Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer.
Breast Neoplasms* ; Breast* ; Cell Line ; Classification ; Epithelial Cells ; Estrogen Receptor alpha ; Estrogens* ; Humans ; Phenobarbital ; Phenotype* ; Progesterone* ; Proto-Oncogenes ; Receptors, Progesterone* ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger* ; Sequence Analysis, DNA

INTRODUCTIONWhile overexpression of syndecan-1 has been associated with aggressive breast cancer in the Caucasian population, the expression pattern of syndecan-1 in Asian women remains unclear. Triple-positive breast carcinoma, in particular, is a unique subtype that has not been extensively studied. We aimed to evaluate the role of syndecan-1 as a potential biomarker and prognostic factor for triple-positive breast carcinoma in Asian women.

METHODSUsing immunohistochemistry, staining scores of 61 triple‑positive breast carcinoma specimens were correlated with patients' clinicopathological variables such as age, ethnicity, tumour size, histological grade, lymph node status, lymphovascular invasion, associated ductal carcinoma in situ grade, recurrence and overall survival.

RESULTSSyndecan-1 had intense staining scores in triple‑positive invasive ductal breast carcinomas when compared to normal breast tissue. On multivariate analysis, syndecan-1 epithelial total percentage and immunoreactivity score showed statistical correlation with survival (p = 0.02).

CONCLUSIONThe intense staining scores of syndecan-1 and their correlation with overall survival in patients with triple-positive breast carcinoma suggest that syndecan-1 may have a role as a biological and prognostic marker in patients with this specific subtype of breast cancer.

Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; Biomarkers, Tumor ; blood ; Breast Neoplasms ; blood ; classification ; mortality ; Estrogen Receptor alpha ; metabolism ; Female ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Middle Aged ; Multivariate Analysis ; Prognosis ; Receptor, ErbB-2 ; metabolism ; Receptors, Progesterone ; metabolism ; Syndecan-1 ; blood ; Tissue Array Analysis ; Treatment Outcome

OBJECTIVETo detect the expression of ERα and ERβ in lung carcinomas and investigate their clinicopathological and prognostic significance by using tissue microarray assay and immunohistochemical staining.

METHODSSix hundred and ninety-eight lung cancer specimens were used in this study, including 651 cases of non-small cell lung carcimomas (NSCLCs) and 47 cases of small cell lung cancers (SCLCs). There were 309 cases of adenocarcimoma and 342 cases of squamous cell carcinoma. The expression of ERα and ERβ was analyzed by immunohistochemistry on paraffin-embedded sections.

RESULTSIn the normal lung tissues, expression of ER&alpha; and ERβ was 0% (0/35) and 25.0% (9/36), respectively. In the tumor tissues, ER&alpha; was expressed in 209 of 295 AC cases (70.8%), 169 of 330 SCC cases (51.2%) and 9 of 47 SCLC cases (19.1%) (P < 0.001). ERβ was expressed in 200 of 297 AC cases (67.3%), 140 of 322 SCC cases (43.5%) and 31 of 47 SCLC cases (66.0%) (P < 0.001). In NSCLC, the expression of ER&alpha; and ERβ was significantly associated with smoking, stage and lymph node metastasis, also with sex refer to ERβ (P < 0.05), but not significantly with age, tumor size and degree of differentiation (P > 0.05). Follow-up was completed in 398 NSCLC cases, and no significant correlation was found between the prognosis and expression of ER&alpha; and ERβ.

CONCLUSIONSThe expression of ER&alpha; and ERβ has significant difference in lung adenocarcinoma, squamous cell carcinoma and small cell lung cancer. In NSCLC, expression of ER&alpha; and ERβ is associated with smoking, stage, and lymph node metastasis. The expression of ERβ is higher in female than in male NSCLC patients.

Adenocarcinoma ; metabolism ; pathology ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Female ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Sex Factors ; Small Cell Lung Carcinoma ; metabolism ; pathology ; Smoking ; Tissue Array Analysis

OBJECTIVETo establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method.

METHODHuman ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed.

RESULTThe recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity.

CONCLUSIONA phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.

Cell Line, Tumor ; Cicer ; chemistry ; metabolism ; Drug Evaluation, Preclinical ; methods ; Estrogen Receptor alpha ; genetics ; metabolism ; Genes, Reporter ; drug effects ; Genetic Vectors ; metabolism ; Genistein ; chemistry ; pharmacology ; Humans ; Luciferases ; drug effects ; metabolism ; Phytoestrogens ; analysis ; pharmacology ; Plant Extracts ; chemistry ; metabolism ; pharmacology ; Plasmids ; drug effects ; metabolism ; Transfection ; methods

OBJECTIVETo investigate phytoestrogenic effects of ferulic acid in ER-positive T47D and ER-negative MDA-MB231 cells in culture.

METHODT47D and MDA-MB231 human breast cancer cells were treated with ferulic acid and examined cell proliferation by means of MTT assay. Cell cycle distribution, ERalpha and ERbeta expression were treated by flow cytometer. The pS2 mRNA expressions were detected by real-time fluorescence quantitative PCR.

RESULTThe proliferations were enhanced significantly by treatment with ferulic acid on T47D cells and the proliferation effects were inhibited by adding Faslodex (1 x 10(-8) mol x L(-1)). However, there was no significant difference on the proliferation in MDA-MB-231 cells compared with solvent control group by both treatment with ferulic acid and co-treatment with Faslodex (1 x 10(-8) mol x L(-1)). Ferulic acid stimulated the amount of T47D cells in phase S and proliferation index increased significantly. The effects were inhibited by treatment with Faslodex (1 x 10(-8) mol x L(-1)), and the amount of cells in phase S and proliferation index decreased, the amount of cells in G0/G1 phase increased, cell cycle of T47D was arrested in G0/G1 phase. Ferulic acid up-regulated pS2 mRNA expressions and increased the level of ERalpha protein expression in T47D cells. Ferulic acid did not show remarkable effect to the level of ERbeta protein expression in T47D cells.

CONCLUSIONFerulic acid possessed phytoestrogenic effect by up-regulating pS2 gene expression and the receptor subtype of ERalpha.

Breast Neoplasms ; chemistry ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coumaric Acids ; pharmacology ; Estrogen Receptor alpha ; analysis ; Estrogen Receptor beta ; analysis ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; RNA, Messenger ; analysis ; Trefoil Factor-1 ; Tumor Suppressor Proteins ; genetics

OBJECTIVETo investigate the expression of a novel metastasis-inducing protein human anterior gradient-2 (AGR2) in breast cancer and its clinical and prognostic significance.

METHODSAGR2 expression was assessed in 160 cases of breast cancer and 20 cases of benign breast diseases by immunohistochemistry using tissue chip technology. In addition the expression of ERa, PR and c-erbB-2 in breast cancer was also evaluated. Follow-up information of 5-year duration was available in 127 patients with breast cancer. Kaplan-Meier analysis and COX regression model were used to analyze the correlation between AGR2 expression and the follow-up clinical data.

RESULTSThe expression of AGR2 was significantly higher in breast cancers than that in benign diseases (68.3% vs. 25.0% , P < 0.01). There was a negative correlation between AGR2 expression and the histological grade of breast cancer (P <0.05) , whereas positive correlations was found between the expression of AGR2 and ERalpha (P <0.05), and between the expression of AGR2 and PR (P <0.01). In the subgroup of ERalpha-positive breast cancer, Logistic regression model demonstrated AGR2 and TNM stage were important factors affecting lymph node metastasis (both P < 0.01). Kaplan-Meier analysis demonstrated that a positive expression of AGR2 was associated with poor overall survival and relapse-free survival (both P <0.01). Moreover, COX regression model confirmed the expression of AGR2 as an independent prognostic factor among patients with ERa-positive breast cancer (P <0.01).

CONCLUSIONSThe abnormal expression of AGR2 may play a role in the pathogenesis and progression of breast cancer. The metastasis-inducing capability of AGR2 may be partly regulated through the ER pathway. Therefore, AGR2 may be a useful molecular marker for prognostication for patient with hormone-responsive breast cancer.

Antineoplastic Agents, Hormonal ; analysis ; BRCA2 Protein ; genetics ; metabolism ; Biomarkers, Tumor ; analysis ; Breast Neoplasms ; diagnosis ; genetics ; metabolism ; Estrogen Receptor alpha ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; Immunohistochemistry ; Neoplasm Metastasis ; diagnosis ; Neoplasm Staging ; Prognosis ; Proteins ; genetics ; metabolism ; Receptor, ErbB-2 ; analysis ; metabolism

OBJECTIVETo investigate the distribution of ER isoforms in endometriosis and eutopic endometrium.

METHODSTissue samples of patients with ovarian endometriosis, treated in People's Liberation Army General Hospital from January 2004 to December 2006, were retrieved. A total of 60 cases of ovarian endometriotic cysts with their corresponding eutopic endometrium (30 cases of proliferation phase and 30 of secretary phase eutopic endometrium) and 30 cases of normal endometrium (15 proliferative and 15 secretary phase endometrial samples respectively) were included. Expressions of ERalpha and ERbeta were analyzed using immunohistochemistry and the expression ratio was statistically analyzed by using SPSS 12.0 software.

RESULTSExpressions of both ERalpha and ERbeta in epithelial cells were positively correlated with that of the stromal cells. The expression of ERalpha in eutopic endometrium (73.3% in epithelium and 76.7% in stroma) was significantly higher than that in ovarian endometriotic cysts (43.3% in epithelium and 46.7% in stroma), or normal control (56.7% in epithelium and 50.0% in stroma, respectively, each P < 0.05. However, the expression of ERbeta (90.0% in epithelium and 76.7% in stroma) was higher in ovarian endometriotic cysts than that in the eutopic endometrium (68.0% in epithelium and 63.3% in stroma respectively, P < 0.05), and ERbeta expression in eutopic endometrium was higher than that in the normal control endometrium (36.7% in epithelium and 26.7% in stroma, respectively, P < 0.05). The expressions of both ERalpha and ERbeta changed periodically in eutopic and normal endometrium, whereas ERalpha and ERbeta level were less variable in the ectopic endometrium. The expression of ERbeta was statistically higher than that of ERalpha (P < 0.05) in ectopic endometrium, whereas no significant difference was seen between the two isoforms in the eutopic or normal endometrium.

CONCLUSIONSBoth ERalpha and ERbeta have higher expression levels in eutopic endometrium of patients with ovarian endometriotic cysts. ERbeta is predominantly expressed in endometriotic cysts, where the expression of ERalpha is limited. The different distribution of ERalpha and ERbeta may play an important role in the development of ovarian endometriosis.

Adult ; Choristoma ; pathology ; Endometriosis ; metabolism ; pathology ; Endometrium ; metabolism ; pathology ; Epithelium ; Estrogen Receptor alpha ; analysis ; Estrogen Receptor beta ; analysis ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Protein Isoforms ; analysis ; Receptors, Estrogen ; analysis ; Stromal Cells ; metabolism

We developed the recombinant green fluorescent protein gene yeast cell to screen estrogenic compounds based on two episomal vectors. In the expression vector the expression of human estrogen receptor alpha(hERalpha) was driven by 3-glyceraldehydephosphate dehydrogenase (GPD) promoter; in the reporter vector the expression of the yeast enhanced green fluorescent protein (yEGFP) gene was under the control of the estrogen response element (ERE). The vectors were transformed into yeast cell (W303-1A) to construct GFP recombinant yeast cell. Incubation of the yeast cell with various concentrations of the estrogenic compounds led to expression of the reporter gene product GFP in a dose dependent manner. Compared to other yeast bioassays, the yeast cell for environmental estrogen bioassay based on yEGFP reporter gene did not need cell wall disruption or the addition of a substrate or reagent. This yEGFP assay was performed completely in 96 well plates. So this test system can be used as a rapid and high throughput system for screening estrogenic chemical products, which has the characteristics of the sensitivity, reproducibility and cheapness.
Biological Assay ; methods ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogens ; analysis ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism

OBJECTIVETo study on the inhibitory action of combined acupuncture and medicine therapy on the model rat of hyperplasia of mammary glands and the mechanism.

METHODSThe model rat of hyperplasia of mammary glands were prepared. After modelling, they were randomly divided into an acupuncture group, a Chinese drug group and a combined acupuncture and drug group, a control group and a model group. Except both the control group and the model group, other 4 groups were treated respectively with acupuncture, Chinese drug, combined acupuncture and Chinese drug, and Premormine, once each day, 9 sessions constituting one course. After treatment of 3 courses (30 days), changes of the breast tissue form were observed, and the diameter and the area of the acina cavity were determined and expressions of estrogen receptor subgroups (ERalpha and ERbeta) were detected with immunohistochamical methods.

RESULTSThe diameter and the area of the acina cavity were increased in the model group as compared with those in the normal group (both P < 0.01), and in the treatment group they were decreased as compared with those in the model group (P < 0.05 or P < 0.01)); both acupuncture and Chinese drug could up-regulate the expression of ERbeta and down-regulate the expression of ERalpha.

CONCLUSIONBoth acupuncture and moxibustion, and Chinese medicine have inhibitory action on hyperplasia of mammary glands in the rat, with the strongest inhibitory action of the combined acupuncture and medicine treatment which is basically close to the level of Premormine. The mechanism is possily related with the up-regulation of ERbeta expression and down-regulation of ERalpha expression.

Acupuncture Points ; Acupuncture Therapy ; Animals ; Combined Modality Therapy ; Estrogen Receptor alpha ; analysis ; Estrogen Receptor beta ; analysis ; Female ; Hyperplasia ; Mammary Glands, Animal ; chemistry ; drug effects ; pathology ; Medicine, Chinese Traditional ; Rats ; Rats, Wistar

BACKGROUNDEstrogen might play an important role in type 2 diabetes mellitus pathogenesis. A number of polymorphisms have been reported in the estrogen receptor alpha (ERalpha) gene (also named ESR1), including the XbaI and PvuII restriction enzyme polymorphisms of ESR1, which may be involved in disease pathogenesis. The aim of this study was to determine whether ERX gene polymorphisms are associated with type 2 diabetes mellitus and serum lipid level.

METHODSTwo hundred and ninety-nine patients with type 2 diabetes mellitus were compared with three hundred and forty-one health controls of Guangzhou in China, both were male and postmenopausal female residents at 51 - 70 years. ESR1 genotyping was performed using polymerase chain reaction (PCR) and PvuII and XbaI restriction fragment length polymorphism (PCR-RFLP) analysis.

RESULTSESR1 allelic frequencies of P, p and X, x alleles were 0.408, 0.592; 0.360, 0.640 in the type 2 diabetes mellitus group and 0.318, 0.682; 0.328, 0.672 in the control group, respectively. In case-control study, there was significant difference in PvuII, but not XbaI, allele frequency between the type 2 diabetes mellitus and control groups (P = 0.001 and P = 0.122). When the group was separated into men and women, the difference was significant in women (P < 0.001) but not in men (P = 0.854) with the PvuII genotype, and the effect of PvuII variant on the development of type 2 diabetes mellitus was improved with aging. In addition, PvuII genotype was associated with blood glucose [fasting blood glucose (FBG), postprandial blood glucose (PBG)] and serum lipid [total cholesterol (TC) and low density lipoprotein (LDL)-c] concentration in healthy women.

CONCLUSIONSPvuII polymorphism of ESR1 increases susceptibility to type 2 diabetes mellitus in Chinese Guangzhou women. ESR1 variants may also impact serum lipid metabolism, which might provide a mechanism connecting ESR1 to type 2 diabetes.

Aged ; Blood Glucose ; analysis ; Cholesterol, LDL ; blood ; Diabetes Mellitus, Type 2 ; blood ; genetics ; Estrogen Receptor alpha ; genetics ; Female ; Genotype ; Humans ; Lipids ; blood ; Logistic Models ; Middle Aged ; Polymorphism, Genetic