Estrogen participates in many life activities through combination with estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) in the body. In order to establish an in vitro estrogen-like compound screening model, the coding region of human ERalpha and ERbeta was separately constructed into pET32-ERalpha and pET43-ERbeta prokaryotic expression vector and water-soluble recombinant ERalpha and ERbeta proteins were expressed in Escherichia coli strain BL21. Western blotting revealed that both recombinant proteins have estrogen receptor binding sites. The proteins were purified using S-Tag affinity Purification Kit and digested with enterokinase to get the ERalpha and ERbeta proteins. About 0.90 mg of ERalpha and 0.65 mg of ERbeta were obtained at the concentration of 0.181 and 0.131 mg x mL(-1), respectively.
Binding Sites ; Escherichia coli ; metabolism ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Genetic Vectors ; Humans ; Protein Binding ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVE: To observe the effect of different concentrations of nylestriol (NYL) and levonorgestrel (LNG) on the expression of ERα and ERβ in human osteoscarcoma MG-63 cell lines, and to explore the impact of paracrine effect on the gene expression. METHODS: MG-63 cells were treated with 3 concentrations (10(-10),10(-8), and 10(-6) mol/L) of NYL or LNG. The untreated control group and the positive control group were also established. The 2 groups treated with NYL (10(-10) mol/L) or LNG (10(-8) mol/L) were designed to renew the medium every 12 h. Semi-quantitative RT-PCR was conducted to detect the mRNA expression of ERα and ERβ on the MG-63 cells treated with different concentrations of the 2 drugs, respectively. RESULTS: Both drugs up-regulated ERα and ERβ mRNA expression. The best concentration for both NYL and LNG was 10(-6) mol/L for ERα expression. As for ERβ, the best concentration of NYL and LNG was 10(-10) mol/L and 10(-8) mol/L. The role of medium replacement on the expression of ERα was not observed, but medium replacement inhibited ERβ expression. CONCLUSION Both NYL and LNG can up-regulate the mRNA expression of ER subtypes in MG-63 cells, with mutual restriction between the 2 subtypes. The paracrine effect on MG-63 cell lines may be involved in the regulation process of mRNA expression of ERβ.
Cell Line, Tumor ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Humans ; Levonorgestrel ; pharmacology ; Osteosarcoma ; metabolism ; pathology ; Quinestrol ; analogs & derivatives ; pharmacology ; RNA, Messenger ; genetics ; metabolism

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.
Adenoviridae/genetics* ; Cell Proliferation ; Estrogen Receptor alpha/metabolism* ; Recombinant Proteins ; Transfection

BACKGROUNDRecent studies have suggested that estrogens are involved in normal and abnormal prostate growth, though their exact role is still controversial. Oestrogens exert inhibitory and stimulatory effects on prostate gland, but the expression of oestrogen receptor-alpha (ERalpha) and oestrogen receptor-beta (ERbeta) in malignant prostate tissue remains unresolved. We determined ERalpha and ERbeta in prostate cancer and investigated the relationship between expression of ER and pathological features of prostate carcinoma.

METHODSThirty-two cases of prostate cancer, 12 cases of normal prostate tissue and 32 cases of benign prostate hyperplasia were analyzed for the expression of ERalpha and ERbeta using semiquantitative, reverse transcription polymerase chain reaction (RT-PCR) and the products sequenced.

RESULTSComparisons of the normal, hyperplastic and tumour prostate tissues indicated an overexpression of ERalpha in tumour specimens (P < 0.01). However, the expression of ERbeta significantly reduced in tumour tissues compared with normal and hyperplastic specimens (P < 0.01), suggesting that severe pathological features of prostate cancer were associated with lower ERbeta expression. Spearman analysis showed negative correlation between ERbeta expression and tumour stage, grade (-0.67, -0.43, respectively, both P < 0.05), and a positive correlation between ERalpha expression and tumour stage, grade (0.51, 0.57, respectively, both P < 0.01). Our analysis also showed that hormone refractory, prostate cancer, compared with hormone dependent, prostate cancer, displayed a decreased expression of ERbeta (P < 0.01) and an increased expression of ERalpha.

CONCLUSIONSERalpha and ERbeta may play important roles in the development of prostate cancer. The decrease in ERbeta expression is associated with higher Gleason grade tumours and prostate cancer with higher metastatic potential. The loss of ERbeta could be one of the key processes leading to uncontrolled growth of prostate epithelial cells.

Estrogen Receptor alpha ; genetics ; Estrogen Receptor beta ; genetics ; Humans ; Male ; Prostate-Specific Antigen ; blood ; Prostatic Neoplasms ; blood ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction

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 detect the estrogenic activity of genistein and apigenin with ER-positive cell line MCF-7 human breast cancer cells.

METHODMTT method was adopted to study the impact of genistein and apigenin on MCF-7 proliferation in vitro. Real-time RT-PCR method was used to detect their impact on ERalpha, ERbeta, PR and PS2 mRNA expression levels.

RESULTGenistein and apigenin promoted the proliferation of MCF-7. Genistein 1 x 10(-10) mol x L(-1) group showed a significant increase in the expression of ERa mRNA levels or a 17. 76 times more than the control group and a 1.75 times more than the E2 group. Apigenin notably promoted the PR mRNA expression or a 4. 57 times more than the control group and a 1.11 times more than the E2 group. Both of them had different effect in promoting ERalpha, ERbeta, PR or PS2 mRNA.

CONCLUSIONBoth genistein and apigenin have a strong estrogen-like effect. Although they have different effect in promoting estrogenic response genes (such as ERa, ERbeta, PR and PS2 mRNA), genistein shows a stronger activity than apigenin. It also suggests that the signaling pathways of phytoestrogens showing estrogen-like effect are not completely identical with estrogen pathways. The B-cycle position of flavonoids is one of the key sites to estrogen-like activity, and isoflavones (cycle B on site 3) show stronger estrogen-like activity than flavones (B-cycle lies in site 2).

Apigenin ; pharmacology ; Cell Proliferation ; drug effects ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Gene Expression ; drug effects ; Genistein ; pharmacology ; Humans ; MCF-7 Cells ; Phytoestrogens ; pharmacology ; Presenilin-2 ; genetics ; metabolism

OBJECTIVETo explore the expression of estrogen receptors in the human periodontal ligament fibroblasts in vitro.

METHODSHuman periodontal ligament fibroblasts were cultured in vitro. The estrogen receptors (ER)-alpha, ER-beta were detected with immunocytochemistry staining; The mRNA of ER-alpha, ER-beta were measured by reverse transcription polymerase chain reaction; The expression of ER-alpha, ER-beta protein were detected by Western blot.

RESULTSThe mRNA and protein expression of two subtype of ER were observed on human periodontal ligament fibroblasts (HPLF). The intensity of the ER-beta bands were stronger than those of ER-alpha, indicating that ER-beta expression in HPLF was higher.

CONCLUSIONSER may play an important role in the function of human periodontal ligament fibroblasts.

Adolescent ; Blotting, Western ; Cells, Cultured ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Fibroblasts ; metabolism ; Humans ; Immunohistochemistry ; Periodontal Ligament ; cytology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the estrogenic activity of icariin and genistein with estrogen-dependent human breast cancer (MCF-7) cells.

METHODMCF-7 cells were incubated with media containing 5% charcoal dextran-treated FBS in phenol red-free media for 48 h. CCK-8 kit was used to study the impact of defferent concentration of icariin and genistein on MCF-7 proliferation in vitro. Optimal concentration icariin and genistein were added into medium and total RNA was isolated after 12, 24, 36, 48 h. The gene expression of ERalpha, ERbeta, PS2, and PR were investigated by Real-time RT-PCR Total protein was also isolated and secretion of ERalpha, ERbeta, PS2, and PR were examined by Western blot.

RESULT10 micromol x L(-1) icariin and genistein could promote the proliferation of MCF-7 evidently. However, the ability of genistein to promote the proliferation was better than icariin. With the concentration of 10 micromol x L(-1), genistein group had a stronger expression of ERa, PS2 and PR mRNA levels than icariin while ERbetaexpression had no significant difference in two group. The same effects were detected by western blotting.

CONCLUSIONBoth genistein and icariin have a strong estrogen-like effect, but the estrogenic activity of genistein is stronger than icariin. It showed that the activity of icariin is stron-ger than genistein to promote ROB maturation. So it must be that icariin promotes the maturation of osteoblasts in vitro by a estogen-independent mechanism.

Cell Proliferation ; drug effects ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Estrogens ; pharmacology ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; Genistein ; pharmacology ; Humans ; MCF-7 Cells ; Osteoblasts ; cytology ; drug effects ; metabolism ; Presenilin-2 ; metabolism

OBJECTIVETo investigate the expression of the miR-155 in human primary breast cancer and its clinical significance.

METHODSFrom February to June 2009, 45 pairs of specimens of human primary breast cancer and matched nontumor breast tissues were collected from the patients who received operation for breast cancer. Real-time polymerase chain reaction (RT-PCR) was used to detect the miR-155 expression in those specimens.

RESULTSThe stem-loop RT-PCR was sensitive and specific enough to detect the expression of the miR-155. The median relative expression of miR-155 was 0.360 in tumor samples, and it was 0.135 in matched nontumor breast tissues, the difference was statistically significant (P < 0.05). It's indicated that the up-regulation of miR-155 expression was associated with advanced TNM clinical stage (median 0.316, 0.358 and 0.417 respectively for stage I, II and III tumor, P = 0.002), lymph node metastasis (median 0.383 and 0.355 respectively for cases with positive and negative lymph nodes, P = 0.034), higher proliferation index [median 0.387 and 0.353 respectively for cases with high proliferation index (Ki67 > 10%) and low proliferation index (Ki67 ≤ 10%), P = 0.019], estrogen receptor-positive (0.367 and 0.318 respectively for cases with positive estrogen receptor and negative group, P = 0.041) and progesterone receptor-positive (0.398 and 0.335 respectively for cases with positive progesterone receptor and negative group, P = 0.029) in patients with breast cancer.

CONCLUSIONSThe expression of miR-155 is up-regulated in primary breast cancer, especially in patients with positive estrogen and progesterone receptor. miR-155 may play an important role in the proliferation, invasion and metastasis of human primary breast cancer, and it could be a indicator in the diagnosis and prognosis of primary breast cancer.

Adult ; Aged ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Estrogen Receptor alpha ; metabolism ; Female ; Humans ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Receptors, Progesterone ; metabolism

OBJECTIVETo investigate the possible mechanism by which estrogen regulates apoptosis through the estrogen receptor.

METHODSBy means of fluorescence immunocytochemistry, the present study investigated the distribution of Bcl-2 and the colocolization of Bcl-2 and ERalpha immunoreactivity in the hippocampus of 10 Alzheimer's disease (AD) patients and 10 aged controls.

RESULTSBcl-2 immunoreactivity was widely distributed in neurons, concentrating predominantly on the subfields CA3 and CA4 in the stratum pyramidale of hippocampus both in controls and in AD patients. Bcl-2 staining in the labeled neuron was observed mainly in the cytoplasm and neuritic processes, but a few nuclei were also positive. Bcl-2 labeling was also detected in the astrocytes mainly in AD, but sparsely in controls. Double-labeled fluorescence immunocytochemistry showed that most Bcl-2-immunolabeled neurons also exhibited positive staining for ERalpha.

CONCLUSIONSEstrogen may function as a regulator of apoptosis to modulate the expression of Bcl-2 in neurons and astrocytes in hippocampus of AD through ERalpha.

Alzheimer Disease ; genetics ; metabolism ; Apoptosis ; Astrocytes ; metabolism ; Estrogen Receptor alpha ; Female ; Hippocampus ; metabolism ; pathology ; Humans ; Neurons ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Receptors, Estrogen ; genetics ; metabolism ; physiology