Affinity For Estrogen Receptor Research Articles

156 EFFECT OF 2-METHOXYESTRADIOL (2-ME) ON HUMAN UTERINE LEIOMYOSARCOMA

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17β-oestradiol (E2) and has affinity for oestrogen receptors. It was reported as a promising antitumour drug due to antiproliferative activity in many tumour cell types. It has also been used in several preclinical and clinical studies for treatment of solid tumours. Thus, several studies have been conducted to investigate the cytotoxic effect of 2-ME on tumour cell lines in which it induced G2/M cell cycle arrest and subsequent apoptosis. Uterine leiomyosarcoma is a rare form of malignant smooth muscle cell tumour in the uterus muscle layer. This tumour is also influenced by oestrogen, which acts as a promoter. The purpose of this study was to examine the antiproliferative effect of 2-ME in vitro and in vivo. To perform in vitro experiments, we evaluated the antiproliferative effect of 2-ME on SK-LMS-1 cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and Western blot. In vivo experiment also investigated the effect of 2-ME on uterine leiomyoma by measuring tumour size and Western blotting. Employing the mouse xenograft model using human leiomyosarcoma SK-LMS-1 cell line compared with the positive control flavopiridol for 2 weeks, mice were divided into 4 groups (6 mice per each group); VE, 2-ME (75 or 150 mg kg–1), or flavopiridol (7.5 mg kg–1). All experimental procedures were performed in triplicate. P-values were calculated using one-way ANOVA, followed by Tukey’s test for multiple comparisons of columns. Data were considered statistically significant at P < 0.05. In vitro, we confirmed that 2-ME at 10–5 M and flavopiridol have an antiproliferative influence on SK-LMS-1 cells, but the same effect at 10–7 M or 10–6 M dose of 2-ME were not detected. These 2 doses showed little proliferative response in the MTT assay. Also, BAX/BCL-2 and LC3 expression were increased by 2-ME (10–5 M) treatment in Western blot analysis. In the in vivo xenograft study, application of 2-ME (75 mg kg–1) resulted in an increased tumour size but 150 mg kg–1 of 2-ME and flavopiridol decreased the size. In a previous study, we found that 2-ME has 2 types of action in an in vitro and an in vivo model. The 2-ME, which is used as a therapeutic agent for solid cancers, has not only apoptotic but also a proliferative effect, depending on the dose. We expect that 2-ME may be a potential therapeutic reagent for human uterine leiomyoma, but the appropriate dose of 2-ME should be used for oestrogen-response tumour treatment.

Licorice root components in dietary supplements are selective estrogen receptor modulators with a spectrum of estrogenic and anti-estrogenic activities

Licorice root extracts are often consumed as botanical dietary supplements by menopausal women as a natural alternative to pharmaceutical hormone replacement therapy. In addition to their components liquiritigenin (Liq) and isoliquiritigenin (Iso-Liq), known to have estrogenic activity, licorice root extracts also contain a number of other flavonoids, isoflavonoids, and chalcones. We have investigated the estrogenic activity of 7 of these components, obtained from an extract of Glycyrrhiza glabra powder, namely Glabridin (L1), Calycosin (L2), Methoxychalcone (L3), Vestitol (L4), Glyasperin C (L5), Glycycoumarin (L6), and Glicoricone (L7), and compared them with Liq, Iso-Liq, and estradiol (E2). All components, including Liq and Iso-Liq, have low binding affinity for estrogen receptors (ERs). Their potency and efficacy in stimulating the expression of estrogen-regulated genes reveal that Liq and Iso-Liq and L2, L3, L4, and L6 are estrogen agonists. Interestingly, L3 and L4 have an efficacy nearly equivalent to E2 but with a potency ca. 10,000-fold less. The other components, L1, L5 and L7, acted as partial estrogen antagonists. All agonist activities were reversed by the antiestrogen, ICI 182,780, or by knockdown of ERα with siRNA, indicating that they are ER dependent. In HepG2 hepatoma cells stably expressing ERα, only Liq, Iso-Liq, and L3 stimulated estrogen-regulated gene expression, and in all cases gene stimulation did not occur in HepG2 cells lacking ERα. Collectively, these findings classify the components of licorice root extracts as low potency, mixed ER agonists and antagonists, having a character akin to that of selective estrogen receptor modulators or SERMs.

2-Methoxyestradiol blocks the RhoA/ROCK1 pathway in human aortic smooth muscle cells.

2-Methoxyestradiol (2-ME), a metabolite of estradiol with little affinity for estrogen receptors, inhibits proliferation of vascular smooth muscle cells; however, the molecular mechanisms underlying this effect are incompletely understood. Our previous work shows that 2-ME inhibits initiation (blocks phosphorylation of ERK and Akt) and progression (reduces cyclin expression and increases expression of cyclin inhibitors) of the mitogenic pathway and interferes with mitosis (disrupts tubulin organization). Because the RhoA/ROCK1 pathway (RhoA → ROCK1 → myosin phosphatase targeting subunit → myosin light chain) is involved in cytokinesis, herein we tested the concept that 2-ME also blocks the RhoA/ROCK1 pathway. Because of the potential importance of 2-ME for preventing/treating vascular diseases, experiments were conducted in female human aortic vascular smooth muscle cells. Microarray transcriptional profiling suggested an effect of 2-ME on the RhoA/ROCK1 pathway. Indeed, 2-ME blocked mitogen-induced GTP-bound RhoABC expression and membrane-bound RhoA, suggesting interference with the activation of RhoA. 2-ME also reduced ROCK1 expression, suggesting reduced production of the primary downstream signaling kinase of the RhoA pathway. Moreover, 2-ME inhibited RhoA/ROCK1 pathway downstream signaling, including phosphorylated myosin phosphatase targeting subunit and myosin light chain; the ROCK1 inhibitor H-1152 mimicked these effects of 2-ME; both 2-ME and H-1152 blocked cytokinesis. 2-ME also reduced the expression of tissue factor, yet another downstream signaling component of the RhoA/ROCK1 pathway. We conclude that 2-ME inhibits the pathway RhoA → ROCK1 → myosin phosphatase targeting subunit → myosin light chain, and this likely contributes to the reduced cytokinesis in 2-ME treated HASMCs.

Estrogenic activity of bis(4-hydroxyphenyl)methanes with cyclic hydrophobic structure

Monoalkylated bis(4-hydroxyphenyl)methanes (e.g., 1) are reported to show weak binding affinity for estrogen receptor (ER). We hypothesized that introduction of appropriately located hydrophobic substituents in these compounds would increase the binding affinity. Indeed, we found that bis(4-hydroxyphenyl)methane bearing a 3,3-dimethylcyclohexyl group (7) shows potent ERα binding affinity, comparable to that of estradiol. Bulkier substituents could be introduced at the 3,3-position without decreasing the affinity. However, the position of the substituents was critical: the 4,4-dimethylcyclohexyl derivative (2) showed very weak binding affinity. The compounds with high ER-binding affinity showed predominantly agonistic activity, together with weak antagonistic activity at high concentration, in cell proliferation assay with human breast cancer cell line MCF-7. Further structure–function studies of these compounds and their derivatives might lead to the development of more selective and potent estrogen receptor modulators.

Isoguaiacins, Arylnaphthalene Types Identified as Novel Potent Estrogenic Signaling Molecules from Larrea nitida

In our study on the biological activities of isoguaiacins, we demonstrated lignans with a rigid structure from Larrea nitida as selective estrogen receptor modulators (SERMs). Estrogenic activity‐guided isolation of the L. nitida extract led to the identification of four lignans compounds. All four lignans displayed high binding affinity for human estrogen receptor (hER) α and β as well as transcriptional activities for estrogen response element (ERE). Furthermore, arylnaphthalene type lignans 1 (5,6,7,8‐tetrahydro‐5‐(3,4‐dihydroxyphenyl)‐6,7‐dimethylnaphthalene‐2,3‐diol) and 2 (5,6,7,8‐tetrahydro‐5‐(4‐hydroxy‐3‐methoxyphenyl)‐6,7‐dimethylnaphthalene‐2,3‐diol) showed especially high potencies for ERβ and ERE with IC50 of 0.045 and 0.85 μM, respectively. Arylnaphthalene type lignans 1 and 2 and bibenzyl butane type lignans 3 (4‐(4‐(4‐hydroxyphenyl)‐2,3‐dimethylbutyl) benzene‐1,2‐diol) increased the proliferation of MCF‐7 cells and the ERα target gene pS2 . Our results indicate that the four active lignans have selective affinities for hERβ and exhibit SERM properties. For the first time, we have discovered the estrogenic activities of isoguaiacins from L. nitida, which are promising candidates for the treatment of estrogen‐related conditions and warrant further preclinical evaluation.

Two faces of the estrogen metabolite 2-methoxyestradiol in vitro and in vivo.

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-estradiol (E2), interacts with estrogen receptors (ERs) and microtubules, however, 2-ME has a low affinity for ERs. Furthermore, 2‑ME has been identified as a potential novel antitumor agent, combining its anti‑proliferative effects on a variety of tumor cell types with its anti‑angiogenic action. Therefore, 2‑ME is of interest due to its potential anticancer therapeutic effects. In the current study, the estrogenic effect of 2‑ME on CaBP‑9k, ERα, and progesterone receptor (PR) mRNA levels in the absence and presence of E2 and progesterone (P4) in in vivo and in vitro models was examined. In GH3 cells, the mRNA level of CaBP‑9k was induced in the E2 treatment group (concentration, 10‑9 M), and the expression of CaBP‑9k was also upregulated in the 2‑ME‑treated group (concentration, 10‑7 M). Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2‑ME group [dose, 40 mg/kg body weight (BW)], which was comparable to the response with E2 (dose, 40 µg/kg BW) observed in mice. As inhibitors of ER and PR activity, ICI 182,780 and mifepristone (RU486) were observed to reverse the E2 or 2‑ME mediated increase of CaBP‑9k and Ltf mRNA expression. In addition, it was found that 2‑ME significantly decreased the levels of ERα and increased PR transcripts. Consistent with the in vitro results, the mRNA levels revealed decreased ERα and increased PR in in vivo treatment of E2 and 2‑ME. These findings demonstrate that the expression of estrogenic markers, CaBP‑9k and Ltf, is regulated by 2‑ME in in vitro and in vivo models, therefore, estrogenic activi-ties of 2-ME may be increased in females during the estrous cycle via the ER and/or PR-mediated signaling pathway.

Zearalenone metabolism in human placental subcellular organelles, JEG-3 cells, and recombinant CYP19A1.

Zearalenone (ZEN) and its derivative, zearalanone (ZAN), are endocrine disruptive mycotoxins produced by Fusarium species. We investigated the human placental metabolism of ZEN and ZAN invitro in JEG-3 cells, human term placental subcellular fractions and recombinant enzymes. Human placental enzymes were capable of metabolizing ZEN and ZAN to their primary OH-metabolites which have higher affinity for estrogen receptors than their parent compounds. These metabolites may interfere with physiological placental estrogen signaling and thus disrupt the progress of gestation.

Formulation of Anti-miR-21 and 4-Hydroxytamoxifen Co-loaded Biodegradable Polymer Nanoparticles and Their Antiproliferative Effect on Breast Cancer Cells.

Breast cancer is the second leading cause of cancer-related death in women. The majority of breast tumors are estrogen receptor-positive (ER+) and hormone-dependent. Neoadjuvant anti-estrogen therapy has been widely employed to reduce tumor mass prior to surgery. Tamoxifen is a broadly used anti-estrogen for early and advanced ER+ breast cancers in women and the most common hormone treatment for male breast cancer. 4-Hydroxytamoxifen (4-OHT) is an active metabolite of tamoxifen that functions as an estrogen receptor antagonist and displays higher affinity for estrogen receptors than that of tamoxifen and its other metabolites. MicroRNA-21 (miR-21) is a small noncoding RNA of 23 nucleotides that regulates several apoptotic and tumor suppressor genes and contributes to chemoresistance in numerous cancers, including breast cancer. The present study investigated the therapeutic potential of 4-OHT and anti-miR-21 coadministration in an attempt to combat tamoxifen resistance, a common problem often encountered in anti-estrogen therapy. A biodegradable poly(d,l-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) copolymer was utilized as a carrier to codeliver 4-OHT and anti-miR-21 to ER+ breast cancer cells. 4-OHT and anti-miR-21 co-loaded PLGA-b-PEG nanoparticles (NPs) were developed using emulsion-diffusion evaporation (EDE) and water-in-oil-in-water (w/o/w) double emulsion methods. The EDE method was found to be best method for 4-OHT loading, and the w/o/w method proved to be more effective for coloading NPs with anti-miR-21 and 4-OHT. The optimal NPs, which were prepared using the double emulsion method, were evaluated for their antiproliferative and apoptotic effects against MCF7, ZR-75-1, and BT-474 human breast cancer cells as well as against 4T1 mouse mammary carcinoma cells. We demonstrated that PLGA-b-PEG NP encapsulation significantly extended 4-OHT’s stability and biological activity compared to that of free 4-OHT. MTT assays indicated that treatment of MCF7 cells with 4-OHT–anti-miR-21 co-loaded NPs resulted in dose-dependent antiproliferative effects at 24 h, which was significantly higher than what was achieved with free 4-OHT at 48 and 72 h post-treatment. Cell proliferation analysis showed that 4-OHT and anti-miR-21 co-loaded NPs significantly inhibited MCF-7 cell growth compared to that of free 4-OHT (1.9-fold) and untreated cells (5.4-fold) at 1 μM concentration. The growth rate of MCF7 cells treated with control NPs or NPs loaded with anti-miR-21 showed no significant difference from that of untreated cells. These findings demonstrate the utility of the PLGA-b-PEG polymer NPs as an effective nanocarrier for co-delivery of anti-miR-21 and 4-OHT as well as the potential of this drug combination for use in the treatment of ER+ breast cancer.

The active tamoxifen metabolite endoxifen (4OHNDtam) strongly down-regulates cytokeratin 6 (CK6) in MCF-7 breast cancer cells.

IntroductionTamoxifen is an anti-estrogen drug used in treatment of Estrogen Receptor (ER) positive breast cancer. Effects and side effects of tamoxifen is the sum of tamoxifen and all its metabolites. 4-Hydroxytamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam, endoxifen) both have ER affinity exceeding that of the parent drug tamoxifen. 4OHNDtam is considered the main active metabolite of tamoxifen. Ndesmethyltamoxifen (NDtam) is the major tamoxifen metabolite. It has low affinity to the ER and is not believed to influence tumor growth. However, NDtam might mediate adverse effects of tamoxifen treatment. In this study we investigated the gene regulatory effects of the three metabolites of tamoxifen in MCF-7 breast cancer cells.Material and MethodsUsing concentrations that mimic the clinical situation we examined effects of 4OHtam, 4OHNDtam and NDtam on global gene expression in 17β-estradiol (E2) treated MCF-7 cells. Transcriptomic responses were assessed by correspondence analysis, differential expression, gene ontology analysis and quantitative real time PCR (Q-rt-PCR). E2 deprivation and knockdown of Steroid Receptor Coactivator-3 (SRC-3)/Amplified in Breast Cancer 1 (AIB1) mRNA in MCF-7 cells were performed to further characterize specific effects on gene expression.Results4OHNDtam and 4OHtam caused major changes in gene expression compared to treatment with E2 alone, with a stronger effect of 4OHNDtam. NDtam had nearly no effect on the global gene expression profile. Treatment of MCF-7 cells with 4OHNDtam led to a strong down-regulation of the CytoKeratin 6 isoforms (KRT6A, KRT6B and KRT6C). The CytoKeratin 6 mRNAs were also down-regulated in MCF-7 cells after E2 deprivation and after SRC-3/AIB1 knockdown.ConclusionUsing concentrations that mimic the clinical situation we report global gene expression changes that were most pronounced with 4OHNDtam and minimal with NDtam. Genes encoding CytoKeratin 6, were highly down-regulated by 4OHNDtam, as well as after E2 deprivation and knockdown of SRC-3/AIB1, indicating an estrogen receptor-dependent regulation.

Role of 2-methoxyestradiol, an Endogenous Estrogen Metabolite, in Health and Disease.

Estradiol (E2) is a steroid hormone whose physiological actions are mainly mediated by its interaction with intracellular estrogen receptors (ER) leading to modification on the mRNA and protein synthesis in its target cells. However, estrogens can also activate several intracellular signal transduction cascades by non-genomic mechanisms. Estrogens must be inactivated and removed from blood through its conversion to soluble compounds with an apparent low estrogenic activity and decreased affinity for ER. In this context, 2-methoxyestradiol (2ME2) is generated by a sequential hydroxylation of E2 via the enzyme cytochrome P450 isoform 1A1 to produce 2-hydroxyestradiol (2OHE2) followed by a conjugation reaction catalyzed by the enzyme Catechol-O-Methyltransferase generating 2ME2 from 2OHE2. Recent evidence indicates that physiological concentration of 2ME2 may regulate several biological processes while high concentrations of this metabolite may induce pathophysiological alterations in several tissues. In the last years, 2ME2 has also been described as a promising anticancer drug although its cellular and molecular mechanisms are still being disclosed. Herein, we will review the available literature concerning the role of 2ME2 in health and disease. We will focus on to describing the intracellular mechanisms by which 2ME2 exerts its effects on reproductive and non-reproductive tissues. The promising anticancer effects of 2ME2 and its synthetic derivatives will also be discussed. Finally, a group of 2ME2-target genes that could be used as biomarkers of 2ME2 under physiological or pathophysiological conditions will be reviewed.

Sequence to structure approach of estrogen receptor alpha and ligand interactions.

Estrogen receptors (ERs) are steroid receptors located in the cytoplasm and on the nuclear membrane. The sequence similarities of human ERα, mouse ERα, rat ERα, dog ERα, and cat ERα are above 90%, but structures of ERα may different among species. Estrogen can be agonist and antagonist depending on its target organs. This hormone play roles in several diseases including breast cancer. There are variety of the relative binding affinity (RBA) of ER and estrogen species in comparison to 17β-estradiol (E2), which is a natural ligand of both ERα and ERβ. The RBA of the estrogen species are as following: diethyl stilbestrol (DES)>hexestrol>dienestrol>17β-estradiol (E2)>17-estradiol>moxestrol>estriol (E3)>4-OH estradiol>estrone-3-sulfate. Estrogen mimetic drugs, selective estrogen receptor modulators (SERMs), have been used as hormonal therapy for ER positive breast cancer and postmenopausal osteoporosis. In the postgenomic era, in silico models have become effective tools for modern drug discovery. These provide three dimensional structures of many transmembrane receptors and enzymes, which are important targets of de novo drug development. The estimated inhibition constants (Ki) from computational model have been used as a screening procedure before in vitro and in vivo studies.

Estimation of safety/risk profile of the phytoestrogens 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland

Evaluation of stimulation of proliferation in female reproductive organs by phytoestrogens of reproductive tract organs is important to estimate their safety/risk profile if used as an alternative to hormone therapy. To assess the proliferative capacity of the flavanones 8-Prenylnaringenin (8-PN), 6-(1.1-dimethylallyl)naringenin (6-DMAN) and Naringenin (Nar) on the mammary gland we compared their estrogenic properties to 17β-Estradiol (E2) in three independent assays. We performed competitive estrogen receptor (ER) ligand binding assays, MCF-7 proliferation studies and a three day exposure experiment in young adult ovariectomized female rats. 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα (19.46%) compared to ERβ (6.5%). Among the tested flavanones 8-PN had the strongest mitotic effect on MCF-7 cells. In vivo 8-PN increased the number of terminal end buds in the mammary gland and the protein expression of the proliferation markers Ki-67 and PCNA in epithelial ductal cells and further led to an increase of Amphiregulin (Areg) mRNA expression, endpoints correlated to mammary gland proliferation. 6-DMAN also possesses a higher relative binding affinity for the ERα than for the ERβ and stimulated the cell proliferation of MCF-7 cells. It enhanced Ki-67 protein expression and β-Casein mRNA expression in vivo. Nar displayed the weakest estrogenic properties and was the only tested compound with a higher affinity for the ERβ than the ERα. It displayed proliferative effects on MCF-7 cells and slightly induced Ki-67 protein expression in vivo.

Design and synthesis of norendoxifen analogues with dual aromatase inhibitory and estrogen receptor modulatory activities.

Both selective estrogen receptor modulators and aromatase inhibitors are widely used for the treatment of breast cancer. Compounds with both aromatase inhibitory and estrogen receptor modulatory activities could have special advantages for treatment of breast cancer. Our previous efforts led to the discovery of norendoxifen as the first compound with dual aromatase inhibitory and estrogen receptor binding activities. To optimize its efficacy and aromatase selectivity versus other cytochrome P450 enzymes, a series of structurally related norendoxifen analogues were designed and synthesized. The most potent compound, 4'-hydroxynorendoxifen (10), displayed elevated inhibitory potency against aromatase and enhanced affinity for estrogen receptors when compared to norendoxifen. The selectivity of 10 for aromatase versus other cytochrome P450 enzymes was also superior to norendoxifen. 4'-Hydroxynorendoxifen is therefore an interesting lead for further development to obtain new anticancer agents of potential value for the treatment of breast cancer.

Analytical Procedure for the Determination of Zearalenone in Environmental and Biological Samples

The metabolism of zearalenone (ZEA) and analytical methods for determining the presence of ZEA and its metabolites are discussed in this study. Similar to phytoestrogens, solid metaloestrogens, pharmaceuticals, and selected pesticides, ZEA is a substance that displays endocrine activity. ZEA is accumulated in living organisms, and it is capable of contaminating all trophic levels of the food chain, from grain, maize, and other crop plants to human consumers. Zearalenone has a structure similar to that of estrogen (the presence of a macrocyclic lactone ring), it has an affinity for estrogen receptors, and it competes with 17β-estradiol for binding the estrogen receptor in natural pathways. As endocrine disruptors, zearalenone and its metabolites can also contribute to carcinogenic mutations associated with female secondary sex characteristics. The determination of zearalenone and its metabolites in various matrices, first of all biological and environmental samples, poses significant problems. A variety ways of extracting and purifying zearalenone, including liquid-liquid extraction and solid-phase extraction, are described. Furthermore, it describes the possibility of applying a plurality of sensitive and specific instrumental methods, chromatographic techniques (TLC, HPLC, GC) as well as other methods (immunoaffinity chromatography).

220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

Abstract 625: Proliferative effects of the phytoestrogens 8-Prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and Naringenin in MCF-7 cells and the rat mammary gland

Abstract Evaluation of stimulation of proliferation in female reproductive organs by phytoestrogens of reproductive tract organs is important to estimate their safety/risk profile if used as an alternative to hormone therapy. To assess the proliferative capacity of the flavanones 8-Prenylnaringenin (8-PN), 6-(1.1-dimethylallyl)naringenin (6-DMAN) and Naringenin (Nar) on the mammary gland we compared their estrogenic properties to 17β-Estradiol (E2) in three independent assays. We performed competitive estrogen receptor (ER) ligand binding assays, MCF-7 proliferation studies and a three day exposure experiment in young adult ovariectomized female rats. 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα (19.46 %) compared to ERβ (6.5 %). Among the tested flavanones 8-PN had the strongest mitotic effect on MCF-7 cells. In vivo 8-PN increased the number of terminal end buds (TEB) in the mammary gland and the protein expression of the proliferation markers Ki-67 and PCNA in epithelial ductal cells and further led to an increase of Amphiregulin (Areg) mRNA expression, endpoints correlated to mammary gland proliferation. 6-DMAN also possesses a higher relative binding affinity for the ERα than for the ERβ (1.46 %, 0.84 %) and stimulated the cell proliferation of MCF-7 cells. It enhanced Ki-67 protein expression and β-Casein mRNA expression in vivo. Nar displayed the weakest estrogenic properties and was the only tested compound with a higher affinity for the ERβ than the ERα (0.5 %, 0.08 %). It displayed proliferative effects on MCF-7 cells and slightly induced Ki-67 protein expression in vivo. Citation Format: Janina Helle, Oliver Zierau, Kristin Kräker, Annekathrin Keiler, Günter Vollmer, JoEllen Welsh, Georg Kretzschmar. Proliferative effects of the phytoestrogens 8-Prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and Naringenin in MCF-7 cells and the rat mammary gland. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2014-625

Assessment of the proliferative capacity of the flavanones 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland

8-Prenylnaringenin (8-PN) and naringenin (Nar) are phytoestrogens found in food items and nutritional supplements, while 6-(1.1-dimethylallyl)naringenin (6-DMAN) is a component of an African plant. Besides their assumed beneficial effects they may promote mammary and endometrial cancer. We therefore assessed their proliferative and estrogenic potential on the mammary gland in vitro and in vivo. In competitive estrogen receptor (ER) ligand binding assays 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα and had the strongest mitotic effect on MCF-7 cells among the test substances. In a three day exposure in young adult ovariectomized female rats 15mg/kg 8-PN had the highest capacity to increase the number of terminal end buds (TEB) in the mammary gland and stimulated expression of proliferation markers in epithelial ductal cells, followed by 6-DMAN and Nar, but overall their capacity to stimulate proliferation was weak in comparison to 17β-Estradiol (E2).

17α-Oestradiol-induced neuroprotection in the brain of spontaneously hypertensive rats.

17β-oestradiol is a powerful neuroprotective factor for the brain abnormalities of spontaneously hypertensive rats (SHR). 17α-Oestradiol, a nonfeminising isomer showing low affinity for oestrogen receptors, is also endowed with neuroprotective effects in vivo and in vitro. We therefore investigated whether treatment with 17α-oestradiol prevented pathological changes of the hippocampus and hypothalamus of SHR. We used 20-week-old male SHR with a blood pressure of approximately 170 mmHg receiving s.c. a single 800 μg pellet of 17α-oestradiol dissolved in cholesterol or vehicle only for 2 weeks Normotensive Wistar-Kyoto (WKY) rats were used as controls. 17α-Oestradiol did not modify blood pressure, serum prolactin, 17β-oestradiol levels or the weight of the testis and pituitary of SHR. In the brain, we analysed steroid effects on hippocampus Ki67+ proliferating cells, doublecortin (DCX) positive neuroblasts, glial fibrillary acidic protein (GFAP)+ astrocyte density, aromatase immunostaining and brain-derived neurotrophic factor (BDNF) mRNA. In the hypothalamus, we determined arginine vasopressin (AVP) mRNA. Treatment of SHR with 17α-oestradiol enhanced the number of Ki67+ in the subgranular zone and DCX+ cells in the inner granule cell layer of the dentate gyrus, increased BDNF mRNA in the CA1 region and gyrus dentatus, decreased GFAP+ astrogliosis in the CA1 subfield, and decreased hypothalamic AVP mRNA. Aromatase expression was unmodified. By contrast to SHR, normotensive WKY rats were unresponsive to 17α-oestradiol. These data indicate a role for 17α-oestradiol as a protective factor for the treatment of hypertensive encephalopathy. Furthermore, 17α-oestradiol is weakly oestrogenic in the periphery and can be used in males.

Estrogen receptor affinity chromatography: A new method for characterization of novel estrogenic disinfection by-products

To identify the unknown estrogenic disinfection by-products (DBPs) from the chlorination extract, an effective method based on affinity chromatography with immobilized human recombinant estrogen receptor α (ERα) was developed, which has an advantage in targeting different potential estrogenic compounds from mixed sample simultaneously by comparing their relative binding activities to ER. The new method worked well for six known environmental estrogens. To further test the validity of this method for unknown chemicals, six DBPs of diethylstilbestrol (DES) with relatively strong ER binding affinity after chlorination were isolated and identified. It was found that except for 2-chloro-DES which showed 1.36 times stronger binding affinity than DES, most of the by-products bound to ER much more weakly than DES. All these seven by-products induced a dose-dependent transcriptional activation in two-hybrid-yeast assays. Z,Z-dienestrol (DE) and 2-chloro-DES, which exhibiting the weakest and the strongest binding affinity, were further tested for their transcriptional potential as 0.00243 and 0.014 compared to DES, respectively. However, they were still potential harmful environmental estrogenic disruptors as their estrogenic activities were much stronger than that of bisphenol A (BPA). These results demonstrated that the new method can help to screen unknown estrogenic compounds from mixture more efficiently.

The estrogenic effects of apigenin, phloretin and myricetin based on uterotrophic assay in immature Wistar albino rats

Chemicals that occur in vegetal food and known as phytoestrogens, because of their structures similarity to estrogen, have benefits on chronic diseases. Despite this, when they are taken at high amounts, they can cause harmful effects on endocrine system of human and animals. In this study, it has been intended to determine the estrogenic potencies of phytoestrogens apigenin, phloretin and myricetin whose affinities for estrogen receptors in vitro. The female rats divided into 17 groups, each containing six rats. There was a negative control group and there were positive control dose groups which contains ethinyl estradiol, ethinyl estradiol+tamoxifen and genistein. The other dose groups which were tested for estrogenic activity contains apigenin, myricetin and phloretin All chemicals have been given to Wistar immature female rats with oral gavage for 3 consecutive days. By using uterotrophic analysis, uterus wet and blotted weights, vaginal opening, uterus length of female rats has been recorded at the end of the experiment. For detect of cell response, luminal epithelium height, gland number and lactoferrin intensity in luminal epithelium of uterus were evaluated. Biochemical analysises in blood were performed.Relative uterus weights of rats in 100mg/kg/day dose group of myricetin were statistically increased according to vehicle control and positive control groups. In dose groups of apigenin and phloretin it was found that there were cell responses in uterus. All treatment groups had a significant difference in the high intensity of lactoferrin and uterine gland count compared to oil control group. There was no difference between phloretin and apigenin treatment groups in uterine weight statictically. Uterine heights were increased in positive control groups and 100mg/kg/day dose group of myricetin. Epithelial cell heights were increased in treatment groups except apigenin and phloretin dose groups. There was no difference between all treatment groups in vaginal opening values according to positive control.