The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells.

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ERα) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ERα in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ERα expression. GPER mRNA, and protein expression were analyzed in ERα-positive or -negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17ß-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ERα agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ERα knockdown. ERα-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ERα expression in breast tumors. The median GPER expression in ERα-positive tumors was significantly higher than ERα-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ERα-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ERα-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ERα occupancy in the upstream region of GPER. Furthermore, treatment with 17ß-estradiol or PPT significantly reduced the IC50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ERα in breast tumors, and induced by estrogen-ERα signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ERα co-expression, and their interplay in breast tumor development, progression, and treatment.

Transcriptomic data of MCF-7 breast cancer cells treated with 10 µM enterolactone.

Enterolactone (EL) is a mammalian enterolignan produced in the intestine as a result of the microbial biotransformation of the dietary lignans. EL is a potential nutraceutical, with several health benefits, including anticancer and antimetastatic properties. Epidemiological data suggest a possible link between EL exposure and breast cancer risk. However, EL binds to estrogen receptor-α, produces estrogen-like effects on gene expression, and induces proliferation of MCF-7 breast cancer cells at a concentration of 10 µM. Here, we present RNA-seq data obtained from MCF-7 breast cancer cells treated with 10 µM EL for a period of 72 h, which captures the transcriptomic alterations associated with cell proliferation. The data are available from Gene Expression Omnibus (GEO, accession number GSE216876).

Transcriptomic data of MCF-7 breast cancer cells treated with G1, a G-protein coupled estrogen receptor (GPER) agonist.

Besides short-term non-genomic effects, the G-protein coupled estrogen receptor (GPER) also mediates long-term genomic effects of estrogen. The genomic effects of GPER activation are not completely understood. G1 is a selective GPER agonist, which is popularly used for addressing the effects of GPER activation. Here, we present transcriptomic (RNA-seq) data on MCF-7 cells treated with 100 nM, or 1 µM G1 for a period of 48 h. The data are available from GEO (accession number GSE188706).

RUNX1T1, a potential prognostic marker in breast cancer, is co-ordinately expressed with ERα, and regulated by estrogen receptor signalling in breast cancer cells.

BACKGROUND: RUNX1T1 is extensively studied in the context of AML1-RUNX1T1 fusion protein in acute myeloid leukemia. Little is known about the function of RUNX1T1 itself, although data on its function and regulation have begun to emerge from clinical, and in vitro studies. It is a putative tumor suppressor, whose expression is altered in a variety of solid tumors. Recently, reduced expression of RUNX1T1 in triple-negative breast tumors, and its influence on prognosis was reported. METHODS AND RESULTS: The Kaplan-Meier Plotter online tool was used to study the relationship between RUNX1T1 expression and survival of breast cancer patients. High RUNX1T1 expression was associated with longer overall survival (OS), relapse-free survival (RFS) and distant metastasis free survival (DMFS). RUNX1T1 expression positively and negatively influenced OS of patients with ERα-positive and ERα-negative breast tumors, respectively. It was also associated with prolonged RFS, and DMFS in tamoxifen-treated patients. Expression of RUNX1T1 and ERα mRNA was analyzed in 40 breast tumor samples, and breast cancer cell lines using RT-PCR. TCGA-BRCA data was mined to study the relationship between RUNX1T1 and ERα mRNA expression. ERα-positive breast tumors showed significantly higher RUNX1T1 mRNA expression compared to ERα-negative tumors. RUNX1T1 mRNA expression was analyzed by qRT-PCR in MCF-7 or T47D cells, which were treated with 17ß-estradiol, or the ERα agonist PPT, alone or in combination with 4-hydroxytamoxifen. Effect of ERα knockdown was also investigated. Results indicate that estrogen downmodulated RUNX1T1 mRNA expression via ERα. CONCLUSION: Higher expression of RUNX1T1 in breast tumors is associated with favourable prognosis. RUNX1T1 and ERα show co-ordinated expression in breast tumors, and breast cancer cell lines. Estrogen-ERα signalling downmodulates the expression of RUNX1T1 mRNA in ERα-positive breast cancer cells. In-depth investigations on the interaction between RUNX1T1 and ERα are warranted to unravel the role and relevance of RUNX1T1 in breast cancer.

A novel transcript variant of human G-protein coupled estrogen receptor.

The G-protein coupled estrogen receptor (GPER) mediates short-term non-genomic effects of estrogen in diverse cell types and tissues. According to the NCBI nucleotide database, three variants of GPER are known. They are NM_001505.2 (GPER-v2), NM_001039966.1 (GPER-v3), and NM_001098201.1 (GPER-v4). Investigations on GPER expression are key to understand its physiological and pathological roles. However, most studies on GPER mRNA expression have considered total GPER mRNA expression regardless of the individual variants. The present study is motivated by a novel transcript observed in the UCSC Genome Browser (uc010ksd.1), which is annotated as GPER. The novel variant is similar to the known transcript variants of GPER in terms of the protein-coding sequence and the 3'UTR. However, it has a unique 5'UTR, which distinguishes it from other GPER variants. Using primers specific for uc010ksd.1, we have performed RT-PCR to show that the novel GPER transcript (hereafter referred to as GPER-v5) is expressed in human cancer cell lines, such as MCF-7, SW-620, COLO-205, and HT-29. Preliminary evidences indicate that GPER-v5 is a novel GPER mRNA variant. The expression of GPER-v5 in primary cells and tissues should be investigated before probing into its role and relevance in physiological and pathological conditions.

DNA methylation in the upstream CpG island of the GPER locus and its relationship with GPER expression in colon cancer cell lines.

The G-protein coupled estrogen receptor (GPER), a proposed tumor suppressor, relays short-term non-genomic responses in target cells and tissues. It frequently undergoes down-modulation in primary tumors of the breast, ovary, and endometrium. Liu and co-workers recently reported loss of GPER expression in colorectal cancer and attributed it to DNA methylation-dependent silencing. We hypothesized that GPER expression is inversely correlated with methylation in the upstream CpG island (upCpGi) in the GPER locus. Methylation in the upCpGi was analysed by bisulfite sequencing and correlated with GPER expression in a panel of colon cancer cell lines. Eight downstream CpGs of the upCpGi was differentially methylated across the cell lines. Methylation in this differentially methylated region (DMR) correlated inversely with GPER expression. Two cell lines, namely SW620 and COLO-320DM, were compared in terms of their viability in response to varying concentrations of G1, a GPER specific agonist. SW-620 cells, which had the least methylated DMR and the highest level of GPER expression, showed significant loss of viability with 1 µM G1. COLO-320DM, which had the most methylated DMR and the lowest level of GPER expression, did not show a significant response to 1 µM G1. At 5 µM G1, SW620 cells showed a greater reduction in viability than COLO-320DM cells. DNA methylation in the DMR is inversely correlated with GPER expression. DNA methylation-dependent silencing of GPER may be, at least in part, the underlying reason behind the loss of estrogen's oncoprotective effect via GPER in the colon.

Unveiling the cytotoxicity of phytosynthesised silver nanoparticles using Tinospora cordifolia leaves against human lung adenocarcinoma A549 cell line.

Biosynthesis of silver nanoparticles (AgNPs) using plant extract is a cheap, easily accessible and natural process in which the phyto-constituents of the plants act as capping, stabilising and reducing agent. The present study explored the biosynthesis of AgNPs using aqueous leaf extract of Tinospora cordifolia and characterised via various techniques such as Fourier transform infrared, scanning electron microscopy, transmission electron microscopy (TEM), energy dispersive X-ray analysis and X-ray diffraction. Here, TEM confirmed the spherical morphology with 25-50 nm size of synthesised AgNPs. Further, anticancer efficiency of AgNPs synthesised using T. cordifolia leaves were evaluated against human lung adenocarcinoma cell line A549 by MTT, trypan blue assay, apoptotic morphological changes using Annexin V-FITC and Propidium iodide (PI), nuclear morphological changes by DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, reactive oxygen species generation and mitochondrial membrane potential determination. Results confirmed the AgNPs synthesised using T. cordifolia leaves are found to be highly toxic against human lung adenocarcinoma cell line A549.

Cholesterol-lowering drug, in combination with chromium chloride, induces early apoptotic signals in intracellular L. donovani amastigotes, leading to death.

Leishmania establishes a successful parasitism by evading both oxidative and non-oxidative killing pathways, and its drug resistance against the currently available therapeutics demands for a safe and cheap drug. Since the parasite synthesizes ergosterol instead of cholesterol, using the same biochemical pathway and enzymes, an inhibitor of HMG-CoA-Reductase, Lovastatin, has been tried for its anti-Leishmanial effect. Lovastatin, being an inhibitor of HMG-CoA-Reductase, inhibits infection by cholesterol depletion, while chromium chloride complexes, at their higher concentrations, are reported to exhibit cytotoxicity. In intracellular amastigotes, cytotoxicity has been checked by assessing various manifestation of cell death, viz. DNA fragmentation, AnnexinV-FITC binding and JC-1 fluorescence ratio. Release of hydrogen peroxide (HPO) and nitric oxide (NO) has been assessed in live cell. Lovastatin and CrCl3.6H2O in combination has appeared to be ineffective on promastigotes but has induced cytotoxic effect on the intracellular amastigotes through up-regulation of cellular signalling mechanisms. CrCl 3.6H2O stimulates generation of NO, leading to reduction of the number of intracellular amastigote, while Lovastatin shows HPO-mediated killing of the same, keeping the host cell unaffected. This novel therapeutic approach, involving two known safe compounds in suboptimal doses, may resolve human visceral Leishmaniasis.