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 effect of the overexpression of the ERbeta gene on the penile vascular endothelium of ERbeta knockout (ERbetaKO) mice and its molecular mechanisms.

METHODSWe randomly divided 12 ERbetaKO male mice into groups A (ERbetaKO + TNFalpha + pAdxsi-ERbeta) and B (ERbetaKO + TNFalpha + empty virus), the former treated by pAdxsi-ERbeta adenovirus transfection, the latter with empty virus, and meanwhile both injected intraperitoneally with TNFalpha at 6 microg per kg body weight per d for 14 days. Then we observed the erectile function of the mice by APO, determined the changes of the endothelial markers CD34 and vWF by immunohistochemical staining, and detected the expressions of the relevant molecules in the eNOS-NO pathway by RT-PCR, Western blot and immunohistochemistry.

RESULTSCompared with group B, group A showed a significantly increased number of penile erections (0.50 +/- 0.55 vs 2.17 +/- 0.41, P < 0.05), shortened erectile latency ([28.83 +/- 1.33] min vs [24.00 +/- 1.27] min, P < 0.05), enriched CD34 and vWF markers (0.67 +/- 0.52 vs 1.50 +/- 0.55 and 0.50 +/- 0.55 vs 1.33 +/- 0.52, both P < 0.05), elevated expressions of eNOS and Cam (RT-PCR: 1.38 +/- 0.03 vs 1.62 +/- 0.05 and 1.02 +/- 0.09 vs 1.42 +/- 0.05, both P < 0.05; Western blot: 1.27 +/- 0.04 vs 1.55 +/- 0.07 and 0.76 +/- 0.05 vs 0.95 +/- 0.08, both P < 0.05), and reduced expression of caveolin-1 (RT-PCR: 2.13 +/- 0.13 vs 1.72 +/- 0.08, P < 0.05; Western blot: 3.99 +/- 0.16 vs 3.40 +/- 0.14, P < 0.05). The results of RT-PCR were consistent with those of Western blot.

CONCLUSIONThe ERbeta gene protects the penile vascular endothelium via the eNOS-NO pathway.

Adenoviridae ; genetics ; Animals ; Endothelium, Vascular ; metabolism ; Estrogen Receptor beta ; genetics ; Male ; Mice ; Mice, Inbred Strains ; Mice, Knockout ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Penis ; blood supply ; metabolism ; Transfection

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

Estrogen has anti-colorectal cancer effects which are thought to be mediated by mismatch repair gene (MMR) activity. Estrogen receptor (ER) expression is associated with microRNA (miRNA) expression in ER-positive tumors. However, studies of direct link between estrogen (especially estradiol E2), miRNA expression, and MMR in colorectal cancer (CRC) have not been done. In this study, we first evaluated the effects of estradiol (E2) and its antagonist ICI182,780 on the expression of miRNAs (miR-31, miR-155 and miR-135b) using COLO205, SW480 and MCF-7 cell lines, followed by examining the association of tissue miRNA expression and serum E2 levels using samples collected from 18 colorectal cancer patients. E2 inhibited the expressions of miRNAs in COLO205 cells, which could be reversed by E2 antagonist ICI 182.780. The expression of miR-135b was inversely correlated with serum E2 level and ER-beta mRNA expression in CRC patients' cancer tissues. There were significant correlations between serum E2 level and expression of ER-beta, miR-135b, and MMR in colon cancer tissue. This study suggests that the effects of estrogen on MMR function may be related to regulating miRNA expression via ER-beta, which may be the basis for the anti-cancer effect in colorectal cells.
Adaptor Proteins, Signal Transducing/genetics/metabolism ; Adult ; Aged ; Cell Line, Tumor ; Colorectal Neoplasms/*genetics/metabolism ; DNA Mismatch Repair/*genetics ; Estradiol/analogs & derivatives/blood/*pharmacology ; Estrogen Antagonists/pharmacology ; Estrogen Receptor beta/genetics/*metabolism ; Female ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics/*metabolism ; Middle Aged ; MutS Homolog 2 Protein/genetics/metabolism ; Nuclear Proteins/genetics/metabolism ; RNA, Messenger/biosynthesis

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

The temporal expression of estrogen receptor (ER)-alpha and ER-beta mRNA was examined in male Japanese quails. Femurs of quails receiving 17beta-estradiol underwent RTPCR and histochemical analysis 1 to 15 days after treatment. Untreated quails were used as controls (day 0). Between days 0 and 5, cells lining the bone endosteal surface differentiated into osteoblasts, which in turn formed medullary bone. Expression of ER-alpha was already observed on day 0 and increased slightly during bone formation whereas ER-beta was hardly detected throughout this process. After osteoclasts appeared on the medullary bone surface, this type of bone disappeared from the bone marrow cavity (days 7~15). ER-alpha expression simultaneously decreased slightly and ER-beta levels remained very low. These results suggest that estrogen activity mediated by ER-alpha not only affects medullary bone formation but also bone resorption.
Animals ; Bone Resorption/genetics ; Bone and Bones/chemistry/cytology/*metabolism ; Cells, Cultured ; Coturnix/*metabolism ; Estradiol/*pharmacology ; Estrogen Receptor alpha/genetics/*metabolism ; Estrogen Receptor beta/genetics/*metabolism ; Gene Expression Regulation ; Male ; Osteoblasts/chemistry/cytology/*metabolism ; Osteogenesis/genetics ; RNA, Messenger/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDIt is essential to clarify the interactions of hormones during the progression of human breast cancer. This study examined the effects of exogenous human leptin on estrogen receptor (ER) alpha and beta in human breast tumor tissue in a nude mouse xenograft model.

METHODSWe created nude mice xenografts of MCF-7 human breast cancer cells, and randomly divided them into an experimental group and a control group. The mice in experimental group were injected subcutaneously around tumors with human leptin, while the control group were injected with the same dose of normal saline. A real-time RT-PCR assay was developed to quantify the mRNA of ERalpha, beta in the tumor tissues. Western blotting analyses were used to assess the relative quantities of the ERalpha, beta proteins.

RESULTSLeptin-treated xenografted nude mice were successfully established. The amount of ERalpha mRNA was significantly higher in the leptin group than in the control group (P < 0.01), while the amount of ERbeta mRNA was significantly lower in the leptin group than in the control group (P < 0.01). Western blotting analyses revealed that the ERalpha protein level was significantly higher in the leptin group than in the control group (P < 0.01), while the ERbeta protein level was significantly lower in the leptin group than in the control group (P < 0.01).

CONCLUSIONSNude mouse xenograft model can be safely and serviceably treated with human leptin by subcutaneous injections around tumor. ERalpha, beta were both targets of leptin in breast cancer. Leptin can up-regulate the expression of ERalpha and down-regulate the expression of the ERbeta in human breast tumor.

Animals ; Blotting, Western ; Breast Neoplasms ; drug therapy ; genetics ; metabolism ; Cell Line, Tumor ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Humans ; Leptin ; therapeutic use ; Mice ; Mice, Nude ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; Random Allocation ; Xenograft Model Antitumor Assays

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

OBJECTIVETo study the phytoestrogenic effects and their possible mechanisms of Jiaoai tang and Shenqi Jiaoai tang through the tests in mice and ER (+) MCF7 cells.

METHODSixty kunming mice weighing 9-12 g were randomly divided into 6 groups: solvent control group (administrated equal dose of diswater), diethylstilbestrol control group (administrated diethylstilbestrol at a dose of 0.35 mg x kg(-1) x d(-1) and 4 Chinese medicine treated groups (administrated low or high doses of Jiaoai tang and Shenqi Jiaoai tang at 2.5 g x kg(-1) x d(-1) or 5.0 g x kg(-1) x d(-1) respectively). After administration for 4 days, the mice were sacrificed; uterus was removed and weighed, uterus rate was calculated. The blood serum was also separated. The proliferation rate of MCF7 cells influenced by Jiaoai tang and Shenqi Jiaoai tang was determined by MIT assay. PS2, ERalpha and ERbeta mRNA expression was quantified by Real-time PCR assay. Estrogen receptor antagonist ICI182, 780 was employed as a tool.

RESULTAdministration of Jiaoai tang and Shenqi Jiaoai tang at high dose significantly increased uterus rate in mice (P < 0. 05). The pharmacological serum from two high-dosage groups of Chinese herbal medicine decoction significantly enhanced proliferation rate of MCF7 cells (P < 0.05 or 0.01), while their effects were blocked by ICI182, 780 (P < 0.05 or 0.01). The pharmacological serum could cause elevation of pS2 level (P < 0.01) which would be obviously inhibited by ICI182, 780 (P < 0.01). ERalpha and ERbeta mRNA levels were also elevated significantly (P < 0.05 and P < 0.01 respectively).

CONCLUSIONJiaoai tang and Shenqi Jiaoai tang have phytoestrogenic effects, which were attained via ER pathway. They can also increase the mRNA levels of estrogen receptor subtypes, especially ERbeta.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; pharmacology ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Gene Expression ; drug effects ; Mice ; Phytoestrogens ; pharmacology ; Random Allocation ; Signal Transduction ; drug effects ; Uterus ; drug effects ; 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