ER Agonists Research Articles

Abstract P3-05-14: Modeling chemoendocrine therapy for ER+/p53wt luminal breast cancer

Abstract Background ER+ breast cancers are predominantly p53 wildtype (wt). Despite this genotype, these cancers are relatively resistant to chemotherapy-induced apoptosis in the presence of estrogen. Thus, current clinical practice is to administer hormonal therapy and chemotherapy sequentially rather than concurrently. Using the ER+/p53wt cell line MCF-7, we previously demonstrated that ER bound by either the ER agonist estradiol or partial antagonist tamoxifen inhibits the expression of a set of proapoptotic p53 target genes, whereas the full ER antagonist fulvestrant, a selective ER downregulator (SERD), removes the ER-mediated suppression of these genes, sensitizing breast cancer cells to p53-mediated cell death .We hypothesize that effective chemoendocrine therapy requires complete ER antagonism resulting in ER downregulation, increased p53 activity, and ultimately apoptosis. Methods: To model mechanisms of tumor sensitivity and resistance to therapy in vivo, therapeutic experiments were performed in immunodeficient mice bearing ER+/p53wt tumor xenografts derived from a 61-year-old African-American female with grade 2 invasive ductal carcinoma. The mice were randomly assigned to the following treatment groups: A) fulvestrant B) tamoxifen, C) doxorubicin, D) fulvestrant plus doxorubicin, E) tamoxifen plus doxorubicin, and F) control. Tumors were measured weekly for 42 days to determine treatment effects on growth. In addition, RNA was harvested from tumors at an early time point for RNA sequencing to determine genes regulated by the treatments. Results: Combination therapy comprising doxorubicin plus fulvestrant resulted in tumor regression compared with single-agent or doxorubicin plus tamoxifen treatment, which inhibited the growth of tumors but did not lead to their regression. Conclusions: Current paradigms for the treatment of ER+ breast cancer either in the adjuvant or advanced setting have involved the sequential use of endocrine therapy and chemotherapy. This protocol is partially based on evidence of potential antagonism between tamoxifen and chemotherapy as observed in early studies. Our published work in vitro and the preclinical study presented here suggest that complete ER antagonism with SERDs such as fulvestrant is required to overcome the ability of ER to block p53-mediated apoptosis. Our results suggest treatments involving fulvestrant concurrent with chemotherapies involving p53 activation should be considered for the treatment of patients with ER+/p53wt breast cancer. Citation Format: Shiliang A Cao, Elgene Lim, Myles Brown, Shannon T Bailey. Modeling chemoendocrine therapy for ER+/p53wt luminal breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-14.

ER‐α Receptors Have More Prominent Depressor Role On Vasoconstractile Sensitivity in Treated And Untreated‐Ovariectomized Rat Mesentery Artery

Natural estrogens have cardioprotective effects in premenopausal women. Nuclear estrogen receptors mediate genomic effects of estrogens. G‐protein coupled estrogen receptors are also defined cardiovascular system and mediate rapid non‐genomic effects. The role of estrogen receptor ER‐α and ER‐β on vasculature system is not completely understood. In this study, we investigated the effects of ER‐α and β on α1‐adrenergic receptor mediated vasoconstraction in control (C), 16‐week ovariectomized (O) and 17‐β estradiol (E2) treatment ovariectomized‐rat (OE) mesentery arteries. Phe‐mediated concentration response curves (CRC) were obtained from C, O and OE groups in the presence and absence of non‐selective ER agonist E2 (0.1μM), selective ER‐α agonist PPT (0.1μM) and selective ER‐β agonist DPN (0.1μM). Phe‐mediated CRC shifted to right presence of PPT in C (pD2:6.32 vs pD2: 5.80), O (pD2:6.40 vs pD2: 5.79) and OE (pD2: 6.52 vs pD2:6.08) groups. After E2 incubation, Phe‐mediated CRC only shifted to right in C group (pD2:6.40; pD2: 5.80), but insignificantly shifted to right in O (pD2:6.40 vs pD2:6.13) and OE (6.52 vs. 6.21) groups. On the other hand, DPN incubation did not change Phe‐mediated CRC in all groups.In conclusion, ER‐α receptor mediated responsiveness have more prominent depressor effects on vasocontractile sensitivity in all group.

Bisphenol A depresses monosynaptic and polysynaptic reflexes in neonatal rat spinal cord in vitro involving estrogen receptor-dependent NO-mediated mechanisms

Bisphenol A (BPA), a toxic chemical from plastics, is known to produce locomotor abnormalities which may imply the alteration in synaptic activity at Ia-α motoneuron synapse also. However the effect of BPA on this synapse is not known. Therefore, this study was undertaken to examine the effect of BPA on reflexes originating at Ia-α motoneuron synapse in the spinal cord. The experiments were performed on isolated hemisected spinal cords from 4 to 6d rats. Stimulation of a dorsal root evoked segmental monosynaptic (MSR) and polysynaptic (PSR) reflex potentials in the corresponding ventral root. Nitrite content (indicator of NO activity) of cords was estimated in the presence of BPA with/without antagonists. Superfusion of BPA (3–100μM) depressed the reflexes in a concentration- and time-dependent manner. The depression was ∼20, ∼50 and ∼70% at 10, 30 and 100μM of BPA, respectively. The 50% depression occurred around 15min at 30μM of BPA. Pretreatment with estrogen receptor (ERα) antagonist, tamoxifen, blocked the BPA-induced depression of reflexes, whereas, 17β-estradiol, ER agonist, did not depress the reflexes even up to 10μM. Further, pretreatment with Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME) or hemoglobin (Hb) blocked the BPA-induced depression of spinal reflexes. Nitric oxide (NO) donor sodium-nitroprusside depressed the MSR and PSR in a concentration-dependent manner. The nitrite concentration of the cords exposed to BPA was 733μM/gm of tissue (three times the saline group). Pretreatment with tamoxifen/l-NAME/Hb blocked the BPA-induced increase of nitrite levels. The present observations indicate that BPA depressed spinal synaptic transmission through ERα-dependent NO-mediated mechanisms. The altered synaptic activity may implicate for neurobehavioral locomotor abnormalities after exposure to BPA.

First-time fathers’ making a difference during labour and birth

Bisphenol A (BPA), a toxic chemical from plastics, is known to produce locomotor abnormalities which may imply the alteration in synaptic activity at Ia-α motoneuron synapse also. However the effect of BPA on this synapse is not known. Therefore, this study was undertaken to examine the effect of BPA on reflexes originating at Ia-α motoneuron synapse in the spinal cord. The experiments were performed on isolated hemisected spinal cords from 4 to 6 d rats. Stimulation of a dorsal root evoked segmental monosynaptic (MSR) and polysynaptic (PSR) reflex potentials in the corresponding ventral root. Nitrite content (indicator of NO activity) of cords was estimated in the presence of BPA with/without antagonists. Superfusion of BPA (3–100 μM) depressed the reflexes in a concentration- and time-dependent manner. The depression was ∼20, ∼50 and ∼70% at 10, 30 and 100 μM of BPA, respectively. The 50% depression occurred around 15 min at 30 μM of BPA. Pretreatment with estrogen receptor (ERα) antagonist, tamoxifen, blocked the BPA-induced depression of reflexes, whereas, 17β-estradiol, ER agonist, did not depress the reflexes even up to 10 μM. Further, pretreatment with Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME) or hemoglobin (Hb) blocked the BPA-induced depression of spinal reflexes. Nitric oxide (NO) donor sodium-nitroprusside depressed the MSR and PSR in a concentration-dependent manner. The nitrite concentration of the cords exposed to BPA was 733 μM/gm of tissue (three times the saline group). Pretreatment with tamoxifen/l-NAME/Hb blocked the BPA-induced increase of nitrite levels. The present observations indicate that BPA depressed spinal synaptic transmission through ERα-dependent NO-mediated mechanisms. The altered synaptic activity may implicate for neurobehavioral locomotor abnormalities after exposure to BPA.

Evaluation of the sensitivity, responsiveness and reproducibility of primary rainbow trout hepatocyte vitellogenin expression as a screening assay for estrogen mimics

Vitellogenin (Vtg) induction in primary fish hepatocytes has been proposed as an in vitro screening assay for ER agonists and antagonists, but has not yet been used extensively as a high-throughput screening tool due to poor reproducibility, sensitivity and overall feasibility. The present work has evaluated the role of seasonal variation, normalization, optimal culture and assay conditions on the sensitivity, responsiveness and reproducibility of in vitro vtg gene mRNA and protein expression in rainbow trout (Oncorhynchus mykiss) primary hepatocytes using the xenoestrogen 17α-ethynylestradiol (EE2) as a test chemical. The results show that primary hepatocytes display a relatively high individual and seasonal variation in both Vtg mRNA and protein induction potential, although less variance was observed in assay sensitivity. Data normalization of assay response to maximum (3nM EE2) and minimum (DMSO) Vtg production dramatically reduced this variance and led to improved assay reproducibility. A time-dependent response was observed both for mRNA and protein expression, reaching maximum Vtg induction after 96h of exposure, although reproducible concentration response curves for both Vtg mRNA and protein could be obtained already after 48h. A need for chemical re-exposure of the hepatocytes was identified to be important for sustaining exposure concentrations in extended studies (>48h), whereas different plate formats (96, 24 or 6 wells) did not affect the bioassay performance. In conclusion, standardization of hepatocyte bioassay and test conditions as well as data-normalization procedures are proposed to be instrumental for more consistent and comparable results in future use of this in vitro assay.

Abstract 2710: Lead based development and evaluation of selective estrogen mimics in tamoxifen resistant breast cancer

Abstract Tamoxifen is the standard of care for many patients with ER+ breast cancer and works by antagonizing the actions of estrogen at ER. However it poses an increased risk of endometrial cancer and thrombotic events, and most significantly, 30-50% women develop resistance to tamoxifen therapy, underlining the need for superior therapeutic options. Paradoxically, prior to tamoxifen therapy, estradiol (E2) and the ER agonist, diethylstilbestrol, had been used in breast cancer therapy, though with serious side effects. Development of Selective Estrogen Mimics (SEMs) that cause regression of tamoxifen-resistant breast cancer, but without the side effects of E2, represents a rational therapeutic strategy. Novel SEMs were developed, which in vitro in 3D cultures and in vivo caused complete regression of tamoxifen-resistant xenografts, with characteristics similar to those of E2. These SEMs did not fuel growth of estrogen-dependent T47D xenografts and did not cause uterine growth. SEM mediation of classical ER-signaling was profiled in MCF7 and MDA-MB231:β41 cells. A tamoxifen-resistant cell line, MCF-7: 5C, was used to assay induction of cell death by both E2 and the SEMs and to probe the mechanism of action. Several SEMs were observed to act as partial agonists at ERα , suggesting ideal characteristics as therapeutics. Structure-activity relationships were explored, suggesting that partial ERα agonist have the capacity to cause regression of tamoxifen-resistant tumors, without adverse effects associated with estrogenic actions in normal gynecological tissues of the breast and uterus, which might contribute to carcinogenesis. The ER-mediated agonist/antagonist activity, regression efficacy, and pharmacokinetic profiles of these SEMs were examined to obtain lead compounds that are effective with minimal estrogenic side effects. In MCF-7:5C cells, SEM-induced cell death was mediated by activation of classical nuclear ERα signaling, via induction of apoptosis. This research paves the way for use of SEMs with partial agonist activity at ER for use in tamoxifen-resistant breast cancer with enhanced safety profiles. Citation Format: Hitisha Patel, Rui Xiong, Jiong Zhao, Mary Ellen Molloy, Debra Tonetti, Gregory R.J Thatcher. Lead based development and evaluation of selective estrogen mimics in tamoxifen resistant breast cancer. [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 2710. doi:10.1158/1538-7445.AM2014-2710

A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals.

The fish short-term reproduction assay (FSTRA) is a key component of the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), which uses a weight-of-evidence analysis based on data from several assays to identify the potential for chemicals to act as agonists or antagonists of the estrogen or androgen receptors (ER and AR), or inhibitors of steroidogenic enzymes. The FSTRA considers a variety of mechanistic and apical responses in 21-d exposures with the fathead minnow (Pimephales promelas), including plasma steroid and vitellogenin (VTG; egg yolk protein) concentrations, secondary sex characteristics, gonad size and histopathology, and egg production. Although the FSTRA initially was described several years ago, recent data generation associated with implementation of the EDSP highlighted the need for more formal guidance regarding evaluation of information from the assay. The authors describe a framework for interpretation of FSTRA data relative to perturbation of endocrine pathways of concern to the EDSP. The framework considers end points individually and as suites of physiologically related responses relative to pathway identification. Sometimes changes in single end points can be highly diagnostic (e.g., induction of VTG in males by ER agonists, production of male secondary sex characteristics in females by AR agonists); in other instances, however, multiple, related end points are needed to reliably assess pathway perturbation (e.g., AR antagonism, steroid synthesis inhibition). In addition to describing an interpretive framework, the authors demonstrate its practical utility using publicly available FSTRA data for a wide range of known and hypothesized endocrine-disrupting chemicals. Environ Toxicol Chem 2014;33:2529-2540. Published 2014 Wiley Periodicals Inc., on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Identification of coregulators influenced by estrogen receptor subtype specific binding of the ER antagonists 4-hydroxytamoxifen and fulvestrant

The aim of the present study was to investigate modulation of the interaction of ERα and ERβ with coregulators in the ligand dependent responses induced by the ER antagonistic compounds 4OHT and fulvestrant. Comparison with the modulation index (MI) profiles for the ER agonist estradiol (E2) will elucidate whether differences in the (ant)agonist dependent interaction of ERα and ERβ with coregulators expressed in MI profiles contribute to the differences in (ant)agonist responses.To this end, the selected ER antagonistic compounds were first characterized for intrinsic relative potency and efficacy towards ERα and ERβ using ER selective U2OS reporter gene assays, and subsequently tested for ligand dependent modulation of the interaction of ERα and ERβ with coregulators using the MARCoNI assay. Results obtained indicate a preference of 4OHT to antagonize ERβ and find fulvestrant to be less ER specific. MARCoNI assay responses reveal that ERα and ERβ mediated interaction with coregulators expressed in MI profiles are similar for 4OHT and fulvestrant and generally opposite to the MI profile of the ER agonist E2. Hierarchical clustering based on the MI profiles appeared able to clearly discriminate the two compounds with ER antagonistic properties from the ER agonist E2. Taken together the data reveal that modulation of the interaction of ERs with coregulators discriminates ER agonists from antagonists but does not discriminate between the less specific ER antagonist fulvestrant and the preferential ERβ antagonistic compound 4OHT. It is concluded that differences in modulation of the interaction of ERα and ERβ with coregulators contribute to the differences in ligand dependent responses induced by ER agonists and ER antagonists but the importance of the subtle differences in modulation of the interaction of ERs with coregulators between the ER antagonistic compounds 4OHT and fulvestrant for the ultimate biological effect remains to be established.

Cell proliferation and modulation of interaction of estrogen receptors with coregulators induced by ERα and ERβ agonists

The aim of the present study was to investigate modulation of the interaction of the ERα and ERβ with coregulators in the ligand responses induced by estrogenic compounds. To this end, selective ERα and ERβ agonists were characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ERα and ERβ mediated response in ER selective reporter gene assays, and subsequently tested for induction of cell proliferation in T47D-ERβ cells with variable ERα/ERβ ratio, and finally for ligand dependent modulation of the interaction of ERα and ERβ with coregulators using the MARCoNI assay, with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ERα/ERβ ratio and receptor selectivity of the compounds tested on induction of cell proliferation. ERα agonists activate cell proliferation whereas ERβ suppresses ERα mediated cell proliferation. The responses in the MARCoNI assay reveal that upon ERα or ERβ activation by a specific agonist, the modulation of the interaction of the ERs with coregulators is very similar indicating only a limited number of differences upon ERα or ERβ activation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists were more pronounced. Based on ligand dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay was shown to be able to classify the ER agonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER selective reporter gene or proliferation assays.It is concluded that the ultimate effect of the model compounds on proliferation of estrogen responsive cells depends on the intrinsic relative potency of the agonist towards ERα and ERβ and the cellular ERα/ERβ ratio whereas differences in the modulation of the interaction of the ERα and ERβ with coregulators contribute to the ligand dependent responses induced by estrogenic compounds.

Age- and sex-dependent hormonal modulation of the expression of vitamin D receptor and 25-hydroxyvitamin D 1α hydroxylase in human bone cells

Background: Human cultured osteoblasts from pre-Ob, post-Ob or m-Ob express different mRNAs and respond to different hormones. Aims: To test expression and hormonal modulation of VDR and 1OHase and 1,25D production in hObs. Methods: hObs obtained from bone explants were prepared, treated and analyzed as before. Results: (i) VDR and 1OHase were expressed in all hObs. (ii) 1,25D was produced similarly. (iii) Treatment with E2, DPN (ER agonist), but not PPT (ER agonist) increased VDR. (iv) These hormones increased 1OHase. (v) Vitamin D analog JKF and PTH 1-84 increased similarly mRNAs. (vi) Treatment with E2, DPN and PPT increased 1,25D. (vii) JKF and PTH increased similarly 1,25D. (viii) DNA synthesis and CK were stimulated by all hormones in hObs. Conclusions: Hormonal modulation of VDR and 1OHase and 1,25D production is important for bone physiology but their correlation, activity and bone physiology is not yet clear.

Diosgenin does not express estrogenic activity: a uterotrophic assay

This study assessed the effects of diosgenin on estrogenic activity using a uterotrophic assay. Immature female rats received diosgenin orally at doses of 200, 100, or 20 mg/kg body mass; and 17α ethynylestradiol at doses of 1 or 0.3 μg/kg, daily, for 3 consecutive days from day 19 to day 21. Controls were distributed among 2 groups: an intact control group and a vehicle control group. Animals were sacrificed 24 h after the last application of diosgenin, estradiol, or vehicle (22nd day of life). Uterine wet weight, stereological and histomorphometrical changes, immunohistochemical expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and the expression of lactoferrin (LF) were examined. Diosgenin did not affect the uterine wet weight, epithelium height, volume densities of endometrium, endometrial epithelia, number of endometrial glands, or histological appearance of vaginal epithelia. ERα, PR, and LF immunostaining intensity were not altered in the animals that received diosgenin. High-potency reference ER agonist 17α-ethynylestradiol induced a significant increase in all of the measured parameters, and as expected, decreased ERα immunostaining intensity. Based on these data, it can be concluded that diosgenin, at doses of 20-200 mg/kg, did not act as an estrogen agonist in the immature rat uterotrophic assay.

Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer.

Rapid Oestrogenic Regulation of Social and Nonsocial Learning

Much research on oestrogens has focused on their long-term action, exerting behavioural effects within hours to days through gene transcription. Oestrogens also affect behaviour on a much shorter time scale. These rapid effects are assumed to occur through cell signalling and can elicit a behavioural effect as early as 15min after treatment. These effects on behaviour have primarily been explored through the action of oestradiol at three well-known oestrogen receptors (ERs): ERα, ERβ and the more recently described G protein-coupled ER1 (GPER1). The rapid effects of oestradiol and ER agonists have been tested on both social and nonsocial learning paradigms. Social learning refers to a paradigm in which an animal acquires information and modifies its behaviour based on observation of another animal, commonly studied using the social transmission of food preferences paradigm. When administered shortly before testing, oestradiol rapidly improves social learning on this task, although no ER agonist has definitive, comparable improving effects. Some evidence points to GPER1, whereas ERα impairs, and ERβ activation has no effect on social learning. Conversely, ERα and GPER1 play a larger role than ERβ in the rapid improving effect of oestrogens on nonsocial learning, including social and object recognition. In addition, when administered immediately post-acquisition, oestrogens also rapidly improve memory consolidation in a variety of learning paradigms: object recognition, object placement, inhibitory avoidance and the Morris water maze, indicating that oestradiol affects the consolidation of multiple types of memory. Evidence suggests that these improvements are the result of oestrogens acting in the dorsal hippocampus where selective activation of all three ERs shows rapid improving effects on spatial learning comparable to oestradiol. However, the hippocampus is not necessary for rapid oestradiol improvements on social recognition. Although acute treatment with oestradiol enhances learning and memory on various social and nonsocial learning paradigms, the specific ERs play different roles in each type of learning. Future research should aim to further determine the roles of ERs with respect to the enhancing effects of oestradiol on learning and memory, and also determine where in the brain oestradiol acts to affect social and nonsocial learning.

Estrogen, vascular estrogen receptor and hormone therapy in postmenopausal vascular disease

Cardiovascular disease (CVD) is less common in premenopausal women than men of the same age or postmenopausal women, suggesting vascular benefits of estrogen. Estrogen activates estrogen receptors ERα, ERβ and GPR30 in endothelium and vascular smooth muscle (VSM), which trigger downstream signaling pathways and lead to genomic and non-genomic vascular effects such as vasodilation, decreased VSM contraction and growth and reduced vascular remodeling. However, randomized clinical trials (RCTs), such as the Women's Health Initiative (WHI) and Heart and Estrogen/progestin Replacement Study (HERS), have shown little vascular benefits and even adverse events with menopausal hormone therapy (MHT), likely due to factors related to the MHT used, ER profile, and RCT design. Some MHT forms, dose, combinations or route of administration may have inadequate vascular effects. Age-related changes in ER amount, distribution, integrity and post-ER signaling could alter the vascular response to MHT. The subject's age, preexisting CVD, and hormone environment could also reduce the effects of MHT. Further evaluation of natural and synthetic estrogens, phytoestrogens, and selective estrogen-receptor modulators (SERMs), and the design of appropriate MHT combinations, dose, route and ‘timing’ could improve the effectiveness of conventional MHT and provide alternative therapies in the peri-menopausal period. Targeting ER using specific ER agonists, localized MHT delivery, and activation of specific post-ER signaling pathways could counter age-related changes in ER. Examination of the hormone environment and conditions associated with hormone imbalance such as polycystic ovary syndrome may reveal the causes of abnormal hormone-receptor interactions. Consideration of these factors in new RCTs such as the Kronos Early Estrogen Prevention Study (KEEPS) could enhance the vascular benefits of estrogen in postmenopausal CVD.

Discovery of natural estrogen receptor modulators with structure-based virtual screening

Eleven compounds were identified as estrogen receptor modulators from an in-house natural product database (NPD) by structure-based virtual screening for ERα and ERβ. Among them, 3 compounds were confirmed as ER agonists and 8 compounds were confirmed as ER antagonists by yeast two-hybrid (Y2H) assay, with EC50 values ranging from several micromolar to 100 micromolar. In this study, a novel series of cycloartane triterpenoids isolated from Schisandra glaucescens Diels was found to have ER antagonistic effect, the most potent antagonist of which exhibited activity with EC50 value of 2.55 and 4.68μM for ERα and ERβ, respectively. Moreover, the types of modulation and subtype selectivity were also investigated through molecular docking simulation.

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To further develop an integrated in vitro testing strategy for replacement of in vivo tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulators with estrogen receptor α was assessed using a PamChip® plate. The relative estrogenic potencies determined, based on ERα binding to coregulator peptides in the presence of ligands on the PamChip® plate, were compared to the relative estrogenic potencies as determined in the in vivo uterotrophic assay. The results show that the estrogenic potencies predicted by the 57 coactivators on the peptide microarray for 18 compounds that display a clear E2 dose-dependent response (goodness of fit of a logistic dose-response model of 0.90 or higher) correlated very well with their in vivo potencies in the uterotrophic assay, i.e., coefficient of determination values for 30 coactivators higher than or equal to 0.85. Moreover, this coregulator binding assay is able to distinguish ER agonists from ER antagonists: profiles of selective estrogen receptor modulators, such as tamoxifen, were distinct from those of pure ER agonists, such as dienestrol. Combination of this coregulator binding assay with other types of in vitro assays, e.g., reporter gene assays and the H295R steroidogenesis assay, will frame an in vitro test panel for screening and prioritization of chemicals, thereby contributing to the reduction and ultimately the replacement of animal testing for (anti-)estrogenic effects.

Small-animal PET of steroid hormone receptors predicts tumor response to endocrine therapy using a preclinical model of breast cancer.

Estrogen receptor-α (ERα) and progesterone receptor (PR) are expressed in most human breast cancers and are important predictive factors for directing therapy. Because of de novo and acquired resistance to endocrine therapy, there remains a need to identify which ERα-positive (ERα(+))/PR-positive (PR(+)) tumors are most likely to respond. The purpose of this study was to use estrogen- and progestin-based radiopharmaceuticals to image ERα and PR in mouse mammary tumors at baseline and after hormonal therapy and to determine whether changes in these imaging biomarkers can serve as an early predictive indicator of therapeutic response. Mammary adenocarcinomas that spontaneously develop in aged female mice deficient in signal transducer and activator of transcription-1 (STAT1) were used. Imaging of ERα and PR in primary tumor-bearing mice and mice implanted with mammary cell lines (SSM1, SSM2, and SSM3) derived from primary STAT1-deficient (STAT1(-/-)) tumors was performed. Hormonal treatments consisted of estradiol, an ER agonist; letrozole, an aromatase inhibitor; and fulvestrant, a pure ER antagonist. Small-animal PET/CT was performed using (18)F-fluoroestradiol ((18)F-FES) for ER, (18)F-fluoro furanyl norprogesterone ((18)F-FFNP) for PR, and (18)F-FDG for glucose uptake. Tracer uptake in the tumor was quantified and compared with receptor concentration determined by in vitro assays of resected tumors. Primary STAT1(-/-) mammary tumors and implanted SSM2 and SSM3 tumors showed high (18)F-FES and (18)F-FFNP uptake and were confirmed to be ERα(+)/PR(+). Classic estrogen-induced regulation of the progesterone receptor gene was demonstrated by increased (18)F-FFNP uptake of estradiol-treated SSM3 tumors. Treatment with fulvestrant decreased (18)F-FFNP, (18)F-FES, and (18)F-FDG uptake and inhibited growth of SSM3 tumors but decreased only (18)F-FES uptake in SSM2 tumors, with no effect on growth, despite both tumors being ERα(+)/PR(+). Decreased (18)F-FFNP uptake by SSM3 tumors occurred early after initiation of treatment, before measurable tumor growth inhibition. Using small-animal PET, a profile was identified that distinguished fulvestrant-sensitive from fulvestrant-resistant ERα(+)/PR(+) tumors before changes in tumor size. This work demonstrates that imaging baseline tumoral (18)F-FES uptake and initial changes in (18)F-FFNP uptake in a noninvasive manner is a potentially useful strategy to identify responders and nonresponders to endocrine therapy at an early stage.

Estrogenic activity of B-fluorinated o-carborane-1,2-bisphenol synthesized via SNAr reaction

We previously identified o-carborane bisphenol BE360 (4) as a selective estrogen receptor modulator (SERM), which ameliorated bone loss without inducing estrogenic action in uterus of OVX and ORX mice. Here, we synthesized a fluorinated derivative, B-fluorinated o-carborane bisphenol BE310 (5) by means of SNAr reaction. Compound 5 was a partial ER agonist, like 4, with little change of ERα and ERβ selectivity as compared with 4. However, its agonistic activity was 40 times weaker than that of 4. Thus, 5 is a novel SERM candidate with potential for reduced estrogenic side effects, and in vivo evaluation as an anti-osteoporosis agent seems warranted.

Anatomically‐Specific Actions of Oestrogen Receptor in the Developing Female Rat Brain: Effects of Oestradiol and Selective Oestrogen Receptor Modulators on Progestin Receptor Expression

Steroid hormones largely exert their actions by activating nuclear receptors, which, as transcription factors, powerfully influence fundamental processes of neural development. Often, steroid receptor action demonstrates remarkable specificity under different developmental, anatomical or hormonal conditions. Yet, the mechanisms underlying such specificity are poorly understood. The present study examined the anatomically-specific regulation of progestin receptor (PR) expression by oestrogen receptor (ER) activation in the ventromedial nucleus (VMN) of the hypothalamus and the medial preoptic nucleus (MPN) of the neonatal female rat brain, using the selective oestrogen receptor modulators (SERMs), tamoxifen and ICI 182780 (ICI), in the presence or absence of oestradiol benzoate (EB) treatment. The results demonstrate that PR immunoreactivity (PR-ir) in the neonatal female MPN was significantly increased by EB and this increase was abolished by either tamoxifen or ICI treatment. In contrast, within the VMN of the same animals, EB had no effect on PR-ir and the SERMs only modestly decreased PR-ir. Interestingly, ICI acted as a true antagonist regardless of EB treatment, whereas tamoxifen acted as an ER agonist in the absence of EB in the MPN, but not the VMN, representing one of the first in vivo demonstrations of tissue-specific and oestradiol-independent effects of tamoxifen on ER activation. The present results indicate that PR expression is highly dependent on oestradiol and its receptor in the MPN, although it is independent of both oestradiol and ER activation within the neonatal VMN. These findings demonstrate the anatomically-specific actions of oestradiol and its receptor to induce PR in two brain regions controlling different aspects of female reproductive behaviours in adulthood.

Estrogens regulate neuroinflammatory genes via estrogen receptors α and β in the frontal cortex of middle-aged female rats

BackgroundEstrogens exert anti-inflammatory and neuroprotective effects in the brain mainly via estrogen receptors α (ERα) and β (ERβ). These receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors. This study was aimed at the elucidation of the effects of ERα and ERβ agonists on the expression of neuroinflammatory genes in the frontal cortex of aging female rats.MethodsTo identify estrogen-responsive immunity/inflammation genes, we treated middle-aged, ovariectomized rats with 17β-estradiol (E2), ERα agonist 16α-lactone-estradiol (16α-LE2) and ERβ agonist diarylpropionitrile (DPN), or vehicle by Alzet minipump delivery for 29 days. Then we compared the transcriptomes of the frontal cortex of estrogen-deprived versus ER agonist-treated animals using Affymetrix Rat230 2.0 expression arrays and TaqMan-based quantitative real-time PCR. Microarray and PCR data were evaluated by using Bioconductor packages and the RealTime StatMiner software, respectively.ResultsMicroarray analysis revealed the transcriptional regulation of 21 immunity/inflammation genes by 16α-LE2. The subsequent comparative real-time PCR study analyzed the isotype specific effects of ER agonists on neuroinflammatory genes of primarily glial origin. E2 regulated the expression of sixteen genes, including down-regulation of complement C3 and C4b, Ccl2, Tgfb1, macrophage expressed gene Mpeg1, RT1-Aw2, Cx3cr1, Fcgr2b, Cd11b, Tlr4 and Tlr9, and up-regulation of defensin Np4 and RatNP-3b, IgG-2a, Il6 and ER gene Esr1. Similar to E2, both 16α-LE2 and DPN evoked up-regulation of defensins, IgG-2a and Il6, and down-regulation of C3 and its receptor Cd11b, Ccl2, RT1-Aw2 and Fcgr2b.ConclusionsThese findings provide evidence that E2, 16α-LE2 and DPN modulate the expression of neuroinflammatory genes in the frontal cortex of middle-aged female rats via both ERα and ERβ. We propose that ERβ is a promising target to suppress regulatory functions of glial cells in the E2-deprived female brain and in various neuroinflammatory diseases.