pS2 Expression Research Articles

Total ginsenosides decrease Aβ production through activating PPARγ

IntroductionTotal ginsenosides (TG), the major active constituents of ginseng, have been proven to be beneficial in treatment of Alzheimer’s disease (AD). However, the underlying mechanism of TG remains unclear. MethodsAPP/PS1 mice and N2a/APP695 cells were used as in vivo and in vitro model, respectively. Morris water maze (MWM) was used to investigate behavioral changes of mice; neuronal pathological changes were assessed by hematoxylin and eosin (H&E) and nissl staining; immunofluorescence staining was used to examine amyloid beta (Aβ) deposition; Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to examine the expression of relative amyloidogenic genes and proteins. Moreover, the antagonist of PPARγ, GW9662, was used to determine whether the effects of TG on Aβ production were associated with PPARγ activity. ResultsTG treatment increased the spatial learning and memory abilities of APP/PS1 mice while decreasing the Aβ accumulation in the cortex and hippocampus. In N2a/APP695 cells, TG treatment attenuated the secretion of Aβ1–40 and Aβ1–42 acting as an PPARγ agonist by inhibiting the translocation of NF-κB p65. Additionally, TG treatment also decreased the expression of amyloidogenic pathway related gene BACE1, PS1 and PS2. ConclusionsTG treatment reduced the production of Aβ both in vivo and in vitro. Activating PPARγ might be a potential therapeutic target of TG in facilitating Aβ clearance and ameliorating cognitive deficiency in APP/PS1 mice.

Phloretin Inhibits the Proliferation of Breast Cancer Cells Through the Down-regulation of Estrogen Receptor α.

Phloretin is a natural flavonoid compound found in some plants, such as apples and pears, as well as in the bark of apple trees. Phloretin has been shown to have inhibitory effects on glucose transporters in cells and can potentially inhibit the growth of cancer cells. However, the mechanism by which phloretin regulates the expression of estrogen receptor alpha (ERα), a key transcription factor in breast cancer, is still unclear. This study investigated how phloretin affects the growth of ERα positive human breast cancer cells. The growth of breast cancer cell lines, including MCF7 and T47D, was examined using cell proliferation and colony formation assays. Western blotting and semi-quantitative RT-PCR were used to examine protein and mRNA levels, respectively. Localization of cellular proteins was analyzed using subcellular fractionation. Transient transfection and reported gene assays were used to elucidate the impact of phloretin on cell proliferation and ERα transactivation. Phloretin decreased ERα expression at the mRNA and protein levels in MCF7 and T47D cells. It also inhibited the binding of ERα to the estrogen response element present in the promoter of target genes. Moreover, treatment with phloretin inhibited the expression of cyclin D1 and breast cancer marker gene pS2, which are known ERα target genes. Consequently, it inhibited the growth of ERα-positive human breast cancer cells. Furthermore, inhibition of breast cancer growth by phloretin was found to be mediated through both the ERα and ERK1/ERK2 pathways. Phloretin, a dihydrochalcone extracted from natural sources, exhibits the ability to regulate ERα function and suppress breast cancer cell proliferation.

The effect of hesperetin on estrogen receptor gene expression and its relationship with the downstream pathways of estrogen receptor alpha.

Estrogen receptor (ER) is a transcription factor that affects the expression of some genes involved in the progression and development of breast cancer (BC). Hesperetin (Hst) is a flavonoid that inhibits the proliferation of BC cells. In this study, we investigated the effect of Hst on the cell viability of MCF-7 cells and the gene expression of the ERα, ERβ, IL-6, Ps2, and Cyclin D1. In this study, cell viability was determined by MTT assay. The cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, 200, and 400 µM) for 24h, and IC50 was calculated. Real-time PCR was used to assess the expression of ERα, ERβ, pS2, Cyclin D1, and IL-6 mRNA. MCF-7 cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, and 200 µM) for 24h. Real-time PCR was carried out using a Step One Real-Time PCR System (ABI, USA) and Amplicon SYBR Green reagents. The MTT assay revealed increased cytotoxicity with higher concentrations of Hst, and the IC50 was calculated at 200 µM. Real-time PCR analysis following treatment with Hst showed a significant increase in ERα gene expression at 25 µM of Hst and a decrease in expression at 50, 100, and 200 µM of Hst (p < 0.0001). ERβ gene expression significantly decreased across all concentrations of Hst (p < 0.0001), while IL-6 gene expression decreased significantly in all concentrations (p < 0.0001). pS2 gene expression increased significantly with all concentrations of Hst (p < 0.0001), while Cyclin D1 gene expression did not significantly decrease upon Hst exposure (p > 0.05). The results of our study demonstrate that Hst has the ability to induce cell death in MCF-7 cells. Furthermore, it was observed that Hst reduces the expression of the ER gene and enhances its activity, which can affect the downstream pathways of the ER.

Molecular docking simulation and in vitro studies on estrogenic activities of flavonoids from leaves of Carya cathayensis Sarg

The main purpose of this study was to evaluate the estrogenic properties of total flavonoids (TFs) and five flavonoid monomers (cardamonin (Car), pinostrobin chalcone (PC), wogonin (Wo), chrysin (Chr) and Pinocembrin (PI)) from leaves of Carya cathayensis Sarg (LCC). TFs from LCC were isolated and determined using HPLC. The 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were performed to assess the effects of flavonoids on cell proliferation and cell cycle, respectively. The molecular docking technique was applied to investigate binding conformations of the monomers from LCC to the estrogen receptor ERα and ERβ. Gene and protein expression patterns were assessed using quantitative real-time PCR (qRT-PCR) and western blot, respectively. The results showed that TFs, Car, PC, Wo and Chr promoted proliferation of MCF-7 cells and cell transition from the G1 to S phase, and inhabitation of MCF-7 cell proliferation was observed after the treatment of PI. Molecular docking studies confirmed ERs as molecular targets for the monomers. TFs, Car, PC, Wo and Chr from LCC promoted gene expression of ERα, ERβ, progesterone receptor (PR) and pS2. Our collective results demonstrated that TFs and monomers from LCC may exert ER agonist activity through competitively bind to ER, inducing ER upregulation and active ER to estrogen response element (ERE)- independent gene regulation. As an abundant natural product, LCC may provide a novel medicinal source for treatment of diseases caused by estrogen deficiency.

The potential endocrine disruption of pesticide transformation products (TPs): The blind spot of pesticide risk assessment

The ecological and health risk assessment of environmental pesticide residues have attracted ever-growing attention; however, their transformation products (TPs) have seldom been considered. Herein, we examined the endocrine-disrupting effects of 4 widely used pesticides as pyriproxyfen (Pyr), malathion (ML), benalaxyl (BX), and fenoxaprop-ethyl (FE), together with their 21 TPs through in vitro and in silico approaches, and found approximately 50% of the TPs exhibited stronger endocrine-disrupting effects than their corresponding parent compounds. Specifically, Pyr and 9 TPs (five TPs of Pyr, one of ML, one of BX, and two of FE) exhibited estrogen-disrupting effects, which were also confirmed by results of E-screen and pS2 expression assays, and molecular docking showed that certain hydroxylated TPs could well mimic the binding mode of estrogen with ERα. Meanwhile, two TPs of Pyr, ML and its TP demonstrated weak glucocorticoid antagonistic activities partially contributed by hydrogen bonds. We also discovered that in H295R cells, all the endocrine disruptors increased hormone secretion and the related gene expression levels. Conclusively, since an increasing number of pesticide TPs have been being detected in various environmental media, a more comprehensive understanding of the ecological risk of pesticide TPs is imperative for risk assessments more extensively and regulatory policy-making on pesticide restriction in the future.

Short Term Exposure to Bilirubin Induces Encephalopathy Similar to Alzheimer's Disease in Late Life.

Hyperbilirubinemia may increase the risk of Alzheimer's disease (AD) but its mechanistic role in AD pathogenesis remains obscure. Here, we used animal models to investigate the short- and long-term effects of neonatal systemic exposure to bilirubin on brain histology and function as well as the acute effect of lateral ventricle injection of bilirubin in adult rats. We found that three days exposure to bilirubin in newborn rats could induce AD-like pathological changes in late life, including tau protein hyperphosphorylation at multiple sites, increased Aβ production in brain tissues, and spatial learning and memory injury. Bilirubin activated the activities of several protein kinases (GSK-3β, CDK5, and JNK), which were positively correlated with hyperphosphorylated tau; simultaneously increased the expression of AβPP γ-secretase PS2 and decreased the expression of α-secretase ADAM17, which were positively correlated with Aβ production. The above results were well replicated in primary hippocampal cell cultures. These data demonstrate that bilirubin encephalopathy is an AD-like disease, suggesting a potent role of bilirubin in AD.

Short Term Exposure to Bilirubin Induces Encephalopathy Similar to Alzheimer's Disease in Late Life

Background: Hyperbilirubinemia may increase the risk of Alzheimer's disease (AD) but its mechanistic role in AD pathogenesis remains obscure. Methods: Three animal models and primary hippocampal cells were used to explore in this study: (1) acute bilirubin treatment; (2) lateral ventricle injection; (3) long term observation. Results: Three days exposure to bilirubin in newborn rats could induce AD-like pathological changes in late life, including tau protein hyperphosphorylation at multiple sites, increased Aβ production in brain tissues, and spatial learning and memory injury. Bilirubin activated the activities of several protein kinases (GSK-3β, CDK5 and JNK), which were positively correlated with hyperphosphorylated tau; simultaneously increased the expression of APP γ-secretase PS2 and decreased the expression of α-secretase ADAM17, which were positively correlated with Aβ production. The above results were well replicated in primary hippocampal cells. Conclusions: Bilirubin may be an upstream activator to induce AD, and bilirubin encephalopathy is an AD-like disease. Funding Statement: The study was supported in part by the National High Technology Research and Development Program of China (2006AA02Z4B5); the National Natural Science Foundation of China(81170608, 81570278, 81602427, 81600089, 81670156); the Science and Technology Program of Guangzhou (201707010046, 201604020001); The National Funds for Developing Local Colleges and Universities (B16056001); Natural Science Foundation research team of Guangdong Province (2018B030312001); Guangzhou Education System (1201610014, 1201620494); Natural Science Foundation of Guangdong Province (2017A030313662). Declaration of Interests: All authors declare that there are no conflicts of interest. Ethics Approval Statement: The protocol for the care and use of all animals in this study was in accordance with the Guangdong Animal Center for the ethical treatment of animals and approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (SYXK2016-0168, Guangzhou, China).

Long-time qingyan formula extract treatment exerts estrogenic activities on reproductive tissues without side effects in ovariectomized rats and via active ER to ERE-independent gene regulation.

The reproductive tissues are negatively influenced by estrogens in hormone therapy. Qingyan formula ethanol extract (QYFE)’s estrogenic effects and safety on reproductive tissues after long-term administration and its mechanism via estrogen receptor (ER) pathway haven’t been studied. Here, we characterized its estrogenic effects using ovariectomized rats together with in vitro studies for further molecular characterization. Ovariectomized rats were treated with QYFE at doses of 0.7, 1.4, and 2.8g/kg for 12 weeks. The results showed QYFE has a potent estrogenic activity, as indicated by restoring the disappeared estrous cycle, antagonizing the atrophy of uterus, vagina and mammary gland, and the estrogen decline in circulation caused by ovariectomy. In addition, QYFE upregulated ERα and ERβ expressions and had a less stimulatory effect on PCNA and ki-67 antigen in reproductive tissues compared with estradiol valerate. QYFE components can bind to ERα and ERβ, significantly increased ERα/β-ERE luciferase reporter gene expression, upregulated the expressions of ERs, PR and pS2 in MCF-7 cells at protein and gene level. All these activities were significantly inhibited by the ER antagonist ICI182,780. QYFE’s estrogenic activity maybe mediated by stimulating biosynthesis of estrogen and increasing the quantity of ERs in target tissue and via active ER to ERE-independent gene regulation.

Estrogenic effects of phytoestrogens derived from Flemingia strobilifera in MCF-7 cells and immature rats.

Phytoestrogen (PE) has received considerable attention due to the physiological significance of its estrogenicity. Flemingia strobilifera (FS) has been used as a folk medicine in Asia for the treatment of inflammation, cancer, and infection; however, the estrogenic effects and chemical components of FS have not yet been reported. We aimed to uncover the estrogenic properties and PEs derived from FS using phytochemical and pharmacological evaluation. PEs from FS extract (FSE) were analyzed by NMR, HPLC, and MS. To evaluate estrogenic activity, FSE and its compounds were evaluated by in vitro and in vivo assays, including human estrogen receptor alpha (hERα) binding, estrogen response element (ERE)-luciferase reporter assays, and uterotrophic assays. FSE and its compounds 1-5 showed binding affinities for hERα and activated ERE transcription in MCF-7 cells. Additionally, FSE and compounds 1-5 induced MCF-7 cell proliferation and trefoil factor 1 (pS2) expression. In immature female rats, significant increases in uterine weight and pS2 gene were observed in FSE-treated groups. We identified estrogenic activities of FSE and its bioactive compounds, suggesting their possible roles as PEs via ERs. PEs derived from FSE are promising candidates for ER-targeted therapy for post-menopausal symptoms.

Phytoestrogenic effect of Inula racemosa Hook f – A cardioprotective root drug in traditional medicine

Ethnopharmacological relevanceRoots of Inula racemosa are used as a cardio protective in Ayurveda in India, being prescribed as a medicine for precordial chest pain, cough and dyspnoea, both singly and as a poly herbal. AimEvaluation of Phytoestrogenic activity of the root extracts of Inula racemosa and compounds isolated therefrom in vivo, in silico and in vitro. Materials and methodsAlcohol (IrA) and hexane (IrH) extracts characterized by HPTLC/GC-MS analysis respectively and processed for compound isolation were evaluated for estrogenic activity (100 & 250mg/kg bw) by the Immature rat uterotrophic assay using ethinylestradiol (EE −30µg/kg bw) as standard drug. Alantolactone (ALT), Isoalantolactone (IALT) and Stigmasterolglucoside (SG) isolated from the extracts were characterized and screened in silico for ERα, ERβ binding affinity, assessed in vitro for growth modulatory effects on MCF-7 cells by MTT assay and cell cycle distribution analysis using Flow cytometry. RT-PCR analysis evaluated the mRNA expression of pS2 in these cells post exposure to ALT, IALT and SG. ResultsIn the IrA treated groups there has been a statistically significant increase (P < 0.05) in absolute and normalised uterine weight, uterine diameter, endometrial thickness, luminal epithelial cell height,diameter of ovary and in the number of primary and secondary ovarian follicles relative to untreated controls. Presence of ciliated epithelial cells in the oviduct, elevated number of early growing follicles characterized by an increased oocyte diameter, and signs of vascularization in the cortex of ovarian sections in this group relative to EE treated group are indicative of pervasive activation of follicular growth and initiation. Virtual docking demonstrated ERα affinity for IALT, ERβ affinity for ALT, while SG showed a high binding affinity to both with a relatively greater ERβ binding affinity. Dose dependent decrease in cell viability mediated by IALT and SG in the MTT assay is corroborated by a statistically significant increase (p < 0.05) in sub G0-G1 cells by SG at 200 and 400µM in cell cycle analysis and there has been an induction of pS2 by IALT and SG in the ER regulated MCF-7 cells. ConclusionsDemonstration of classical morphological changes induced by estrogen stimulation mediated by IrA in vivo at both the tested doses, isolation of the antioxidant SG from IrA and its dose dependent growth inhibitory effect on estrogen sensitive MCF-7 cells through apoptotic induction and an up regulation of pS2 are suggestive of an anti-estrogenic effect through estrogen receptor binding affinity, typical of phytoestrogens that bind to ER but do not elicit a full estrogenic response. The observed estrogenic effect of IrA suggests a multi mechanistic molecular action involving antioxidant as well as redox signalling pathways acting in consonance with their anti-estrogenic effects owing to the weak estrogen like competitive receptor binding of SG.

Urolithin A suppresses the proliferation of endometrial cancer cells by mediating estrogen receptor‐α‐dependent gene expression

Obese and overweight women are at high risk of developing endometrial cancer; indeed, many of endometrial cancer patients are obese. The increased number and size of adipocytes due to obesity elevate levels of circulating estrogens that stimulate cell proliferation in the endometrium. However, black raspberries are a promising approach to preventing endometrial cancer. We examined 17 black raspberry constituents and metabolites (10 μM or 10 μg/mL, 48 h) for their ability to prevent endometrial cancer cells from proliferating. Urolithin A (UA) was most able to suppress proliferation in a time- and dose-dependent manner (p < 0.05). It arrested the G2/M phase of the cell cycle by upregulating cyclin-B1, cyclin-E2, p21, phospho-cdc2, and CDC25B. UA also acted as an estrogen agonist by modulating estrogen receptor-α (ERα) dependent gene expression in ER-positive endometrial cancer cells. UA enhanced the expression of ERβ, PGR, pS2, GREB1 while inhibiting the expression of ERα and GRIP1. Coincubating UA-treated cells with the estrogen antagonist ICI182,780 abolished UA's estrogenic effects. Knocking down ERα suppressed PGR, pS2, and GREB gene expression but increased GRIP1 expression. Thus, UA's actions appear to be mediated through ERα. This study suggests that UA modulates ERα-dependent gene expression, thereby inhibiting endometrial cancer proliferation.

Presenilin 2 overexpression is associated with apoptosis in Neuro2a cells.

Presenilin 1 (PS1) and PS2 are evolutionarily conserved transmembrane proteins of the aspartyl protease family. Initially, they were reported to be associated with the early onset of familial, early-onset Alzheimer’s disease. PS1 has been implicated in several crucial brain functions including developmental processes, synaptic plasticity, and processing of various molecules, while PS2 has been poorly studied and is considered to be a compensatory partner of PS1. Certain controversial reports have suggested that PS2 has a role in apoptosis, though the underlying mechanism is not clear. To ascertain the role of PS2 in apoptosis, mouse neuroblastoma cells (Neuro2a) were transfected with a cDNA construct encoding full length mouse PS2 and analyzed for viability, expression of PS1, PS2, Bax and p53, Bax protein, and status of chromatin condensation. Our results showed reduced viability, condensed chromatin and higher expression of Bax at mRNA and protein levels, but no change in the expression of p53 and PS1 in PS2-overexpressing Neuro2a cells. Thus, it is evident that PS2, independent of PS1, is associated with apoptosis via a Bax-mediated pathway. These findings might help in the understanding of the involvement of PS2 in apoptosis and its associated brain disorders.

Novel CARM1-Interacting Protein, DZIP3, Is a Transcriptional Coactivator of Estrogen Receptor-α.

Coactivator-associated arginine methyltransferase 1 (CARM1) is known to promote estrogen receptor (ER)α-mediated transcription in breast cancer cells. To further characterize the regulation of ERα-mediated transcription by CARM1, we screened CARM1-interacting proteins by yeast two-hybrid. Here, we have identified an E3 ubiquitin ligase, DAZ (deleted in azoospermia)-interacting protein 3 (DZIP3), as a novel CARM1-binding protein. DZIP3-dependent ubiquitination of histone H2A has been associated with repression of transcription. However, ERα reporter gene assays demonstrated that DZIP3 enhanced ERα-mediated transcription and cooperated synergistically with CARM1. Interaction with CARM1 was observed with the E3 ligase RING domain of DZIP3. The methyltransferase activity of CARM1 partially contributed to the synergy with DZIP3 for transcription activation, but the E3 ubiquitin ligase activity of DZIP3 was dispensable. DZIP3 also interacted with the C-terminal activation domain 2 of glucocorticoid receptor-interacting protein 1 (GRIP1) and enhanced the interaction between GRIP1 and CARM1. Depletion of DZIP3 by small interfering RNA in MCF7 cells reduced estradiol-induced gene expression of ERα target genes, GREB1 and pS2, and DZIP3 was recruited to the estrogen response elements of the same ERα target genes. These results indicate that DZIP3 is a novel coactivator of ERα target gene expression.

Abstract 5006: Elucidating the mechanism of bile acid-induced activation of estrogen receptor β in colorectal adenocarcinoma

Abstract In previous studies, Ursodeoxycholic Acid (UDCA) was investigated as a chemopreventive drug for the prevention of colorectal adenoma recurrence. These studies revealed that the efficacy of UDCA was gender-specific. The use of UDCA in men significantly lowered the odds of advanced lesions in men, but significantly increased the odds of advanced lesions in women, more specifically, in females who were younger, obese, or whose diet was high in fat. The gender specific role of UDCA led us to investigate this bile acids’, along with deoxycholic acid's (DCA), ability to activate the estrogen receptor in colon cancer. Estrogen receptor β (ERβ) is the predominant estrogen receptor in both normal and malignant human colon tissue, and loss of ERβ has been shown to be critical for tumor cell differentiation. Elevated concentrations of bile acids have previously been shown to promote colon cancer and growing evidence indicates that these molecules play a vital role in regulating cell signaling and gene expression. In our current studies, through western blot and qRT-PCR analysis, we have shown that DCA-treated HT-29 colorectal adenocarcinoma cells significantly induce the phosphorylation of ERβ, as well as increase gene expression of the pS2 estrogen response element (ERE). UDCA was also shown to induce phosphorylation of ERβ, but much later than DCA. However, UDCA increases gene expression of pS2 at much earlier time points than DCA. In addition, we have also shown that when pre-treating HT-29 cells with the MEK 1/2 inhibitor, PD98059, bile acid-induced phosphorylation of ERβ is significantly reduced, along with ERE gene up-regulation. Collectively, this data suggests that bile acids are inducing the activation of ERβ through a ligand-independent mechanism via the MAPK signaling cascade. Citation Format: Jesse Trujillo. Elucidating the mechanism of bile acid-induced activation of estrogen receptor β in colorectal adenocarcinoma. [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 5006. doi:10.1158/1538-7445.AM2015-5006

Structural development of stabilized helical peptides as inhibitors of estrogen receptor (ER)-mediated transcription

Three types of stabilized helical peptides containing disulfide bonds, C–C cross-linked side chains, or α,α-disubstituted amino acids (2-aminoisobutyric acid (Aib)) were designed and synthesized as inhibitors of estrogen receptor (ER)-coactivator interactions. Furthermore, heptaarginine (R7)-conjugated versions of the peptides were prepared, and their effects on ER-mediated transcription were evaluated at the cellular level (in ER-positive T47D cells). Among them, the R7-conjugated peptides 11 and 12 downregulated the mRNA expression of pS2 (an ER-mediated gene whose expression is upregulated by 17β-estradiol) by 95% (at a dose of 10μM) and 87% (at a dose of 3μM), respectively.

Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha

Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy.

In silico discovery and validation of potent small-molecule inhibitors targeting the activation function 2 site of human oestrogen receptor α.

IntroductionCurrent approaches to inhibit oestrogen receptor-alpha (ERα) are focused on targeting its hormone-binding pocket and have limitations. Thus, we propose that inhibitors that bind to a coactivator-binding pocket on ERα, called activation function 2 (AF2), might overcome some of these limitations.MethodsIn silico virtual screening was used to identify small-molecule ERα AF2 inhibitors. These compounds were screened for inhibition of ERα transcriptional activity using stably transfected T47D-KBluc cell line. A direct physical interaction between the AF2 binders and the ERα protein was measured using biolayer interferometry (BLI) and an ERα coactivator displacement assay. Cell viability was assessed by MTS assay in ERα-positive MCF7 cells, tamoxifen-resistant (TamR) cell lines TamR3 and TamR6, and ERα-negative MDA-MB-453 and HeLa cell lines. In addition, ERα inhibition in TamR cells and the effect of compounds on mRNA and protein expression of oestrogen-dependent genes, pS2, cathepsin D and cell division cycle 2 (CDC2) were determined.ResultsFifteen inhibitors from two chemical classes, derivatives of pyrazolidine-3,5-dione and carbohydrazide, were identified. In a series of in vitro assays, VPC-16230 of the carbohydrazide chemical class emerged as a lead ERα AF2 inhibitor that significantly downregulated ERα transcriptional activity (half-maximal inhibitory concentration = 5.81 μM). By directly binding to the ERα protein, as confirmed by BLI, VPC-16230 effectively displaced coactivator peptides from the AF2 pocket, confirming its site-specific action. VPC-16230 selectively suppressed the growth of ERα-positive breast cancer cells. Furthermore, it significantly inhibited ERα mediated transcription in TamR cells. More importantly, it reduced mRNA and protein levels of pS2, cathepsin D and CDC2, validating its ER-directed activity.ConclusionWe identified VPC-16230 as an ERα AF2-specific inhibitor that demonstrated promising antiproliferative effects in breast cancer cell lines, including TamR cells. VPC-16230 reduced the expression of ERα-inducible genes, including CDC2, which is involved in cell division. We anticipate that the application of ERα AF2 inhibitors will provide a novel approach that can act as a complementary therapeutic to treat ERα-positive, tamoxifen-resistant and metastatic breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0529-8) contains supplementary material, which is available to authorized users.

Epigenetic regulation of presenilin 1 and 2 in the cerebral cortex of mice during development

Previously, we reported differential expression profile of Presenilin (PS)1 and 2 and their interacting partners in mouse cerebral cortex during development. Our findings indicated crucial involvement of these proteases in prenatal and postnatal development of mouse cerebral cortex. However, the mechanisms that precisely control their expression in stage-specific manner during brain development are still elusive. In this regard, epigenetic modifications by DNA methylation and histone acetylation during brain development deserve major attention. Therefore, we have analyzed the epigenetic regulation of PS1 and PS2 in mouse cerebral cortex during development. The data demonstrated a good correspondence of H3K9/14 Ac level in PS1 and PS2 promoter with their expression profile during cerebral cortical development. H3K9/14 Ac level was high at embryonic day (E)12.5, declined at E18.5, increased from postnatal day (P)0 to P45 and decreased again at 20 weeks (w) in PS1 promoter. For PS2, H3K9/14 Ac level was high at E12.5, thereafter, reduced upto P20 and increased at P45 and 20 weeks. DNA methylation sites also varied in number and position at different developmental stages, and some of them are putative sites for binding of transcription factors like HSF-1, Ets-1, and Sp1 that are crucial for brain developmental processes, as revealed by in silico analysis. Though MeCP2 level also altered during development, they did not correlate with PS1 and PS2 expression profile. Taken together, our findings provide the first evidence of epigenetic regulation of PS1 and PS2 by H3K9/14 histone acetylation and DNA methylation in mouse cerebral cortex during development.

A precisely substituted benzopyran targets androgen refractory prostate cancer cells through selective modulation of estrogen receptors

Dietary consumption of phytoestrogens like genistein has been linked with lower incidence of prostate cancer. The estradiol-like benzopyran core of genistein confers estrogen receptor-β (ER-β) selectivity that imparts weak anti-proliferative activity against prostate cancer cells. DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran (BP), a SERM designed with benzopyran core, targeted androgen independent prostate cancer (PC-3) cells 14-times more potently than genistein, ~25% more efficiently than tamoxifen and 6.5-times more actively than ICI-182780, without forfeiting significant specificity in comparison to genistein. BP increased apoptosis (annexin-V and TUNEL labeling), arrested cell cycle, and significantly increased caspase-3 activity along with mRNA expressions of estrogen receptor (ER)-β and FasL (qPCR) in PC-3 cells. In classical ERE-luc reporter assay BP behaved as a potent ER-α antagonist and ER-β agonist. Accordingly, it decreased expression of ER-α target PS2 (P<0.01) and increased expression of ER-β target TNF-α (P<0.05) genes in PC-3. ER-β deficient PC-3 (siRNA-transfected) was resistant to apoptotic and anti-proliferative actions of SERMs, including stimulation of FasL expression by BP. BP significantly inhibited phosphorylation of Akt and ERK-1/2, JNK and p38 in PC-3 (immunoblotting), and thus adopted a multi-pathway mechanism to exert a more potent anti-proliferative activity against prostate cancer cells than natural and synthetic SERMs. Its precise ER-subtype specific activity presents a unique lead structure for further optimization.

Development of cell-penetrating R7 fragment-conjugated helical peptides as inhibitors of estrogen receptor-mediated transcription.

The heptaarginine (R7)-conjugated peptide 5 was designed and synthesized as an inhibitor of ER-coactivator interactions and ER-mediated transcription at the cellular level. The R7-conjugated peptide 5 was able to enter ER-positive T47D cells efficiently, and treatment with 3 μM of 5 downregulated the mRNA expression of pS2 (an ER-mediated gene) by 87%.