Estrogen Response Element Activity Research Articles

Glyphosate induces human breast cancer cells growth via estrogen receptors

Glyphosate is an active ingredient of the most widely used herbicide and it is believed to be less toxic than other pesticides. However, several recent studies showed its potential adverse health effects to humans as it may be an endocrine disruptor. This study focuses on the effects of pure glyphosate on estrogen receptors (ERs) mediated transcriptional activity and their expressions. Glyphosate exerted proliferative effects only in human hormone-dependent breast cancer, T47D cells, but not in hormone-independent breast cancer, MDA-MB231 cells, at 10⁻¹² to 10⁻⁶M in estrogen withdrawal condition. The proliferative concentrations of glyphosate that induced the activation of estrogen response element (ERE) transcription activity were 5-13 fold of control in T47D-KBluc cells and this activation was inhibited by an estrogen antagonist, ICI 182780, indicating that the estrogenic activity of glyphosate was mediated via ERs. Furthermore, glyphosate also altered both ERα and β expression. These results indicated that low and environmentally relevant concentrations of glyphosate possessed estrogenic activity. Glyphosate-based herbicides are widely used for soybean cultivation, and our results also found that there was an additive estrogenic effect between glyphosate and genistein, a phytoestrogen in soybeans. However, these additive effects of glyphosate contamination in soybeans need further animal study.

Abstract 3079: Estrogen signaling promotes cancer stem cell expansion via microRNA-140 targeting of pluripotency-associated factor SOX2 in ERα positive breast cancer cells.

Abstract Multiple reports indicate that the prospective tumor suppressor miR-140 is down regulated in breast tumors compared to normal breast tissues; however, the role of miR-140 in breast tumorigenesis remains unclear. We have identified a novel target for miR-140 in mammary epithelium, the core pluripotency transcription factor SOX2. It is well known that SOX2 regulates the self-renewal of embryonic stem cells, however, less is known concerning possible oncogenic roles of SOX2 in breast cancer. We found that miR-140 and SOX2 target mRNA exhibited inverse expression in breast tumors and normal breast tissues, which was also correlated with estrogen receptor alpha (ERα) status. We investigated the role of ERα signaling in the regulation of miR-140 and SOX2 in breast tumors and found that estrogen stimulation resulted in decreased miR-140 and increased SOX2 expression in mammary epithelium and breast cancer cells. We performed bioinformatics promoter analyses of the miR-140 promoter revealing several predicted estrogen response elements (ERE) in the miR-140 promoter. We examined estrogen-induced activation of these prospective EREs using luciferase reporters for a miR-140 promoter deletion series. Furthermore, we used chromatin-immunoprecipitation to reveal that ERα binds to an ERE flanking the miR-140 promoter, consequently suppressing miR-140 transcription. As estrogen signaling was previously implicated in the regulation of breast cancer stem cells (CSCs), we investigated whether the miR-140/SOX2 pathway might provide a mechanistic link between ERα signaling and CSC maintenance. We found that forced activation of the miR-140/SOX2 pathway decreased mammosphere formation (a surrogate measure of CSC self-renewal) and opposed estrogen-induced expansion of the well-characterized CD44(high)/CD24(low) CSC subpopulation in ERα+ breast cancer cells. These results indicate that an ERα/miR-140/SOX2 pathway critically regulates breast CSC maintenance in ERα+ breast tumors. Citation Format: Yongshu Zhang, Gabriel L. Eades, Yuan Yao, Qinglin Li, Qun Zhou. Estrogen signaling promotes cancer stem cell expansion via microRNA-140 targeting of pluripotency-associated factor SOX2 in ERα positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2013-3079

Transcriptional profiling of Chinese medicinal formula Si-Wu-Tang on breast cancer cells reveals phytoestrogenic activity

BackgroundSi-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women’s diseases. In our previous study, the microarray gene expression profiles of SWT on breast cancer cell line MCF-7 were found similar to the effect of β-estradiol (E2) on MCF-7 cells in the Connectivity Map database.MethodsFurther data analysis was conducted to find the main similarities and differences between the effects of SWT and E2 on MCF-7 gene expression. The cell proliferation assay on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells were used to examine such estrogenic activity. The estrogenic potency of SWT was further confirmed by estrogen-responsive element (ERE) luciferase reporter assay in MCF-7 cells.ResultsMany estrogen regulated genes strongly up-regulated by E2 were similarly up-regulated by SWT, e.g., GREB1, PGR and EGR3. Of interest with regard to safety of SWT, the oncogenes MYBL1 and RET were strongly induced by E2 but not by SWT. Quantitative RT-PCR analysis revealed a highly concordant expression change in selected genes with data obtained by microarrays. Further supporting SWT’s estrogenic activity, in MCF-7 but not in MDA-MB-231 cells, SWT stimulated cell growth at lower concentrations (< 3.0 mg/ml), while at high concentrations, it inhibits the growth of both cell lines. The growth inhibitory potency of SWT was significantly higher in MDA-MB-231 than in MCF-7 cells. The SWT-induced cell growth of MCF-7 could be blocked by addition of the estrogen receptor antagonist tamoxifen. In addition, SWT was able to activate the ERE activity at lower concentrations. The herbal components Angelicae, Chuanxiong and Rehmanniae at lower concentrations (< 3.0 mg/ml) also showed growth-inducing and ERE-activating activity in MCF-7 cells.ConclusionsThese results revealed a new mechanism to support the clinical use of SWT for estrogen related diseases and possibly for cancer prevention. This study also demonstrated the feasibility of using microarray transcriptional profiling to discover phytoestrogenic components that are present in natural products.

Abstract B42: Estrogenic activities of Chinese medicinal formula Si-Wu-Tang on breast cancer cells

Abstract Chinese medicinal herbs provide a promising source to develop alternative and complementary medicine for cancer prevention. Si-Wu-Tang (SWT, or Si-Wu decoction) comprises the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae. It is one of the most popular traditional medicines for women's health although the mechanism of the pharmacological action has not been clarified. The microarray gene expression profiles of SWT on human breast cancer cell line MCF-7 showed high similarity to that of β-estradiol (E2)-treated MCF-7 cells in the “Connectivity Map” (cMAP) database in our previous study (Wen et al, PLoS ONE, 2011), indicating novel estrogen-like activity. The objective of this study is to investigate the effects of SWT and its individual herbal components on the estrogen response pathways. The Sulforhodamine B (SRB) cell proliferation assay and luciferase estrogen-responsive element (ERE) reporter assay on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells were used to examine such estrogenic effect. The cells in 96-well plates were treated with SWT or its components for 48 hours before staining with SRB solution. SWT stimulated cell growth in the MCF-7 cell line at low concentrations (1.5 and 3.0 mg/ml), while at high concentrations (&amp;gt; 6.0 mg/ml) SWT inhibited the growth of both cell lines. This growth inhibitory effect of SWT was significantly higher in MDA-MB-231 cells than in MCF-7 cells. The combination of SWT with tamoxifen showed enhanced growth inhibitory effects on MCF-7 cells but did not show the same effects in MDA-MB-231 cells. All herbal components of SWT except for Paeoniae similarly induced growth of MCF-7 cells at low concentrations (1.5 and 3.0 mg/ml). At higher concentrations, all of the components showed cytotoxicity. In MCF-7 cells, the cytotoxicity is in the order of Paeoniae (IC50 = 2.6 + 0.16) &amp;gt;Angelicae (IC50 = 3.1 + 0.073) &amp;gt;Chuanxiong (IC50 = 5.7 + 0.57) &amp;gt;Rehmanniae (IC50&amp;gt;12 mg/ml). In MDA-MB-231 cells, the cytotoxicity is in the same order of Paeoniae (IC50 = 1.1 + 0.21) &amp;gt;Angelicae (IC50 = 2.8 + 0.20) &amp;gt;Chuanxiong (IC50 = 5.9 + 0.47) &amp;gt;Rehmanniae (IC50&amp;gt;12 mg/ml). To quantify the effect on ERE when exposed to SWT and components, MCF-7 cells were plated in 96-well plates and co-transfected with plasmids containing ERE and renilla luciferases. Then the cells were treated with various concentrations of SWT (0.256 - 5.12 mg/ml). The results of the dual luciferase assays revealed that SWT induced a dose dependent increase in the activation of ERE in MCF-7 cells. Except for Paeoniae, all of the herbal components of SWT also induced a significant increase in the ERE activity. In conclusion, the data obtained in this study indicate that SWT possesses estrogenic activity. Our results support the clinical use of SWT for estrogen related diseases and suggest a potential new application for chemoprevention of estrogen-dependent or –independent types of breast cancer. Citation Format: Jeffery Fan, Mandy Liu, Zhijun Wang, Zhong Zuo, Moses Chow, Ying Huang. Estrogenic activities of Chinese medicinal formula Si-Wu-Tang on breast cancer cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B42.

Abstract B30: Estrogen-ERβ axis: Implications for racial disparity of prostate cancer.

Abstract Introduction: The roles of estrogens and estrogen receptors (ERs) in the disproportionate incidence and mortality of prostate cancer (PCa) among African Americans (AA) as opposed to Caucasian Americans (CA) have not been established. Although estrogens have been shown to be involved in normal and pathologic growth of prostate gland including prostate cancer (PCa). Because ERα and ERβ are different in terms of ligand binding, heterodimerization, transactivation, and estrogen response element activity, their aberrant expression may act as a key factor to determine the ultimate estrogen effects on prostate cancer cells. Although recent studies have reported a role for ERβ in PCa, its ethnic-based expression and underlying mechanisms are not fully understood. Methods: Differential expression of ERβ was assessed using an NCI-designed ethnicity-based tissue microarray (TMA) slide encompassing 150 each of AA and CA prostate cancer tissue cores, 17 BPH, 13 normal biopsies, and 3 PCa cell lines (LNCaP, DU-145 and PC-3). In another set of experiments, androgen dependent (LNCaP) and independent (C4-2B and PC-3) cell lines were treated with various concentrations (up to 10 nM) of dihydrotestosterone (DHT) and estradiol (E2). Cell proliferation, expression of hormone receptors (qPCR and Western blot analysis), and crosstalk of AR and ERs transactivation using specific pharmacologic inhibitors and ERα and ERβ shRNA-stable-transfected COS-7 cells in presence or absence of hormones were performed. Results: In comparison to normal subjects, circulating E2 levels were significantly elevated (p=0.016) in all PCa patients. Further analysis demonstrates an increase in circulating E2 levels in AA (p=0.002) in comparison to CA men with PCa. Histoscore analysis demonstrates intense nuclear immunoreactivity (p=0.002) for ERβ in tumor cores of AA than in CA men. The biracial expression of ERβ was validated by qRT-PCR on LCM-procured tumor cells (p=0.032). Adjusted multivariate analysis showed a positive correlation (p&amp;lt;0.042) between ERβ expression and the age at diagnosis in AA men. However, no correlation found between ERβ and other clinical parameters in CA men. At the molecular level, mRNA and protein expression of AR were elevated in E2-treated PCs cells. Cell proliferation rate was accelerated after 72h of E2 treatment, especially in AR-independent PC cells. In addition, the activity of AR was increased by E2 treatment, an effect that was reversed by specific ER-inhibitors (ICI-182780 and Tamoxifen). Intriguingly, an AR ligand-independent increase in psPSA-luc promoter activity was observed in response to E2 in ERα/β-transfected COS-7 cells. Treatment of COS-7 cells with MPP or ERα shRNA apparently decreased the promoter activity in ERα-transfected CO7 cells after E2 treatment. Conclusion: Taken together, estrogen-ERβ axis has a distinctive role in activating AR-signaling in prostate cancer cells. Importantly, high circulating levels of estrogens coupled with differential expression of ERβ in tumor cells suggest a potential role for estrogen-ERβ axis in development of castrate-resistant prostate cancer (CRPC) in AA men. Citation Format: Krzysztof Moroz, Zakaria Abd Elmageed, Sudesh Srivastav, Krishnarao Moparty, Krishnarao Moparty, Raju Thomas, Asim B. Abdel-Mageed. Estrogen-ERβ axis: Implications for racial disparity of prostate cancer. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B30.

Abstract 2152: p38α MAPK regulates signal transduction between estrogen receptor (ER) and its antagonist by switching ER signaling from the classical to the non-classical pathway

Abstract Estrogen receptor ≤ (ER) plays a decisive role in breast cancer growth through regulating gene expression via the classical {ERE (estrogen-response element) binding} and the non-classical (AP-1 recruitment by interacting with c-Jun) pathways and serves as the only target for hormone therapy. However, anti-estrogen acts on several cascades beside ER and the implications of these “off-target” effects on hormone sensitivity remain unexplored. Here we report that p38α MAPK is selectively activated by treatment of breast cancer cells with the antiestrogen tamoxifen (TAM) and activated p38α in turn switches ER signaling from the classical to the non-classical pathway leading to increased hormone sensitivity. This occurs primarily through p38α phosphorylating ER/S118 leading to its S118-dependent proteasome-degradation through the E3 ligase E6AP resulting in decreased ERE activity. Unexpectedly, Ser-118 is also required for ER to form a complex with p38α, for p38α to stimulate AP-1, to inhibit ERE, and to sensitize breast cancer cells to TAM-induced growth inhibition and ER/S118 phosphorylation alone is both necessary and sufficient for ER binding to the AP-1 DNA. These results indicate that Ser-118 phosphorylation serves as a central switch for p38α to regulate the ER signaling distribution, which may be targeted to increase breast cancer hormone sensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2152. doi:1538-7445.AM2012-2152

P4-01-15: Alteration of Y-box Binding Protein-1 Expression Modifies the Response to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer.

Abstract Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in ER-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to the ER and growth factor receptor signaling pathways, response to the antiestrogens (tamoxifen and fulvestrant), and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of the YB-1 and the factors related to the ER and growth factor receptor signaling pathways were evaluated in the clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk and c-Myc were increased in the MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors, but increased the expression of ERα in the wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to those in the MCF7-vector cells. In the MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when the cells were treated either with tamoxifen or fulvestrant. The ERE activity was decreased in the MCF7-YB-1 cells in comparison to the MCF7-vector cells, and the ERE activity of the MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity of the MCF7-vector cells. In the ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more of the specimens that showed increased or positive nuclear YB-1 expression after the chemotherapy were positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently may alter the response to endocrine therapy in these cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-15.

Polyphenols, isothiocyanates, and carotenoid derivatives enhance estrogenic activity in bone cells but inhibit it in breast cancer cells

While exposure to estrogens is a major risk factor of breast and endometrial cancer, it well established that estrogens are beneficial for bone health. We have previously shown that carotenoids inhibit estrogen signaling in breast and endometrial cancer cells. The aim of this study was to compare the effects of various phytonutrients, (carotenoid derivatives, polyphenols, isothiocyanates) on estrogenic activity in breast cancer cells and osteoblast-like cells. All the tested phytonutrients inhibited estrogen response element (ERE) transactivation in breast cancer cells. In contrast, these compounds either did not affect or enhanced ERE activity and the expression of several bone-forming genes. These results were obtained using two osteoblast-like cell lines, MG-63 human osteosarcoma cells stably transfected with estrogen receptor-α (ERα) and MC3T3-E1 mouse calvaria-derived cells expressing endogenous ER. Phytonutrients-induced ERE inhibition in breast cancer cells, and its potentiation in osteoblast-like cells were associated both with a decrease and a rise in total and nuclear ERα levels, respectively. Phytonutrients activated the electrophile/antioxidant response element (EpRE/ARE) transcription system to various extents in both cancer and bone cell lines. Overexpression of Nrf2, the major EpRE/ARE activating transcription factor, mimicked the effects of phytonutrients, causing inhibition and enhancement of ERE transactivation in breast cancer cells and in osteoblast-like cells, respectively. Moreover, reduction in Nrf2 levels by RNAi led to a decrease in the phytonutrient potentiation of ERE activity transactivation in osteoblast-like cells. These findings suggest that the enhancement and inhibition of estrogen signaling by phytonutrients in bone-derived cells and breast cancer cells, respectively, is partially mediated by the activation of the Nrf2/ARE system.

Alteration of Y-box binding protein-1 expression modifies the response to endocrine therapy in estrogen receptor-positive breast cancer

Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in estrogen receptor (ER)-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to ER and growth factor receptor signaling pathways and responses to antiestrogens (tamoxifen and fulvestrant) and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of YB-1 and the factors related to ER and growth factor receptor signaling pathways were evaluated in clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk, and c-Myc was increased in MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors but increased the expression of ERα in wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to that in MCF7-vector cells. The introduction of YB-1 into MCF7 cells inhibited apoptosis and cell cycle arrest at G1 phase induced by antiestrogens. In MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when cells were treated with either tamoxifen or fulvestrant. The ERE activity was reduced in MCF7-YB-1 cells in comparison to MCF7-vector cells, and the ERE activity in MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity in MCF7-vector cells. In ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more number of specimens that showed increased or positive YB-1 expression after chemotherapy was positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently, may alter the response to endocrine therapy in ER-positive breast cancer cells.

Antiosteoporotic compounds from seeds of Cuscuta chinensis

The seeds of Cuscuta chinensis (Tu-Si-Zi, TSZ) have long been used for the treatment of osteoporosis in China and some Asian countries. The compounds in TSZ responsible for the antiosteoporotic activity are still poorly understood. The present study was designed to investigate the osteogenic compounds in TSZ, and to evaluate their antiosteoporotic effects in osteoblastic cells. Osteoblast-like UMR-106 cells were used for bioactivity-guided isolation of the active compounds. The activity of alkaline phosphatase (ALP) in UMR-106 cells was measured by p-nitrophenyl sodium phosphate assay. The proliferation of UMR-106 cells was assayed by Alamar-Blue method. Estrogenic activity of the extracts and isolated compounds was evaluated by activation of estrogen response element (ERE) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 5×ERE luciferase reporter plasmid. Antiestrogenic activity of the extracts and isolated compounds were evaluated by activation of activator protein-1 (AP-1) luciferase reporter expression in HeLa cells co-transfected with human estrogen receptor subtypes (ERα or ERβ) expression vectors and 6×AP-1 luciferase reporter plasmid. ALP-guided fractionation led to the isolation of five known flavonoids, quercetin, kaempferol, isorhamnetin, hyperoside and astragalin from the crude ethanolic extract of TSZ. Further study showed that kaempferol and hyperoside significantly increased the ALP activity in UMR-106 cells. Astragalin promoted the proliferation of UMR-106 cells whereas other compounds had no such effect. The isolated compounds showed estrogenic activity but quercetin, kaempferol and isorhamnetin showed more potent ERβ agonist activity. However, compared with their ER agonist activity, only quercetin and kaempferol showed potent ER antagonist activity by activating ERα/β-mediated AP-1 reporter expression. Our findings validated the clinical use of TSZ in the treatment of osteoporosis, and demonstrated that kaempferol and hyperoside are the active compounds in TSZ for the osteogenic effect.

Role of 2-methoxyestradiol as inhibitor of arthritis and osteoporosis in a model of postmenopausal rheumatoid arthritis

In postmenopausal rheumatoid arthritis, both the inflammatory disease and estrogen deficiency contribute to the development of osteoporosis. As hormone replacement therapy is no longer an option, we hypothesized that 2-methoxyestradiol (2me2) could be beneficial, and asked if such therapy was associated with effects on reproductive organs. Mice were ovariectomized and arthritis was induced, whereafter mice were administered 2me2, estradiol, or placebo. Clinical and histological scores of arthritis, together with bone mineral density were evaluated. Uteri weight, reactive oxygen species (ROS) from spleen cells, and characterization of cells from joints and lymph nodes were analyzed. In addition, in vivo activation of estrogen response elements (ERE) by 2me2 was evaluated. Treatment with 2me2 and estradiol decreased the frequency and severity of arthritis and preserved bone. Joint destruction was reduced, neutrophils diminished and ROS production decreased. The uterine weight increased upon long-term 2me2 exposure, however short-term exposure did not activate ERE in vivo.

GRIP1 enhances estrogen receptor α-dependent extracellular matrix gene expression in chondrogenic cells

The role of postmenopause on the pathogenesis of cartilage degeneration has been an open question. We assessed cartilage degeneration in estrogen receptor (ER)alpha null mice and examined the role of glucocorticoid receptor-interacting protein 1 (GRIP1) in the ERalpha-dependent transcription of a type II collagen gene (col2a1) with special reference to a crosstalk with the transforming growth factor (TGF)-beta signaling pathway. The vertebral cartilaginous endplate from female ERalpha null mice was subjected to histological analyses. Col2a1 expression of primary chondrocytes (PCs) obtained from ERalpha null mice after 17beta-estradiol (E(2)) and TGF-beta1 stimulation was examined by reverse transcription polymerase chain reaction (RT-PCR). Estrogen response element (ERE) or col2a1 promoter-enhancer luciferase reporter system was used to investigate the crosstalk among ERalpha, GRIP1, and MKK6. Col2a1 expression and glycosaminoglycan (GAG) content were measured in ATDC5 cells treated with GRIP1 small interfering RNA (siRNA). ERalpha deficiency clearly accelerated impairment of the vertebral cartilaginous endplate. E(2) and TGF-beta1 stimulation increased col2a1 expression in PC from wild-type mice, but not that from ERalpha null mice. The same stimulation increased the col2a1 promoter-enhancer reporter activity, and the elevated activity was decreased by dominant-negative ERalpha and p38 mitogen-activated protein kinase (MAPK) inhibitor. GRIP1 increased the E(2)-dependent ERE activation in the presence of ERalpha and constitutive-active MKK6. GRIP1 siRNA repressed col2a1 expression and GAG production in ATDC5 cells. Crosstalks between ERalpha/GRIP1 and TGF-beta/MKK6/p38 MAPK pathway have protective roles on cartilage metabolism via regulating the extracellular matrices expression. The finding may lead to the development of a novel therapeutic approach for cartilage degeneration.

An additive interaction between the NFkappaB and estrogen receptor signalling pathways in human endometrial epithelial cells.

BACKGROUNDHuman embryo implantation is regulated by estradiol (E2), progesterone and locally produced mediators including interleukin-1β (IL-1β). Interactions between the estrogen receptor (ER) and NF kappa B (NFκB) signalling pathways have been reported in other systems but have not been detailed in human endometrium.METHODS AND RESULTSReal-time PCR showed that mRNA for the p65 and p105 NFκB subunits is maximally expressed in endometrium from the putative implantation window. Both subunits are localized in the endometrial epithelium throughout the menstrual cycle. Reporter assays for estrogen response element (ERE) activity were used to examine functional interactions between ER and NFκB in telomerase immortalized endometrial epithelial cells (TERT-EEC). E2 and IL-1β treatment of TERT-EECs enhances ERE activity by a NFκB and ER dependent mechanism; this effect could be mediated by ERα or ERβ. E2 and IL-1β also positively interact to increase endogenous gene expression of prostaglandin E synthase and c-myc. This is a gene-dependent action as there is no additive effect on cyclin D1 or progesterone receptor expression.CONCLUSIONIn summary, we have established that NFκB signalling proteins are expressed in normal endometrium and report that IL-1β can enhance the actions of E2 in a cell line derived from healthy endometrium. This mechanism may allow IL-1β, possibly from the developing embryo, to modulate the function of the endometrial epithelium to promote successful implantation, for example by regulating prostaglandin production. Aberrations in the interaction between the ER and NFκB signalling pathways may have a negative impact on implantation contributing to pathologies such as early pregnancy loss and infertility.

Caffeic acid phenethyl ester, a component of beehive propolis, is a novel selective estrogen receptor modulator

Caffeic acid phenethyl ester (CAPE) is an active ingredient of beehive propolis with a structure similar to phenolic acid. The estrogenic effects of propolis were previously demonstrated through the activation of an estrogen receptor. To identify the estrogenic properties of propolis, CAPE was evaluated using in vitro and in vivo methods. CAPE showed selective binding affinity to human estrogen receptor beta (hERbeta) rather than hERalpha. CAPE also reduced ERalpha expression in MCF-7 and MDA 231 cells. In the yeast estrogen receptor transcription assay, CAPE produced the transcriptional activity of estrogen-responsive element with EC(50) values of 3.72 x 10(-6) M. CAPE did not increase the growth of MCF-7 estrogen receptor-positive breast cancer cells in doses ranging from 10(-7) to 10(-5) M. In order to understand how CAPE acts in animals, CAPE was tested by a uterotrophic bioassay. Treatment with CAPE (100, 500 mg/kg) did not increase the uterine weight relative to 3 microg/kg 17beta-estradiol treatment. The results indicate that CAPE, which is a selective agonist to hERbeta, but does not show any estrogenic effect on estrogen receptor-positive breast cancer cells and in immature rat uterine tissue, is a potential selective estrogen receptor modulator.

Inhibition of Peroxisome Proliferator-Activated Receptor γ Increases Estrogen Receptor–Dependent Tumor Specification

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates gene transcription associated with intermediary metabolism, adipocyte differentiation, and tumor suppression and proliferation. To understand the role of PPARgamma in tumorigenesis, transgenic mice were generated with mammary gland-directed expression of the dominant-negative transgene Pax8PPARgamma. Transgenic mice were phenotypically indistinguishable from wild-type (WT) mice, but mammary epithelial cells expressed a greater percentage of CD29(hi)/CD24(neg), CK5(+), and double-positive CK14/CK18 cells. These changes correlated with reduced PTEN and increased Ras and extracellular signal-regulated kinase (ERK) and AKT activation. Although spontaneous tumorigenesis did not occur, transgenic animals were highly susceptible to progestin/7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis, which in contrast to WT mice resulted in a high tumor multiplicity and, most importantly, in the appearance of predominantly estrogen receptor alpha-positive (ER(+)) ductal adenocarcinomas. Tumors expressed a similar PTEN(lo)/pERK(hi)/pAKT(hi) phenotype as mammary epithelium and exhibited high activation of estrogen response element-dependent reporter gene activity. Tumorigenesis in MMTV-Pax8PPARgamma mice was insensitive to the chemopreventive effect of a PPARgamma agonist but was profoundly inhibited by the ER antagonist fulvestrant. These results reveal important new insights into the previously unrecognized role of PPARgamma in the specification of mammary lineage and the development of ER(+) tumors.

Activation of estrogen response elements is mediated both via estrogen and muscle contractions in rat skeletal muscle myotubes

The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction on the mRNA levels of ERalpha and ERbeta was studied to determine the functional consequence of the transactivation. Myoblasts were isolated from rat skeletal muscle and transfected with a vector consisting of sequences of EREs coupled to the gene for luciferase. The transfected myoblasts were then differentiated into myotubes and subjected to either estrogen or electrical stimulation. Activation of the ERE sequence was determined by measurement of luciferase activity. The results show that both ERalpha and ERbeta are expressed in myotubes from rats. Both estrogen stimulation and muscle contraction increased (P < 0.05) transactivation of the ERE sequence and enhanced ERbeta mRNA, whereas ERalpha was unaffected by estrogen and attenuated (P < 0.05) by muscle contraction. Use of ER antagonists showed that, whereas the estrogen-induced transactivation is mediated via ERs, the effect of muscle contraction is ER independent. The muscle contraction-induced transactivation of ERE and increase in ERbeta mRNA were instead found to be MAP kinase (MAPK) dependent. This study demonstrates for the first time that muscle contractions have a similar functional effect as estrogen in skeletal muscle myotubes, causing ERE activation and an enhancement in ERbeta mRNA. However, in contrast to estrogen, the effect is independent of ERs and dependent on MAPK, suggesting activation via the estrogen related receptor (ERR).

Cell Growth of Ovarian Cancer Cells is Stimulated by Xenoestrogens through an Estrogen-Dependent Pathway, but Their Stimulation of Cell Growth Appears not to be Involved in the Activation of the Mitogen-Activated Protein Kinases ERK-1 and p38

Although endocrine disrupting chemicals (EDCs) may interfere with the endocrine system(s) of our body and have estrogenicity or androgenicity, the exact mechanism(s) underlying their detrimental effects is not clearly understood. Thus, in this study, we evaluated the effects of EDCs on proliferation and regulation of transcription of estrogen receptor (ER)-positive BG-1 ovarian cancer cells, and their possible mechanisms were further examined. Treatment with bisphenol A (BPA), nonylphenol (NP), octylphenol (OP) and methoxychlor (MXC) for 24 h resulted in an increase of cell proliferation. Treatment with BPA, NP, OP and MXC increased the estrogen response element (ERE) activity. The increase of cell proliferation and activation of ERE were reversed in the presence of an estrogen receptor antagonist, ICI 182780. These results suggest that ER is involved in EDC-mediated pathway in ovarian cancer cells. Based on this, we further investigated the involvement of EDCs in activation of mitogen-activated protein kinase (MAPK) in relation to cell growth. BPA rapidly induced activation of extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK at 15 min, but the effect of BPA (10 microM) on stimulation of cell growth was not blocked by pretreatment with inhibitors of MEK (PD98059) or p38 (SB203580) in a dose-dependent manner. Taken together, EDC-induced proliferation is mediated by a genomic effect through ERs and ERE, but EDC-activated MAPK is unlikely to be involved in EDC-induced cell growth in estrogen-responsive ovarian cancer cells.

Selective estrogen receptor modulators regulate IL‐6 inflammatory response in ARPE‐19 cells

Abstract Purpose Recent findings reveal that Toll‐like receptors (TLRs) and innate immunity participate in the pathology of age‐related macular degeneration (AMD). Many studies indicate that estrogens and selective estrogen modulators (SERMs) modulate inflammatory responses, but their effect on the development of AMD is weakly understood. In this study, we investigated the regulatory role of various SERMs (provided by Hormos Medical Ltd.) on IL‐6 expression in human retinal pigment epithelial cells (ARPE‐19). Methods ARPE‐19 cells were exposed to lipopolysaccharide (LPS; TLR 4 agonist) with simultaneous exposure to various SERMs and the secretion of IL‐6 cytokine was analyzed by ELISA. The estrogen receptor alpha and beta were qualitatively measured by RT‐PCR. To study the effect of various SERM treatments of estrogen response element (ERE) ‐mediated transcription, the ARPE‐19 cells were transiently transfected with ERE‐luciferase vector. The activity of ERE was measured by Luciferase assay. Results Simultaneous exposures to LPS and SERM‐320 reduced the IL‐6 expression levels in ARPE‐19 cells compared to LPS exposure alone. The RT‐PCR analysis showed that ARPE‐19 cells expressed estrogen receptor alpha but not beta proteins. Interestingly, SERM‐320 did not increase the activity of ERE in ARPE cells. This reveals that SERM‐320 is implicated in regulation of IL‐6 expression, but is not mediated through estrogen response element. Conclusion Our findings reveal that SERM‐320 is a novel compound to suppress innate immunity response in human retinal pigment epithelial cells.

Estrogen signaling ability in human endometrial cancer through the cancer-stromal interaction

The estrogen pathway plays an important role in the etiology of human endometrial carcinoma (EC). We examined whether estrogen biosynthesis in the tumor microenvironment promotes endometrial cancer. To examine the contribution of stromal cells to estrogen signaling in EC, we used reporter cells stably transfected with the estrogen response element (ERE) fused to the destabilized green fluorescent protein (GFP) gene. In this system, the endometrial cancer stromal cells from several patients activated the ERE of cancer cells to a variable extent. The GFP expression level increased when testosterone, a substrate for aromatase, was added. The effect was variably inhibited by aromatase inhibitors (AIs), although the response to AIs varied among patients. These results suggest that GFP expression is driven by estrogen synthesized by aromatase in the endometrial cancer stromal cells. In a second experiment, we constructed an adenovirus reporter vector containing the same construct as the reporter cells described above, and visualized endogenous ERE activity in primary culture cancer cells from 15 EC specimens. The GFP expression levels varied among the cases, and in most primary tissues, ERE activities were strongly inhibited by a pure anti-estrogen, fulvestrant. Interestingly, a minority of primary tissues in endometrial cancer showed ERE activity independent of the estrogen-ER pathway. These results suggest that AI may have some therapeutic value in EC; however, the hormonal microenvironment must be assessed prior to initiating therapy.

Estrogen and prostate cancer: An eclipsed truth in an androgen‐dominated scenario

Prostate cancer is the commonest non-skin cancer in men. Incidence and mortality rates of this tumor vary strikingly throughout the world. Although several factors have been implicated to explain this remarkable variation, lifestyle and dietary factors may play a dominant role, with sex hormones behaving as intermediaries between exogenous factors and molecular targets in development and progression of prostate cancer. Human prostate cancer is generally considered a paradigm of androgen-dependent tumor; however, estrogen role in both normal and malignant prostate appears to be equally important. The association between plasma androgens and prostate cancer remains contradictory and mostly not compatible with the androgen hypothesis. Similar evidence apply to estrogens, although the ratio of androgen to estrogen in plasma declines with age. Apart from methodological problems, a major issue is to what extent circulating hormones can be considered representative of their intraprostatic levels. Both nontumoral and malignant human prostate tissues and cells are endowed with key enzymes of steroid metabolism, including 17betahydroxysteroid dehydrogenase (17betaHSD), 5beta-reductase, 3alpha/3betaHSD, and aromatase. A divergent expression and/or activity of these enzymes may eventually lead to a differential prostate accumulation of steroid derivatives having distinct biological activities, as it occurs for hydroxylated estrogens in the human breast. Locally produced or metabolically transformed estrogens may differently affect proliferative activity of prostate cancer cells. Aberrant aromatase expression and activity has been reported in prostate tumor tissues and cells, implying that androgen aromatization to estrogens may play a role in prostate carcinogenesis or tumor progression. Interestingly, many genes encoding for steroid enzymes are polymorphic, although only a few studies have supported their relation with risk of prostate cancer. In animal model systems estrogens, combined with androgens, appear to be required for the malignant transformation of prostate epithelial cells. Although the mechanisms underlying the hormonal induction of prostate cancer in experimental animals remain uncertain, there is however evidence to support the assumption that long term administration of androgens and estrogens results in an estrogenic milieu in rat prostates and in the ensuing development of dysplasia and cancer. Both androgen and estrogen have been reported to stimulate proliferation of cultured prostate cancer cells, primarily through receptor-mediated effects. As for estrogens, the two major receptor types, ERalpha and ERbeta, are expressed in both normal and diseased human prostate, though with a different cellular localization. Since these two receptors are different in terms of ligand binding, heterodimerization, transactivation, and estrogen response element activity, it is likely that an imbalance of their expression may be critical to determine the ultimate estrogen effects on prostate cancer cells. In prostate cancer, ERbeta activation appears to limit cell proliferation directly or through ERalpha inhibition, and loss of ERbeta has been consistently associated with tumor progression. Several splicing variants of both ERalpha and ERbeta exist. Little is known about their expression and function in the human prostate, although reciprocal regulation and interaction with gene promoter both warrant further investigation. In summary, although multiple consistent evidence suggests that estrogens are critical players in human prostate cancer, their role has been only recently reconsidered, being eclipsed for years by an androgen-dominated interest.