Benzothiepin-derived molecular scaffolds for estrogen receptor modulators: synthesis and antagonistic effects in breast cancer cells

A series of novel benzothiepin-derived compounds are described as potent selective modulators of the human estrogen receptor (SERMs). The objective of the study is to evaluate the antiproliferative effects of the compounds on human MCF-7 breast tumor cells. These heterocyclic compounds contain the traditional triarylethylene arrangement exemplified by tamoxifen, conformationally restrained through the incorporation of the benzothiepin ring system. The compounds demonstrated potency at nanomolar concentrations in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity. The compounds exhibited low nanomolar binding affinity for the estrogen receptor (ER) with some specificity for ERβ, and also demonstrate potent antiestrogenic properties in the human uterine Ishikawa cell line. The effect of a number of functional group substitutions on the ER binding properties of the benzothiepin molecular scaffold is explored through a brief computational structure-activity relationship investigation with molecular simulation.     

雌激素受体α真核表达载体的构建及其转录活性

目的：构建雌激素受体α（ERα）高效真核表达载体,并检测其转录活性。方法：用常规PCR方法特异扩增带有Flag标签的人ERα全长编码序列,经双酶切后将其克隆到真核表达载体pIRESpuro2中,构建重组质粒pIRESpuro2-Flag-ERα;用Westernblot鉴定该重组质粒介导的Flag-ERα在人胚肾细胞株293T中的表达情况;用含雌激素应答元件的报告基因系统检测ERα的转录活性。结果：构建了pIRESpuro2-Flag-ERα重组质粒,该质粒介导的融合蛋白在293T细胞中得到表达,并可以激活含雌激素应答元件的报告基因的活性。结论：ERα高效真核表达载体的构建为进一步研究ERα在乳腺癌中的作用奠定了基础。     

ERα与Sp1相互作用激活LRP16启动子转录活性

根据已报道的LRP1 6启动子序列 (2 6kb) ,采用PCR反应获得 6个启动子 5′删除突变体 ,分别插入pGL3 Basic载体 ,构建 6种 5′缺失报告基因表达载体 (pS1 ～pS6) .分别与ERα真核表达载体共转染MCF 7细胞 .用双荧光素酶报告系统测定荧光素酶活性 ,以明确LRP1 6基因上游启动子区域中的雌激素反应序列 .结果显示 ,pS1 ～pS6均有雌二醇反应性 .进而对pS5的 3′端缺失分析发现LRP1 6基因翻译起始位点上游 - 2 1 4至 - 2 5 1位置的序列具有雌激素应答 ,序列分析发现该片段序列中包含了一个供转录因子Sp1结合的GC富含位点和一个ERα反应元件的半位点 (1 2ERE Sp1 ) ,进一步突变分析显示这两个元件均为雌激素反应性所必需 .以含这两个顺式元件的序列 (- 2 5 3bp至 - 2 2 4bp)作为探针 ,超级迁移凝胶电泳试验结果表明了ERα和Sp1蛋白均可以和探针结合 .研究发现了雌激素上调LRP1 6基因表达的一个增强子元件— 1 2ERE Sp1 ,ERα和Sp1蛋白需要与DNA结合形成复合体 ,通过其相互作用激活转录     

GAS6 is an estrogen-inducible gene in mammary epithelial cells

To identify estrogen-responsive genes in mammary glands, microarray assays were performed. Twenty genes were found to be up-regulated while 16 genes were repressed in the 9h estrogen treated glands. The induction of GAS6, one of the genes up-regulated by estrogen, was confirmed by RNase protection assay. Furthermore, GAS6 was also demonstrated to be induced by estrogen in ER positive breast cancer cells. Analysis of GAS6 promoter revealed that GAS6 promoter was regulated by estrogen. An estrogen response element (ERE) was identified in the GAS6 promoter. Electrophoretic mobility shift assay revealed that ERalpha interacted with the ERE in the GAS6 promoter. Chromatin immunoprecipitation demonstrated that ERalpha was recruited to the GAS6 promoter upon estrogen stimulation. These results suggested that GAS6 is an estrogen target gene in mammary epithelial cells.     

Psoralidin,a coumestan analogue,as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERβ agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERβ. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10 μM was able to induce the maximum reporter gene expression corresponding to that of E₂-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin–ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E₂, and thus, it could act as an ER agonist in the cellular environment.     

雌激素受体β潜在磷酸化位点突变体的构建及其活性测定

目的：构建雌激素受体（ER）β潜在磷酸化位点突变体,并在人胚肾细胞293T中检测其对ERβ下游基因转录的影响。方法：通过ERα和ERβ序列同源性比对,寻找ERβ的潜在磷酸化位点;以pcDNA3-ERβ-FLAG为模板,通过重组PCR,将ERβ264位、469位Ser编码基因突变为Ala编码基因,将突变片段连接到同样双酶切的pcDNA3-FLAG载体中,Western blot检测其表达;将重组质粒转入293T细胞中,检测突变体对含雌激素应答元件（ERE）的报告基因转录活性的影响。结果：ERα和ERβ序列同源性比对发现ERβ的264位Ser和469位Ser可能是其潜在的磷酸化位点;构建了ERβ264位和469位2个点突变体载体pcDNA3-FLAG-ERβ（S264A）和pcDNA3-FLAG-ERβ（S469A）,Western blot可检测到ERβ突变体融合蛋白在293T细胞中表达。萤光素酶活性检测表明,在没有雌激素刺激的情况下,2个突变体的活性较野生型没有变化;加入雌激素后,突变体的活性较野生型略有升高。结论：ERβ的264位和469位Ser位点的磷酸化可能不是ERβ调节下游基因转录所必需的,活性升高可能是由于ERβ构象变化造成的。     

Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.