mRNA Expression Levels Of ERα Research Articles

Effect of quercetin on granulosa cells development from hierarchical follicles in chicken

ABSTRACT 1. The bioflavonoid quercetin is a biologically active component, but its functional regulation of granulosa cells (GCs) during chicken follicular development is little studied. To investigate the effect of quercetin on follicular development in laying hens, an in vitro study was conducted on granulosa cells from hierarchical follicles treated with quercetin. 2. The effect of quercetin on cell activity, proliferation and apoptosis of granulosa cells was detected by CCK-8, EdU and apoptosis assays. The effect on progesterone secretion from granulosa cells was investigated by enzyme-linked immunosorbent assay (ELISA). Expression of proliferating cell nuclear antigen (PCNA) mRNA and oestrogen receptors (ERs), as well as the expression of steroid acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA during progesterone synthesis, were measured by real-time quantitative polymerase chain reaction (RT-qPCR). PCNA, StAR and CYP11A1 protein expression levels were detected using Western blotting (WB). 3. The results showed that treatment with quercetin in granulosa cells significantly enhanced cell vitality and proliferation, reduced apoptosis and promoted the expression of gene and protein levels of PCNA. The levels of progesterone secretion increased significantly following quercetin treatment, as did the expression levels of StAR and CYP11A1 using the Western Blot (WB) method. 4. The mRNA expression levels of ERα were significantly upregulated in the 100 ng/ml and 1000 ng/ml quercetin-treated groups, while there was no significant difference in expression levels of ERβ mRNA.

Effect of berberine on serum hormonal levels and endometrial gene and protein signaling in a rat model of polycystic ovary syndrome

Purpose: To study the effect of berberine on serum hormonal levels, endometrial gene, and protein signaling in a rat model of polycystic ovary syndrome (PCOS).Methods: Eighty-seven healthy female Sprague Dawley (SD) rats were assigned to control group, PCOS model group, and berberine group, each with 29 rats. The rats were assigned to three groups: control, model, and berberine groups, each with 29 rats. A rat model of PCOS was established via the administration of letrozole (1 mg/kg) to rats in the model group continuously for 21 days through gavage. Rats in berberine group were also given letrozole at the same dose for the same duration through gavage, while rats in control group received physiological saline in place of berberine. Estrus cycle, serum hormonal levels, ovarian tissue morphology, estrogen receptor α (ERα) levels, and mRNA expression levels of protein kinase B subtype 3 (Akt-3β) were evaluated. RAS-C3 botulinum toxin substrate 1 (Rac1), human chromosome 10 deleted phosphatase (PTEN) and mouse sarcoma-like virus oncogene (KRAS) were also determined using appropriate procedures.Results: Berberine treatment resulted in a significant decrease in the mean body weight of PCOS rats, relative to model rats (p < 0.05). Estrus interval in model rats was significantly prolonged. Levels of testosterone and estradiol, and mRNA expression levels of ERα, Akt3, Racl, PTEN, and KRAS were significantly increased, while progesterone level was significantly decreased, relative to control (p < 0.05). Estrus interval in the berberine group was shortened and estrus cycle gradually returned to normal, while the levels of testosterone and estradiol, and mRNA levels of ERα were gradually restored, relative to untreated PCOS rats (p < 0.05). Moreover, mRNA levels of Akt3, RAC1, PTEN, and KRAS were significantly decreased, while progesterone level was significantly increased (p < 0.05).Conclusion: Berberine appreciably restores heat cycle and ovary morphology in PCOS rats, and regulates serum hormonal levels, and endometrium-related gene and protein signaling.

Therapeutic effects and mechanism of ferulic acid and icariin in mammary gland hyperplasia model rats via regulation of the ERK pathway

BackgroundFerulic acid (FA) and icariin (ICA), as the active compounds derived from Angelica sinensis and Epimedium brevicornum Maxim, respectively, have been shown to promote blood circulation, regulate menstruation, exhibit anti-inflammatory effects, and modulate estrogen levels. The aim of this study was to elucidate the possible mechanism of FA, ICA, and FA-ICA on estrogen-induced mammary gland hyperplasia (MGH) in rats.MethodsHematoxylin and eosin (HE) staining was performed to record the pathological changes in breast tissue, and enzyme-linked immunosorbent assay (ELISA) was utilized to determine the serum levels of luteinizing hormone (LH), PRL (prolactin), testosterone (T), estradiol (E2), follicular stimulating hormone (FSH), and progesterone (P). The message RNA (mRNA) expression levels of extracellular signal-regulated kinase 1 (ERK1), extracellular signal-regulated kinase 2 (ERK2), estrogen receptor (ER), and progesterone receptor (PR) were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, ER, phosphorylated-ER, PR, p-PR, ERK1/2, and p-ERK1/2 protein factors in the ERK signaling pathway were evaluated by Western blotting. Simultaneously, changes in breast height, diameter, body weight, and organ index were all recorded.ResultsWe found that FA and ICA could modulate the degree of breast swelling, and reduce the body weight, thymus index, and uterus index. Furthermore, it could also block the pathological changes of MGH, including the number of mammary lobules, and the proliferation or expansion of acini and ducts. Moreover, treatment with FA and ICA remarkably down-regulated the serum expression levels of LH, PRL, T, and E2, as well as the mRNA expression levels of ERα, PR, ERK1, and ERK2. Additionally, protein levels of ERα, p-ERα, PR, p-PR, ERK1/2, and p-ERK1/2 in breast tissue were down-regulated, however the serum content of FSH and P was up-regulated.ConclusionsOur outcomes revealed that FA and ICA might potentially inhibit ERα, PR, ERK1/2, and their phosphorylated proteins via the ERK signaling pathway, thus indicating a positive feedback control for the degree of breast hyperplasia.

Methylation of ERβ 5'-untranslated region attenuates its inhibitory effect on ERα gene transcription and promotes the initiation and progression of papillary thyroid cancer.

Normal thyroid tissue displays a prevalent expression of ERβ than ERα, which drastically turns upside down in the initiation and progression of papillary thyroid cancer (PTC). The underlying molecular mechanism of this phenomenon remains unclear. Here, we demonstrated that ERα and ERβ were coexpressed in human thyroid tissues and cells. ERα mRNA (A-1) and ERβ mRNA (0N-1), transcribed from Promoter A of ERα gene and Promoter 0N of ERβ gene, respectively, were the major mRNA isoforms which mainly contributed to total ERα mRNA and total ERβ mRNA in human thyroid-derived cell lines and tissues. The expression levels of ERα mRNA (A-1) and total ERα mRNA were gradually increased, and those of ERβ mRNA (0N-1) and total ERβ mRNA were decreased by degree in the initiation and progression of PTC. No aberrant DNA methylation of ERα 5'-untranslated region was involved in its up-regulation; however, aberrant DNA methylation in Promoter 0N and Exon 0N of ERβ gene was found to be involved in its down-regulation in the initiation and progression of PTC. ERβ can repress ERα gene transcription via recruitment of NCoR and displacement of RNA polymerase II at the Sp1 site in ERα Promoter A-specific region in thyroid-derived cells. It is suggested that DNA methylation of CpG islands in Promoter 0N and Exon 0N of ERβ gene leads to a decreased ERβ gene expression, which attenuates its inhibitory effect on ERα gene transcription and results in an increased ERα gene expression, cell proliferation, initiation, and progression of PTC.

Seasonal expressions of androgen receptor, estrogen receptors and cytochrome P450 aromatase in the uteri of the wild Daurian ground squirrels (Spermophilus dauricus).

The reproductive tissues including the uterus undergo dramatic changes in seasonal breeders from the breeding to non-breeding seasons. Classically, sex steroid hormones play important roles in the uterine morphology and functions. To clarify the relationship between sex steroid hormones and seasonal changes in the uterine morphology and functions, the wild Daurian ground squirrels (Spermophilus dauricus) were used as seasonal breeder model. And the immunolocalizations and expression levels of androgen receptor (AR), estrogen receptors α and β (ERα and ERβ) and cytochrome P450 aromatase (P450arom) were investigated in the uteri of the wild Daurian ground squirrels in the breeding (April) and the non-breeding (June) seasons via immunohistochemistry, Western blot and RT-PCR. Histologically, the uterine weight, the thickness of endometrium and the glandular density were significantly higher in the uteri of the breeding season than those of the non-breeding season. In both seasons, the immunostaining of AR was only presented in stromal cells of the uteri; the positive staining of ERα and ERβ were localized in stromal cells and glandular cells; P450arom was merely immunolocalized in glandular cells. The protein and mRNA expression levels of ERα, ERβ and P450arom were higher in the uteri of the breeding season than those of the nonbreeding season; conversely, the expressions of AR were higher in the uteri of the non-breeding season comparing with those of the breeding season in both protein and mRNA levels. The AR: ER ratio in the uteri of the non-breeding season exceeded the AR: ER ratio in the uteri of the breeding season in the wild Daurian ground squirrels. These results suggested that seasonal changes in the expression levels of AR, ERs and P450arom might be correlated with the uterine morphology and histology changes, and estrogen may play an important autocrine/paracrine role in regulating the uterine functions of the wild Daurian ground squirrels.

MP36-15 EFFECTS OF DUTASTERIDE ON PROSTATIC INFLAMMATION MEDIATED BY ESTROGEN RECEPTOR β IN A RAT MODEL OF NONBACTERIAL PROSTATITIS

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Prostate & Genitalia II1 Apr 2016MP36-15 EFFECTS OF DUTASTERIDE ON PROSTATIC INFLAMMATION MEDIATED BY ESTROGEN RECEPTOR β IN A RAT MODEL OF NONBACTERIAL PROSTATITIS Shinsuke Mizoguchi, Kenichi Mori, Fuminori Sato, Naoki yoshimura, and Hiromitsu Mimata Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Kenichi MoriKenichi Mori More articles by this author , Fuminori SatoFuminori Sato More articles by this author , Naoki yoshimuraNaoki yoshimura More articles by this author , and Hiromitsu MimataHiromitsu Mimata More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1649AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Estrogen receptor β (ERβ) stimulation can improve local inflammation whereas ERα stimulation could induce local inflammation. It has been reported that dutasteride, which not only inhibits 5α-reductase, but also stimulates ERβ, might have anti-inflammatory effects in the prostate of patients with lower urinary tract symptoms (Am J Mens Health September 7, 2015 doi:10.1177/1557988315604019). Therefore we investigated the effect of dutasteride on prostatic inflammation using a rat model of nonbacterial prostatitis. METHODS Male SD rats at 10 weeks old were used. Prostatic inflammation was induced by 5% formalin injection into ventral lobes of the prostate and saline was injected in the control group (CG, n=5). Rats with prostatitis were divided into dutasteride therapy (TG, n=5) and placebo groups (PG, n=5). TG rats were treated with dutasteride at a dose of 0.5mg/kg daily from 2 days before induction of prostatitis whereas PG rats received vehicle only. Twenty-eight days later, conscious cystometry was performed to measure non-voiding contractions (NVCs), voiding interval (VI) and postvoid residual volume (RV). After cystometry, the prostate was excised for analysis of mRNA expression levels of ERα, ERβ, IL1B, IL18 by real-time PCR. RESULTS The mean number of NVCs was significantly greater in PG rats than that of CG rats (P<0.05), and VI were significantly decreased in PG rats compared to CG rats (P<0.05), but not significantly different between CG and TG rats. There was no significant difference in RV among groups. mRNA expression of ERα, IL1B and IL18 were significantly increased in PG rats compared to CG rats (P<0.05), but not significantly different between CG and TG rats. On the other hand, mRNA expression level of ERβ was significantly decreased in PG rats compared to CG rats (P<0.05), but not significantly different between CG and TG rats. The relative mRNA expression ratio of ERβ against ERα (ERβ/ERα) in PG rats was significantly decreased compared to CG rats (P<0.05), whereas ERβ/ERα ratio in TG rats was significantly increased compared to PG rats (P<0.05). CONCLUSIONS Dutasteride improved not only prostatic inflammation evidenced by the decrease of mRNA IL1B and IL18 levels, which are reported as nonbacterial prostatitis markers, but also bladder overactivity as shown by cystmetric investigation. These therapeutic effects were associated with the increased ERβ/ERα ratio. Therefore, dutasteride might be effective via ERβ for the treatment of nonbacterial prostatic inflammation in addition to benign prostatic hyperplasia. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e495 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Shinsuke Mizoguchi More articles by this author Kenichi Mori More articles by this author Fuminori Sato More articles by this author Naoki yoshimura More articles by this author Hiromitsu Mimata More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis

BackgroundThe up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. MethodsIn this study, we enrolled 107 women older than 45years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. ResultsERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (>55years), and low ERα mRNA levels in PBLs (≦250.39 copies/μg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. ConclusionWe conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. General significanceOur findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels.

Effects of 4-nonylphenol isomers on cell receptors and mitogen-activated protein kinase pathway in mouse Sertoli TM4 cells.

In the present study, experiments were performed to investigate the effects of nonylphenol (NP) isomers (4-[1,2, 4-trimethylhexyl]-phenol (NP41), 4-[1,2, 5-trimethylhexyl]-phenol (NP42)) on Sertoli TM4 cells. NP41 decreased mRNA expression levels of androgen receptor and toll-like receptor (TLR)-4 in 20-40μM (P<0.05), and increased mRNA levels of estrogen receptor (ER)-α and progesterone receptor in 1-40μM (P<0.05). NP42 treatment only evoked significant decrease in mRNA expression levels of ER-α in 20-40μM (P<0.05). Similarly, NP41 (1-40μM) drastically increased the protein expression of ER-α, which was significantly decreased in 20-40μM NP42 groups (P<0.01). Both NP41 and NP42 showed no effect on the expression of ER-β. Protein levels of follicle stimulating hormone receptor were increased significantly in high concentrations of NP41 (40μM) and NP42 (10-40μM) challenged cells. Furthermore, NP41 and NP42 showed various effects on the expression of junction-associated molecules and inhibin B secretion in TM4 cells. Additionally, activation of JNK1/2 pathway was induced by NP41 and NP42. However, ERK1/2 and p38 pathways were inhibited in TM4 cells exposed to low concentrations of NP41 (0.1-20μM) and NP42 (0.1-1μM), and high concentrations of NP41 (40μM) and NP42 (10-40μM) resulted in a return of p-ERK1/2 and p-p38 to control levels. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signaling pathways, and the effects may be related to the structure of NP isomer.

Unilateral Lesion Increases Oestrogen Receptor α Expression in the Intact Side of the Ventromedial Hypothalamic Nucleus in Ovariectomised Rats

To determine the relationship between the right and left sides of the ventrolateral ventromedial hypothalamic nucleus (vlVMN) in regulating the expression of oestrogen receptor (ER)α, the unilateral vlVMN was lesioned and the number of ERα-immunoreactive cells and the ERα mRNA level in the intact side of the vlVMN and arcuate nucleus (ARC) were measured in ovariectomised rats. Twenty-four hours after lesioning, brain samples were collected for analysis of ERα expression by immunohistochemistry and the real-time reverse transcriptase-polymerase chain reaction. The number of ERα-immunoreactive cells in the intact side of the vlVMN but not the ARC in the unilateral lesioned group was significantly higher than that in the control or sham-lesioned group. Expression levels of ERα mRNA in the intact side of the vlVMN but not the ARC in unilateral lesioned rats were significantly higher than those in the sham-lesioned group. Of transcript variants with alternative 5'-untranslated regions (0S, 0N, 0, 0T and E1), the ERα 0 transcript level was significantly increased. These results indicate that unilateral damage of vlVMN induces an increase in ERα in the intact side by increasing ERα transcription in a promoter-specific manner. The findings also suggest the existence of new neuroendocrine control system between the right and left sides for the expression of ERα in the vlVMN.

Oestrogen action on thyroid progenitor cells: relevant for the pathogenesis of thyroid nodules?

Benign and malignant thyroid nodules are more prevalent in females than in males. Experimental data suggest that the proliferative effect of oestrogen rather than polymorphisms is responsible for this gender difference. This study analysed whether both differentiated thyroid cells and thyroid stem and progenitor cells are targets of oestrogen action. In thyroid stem/progenitor cells derived from nodular goitres, the ability of 17β-oestradiol (E₂) to induce the formation of thyrospheres and the expression of oestrogen receptors (ERs) and the effect of E₂ on the growth and expression of markers of stem cells and thyroid differentiation (TSH receptor, thyroperoxidase, thyroglobulin and sodium iodide symporter (NIS)) were analysed. E₂ induced thyrosphere formation, albeit to a lower extent than other growth factors. Thyroid stem and progenitor cells expressed ERα (ESR1) and ERβ (ESR2) with eight times higher expression levels of ERα mRNA compared with the differentiated thyrocytes. E₂ was a potent stimulator of the growth of thyroid stem/progenitor cells. In contrast, TSH-induced differentiation of progenitor cells, in particular, the expression of NIS, was significantly inhibited by E₂. In conclusion, oestrogen stimulated the growth and simultaneously inhibited the differentiation of thyroid nodule-derived stem/progenitor cells. From these data and based on the concept of cellular heterogeneity, we hypothesize a supportive role of oestrogen in the propagation of thyroid stem/progenitor cells leading to the selection of a progeny of growth-prone cells with a decreased differentiation. These cells may be the origin of hypofunctioning or non-functioning thyroid nodules in females.

Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression

Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α- and ERβ-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERβ mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERβ in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

Effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils (Meriones unguiculatus)

Quinestrol, a synthetic estrogen with marked estrogenic effects and prolonged activity, has potential as a contraceptive for Mongolian gerbils. The objective of this study was to describe the effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils. Serum and pituitary concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were decreased, whereas serum concentrations of estradiol (E2) and progesterone (P4) were increased after quinestrol treatment; the effects were both time- and dose-dependent. Furthermore, quinestrol downregulated expression of FSHβ and LHβ mRNA in the pituitary gland, as well as FSH receptor (FSHR) and estrogen receptor (ER) β in the ovary. However, it up-regulated mRNA expression levels of ERα and progesterone receptor (PR) in the pituitary gland and uterus, as well as mRNA for LH receptor (LHR) and PR in the ovary (these effects were time- and dose-dependent). In contrast, quinestrol had no significant effects on the mRNA expression levels of ERα in the ovary, or the gonadotropin α (GtHα) subunit in the pituitary gland. We inferred that quinestrol impaired synthesis and secretion of FSH and LH and that the predominant ER subtype in the pituitary gland of Mongolian gerbils may be ERα. Overall, quinestrol disrupted reproductive hormone receptor expression at the mRNA level in the pituitary-gonadal axis of the Mongolian gerbil.

PFOS elicits transcriptional responses of the ER, AHR and PPAR pathways in Oryzias melastigma in a stage-specific manner

Perfluorooctane sulfonate (PFOS) is widely distributed in seawater. However, the effects of PFOS on important receptors involved in the toxicity of POPs have not been fully defined, especially for the embryonic stages of marine fish. In this study, we examined the transcriptional responses and PFOS accumulation in the marine medaka embryos at the early and late developmental stages of 4 and 10 dpf upon PFOS exposure (1, 4, and 16 mg/L). PFOS accumulated in the embryos, and the embryonic burdens of PFOS at 10 dpf were markedly higher than those at 4 dpf. Moreover, thirteen genes involved in three important POPs-related receptor pathways, including ER, AHR and PPAR, were cloned and investigated. The mRNA expression levels of ERα and ERγ were not significantly altered, but the estrogenic marker genes were downregulated upon PFOS exposure at 4 dpf. Conversely, ERs and related marker genes all were significantly upregulated at 10 dpf. The expressions of ARNT and cyp1a were both upregulated at 4 dpf, while no obvious changes were detected at 10 dpf. The expressions of cyp19a and cyp19b were regulated by PFOS in a stage-specific manner. PFOS produced different effects on three isoforms of PPAR. PPARα and PPARβ were first inhibited at 4 dpf and were induced at 10 dpf. PFOS did not elicit a change in PPARγ expression at either stage. In conclusion, this study showed that PFOS has an estrogenic activity and endocrine-disruptive properties. Meanwhile, PFOS could elicit transcriptional responses on POPs-related pathways in a stage-specific manner.

Effects of soy isoflavones and major active component genistein on the expression of ovarian estrogen receptor-α in rats

To investigate the effects of soy isoflavones (SI) on the expression of estrogen receptor-α (ER-α) in senile rat ovaries and ovarian granulosa cell cultured in vitro treated with genistein, a major active component of SI. The animal model of perimenopause rats was established by unforced aging. The animals were treated by intragastric administration (ig) with low (50 mg/kg), middle (158 mg/kg) and high (500 mg/kg) dose of SI for 8 weeks. The expressions of ER-α mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry respectively. The granulosa cells of rat ovaries were isolated and administered with genistein (0, 0.1, 1, 5, 10, 100 µmol/L) for 48 h and the expression levels of ER-α mRNA detected by RT-PCR. The ER-α mRNA expression levels of the low, middle and high dose groups of SI (0.207 ± 0.014, 0.316 ± 0.073 and 0.402 ± 0.170 respectively) were higher than those of the model group (0.671 ± 0.170) (all P < 0.01). The expression levels of ER-α protein for the low, middle and high dose groups of SI (7.35 ± 4.90, 13.90 ± 5.12 and 23.79 ± 10.31 respectively) were higher than those of the model group (2.74 ± 0.09) (all P < 0.01). The expression levels of ER-α mRNA in granulosa cells treated with 1, 5, 10 µmol/L genistein for 48 h were 0.927 ± 0.232, 1.067 ± 0.154, 1.118 ± 0.126 respectively (all P < 0.01). They were higher than those of the control group (0.671 ± 0.170). But the expression levels of 100 µmol/L genistein group were lower than those of the control group (P < 0.05). Soy isoflavones can up-regulate the expressions of ER-α mRNA and protein in senile rat ovaries. As a major active component of soy isoflavones, genistein can regulate the expressions ER-α mRNA in granulosa cells of rat ovaries. Such an effect is concentration-dependent. And 1-10 µmol/L genistein may up-regulate the expression of ER-α mRNA.

Oral fluoropyrimidine may augment the efficacy of aromatase inhibitor via the down-regulation of estrogen receptor in estrogen-responsive breast cancer xenografts

The present preclinical study was designed to evaluate a new combination therapy comprised of the aromatase inhibitor anastrozole (ANA) and the oral fluoropyrimidines, UFT and S-1 against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7/Arom 14, which was stably transfected with the cDNA of human aromatase. MCF-7/Arom 14 cells showed a high aromatase activity and notably were able to grow in the presence of testosterone and estradiol (E(2)) in vitro. ANA and 5-fluorouracil (5-FU) inhibited cell growth at concentrations of 0.005-10 and 0.2-5 μM, respectively, and the combination of both drugs additively inhibited cell growth. The growth of MCF-7/Arom 14 tumors was significantly inhibited by ANA and S-1 or UFT in vivo. The combination of ANA with S-1 or UFT administered using a 21-day consecutive, metronomic-like regimen significantly enhanced the antitumor efficacy, suppressing tumor growth for 2-4 times longer than monotherapy. To investigate the mechanisms by which S-1 enhances the antitumor activity of ANA, the protein and mRNA expression levels of ER-α in tumor tissue after treatment with S-1, ANA, and the typical chemotherapeutic agents doxorubicin (ADM) or paclitaxel (TXL) were analyzed. The protein and mRNA expression levels of ER-α in the tumor tissue were markedly decreased after treatment with S-1 or S-1 + ANA, but not after treatment with either ADM or TXL. The reduced ER-α level after S-1 treatment might contribute to the increased antitumor activity of ANA by reducing ER-α-induced growth signaling in addition to the decrease in estrogen production induced by ANA. Based on these results, the combination of ANA and S-1 might yield a greater benefit than other chemotherapeutic agents in postmenopausal women with ER-positive breast cancer.

Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Neonatal Imprinting and Regulation of Estrogen Receptor Alpha and Beta mRNA Expression by Estrogen in the Pituitary and Hypothalamus of the Male Rat

In male rodents, complete reproductive function is critically dependent on adequate estrogen action at different levels of the hypothalamic-pituitary-testicular axis. However, administration of high doses of estrogen during the critical period of neonatal differentiation results in multiple defects in the reproductive axis that disrupt male fertility, the molecular mechanism(s) behind such a phenotype remaining poorly characterized. The aim of this study was twofold: (1) to characterize the effects of neonatal estrogenization upon the pattern of estrogen receptors ERα and ERβ mRNA expression in the pituitary and hypothalamus of the male rat, and (2) to evaluate the ability of estrogen to acutely regulate pituitary and hypothalamic ERα and ERβ mRNA expression in the male rat. To achieve the first goal, groups of male rats were treated on the first day of life with estradiol benzoate (EB; 500 µg/rat), and pituitary and hypothalamic expression of ERα and ERβ mRNA levels were assayed by semiquantitative RT-PCR at different ages from the neonatal period until adulthood (days 1–75 of life). In addition, the mechanism(s) of altered pituitary expression of ERα and ERβ messages in neonatally estrogenized rats was explored by comparison of the effects of neonatal treatment with estrogen or a potent gonadotropin-releasing hormone (GnRH) antagonist. For the second goal, the acute effects of a single dose of EB (12.5–25 µg/rat) on hypothalamic and pituitary ERα and ERβ mRNA expression were assessed in prepubertal and adult male rats. Neonatal estrogenization permanently decreased pituitary ERα and ERβ mRNA expression levels respective to control values at all ages studied. This pattern of response was similar to the short-term effects of neonatal blockade of endogenous GnRH actions. In contrast, neonatal exposure to estrogen transiently increased the hypothalamic expression levels of ERα and ERβ messages. This effect was detected in neonatal (5-day-old), infantile (15-day-old) and prepubertal (30-day-old) rats, and it disappeared at puberty. In addition, estrogen was able to acutely regulate mRNA expression levels of its cognate receptor subtypes in the pituitary and hypothalamus of intact male rats. In adult (75-day-old) males, EB administration (25 µg/rat) induced a significant time-dependent decrease in pituitary ERα and ERβ mRNA levels. In contrast hypothalamic expression of ERα and ERβ messages was increased after acute exposure to EB (12.5 µg/rat) in prepubertal males (30 days old); yet differences in the time course of the response to estrogen were noticed between targets. In conclusion, our data indicate that estrogen is able to neonatally imprint and acutely regulate mRNA expression levels of ERα and ERβ in the pituitary and hypothalamus of the male rat. Regulation of pituitary and hypothalamic ERα and ERβ gene expression by the cognate ligand represents a novel mechanism whereby estrogen modulates its own biological actions upon different levels of the male reproductive axis throughout the life span.