Activation Of Estrogen Receptor-α Research Articles

Abstract PO2-14-11: Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment

Abstract NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader (SERD), together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ PDX models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pull-down. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An immunohistochemistry (IHC) assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Citation Format: Beom-Jun Kim, Ze-Yi Zheng, Jonathan Lei, Matthew Holt, Anran Chen, Jianheng Peng, Diana Fandino, Purba Singh, Hilda Kennedy, Yongchao Dou, M Rosario Chica-Parrado, Emmanuel Bikorimana, Dan Ye, Yunguan Wang, Ariella Hanker, Nada Mohamed, Susan Hilsenbeck, Bora Lim, Jaya Ruth Asirvatham, Arun Sreekumar, Bing Zhang, George Miles, Meenakshi Anurag, Matthew Ellis, Eric Chang. Proteogenomic approaches for the identification of NF1/neurofibromin-depleted estrogen receptor positive breast cancers for targeted treatment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-11.

Quality Control for Single Cell Imaging Analytics Using Endocrine Disruptor-Induced Changes in Estrogen Receptor Expression.

Background:Diverse toxicants and mixtures that affect hormone responsive cells [endocrine disrupting chemicals (EDCs)] are highly pervasive in the environment and are directly linked to human disease. They often target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses.Objective:We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen (ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner.Methods:We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with toxicants, over a large number of biological and technical replicates.Results:We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence in situ hybridization (RNA FISH).Discussion:We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants. https://doi.org/10.1289/EHP9297

Prediction of Compounds from 96% Ethanol Extract of Marsilea crenata Presl. Leaves in Increasing Estrogen Receptor-α Activation

Women who experience menopause will experience estrogen deficiency, which will impact their health, one of which will increase the risk of neurodegenerative. Phytoestrogen compounds in Marsilea crenata can provide activity after binding to their receptors or ER-dependent pathways. The research was conducted in silico with the molecular docking method using the ERα (1A52) receptor. In silico analysis was carried out on the metabolite profiling compound of the 96% ethanol extract of M. crenata leaves from the previous study. Sample preparation was carried out using the Biovia Discovery Studio 2021 application to separate macromolecules and native ligands and prepared to get a 3D structure using ChemDraw Ultra 12.0. then analyzed its pharmacokinetics and pharmacodynamics with the SwissADME webtool. Furthermore, the geometry of the compound was optimized using Avogadro 1.0.1, and molecular docking of the compound to the 1A52 receptor was carried out using Autodock vina (PyRx 0.8). The interaction visualization stage was carried out with Biovia Discovery Studio 2021, and a toxicity test was carried out using the ProTox II online tool. The results of the in-silico study showed that six compounds met the pharmacokinetic and pharmacodynamic criteria, toxicity, and had similar pharmacophore distances and amino acid binding with native 17β-estradiol, a 1A52 agonist with anti-neuroinflammatory effect. So, 96% ethanol extract of M. crenata leaves is predicted to potentially inhibit PD progression with an anti-neuroinflammatory mechanism.

Endocrine disrupting chemicals differentially alter intranuclear dynamics and transcriptional activation of estrogen receptor-α

SummaryTranscription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor.

Dual-mechanism estrogen receptor inhibitors

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Regulation of mRNA Translation by Hormone Receptors in Breast and Prostate Cancer.

Simple SummaryThe estrogen and androgen receptors (ER, AR) are key oncogenic drivers and therapeutic targets in breast and prostate cancer, respectively. These receptors bind to DNA and regulate gene expression but emerging evidence indicates that they also play important roles in controlling the process of mRNA translation, which dictates cellular protein production. Here, we review the mechanisms by which abnormal activities of ER and AR can dysregulate mRNA translation in breast and prostate cancer cells. Specifically, we explore how the intricate cellular signalling pathways that keep mRNA translation in check are perturbed by aberrant ER and AR signalling, which can lead to enhanced cancer cell growth. We also discuss the potential of targeting mRNA translation as a strategy to treat patients with breast and prostate cancer.Breast and prostate cancer are the second and third leading causes of death amongst all cancer types, respectively. Pathogenesis of these malignancies is characterised by dysregulation of sex hormone signalling pathways, mediated by the estrogen receptor-α (ER) in breast cancer and androgen receptor (AR) in prostate cancer. ER and AR are transcription factors whose aberrant function drives oncogenic transcriptional programs to promote cancer growth and progression. While ER/AR are known to stimulate cell growth and survival by modulating gene transcription, emerging findings indicate that their effects in neoplasia are also mediated by dysregulation of protein synthesis (i.e., mRNA translation). This suggests that ER/AR can coordinately perturb both transcriptional and translational programs, resulting in the establishment of proteomes that promote malignancy. In this review, we will discuss relatively understudied aspects of ER and AR activity in regulating protein synthesis as well as the potential of targeting mRNA translation in breast and prostate cancer.

Vulnerability to psychological stress‐induced anorexia in female mice depends on blockade of ghrelin signal in nucleus tractus solitarius

Background and PurposeWomen have a higher incidence of eating disorders than men. We investigated whether the effects of ghrelin on feeding are affected by sex and stress, and to elucidate the mechanisms that may cause sex differences in stress‐mediated anorexia, focusing on ghrelin.Experimental ApproachAcylated ghrelin was administered to naïve and psychologically stressed male and female C57BL/6J mice, followed by measurements of food intake and plasma hormone levels. Ovariectomy was performed to determine the effects of ovary‐derived oestrogen on stress‐induced eating disorders in female mice. The numbers of Agrp or c‐Fos mRNA‐positive cells and estrogen receptor α/c‐Fos protein‐double‐positive cells were assessed.Key ResultsGhrelin administration to naïve female mice caused a higher increase in food intake, growth hormone secretion, Agrp mRNA expression in the arcuate nucleus and c‐Fos expression in the nucleus tractus solitarius (NTS) than in male mice. In contrast, psychological stress caused a more sustained reduction in food intake in females than males. The high sensitivity of naïve females to exogenous ghrelin was attenuated by stress exposure. The stress‐induced decline in food intake was not abolished by ovariectomy. Estrogen receptor‐α but not ‐β antagonism prevented the decrease in food intake under stress. Estrogen receptor‐α/c‐Fos‐double‐positive cells in the NTS were significantly increased by stress only in females.Conclusion and ImplicationsStress‐mediated eating disorders in females may be due to blockade of ghrelin signalling via estrogen receptor‐α activation in the NTS. Targeting the ghrelin signal in the brain could be a new treatment strategy to prevent these disorders.

Ovariectomy provokes inflammatory and cardiovascular effects of endotoxemia in rats: Dissimilar benefits of hormonal supplements

The female gender is protected against immunological complications of endotoxemia. Here we investigated whether gonadal hormone depletion by ovariectomy (OVX) uncovers inflammatory and cardiovascular effects of endotoxemia and whether these effects are reversed by hormone replacement therapies. Changes in inflammatory cytokines, blood pressure (BP), left ventricular (LV) function, and cardiac autonomic activity caused by lipopolysaccharide (LPS) in conscious female rats with different hormonal states were determined. In contrast to no effects in sham-operated females, treatment of OVX rats with LPS (i) decreased BP, (ii) increased spectral low-frequency/high-frequency ratio of HRV, denoting enhanced cardiac sympathetic dominance, (iii) attenuated reflex tachycardic responses to sodium nitroprusside, and (iv) increased systolic contractility (dP/dtmax). The developed hypotension was (i) fully eliminated in estrogen (E2)-pretreated OVX rats, (ii) partially counteracted after selective activation of estrogen receptor-α (PPT) or β (DPN). All estrogenic compounds abrogated LPS enhancement of cardiac sympathetic drive. However, PPT was more successful than E2 or DPN in compromising LPS depression in baroreflex activity and elevation in dP/dtmax. Molecular studies showed that PPT was most effective in attenuating the upregulated myocardial expressions of NF-κB and iNOS in endotoxic OVX rats. Myocardial expression of the defensive HSP70 was comparably increased by all estrogenic products. Except for improved cardiac spectral activity, none of these functional or molecular entities was affected by medroxyprogesterone acetate (MPA). Overall, our data suggest diverse therapeutic advantages for gonadal hormones in the worsened endotoxic complications in rats with surgical menopause, with probably more favorable role for ERα agonism within this context.

Abstract 1883: Ovarian cancer cells overcome the cytotoxic effect of cisplatin by hyper activating CCR9-mediated signaling in response to cisplatin

Abstract Ovarian cancer (OC) has highest mortality rate among the gynecological malignancies. Chemotherapies, in combination with surgery, are standard treatment offered to the patients with advanced OC. Although 80% patients initially respond to platinum-based chemotherapy, clinical responses remain short-lived due to chemoresistance. Most patients experience relapse and 50-60% of them die within 5 years. Therefore, development of novel therapies for OC remains a high priority. Hence, the major focus of this study is to define the biology of CCR9, which is highly expressed in OC in response to platinum drug. Effect of cisplatin on CCR9 expression and impact of CCR9 activation on molecular and cellular changes involved in overcoming the effect of cisplatin was determined by flow cytometric and western blot analyses. Our data show higher expression of CCR9 and CCL25 in OC cells in response to cisplatin treatment. Additionally, our data show higher expression and activation of estrogen receptor-α (ER- α) in OC cell after CCR9 activation. In addition to this, we show CCR9-mediated expression of Smoothened (SMO) known to support stem cell like phenotype by activating Hedgehog signaling and co-expression of CD44 and CCR9 as well as higher CD44 expression in OC cells following CCL25 treatment. CCR9-CCL25 axis promoted epithelial to mesenchymal transition. These observations strongly show an association of CCR9-CCL25 axis in OC pathogenesis, therapeutic resistance, and chemotherapeutic efficacy. Citation Format: Hina Mir, Neeraj Kapur, Shailesh Singh. Ovarian cancer cells overcome the cytotoxic effect of cisplatin by hyper activating CCR9-mediated signaling in response to cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1883.

Genome-wide Estrogen Receptor-α activation is sustained, not cyclical.

Estrogen Receptor-alpha (ER) drives 75% of breast cancers. Stimulation of the ER by estra-2-diol forms a transcriptionally-active chromatin-bound complex. Previous studies reported that ER binding follows a cyclical pattern. However, most studies have been limited to individual ER target genes and without replicates. Thus, the robustness and generality of ER cycling are not well understood. We present a comprehensive genome-wide analysis of the ER after activation, based on 6 replicates at 10 time-points, using our method for precise quantification of binding, Parallel-Factor ChIP-seq. In contrast to previous studies, we identified a sustained increase in affinity, alongside a class of estra-2-diol independent binding sites. Our results are corroborated by quantitative re-analysis of multiple independent studies. Our new model reconciles the conflicting studies into the ER at the TFF1 promoter and provides a detailed understanding in the context of the ER's role as both the driver and therapeutic target of breast cancer.

The Provoked Cardiovascular and Autonomic Effects of Endotoxemia in Ovariectomized Rats Are Distinctly Affected by Estrogen and Progesterone Supplementation

The female gender is protected against immunological and cardiovascular complications of sepsis. This study investigated whether gonadal hormone depletion by bilateral ovariectomy (OVX) could uncover the cardiovascular and autonomic effects of endotoxemia and whether these effects can be reversed by hormone replacement therapies. Changes in blood pressure (BP), heart rate variability (HRV), and left ventricular (LV) function caused by i.v. administration of lipopolysaccharide (LPS, 10 mg/kg) were evaluated in conscious female rats with different hormonal states. While LPS caused no specific cardiovascular effects in intact female rats, LPS‐treated OVX rats exhibited significant decreases in blood pressure (BP) and increases in spectral low‐frequency/high‐frequency ratio (LF/HF) of HRV, denoting enhanced cardiac sympathetic dominance. Moreover, LPS increased the rate of rise in LV pressure (dP/dtmax, LV contractility) without affecting the isovolumic relaxation constant (Tau, a measure of diastolic function). LPS hypotension was totally eliminated in OVX rats pretreated with estrogen but not medroxyprogesterone acetate (MPA). Prior selective activation of estrogen receptor‐α (4,4′,4″‐(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol, PPT) or estrogen receptor‐β (Diarylpropionitrile, DPN) partially counteracted LPS hypotension. Each of the abovementioned drug treatments (estrogen, MPA, PPT, or DPN) abrogated the LPS‐evoked exaggeration in cardiac sympathetic control (LF/HF ratio). By contrast, the elevated dP/dtmax in LPS‐treated rats was largely compromised by PPT only. The data suggest differential modulatory roles for gonadal hormones (estrogen and progesterone) and estrogen receptors (α and β) on endotoxic cardiovascular and autonomic manifestations in female rats.Support or Funding InformationSupported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of Hormonal Carcinogenesis Genes and Related Regulatory microRNAs in Uterus and Ovaries of DDT-Treated Female Rats.

The insecticide dichlorodiphenyltrichloroethane (DDT) is a nonmutagenic xenobiotic compound able to exert estrogen-like effects resulting in activation of estrogen receptor-α (ERα) followed by changed expression of its downstream target genes. In addition, studies performed over recent years suggest that DDT may also influence expression of microRNAs. However, an impact of DDT on expression of ER, microRNAs, and related target genes has not been fully elucidated. Here, using real-time PCR, we assessed changes in expression of key genes involved in hormonal carcinogenesis as well as potentially related regulatory oncogenic/tumor suppressor microRNAs and their target genes in the uterus and ovaries of female Wistar rats during single and chronic multiple-dose DDT exposure. We found that applying DDT results in altered expression of microRNAs-221, -222, -205, -126a, and -429, their target genes (Pten, Dicer1), as well as genes involved in hormonal carcinogenesis (Esr1, Pgr, Ccnd1, Cyp19a1). Notably, Cyp19a1 expression seems to be also regulated by microRNAs-221, -222, and -205. The data suggest that epigenetic effects induced by DDT as a potential carcinogen may be based on at least two mechanisms: (i) activation of ERα followed by altered expression of the target genes encoding receptor Pgr and Ccnd1 as well as impaired expression of Cyp19a1, affecting, thereby, cell hormone balance; and (ii) changed expression of microRNAs resulting in impaired expression of related target genes including reduced level of Cyp19a1 mRNA.

Schizandra chinensis exhibits phytoestrogenic effects by regulating the activation of estrogen receptor-α and -β.

To investigate the phytoestrogenic effects of Schizandra chinensis (SC) extract by regulating the activation of estrogen receptor. Western blotting assay was performed to investigate the effect of SC extract (1, 10, 100 μg/mL) on the expression of estrogen receptor (ER)-α and -β in MCF-7 human breast cancer cells. Cell viability and the levels of c-fos and c-Jun were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis, respectively, to further confirm the anti-cancer effect of SC extract. SC extract increased the expressions of ER-α and -β (P<0.001), whereas cell viability and the expressions of growth factors (c-fos and c-Jun) were inhibited (P< and <0.001, respectively) following treatment. SC extract has phytoestrogenic effects, and its biological action includes ER binding ability with low cancer risk. Therefore, SC might be a potential source for the development of a new alternative to hormone therapy in menopause.

PI3K in the ventromedial hypothalamic nucleus mediates estrogenic actions on energy expenditure in female mice.

Estrogens act in the ventromedial hypothalamic nucleus (VMH) to regulate body weight homeostasis. However, the molecular mechanisms underlying these estrogenic effects are unknown. We show that activation of estrogen receptor-α (ERα) stimulates neural firing of VMH neurons expressing ERα, and these effects are blocked with intracellular application of a pharmacological inhibitor of the phosphatidyl inositol 3-kinase (PI3K). Further, we demonstrated that mice with genetic inhibition of PI3K activity in VMH neurons showed a sexual dimorphic obese phenotype, with only female mutants being affected. In addition, inhibition of VMH PI3K activity blocked effects of 17β-estradiol to stimulate energy expenditure, but did not affect estrogen-induced anorexia. Collectively, our results indicate that PI3K activity in VMH neurons plays a physiologically relevant role in mediating estrogenic actions on energy expenditure in females.

Regulation of hematopoietic progenitors by estrogens as a basis for new antileukemic strategies

We recently reported that estrogens regulate survival, proliferation, and self-renewal of hematopoietic stem cells and progenitors via estrogen receptor-α activation. Through its proapoptotic effect on malignant progenitors, tamoxifen treatment blocks the development of JAK2V617F-induced myeloproliferative neoplasms in mice and sensitizes MLL-AF9-induced leukemias to chemotherapy, without detrimental effects on normal hematopoiesis.

G protein-coupled estrogen receptor regulates mammary tumorigenesis and metastasis.

The role of 17β-estradiol (E2) in breast cancer development and tumor growth has traditionally been attributed exclusively to the activation of estrogen receptor-α (ERα). Although targeted inhibition of ERα is a successful approach for patients with ERα(+) breast cancer, many patients fail to respond or become resistant to anti-estrogen therapy. The discovery of the G protein-coupled estrogen receptor (GPER) suggested an additional mechanism through which E2 could exert its effects in breast cancer. Studies have demonstrated clinical correlations between GPER expression in human breast tumor specimens and increased tumor size, distant metastasis, and recurrence, as well as established a proliferative role for GPER in vitro; however, direct in vivo evidence has been lacking. To this end, a GPER-null mutation [GPER knockout (KO)] was introduced, through interbreeding, into a widely used transgenic mouse model of mammary tumorigenesis [MMTV-PyMT (PyMT)]. Early tumor development, assessed by the extent of hyperplasia and proliferation, was not different between GPER wild-type/PyMT (WT/PyMT) and those mice harboring the GPER-null mutation (KO/PyMT). However, by 12 to 13 weeks of age, tumors from KO/PyMT mice were smaller with decreased proliferation compared with those from WT/PyMT mice. Furthermore, tumors from the KO/PyMT mice were of histologically lower grade compared with tumors from their WT counterparts, suggesting less aggressive tumors in the KO/PyMT mice. Finally, KO/PyMT mice displayed dramatically fewer lung metastases compared with WT/PyMT mice. Combined, these data provide the first in vivo evidence that GPER plays a critical role in breast tumor growth and distant metastasis. This is the first description of a role for the novel estrogen receptor GPER in breast tumorigenesis and metastasis, demonstrating that it represents a new target in breast cancer diagnosis, prognosis, and therapy.

Delay in post‐ovariectomy estrogen replacement negates post‐exercise estrogen augmentation of muscle satellite cell population in rats (1163.23)

17β‐Estradiol (E2) enhances activation, proliferation and differentiation of muscle satellite cells (SCs) following exercise via activation of estrogen receptor‐α (ER) and phosphatidylinositol 3‐kinase (PI3K) signaling. Supplementation of E2 through hormone replacement has been shown to ameliorate muscle atrophic effects of menopause in women. Delay in post‐menopause E2 replacement in humans or following ovariectomy in rodents has resulted in negation of positive health or regenerative effects of E2 on various tissues. To determine if delay in E2 replacement influenced E2 effects on skeletal muscle, 64, nine‐week old, ovariectomized Sprague‐Dawley rats were divided into eight groups based on: E2 status (0.25 mg estrogen pellet or sham), exercise status (90 min run @ 17 m/min, ‐13.5° or unexercised) and E2 replacement (“proximal”; E2 replacement within one week or “delayed”; eleven‐weeks following ovariectomy). Significant increases in total SCs were found in soleus muscle (immunofluorescent co‐localization of nuclei with Pax7) 72 hr following eccentric exercise (p &lt; 0.05). Only “proximal” estrogen‐replacement resulted in further enhancement in total muscle SCs in exercised rats (p &lt; 0.05). Delayed estrogen‐supplementation did not produce a further augmentation in post‐exercise SCs. This suggests the existence of a short window of opportunity where E2 replacement must commence following loss of endogenous estrogen production in order for benefits to skeletal muscle regeneration to manifest.Grant Funding Source: Supported by NSERC

Diallyl trisulfide inhibits estrogen receptor-α activity in human breast cancer cells

Organosulfur compounds from garlic effectively inhibit growth of transplanted as well as spontaneous cancers in preclinical animal models without any adverse side effects. However, the mechanisms underlying anticancer effect of this class of compounds are not fully understood. This study reports, for the first time, that garlic organosulfide diallyl trisulfide (DATS) inhibits estrogen receptor-α (ER-α) activity in human breast cancer cells. Exposure of MCF-7 and T47D cells to DATS resulted in downregulation of ER-α protein, which peaked between 12- and 24-h post-treatment. DATS was relatively more effective in suppressing ER-α protein expression compared with its mono and disulfide analogs. The 17β-estradiol (E2)-induced expression of pS2 and cyclin D1, ER-α target gene products, was also decreased in the presence of DATS. Downregulation of ER-α protein expression resulting from DATS treatment was accompanied by a decrease in nuclear levels of ER-α protein, ER-α mRNA suppression, and inhibition of ERE2e1b-luciferase reporter activity. DATS-mediated inhibition of cell viability and apoptosis induction were not affected in the presence of E2. In agreement with these results, ectopic expression of ER-α in MDA-MB-231 cell line failed to confer any protection against cell proliferation inhibition or apoptosis induction resulting from DATS exposure. DATS treatment caused a decrease in protein levels of peptidyl-prolyl cis-trans isomerase (Pin1), and overexpression of Pin1 partially attenuated ER-α downregulation by DATS. DATS-induced apoptosis was modestly but significantly augmented by overexpression of Pin1. In conclusion, this study identifies ER-α as a novel target of DATS in mammary cancer cells.

Insulin-like growth factor binding protein-4 and -5 modulate ligand-dependent estrogen receptor-α activation in breast cancer cells in an IGF-independent manner

Insulin-like growth factor binding proteins (IGFBPs) are modulators of numerous cellular processes including cell proliferation. Although IGFBPs classically act by sequestration of extracellular insulin-like growth factors (IGFs), thereby contributing to the fine-tuning of growth factor signals, IGF-independent actions of IGFBPs have also been described. In the breast, growth factor signaling in association with estradiol (E2)-stimulated estrogen receptor function is organized in a complex cross-talk. The importance of phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) pathway components for the E2-induced activation of estrogen receptor-alpha (ERα) is well accepted. Here we show that in the absence of IGFs, IGFBP-4 or IGFBP-5, either overexpressed in MCF-7 breast cancer cells or added exogenously, decreased the capability of E2 to induce ERα transcriptional activity. In addition, overexpression or addition of recombinant IGFBP-4 or IGFBP-5 resulted in reduction of E2-induced phosphorylation of Akt/PKB, GSK-3α/β and ERα in MCF-7 cells. The activation of the Akt/PKB-pathway describes a non-genomic effect of E2, which did not involve activation/phosphorylation of the IGF-I receptor (IGF-IR). Furthermore, knockdown of the IGF-IR did not affect the inhibition of E2-induced ERα phosphorylation by IGFBP-4 and 5. Moreover, IGFBP-4 and IGFBP-5 strongly decreased E2-triggered growth of MCF-7 cells. Our data suggest that IGFBPs interfere with the E2-induced activation of the Akt/PKB-pathway and prevent full hormone-dependent activation of ERα and breast cancer cell growth in an IGF- and IGF-IR-independent manner.

Gender differences in brain susceptibility to oxidative stress are mediated by levels of paraoxonase-2 expression

Paraoxonase 2 (PON2), a member of a gene family that also includes PON1 and PON3, is expressed in most tissues, including the brain. In mouse brain, PON2 levels are highest in dopaminergic areas (e.g., striatum) and are higher in astrocytes than in neurons. PON2 is primarily located in mitochondria and exerts a potent antioxidant effect, protecting mouse CNS cells against oxidative stress. The aim of this study was to characterize PON2 expression and functions in the brains of male and female mice. Levels of PON2 (protein, mRNA, and lactonase activity) were higher in brain regions and cells of female mice. Astrocytes and neurons from male mice were significantly more sensitive (by 3- to 4-fold) to oxidative stress-induced toxicity than the same cells from female mice. Glutathione levels did not differ between genders. Importantly, no significant gender differences in susceptibility to the same oxidants were seen in cells from PON2−/− mice. Treatment with estradiol induced a time- and concentration-dependent increase in the levels of PON2 protein and mRNA in male (4.5-fold) and female (1.8-fold) astrocytes, which was dependent on activation of estrogen receptor-α. In ovariectomized mice, PON2 protein and mRNA were decreased to male levels in brain regions and in liver. Estradiol protected astrocytes from wild-type mice against oxidative stress-induced neurotoxicity, but did not protect cells from PON2−/− mice. These results suggest that PON2 is a novel major intracellular factor that protects CNS cells against oxidative stress and confers gender-dependent susceptibility to such stress. The lower expression of PON2 in males may have broad ramifications for susceptibility to diseases involving oxidative stress, including neurodegenerative diseases.