1.Expression of human estrogen receptor alpha and beta in Escherichia coli.
Lu LUO ; Shu-juan ZHAO ; Zheng-tao WANG ; Zhi-bi HU
Acta Pharmaceutica Sinica 2012;47(10):1399-1402
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
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Escherichia coli
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metabolism
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Estrogen Receptor alpha
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genetics
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metabolism
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Estrogen Receptor beta
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genetics
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metabolism
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Genetic Vectors
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Humans
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Protein Binding
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Recombinant Proteins
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genetics
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metabolism
2.Effect of nylestriol and levonorgestrel on the expression of estrogen receptor subtypes in human osteosarcoma MG-63 cell lines.
Kaichu YANG ; Eryuan LIAO ; Houde ZHOU
Journal of Central South University(Medical Sciences) 2010;35(12):1248-1253
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
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Estrogen Receptor alpha
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genetics
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metabolism
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Estrogen Receptor beta
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genetics
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metabolism
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Humans
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Levonorgestrel
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pharmacology
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Osteosarcoma
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metabolism
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pathology
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Quinestrol
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analogs & derivatives
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pharmacology
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RNA, Messenger
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genetics
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metabolism
3.Expression of oestrogen receptor-alpha and oestrogen receptor-beta in prostate cancer.
Guo-Sheng YANG ; Ying WANG ; Ping WANG ; Zhao-Dian CHEN
Chinese Medical Journal 2007;120(18):1611-1615
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
4.Effects of exogenous ER beta expression on the cell growth properties of MCF-7 breast cancer cell line.
Jian-hua ZHU ; Qi-nong YE ; San-tai SONG ; Ze-fei JIANG ; Jing-hua YAN ; Chun-fang HAO ; Cui-fen HUANG
Chinese Journal of Oncology 2006;28(2):103-106
OBJECTIVETo study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment.
METHODSAn eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed.
RESULTSA stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed.
CONCLUSIONExogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estradiol ; pharmacology ; Estrogen Antagonists ; pharmacology ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Humans ; Tamoxifen ; analogs & derivatives ; pharmacology ; Transfection
5.Study on estrogenic effect of genistein and apigenin in vitro.
Ruiqing ZHU ; Baofeng GE ; Bin YANG ; Keming CHEN ; Yimin WEN ; Jian ZHOU ; Guiqiu HAN ; Guozheng CHENG ; Yuankun ZHAI
China Journal of Chinese Materia Medica 2012;37(15):2317-2322
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
6.The expression of estrogen receptors in the human periodontal ligament fibroblasts in vitro.
Meng CAO ; Yin DING ; Lei SHU ; Jia LI ; Wei ZHANG ; Tao GUO
Chinese Journal of Stomatology 2007;42(9):533-536
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
7.Icariin promote maturation of osteoblasts in vitro by an estrogen-independent mechanism.
Wen-Gui SHI ; Xiao-Ni MA ; Yan-Fang XIE ; Jian ZHOU ; Jian ZHOU
China Journal of Chinese Materia Medica 2014;39(14):2704-2709
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
8.Expression and significance of estrogen receptor isoforms in the development of human breast carcinoma.
Shu WANG ; Shan WANG ; Jia-qing ZHANG ; Xin-min QIAO ; De-qi YANG ; Fu-zhong TONG ; Ying-jiang YE ; Zhi-rong CUI
Chinese Journal of Surgery 2004;42(13):792-794
OBJECTIVETo investigate the estrogen receptors (ER)alpha and ERbeta expression and their relationship with clinicopathological parameters in human breast carcinoma.
METHODSSamples were obtained from 30 breast carcinoma, reverse transcriptase polymerase chain reaction was used to measure the expression of ERalpha and ERbeta mRNA.
RESULTSERalpha mRNA level was up-regulated in breast carcinoma tissue compared with adjacent normal tissue (t = 7.399, P < 0.01) while down-regulated in ERbeta. The relative ratio of ERalpha and ERbeta was decreased in normal tissue vs. carcinoma (t = 6.385, P < 0.01), in patients with lymph node metastasis vs. those without lymph node metastasis (t = 2.602, P < 0.05), in late stage carcinoma vs. early stage (t = 3.754, P < 0.05).
CONCLUSIONERalpha and ERbeta play divergent role in the development of human breast carcinoma.
Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; metabolism ; pathology ; physiopathology ; Cell Line, Tumor ; Estrogen Receptor alpha ; biosynthesis ; genetics ; physiology ; Estrogen Receptor beta ; biosynthesis ; genetics ; physiology ; Female ; Humans ; Middle Aged ; RNA, Messenger ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction
9.Inhibitory effect of isoflavones on prostate cancer cells and PTEN gene.
Feng CAO ; Tai-Yi JIN ; Yuan-Fen ZHOU
Biomedical and Environmental Sciences 2006;19(1):35-41
OBJECTIVETo explore the mechanisms by which genistein and daidzein inhibit the growth of prostate cancer cells.
METHODSLNCaP and PC-3 cells were exposed to genistein and daidzein and cell viability was determined by MTT assay and cytotoxicity of the drugs by LDH test. Flow cytometry (FCM) was used to assess the cell cycle in LNCaP and PC-3 cells. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to examine the expression of PTEN gene (a tumor suppressor gene), estrogen receptor alpha gene (ERalpha), estrogen receptor beta gene (ERbeta), androgen receptor gene (AR) and vascular endothelial growth factor gene (VEGF).
RESULTSThe viability of PC-3 and LNCaP cells decreased with increasing concentrations and exposure time of genistein and daidzein. Genistein increased G2/M phase cells in PC-3 cells while decreased S phase cells in LNCaP cells in a dose-dependent manner. Daidzein exerted no influence on the cell cycle of LNCaP and PC-3 cells, but the apoptosis percentage of LNCaP cells was elevated significantly by daidzein. Genistein induced the expression of PTEN gene in PC-3 and LNCaP cells. Daidzein induced the expression of PTEN gene in LNCaP but not in PC-3 cells. The expression of VEGF, ERalpha and ERbeta genes decreased and AR gene was not expressed after incubation with genistein and daidzein in PC-3 cells. In LNCaP cells, the expression of VEGF and AR gene decreased but there was no change in the expression of ERalpha and ERbeta gene after incubation with genistein and daidzein. Conclusion Genistein and daidzein exert a time- and dose-dependent inhibitory effect on PC-3 and LNCaP cells. The down-regulation of ER gene by daidzein influences the growth of PC-3 cells directly. The inhibition of PC-3 cells by genistein and that of LNCaP cells by genistein and daidzein may be via Akt pathway that is repressed by PTEN gene, which subsequently down-regulates the expression of AR and VEGF genes. Our results suggest that the expression of PTEN gene plays a key role and several pathways may be involved in the suppression of prostate cancer cells by genistein and daidzein.
Antineoplastic Agents ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Gene Expression Regulation ; Genistein ; pharmacology ; Humans ; Isoflavones ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Male ; PTEN Phosphohydrolase ; genetics ; metabolism ; Prostatic Neoplasms ; Receptors, Androgen ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism
10.Establishment of a reporter gene-based cell screening model for discovering new agonists of estrogen receptor beta subtype.
Li-min CHEN ; Qiu-jun LÜ ; Inoue SATOSHI ; Guang-xing BIAN ; Zhen-hua CHEN ; Li-qing WEN
Acta Pharmaceutica Sinica 2006;41(8):721-726
AIMTo establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype.
METHODSA recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector. pTAL-ERE-SEAP was then transfected into human embryonic kidney (HEK293) cells. G418 (200 microg x mL(-1)) was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERbeta expression after transfection were observed. 2 622 compounds were screened by using this model.
RESULTSStably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and time-effect relationship manners. The Z' factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERbeta. E2 had no proliferating effects on stably transfected clones.
CONCLUSIONStably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor beta subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.
Alkaline Phosphatase ; genetics ; metabolism ; Cell Line ; Estradiol ; pharmacology ; Estrogen Receptor beta ; agonists ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; Humans ; Immunohistochemistry ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Response Elements ; genetics ; Transfection