Project description:The fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer risk. Despite estrogenâ??s influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20% response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen receptor signaling in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors that arise from this cell type, such as high-grade serous cancer. Using normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in the oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation in CD1, but not in FVB derived cell lines. Further, using RNAseq, the oviduct specific transcriptional genes targets of estrogen and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated highlighting the need for better models of estrogen responsive HGSC cell lines. Murine oviductal epithelial cells from the FVB background were hormone starved for 48 hours (with a media change after 24 hours), then treated in triplicate with solvent control (DMSO) (0.1%), 1 nM 17-betaestradiol or 100 nM 4-hydroxytamoxifen for 24 hours. Following treatment, RNA was was isolated, libraries were prepped and sequenced using the Illumina HiSeq 2500 platform.

Project description:E2 exposure significantly decreased peak viral titer in hNECs from female donors. We used microarray analyses to identify global gene expression patterns between E2 and vehicle exposed hNECs from female donors Influenza causes an acute infection characterized by virus replication in respiratory epithelial cells. The severity of influenza and other respiratory diseases changes over the life course and during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult male and female donors, we exposed cultures to the endogenous 17Î²-estradiol (E2) or select estrogen receptor modulators (SERMs), then infected cultures with a seasonal influenza A virus (IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and whether these effects where sex-dependent. Estradiol, raloxifene, and bisphenol A decreased IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was dependent on the genomic estrogen receptor- 2 (ESR2) as neither genomic ESR1 nor non- genomic GPR30 were expressed in hNEC cultures and addition of the genomic ER antagonist ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on interferon or chemokine secretion, but significantly downregulated cell metabolic processes, including genes that encode for zinc finger proteins, many of which contain estrogen response elements in their promoters. These data provide novel insights into the cellular and molecular mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial cells derived from humans. Primary human nasal epithelial cells from females were exposed to E2 for 24h prior to infection, then infected with an H3N2 strain of influenza a virus for 2 hours. At 24 and 48h post infection, hNECs were collected in Trizol for RNA extraction and hybridization on Affymetrix Human Gene ST 2.0 microarrays.

Project description:Estrogen is a reproductive steroid hormone that has both beneficial and detrimental effects on the cardiovascular system. Selective estrogen receptor modulators (SERMs) exhibit partial estrogen agonist/antagonist activity in estrogen target tissues. Since we still have an incomplete picture of the gene targets of estrogen in the vasculature, and our understanding of SERM gene targets in the vasculature is even less well-developed, it is important to broaden the available information on this subject. The present study tested the hypothesis that estrogens (ethinyl estradiol, estradiol benzoate, and equilin) and SERMs (tamoxifen and raloxifene) cause differential gene and protein expression in the vasculature. DNA microarray and real-time RT-PCR were used to investigate gene expression in the mesenteric arteries of estrogen and SERM treated ovariectomized rats. The genes shown to be differentially expressed included stearoyl-CoA desaturase (SCD), soluble epoxide hydrolase (sEH), secreted frizzled related protein-4 (SFRP-4), insulin-like growth factor-1 (IGF-1), phospholipase A2 group 1B (PLA2-G1B), and fatty acid synthase (FAS). Western blot further confirmed the differential expression of sEH, SFRP-4, FAS, and SCD protein. These results reveal that estrogens and SERMs cause differential gene and protein expression in the mesenteric artery. Consequently, the use of these agents may be associated with a unique profile of functional and structural changes in the mesenteric arterial circulation. Goal of the experiment: To examine the effects of three formulations of estrogen (ethinyl estradiol, equilin, and estradiol benzoate) and of the selective estrogen receptor modulators, tamoxifen and raloxifene, on gene expression in the mesenteric arteries of the rat. Keywords: rat, artery, gene expression, mesentery, estrogen, tamoxifen, raloxifene Experimental factors: hormone treatment, drug treatment Experimental design: Prepubertal female Sprague Dawley rats (Harlan, Indianapolis, IN) were ovariectomized at 4 weeks of age, prior to the initiation of regular ovarian cycles. The animals were allowed to recover for 2 weeks before the experimental protocol was initiated. The rats were randomly divided into 6 groups (n=3-4/group) and treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After 3 weeks of treatment, the rats were euthanized and the mesentery obtained. Total RNA was extracted from each individual sample and used for the synthesis of labeled cRNA for microarray analysis. After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated and harvested and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Mesenteric artery RNA was extracted and purified using an RNeasy Mini Kit (Qiagen). Any potentially contaminating DNA was removed by treatment with RNase-free DNase (Qiagen) on the RNeasy column. Total RNA was quantified and RNA integrity was assessed using the Agilent Bioanalyzer (Santa Clara, CA). Following analysis, the RNA was stored at -80ºC until it was used for gene expression studies. DNA microarray: CodeLink Whole Rat Genome Bioarrays (Applied Microarrays, Tempe, AZ) were used. Five μg total RNA was used from each mesenteric arterial sample for the microarray analysis. Complementary RNA was synthesized per company protocol (Ambion). A set of bacterial RNAs was added to each total RNA sample and carried through the synthesis and labeling reactions as controls and for nor¬malization of the data. The microarrays were scanned with the GenePix 4000B (Axon Instruments, Union City, CA) with GenePix Pro 5.0 software (Axon Instruments). Analysis of the microar¬rays was performed with the CodeLink Expression Analysis software (Applied Microarrays) and Acuity 3.1 software (Axon), and statistical analysis was performed with GeneSpring 7.1 software (Agilent, Santa Clara, CA). A sample of n=3 was completed with the microarrays for each treatment. Quality control steps: The cRNA that was synthesized from each mesenteric artery treatment sample was used for hybridization to a single CodeLink (Applied Microarrays) whole rat genome microarray. Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were used. Bacterial control spikes were used per manufacturer's instructions. Samples used, extract preparation and labeling: The origin of each biological sample: The samples were isolated mesenteric arteries from individual Sprague Dawley rats that had been treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). Manipulations of biological samples and protocols used: Ovariectomized female rats were treated by daily gavage for 3 weeks with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, Sigma, St Louis, MO), ethinyl estradiol (EE, 0.15 mg/kg; Sigma), estradiol benzoate (EB, 0.15 mg/kg; Sigma), equilin (EQ, 0.15 mg/kg; Sigma), tamoxifen (TAM, 3 mg/kg; Sigma), or raloxifene (RAL, 3 mg/kg; Eli Lilly & Co., Indianapolis, IN). After the final gavage treatment on day 21, the rats were deeply anesthetized with isoflurane and euthanized by decapitation. The mesenteric arterial tree was isolated, harvested, and stored in RNAlater (Ambion) at -80ºC. The mesenteric arteries were dissected free from veins and surrounding fat and homogenized in TRI reagent (MRC, Cincinnati, OH). Experimental factor: hormone treatment, drug treatment Technical protocols: The mesenteric arteries were minced with scalpels and TRI reagent (MRC) was added. The tissue was homogenized with a Polytron homogenizer. Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labeled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol. 5.0 microgram of rat mesenteric artery total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on spin columns included in the Ambion kit. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on spin columns from the Ambion kit. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of biotinylated cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides (CodeLink Whole Rat Genome Bioarrays), and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

Project description:The efficacy and exceptionally good tolerance of estrogen blockade in the treatment of breast cancer is well recognized but novel agents are required, especially to take advantage of the multiple consecutive responses obtained in breast cancer progressing following previous hormone therapy, thus delaying the use of cytotoxic chemotherapy with its usually serious side effects. Acolbifene (ACOL) is a novel and unique antiestrogen completely free of estrogen-like activity in both the mammary gland and uterus while preventing bone loss. From the preclinical and clinical data so-far available, this new antiestrogen represents a unique opportunity for a highly potent and specific blockade of estrogen action in the mammary gland and uterus while exerting estrogen-like beneficial effects in other tissues (selective estrogen receptor modulator or SERM activity). In order to better understand the specificity of action of acolbifene, we have used Affymetrix GeneChips containing 45,000 probe sets to analyze 34,000 genes to determine the specificity of this compound compared to the pure antiestrogen fulvestrant, as well as the mixed antagonists/agonists tamoxifen and raloxifene to block the effect of estradiol (E2) and to induce effects of their own on gene expression in the mouse mammary gland. The genes modulated by E2 were those identified in two separate experiments and validated by quantitative real-time PCR (Q_RT-PCR). Three hours after the single subcutaneous injection of E2 (0.05 ug), the simultaneous administration of acolbifene, fulvestrant, tamoxifen and raloxifene blocked by 98%, 62%, 43% and 92% the number of E2-upregulated genes, respectively. On the other hand, 70%, 10%, 25% and 55% of the genes down-regulated by E2 were blocked by the same compounds. Acolbifene was also the compound which, when used alone, modulated the smallest number of genes also influenced by E2, namely 4%, thus possibly explaining the potent tumoricidal action of this compound in human breast cancer xenografts where 61% of tumors disappeared, thus bringing a new paradigm in the hormonal therapy of breast cancer. Female C57BL6 mice were ovariectomized (OVX). One week after OVX, mice were treated with EM-652.HCl (acolbifene), tamoxifen citrate, raloxifene or ICI 182780 (fulvestrant). Compounds were administered to OVX mice or to OVX mice simultaneously treated with 17b-estradiol (E2). Control groups received an injection of the vehicle alone. All animals were sacrificed after 3h of treatment.Mammary glands from all mice of the same group were collected and pooled. Total mRNA was isolated and converted to biotinylated cRNA.A microarray analysis was performed using Murine U74Av2 Affymetrix microarrays.

Dataset Information

Differential regulation of transcription in Beta-estrodial treated Osteoblasts

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets