The Effects of Estradiol, Androgens and the Selective Estrogen Modulators: Ospemifene, Raloxifene and Tamoxifen on Explant Cultures of Human Breast Tissue

The impact of menopausal hormone therapy (MHT) on increasing the risk for breast cancer (BC) remains controversial. To understand MHT-elicited cellular breast effects and the potential risks, included with using this therapy, a further investigation into this controversy is the subject of this thesis. In this thesis, to study the effects of estrogen, progestin, androgens and selective estrogen receptor modulators (SERMs), a modified tissue explant culture system was used. The different types of human breast tissues (HBTs) used in this study were normal HBTs, obtained from reduction mammoplasties of premenopausal women (prem-HBTs) or postmenopausal (postm-HBTs) women and peritumoral HBTs (peritum-HBTs) which were obtained from surgeries on postmenopausal BC patients. The explants were cultured up to three weeks in the presence or absence of estradiol (E2), medroxyprogesterone acetate (MPA), testosterone (T), dihydrotestosterone (DHT) and SERMs - ospemifene (OSP), raloxifene (RAL) and tamoxifen (TAM). The cultured HBTs maintained morphological integrity and responded to hormonal treatment in vitro. E2, MPA or E2/MPA increased proliferative activity and was associated with increased cyclin-D1 and caused changes in the cell cycle inhibitors p21 and p27, whereas the androgens T and DHT inhibited proliferation and increased apoptosis in HBT epithelia and opposed E2-stimulated proliferation and cell survival. The postm-HBTs were more sensitive to E2 than prem-HBTs. The effects of OSP, RAL and TAM on HBT epithelium were antiproliferative. E2, androgens and SERMs were associated with marked changes in the proportions of epithelial cells expressing steroid hormone receptors: E2 increased ERα expressing cells and decreased androgen receptor (AR) positive cells, whereas T and DHT had opposite effects. The OSP, RAL and TAM, also decreased a proportion of ERα positive cells in HBT epithelium. At 100 nM, these compounds maintained the relative number of AR positive cells, present at control level, which may partly explain proliferative inhibition. In conclusion, the proliferative activity of E2, in the epithelium of postm-HBTs, is opposed by T and DHT, which suggests that the inclusion of androgens in MHT may decrease the risk for developing BC.

Roles of estrogen and progesterone in modulating renal nerve function in the rat kidney

The maintenance of extracellular Na+ and Cl- concentrations in mammals depends, at least in part, on renal function. It has been shown that neural and endocrine mechanisms regulate extracellular fluid volume and transport of electrolytes along nephrons. Studies of sex hormones and renal nerves suggested that sex hormones modulate renal function, although this relationship is not well understood in the kidney. To better understand the role of these hormones on the effects that renal nerves have on Na+ and Cl- reabsorption, we studied the effects of renal denervation and oophorectomy in female rats. Oophorectomized (OVX) rats received 17β-estradiol benzoate (OVE, 2.0 mg·kg-1·day-1, sc) and progesterone (OVP, 1.7 mg·kg-1·day-1,sc). We assessed Na+ and Cl-fractional excretion (FENa+ and FECl-, respectively) and renal and plasma catecholamine release concentrations. FENa+, FECl-, water intake, urinary flow, and renal and plasma catecholamine release levels increased in OVX vs control rats. These effects were reversed by 17β-estradiol benzoate but not by progesterone. Renal denervation did not alter FENa+, FECl-, water intake, or urinary flow values vs controls. However, the renal catecholamine release level was decreased in the OVP (236.6±36.1 ng/g) and denervated rat groups (D: 102.1±15.7; ODE: 108.7±23.2; ODP: 101.1±22.1 ng/g). Furthermore, combining OVX + D (OD: 111.9±25.4) decreased renal catecholamine release levels compared to either treatment alone. OVE normalized and OVP reduced renal catecholamine release levels, and the effects on plasma catecholamine release levels were reversed by ODE and ODP replacement in OD. These data suggest that progesterone may influence catecholamine release levels by renal innervation and that there are complex interactions among renal nerves, estrogen, and progesterone in the modulation of renal function.

Growth factor activation of ErbB2/ErbB3 signaling pathways regulate the activity of Estrogen Receptors (ER)

La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance. Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles.

Osteocalcin immunolabeling during the alveolar healing process in ovariectomized rats treated with estrogen or raloxifene

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunohistochemical detection of estrogen receptor beta in alveolar bone cells of estradiol-treated female rats: possible direct action of estrogen on osteoclast life span

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of estradiol and triiodothyronine on breast cancer cell lines proliferation and expression of estrogen and thyroid hormone receptors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructural and proliferative features of the ventral lobe of the prostate in non-obese diabetic mice (NOD) following androgen and estrogen replacement associated to insulin therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Exposure of young rats to high estrogen doses leads to degeneration of elongated spermatids

Single high doses of estrogen (35 mg/kg body weight) were administered to young rats aiming to exacerbate its effects on germ cell populations. The short-term (1 week) and medium-term (7 weeks) consequences of this estrogenic treatment (ET) on the testis were evaluated using light and electron microscopies, quantitative methods and TUNEL reaction. Short-term ET led to 50% atrophy of the testis, however, in the medium term the gonado-somatic index was recovered. No histopathological alterations were found at seminiferous epithelium except for short-term severe degeneration of elongated spermatids (EL) and low frequency of these cells in both time intervals. Two morphologically distinct patterns of degeneration were observed: (1) clusters of EL which were TUNEL-negative and exhibited bizarre appearance and nuclear fragmentation, (2) isolated apoptotic EL within the cytoplasm of Sertoli cells (SC). Both degenerative phenomena were more frequent in stages III - VIII of seminiferous cycle, whereas at stages I and II only coiling of flagellum was observed. One week after ET, small amounts of EL were detected in stages IX - XII, suggesting spermiation failure. Signs of functional SC damage such as an accumulation of myelin-like inclusions in their cytoplasm were observed in the short but not medium-term. However, the apoptotic rates still remained five times higher and the number of elongated spermatids was three-fold lower. Our data indicate that exposure to a high dose of estrogen around puberty has stage-specific effects on the testis and causes massive degeneration of elongated spermatids. (c) 2007 Elsevier Ltd. All rights reserved.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Intraepithelial alterations in the Guinea pig lateral prostate at different ages after estradiol treatment

The prostate is an accessory gland of the mammal reproductive system with great volume and high functional importance. Many works infer that, in addition to the androgenic ones, the estrogen can be associated with benign prostatic hyperplasia and prostatic cancer, but no conclusive evidence exists on the role of estrogen in normal prostatic and neoplastic tissue. The objective of this work was to evaluate the effects of chronic administration of estradiol benzoate on the lateral prostate of guinea pigs in the pre-pubescent, pubescent, post-pubescent and adult phases, with emphasis on the modifications provoked by this hormone on the glandular epithelium. The analyses of the estradiol-treated and control groups were investigated using histological procedures and transmission electron microscopy. The histopathological analysis of the lateral prostate in the treated group revealed areas where epithelial dysplasia was observed, assuming at some places a pattern of epithelial stratification characteristic of prostatic intraepithelial neoplasia. After ultrastructural analysis, the following were observed: enlargement of the internal membranes, heterogeneity in the cellular types, hypertrophy of the basal cells and apparent decrease of cytoplasmic organelles in some cells of the prostatic intraepithelial neoplasia. Still, a loss of cellular polarity was observed, along with nuclei of various forms, sizes and heights - as well as irregular chromatin distribution patterns. Such alterations were found mainly in pubescent, post-pubescent and adult animals subject to the chronic administration of estradiol. These findings reinforce the already existent data in understanding the role of estrogen in the etiology of prostatic diseases.

Efficacy of hormone therapy with and without methyltestosterone augmentation of venlafaxine in the treatment of postmenopausal depression: A double-blind controlled pilot study

Objective: This study evaluated the augmentation of venlafaxine with hormone therapy in the treatment of postmenopausal depression. The hormones evaluated were estrogen (0.625 mg) in combination with medroxyprogesterone acetate (2.5 mg) and methyltestosterone (2.5 mg). Design: Seventy-two menopausal women (mean age: 53.6 ± 4.27 years) diagnosed with depression (Montgomery-Åsberg Depression Rating Scale [MADRS] scores ≥ 20) were treated with venlafaxine and one of the following hormone therapy combinations, in a double-blind regimen: estrogen + medroxyprogesterone + methyltestosterone (group 1, n = 20); estrogen + medroxyprogesterone acetate (group 2, n = 20); methyltestosterone only (group 3, n = 16); and no hormone therapy (group 4, n = 16). Study duration was 24 weeks. Primary efficacy outcome was remission according to the MADRS, whereas secondary efficacy measures included the Clinical Global Impression (CGI), Blatt-Kupperman Index, and Women's Health Questionnaire (WHQ). Results: Forty-eight patients completed the study. All groups showed significant improvement from baseline. Group 3 demonstrated significant improvement on the MADRS compared with placebo (group 4) at weeks 20 (P = 0.048) and 24 (P = 0.030); effect size 8.04 (0.83; 15.26) (P = 0.029), but also had the highest dropout rate. Groups 1 and 3 had significant CGI improvement rates compared with placebo: 42.23% (P = 0.012) and 44.45% (P = 0.08), respectively. There were no differences in the WHQ or BKI scores among the groups. Conclusions: Methyltestosterone 2.5 mg had the highest effect size compared with placebo, but the high dropout rate prevented its efficacy from being determined. Estrogen plus medroxyprogesterone, combined with methyltestosterone or otherwise, demonstrated a trend toward increased efficacy of venlafaxine. Further larger-scale clinical trials are needed to elucidate the findings of this pilot study. © 2006 by The North American Menopause Society.

Adjunctive antimicrobial photodynamic treatment of experimentally induced periodontitis in rats with ovariectomy

Background: The aim of this study is to compare antimicrobial photodynamic therapy (aPDT) as an adjunctive therapy to scaling and root planing (SRP) for the treatment of experimentally induced periodontitis in rats with ovariectomy (OVX) that are or are not treated with estrogen replacement. Methods: A total of 270 female rats were divided into three groups: 1) normal rats; 2) rats with OVX; and 3) rats with OVX with estrogen replacement. Periodontal disease was induced through the introduction of a cotton thread around the mandibular left first molar. After 7 days, the ligature was removed, and the rats were randomly divided into the following treatment groups: 1) SRP plus saline solution; 2) SRP plus low-level laser therapy (LLLT); and 3) SRP plus toluidine blue O irrigation followed by LLLT. Ten rats from each group were euthanized at days 7, 15, and 30 after dental treatment. Bone loss (BL) in the furcation region was evaluated using histometric and immunohistochemical analyses. Results: aPDT treatment resulted in reduced BL compared with SRP treatment at all time points. Additionally, rats treated with aPDT exhibited reduced numbers of tartrate-resistant acid-phosphatase-positive cells and more proliferating cell nuclear antigen-positive cells in all treatment groups regardless of estrogen status. Whereas rats treated with aPDT showed weak immunoreactivity to the receptor activator of nuclear factor-k B ligand at day 7 post-treatment, strong osteoprotegerin immunoreactivity was observed at day 15 post-treatment. Conclusion: aPDT is an effective adjunctive therapy for the treatment of periodontitis in rats with OVX that are or are not given estrogen replacement therapy.

Estrogen-Responsive Genes Overlap with Triiodothyronine-Responsive Genes in a Breast Carcinoma Cell Line

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of melatonin treatment in primary culture of canine mammary tumors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene expression of estrogen receptor-alpha in orbital fibroblasts in Graves’ ophthalmopathy

Graves’ ophthalmopathy (GO) is one of the most severe clinical manifestations of Graves’ disease (GD), and its treatment might involve high-dose glucocorticoid therapy. The higher incidence of GO among females, and the reported association between polymorphisms of estrogen receptor (ER) and GD susceptibility have led us to question the role of estrogen and its receptor in GO pathogenesis. We, thus, assessed estrogen receptor-alpha (ERA) gene expression in cultures of orbital fibroblasts from a patient with GO before (controls) and after treatment with 10 nM and 100 nM dexamethasone (DEX). Orbital fibroblasts showed ERA gene expression. In the cells treated with 10 nM and 100 nM DEX, ERA gene expression was, respectively, 85% higher and 74% lower, than in the control group. We concluded that ERA gene expression is found in the orbital fibroblasts of patient with GO, which may be affected by glucocorticoids in a dose-related manner. Arch Endocrinol Metab. 2015;59(3):273-6