Preparation and characterization of Ad-ERα-36-Fc-GFP / 生物工程学报

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 AdMaxTM 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.

Exploration on Effect of Yinhuotang on Depression-like Behavior in Mice with Bilateral Ovariectomy Induced by Chronic Stress Based on E2/ERβ Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effect of Yinhuotang （YHT） on the depression-like behavior of mice with bilateral ovariectomy （OVX） induced by chronic stress and explore its action mechanism based on estradiol （E₂）-estrogen receptor <italic>β </italic>（ER<italic>β</italic>） pathway. Method:The experiment was divided into two parts. In the first part， mice were randomly divided into the sham operation （Sham） group， model （OVX） group， positive drug （E₂， 0.13 mg·kg^-1） group， and YHT （23.4 g·kg^-1） group. The OVX model was reproduced by OVX combined with chronic unpredictable mild stress （CUMS）. On the 8th day after OVX， the mice in each group were exposed to CUMS and treated with drugs. The changes in immobility time， horizontal movement score， and vertical movement score of mice in each group were observed in forced swimming test （FST）， tail suspension test （TST） and open-field test （OFT）， respectively. Serum and brain E₂ levels were detected by enzyme-linked immunosorbent assay （ELISA）， the aromatizing enzyme （Cyp19） mRNA expression by real-time fluorescence polymerase chain reaction （Real-time PCR）， the expression of ER<italic>α</italic> and ER<italic>β</italic> in dentate gyrus of hippocampus by immunohistochemistry （IHC）， and the total ER<italic>α</italic> and ER<italic>β</italic> levels in the brain by Western blotting. In the second part， the mice were divided into the Sham group， OVX group， YHT group， and blocker （Y+B， 23.4 g·kg^-1+100 μg·kg^-1） group. Mice in the Y+B group were first treated with intragastric administration of YHT and then with intraperitoneal injection of ER<italic>β</italic> blocker （PHTPP） on the next day. The changes in immobility time， horizontal motor score， and vertical motor score were observed in the three behavioral tests. Result:Compared with the Sham group， the OVX group displayed significantly increased immobility time， decreased horizontal and vertical movement scores （<italic>P</italic><0.01）， down-regulated serum and brain E₂levels （<italic>P</italic><0.01） and Cyp19 mRNA expression in the brain （<italic>P</italic><0.01）， and up-regulated total ER<italic>β</italic> in dentate gyrus and brain （<italic>P</italic><0.01）. However， there was no significant change in total ER<italic>α</italic> expression in the dentate gyrus or brain. As revealed by comparison with the OVX group， the immobility time of the E₂ group was decreased significantly， while the horizontal and vertical movement scores were increased significantly （<italic>P</italic><0.01）. The E₂ levels in the serum was significantly elevated （<italic>P</italic><0.01）. The Cyp19 mRNA expression in the brain and the total ER<italic>α</italic> expression in the dentate gyrus and brain were not significantly changed， while the expression levels of total ER<italic>β</italic> in dentate gyrus and brain were significantly increased （<italic>P</italic><0.01）. In the YHT group， the immobility time declined significantly， and the horizontal and vertical movement scores rose significantly （<italic>P</italic><0.01）. The serum E₂ did not increase， whereas the brain E₂ increased significantly （<italic>P</italic><0.01）. The expression of Cyp19 gene in the brain was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. There was no significant change in the total ER<italic>α</italic> of dentate gyrus and brain， but the expression levels of total ER<italic>β</italic> in dentate gyrus and brain were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. PHTPP reversed the effects of YHT on OVX mice in FST， TST and OFT. Conclusion:YHT promotes the synthesis and release of endogenous estrogen in brain and improves the depression-like behavior of OVX mice induced by chronic stress， which is possibly related to the activation of E₂/ER<italic>β</italic> pathway.

Norlichexanthone purified from plant endophyte prevents postmenopausal osteoporosis by targeting ER

Although different types of drugs are available for postmenopausal osteoporosis, the limitations of the current therapies including drug resistances and adverse effects require identification of novel anti-osteoporosis agents. Here, we defined that norlichexanthone (NOR), a natural product, is a ligand of estrogen receptor-alpha (ER

ERα promotes transcription of tumor suppressor gene / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Apolipoprotein A-I (ApoA-I), the main protein component of high-density lipoprotein (HDL), plays a pivotal role in reverse cholesterol transport (RCT). Previous studies indicated a reduction of serum ApoA-I levels in various types of cancer, suggesting ApoA-I as a potential cancer biomarker. Herein, ectopically overexpressed ApoA-I in MDA-MB-231 breast cancer cells was observed to have antitumor effects, inhibiting cell proliferation and migration. Subsequent studies on the mechanism of expression regulation revealed that estradiol (E2)/estrogen receptor α (ERα) signaling activates

Immuno Localization of Estrogen Receptor (ERα) and Progesterone Receptor (PR) in Uterus of Buffalo during Follicular and Luteal Phases of Estrous Cycle

The tissue distribution of estrogen receptor alpha (ERα) and progesterone receptor (PR) was examined using immunohistochemical technique. Image analysis was done to quantify the immune reactivity. The ERα and PR was localized in luminal epithelium, glandular epithelium, stromal cells, endothelial cells and myometrium and few cells in perimetrium. The immuno staining was observed in the nuclei of cells, however, faint cytoplasmic staining for PR was also observed. Variations were seen in the different tissue compartments of the uterus and during the different phases of the estrous cycle. Significantly higher number of ERα positive cells was observed in lamina epithelialis as compared to stromal cells and smooth muscle cells in myometrium. Significantly higher percentage of ERα positive cells was observed in the lamina epithelialis and lining epithelium of endometrial glands during follicular phase as compared to the luteal phases of estrous cycle (P < 0.05). Higher number of PR positive cells was observed in lamina epithelialis as compared to stromal cells and smooth muscle cells in myometrium (P < 0.05). Higher percentage of PR positive cells was observed in the lamina epithelialis during follicular phase as compared to the luteal phases of estrous cycle (P < 0.05). There was no significant difference in the immuno reactivity in stromal cells, lining epithelium of endometrial glands and smooth muscle cells of myometrium during the phases of estrous cycle. The study concluded that ERα and PR expressions were higher during follicular phase as compared to the luteal phase

Expression and clinical significance of LMTK3 in prostate cancer tissues / 中国免疫学杂志

Objective: To investigate the expression and clinical significance of LMTK3 in patients with prostate carcer. Methods: Immunohistochemistry was used to detect the expression of LMTK3 and ERα in 55 cases of prostate cancer tissues and 25 cases of benign prostatic hyperplasia tissues. The relationship between the expression of LMTK3 and ERα and clinicopathological parameters was evaluated by square test and Fisher exact test. The association between LMTK3 and ERα expression was analyzed with Pearson and Spearman rank correlation. Results: The results of immunohistochemistry demonstrated that the LMTK3 and ERα protein positive expression rate in 55 cases of prostate cancer tissues was 36. 36％ and 32. 73％, whereas was 64. 00％ and 56. 00％ in the benign prostatic hyperplasia, respectively, showed a significant difference of comparison within this result ( P ＜ 0. 05). The expression of LMTK3 in prostate cancer tissues was inversely related with the level of Gleason grade ( P＜0. 05), but no relation with the levels of age, TPSA, TNM stage and lymph node metastasis ( P＞0. 05). Moreover, the expression of ERα in prostate cancer tissues was oppositely related with the levels of gleason grade and TPSA ( P＜0. 05), but no relation with the levels of age, TNM stage and lymph node metastasis ( P＞0. 05). Pearson and Spearman rank correlation analysis revealed, to some extent, there was positively correlated with the two proteins ( r = 0. 296, P＜0. 05). Conclusion: The expression of LMTK3 in prostate cancer tissues was decreased compared with benign prostatic hyperplasia tissues and negatively related with the level of gleason grade. In some degree, there is a positively correlation between the LMTK3 and ERα proteins.

Chemotherapeutic drugs affect methylation of ER-α in breast cancer cells by down-regulating miR-148b / 中华内分泌外科杂志

Objective To investigate the effects of chemotherapeutic drugs on ER-α expression and methylation in breast cancer cells.Methods Human breast cancer cells MCF-7(ER+,Luminal A) were induced by paclitaxel(PTX) and epirubicin(EPI) for more than 6 months,with an incremental dose,respectively.The expression and methylation status of ER-α in MCF-7 cells were detected before and after drug treatment.miRNAs with consistent expression changes in MCF-7 cells after two drugs' treatment were screened by microarray,and verified by quantitative PCR (qPCR).Targets of the most significantly down-regulated miRNA were analyzed by bioinformatics.miRNA inhibitor was transfected into MCF-7 cells,miRNA mimic was transfected into MCF-7/PTX and MCF-7/EPI cells,then ER-α and DNA methyltransferase 1 (DNMT1) expression were detected by Western blot,and ER-α methylation was detected by quantitative methylation-specific PCR (qMSP).Results PTX resistant MCF-7/PTX cell line and EPI resistant MCF-7/EPI cell line were established.Both drug treatments caused a decrease in ER-α protein expression and an increase in methylation levels,with up-regulation of DNMT1 and his tone deacetylase 1 (HDAC 1) expression.miRNAs with consistent expression changes in MCF-7 cells after drug treatments were screened and verified by qPCR,the most significant down-regulation among which was miR-148b.Bioinformatics analysis,and further confirmed by luciferase reporter gene assay (Luciferas) that DNMT1 was a direct target of miR-148b.miR-148b inhibitor induced decreased expression of ER-α and increased methylation level in MCF-7 cells,accompanied by increased expression of DNMT1;whereas miR-148b mimic caused an increased expression of ER-α and decreased methylation level in MCF-7/PTX and MCF-7/EPI cells,with a decreased expression of DNMT1.Conclusion Chemotherapeutic drugs (represented by PTX and EPI) induce aberrant miRNA expression in breast cancer MCF-7 cells,and down-regulate miR-148b further to attenuate the inhibition of DNMT1 expression,which promote,hypermethylation and down-regulation of ER-α.

Effects of estrogen receptor antagonist on the expressions of Erα, Erβ and p57kip2 in JEC cells of human endometrial carcinoma / 临床与实验病理学杂志

Purpose To explore the effects of estrogen receptor antagonist on the expression of estrogen receptor subtype (ERα, ERβ), and p57kip2 protein in human endometrioid carcinoma cells named JEC. Methods The JEC cells (moderately differentiated EC cells) cultured in vitro were treated with β-Estradiol (E2) (10～6 mol/L) and two types of estrogen receptor antagonists, tamoxifen (TAM) and fulvestrant (ICI182780) (10-6 mol/L). After 24, 48, 72 h, MTT was used to detect the growth condition of JEC cells, and the light microscopy and electron microscopy were used to observe the growth condition and morphological changes of cells, Western blot was used to detect the expression of ERα, ERβ, PR-A, PR-B and P57kip2 protein in JEC cells. Results MTT results: Compared with the control group, E2 could promote the proliferation of JEC cells significantly (P＜0.05), and ICI182780 could inhibit the proliferation of JEC cells obviously (P＜0.05). Compared with the E2 group, the proliferation ability of JEC cells in E2 + ICI182780 group were lower(P＜0.05). Morphological change: Compared with the control group, the cells density of E2 group increased obviously, and the pathologic mitosis was easy to seen in some cells. The cells density decreased obviously in ICI182780 group. Compared with E2 group, the cells density of E2 + TAM group and E2 + ICI182780 group were decreased, and pathological mitotic figures were difficult to seen. Western blot results: Compared with the control group, the expression of ERβ protein increased, and the expression of p57kip2 protein decreased in E2 group (P＜0.05). The expression of ERβ protein decreased, and the expression of p57kip2 protein increased in ICI182780 group and TAM group, and the difference was statistically significant between ICI182780 group and control group (P＜0.05). Compared with the E2 group, the expression of ERβ protein decreased, and the expression of p57kip2 protein increased in E2 + ICI182780 group and E2 + TAM group, and the difference was statistically significant between E2 + ICI182780 group and E2 group (P＜0.05). ERa protein of JEC cells did not expressed in experimental group or control group. Conclusion ERa protein are not expressed in JEC cells. ICI182780 have a stronger role in antagonizing estrogen, and may induce the expression of p57kip2 protein by down-regulating the expression of ERβ protein in JEC cells, block the cell cycle progression and inhibit the growth of tumor cells. TAM has a weaker estrogen like effect on the growth of JEC cells. It is possible that combined detection of the expression of ERa and p57kip2 protein in EC has an important reference value for individualized selection of endocrine therapy for EC patients.

Effects of miR-143 target-regulating ER-α36 on the invasion of gastric cancer cell line SGC 7901 / 基础医学与临床

Objective To investigate the effects of ER-α36 on invasion of human gastric cancer cell lines SGC7901 by miR-143. Methods Lentiviral vectors were constructed to generate up-and down-regulations of miR-143 lentiviruses (LV-miR-143 and LV-anti-miR-143,respectively).The viruses were used to infect human gastric cancer cell lines SGC7901.The ER-α36 protein expression level and the invasion of constructed cells were detected by Western blot and transwell. The target gene of miR-143 was predicted by bioinformatics tools.Luciferase reporter assay was carried out to confirm the predicted target gene. Results The infection efficiency of the lentivirus titers of LV-miR-143 and LV-anti-miR-143 were over 80% shown by the green fluorescence. The ER-α36 expression level,the cell invasion in LV-miR-21 group were significantly lower than those in LV-anti-miR-21 group(P<0.05). Conclusions miR-143 plays an important role in the negative control of gastric cancer invasion by the regulation of ER-α36.

Study on the regulatory mechanism of electroacupuncture based on thyroid pathway for mammary gland hyperplasia rats / 中国针灸

<p><b>OBJECTIVE</b>To study the regulating effect of thyroid pathway on electroacupuncture (EA) for mammary gland hyperplasia (MGH) so as to provide new research ideas for the mechanism of EA for MGH and to provide the evidence for clinical application.</p><p><b>METHODS</b>Sixty adult female SD rats were randomly divided into a blank group, a model group, an EA group, an EA with thyroidectomy group, an EA with sham operation group, 12 rats in each one. Except the blank group, the MGH model was established. Thyroid ablation was performed in the EA with thyroidectomy group, and sham operation was used in the EA with sham operation group, exposing thyroid without excision, 1 day after model establishment. EA was applied in the EA, the EA with thyroidectomy, and the EA with sham operation groups on the 4th day after model establishment, and not used in the other groups, but catching, routine disinfection and fixation were all the same as the above groups. The acupoints in the group A were bilateral "Tianzong" (SI 11), "Ganshu" (BL 18) and "Zusanli" (ST 36); and those in the group B were bilateral "Wuyi" (ST 15), "Hegu" (LI 4) and "Danzhong" (CV 17). The two groups of points were alternately used. EA, continuous wave, 2 Hz and 1 mA, was connected at "Tianzong" (SI 11) and "Ganshu" (BL 18), "Wuyi" (ST 15) and "Hegu" (LI 4) at the same side, 2 pairs EA a time, 20 min a time, once a day. All the intervention was given for 4 courses, 5 times as 1 course with 2 days between courses. After intervention, the height and diameter of the rat papilla were measured. Estrogen (E) and progestational hormone (P) in the serum were detected by enzyme linked immunosorbent assay (ELISA), and the contents and protein expression of estrogen receptor α (ERα) and progesterone receptor (PR) in the mammary glands were detected by immunofluorescence and Western-blot.</p><p><b>RESULTS</b>(1) The height and diameter of papilla in the model group increased compared with those in the blank group (both <0.01). The height and diameter of papilla in the EA, EA with sham operation groups reduced compared with those in the model group (all <0.01). Those in the EA with thyroidectomy group were lower than those in the model group, without statistical significance (both >0.05). (2) Compared with the blank group, E increased and P decreased in the model group (both <0.01). Compared with the model group, E decreased and P increased in the EA and EA with sham operation groups (all <0.01). The contents of E and P had no statistical significance between the model and the EA with thyroidectomy groups (both >0.05). (3) Compared with the blank group, the ERα content and protein expression increased and the PR content and protein expression decreased in the model group (all <0.01). Compared with the model group, the ERα content and protein expression decreased and the PR content and protein expression increased in the EA and EA with sham operation groups (all <0.01). The ERα and PR content and protein expression had no statistical significance between the model and the EA with thyroidectomy groups (all >0.05).</p><p><b>CONCLUSION</b>The effect of EA for MGH may be closely related to the regulation of thyroid.</p>

Effects of total flavonoids in Astragali Complanati Semen on liver lipid level and ERα expression on liver in hyperlipidemia rats with kidney-Yang deficiency pattern / 中国中药杂志

Menopausal women appear lipid metabolism disorder with the ovarian function decline and the estrogen levels decreased. Modern clinical usually use estrogen replacement therapy and with long time application with lots of side effect appear. Traditional Chinese medicine has more secure and effective methords,using warming Yang drugs and methods. And the previous study proves the Chinese medicine Astragali Complanati Semen water extraction has a good role in regulation of blood lipids. Because of the liver is the most important organ on regulating metabolism, therefore this study aimed to evaluate the effects of total flavonoids in Astragali Complanati Semen(TFS)on liverlipid level and ERα expressionon liver in hyperlipidemia rats with kidney-Yang deficiency pattern to explore the substance basis and mechanism of Astragali Complanati Semen in regulate lipid effect and clarify traditional Chinese medicine advantages and features. This experiment uses hyperlipidemia rats with kidney-Yang deficiency pattern with bilateral ovariectomized and fed with high fat diet for 6 weeks. And rats of sham operation group and model group rats were intragastrilly(ig) with saline, estrogen group were intragastrilly with estrogen(0.2 mg·kg⁻¹). And three TFS group were intragastrilly with TFS at dose 28.5, 57, 114 mg·kg⁻¹ for 8 weeks. At the same time, TC, TG, LDL-C,HDL-C liver weight, liver index, uterine weight, uterine index, serum estrogen level, FSH levels and liver pathology, liver estrogen receptor expression were detected, weighting and calculating their organ index. The experimental results compared with the model group, TFS 114 mg·kg⁻¹ decreased the level of liver TG (<0.05), TC (<0.001) and LDL-C (<0.001) and increased the level of HDL-C (<0.05). Compared with the model group, estrogen group increased the level of blood serum (<0.001) and decreased the level of FSH (<0.001). In addition, compared with sham operation group,model group decreased the protein expression of ERα（<0.01）. Compared with the model group, estrogen group increased the protein expression of ERα significantly（<0.001）.TFS mid-dose group and TFS high-dose group is increased the protein expression of ERα（<0.01, <0.001）.In a conclusion,Flavonoids is the main active ingredient of Astragali Complanati Semen. The mechanism of it maybe is enhancing the estrogen receptor sensitivity or the number of estrogen receptors, amplifying the signal after the receptor conduction, which could result in lipid-lowering effect.

Electroacupuncture at intercostal nerve for mammary gland hyperplasia in rats / 中国针灸

<p><b>OBJECTIVE</b>To explore the correlation between efficacy of electroacupuncture (EA) on mammary gland hyperplasia (MGH) and the regulatory pathway of intercostal nerve.</p><p><b>METHODS</b>Fifty female SD rats were randomly divided into a blank group (group A, 12 rats), a model group (group B, 12 rats), an EA group (group C, 13 rats) and an intercostal nerve transection group (group D, 13 rats). The rats in the group B, group C and group D were prepared into MGH model; after model was successfully prepared, the 7th intercostal nerve was cut off in the group D. EA was applied at back acupoints including bilateral "Tianzong" (SI 11), "Ganshu" (BL 18) and "Shenshu" (BL 23) as well as chest acupoints including bilateral "Wuyi" (ST 15), "Hegu" (LI 4) and "Danzhong" (CV 17) in the group C and D. The two groups of acupoints were selected alternately. EA was given for 20 min, once a day; 5-day treatment was taken as one course; there was an interval of 2 days between course; totally 20 treatments were given. After treatment, the height and diameter of papilla were observed; the contents of serum estradiol (E) and progestin (P), the expression of estrogen receptor α (ERα) and progestrone receptor (PR) in mammary gland were measured.</p><p><b>RESULTS</b>(1) The height and diameter of papilla: after treatment, the height and diameter of papilla in the group C were significantly smaller than those in the group B (both<0.05); the height and diameter of left-side papilla in the group D were significantly bigger than those in the group C (both<0.05). (2) Serum Eand P: after treatment, compared with the group B, the contents of Eand E/P were reduced and the content of P was increased in the group C and group D (all<0.05). Compared with the group C, the contents of Eand E/P were increased and the content of P was reduced in the group D (all<0.05). (3) ERα and PR in mammary gland: compared with the group B, the content of ERαwas decreased and the content of PR was increased in the group C (both<0.05). Compared with the group C, the content of ERαwas increased and the content of PR was decreased in the group D ((both<0.05).</p><p><b>CONCLUSION</b>The efficacy mechanism of EA for MGH is likely to be related with the pathway of intercostal nerve; the mechanism may be acupuncture regulating the contents of serum Eand P as well as contents of ERα and PR in mammary gland.</p>

Therapeautic Mechanism of Electroacupuncture with Unblocking and Regulating Needling Therapy for Liver-stagnation Mammary Gland Hyperplasia Rats and Its Relationship with Vagus Nerve Regulation / 广州中医药大学学报

Objective To explore the actions of vagus nerve in electroacupuncture (EA) with unblocking and regulating needling therapy for the treatment of mammary gland hyperplasia(MGH)with liver stagnation type, and to provide experimental evidences for clinical usage of EA with unblocking and regulating needling therapy. Methods Sixty healthy SD rats were randomly divided into 5 groups,namely blank control group,model control group,EA group,EA with disconnection of vagus nerve group,and sham-operation EA group,12 rats in each group. Except for the blank control group, the rats in the remaining groups were modeled. After successful establishment of the model of mammary gland hyperplasia (MGH) with liver stagnation type, the rats in EA group with disconnection of vagus nerve were given disconnection of unilateral vagotomy operation, the rats in sham-operation EA group were given sham operation, and EA group had no treatment, and then all of the 3 treatment groups were given EA with unblocking and regulating needling therapy. At the end of the experiment, the diameter and height of rat nipple were measured,the serum contents of estradiol (E2)and progestin (P)in the rats were detected by enzyme-linked immunoassay(ELISA),and the contents of anti-estrogen receptor alpha (ERα)and anti-progesterone receptor(PR)in the rat breast tissue were detected by immunofluorescence double staining and Western blotting method. Results (1)EA treatment can obviously improve the diameter and height of MGH rat nipple, but the therapeutic effect of EA group with disconnection of vagus nerve was not obvious. (2)Compared with the model control group,the levels of serum E2 and mammary ERαwere markedly reduced(P<0.01),and serum P level and mammary PR content were obviously increased in EA group and sham-operation EA group (P < 0.01). However,the above indexes had no obvious changes in EA group with disconnection of vagus nerve (P>0.05). Conclusion The therapeutic mechanism of EA with unblocking and regulating needling therapy for liver-stagnation MGH may be closely related with the regulation of vagus nerve.

Synergistic effect of ERα on HDAC1 regulatingtranscription of p21WAF1/CIP1 in breast cancer MCF-7 cells / 中国药理学通报

Aim To investigate the specific binding sites that histone deacetylases 1(HDAC1) and estrogen receptor α(ERα)can be recruited to regulate the transcriptional activity of p21WAF1/CIP1 promoter in the breast cancer MCF-7 cells, and to clarify the molecular mechanism of suberoylanilide hydroxamic acid(SAHA) and leptin regulating p21WAF1/CIP1 promoter function.Methods The breast cancer MCF-7 cells in logarithmic growth phase were starved with FBS free medium for 24 hours, and treated with 20 μmol·L-1 SAHA(SAHA group) or 0.625 nmol·L-1 leptin(Leptin group) for 24 hours.The cells that were cultured in complete RPMI 1640 medium without any treatment were assigned as control group(Basal group).The cell lysis was prepared and incubated respectively with anti-HDAC1 and anti-ERα antibody by chromatin-immunoprecipitation(ChIP) method overnight at 4℃.The DNA-ChIP was followed the manufacturer′s protocol for the assay.DNA fragments binding anti-HDAC1 and anti-ERα antibody were gathered and purified.The relative expression level of DNA fragments from TSS to the upstream of the p21WAF1/CIP1 promoter region(+2~-4 000 bp) binding with antibody was detected by real-time PCR and analyzed by 2-△△CT method.Results In basal group, HDAC1 and ERα had high affinity with the f1 and f8 fragments of p21WAF1/CIP1 promoter compared to the f4 fragment.In SAHA group, the binding ability of HDAC1 and ERα to the f1 and f8 fragments of p21WAF1/CIP1 promoter was significantly lower than that of the control, while reversing to reach the peak after leptin treatment.Conclusions HDAC1 and ERα can be recruited to p21WAF1/CIP1 promoter by the cell proliferation signal during the proliferation of breast cancer MCF-7 cells.The DNA f1(from 0 to-400 bp) and f8(from-2 800 to-3 200 bp) fragment in the upstream of p21WAF1/CIP1 promoter are the target functional region for the binding with HDAC1and ERα.

Bidirectional regulation of angiogenesis by phytoestrogens through estrogen receptor-mediated signaling networks / 中国天然药物

Sex hormone estrogen is one of the most active intrinsic angiogenesis regulators; its therapeutic use has been limited due to its carcinogenic potential. Plant-derived phytoestrogens are attractive alternatives, but reports on their angiogenic activities often lack in-depth analysis and sometimes are controversial. Herein, we report a data-mining study with the existing literature, using IPA system to classify and characterize phytoestrogens based on their angiogenic properties and pharmacological consequences. We found that pro-angiogenic phytoestrogens functioned predominantly as cardiovascular protectors whereas anti-angiogenic phytoestrogens played a role in cancer prevention and therapy. This bidirectional regulation were shown to be target-selective and, for the most part, estrogen-receptor-dependent. The transactivation properties of ERα and ERβ by phytoestrogens were examined in the context of angiogenesis-related gene transcription. ERα and ERβ were shown to signal in opposite ways when complexed with the phytoestrogen for bidirectional regulation of angiogenesis. With ERα, phytoestrogen activated or inhibited transcription of some angiogenesis-related genes, resulting in the promotion of angiogenesis, whereas, with ERβ, phytoestrogen regulated transcription of angiogenesis-related genes, resulting in inhibition of angiogenesis. Therefore, the selectivity of phytoestrogen to ERα and ERβ may be critical in the balance of pro- or anti-angiogenesis process.

Expression and significance of STIP1 and ER-αin papillary thyroid carcinomas / 临床与实验病理学杂志

Purpose To detect the expressions of stress induced phosphoprotein 1 (STIP1) and estrogen receptor-α(ER-α) in papil-lary thyroid carcinoma and to analyse the relationship between STIP1 and ER-α. Methods 54 cases of paraffin-embedded tissues of papillary thyroid carcinoma, 18 cases of papillary thyroid carcinoma with lymph node metastasis, 15 cases of Hashimoto’ s thyroiditis, 10 cases of adjacent normal thyroid tissue were collected. The expressions of STIP1 and ER-αwere detected by immunohistochemistry, and the relationship between the expressions and clinicopathological features of papillary thyroid carcinoma was analyzed. Results The expression of STIP1 and ER-α in papillary thyroid cancer group ( 55. 6% and 44. 4%) were higher than that of normal thyroid group (10% and 0) and Hashimoto’s thyroiditis group (8. 3% and 0, all P0. 05). The expressions of STIP1 and ER-α in papillary thyroid carcinoma were not related to patients’ age , tumor location, number of tumors, tumer size, invasion of capsule, the concomitant Hashimoto’ s thyroiditis and TPO-Ab ( all P>0. 05). And the expressions of STIP1 was not related to gender, TG-Ab and the merger of nodular goiter (all P>0. 05). A positive correlation was found between the expressions of STIP1 and ER-αin thyroid papillary carcinoma (P<0. 05). Conclusion STIP1 and ER-α in papillary thyroid carcinoma may be related with lymph node metastasis.

The association of DNMT3a, DNMT3b expression with the state of promoter methylation of ERαgene and ERαexpression in sporadic breast cancer / 天津医药

Objective To investigate the correlationship between DNMT3a, DNMT3b protein expressions and the state of promoter methylation of ERα gene and ERα protein expression in the development of sporadic breast cancer. Methods A total of 180 patients with sporadic breast cancer and 30 patients with breast fibroadenoma were included in this study. The expressions of DNMT3a and DNMT3b protein were detected by immunohistochemical method. The state of promoter methylation of ERα gene was detected by methylation specific PCR in 97 patients with sporadic breast cancer. Results There were no significant differences in positive expression rates of DNMT3a and DNMT3b protein between breast fibroadenoma and breast cancer. There were higher expression levels of DNMT3a and DNMT3b in breast cancer patient of Ⅲ～Ⅳstages than those of Ⅰ～Ⅱstages. The expression of DNMT3a was significantly higher in patients with lymph node metastasis than that of patients without lymph node metastasis (P＜0.05). Of 97 cases of breast cancer patients, ERα gene promoter methylation occurred in 39 cases (40.2%). The positive expression of DNMT3a protein was positively correlated with the ERα gene methylation (rS=0.250). The DNMT3a protein expression showed a significant influence to the overall survival (OS) in patients of breast cancer (P=0.035), no significant influence to the disease-free survival (DFS) (P=0.064). DNMT3b protein expression showed no significant influence to OS and DFS of patients with breast cancer (P=0.914 and 0.961). Conclusion The positive expressions of DNMT3a and DNMT3b are correlated with the invasion, metastasis and poor prognosis of sporadic breast cancer. DNMT3a was positively correlated with the state of ERα gene promoter methylation. The inhibition of DNMT3a and DNMT3b may have advantages in the prevention and treatment of sporadic breast cancer.

Correlation of estrogen receptor-alpha 36 and cancer / 肿瘤

Estrogen receptor-alpha 36 (ER-α36), which is a new type of ER with a relative molecular weight of 3.57×104, has been demonstrated to have many important physiological functions in different tissues and organs in recent years. Many studies have shown that the occurrence and development of cancer are closely related to the expression level of ER-α36, which suggests that ER-α36 may serve as a potential therapeutic target in the treatment of some cancers. This paper reviews the advances in research on the correlation between ER-α36 and some cancers such as breast cancer, gastric cancer, liver cancer, and so on.

Molecular mechanism for inhibitory effect of puerarin on IL-6 secretion in human osteoblast-like MG-63 cells / 中国药理学通报

Aim To investigate the molecular mecha-nism for the inhibitory effect of puerarin on IL-6 secre-tion in human osteoblast-likeMG-63 cells. Methods According to our previous studies, human osteoblast-like MG-63 cells containing two estrogen receptor ( ER) isoforms are a suitable model for this study. En-zyme-linked immnosorbent assay (ELISA), RT-PCR, luciferase reporter assay and small interfering RNA were performed to investigate the effect of puerarin on IL-6 expression in osteoblast-derived cells and underly-ing molecular mechanism. Results Puerarin could obviously inhibit IL-6 expression and IL-6 promoter ac-tivity by human osteoblastic MG-63 cells. Treatment with the ER antagonist ICI 182,780 abrogates the a-bove actions of puerarin on osteoblast-derived cells. U-sing siRNA technology, we further demonstrated that the effects of puerarin on IL-6 production were media-ted by ERα. Conclusion The effect of puerarin on IL-6 production in osteoblast is mediated by ERα.

Estrogen receptor ERα36 gene silencing impacts the expression of growth-associated protein in PC12 cells / 中华行为医学与脑科学杂志

Objective To investigate the effects of ERα36 (a novel subtype of estrogen receptor alpha) on growth and proliferation in PC12 cells via examining the expression of growth-associated protein in differentiated PC12 cells after ERα36 gene silencing.Methods Transfection of ERα36-shRNA plasmid into PC12 cells was performed to establish the ERoα36 gene silencing cells model (PC12-36L1,PC12-36L2).Immunocytofluorescence was used to examine the expression of ERα36,and Western blot was used to analyze the expression of PCNA,cyclinD1 and MAPK in the PC12 cells.Results ① ERα36 was expressed in both cell types(PC12-36C1,PC12-36L1 and PC12-36L2).Compared with PC12-36C1,PC12-36L2 cells(OD value were respectively 0.95±0.05,0.78±0.10),PC12-36L1 cells significantly decreased expression of ERα36(OD value 0.47±0.12,P＜0.01).② Compared with PC12 and PC12-36C1 cells,PC12-36L1 cells were significantly higher expression of PCNA,CyclinD1 and p-MAPK(P＜0.01)(OD value of PCNA,CyclinD1 and p-MAPK:PC12 cells were respectively 1.00±0.05,1.00± ±0.11,1.00±0.05,PC12-36C1 cells were respectively 1.09±0.15,0.92±0.23,1.12± 0.08,PC12-36L1 cells were respectively 1.74±0.12,2.20±0.25,1.77±0.06).Conclusion ERα36 gene silencing can promote the growth and proliferation in PC12 cells.It suggests that the lower expression of ERα36 may be related to the diseases in nervous system such as brain tumor.