A rapid decline in egg production of laying hens begins after 480 d of age. Such a rapid decrease results predominantly from the ovarian aging, accompanied by endocrine changes, decreased yolk synthesis and accumulation, and the reduction in follicles selected into the preovulatory hierarchy. In this study, hens at 90, 150, 280, and 580 d old (D90, D150, D280, and D580, respectively) were compared for yolk precursor formation in the liver to elucidate effects of aging on laying performance. The results showed that liver lipid synthesis increased remarkably in hens from D90 to D150, but decreased sharply at D580 as indicated by the changes in triglyceride (TG) levels. This result was consistent with the age-related changes of the laying performance. The levels of liver antioxidants and total antioxidant capacity decreased significantly in D580 hens and the methane dicarboxylic aldehyde in D580 hens was much higher than that at other stages. The serum 17β-estradiol level increased from D90 to D280, but decreased at D580 (P<0.05). The expression of estrogen receptor α and β mRNAs in the liver displayed similar changes to the serum 17β-estradiol in D580 hens. Expressions of the genes related to yolk precursor formation and enzymes responsible for fat acid synthesis were all decreased in D580 hens. These results indicated that decreased yolk precursor formation in the liver of the aged hens resulted from concomitant decreases of serum 17β-estradiol level, transcription levels of estrogen receptors and critical genes involved in yolk precursor synthesis, and liver antioxidant status.
Age Factors ; Animals ; Antioxidants ; metabolism ; Chickens ; Egg Yolk ; metabolism ; Estradiol ; blood ; Female ; Lipids ; biosynthesis ; Liver ; metabolism ; Oviposition ; Receptors, Estrogen ; genetics

Estrogen-related receptor alpha (ERRα) plays an important role in the development of hormone-dependent cancers, but its roles in lung cancer remain elusive. The present study was aimed to investigate the effects of ERRα on the proliferation and metastasis of lung cancer A549 cells. The mRNA and protein levels of ERRα were detected in lung cancer A549 and MCF-7 cells and bronchial epithelial BEAS-2B cells by qRT-PCR and Western blotting, respectively. ERRα plasmid transfection and XCT-790 (an inverse agonist of ERRα) were used to up-regulate or down-regulate ERRα expression in A549 cells, respectively. The viability of A549 cells was measured by cell counting kit-8 (CCK-8) and the motility of A549 cells by wound healing assay and Transwell migration/invasion assay. The epithelial markers E-cadherin (E-Cad) and zona occludin-1 (ZO-1), the mesenchymal markers fibronectin (FN) and vimentin (Vim) and the transcription factors (Snail, Zeb1 Twist and Slug) were further detected at mRNA and protein levels by qRT-PCR and Western blotting, respectively. The results showed that ERRα promoted the growth of lung cancer A549 cells in vitro. XCT-790 significantly inhibited the migration and invasion of A549 cells. Over-expression of ERRα promoted the epithelial-to-mesenchymal transition (EMT) of A549 cells, down-regulated the epithelial makers E-Cad and ZO-1, and up-regulated the mesenchymal makers FN and Vim. Silencing of Slug, but not other transcription factors, significantly abolished the ERRα-induced EMT of A549 cells. It was suggested that ERRα promoted the migration and invasion of A549 cells by inducing EMT, and Slug was involved in the process. Targeting ERRα might be an efficient approach for lung cancer treatment.
Cell Line, Tumor ; Cell Proliferation ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation ; Humans ; Lung Neoplasms ; genetics ; metabolism ; prevention & control ; Neoplasm Metastasis ; Neoplasm Proteins ; biosynthesis ; genetics ; Receptors, Estrogen ; biosynthesis ; genetics

OBJECTIVETo establish a bioluminescent MDA-MB-231 cell line which can stably express luciferase and green fluorescent protein to allow bioluminescent imaging in nude mouse models bearing human triple-negative breast cancer xenografts.

METHODSThe lentivirus carrying luc2, eGFP and neo fusion genes were packaged in 293T cells via calcium phosphate co-precipitation. Human triple-negative breast cancer cell line MDA-MB-231 was infected by the lentivirus, and the positive cell clones were tested for eGFP and luc2 expressions by fluorescence microscopy and Xenogen IVIS200 bioluminescent imaging system, respectively. MTT assay, transwell invasion assay and wound healing assay were performed to evaluate the changes in the proliferation, invasion and migration abilities of the infected cells. The cells were then orthotopically implanted into the right second mammary fat pat of female BALB/c nude mice. The tumor growth was monitored by the in vivo imaging system every week, and the tumor tissues were harvested to evaluate the in vivo stability and tumorigenicity of the modified cells using cryosection and HE staining.

RESULTSThe lentivirus-infected MDA-MB-231cells could stably express luc2 and eGFP, and the luciferase activity reached 9689 phontons/s/per cell. No significant changes occurred in the biological activities of the lentivirus-infected MDA-MB-231 cells. We successfully established the nude mouse model bearing orthotopically implanted human triple-negative breast cancer cells.

CONCLUSIONThe modified MDA-MB-231 cell line can be detected sensitively at the primary implantation site and distant metastasis site in nude mice, which provides a convenient and sensitive platform for the research of metastatic mechanism and new antitumor drugs of human triple-negative breast cancer. The combination of eGFP and luc2 is superior to single reporter gene.

Animals ; Brain Neoplasms ; secondary ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Genes, Reporter ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; biosynthesis ; genetics ; Luminescent Measurements ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

Estrogen-related receptor alpha (ERR alpha) is an orphan nuclear receptor and functions as a key regulator of energy metabolism in high energy demand tissues. However, its role in white adipose tissue is largely unknown. In this study, we aim to clone the ORF sequence of pig ERR alpha with touch down-PCR, analyze the expression pattern of ERR alpha protein and its subcellular localization with Western blotting and cell immunofluorescence method respectively, and identify the effect of ERR alpha on lipid accumulation in mature porcine adipocytes with its specific inhibitor XCT790. The results showed that the ERR alpha ORF sequence is 1269 bp (GenBank Accession No. FJ446485, not published), and encode 422 amino acids. The homologies of ERR alpha nucleotide and amino acids sequences are high in different species. ERR alpha protein is highly expressed in pig white adipose tissue, kidney and heart, while remarkably lower in spleen. Cell immunofluorescence results indicated that ERR alpha protein distribute widely in adipocytes nucleus and cytoplasm. XCT790 significantly inhibited the expression of ERR alpha and lipid accumulation in porcine mature adipocyte. This study will provide new target and theoretical reference for fat deposition control.
Adipocytes ; metabolism ; Animals ; Animals, Newborn ; Cloning, Molecular ; Energy Metabolism ; Gene Expression Regulation ; Lipid Metabolism ; Molecular Sequence Data ; Nitriles ; pharmacology ; Open Reading Frames ; Polymerase Chain Reaction ; methods ; Receptors, Estrogen ; biosynthesis ; genetics ; Swine ; Thiazoles ; pharmacology

AIMTo investigate the effects of 17beta-estradiol (E2), Peganum harmala extract (PHE) and caloric restriction (CR) on various testis parameters during aging.

METHODSTwelve month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR (40%).

RESULTSOur results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes: gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats.

CONCLUSIONBesides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species (ROS) produced in large quantities in the aged testis.

Aging ; physiology ; Animals ; Antioxidants ; metabolism ; Aromatase ; biosynthesis ; genetics ; Caloric Restriction ; Estradiol ; metabolism ; pharmacology ; Estrogens ; pharmacology ; Lipid Peroxidation ; drug effects ; Male ; Oxidative Stress ; drug effects ; Peganum ; chemistry ; Plant Extracts ; pharmacology ; RNA ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Receptors, Estrogen ; biosynthesis ; genetics ; Testis ; drug effects ; enzymology ; growth & development ; Testosterone ; metabolism ; Thiobarbituric Acid Reactive Substances ; metabolism

OBJECTIVETo investigate the correlation between the lack of estrogen receptor (ER) gene expression and hypermethylation of ER gene, and detect whether re-expressed ER protein is activated.

METHODSThe methylation status of ER gene promoter in the ER-negative breast cancer cells was evaluated by methylation specific PCR (MSP) and genomic sequencing. The expression of ER and progesterone receptor (PR) mRNA as well as the production of ER protein were detected by RT-PCR and Western blot method, respectively. MTI assay was used to examine the function of re-expressed ER protein.

RESULTSThe ER gene promoter was highly methylated, while ER mRNA and ER protein were not expressed in the ER-negative breast cell line MDA-MB-231. The ER-negative breast cells treated with demethylating agent 5 -aza-2'-deoxycytidine (5-AZA-2'-deoxyC) restored the expression of ER mRNA and ER protein. Expression of the endogenous ER-responsive PR gene was activated and the methylation of ER gene was simultaneously decreased. After MDA-MB-231 was treated with 5-AZA-2'-deoxyC, the protein of ER was re-expressed and the growth of cells treated with tamoxifen were inhibited significantly (P < 0.05).

CONCLUSIONinactivation of ER gene has a close relationship with the abnormal methylation of ER gene promoter. 5-AZA-2'-deoxyC may effectively cause demethylation and restore functional expression of ER silenced by aberrant hypermethylation. The result may offer a new measure and theory for breast cancer patients with ER-negative expression to receive endocrine therapies.

Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents, Hormonal ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Base Sequence ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Methylation ; Estrogen Receptor alpha ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Progesterone ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Selective Estrogen Receptor Modulators ; pharmacology ; Tamoxifen ; pharmacology

OBJECTIVETo investigate the expression of ER alpha in chemically induced, ER alpha-negative human breast cancer MDA-MB-435 cells and its restoration of the responsiveness to endocrine therapy.

METHODSMDA-MB-435 cells were treated with HDAC inhibitor trichostatin A(TSA)and DNMT1 inhibitor 5-AZA-CdR (AZA). The mRNA level of ER alpha, PR and PS2 in treated MDA-MB-435 cells was detected by RT-PCR. The WST-8 (water-soluble tetrazolium salt-8) method was used to analyze the proliferation rate of the cells. Xenograft in female nude mice was used to further explore the change of proliferation rate of treated MDA-MB-435 cells in vivo.

RESULTSAfter treatment with AZA and TSA, mRNA expression of ER alpha, PR and pS2 was up-regulated in MDA-MB-435 cells. The mRNA level of ER alpha was the hightest when MDA-MB-435 cells were treated with 2.5 micromol/L AZA and 100 ng/ml TSA. The treated MDA-MB-435 cells showed different proliferation rate in various media containing different concentration of estrodial. The MDA-MB-435 cells showed down-regulated proliferation rate after treatment with the combination of 2.5 micromol/L AZA and 100 ng/ml TSA, and 4-OH tamoxifen could suppress the growth rate of the induced MD-MBA-435 cells but not the untreated cells. The treated MDA-MB-435 cells showed slower proliferation rate than that of untreated cells in vivo (P <0. 01), and the proliferation rate of the treated MDA-MB-435 cells became lower when the nude mice were deprived of estrogen by castration (P <0. 01).

CONCLUSIONAfter treatment with TSA and AZA, ER alpha-negative MDA-MB-435 cells can express functional ER alpha and regain responsiveness to estrogen both in vitro and in vivo. HDAC inhibitor and DNMT1 inhibitor may play an important role in restoration of sensitivity of ER alpha-negative breast cancers to endocrine therapy.

Animals ; Azacitidine ; analogs & derivatives ; pharmacology ; Breast Neoplasms ; genetics ; pathology ; prevention & control ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Modification Methylases ; antagonists & inhibitors ; Enzyme Inhibitors ; pharmacology ; Estrogen Receptor alpha ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; Mammary Neoplasms, Experimental ; genetics ; pathology ; prevention & control ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Ovariectomy ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Progesterone ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Trefoil Factor-1 ; Tumor Suppressor Proteins ; genetics ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the expression of interleukin-8 (IL-8) and its prognostic significance in breast cancer.

METHODSExpression of IL-8 in 113 breast cancers, 19 breast benign tumors and 20 breast normal tissues was examined by tissue microarray using immunohistochemistry, and the association of IL-8 expression with patient's clinico-pathological characteristics and prognosis was further analyzed.

RESULTSThe positive rate of IL-8 expression in breast cancer was 27.4%, which was significantly higher than that in benign tumor and normal tissue of breast (P = 0.002). IL-8 expression related to histological type (P = 0.040) and lymph node status (P = 0.021). The expression of IL-8 was observed to correlate negatively with ER and PR status (P = 0.015 and P = 0.034), and correlate positively with C-erbB-2 status (P = 0.002). In addition, Kaplan-Meier curves of disease-free survival analysis showed a significant difference between IL-8 positive groups and negative group (P = 0.031).

CONCLUSIONSIL-8 might be a poor prognostic factor for human breast cancer, and also might be a novel molecular marker to predicate the occurrence and progression of breast cancer.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; diagnosis ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Interleukin-8 ; biosynthesis ; Lymphatic Metastasis ; Middle Aged ; Prognosis ; Receptor, ErbB-2 ; biosynthesis ; Receptors, Estrogen ; biosynthesis ; Receptors, Progesterone ; biosynthesis

OBJECTIVETo investigate the effects of epidermal growth factor (EGF) on estrogen receptor (ER) and androgen receptor (AR) in mouse prostate cells and explore the putative role of EGF in prostatic hyperplasia.

METHODSSixty male Kunming mice were randomly divided into two EGF groups and one control group (n=20) and subjected to subcutaneous injection of 1 and 2 microg/day EGF and distilled water, respectively, for 28 consecutive days. The cellular expression of ER and AR in the prostate of mice in different groups was evaluated by flow cytometry.

RESULTSCompared with the control group, the positivity rate of ER and its expression level were significantly increased in the mouse prostate after EGF treatment (P<0.01), and the ER expression level was significantly higher in mouse with 2 microg/day EGF treatment than in those treated with 2 microg/day EGF (P<0.01). AR positivity rate and expression level also increased significantly in comparison with the control group (P<0.05), but no significant variation was found between 1 microg/day and 2 microg/day EGF groups.

CONCLUSIONEGF can increase the cellular expression of ER and AR in mice prostate and may play a role in the pathogenesis of prostatic hyperplasia.

Animals ; Epidermal Growth Factor ; administration & dosage ; pharmacology ; Flow Cytometry ; Injections, Subcutaneous ; Male ; Mice ; Prostate ; cytology ; metabolism ; Prostatic Hyperplasia ; metabolism ; pathology ; Random Allocation ; Receptors, Androgen ; biosynthesis ; Receptors, Estrogen ; biosynthesis

OBJECTIVETo detect the expressions of human epidermal growth factor receptor 2 (Her-2), epidermal growth factor receptor (EGFR), presenilin 2 (PS-2) and estrogen receptor (ER) in breast cancer and discuss their clinical implications.

METHODSThe expressions of Her-2, EGFR, PS-2 and ER were measured immunohistochemically in 108 patients with breast cancer.

RESULTSThe positive expression rates of Her-2, EGFR, PS-2 and ER were 37.0%, 40.7%, 57.4% and 53.7% respectively in the breast cancer patients. The expression of Her-2 was not correlated with EGFR, but inversely correlated with PS-2 and ER. The expressions of Her-2 and EGFR, PS-2, ER were correlated with the histological grades (P<0.05), and Her-2, EGFR and ER expressions with lymph node metastasis (P<0.05). The expressions of Her-2, EGFR, PS-2 and ER did not correlate to the pathological types, patient's age and tumor size (P>0.05).

CONCLUSIONExpressions of Her-2 and EGFR often suggests an unfavorable prognosis while expressions of PS-2 and ER suggest a more favorable one. Expressions of Her-2, EGFR, PS-2 and ER are useful prognostic factors in breast cancer patients.

Adult ; Aged ; Biomarkers, Tumor ; biosynthesis ; Breast Neoplasms ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Presenilin-2 ; biosynthesis ; Prognosis ; Receptor, Epidermal Growth Factor ; biosynthesis ; Receptor, ErbB-2 ; biosynthesis ; Receptors, Estrogen ; biosynthesis