 4 ng/ L), during early life (from fertilization to 84 days posthatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ERalpha. and ERbeta, in the brain and gonad/ liver. Transactivation assays were developed for both roach ER subtypes and the estrogenic potencies of steroidal estrogens differed markedly at the different ER subtypes. EE2 was by far the most potent chemical, and estrone (E1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ERbeta was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ERalpha. These findings add substantial support to the hypothesis that steroidal estrogens play a significant role in the induction of intersex in roach populations in U.K. rivers and that the molecular approach described could be usefully applied to understand interspecies sensitivity to xenoestrogens.     

Nutritional and health-related compounds in sprouts and seeds of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>), wheat (<Emphasis Type="Italic">Triticum aestivum.L</Emphasis>) and alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis>) treated by a new drying method

Nutritional and health-related compounds of alfalfa, wheat and soybean seeds dried by new drying process called the DIC process (controlled instantaneous pressure release) were evaluated, before and after sprouting. Vitamins (A, B₁, B₂, B₆, C and E), minerals (Fe, Mg, Ca, K, Mn, Na, Cu and Zn) and phytoestrogens (genistein and daidzein ) content were determined. Alfalfa, soybean and wheat seeds dried by DIC showed similar content of biological compounds to seeds dried by traditional processes. Sprouting DIC-seeds significantly increased the levels of vitamins, minerals and phytoestrogens, improving their nutrititional value and health quality compared to fresh products. The increase of 1250-fold and 10-fold of the initial vitamin A and vitamin C content, respectively, of alfalfa seeds due to sprouting is remarkable. Sprouts from DIC-seeds showed a significantly higher vitamin A content than sprouts obtained from seeds dried by other methods. Soybean sprouts obtained from DIC-seeds showed a significant increase in free phytoestrogens, quantified as genistein (70.34LJ.8 mg kg^-1 d.w.) and daidzein (109.17.3ᆥ.3 mg kg^-1 d.w.).     

Combination of low dose of genistein and daidzein has synergistic preventive effects on isogenic human prostate cancer cells when compared with individual soy isoflavone

The reduced incidence of prostate cancer (PCa) in Asia countries has been attributed to high soy diets, and major soy isoflavones, in particular daidzein and genistein, are thought to be the source of the beneficial and anti-cancer effects of soy foods. However, attention has been drawn to the safety of using high levels of soy isoflavones in humans, which is especially the concern for consumers taking regular soy isoflavone dietary supplements. The main objective of this study is thus to identify a soy isoflavone combination with lower levels of daidzein and genistein to be a more efficacious and safer chemo-preventive agent for PCa. The anticancer effects of daidzein and genistein, and their combinations on early-stage androgen-dependent PCa cells (LNCaP) and bone metastatic LNCaP-derivative PCa cells (C4-2B) were compared. Cells were treated with varying concentrations of daidzein, genistein (25–200 μM) or their combinations (25 or 50 μM) and cell proliferation, apoptosis, cell cycles and cellular uptakes of the isoflavones were measured after 48 h. Daidzein and genistein showed a synergistic effect on inhibiting cell proliferation and inducing apoptosis of both PCa cells. Twenty-five μM daidzein/50 μM genistein and 50 μM daidzein/50 μM genistein significantly increased the apoptotic effects on C4-2B cells although they did not show any effect when used individually. Except 50 μM daidzein/50 μM genistein, all other combinations had no impacts on cell cycles. For treatment with soy isoflavone combination, genistein was always better taken up than daidzein by both LNCaP and C4-2B cells.     

大豆异黄酮抑制大鼠乳腺癌细胞增殖的研究

应用MTT法、3H-TdR掺入法及流式细胞术,研究了两种主要的大豆异黄酮棗大豆黄酮和染料木素对乳腺癌细胞增殖的抑制效应,并探讨了其作用机理。结果表明：两种大豆异黄酮均能够抑制乳腺癌细胞的生长,并呈明显的剂量和时间依赖效应;降低3H-TdR掺入量,抑制癌细胞DNA的合成;增加Ｓ期细胞的比例,阻滞细胞的周期活动;但未引起明显的细胞凋亡。     

Estrogen-like activity of Adenophora triphylla var. japonica water extract in MCF-7 cells

This study investigated the estrogen-like activity of Adenophora triphylla var. japonica, which has been shown to have pharmacological activities. Water extracts from A. triphylla (ATWE) could bind to estrogen receptors and displaced binding of E2 to ERα and ERβ. ATWE stimulated the proliferation of estrogen receptor-positive MCF-7 cells in a dose dependent manner (p<0.05). ATWE induced proliferation was blocked by the addition of the estrogen antagonist ICI 182,780 (p<0.05). Moreover, ATWE treatment caused a significant increase in mRNA expression of estrogen-responsive genes (pS2, PR, and cathepsin D; p<0.05). These results indicate that A. triphylla has estrogen-like activity and could be used to improve estrogen deficiency-related menopausal symptoms or diseases in postmenopausal women.     

三羟异黄酮对人乳腺癌细胞株MCF-7增殖的影响机制

研究了三羟异黄酮(geinstein，Gen)对乳腺癌细胞株MCF-7增殖的影响机制。结果发现Gen对MCF-7细胞生长有显著抑制作用，使细胞生长阻滞于G2／M期，并使cyclin B蛋白表达增加，且呈剂量．效应关系；对cyclin E蛋白表达无影响。Gen降低了bcl-2蛋白的表达，促进了bax蛋白的表达并且降低了bcl-2／bax比值。     

Effect of diet on fecal and urinary estrogenic activity

The United States Environmental Protection Agency has identified estrogens from animal feeding operations as a major environmental concern, but few data are available to quantify the excretion of estrogenic compounds by dairy cattle. The objectives of this study were to quantify variation in estrogenic activity in feces and urine due to increased dietary inclusion of phytoestrogens. Ten Holstein heifers were assigned to 2 groups balanced for age and days pregnant; groups were randomly assigned to treatment sequence in a 2-period crossover design. Dietary treatments consisted of grass hay or red clover hay, and necessary supplements. Total collection allowed for sampling of feed refusals, feces, and urine during the last 4 d of each period. Feces and urine samples were pooled by heifer and period, and base extracts were analyzed for estrogenic activity (estrogen equivalents) using the yeast estrogen screen bioassay. Feces and urine samples collected from 5 heifers were extracted and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify excretion of 7 phytoestrogenic compounds. Excretion of 17-β estradiol equivalents in urine was higher and tended to be higher in feces for heifers fed red clover hay (84.4 and 120.2 mg/d for feces and urine, respectively) compared with those fed grass hay (57.4 and 35.6 mg/d). Analysis by LC-MS/MS indicated greater fecal excretion of equol, genistein, daidzein, coumestrol, and formononetin by heifers fed red clover hay (1634, 29.9, 96.3, 27.8, and 163 mg/d, respectively) than heifers fed grass hay (340, 3.0, 46.2, 8.8, and 18.3 mg/d, respectively). Diet had no effect on fecal biochanin A or 2-carbethoxy-5, 7-dihydroxy-4’-methoxyisoflavone. Four phytoestrogens were detected in urine (2-carbethoxy-5, 7-dihydroxy-4’-methoxyisoflavone, daidzein, equol, and formononetin) and their excretion was not affected by diet. Identifying sources of variation in estrogenic activity of manure will aid in the development of practices to reduce environmental estrogen accumulation.     

Determination of unusual soya and non-soya phytoestrogen sources in beer, fish products and other foods

Fish and fish products (14 samples), Indian foods and meals (10 samples), spices (30 samples) and beers (10 samples) were analysed for their phytoestrogen content, and a number of significant non-soya sources of dietary phytoestrogens were identified. No isoflavones were detected in unprocessed, farmed or ocean fish, but some samples of processed fish products contained soya isoflavones, which are assumed to come from coatings or protein addition. Additionally, some processed fish products contained, genistein glycocongugates not derived from soya. Genistein was detected in Indian meals such that, for example, a single portion of a vindalooo curry contained 11 mg genistein. The origin was most likely from the spices used, since the analysis of curry powders, chilli powder, crushed red chillies, garam masala and tandoori powder revealed that some contained genistein at more than 100 mg kg^-1. Cumin was the most likely source material, although not all individual samples of cumin tested contained high levels of genistein. Prenylnaringenin phytoestrogens were determined in UK hop-based beers at mean concentrations of 0.21 mg^-1 6-prenylnaringenin and 0.06 mg^-1 8-prenylnaringenin. The beers also contained traces of daidzein, genistein and biochanin A. The significance of 'hidden soya' in processed foods and these non-soya sources of phytoestrogens is that UK dietary intake of phytoestrogens must be assumed to be higher than estimated previously and that some sources of phytoestrogens remain poorly characterized.     

From Gut to Hormones: Unraveling the Role of Gut Microbiota in (Phyto)Estrogen Modulation in Health and Disease

The human gut microbiota regulates estrogen metabolism through the “estrobolome,” the collection of bacterial genes that encode enzymes like β-glucuronidases and β-glucosidases. These enzymes deconjugate and reactivate estrogen, influencing circulating levels. The estrobolome mediates the enterohepatic circulation and bioavailability of estrogen. Alterations in gut microbiota composition and estrobolome function have been associated with estrogen-related diseases like breast cancer, enometrial cancer, and polycystic ovarian syndrome (PCOS). This is likely due to dysregulated estrogen signaling partly contributed by the microbial impacts on estrogen metabolism. Dietary phytoestrogens also undergo bacterial metabolism into active metabolites like equol, which binds estrogen receptors and exhibits higher estrogenic potency than its precursor daidzein. However, the ability to produce equol varies across populations, depending on the presence of specific gut microbes. Characterizing the estrobolome and equol-producing genes across populations can provide microbiome-based biomarkers. Further research is needed to investigate specific components of the estrobolome, phytoestrogen-microbiota interactions, and mechanisms linking dysbiosis to estrogen-related pathology. However, current evidence suggests that the gut microbiota is an integral regulator of estrogen status with clinical relevance to women's health and hormonal disorders.     

雌马酚对人乳腺癌MCF-7细胞生长的影响

目的：观察雌马酚在不同浓度水平下对雌激素依赖阳性人乳腺癌MCF-7细胞生长的影响。方法：采用MTT法检测细胞增殖,流式细胞术分析细胞周期分布情况。结果：MCF-7细胞经去雌激素处理以后,与对照组相比,雌马酚在10-6～10-5mol/L时可显著抑制细胞增殖,使S期细胞增多,其抑制作用可被雌激素受体特异性抑制剂ICI-182780阻断。结论：雌马酚对人乳腺癌细胞MCF-7的细胞增殖具有抑制作用,可干扰DNA的复制,具有一定的雌激素样作用,且其抑制作用可能是通过雌激素受体（ER）介导的。     

基于GEO芯片评价乳品中胰岛素生长因子-Ⅰ、维生素D3和雌二醇联合对乳腺癌标志因子效应的研究

多项研究表明雌二醇（E2）、维生素D₃（VD₃）和胰岛素生长因子-Ⅰ（IGF-Ⅰ）均对乳腺病发生及身体发育机能调控等具有潜在影响,但目前缺乏三者联合效应的研究报道。本研究主要基于生物信息学方法,分析乳品中E2、VD₃、IGF-Ⅰ联合效应及其对乳腺癌标志因子的调控机制,为科学摄取乳品提供参考依据。依据美国国立生物技术信息中心（NCBI）人乳腺癌细胞（MCF-7）的3套GEO芯片（GSE160497、GSE27220、GSE145787）,使用cytoscape3.7.2筛选关键基因,应用R软件进行GO/KEGG通路富集分析,挖掘E2、VD₃、IGF-Ⅰ联合效应,探究三者对乳腺癌标志因子的调控机制。结果表明E2、VD₃、IGF-Ⅰ共同作用DNA复制、染色体合成等促进机体生长发育信号通路,可使乳腺癌标志因子MUC1表达沉默。E2单独作用可使微小染色体维持蛋白7、核糖体RNA加工蛋白等基因显著下调,可促进MUC1过表达。认为E2可与VD₃、IGF-Ⅰ互作维持平衡机...     

Differential hormonal regulation of estrogen receptors ERalpha and ERbeta and androgen receptor expression in rat efferent ductules

Estrogen receptors, in addition to the androgen receptor (AR), are expressed at high levels in efferent ductules of the male reproductive tract and it is now well recognized that estrogen receptor (ER) alpha is required for the maintenance of normal structure and function of the ductules. However, little is known regarding the hormonal regulation of the receptors themselves in the male. In the present study, efferent ductule ligation and castration, followed by replacement with testosterone, dihydro-testosterone (DHT) or estradiol was used to investigate the relative importance of circulating and luminal sources of steroid for the modulation of ERalpha, ERbeta and AR in rat efferent ductules. Uni- or bilateral castration and ligation did not affect the expression of ERalpha and ERbeta, but bilateral castration caused down-regulation of AR. Replacement with DHT and testosterone alone or in combination with estradiol caused the recovery of AR expression to control levels. A slight recovery of AR was also observed after estrogen replacement. ERalpha expression was decreased to nearly undetectable levels after estrogen replacement. On the other hand, ERbeta did not show evident effects following any of the treatments, suggesting a constitutive expression of this receptor. This differential modulation of the steroid hormone receptors highlights the importance of maintaining a physiological androgen-estrogen balance to regulate the structure and function of efferent ductules in the male.     

Screening of Korean edible wild plants for estrogenic activity using MCF-7 cell proliferation assay

This present study was performed to investigate estrogenic activity of Korean edible wild plants for alternative of estrogen replacement therapy in postmenopausal women. When the estrogenic activity was measured by estrogen induced proliferation of MCF-7 cells, of investigated 29 extracts, 3 samples of kudzu vine (Pueraria thunbergiana), Cardamine leucantha, and David Vetchling (Lathyrus davidii) exhibited a significant proliferation of above 20% at concentration of 100 μg/mL. This stimulation of MCF-7 cell proliferation could be completely blocked by addition of estrogen antagonist ICI 182,780 (100 nM), indicating estrogen receptor-dependent mechanism of these estrogenic effects on MCF-7 cell proliferation.