Local Estrogen Formation Research Articles

Cytotoxic effect of 13α-estrane derivatives on breast, endometrial and ovarian cancer cell lines

Hormone-dependent cancers such as breast, uterine, and ovarian cancers account for more than 35% of all cancers in women. Worldwide, these cancers occur in more than 2.7 million women/year and account for 22% of cancer-related deaths/year. The generally accepted mechanism for the pathophysiology of estrogen-dependent cancers is estrogen receptor-mediated cell proliferation associated with an increased number of mutations. Therefore, drugs that can interfere with either local estrogen formation or estrogen action via estrogen receptors are needed. Estrane derivatives that have low or minimal estrogenic activity can affect both pathways. In this study, we investigated the effect of 36 different estrane derivatives on the proliferation of eight breast, endometrial, and ovarian cancer cell lines and the corresponding three control cell lines. Estrane derivatives 3 and 4_2Cl showed a stronger effect on the endometrial cancer cell lines KLE and Ishikawa, respectively, compared with the control cell line HIEEC, with IC50 values of 32.6 microM and 17.9 microM, respectively. Estrane derivative 4_2Cl was most active in the ovarian cancer cell line COV362 compared to the control cell line HIO80 with an IC50 value of 3.6 microM. In addition, estrane derivative 2_4I showed a strong antiproliferative effect on endometrial and ovarian cancer cell lines, while the effect on the control cell line was slight or absent. The addition of halogen at carbon 2 and/or 4 in estrane derivatives 1 and 2 increased the selectivity for endometrial cancer cells. Overall, these results suggest that single estrane derivatives are efficient cytotoxic agents for endometrial and ovarian cancer cell lines, and thus potential lead compounds for drug development.

Targeting the formation of estrogens for treatment of hormone dependent diseases-current status.

Local formation and action of estrogens have crucial roles in hormone dependent cancers and benign diseases like endometriosis. Drugs that are currently used for the treatment of these diseases act at the receptor and at the pre-receptor levels, targeting the local formation of estrogens. Since 1980s the local formation of estrogens has been targeted by inhibitors of aromatase that catalyses their formation from androgens. Steroidal and non-steroidal inhibitors have successfully been used to treat postmenopausal breast cancer and have also been evaluated in clinical studies in patients with endometrial, ovarian cancers and endometriosis. Over the past decade also inhibitors of sulfatase that catalyses the hydrolysis of inactive estrogen-sulfates entered clinical trials for treatment of breast, endometrial cancers and endometriosis, with clinical effects observed primarily in breast cancer. More recently, inhibitors of 17beta-hydroxysteroid dehydrogenase 1, an enzyme responsible for formation of the most potent estrogen, estradiol, have shown promising results in preclinical studies and have already entered clinical evaluation for endometriosis. This review aims to provide an overview of the current status of the use of hormonal drugs for the major hormone-dependent diseases. Further, it aims to explain the mechanisms behind the -sometimes- observed weak effects and low therapeutic efficacy of these drugs and the possibilities and the advantages of combined treatments targeting several enzymes in the local estrogen formation, or drugs acting with different therapeutic mechanisms.

Local estrogen formation and its regulation in endometriosis.

BackgroundIt has been well established that endometriosis is an estrogen‐dependent disease. Although the exact pathogenesis of the disease is still unclear, it is known to be characterized by estrogen‐dependent growth and maintenance of the ectopic endometrium and increased local estrogen production.MethodsThe authors reviewed studies on local estrogen production and estrogen activities mediated by estrogen receptors in endometriotic tissues.Main findingsAberrant expression of several enzymes in local endometriotic lesions contributed to the production and metabolism of estrogens. Aromatase was one of the key therapeutic targets for the regulation of local estrogen formation. Our findings suggest that PGC‐1a, a transcriptional coactivator‐modulating steroid hormone, regulates aromatase expression and activity. Estrogen activities mediated by different types of estrogen receptors abnormally elevated in local tissues could also be involved in the development of endometriosis. The authors demonstrated that the isoflavone aglycone, a partial agonist of the estrogen receptor, suppressed the formation of endometriotic lesions.ConclusionsLocal estrogen production and estrogen activity mediated by estrogen receptors are important potential therapeutic targets for endometriosis.

STRUCTURAL FEATURES OF THE METABOLIC SYNDROME: CLINICAL AND EPIDEMIOLOGICAL ASPECTS. RELATIONSHIP WITH PROLIFERATIVE PROCESSES AND ENDOMETRIAL CANCER

This literature review is devoted to the relationship of the structural features of the metabolic syndrome in proliferative processes and endometrial cancer (EC), and the identification of the relationship between the individual components of the metabolic syndrome and the development of endometrial cancer. The metabolic syndrome is currently a global medical and social problem, which is due to the wide spread of this symptom complex in the population. Today, the main concept of this syndrome is the concept of a cluster of components associated with an increased risk of developing type 2 diabetes and cardiovascular diseases. It is known that an increase in the mass of adipose tissue above the norm by 20% or more leads to dysfunction of the hypothalamic-pituitary-ovarian system. At the same time, the risk of RE on the background of metabolic syndrome increases by 2-3 times. This probability also largely depends on the severity of proliferation in the endometrium, which is caused by the presence of dyshormonal and metabolic disorders. In addition, this is due to the aggravation of insulin resistance, an increase in the production of androgens by the ovaries, the formation of stable anovulation and, as a result, the progression of pathological changes in the endometrium. With the onset of peri - and postmenopausal endometrial hyperplasia in about 50% of cases progressing to malignant pathology. The role of the system of insulin-like growth factors, adipokines secreted from visceral adipocytes of free fatty acids, local estrogen formation and hyperandrogenization is also recognized in creating a predisposition to the EC formation. According to the results of the study of the functioning of the autocrine-paracrine system of adipose tissue, the risk of endometrial proliferative processes is higher at low levels of adiponectin and high levels of insulin. All this indicates the feasibility of monitoring the status of adipose tissue.

The Significance of the Sulfatase Pathway for Local Estrogen Formation in Endometrial Cancer.

Endometrial cancer (EC) is the most common estrogen-dependent gynecological malignancy in the developed World. To investigate the local formation of estradiol (E2), we first measured the concentrations of the steroid precursor androstenedione (A-dione) and the most potent estrogen, E2, and we evaluated the metabolism of A-dione, estrone-sulfate (E1-S), and estrone (E1) in cancerous and adjacent control endometrium. Furthermore, we studied expression of the key genes for estradiol formation via the aromatase and sulfatase pathways. A-dione and E2 were detected in cancerous and adjacent control endometrium. In cancerous endometrium, A-dione was metabolized to testosterone, and no E2 was formed. Both, E1-S and E1 were metabolized to E2, with increased levels of E2 seen in cancerous tissue. There was no significant difference in expression of the key genes of the aromatase (CYP19A1) and the sulfatase (STS, HSD17B1, HSD17B2) pathways in cancerous endometrium compared to adjacent control tissue. The mRNA levels of CYP19A1 and HSD17B1 were low, and HSD17B14, which promotes inactivation of E2, was significantly down-regulated in cancerous endometrium, especially in patients with lymphovascular invasion. At the protein level, there were no differences in the levels of STS and HSD17B2 between cancerous and adjacent control tissue by Western blotting, and immunohistochemistry revealed intense staining for STS and HSD17B2, and weak staining for SULT1E1 and HSD17B1 in cancerous tissue. Our data demonstrate that in cancerous endometrium, E2 is formed from E1-S via the sulfatase pathway, and not from A-dione via the aromatase pathway.

The Important Roles of Steroid Sulfatase and Sulfotransferases in Gynecological Diseases.

Gynecological diseases such as endometriosis, adenomyosis and uterine fibroids, and gynecological cancers including endometrial cancer and ovarian cancer, affect a large proportion of women. These diseases are estrogen dependent, and their progression often depends on local estrogen formation. In peripheral tissues, estrogens can be formed from the inactive precursors dehydroepiandrosterone sulfate and estrone sulfate. Sulfatase and sulfotransferases have pivotal roles in these processes, where sulfatase hydrolyzes estrone sulfate to estrone, and dehydroepiandrosterone sulfate to dehydroepiandrosterone, and sulfotransferases catalyze the reverse reactions. Further activation of estrone to the most potent estrogen, estradiol, is catalyzed by 17-ketosteroid reductases, while estradiol can also be formed from dehydroepiandrosterone by the sequential actions of 3β-hydroxysteroid dehydrogenase-Δ4-isomerase, aromatase, and 17-ketosteroid reductase. This review introduces the sulfatase and sulfotransferase enzymes, in terms of their structures and reaction mechanisms, and the regulation and different transcripts of their genes, together with the importance of their currently known single nucleotide polymorphisms. Data on expression of sulfatase and sulfotransferases in gynecological diseases are also reviewed. There are often unchanged mRNA and protein levels in diseased tissue, with higher sulfatase activities in cancerous endometrium, ovarian cancer cell lines, and adenomyosis. This can be indicative of a disturbed balance between the sulfatase and sulfotransferases enzymes, defining the potential for sulfatase as a drug target for treatment of gynecological diseases. Finally, clinical trials with sulfatase inhibitors are discussed, where two inhibitors have already concluded phase II trials, although so far with no convincing clinical outcomes for patients with endometrial cancer and endometriosis.

Abstract 1852: Merlin/NF2 is associated with elevated aromatase expression and estrogen formation in human liver tissues and liver cancer cells: An unifying model for hepatocellular carcinoma development and progression

Abstract It is recognized that the product of neurofibromatosis type 2 (NF2) tumor suppressor gene, also referred to as Merlin/neurofibromin, is implicated in the homeostatic regulation of the liver stem cell niche through the control of abundance and signaling of epidermal growth factor receptor (EGFR), with a mechanism independent of the Mst/Lats/Yap Hippo pathway [1]. We have previously reported that locally elevated estrogen formation, driven by abnormally high expression and function of the aromatase enzyme, may be implicated in both development and progression of human hepatocellular carcinoma (HCC) through the activation of a rapid signaling pathway mediated by amphiregulin (AREG) and EGFR [2]. In this framework, we have recently presented a model by which the aromatase-estrogen-amphiregulin-EGFR axis is activated in response to tissue injury and/or inflammatory disease, with its alteration eventually leading to development not only of major human tumors (liver, breast, prostate), but also to other non-neoplastic diseases, including type 2 diabetes, obesity, Alzheimer's disease, heart disease and stroke [3]. In the present work, we have investigated the expression of merlin/neurofibromin (NF2) in different liver cancer cells and liver tissues in relation to aromatase expression/function, estrogen receptor (ER) status, amphiregulin and EGFR. Our data indicate that NF2 expression is strictly associated to aromatase, AREG and EGFR expression, being elevated in HCC tissues and HepG2 cells, intermediate in cirrhotic tissues and Huh7 liver cancer cells, and lower in nontumoral liver and HA22T cells. In addition, NF2 expression is inversely related to that of wild type hERα66 and proportional to the expression of the membrane-associated hERα36 splice variant, as measured by exon-specific RT-PCR analysis, both in vivo and in vitro. Furthermore, incubation with 1 nM estradiol induced a significant decrease of NF2 expression in both HA22T and Huh7 cells (over 54% and 22%, respectively), while no change could be observed in HepG2 cells, this effect being inversely related to aromatase expression and activity in these HCC cell lines. Based on this combined evidence, we propose here that NF2 may behave as a protein sensing tissue damage and, at least in human liver, increasing, via a Hippo-independent mechanism, aromatase-driven local estrogen formation, eventually leading to regulation of oval stem cells differentiation and tissue repair. An alteration of this putative NF2-aromatase-estrogen axis or a deficiency in NF2 may therefore be implicated in HCC development and/or progression. [1] Drvarov O & Cubero FJ 2011. Hepatology 53(5): 1767-70 [2] Carruba G et al. 2011. Horm Mol Biol Clin Invest 5(3): 153-160. [3] Carruba G. 2011. Gordon Research Conference, Invited Paper. Note: This abstract was not presented at the meeting. Citation Format: Letizia Cocciadiferro, Vitale Miceli, Orazia M. Granata, Giuseppe Carruba. Merlin/NF2 is associated with elevated aromatase expression and estrogen formation in human liver tissues and liver cancer cells: An unifying model for hepatocellular carcinoma development and progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1852. doi:10.1158/1538-7445.AM2015-1852

Abstract LB-152: Local estrogen formation and signaling through amphiregulin/EGFR may be implicated in human hepatocellular carcinoma: a unifying hypothesis.

Abstract Although there is evidence that hints at a potential role of estrogen in hepatocellular carcinoma (HCC), the use of antiestrogens has failed to improve survival of patients. We have investigated aromatase-driven estrogen formation in nontumoral and malignant human liver tissues and cells, also in relation to the expression of estrogen receptor (ER) α, ERβ and their splicing variants and of amphiregulin (AREG), an EGFR ligand, aiming to get insights in the potential role of estrogen and the underlying mechanism(s) in HCC. Chromatographic and exon-specific RT-PCR analyses were used to assess activity and expression of aromatase and the expression of wild-type (hERα66, hERβ1) and variant (hERα46, hERα36; hERβ2/Cx, hERβ5) ER, both in vivo and in vitro. Following incubation of tissue minces or cells with either testosterone or androstenedione used as precursor, human HCC tissues and HepG2 hepatoma cells showed elevated aromatase activity, with estrogen formation rates of 20% at 24h and >95% at 72h. By contrast, no aromatase activity could be detected in nontumoral hepatic tissues and HA22T liver cancer. Cirrhotic samples exhibited variable enzyme activity, with average estrogen formation rates of 4-8%, while Huh7 HCC cells gave rise to 34% estrogen formation. Markedly lower aromatase mRNA levels were observed in HA22T cells and nontumoral liver tissues, as compared to HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels. Interestingly, no or low expression of wild type ERα and ERβ could be detected in liver cancer cells and malignant tissues. Conversely, equivalent amounts of the hERα46 variant were observed in both cells and tissues, while hERα36 expression was highest in HepG2 cells and HCC samples, intermediate in Huh7 cells and cirrhotic tissues, and very low in HA22T cells and nontumoral liver. Only occasional expression of the two ERβ variants was observed in liver cancer cells and tissues. It is noteworthy that the pattern of hERα36 expression was strictly related to that of aromatase, suggesting that this ERα variant may be primarily implicated as a mediator of estrogen action in the malignant liver. AREG expression was consistently associated with that of aromatase, with nearly 3-fold and 8-fold higher levels being seen in HepG2 cells than in Huh7 cells (P=0,002) and HA22T cells (P=0,0014), respectively. This evidence suggests that AREG expression may be upregulated by estrogens in human HCC by binding to the membrane-associated ERα36 variant. Based on present and previous experimental evidence, we propose here a unifying hypothetical model whereby locally elevated, aromatase-driven, estrogen formation, combined with a shift in ER wild type/variant expression and the activation of rapid AREG signaling, may be primarily implicated in development and/or progression of human HCC and other neoplastic or chronic human diseases. Citation Format: Giuseppe Carruba, Vitale Miceli, Letizia Cocciadiferro, Lucia M. Polito, Biagio Agostara, Orazia M. Granata. Local estrogen formation and signaling through amphiregulin/EGFR may be implicated in human hepatocellular carcinoma: a unifying hypothesis. [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 LB-152. doi:10.1158/1538-7445.AM2013-LB-152

Direct antiproliferative effect of nonsteroidal 17β-hydroxysteroid dehydrogenase type 1 inhibitors in vitro

Inhibition of the local formation of estrogens seems to be an attractive strategy for pharmacological intervention in hormone-dependent disorders. The direct antiproliferative properties of ten nonsteroidal 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) inhibitors were investigated on human cancer cell lines of gynecological origin. The mechanism of the antiproliferative action was approximated by cell cycle analysis, fluorescent microscopy, BrdU assay, determination of caspase-3 activity and quantification of the expression of cell cycle regulators at mRNA level. Treatment of HeLa cells with some of the compounds resulted in a concentration-dependent inhibition of the G1–S transition and an increase in the apoptotic population. The most effective agents increased the expression of tumor suppressors p21 and p53, while CDK2 and Rb were down-regulated. The reported anticancer actions of the tested compounds are independent of the 17β-HSD1-inhibiting capacity. These results indicate that it is possible to combine direct antiproliferative activity and 17β-HSD1 inhibition resulting in novel agents with dual mode of action.

Impact of testosterone on the expression of organic anion transporting polypeptides (OATP-1A2, OATP-2B1, OATP-3A1) in malignant and non-malignant human breast cells in vitro

ObjectivesPostmenopausal hormone therapy (HT) increases local estrogen formation in breast tissue. The enzymatic substrates depend on transmembrane anion transporting polypeptides (OATPs) to reach intracellular enzymes. The aim of this study was to investigate the effect of testosterone (T) on the expression of OATP-1A2, OATP-2B1, and OATP-3A1 in malignant (MCF-7, BT-474) and non-malignant (HBL-100) breast cells in vitro. Study designCells were incubated in RPMI 1640 medium containing 5% steroid-depleted fetal calf serum for 3d, and subsequently incubated in the absence or presence of T, anastrozole (A), and T+A (10−6M) for 24h at 37°C. Main outcome measuresOATP expression was determined by immunocytochemical staining. Expression intensity was graded as low, moderate, or strong. Hormone receptor (AR, PR, ESR1, ESR2) expression was investigated by qPCR and Western blotting. Rank variance analysis was performed for statistical analysis (p≤0.05). ResultsOATP-1A2, OATP-2B1, and OATP-3A1 expression was present in all untreated breast cell lines examined, with OATP-1A2 and OATP-3A1 being the predominant ones. There was a trend for a higher baseline expression in untreated HBL-100 and BT-474 in comparison to MCF-7 cells, which was significant for OATP-2B1. T treatment led to decreased OATP-1A2, -2B1, and -3A1 expression in BT-474 and HBL-100 cells, respectively. In contrast, in MCF-7 cells, OATP-2B1 expression was significantly increased. T-induced upregulation of AR and PR protein expression in BT-474 and MCF-7 cells was reduced by A treatment. ConclusionsT may constitute a signal for differential regulation of mammary OATP expression. In non-malignant breast cells T seems to have a beneficial effect by reducing the availability of substrates for the intracellular formation of potent estrogens.

Intravaginal dehydroepiandrosterone (prasterone), a highly efficient treatment of dyspareunia

Objective To examine the effect of intravaginal dehydroepiandrosterone (DHEA) on pain at sexual activity (dyspareunia) identified as the most bothersome symptom of vaginal atrophy in postmenopausal women at both screening and day 1.Methods This prospective, randomized, double-blind and placebo-controlled phase III clinical trial studied the effect of prasterone (DHEA) applied locally in the vagina on the severity of dyspareunia in 114 postmenopausal women who had identified dyspareunia as their most bothersome symptom of vaginal atrophy, while meeting the criteria for superficial cells ≤ 5% and pH > 5.0 at both screening and day 1.Results At the standard duration of 12 weeks of treatment, increasing doses of 0.25%, 0.5% and 1.0% DHEA decreased the percentage of parabasal cells by 48.6 ± 6.78%, 42.4 ± 7.36% and 54.9 ± 6.60% (p < 0.0001 vs. placebo for all) with no change with placebo (p = 0.769). The effects on superficial cells and pH were also highly significant compared to placebo at all DHEA doses. The severity score of pain at sexual activity decreased by 0.5, 1.4, 1.6 and 1.4 units in the placebo and 0.25%, 0.5% and 1.0% DHEA groups, respectively, with the p value of differences from placebo ranging from 0.0017 to < 0.0001.Conclusions Intravaginal DHEA, through local estrogen and androgen formation, causes a rapid and highly efficient effect on pain at sexual activity without systemic exposure of the other tissues, thus avoiding the recently reported systemic effects of estrogens.

Abstract LB-203: Aromatase expression promotes breast cancer tumorigenesis and bone metastasis

Abstract Aromatase P450 (CYP19) is the rate-limiting enzyme catalyzing the final step in estrogen biosynthesis, and an important target in breast cancer therapy. In post-menopausal women, ovaries cease to make estrogen, yet the incidence of breast cancer increases with age as aromatase present in breast adipose stroma and epithelium catalyzes local estrogen formation. The circulating estrogen level in postmenopausal women is very low and it reflects the sum of local estrogen biosynthesis in various sites. Although breast cancer cells have been shown to express aromatase prompting the hypothesis that intracrine action of estrogen in breast cancer cells may drive tumor progression, there has been little evidence in addressing the hypothesis. We stably transfected the ER positive (ER+) breast cancer ZR-75-1 cells with an human aromatase expression vector and obtained a few clones expressing a moderately higher level of aromatase in comparison with the control vector-transfected cells. One of the clones called ZR-75-1/Aro/Clone10 (Cl10) was selected for further study due to its increased intracellular estradiol (E2) concentration compared to other clones. The Cl10 cells formed tumors after being xenografted into the inguinal mammary fat pad of female athymic mice while the control cells were incapable of forming tumors without estrogen supplementation. Implantation of testosterone pellet further stimulated the growth of the tumor and a cell line, ZR-75-1/Aro/Clone10/TT1 (TT1) was established from the tumor. Both Cl10 and TT1 cells expressed similar levels of aromatase. Interestingly, the aromatase expression increased when the cells were cultured in suspension suggesting circulating ER+ breast cancer cells may up-regulate intracrine estrogen activity for survival after leaving the estrogen-rich adipose stroma at primary site. To investigate the importance of intracrine estrogen in promoting tumorigenesis and metastasis, we implanted the Cl10 and TT1 cells orthotopically and intracardiacally (I.C.) into female athymic mice. Notably, both cell lines generated orthotopic tumors with no estrogen supplementation after three weeks of inoculation. More interestingly, the mice with I.C. inoculated aromatase-expressing cells presented distant metastasis to bones in mandible and legs after two weeks of inoculation detected by whole mouse fluorescence and bioluminescence imaging as both cells were stably transfected with a luciferase and GFP expression vector. We will determine whether growth of the orthotopic tumors and bone metastases can be inhibited with systemic administration of an aromatase inhibitor and whether bone metastasis is osteoclastic or osteoblastic. Our studies show that a moderate expression of aromatase in breast cancer cells is sufficient to generate intracrine estrogenic action and promote tumorigenesis and skeletal metastasis in ER+ breast cancer cells. These aromatase-expressing models should be useful for future investigation on how ER+ breast cancer cells up-regulate aromatase expression in suspension and induces bone remodeling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-203.

Aromatase expression in the normal and epileptic human hippocampus

Aromatase is a key enzyme in estrogen biosynthesis that is involved in neuronal plasticity in the rodent hippocampus. Although aromatase mRNA expression has been detected in the human hippocampus, its cellular distribution has yet to be determined. Here, we have examined the immunohistochemical distribution of aromatase in the normal and the epileptic and sclerotic human hippocampus. In both the normal and epileptic hippocampus, aromatase was detected in numerous CA1–CA3 pyramidal neurons, in granule cells of the dentate gyrus and in interneurons that co-expressed the calcium-binding proteins calbindin, calretinin or parvalbumin. However, only a small subpopulation of astrocytes was immunoreactive for aromatase in either the normal and epileptic hippocampus. The widespread expression of aromatase in a large population of neurons in the normal and damaged hippocampus suggests that local estrogen formation may play an important role in human hippocampal function.

Effect of testosterone on E1S-sulfatase activity in non-malignant and cancerous breast cells in vitro

Introduction Testosterone (T) is a therapeutic option for women with hypoactive sexual desire disorder. T may have an impact on the mammary gland by altering local estrogen synthesis. The aim of the present study was to measure the effect of T on estrone-sulfate (E1S)-sulfatase (STS) expression, and activity using hormone-dependent BC cells with high and low aggressive potential (BT-474, MCF-7), and HBL-100 as a breast cell line of non-malignant origin. Methods Cells were incubated in RPMI 1640 medium containing 5% steroid-depleted fetal calf serum for 3 d, and subsequently incubated in absence or presence of T alone, and combined with anastrozole (A) at 10 −8 M, and 10 −6 M at 37 °C for either 24 h or directly in cell extracts (“direct”). STS protein expression was measured by dot-blot (immunoblotting), and STS, HSD17B1 and HSD17B2 mRNA levels by quantitative RT-PCR. STS activity was evaluated by incubating homogenized breast cells with [ 3H]-E1S and separating the products E1, and E2 by thin layer chromatography. Results Basal STS mRNA expression did not reveal group differences. However, STS mRNA was decreased by T + A in MCF-7 cells. 17HSDB1 expression was decreased by T + A in BT-474 cells, and 17HSDB2 expression was decreased by A and T + A treatment in MCF-7 cells. Basal and T treated STS protein expression was significantly higher in malignant compared to non-malignant breast cells. However, T did not induce significant intra-cell line differences. Similarly, basal and T treated STS activity was significantly higher in highly malignant compared to non-malignant breast cells. Regardless of cell lines, T slightly decreased STS activity after “direct” incubation, but led to an increase of local estrogen formation after 24 h which was attenuated, and partly reversed by A, respectively. Conclusions The more aggressive the breast cell line, the higher the local estrogen formation. The transition from normal to malignant seems to be accompanied by an altered autoregulation. The given local endocrine milieu seems to be essential for response to T.

Intravaginal dehydroepiandrosterone (Prasterone), a physiological and highly efficient treatment of vaginal atrophy

Because the secretion of dehydroepiandrosterone (DHEA), the exclusive source of sex steroids in postmenopausal women, is already decreased by 60% and continues to decline at the time of menopause, the objective of this study was to examine the effect of intravaginal DHEA on the symptoms and signs of vaginal atrophy. This prospective, randomized, double-blind and placebo-controlled phase III clinical trial studied the effect of Prasterone (DHEA) applied locally in the vagina on the signs and symptoms of vaginal atrophy in 216 postmenopausal women. All three doses (0.25%, 0.5%, and 1.0%) of DHEA ovules applied daily intravaginally induced a highly significant beneficial change in the percentage of vaginal parabasal and superficial cells and pH as well as in the most bothersome symptom at 2 weeks. At the standard 12-week time interval, 0.5% DHEA caused a 45.9 +/- 5.31 (P < 0.0001 vs placebo) decrease in the percentage of parabasal cells, a 6.8 +/- 1.29% (P < 0.0001) increase in superficial cells, a 1.3 +/- 0.13 unit (P < 0.0001) decrease in vaginal pH, and a 1.5 +/- 0.14 score unit (P < 0.0001) decrease in the severity of the most bothersome symptom. Similar changes were seen on vaginal secretions, color, epithelial surface thickness, and epithelial integrity. Comparable effects were observed at the 0.25% and 1.0% DHEA doses. Local Prasterone, through local androgen and estrogen formation, causes a rapid and efficient reversal of all the symptoms and signs of vaginal atrophy with no or minimal changes in serum steroids, which remain well within the normal postmenopausal range. This approach avoids the fear of systemic effects common to all presently available estrogen formulations and adds a novel physiological androgenic component to therapy.

Expression of enzymes involved in synthesis and metabolism of estradiol in human breast as studied by immunocytochemistry and in situ hybridization.

It is well documented that human breast is actively involved in the local formation of estrogens. To determine the site(s) of action of enzymes involved in synthesis and metabolism of the most potent estrogen estradiol (E2), we have studied the expression of the following enzymes: 3beta-hydroxysteroid dehydrogenase (3-HSD), 17beta-HSD types 1, 2, 5, 7 and 12, aromatase, steroid sulfatase (STS) and estrogen sulfotransferase (EST) 1E1 at the cellular level in breast. Both in situ hybridization and immunocytochemistry were used for enzyme localization in normal breast tissues. For immunocytochemistry, we used rabbit antibodies, while in situ hybridization studies were performed using (35S)-labeled cRNA probes. Similar results were obtained with both approaches. All the enzymes (3beta-HSD; 17beta-HSD types 1, 5, 7 and 12; aromatase) involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2 as well as STS which converts estradiol sulfate (E2-S) to E2 have been found to be expressed in epithelial cells of acini and/or ducts as well as the stromal cells. Moreover, 17beta-HSD type 2 and EST1E1, two enzymes which inactivate E2, have been also localized in the same cell types. The present results indicate the enzymes which play a role in the synthesis and metabolism of E2 are expressed in both epithelial and stromal cells in human breast.

Viral Vector–mediated Delivery of Estrogen Receptor-α to the Hippocampus Improves Spatial Learning in Estrogen Receptor-α Knockout Mice

Estrogen, which influences both classical genomic and rapid membrane-associated signaling cascades, has been implicated in the regulation of hippocampal function, including spatial learning. Gene mutation studies suggest that estrogen effects are mediated by estrogen receptor-α (ER-α); however, because gonadal steroids influence the organization of the hippocampus during development, it has been difficult to distinguish developmental effects from those specific to adults. In this study we show that lentiviral delivery of the gene encoding ER-α to the hippocampus of adult ER-α-knockout (ER-αKO) mice restores hippocampal responsiveness to estrogen and rescues spatial learning. We propose that constitutive estrogen receptor activity is important for maintaining hippocampus-dependent memory function in adults.

Effects of hormone therapy on estrogen synthesis from E 1S in the mammary gland of postmenopausal women

Objectives To investigate the effect of hormone therapy (HT) on the expression of hormone receptors and the ex vivo estrogen biosynthesis in the breast. Methods Comparative studies were carried out in breast tissue from 28 postmenopausal women undergoing breast surgery due to breast cancer (BC). Glandular breast tissue at least 1 cm distant from the tumor was analyzed. Groups included patients having received HT ( n = 18), and non-user of HT (controls, n = 10) prior to BC diagnosis. Steroid sulfatase (STS) activity was evaluated by incubating homogenized breast tissue with [ 3H]-estrone sulfate. Thin layer chromatography was performed to separate the products estrone (E 1) and estradiol (E 2). Histomorphometry for breast tissue composition and immunohistochemistry for expression of estrogen receptor (ER) alpha and beta as well as progesterone receptor (PR) were performed. Results In all groups, significantly more E 2 than E 1 was produced. Local E 2 formation was higher in women having been treated with estrogen and progestogen ( p ≤ 0.05). Local E 2 formation was positively correlated with ER alpha expression ( r s = 0.5; p = 0.03). Histomorphometrical and immunohistochemical outcomes did not differ between groups. However, the amount of vessels was higher in women having been treated with HT compared to controls ( p = 0.09). Conclusions Long-term HT increases local estrogen formation in normal human breast tissue.

Effects of black cohosh on estrogen biosynthesis in normal breast tissue in vitro

Objectives To investigate the effect of black cohosh on the estrogen biosynthesis in the breast in vitro. Methods Steroid sulfatase (STS) activity was studied in normal breast tissue obtained from pre- and postmenopausal women undergoing reduction mammoplasty. STS protein expression was studied by immunohistochemistry and western blotting. Breast tissue was incubated in vitro without or with black cohosh (iCR) at concentrations ranging from 0.1 mg/ml to 1 ng/ml. STS activity was evaluated by incubating homogenized breast tissue with [ 3H]-estrone sulfate, separating the formed products, estrone (E 1) and estradiol (E 2), by thin layer chromatography and measuring the amounts of E 1 and E 2 by scintillation counting. Results STS protein expression and enzymatic activity were detected in all specimens investigated. In all groups, significantly more E 1 than E 2 was produced. Local estrogen formation was decreased in premenopausal breast tissue by treatment with iCR at 0.1 mg/ml ( p ≤ 0.05). Conclusions iCR decreases local estrogen formation in normal human breast tissue in vitro. This may contribute to the lack of hormonal effects of black cohosh in breast tissue observed in previous studies.

Differential effect of hormone therapy on E1S-sulfatase activity in non-malignant and cancerous breast cells in vitro

Breast tissue possesses the enzymes for local estrogen biosynthesis. We measured the effect of Estradiol (E2), Tibolone (OrgOD14) and its metabolite Org4094 on estrone sulfate (E1S)-sulfatase (STS) using breast cancer (MCF-7) and non-malignant breast cells (HBL-100). Cells were cultured in 5% steroid depleted fetal calf serum for 3 days and subsequently incubated with each steroid for either 24 h or directly in cell extracts. STS mRNA and protein expression, and its subcellular localization were determined by semi-quantitative RT-PCR, immunoblotting, and confocal immunofluorescence microscopy. STS activity was evaluated by incubating homogenized breast cells with [(3)H]-E1S. The products E1 and E2 were separated by thin layer chromatography. STS was co-localized with the Golgi marker protein GM130 and the endoplasmic reticulum marker protein calnexin. Treatment did not significantly alter STS mRNA expression. STS protein expression was increased by each steroid in HBL-100 cells but by E2 only in MCF-7 cells. 24 h incubation with OrgOD14 and Org4094 did not alter STS activity in both cell lines. However, STS activity was significantly diminished in HBL-100 but slightly increased in MCF-7 cells by 24 h treatment with E2. "Direct" incubation of cell extracts, eliminating cellular regulation of metabolism, reduced estrogen biosynthesis regardless of cell line and treatment. In conclusion, the immediate reduction of estrogen biosynthesis by OrgOD14 is counteracted by an increased STS protein expression. On the contrary, E2 exerts a differential effect on STS in HBL-100 and MCF-7 cells. The transition from normal to malignant breast cells may be accompanied by an abolished autoregulation of local estrogen formation.