Selectively targeting estrogen receptors for cancer treatment

Estrogens regulate growth and development through the action of two distinct estrogen receptors (ERs), ERα and ERβ, which mediate proliferation and differentiation of cells. For decades, ERα mediated estrogen signaling has been therapeutically targeted to treat breast cancer, most notably with the selective estrogen receptor modulator (SERM) tamoxifen. Selectively targeting ERs occurs at two levels: tissue selectivity and receptor subtype selectivity. SERMs have been developed with emphasis on tissue selectivity to target ER signaling for breast cancer treatment. Additionally, new approaches to selectively target the action of ERα going beyond ligand-dependent activity are under current investigation. As evidence of the anti-proliferative role of ERβ accumulates, selectively targeting ERβ is an attractive approach for designing new cancer therapies with the emphasis shifted to designing ligands with subtype selectivity. This review will present the mechanistic and structural features of ERs that determine tissue and subtype selectivity with an emphasis on current approaches to selectively target ERα and ERβ for cancer treatment.     

丹参酮ⅡA抗乳腺癌细胞增殖作用研究

目的利用雌激素受体(estrogenic receptor,ER)阳性乳腺癌T47D细胞和ER阴性乳腺癌MDA-MB-231细胞观察丹参酮ⅡA(TanshinoneⅡA)对细胞增殖活性的影响及其对雌激素受体亚型的调节功能。方法以ER拮抗剂ICI182,780为工具药,采用MTT细胞增殖实验观察1×10-6mol.L-1和1×10-7mol.L-1丹参酮ⅡA对T47D和MDA-MB-231细胞增殖的影响。利用实时荧光定量PCR法及流式细胞术检测其对T47D细胞ERα和ERβmRNA及蛋白表达情况的影响。结果丹参酮ⅡA能够抑制T47D细胞增殖,且该作用可被ICI182,780部分拮抗;对MDA-MB-231细胞增殖的抑制作用较其对T47D细胞的作用更为明显。丹参酮ⅡA可使T47D细胞ERα和ERβmRNA和蛋白表达明显增加,并使ERα/ERβ比值有所上升。结论丹参酮ⅡA具有抑制乳腺癌细胞增殖的作用,抑制强度与对其ER亚型的调节作用相关。     

Validation of a new yeast‐based reporter assay consisting of human estrogen receptors α/β and coactivator SRC‐1: Application for detection of estrogenic activity in environmental samples

Endocrine disruptors are exogenous substances that act like hormones in the endocrine system and disrupt the physiologic function of endogenous hormones. In the present study, we established reporter yeast strains (Saccharomyces cerevisiae) expressing human estrogen receptors, ERα or ERβ. These strains contain a reporter plasmid carrying an estrogen responsive element (ERE) upstream of the β‐galactosidase gene, and a plasmid expressing a steroid receptor coactivator, SRC‐1e. Using these reporter strains, we demonstrated dose‐dependent estrogenic activities of different categories of ligands, a natural hormone, 17β‐estradiol (E2); a synthetic drug, diethylstilbestrol (DES); phytoestrogens, genistein, daizein and emodin; and an environmental endocrine disrupter, bisphenol A. EC₅₀ values of E2 for ERα and ERβ are 5.31 × 10^?10 and 5.85 × 10^?10 M, respectively. We also demonstrated that these yeasts were applicable for measuring estrogenic activities of environmental water samples. Most downstream sites of a river showed similar activity in both ERα and ERβ assays. These yeast strains are useful and convenient for detecting and comparing the estrogenic ligand activities of environmental samples in response to ERα and ERβ. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Novel estrogen receptor ligands and their structure–activity relationship evaluated by scintillation proximity assay for high‐throughput screening

The estrogen receptor (ER) is an important drug target with allosteric characteristics that binds orthotopic hormones and other ligands. A recently developed scintillation proximity (SPA)‐based assay for high‐throughput screening (HTS) of compound libraries was used to identify novel estrogen receptor ligands that might have ER subtype selective binding activity. Radioligand binding was determined in a multi‐detector scintillation counter designed for microtitration plates. Equilibrium binding experiments and kinetic competition tests were performed with [³H]‐estradiol and human ERα and ERβ receptors. A library of 6,000 structurally diverse compounds was screened. From this, several novel ligands were identified that showed pronounced subtype‐selective differences in ligand binding for ERα and ERβ. The observed equilibrium dissociation constant (K_d) for the binding of [³H]estradiol to ERα and ERβ receptors were approximately 0.25 and 0.64 nM, respectively. When 17β‐estradiol, raloxifene and daidzein were tested for binding affinity to ERα in a competition assay, the IC₅₀ values were 0.34, 1.31, and 75.6 nM, respectively. When tested for binding affinity to ERβ, the IC₅₀ values were 1.05, 11.4, and 10.6 nM, respectively. The results obtained show that the methodology is valid in comparison to previously published data regarding estradiol and other standard compounds (raloxifene and daidzein) binding characteristics of estrogen receptors. The assay is also well suited to applied research as a tool in HTS of compound libraries in the search of ER ligands. Several novel active compounds were identified and selected as potent ER subtype ligands. Drug Dev Res 64:203–212, 2005. © 2005 Wiley‐Liss, Inc.     

Effects of dehydroepiandrosterone on proliferation, migration, and death of breast cancer cells

Cancer invasion and metastasis are the leading causes of mortality in patients with breast cancer. Dehydroepiandrosterone (DHEA) has a protective role against cancer, however, the mechanism by which DHEA has this effect remains poorly understood. The present study was aimed at investigating the actions of DHEA on the proliferation, cell cycle, death and migration of breast cancer cell lines. We used MCF-7 cells (estrogen receptors positive) and MDA-MB-231 and Hs578T cells (estrogen receptors negative) for these studies. Cell proliferation was evaluated by crystal violet staining, cell cycle by flow cytometry, and cell death by the carboxyfluorescein FLICA analysis of caspase activation. Migration was evaluated by transwell cell migration and wound assay. We also determined the expression of ECM-1 protein by western blotting and RT-PCR in real time. DHEA inhibited the proliferation of all breast cancer cell lines. This was associated with an arrest in the G1 phase of the cell cycle and death in MCF-7 cells. There was no alteration in any of the cell cycle phases or death in DHEA treated MDA-MB-231 or Hs578T cells. DHEA also suppressed the migration of all breast cancer cell lines, independently of the presence of estrogen receptors and decreased the expression of ECM-1 protein in Hs578T cells. These results suggest that the mechanism of DHEA actions against breast cancer involves the inhibition of cell proliferation and the suppression of migration, indicating that DHEA could be useful in the treatment of breast cancer.     

槲皮素、补骨脂素对人乳腺癌T47D细胞两种受体亚型表达的影响

目的探讨槲皮素和补骨脂素的雌激素作用以及调控雌激素受体亚型表达的作用机制。方法10μmol.L-1槲皮素和10μmol.L-1补骨脂素分别处理T47D细胞48 h,采用RT-PCR和Western blot方法测定对雌激素依赖性乳腺癌细胞T47D雌激素受体亚型ERα和ERβ表达的影响,并以雌激素受体拮抗剂ICI182,780为工具药来评价槲皮素和补骨脂素发挥雌激素样作用与雌激素受体的关系。结果槲皮素、补骨脂素在10μmol.L-1可明显诱导T47D细胞ERαmRNA和蛋白表达,而对ERβ表达没有影响。当与ICI182,780共孵育T47D细胞ERα表达被拮抗。结论槲皮素、补骨脂素产生的促进ER阳性细胞增殖的雌激素样作用是通过增加ERα表达实现的。     

人雌激素受体β绿色荧光蛋白真核表达载体的构建与表达

目的构建人雌激素受体β(ERβ)绿色荧光蛋白(GFP)真核表达载体,并稳定转染乳腺癌MCF-7细胞。方法通过双酶切将pCMV5-hERβ中的人ERβcDNA克隆到pEGFP-C3中,构建并鉴定重组质粒pEGFP-hERβ,然后转染乳腺癌MCF-7细胞,采用G418(500μg/mL)筛选出稳定转染的细胞系,在荧光显微镜下观察融合蛋白质在真核细胞内的表达分布,并采用逆转录-聚合酶链反应(RT-PCR)检测ERβ的mRNA转录水平。结果成功构建重组表达载体pEGFP-hERβ,有效转染并筛选出稳定转染ERβ基因的MCF-7细胞系,外源性ERβ基因在MCF-7细胞中获得了有效转录,并在细胞质和细胞核中均广泛分布。结论建立了稳定表达ERβ的MCF-7细胞系,为筛选ERβ调节剂和进一步研究ERβ在乳腺癌中的作用及其机制奠定基础。     

E0703对雌激素受体(ER)不同亚型的选择性激活作用

目的探讨E0703(雌二醇衍生物)对雌激素受体不同亚型(α和β)的选择性激活作用。方法将报告基因质粒pTAL-SEAP-ERE和校正质粒pSV-β-gal共转染已稳定转染了雌激素受体α/β的人胚肾细胞(HEK293),加入E0703进行报告基因的诱导表达。采用EMSA试验观察E0703对ERα与雌激素反应元件(ERE)的结合增强作用。结果E0703对ERα具有较强的激活作用(最低激活浓度为10-8mol·L-1),激活强度略低于E2;E0703在10-10、10-9、10-8、10-7mol·L-1浓度下对ERβ没有激活作用;EMSA试验证明,E0703能够增强ERα与雌激素反应元件的结合增强作用。结论E0703对雌激素受体α具有选择性激活作用。     

槲皮素、补骨脂素对乳腺癌细胞株MCF-7增殖的影响

目的探讨槲皮素(quercetin,Que)和补骨脂素(psor-alen,Pso)对人类乳腺癌细胞株增殖的影响。方法采用流式细胞术和蛋白印迹法检测槲皮素和补骨脂素对雌激素依赖性乳腺癌细胞MCF-7的影响,并以雌激素受体拮抗剂ICI182,780为工具药来评价槲皮素和补骨脂素发挥雌激素样作用与雌激素受体的关系。结果①槲皮素、补骨脂素在10μmol.L-1可使MCF-7细胞增殖指数明显升高,增加S期细胞的比例,与10-3μmon.L-1E2阳性对照组的变化趋势一致。当ICI182,780分别与E2、金雀异黄素、槲皮素、补骨脂素共孵育48 h后,E2、金雀异黄素、槲皮素、补骨脂素的增殖效应被抑制,细胞周期S期细胞数比例下降,G0/G1期细胞数比例上升。②10μmol.L-1槲皮素和10μmol.L-1补骨脂素均上调MCF-7细胞ERα蛋白水平,而对ERβ蛋白表达没有影响;当分别与ICI182,780共孵育MCF-7细胞ERα蛋白表达被拮抗。结论槲皮素和补骨脂素具有雌激素活性,此作用是通过雌激素受体(ER)介导的;产生的类似金雀异黄素促进MCF-7细胞增殖的作用是通过增加ERα表达实现的。     

Amygdalin Regulates Apoptosis and Adhesion in Hs578T Triple-Negative Breast Cancer Cells

Amygdalin, D-mandelonitrile-β-D-glucoside-6-β-glucoside, belongs to aromatic cyanogenic glycoside group derived from rosaceous plant seed. Mounting evidence has supported the anti-cancer effects of amygdalin. However, whether amygdalin indeed acts as an anti-tumor agent against breast cancer cells is not clear. The present study aimed to investigate the effect of amygdalin on the proliferation of human breast cancer cells. Here, we show that amygdalin exerted cytotoxic activities on estrogen receptors (ER)-positive MCF7 cells, and MDA-MB-231 and Hs578T triple-negative breast cancer (TNBC) cells. Amygdalin induced apoptosis of Hs578T TNBC cells. Amygdalin downregulated B-cell lymphoma 2 (Bcl-2), upregulated Bcl-2-associated X protein (Bax), activated of caspase-3 and cleaved poly ADP-ribose polymerase (PARP). Amygdalin activated a pro-apoptotic signaling molecule p38 mitogen-activated protein kinases (p38 MAPK) in Hs578T cells. Treatment of amygdalin significantly inhibited the adhesion of Hs578T cells, in which integrin α5 may be involved. Taken together, this study demonstrates that amygdalin induces apoptosis and inhibits adhesion of breast cancer cells. The results suggest a potential application of amygdalin as a chemopreventive agent to prevent or alleviate progression of breast cancer, especially TNBC.     

Estrogenic and anti‐estrogenic activity of butylparaben,butylated hydroxyanisole,butylated hydroxytoluene and propyl gallate and their binary mixtures on two estrogen responsive cell lines (T47D‐Kbluc,MCF‐7)

The estrogenic and anti‐estrogenic effects of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) were evaluated for individual compounds as well as for binary mixtures, using an estrogen‐dependent reporter gene assay in T47D‐Kbluc breast cancer cells and an estrogen‐dependent proliferation assay in MCF‐7 breast cancer cells. In terms of estrogenicity the potency of the selected compounds increased from BHA < PG < BuPB in the luciferase assay (with BHT showing no significant estrogenic activity), while in the proliferation assay the following order was observed: BHT < BHA < BuPB (with PG showing no significant estrogenic activity). Non‐monotonic dose–response curves were obtained for BuPB (in both assays) and PG (in the luciferase assay), respectively. In the presence of estradiol, a significant anti‐estrogenic activity was observed in both cell lines for PG, BuPB and BHA, while BHT showed weak anti‐estrogenic activity only in T47D‐Kbluc cells. The evaluation of binary mixtures confirmed the endocrine disruptive potential of the compounds, their individual potency being correlated with that of the mixtures. All mixtures were able to reduce the estradiol‐induced luminescence or cell proliferation, an effect that was accurately predicted by the dose addition mathematical model, suggesting the same (or at least partially overlapping) modes of action for the tested compounds. The results of the present study emphasize the importance of a cumulative risk assessment of endocrine disruptors.     

ER alpha selective chromone,isoxazolylchromones, induces ROS‐mediated cell death without autophagy

Chromones are recognized as privileged structures and useful templates for the design of novel compounds with promising pharmacological activity. Several reports implicate chromone scaffold as an antitumor agent. The present study highlights synthesis, docking, and potential activity of isoxazolylchromones, 3(a–f), a new class of compounds as potential agents exhibiting ERα antagonism and ERβ agonism. Molecular docking studies determined the binding site of compounds 3(a–f) in ERα and ERβ. All the analogues synthesized showed preferential cytotoxicity in ERα+ cell line (MCF‐7) compared to ERα‐ cell line (MDA‐MB‐231). Among the analogues synthesized, analogue 3d exhibited increased cytotoxicity. ERα silencing experiments confirmed the ERα selective nature of ligands. Transactivation assay on compound 3d indicated the down‐regulation of ERα luciferase reporter gene expression and induction of ERβ GFP in the treated cells. Cell cycle analysis revealed an increase in sub‐G0/G1 population on treatment with analogue 3d as compared to control. Similar to tamoxifen, 3d ‐induced cell death is mediated through an increase in ROS as evidenced by change in roGFP ratio. Interestingly, the compound 3d induced mitochondrial trans‐membrane potential loss and caspase activation without indication of autophagy compared to tamoxifen that induced autophagy in the treated cells. Lack of significant autophagy and induction of ERβ signaling by the new compound place them as a better ERα antagonist.     

A-CD estrogens. I. Substituent effects, hormone potency, and receptor subtype selectivity in a new family of flexible estrogenic compounds

Long-term use of estrogen supplements by women leads to an increased risk of breast and uterine cancers. Possible mechanisms include metabolism of estradiol and compounds related to tumor-initiating quinones, and ligand-induced activation of the estrogen receptors ERα and ERβ which can cause cancer cell proliferation, depending on the ratio of receptors present. One therapeutic goal would be to create a spectrum of compounds of variable potency for ERα and ERβ, which are resistant to quinone formation, and to determine an optimum point in this spectrum. We describe the synthesis, modeling, binding affinities, hormone potency, and a measure of quinone formation for a new family of A-CD estrogens, where the A-C bond is formed by ring coupling. Some substituents on the A-ring increase hormone potency, and one compound is much less quinone-forming than estradiol. These compounds span a wide range of receptor subtype selectivities and may be useful in hormone replacement therapy.     

4-氨基-2-三氟甲基苯基维甲酸酯对MCF-7细胞增殖和分化的影响及其机制研究

目的研究4-氨基-2-三氟甲基苯基维甲酸酯(4-ami-no-2-trifluoromethyl-phenyl retinate,ATPR)对人乳腺癌MCF-7细胞增殖和分化的作用及其可能机制。方法不同浓度的ATPR作用MCF-7细胞后,绘制细胞生长曲线,分析细胞增殖情况;瑞氏-吉姆萨染色法观察细胞形态学改变;酶联免疫法(ELISA法)检测粘蛋白(mucin 1,MUC-1)活性;RT-PCR法检测维甲酸受体(RARα、RARβ、RARγ)、维甲酸受体诱导基因1(RRIG1)、雌激素受体(ERα、ERβ)mRNA的表达;Western blot法检测RARα、RARβ、RARγ蛋白的表达。结果 ATPR明显抑制MCF-7细胞增殖,且随浓度和时间增加而逐渐增强;镜下观察ATPR作用72 h后MCF-7细胞形态趋向正常细胞分化;ELISA结果显示ATPR明显降低MCF-7细胞培养上清MUC-1浓度(P<0.05);ATPR作用MCF-7细胞72 h后,RARβ、RRIG1、ERβ表达增强(P<0.05),RARγ表达下调(P<0.05),RARα和ERα表达则无明显变化。结论 ATPR可明显抑制MCF-7细胞增殖并诱导其分化程度增高,其机制可能与调节维甲酸受体和雌激素受体平衡,并上调RRIG1表达有关。     

Differential mechanisms of action are involved in chlorpyrifos effects in estrogen-dependent or -independent breast cancer cells exposed to low or high concentrations of the pesticide

It has reported that many environmental compounds may display estrogenic actions and these findings led to researchers to associate breast cancer risk with the use of some pesticides. The aim of this work was to investigate the effect of chlorpyrifos (CPF) on cell proliferation and the ERα-dependence of this action employing MCF-7 and MDA-MB-231 breast cancer cell lines. We have also analyzed CPF action on the cell cycle distribution and the cyclins that are implicated in G1-S and intra-S checkpoints. Finally, the action on cell death and ROS production were studied. We demonstrated the ability of CPF 0.05μM to induce cell proliferation through ERα in hormone-dependent breast cancer cells. In contrast, CPF 50μM induces intra-S arrest modifying checkpoints proteins, through a mechanism that may involve changes in redox balance in MCF-7. In MDA-MB-231, we have found that CPF 50μM produces an arrest in G2/M phase which could be related to the capacity of the pesticide for binding to tubulin sites altering microtubules polymerization. Altogether, our results provide new evidences on the action of the pesticide CPF as an environmental breast cancer risk factor due to the effects that causes on the mechanisms that modulate breast cell proliferation.