Estrogen Response Pathway Research Articles

Enantioselectivity in Estrogenic Potential and Uptake of Bifenthrin

Despite the fact that the biological processes of chiral compounds are enantioselective, the endocrine disruption activity and uptake of chiral contaminants with respect to enantioselectivity has so far received limited research. In this study, the estrogenic potential and uptake of the enantiomers of a newer pyrethroid insecticide, bifenthrin (BF), were investigated. Significant differences in estrogenic potential were observed between the two enantiomers in the in vitro human breast carcinoma MCF-7 cell proliferation assay (i.e., the E-SCREEN assay) and the in vivo aquatic vertebrate vitellogenin enzyme-linked immunosorbent assay (ELISA). In the E-SCREEN assay, the relative proliferative effect ratios of 1S-cis-BF and 1R-cis-BF were 74.2% and 20.9%, respectively, and the relative proliferative potency ratios were 10% and 1%, respectively. The cell proliferation induced by the two BF enantiomers may be through the classical estrogen response pathway via the estrogen receptor (ER), as the proliferation induced by the enantiomers could be completely blocked when combined with 10-6 mol/L of the ER antagonist ICI 182,780. Measurement of vitellogenin induction in Japanese medaka (Oryzias latipes) showed that, at an exposure level of 10 ng/mL, the response to 1S-cis-BF was about 123 times greater than thattothe Renantiomer. Significant selectivity also occurred in the uptake of BF enantiomers in the liver and other tissues of J. medaka. These results together suggest that assessment of the environmental safety of chiral insecticides should consider enantioselectivity in acute and chronic ecotoxicities such as endocrine disruption.

Estrogen-like effects of ethanol extracts from several Chinese legumes on MCF-7 cell

Ethanol extracts of several Chinese legumes were prepared and their estrogen-like activities were examined using the modified MCF-7 cell proliferation assay method (E-SCREEN assay). The possible mechanisms were addressed using cell cycle analysis and reverse transcription-polymerase chain reaction (RT-PCR) measurement in which a pure estrogen receptor antagonist ICI182,780 was employed as the tool agent. The legumes selected in this study were Mung bean (Vigna radiata (L.) Wilczek), Adzuki bean (Phaseolus angularis Wight.), Black soybean (Glycine max (L.) Merr.), Lima bean (Phaseolus lunatus L.), Broad bean (Vicia faba L.), Pea (Pisum sativum L.), Common bean I (Phaseolus vulgaris L.), and Common bean II (Phaseolus vulgaris var. humilis Alief), respectively. The results showed that ethanol extracts of Adzuki bean (100 μg/ml), Black soybean (1–200 μg/ml) and Lima bean (100 μg/ml), could strongly stimulated the proliferation of MCF-7 cells (P<0.01 vs. control), of which the estrogen-like effects of the one from Adzuki bean and Lima bean were the firstly reported. Cell cycle analysis revealed that the proliferation stimulatory effects of ethanol extracts of Adzuki bean, Black soybean and Lima bean were associated with a significant increase in the number of MCF-7 cells in S phase and in the G2/M population, similar to those of 17β-estradiol (E2). Using RT-PCR measurement, it was found that ethanol extracts of Adzuki bean, Black soybean and Lima bean could induce pS2 transcription in MCF-7 cells. Moreover, these reactions could be significantly inhibited by the specific estrogen receptor antagonist ICI182,780 (P<0.01 vs. the same concentration of compound without treatment of ICI182,780), suggesting that ethanol extracts of Adzuki bean, Black soybean and Lima bean exhibited estrogen-like activities through the estrogen response pathway by interacting with the estrogen receptor.

Melatonin inhibits the growth of DMBA-induced mammary tumors by decreasing the local biosynthesis of estrogens through the modulation of aromatase activity

Melatonin inhibits the growth of breast cancer cells by interacting with estrogen-responsive pathways, thus behaving as an antiestrogenic hormone. Recently, we described that melatonin reduces aromatase expression and activity in MCF-7 human breast cancer cells, thus modulating the local estrogen biosynthesis. To investigate the in vivo aromatase-inhibitory properties of melatonin in our current study, this indoleamine was administered to rats bearing DMBA-induced mammary tumors, ovariectomized (ovx) and treated with testosterone. In these castrated animals, the growth of the estrogen-sensitive mammary tumors depends on the local aromatization of testosterone to estrogens. Ovariectomy significantly reduced the size of the tumors while the administration of testosterone to ovx animals stimulated tumor growth, an effect that was suppressed by administration of melatonin or the aromatase inhibitor aminoglutethimide. Uterine weight of ovx rats, which depends on the local synthesis of estrogens, was increased by testosterone, except in those animals that were also treated with melatonin or aminoglutethimide. The growth-stimulatory effects of testosterone on the uterus and tumors depend exclusively on locally formed estrogens, since no changes in serum estradiol were appreciated in testosterone-treated rats. Tumors from animals treated with melatonin had lower microsomal aromatase activity than tumors of animals from other groups, and incubation with melatonin decreased the aromatase activity of microsomal fractions of tumors. Animals treated with melatonin had the same survival probability as the castrated animals and significantly higher survival probability than the uncastrated. We conclude that melatonin could exert its antitumoral effects on hormone-dependent mammary tumors by inhibiting the aromatase activity of the tumoral tissue.

CSN5/Jab1 Is Involved in Ligand-Dependent Degradation of Estrogen Receptor α by the Proteasome

Here, we show that estrogen receptor alpha (ERalpha) coimmunoprecipitates with CSN5/Jab1, a subunit of the COP9 signalosome (CSN), and that overexpression of CSN5/Jab1 causes an increase in ligand-induced ERalpha degradation. Inhibition of either the kinase activity associated with the CSN complex by curcumin or of nuclear export by leptomycin B (LMB) impaired estradiol-induced ERalpha degradation by the proteasome. Degradation of ERalpha induced by the pure antagonist ICI 182,780 (ICI) was blocked by curcumin but not by LMB, indicating that in the presence of ICI, ERalpha is degraded by a nuclear fraction of the proteasome. In addition, we observed that curcumin inhibited estradiol-induced phosphorylation of ERalpha. The use of three inhibitors of ERalpha degradation that target different steps of the estrogen response pathway (inhibition of the CSN-associated kinase, nuclear export, and proteasome) suggests that a phosphorylation event inhibited by curcumin is necessary for ERalpha binding to its cognate DNA target. Our results demonstrate that transcription per se is not required for ERalpha degradation and that assembly of the transcription-initiation complex is sufficient to target ERalpha for degradation by the proteasome.

Oral Contraceptives and Skin Cancer

The skin expresses estrogen, progesterone, and androgen receptors. In the presence of steroid hormones, such as those contained in oral contraceptives, the skin likely responds to hormonal signals that control the cell cycle, apoptosis, DNA replication, and other cellular functions. Some estrogen-responsive pathways have the potential to promote tumor development, including the augmentation of epidermal growth factor signaling, the expression of proto-oncogenes, and inhibition of apoptosis. The question of whether oral contraceptives increase the risk for the development of skin cancer, particularly melanoma, is still an area of concern. This paper reviews the available evidence, the bulk of which suggests that while the skin responds to estrogens, progestins, and androgens, these responses do not significantly increase the risk of developing skin cancer when estrogen exposure is not excessive.

Differential regulation of estrogen receptor alpha, glucocorticoid receptor and retinoic acid receptor alpha transcriptional activity by melatonin is mediated via different G proteins

Melatonin has been shown to bind to the MT1 G protein-coupled receptor (GPCR) in MCF-7 breast cancer cells to modulate the estrogen response pathway suppressing estrogen-induced estrogen receptor alpha (ERalpha) transcriptional activity, blunting ER/DNA binding activity and suppressing cell proliferation. In these studies we have examined the effect of melatonin on the transcriptional activity of the ERalpha and other members of the steroid/thyroid hormone receptor superfamily, namely, the glucocorticoid receptor (GR) and the retinoic acid receptor alpha (RARalpha). As with the ERalpha, melatonin represses ligand (dexamethasone)-induced activation of the GR. This effect of melatonin on ERalpha and GR is blocked by pertussis toxin (PTX) suggesting that melatonin's actions may be mediated via a PTX-sensitive G(alphai) protein. In contrast, melatonin potentiates the action of all-trans-retinoic acid on RARalpha transcriptional activation and enhances RARalpha/DNA binding activity, an action which is not PTX-sensitive. Expression of a dominant-positive G(alphai2) protein, with which the MT1 receptor has been shown to couple, is able to mimic the effect of melatonin on ERalpha but not RARalpha transcriptional activation in breast cancer cells. This demonstrates that GPCRs can modulate the transcriptional activity of various steroid receptors in response to their ligand through activation of different G protein signaling pathways.

Melatonin modulates aromatase activity in MCF‐7 human breast cancer cells

Most of the current knowledge about the mechanisms by which melatonin inhibits the growth of breast cancer cells point to an interaction of melatonin with estrogen-responsive pathways, thus behaving as an antiestrogenic hormone. However, a possible effect of melatonin on the local synthesis of estrogens had not been examined. The objective of this work was to study whether melatonin may modify the aromatase activity in MCF-7 breast cancer cells thus modulating the local estrogen biosynthesis. In MCF-7 cells cultured with testosterone in estradiol-free media, melatonin (1 nM) counteracts the testosterone-induced cell proliferation dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. We found that melatonin reduces the aromatase activity (measured by the tritiated water release assay) of MCF-7 cells both at basal conditions and when aromatase activity was stimulated by cAMP or cortisol. The greatest inhibition of the aromatase activity was obtained with 1 nm melatonin, the same concentration that gives the highest antiproliferative and anti-invasive effects of MCF-7 cells. Finally, by RT-PCR, we found that melatonin downregulates aromatase expression at the transcriptional level in the MCF-7 cells. We conclude that melatonin, at physiological concentrations, decreases aromatase activity and expression in MCF-7 cells. This aromatase inhibitory effect of melatonin, together with its already known antiestrogenic properties interacting with the estrogen-receptor, makes this indoleamine an interesting tool to be considered in the prevention and treatment of hormone-dependent mammary neoplasias.

Estrogen receptor alpha (ESR1) mutant A908G is not a common feature in benign and malignant proliferations of the breast.

Alterations in estrogen responsive pathways are thought to contribute to benign and malignant breast disease. It has been reported previously that more than a third of typical epithelial hyperplasia lesions harbor the missense mutation A908G in the estrogen receptor alpha (ESR1) gene. This substitution of an arginine for a lysine at codon 303 was reported to confer mitogenic hypersensitivity to estrogen. To explore this finding further, we analyzed ESR1 for this mutation in a series of breast tissues ranging from typical hyperplasia to invasive cancer. In contrast to previous studies, no evidence for this mutation was found in 36 invasive cancers, 11 in situ carcinomas, 14 epithelial hyperplasias with atypia, 11 epithelial hyperplasias without atypia, and 11 breast cancer cell lines. These results indicate that ESR1 mutant A908G does not occur with significant frequency in either benign or malignant proliferations of breast epithelia.

Estrogenic activities of Ginkgo biloba extracts

Ginkgo biloba extracts (GBE) are extracted from the leaves of Ginkgo biloba tree. GBE contains 24% of phytoestrogens, which is kaempferol, quercetin, and isorhamnetin. It has been reported that phytoestrogens could be a part of SERMs (Selective estrogen receptor modulators) and possibly the alternative HRT (Hormone replacement therapy) for postmenopausal women. The goal of this study was to investigate the potencies of GBE and its major components (quercetin, kaempferol, isorhamnetin) for estrogenic effect, which confirms the capacity as an alternative HRP. It was found that GBE and its major components exerted a dual action on ER-α and ER-β in competitive binding assay.The binding affinity of these chemicals to ER-β was higher than to ER-α. In the E-screen assay, these chemicals induced cell proliferation in ER-positive MCF-7 cell, but not in ER-negative MDA-MB-231 cells. The cell proliferation induced by these chemicals was blocked by tamoxifen. Also, GBE and its major components induced pS2 and PR (progesterone receptor) transcription in MCF-7 cells. Therefore these results indicated that GBE and its major components had the weak estrogenic activities through the estrogen response pathway by an interaction with the ER. In conclusion, we provided the evidence of potential estrogenic activities of GBE, which could be useful as an alternative HRP. However, further studies are required to assess the physiological significance of GBE in animals and humans.

Melatonin inhibits estrogen receptor transactivation and cAMP levels in breast cancer cells.

We have previously demonstrated that the pineal hormone, melatonin, can inhibit the growth of estrogen receptor-alpha (ERalpha)-positive breast cancer cells and suppress ERalpha gene transcription. To investigate the relationship between the estrogen response pathway and melatonin's growth inhibition, ERalpha-positive MCF-7 human breast cancer cells were transiently transfected with an estrogen response element (ERE) luciferase reporter construct and then treated with melatonin (10(-9)-10(-6) M) for 30 min followed by 10(-9) M 17-beta-estradiol (E2) or treated with each compound alone. Melatonin pre-treatment significantly reduced E2-induced ERalpha transactivation and ERalpha-ERE binding activity. We also conducted experiments to determine if melatonin modulates cAMP levels in MCF-7 cells. Melatonin inhibited the forskolin-induced and E2-induced elevation of cAMP levels by 57 and 45%, respectively. These data indicate that melatonin can act as a biological modifier to affect ERalpha transcriptional activity by regulating signal transduction pathways which impinge on the ERalpha and by altering E2-mediated ERalpha transactivation and ERalpha DNA binding activity.

Selective estrogen receptor modulators: mechanism of action and clinical experience. Focus on raloxifene.

Selective estrogen receptor modulators (SERMs) are a diverse group of compounds that bind with specific, high-affinity binding to the estrogen receptor (ER). Depending on the tissue, SERMs can act as either ER agonists or antagonists. Recent advances in ER biology have provided insight into possible mechanisms by which SERMs elicit these tissue-specific estrogen agonist and estrogen antagonist activities. The estrogen response pathway has been shown to differ among target tissues depending on various tissue and cellular factors, such as the ER subtype, the structure of the bound receptor-ligand complex, and the tissue-specific cellular transcriptional machinery. Clinically available SERMs include clomiphene, tamoxifen and toremifene, which are triphenylethylenes, and raloxifene, a benzothiophene. Raloxifene has estrogen agonist effects on bone, serum lipids, and arterial vasculature, and estrogen antagonist effects in breast and uterus. Clinical trial data for raloxifene is used to illustrate some of the mechanisms by which SERMs exert their tissue-specific estrogen agonist and estrogen antagonist effects. The complex pharmacology surrounding the tissue selectivity of SERMs is discussed.

DES action in the thymus: inhibition of cell proliferation and genetic variation

Many tissues, including the thymus, represent direct targets of estrogen action. In contrast to many estrogen responsive tissues, the thymus undergoes a profound atrophy in response to elevated levels of estrogens. The mechanism of estrogen-induced atrophy in the thymus is unknown; however, it appears not to involve massive thymocyte apoptosis. Here, we demonstrate that the estrogen diethylstilbestrol (DES) inhibits cell proliferation within the thymic cortex, the primary site of thymocyte proliferation. Unlike glucocorticoid action, the effect of DES on thymocyte proliferation does not appear to be mediated by direct down regulation of cyclin D3. We also demonstrate for the first time that rat strains vary in their sensitivity to DES-induced thymic atrophy. This sensitivity correlates with the ability of DES to inhibit cell proliferation in the thymus. These data suggest that genetic factors may regulate estrogen action within this tissue by affecting estrogen responsive pathways that control cell proliferation.

Differential responsiveness of MCF-7 human breast cancer cell line stocks to the pineal hormone, melatonin.

The estrogen receptor (ER)-positive MCF-7 human breast cancer cell line has been used extensively for the study of estrogen-responsive human breast cancer. However, various levels of estrogen responsiveness have been described in different stocks of MCF-7 cells. Because we have previously shown that the pineal hormone, melatonin, inhibits proliferation of MCF-7 cells and can modulate ER expression and transactivation, we investigated if various stocks of MCF-7 cells exhibit a differential responsiveness to the anti-proliferative effects of melatonin and the possible mechanisms involved. The MCF-7 stocks (M, O, H) were examined for: (1) mitogenic response to estradiol; (2) steady-state ER mRNA levels; (3) expression of the mt1 melatonin membrane receptor; (4) growth inhibition by melatonin; and (5) melatonin's modulation of expression of the ER and the estrogen-regulated genes, PgR, TGFbeta and pS2. For all of these parameters, there was a stock-specific response which showed: MCF-7M > MCF-7O > MCF-7H. These results demonstrate that there are significant differences in the responsiveness of various stocks of MCF-7 breast cancer cells to the growth-inhibitory effects of melatonin which can be correlated with both the level of ER mRNA expression and the degree of estrogen-responsiveness. These findings suggest that not only may these differences have some impact on the cells' estrogen-response pathway, but also that the primary growth-inhibitory effects of melatonin are transduced through the membrane-associated G-protein coupled mt1 melatonin receptor.

Estrogenic responses in estrogen receptor-α deficient mice reveal a distinct estrogen signaling pathway

Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-alpha (ER-alpha). By using ER-alpha "knock-out" (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17beta (4-OH-E2), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-alpha. A primary estrogen, estradiol-17beta (E2), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E2 failed to inhibit uterine LF gene expression induced by 4-OH-E2 or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-alpha and the recently cloned ER-beta. 4-OH-E2, but not E2, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.

Melatonin modulation of estrogen-regulated proteins, growth factors, and proto-oncogenes in human breast cancer.

The growth-inhibitory actions of the pineal hormone, melatonin, on human breast tumor cells and the possible association between this inhibition and melatonin's down-regulation of the estrogen receptor (ER) expression were examined in the ER-positive, estrogen-responsive MCF-7 human breast tumor cell line. As previously reported, melatonin dramatically inhibits the growth of these breast tumor cells and down-regulates ER levels in these cells, suggesting that the modulation of ER may be an important mechanism by which melatonin inhibits breast cancer cell growth. In the present studies, Northern blot analysis was used to examine the expression of estrogen-regulated transcripts known to be involved in estrogen's mitogenic actions. Melatonin, at a physiologic concentration (10(-9) M), rapidly, significantly, and, in some cases, transiently elevated the steady-state mRNA levels of growth stimulatory products such as TGF alpha, c-myc, and pS2, which are normally up-regulated in response to estrogen. Conversely, melatonin decreased the expression of other factors normally up-regulated by estrogen, such as progesterone receptor and c-fos. Significant stimulation of the expression of the growth-inhibitory factor TGF beta was seen with melatonin treatment, potentially supporting the concept that melatonin's growth-inhibitory activity is mediated through the breast tumor cells' estrogen-response pathway. The early regulation of many of these products by melatonin suggests that mechanisms more rapid than the down-regulation of ER are important in melatonin's modulation of their expression. However, the long-term modulation of these transcripts (12-48 hr) may be heavily influenced by melatonin's down-regulation of ER expression. These results clearly define the need for additional in depth studies to dissect the cellular events leading to melatonin-induced growth inhibition in breast tumor cells.

MELATONIN MODULATION OF ESTROGEN-RECEPTOR EXPRESSION IN MCF-7 HUMAN BREAST-CANCER CELLS

MCF-7 human breast cancer cells, which are estrogen receptor (ER)-positive and responsive to the mitogenic actions of estrogen, were used to examine the possible association between the growth-inhibitory activity of melatonin and its ability to modulate the estrogen-response pathway. Melatonin at physiologic concentrations (10(-8)-10(-11) M) significantly decreased estrogen binding activity and the expression of immunoreactive ER in a dose-specific and time-dependent manner. However, melatonin did not alter receptor affinity and was unable to compete with estrogen for binding to the ER. Studies in a yeast trancriptional assay system confirmed that melatonin does not directly bind to the ER to modulate ER expression. Thus, it appears that the antimitotic actions of melatonin may be mediated, at least in part, through the suppression of the estrogen-response pathway of MCF-7 cells.

The growth inhibitory action of melatonin on human breast cancer cells is linked to the estrogen response system

The pineal hormone, melatonin, was examined for its capacity to modulate the proliferation of a panel of human breast cancer cell lines. Melatonin inhibited, to a varying extent, the proliferation of all three estrogen-responsive cell lines, but had no effect on estrogen-insensitive breast tumor cell lines. Melatonin was also able to specifically block estrogen-induced proliferation in MCF-7 breast cancer cells. However, this action was abolished in the presence of tamoxifen. Therefore, it appears that the antiproliferative effects of melatonin are mediated through the estrogen-response pathway.

Prognostic significance of the estrogen-regulated protein, cathepsin D, in breast cancer. An immunohistochemical study

Expression of the estrogen-regulated lysosomal protease, cathepsin D, was studied in a series of 94 breast cancers using an immunohistochemical technique. Granular staining of tumor cell cytoplasm was detected in 62 cases. Positive staining was associated with a significant increase in overall time to relapse and when survival was analyzed in terms of intensity of cathepsin D staining there was a significant trend for both increased time to relapse and increased length of survival. The presence of estrogen receptor was associated with positive cathepsin D immunostaining, and in the subgroup of estrogen receptor-positive tumors cathepsin D staining was associated with significantly prolonged survival; this was not the case for estrogen receptor-negative tumors. Positive cathepsin D immunostaining was associated with significant prognostic advantage in patients with confirmed lymph node metastasis but not in node-negative patients. It is suggested that cathepsin D expression reflects the functional integrity of the estrogen response pathway. Cathepsin D may prove a clinically useful adjunct to assessment of estrogen receptor status.

Progesterone receptors and human breast cancer; ‘wassink lecture’ presented at the 3rd EORTC Breast Cancer Working Conference

INTRODUCTION THE DEVELOPMENT and widespread use of estrogen receptor (ER) assays to predict the endocrine dependence of a patient’s tumor has changed the clinician’s approach to the treatment of breast cancer. When it was discovered that cytoplasmic ER is responsible for the uptake of estrogen into target cells and is necessary for estrogen action, it was reasoned that ER might be useful in determining the endocrine responsiveness of breast cancers in patients with metastatic disease. This hypothesis has been confirmed, and it is now generally accepted that tumors lacking ER rarely respond to endocrine therapy whereas ER+ tumors frequently regress with hormonal manipulations. Determination of the ER status of the tumor is now standard practice in the United States. The ER assay is most helpful when the tumor is ER-. These patients rarely respond to endocrine therapy. On the other hand, a positive ER does not guarantee a response to endocrine therapy, since about 40% of patients fail to responddespite a positive assay. Horwitz et al. [l] suggested that certain ER+ tumors do not regress with endocrine manipulation because of a defect in the estrogen response pathway distal to the binding step, leading to autonomous growth. What was needed was a way of testing the integrity of the entire metabolic sequence that should be triggered by the binding of estrogen to receptor. Based on the observation that progesterone receptor (PgR) is induced by estrogen in normal reproductive tissues and in human breast cancer cells in culture, they hypothesized that PgR might be a better marker than ER for an intact estrogen