Estrogen Receptor Blockade Research Articles

Notoginsenoside R1-mediated neuroprotection involves estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways: A novel mechanism of Nrf2/ARE signaling activation

Notoginsenoside R1 (NGR1), a novel phytoestrogen isolated from Panax notoginseng, has antioxidant and anti-apoptotic properties. Oxidative stress plays a pivotal role in neurodegenerative diseases. To mimic oxidative stress in neurons and explore the neuroprotection of NGR1, H2O2-induced neurotoxicity in NGF-induced differentiation of PC12 cells was used. In this study, NGR1 preconditioning provided neuroprotective effects via suppressing H2O2-induced the intracellular ROS accumulation, the increase in the product of lipid peroxidation (MDA), protein oxidation (protein carbonyl), and DNA fragmentation (8-OHdG), and mitochondrial membrane depolarization as well as caspase-3 activation. Moreover, NGR1 treatment alone potently increased the nuclear translocation of Nrf2, augmented ARE enhancer activity, and upregulated the expression and activity of phase II antioxidant enzymes including HO-1, NQO-1, and γ-GCSc. NGR1 could also increase the ERE activity and activate Akt and ERK1/2 pathways. NGR1-mediated activation of Nrf2/ARE signaling and neuroprotection were abolished by genetic silencing of Nrf2 using siRNA or the pharmacological blockade of estrogen receptors using ICI-182780, and partially inhibited by Akt siRNA or ERK siRNA transfection. In addition, the phosphorylation of ERK1/2 mediated by NGR1 was markedly inhibited in PC12 cells transfected with Akt siRNA. On the contrary, ERK1/2 siRNA transfection hardly had any effect on the phosphorylation of Akt mediated by NGR1. NGR1-mediated activation of Akt and ERK1/2 pathways was blocked by ICI-182780. In conclusion, NGR1 provided neuroprotection via inducing an estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways, subsequently activating Nrf2/ARE signaling and thereby up-regulating phase II antioxidant enzymes.

Investigator-initiated pilot study of bicalutamide and raloxifene in hormone-resistant prostate cancer.

e16065 Background: Estrogens, in addition to androgen may contribute to the progression of prostate cancer.The combination of bicalutamide, a non-steroidal anti-androgen, and raloxifene, a selective estrogen receptor modulator, has a synergistic effect on cell death of prostate cancer cell lines such as DU145 and PC3. We investigated the safety of combined treatment of bicalutamide and raloxifene on quality of life in patients affected by hormone-resistant prostate cancer. Methods: Eligibility requirements included metastatic hormone-resistant prostate cancer, Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the combination of bicalutamide, 50 mg, and raloxifene, 60 mg, daily in 28 day cycles for a maximum of 6 cycles. The primary endpoint was to describe the adverse event profile of combined treatment with bicalutamide and raloxifene. The secondary endpoint was to describe the quality of life of patients receiving the combination. Adverse events were graded by the investigator using the NCI Common Terminology Criteria for Adverse Events version 4.0 and the quality of life was assessed by the patient using aLinear Analog Scale Assessment (scale of 0-100). Serum levels of estradiol and testosterone were monitored during treatment. Results: Eighteen evaluable patients were included in the statistical analysis after an initial run-in safety cohort of six patients. Two patients completed 6 cycles per protocol, the remaining sixteen patients were taken off protocol after disease progression. Patients completed a median of 2 cycles. There were no grade 4 events and grade 3 adverse events consisted of hypertension (5.6%) and back pain (5.6%). Erectile dysfunction (grade 1) was the most common adverse event (77.8%). Patient assessment of quality of life (mental, physical, social, emotional, spiritual) did not reveal any statistically different changes after 2 cycles of treatment. An analysis of testosterone and estradiol levels will be reported. Conclusions: We conclude that combination of bicalutamide and raloxifene is well-tolerated. The combination of androgen and estrogen receptor blockade warrants further study for efficacy in hormone-resistant prostate cancer. Clinical trial information: NCT01050842.

Multicenter Phase II Study of Neoadjuvant Lapatinib and Trastuzumab With Hormonal Therapy and Without Chemotherapy in Patients With Human Epidermal Growth Factor Receptor 2–Overexpressing Breast Cancer: TBCRC 006

We previously reported the eradication of human epidermal growth factor receptor 2 (HER2)- amplified human xenografts in mice by inhibition of the HER2 pathway with lapatinib and trastuzumab to block all homo- and heterodimer signaling as well as by blockade of estrogen receptor (ER) when expressed. In this clinical trial, we sought to translate these findings to patients using targeted therapy without chemotherapy. Women with stages II to III HER2-positive breast cancers were eligible. They received trastuzumab once per week (4 mg/kg loading, then 2 mg/kg) and lapatinib 1000 mg once per day for 12 weeks. Women with ER-positive tumors also received letrozole (plus a luteinizing hormone-releasing hormone [LHRH] agonist if premenopausal). Pathologic response was assessed by ER status. Biopsies were obtained at baseline, weeks 2 and 8, and time of surgery. Sixty-six patients were enrolled, and 64 were eligible and evaluable for response. Median tumor size was 6 cm (range, 1.5 to 30 cm). Adverse events were mainly grades 1 to 2 (GI, 63%; skin, 46%). Grade 3 metabolic, GI, and liver (18%; 12 patients) and grade 4 liver toxicities (one patient) were also observed. Overall, in-breast pathologic complete response (pCR; ypT0-is) was 27% (ER positive, 21%; ER negative, 36%). The rate of low-volume residual disease (ypT1a-b) was 22% (ER positive, 33%; ER negative, 4%). In patients with locally advanced HER2-positive breast cancer, our approach of targeted therapy only resulted in a high pCR rate without chemotherapy. Our data support the hypothesis that selected patients with HER2-positive tumors may not need chemotherapy, and more-complete blockade of HER receptors and ER is an effective strategy worthy of further study.

Abstract 3616: Androgen deprivation therapy and second primary lung cancer risk in prostate cancer patients in the US Veterans.

Abstract Background Previous observational studies have consistently reported decreased risk of lung cancers among prostate cancer patients. Recent literature suggests a role of estrogen receptor (ER) beta and aromatase, which can convert testosterone to estrogen, in development and progression of non small cell lung cancers. Second primary lung cancer risk among breast cancer patients appears to be higher in ER negative patients compared to ER positive patients probably from estrogen receptor blockade. As androgen deprivation therapy (ADT) has been well established for more than 70 years as an effective treatment modality for prostate cancer, we hypothesized the hormone modulation may decrease lung cancer risk and investigated the effect of ADT on second primary lung cancer risk among patients with prostate cancer. Methods We identified 62,589 men in VA Central Cancer Registry (VACCR) who were diagnosed with prostate cancer between 1999 and 2008, with follow-up available through 2010. The primary outcome was development of lung cancer, determined by VACCR. Descriptive statistics were used to compare basic characteristics between the groups. Cox proportional hazards model was used to assess the influence of ADT on the lung cancer incidence, adjusted for patient characteristics and prostate cancer stage. All the statistical analyses were performed using STATA 10 (Stata Corp, College Station, TX). Results Men who had ADT as part of their treatment for prostate cancer tend to be older (median age 71.3 vs 65.7, p < 0.01) and smoke slightly more (72.5% vs 71.4%, p=0.02). As expected, ADT group tend to have higher stage prostate cancers. There were 766 secondary lung cancers diagnosed in the cohort for the follow-up period. ADT group had significantly decreased secondary lung cancer risk by approximately 20% (HR 0.83, 95%CI=0.70-0.97) when adjusted for age, smoking status and prostate cancer stage at presentation. Conclusion Our study suggests that ADT in prostate cancer patients is associated with a decreased risk of second primary lung cancers. Independent validation studies and subgroup analyses to clarify the association is warranted. This work was supported by Veteran's Health Administration 1IK2BX001283-01 and NCI 5P50CA128613-02. Citation Format: Hyunseok Kang, Rathi N. Pillai, Johann C. Brandes. Androgen deprivation therapy and second primary lung cancer risk in prostate cancer patients in the US Veterans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2013-3616

A randomized trial to assess the biological activity of short-term (pre-surgical) fulvestrant 500 mg plus anastrozole versus fulvestrant 500 mg alone or anastrozole alone on primary breast cancer.

IntroductionFulvestrant shows dose-dependent biological activity. Greater estrogen-receptor (ER) blockade may feasibly be achieved by combining fulvestrant with anastrozole. This pre-surgical study compared fulvestrant plus anastrozole versus either agent alone in patients with ER-positive breast cancer.MethodsIn this double-blind, multicenter trial, 121 patients received fulvestrant 500 mg on Day 1 plus anastrozole 1 mg/day for 14 to 21 days (F + A); fulvestrant plus anastrozole placebo (F); or fulvestrant placebo plus anastrozole (A), 2 to 3 weeks before surgery. ER, progesterone-receptor (PgR) and Ki67 expression were determined from tumor biopsies before treatment and at surgery.ResultsA total of 103 paired samples were available (F, n = 35; F+A, n = 31; A, n = 37). All treatments significantly reduced mean ER expression from baseline (F: -41%, P = 0.0001; F + A: -39%, P = 0.0001; A: -13%, P = 0.0034). F and F + A led to greater reductions in ER versus A (both P = 0.0001); F + A did not lead to additional reductions versus F. PgR and Ki67 expression were significantly reduced with all treatments (means were -34% to -45%, and -75% to -85%, respectively; all P = 0.0001), with no differences between groups.ConclusionsIn this short-term study, all treatments reduced ER expression, although F and F + A showed greater reductions than A. No significant differences were detected between the treatment groups in terms of PgR and Ki67 expression. No additional reduction in tumor biomarkers with combination treatment was observed, suggesting that F + A is unlikely to have further clinical benefit over F alone.Trial registrationClinicaltrials.gov NCT00259090.

Novel Approaches for Molecular Targeted Therapy of Breast Cancer: Interfering with PI3K/AKT/mTOR Signaling

Breast cancer is one of the most prevalent and devastating malignant diseases in women worldwide. Fortunately, while breast cancer incidence is still increasing, its death rate is declining. This is mainly due to early diagnosis and potent therapies such as blockade of estrogen receptor- or of ErbB2 (HER2-neu) membrane receptor signaling.In recent years, the PI3K/AKT/mTOR pathway, which transmits signals from the cell membrane into the nucleus and activates multiple oncogenic programs, has been found to play a crucial role in the regulation of breast cancer cell growth. This pathway is densely interconnected with a multitude of other important regulatory systems for glucose-,lipid- and amino acid-metabolism, for energy balance, and for autophagy. It has been found that PI3K/AKT/mTOR signaling modulates estrogen receptor function. Using transverse and feedback regulatory loops the PI3K/AKT/mTOR cascade can communicate with concurrent and with upstream systems. Thus, PI3K/AKT/mTOR is a crucial element within a complicated signaling network. This pathway is hyperactive in more than 70% of breast tumors. Hence, the protein kinases located along this route represent very attractive and promising drug targets for breast cancer therapy. Currently, numerous small molecular drugs that inhibit PI3K, AKT and/or mTOR are being developed in preclinical and clinical models of breast cancer. Some of these compounds are highly selective blocking only one particular kinase complex, whereas others interfere with two (mTORC1+mTORC2) or even three effectors (PI3K+mTORC1+mTORC2) of the pathway. Due to the many interactions with other regulatory systems, silencing of the pathway can cause unexpected results. Therefore, detailed preclinical and clinical evaluation of these compounds as single drugs and in combination is required to achieve optimal results with maximal clinical benefit and acceptable toxicity. Also, reliable biomarkers for the identification of patient subsets that will maximally benefit from PI3K/AKT/mTOR inhibition need to be developed. Thus, selective silencing of PI3K/AKT/mTOR signaling represents a promising approach for breast cancer and might prove useful when combined with other drugs. Here we review the current preclinical and clinical data and compare the potential benefits of multi- versus single-targeting PI3K/AKT/mTOR drugs.

Effect of simultaneous blockade of androgen and estrogen receptors on prostate cancer: In vivo study.

214 Background: In our preliminary in vitro studies, we have demonstrated evidence of enhanced apoptosis and inhibition of cellular proliferation in both hormone sensitive and castrate resistant prostate cancer (PCa) cell lines using a combination of an antiandrogen (Bicalutamide) and a selective estrogen receptor modulator (Raloxifene). The aim of this study was to study the effect of the administration of these two drugs in in vivo models of castrate resistant PCa. Methods: In vivo model consisted on NCr Nude: Mice bearing s.c. human prostate (PC3 cell line) xenografts. Based on the treatment received, mice were divided into 5 groups as follows: Group 1: No drugs (control); Group 2: Bicalutamide 50mg; Group 3: Raloxifene 60 mg; Group 4: Combined administration of Bicalutamide 50 mg and Raloxifene 60 mg; Group 5 Combined administration of Bicalutamide 150 mg and Raloxifene 120 mg. A total of 10 mice where included in each group. All drugs dosages were converted to their equivalent in the mice. Drugs were administered by gavage technique to the mice once per day for a total of 14 days. Results: As expected, Bicalutamide administered alone causes minimal inhibition without reaching statistical significance (Group 2: 0.34 g Vs Group 1: 0.40 g; p=0.073). Although Raloxifene causes some marked growth inhibition, its effect is not statistically significant (Group 3: 0.31 Vs Group 1: 0.40 g; p=0.062). Bicalutamide and Raloxifene, when administered in combination, induced prominent growth inhibition in PC3 tumors when compared to the control group (Group 4: 0.26 g Vs Group 1: 0.40 g; p=0.038). Growth inhibition is significantly more evident when the drugs dosages are increased (Group 5: 0.17 g Vs Group 1: 0.40 g; p=0.024). Conclusions: The simultaneous administration of Bicalutamide and Raloxifene appears to have a synergistic effect on tumor growth inhibition in PC3 xenografts. The pathway(s) responsible for this observation may be independent of the androgen receptor as PC3 cells are AR negative and still affected by the combination over the drugs administered alone. Research is warranted to identify these potential pathways.

Src Inhibition with Saracatinib Reverses Fulvestrant Resistance in ER-Positive Ovarian Cancer Models In Vitro and In Vivo

More effective, less toxic treatments for recurrent ovarian cancer are needed. Although more than 60% of ovarian cancers express the estrogen receptor (ER), ER-targeted drugs have been disappointing due to drug resistance. In other estrogen-sensitive cancers, estrogen activates Src to phosphorylate p27 promoting its degradation and increasing cell-cycle progression. Because Src is activated in most ovarian cancers, we investigated whether combined Src and ER blockade by saracatinib and fulvestrant would circumvent antiestrogen resistance. ER and Src were assayed in 338 primary ovarian cancers. Dual ER and Src blockade effects on cell cycle, ER target gene expression, and survival were assayed in ERα+ ovarian cancer lines, a primary human ovarian cancer culture in vitro, and on xenograft growth. Most primary ovarian cancers express ER. Src activity was greater in ovarian cancer lines than normal epithelial lines. Estrogen activated Src, ER-Src binding, and ER translocation from cytoplasm to nucleus. Estrogen-mediated mitogenesis was via ERα, not ERβ. While each alone had little effect, combined saracatinib and fulvestrant increased p27 and inhibited cyclin E-Cdk2 and cell-cycle progression. Saracatinib also impaired induction of ER-target genes c-Myc and FOSL1; this was greatest with dual therapy. Combined therapy induced autophagy and more effectively inhibited ovarian cancer xenograft growth than monotherapy. Saracatinib augments effects of fulvestrant by opposing estrogen-mediated Src activation and target gene expression, increasing cell-cycle arrest, and impairing survival, all of which would oppose antiestrogen resistance in these ER+ ovarian cancer models. These data support further preclinical and clinical evaluation of combined fulvestrant and saracatinib in ovarian cancer.

Dual Effect of 17β-Estradiol on NMDA-Induced Neuronal Death: Involvement of Metabotropic Glutamate Receptor 1

Pretreatment with 10 nm 17β-estradiol (17βE2) or 100 μm of the metabotropic glutamate 1 receptor (mGlu1R) agonist, dihydroxyphenylglycine (DHPG), protected neurons against N-methyl-d-aspartate (NMDA) toxicity. This effect was sensitive to blockade of both estrogen receptors and mGlu1R by their respective antagonists. In contrast, 17βE2 and/or DHPG, added after a low-concentration NMDA pulse (45 μm), produced an opposite effect, i.e. an exacerbation of NMDA toxicity. Again this effect was prevented by both receptor antagonists. In support of an interaction of estrogen receptors and mGlu1R in mediating a neurotoxic response, exacerbation of NMDA toxicity by 17βE2 disappeared when cultures were treated with DHPG prior to NMDA challenge, and conversely, potentiation of NMDA-induced cell death by DHPG was prevented by pretreatment with 17βE2. Addition of calpain III inhibitor (10 μm), 2 h before NMDA, prevented the increased damage induced by the two agonists, an affect that can be secondary to cleavage of mGlu1R by calpain. Accordingly, NMDA stimulation reduced expression of the full-length (140 kDa) mGluR1, an effect partially reversed by calpain inhibitor. Finally, in the presence of NMDA, the ability of 17βE2 to stimulate phosphorylation of AKT and ERK was impaired. Pretreatment with calpain inhibitor prevented the reduction of phosphorylated ERK but had no significant effect on phosphorylated AKT. Accordingly, the inhibition of ERK signaling by U0126 (1 μm) counteracted the effect of calpain inhibition on 17βE2-induced exacerbation of NMDA toxicity. The present data confirm the dual role of estrogens in neurotoxicity/neuroprotection and highlight the role of the timing of exposure to estrogens.

Effects of gonadal hormones on the peripheral cannabinoid receptor 1 (CB1R) system under a myositis condition in rats

In this study, we assessed the effects of peripherally administered cannabinoids in an orofacial myositis model, and the role of sex hormones in cannabinoid receptor (CBR) expression in trigeminal ganglia (TG). Peripherally administered arachidonylcyclopropylamide (ACPA), a specific CB1R agonist, significantly attenuated complete Freund’s adjuvant (CFA)-induced mechanical hypersensitivity in the masseter muscle in male rats. The ACPA effect was blocked by a local administration of AM251, a specific CB1R antagonist, but not by AM630, a specific CB2R antagonist. In female rats, a 30-fold higher dose of ACPA was required to produce a moderate reduction in mechanical hypersensitivity. CFA injected in masseter muscle significantly upregulated CB1R mRNA expression in TG in male, but not in female, rats. There was a close correlation between the CB1R mRNA levels in TG and the antihyperalgesic effect of ACPA. Interleukin (IL)-1β and IL-6, which are elevated in the muscle tissue following CFA treatment, induced a significant upregulation of CB1R mRNA expression in TG from male rats. The upregulation of CB1R was prevented in TG cultures from orchidectomized male rats, which was restored by the application of testosterone. The cytokines did not alter the CB1R mRNA level in TG from intact as well as ovariectomized female rats. Neither estradiol supplement nor estrogen receptor blockade had any effects on CB1R expression. These data indicate that testosterone, but not estradiol, is required for the regulation of CB1Rs in TG under inflammatory conditions, which provide explanations for the sex differences in the antihyperalgesic effects of peripherally administered cannabinoids.

Involvement of Estrogen in Rapid Pain Modulation in the Rat Spinal Cord

The pivotal role of estrogens in the pain sensitivity has been investigated in many ways. Traditionally, it is ascribed to the slow genomic changes mediated by classical nuclear estrogen receptors (ER), ERα and ERβ, depending on peripheral estrogens. Recently, it has become clear that estrogens can also signal through membrane ERs (mERs), such as G-protein-coupled ER1 (GPER1), mediating the non-genomic effects. However, the spinal specific role played by ERs and the underlying cellular mechanisms remain elusive. The present study investigated the rapid estrogenic regulation of nociception at the spinal level. Spinal administration of 17β-estradiol (E2), the most potent natural estrogen, acutely produced a remarkable mechanical allodynia and thermal hyperalgesia without significant differences among male, female and ovariectomized (Ovx) rats. E2-induced the pro-nociceptive effects were partially abrogated by ICI 182,780 (ERs antagonist), and mimicked by E2-BSA (a mER agonist). Inhibition of local E2 synthesis by 1,4,6-Androstatrien-3,17-dione (ATD, a potent irreversible aromatase inhibitor), or blockade of ERs by ICI 182,780 produced an inhibitory effect on the late phase of formalin nociceptive responses. Notably, lumbar puncture injection of G15 (a selective GPER1 antagonist) resulted in similar but more efficient inhibition of formalin nociceptive responses as compared with ICI 182,780. At the cellular level, the amplitude and decay time of spontaneous inhibitory postsynaptic currents were attenuated by short E2 or E2-BSA treatment in spinal slices. These results indicate that estrogen acutely facilitates nociceptive transmission in the spinal cord via activation of membrane-bound estrogen receptors.

Critical period for estrogen‐dependent motoneuron dendrite growth is coincident with ERα expression in target musculature

The spinal cord of rats contains the sexually dimorphic, steroid-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In males, SNB dendrite growth is dependent on gonadal steroids: dendrite growth is inhibited after castration, but supported in androgen- or estrogen-treated castrated males. Furthermore, estrogenic support of SNB dendrite growth is mediated by estrogen action at the target musculature, inhibited by estrogen receptor (ER) blockade at the muscle and supported by local estradiol treatment. However, this estrogenic support is restricted to the early postnatal period, after which the morphology of SNB dendrites is insensitive to estrogens. To test if the developmentally restricted effects of estrogens on SNB dendrite growth coincide with the transient expression of ER in the target musculature, ERα expression was assessed during development and in adulthood. ERα expression in extra-Muscle fiber cells was greatest from postnatal day 7 (P7) to P14 and declined after P21. Because this pattern of ERα expression coincided with the period of estrogen-dependent dendrite growth, we tested if limiting hormone exposure to the period of maximal ERα expression in extra-muscle fiber cells could fully support estrogen-dependent SNB dendrite growth. We restricted estradiol treatment in castrated males from P7 to P21 and assessed SNB dendritic morphology at P28. Treating castrates with estradiol implants at the muscle from P7 to P21 supported dendrite growth to normal levels through P28. These data suggest that the transient ERα expression in target muscle could potentially define the critical period for estrogen-dependent dendrite growth in SNB motoneurons.

Molecular Pathways: The Role of NR4A Orphan Nuclear Receptors in Cancer

Nuclear receptors are of integral importance in carcinogenesis. Manipulation of classic ligand-activated nuclear receptors, such as estrogen receptor blockade in breast cancer, is an important established cancer therapy. Orphan nuclear receptors, such as nuclear family 4 subgroup A (NR4A) receptors, have no known natural ligand(s). These elusive receptors are increasingly recognized as molecular switches in cell survival and a molecular link between inflammation and cancer. NR4A receptors act as transcription factors, altering expression of downstream genes in apoptosis (Fas-ligand, TRAIL), proliferation, DNA repair, metabolism, cell migration, inflammation (interleukin-8), and angiogenesis (VEGF). NR4A receptors are modulated by multiple cell-signaling pathways, including protein kinase A/CREB, NF-κB, phosphoinositide 3-kinase/AKT, c-jun-NH(2)-kinase, Wnt, and mitogen-activated protein kinase pathways. NR4A receptor effects are context and tissue specific, influenced by their levels of expression, posttranslational modification, and interaction with other transcription factors (RXR, PPAR-Υ). The subcellular location of NR4A "nuclear receptors" is also important functionally; novel roles have been described in the cytoplasm where NR4A proteins act both indirectly and directly on the mitochondria to promote apoptosis via Bcl-2. NR4A receptors are implicated in a wide variety of malignancies, including breast, lung, colon, bladder, and prostate cancer; glioblastoma multiforme; sarcoma; and acute and/or chronic myeloid leukemia. NR4A receptors modulate response to conventional chemotherapy and represent an exciting frontier for chemotherapeutic intervention, as novel agents targeting NR4A receptors have now been developed. This review provides a concise clinical overview of current knowledge of NR4A signaling in cancer and the potential for therapeutic manipulation.

Spinal Synthesis of Estrogen and Concomitant Signaling by Membrane Estrogen Receptors Regulate Spinal κ- and μ-Opioid Receptor Heterodimerization and Female-Specific Spinal Morphine Antinociception

We previously demonstrated that the spinal cord κ-opioid receptor (KOR) and μ-opioid receptor (MOR) form heterodimers (KOR/MOR). KOR/MOR formation and the associated KOR dependency of spinal morphine antinociception are most robust during proestrus. Using Sprague Dawley rats, we now demonstrate that (1) spinal synthesis of estrogen is critical to these processes, and (2) blockade of either estrogen receptor (ER) α-, β-, or G-protein-coupled ER1 or progesterone receptor (PR) substantially reduces KOR/MOR and eliminates mediation by KOR of spinal morphine antinociception. Effects of blocking ERs were manifest within 15 min, whereas those of PR blockade were manifest after 18 h, indicating the requirement for rapid signaling by estrogen and transcriptional effects of progesterone. Individual or combined blockade of ERs produced the same magnitude of effect, suggesting that they work in tandem as part of a macromolecular complex to regulate KOR/MOR formation. Consistent with this inference, we found that KOR and MOR were coexpressed with ERα and G-protein-coupled ER1 in the spinal dorsal horn. Reduction of KOR/MOR by ER or PR blockade or spinal aromatase inhibition shifts spinal morphine antinociception from KOR dependent to KOR independent. This indicates a sex steroid-dependent plasticity of spinal KOR functionality, which could explain the greater analgesic potency of KOR agonists in women versus men. We suggest that KOR/MOR is a molecular switch that shifts the function of KOR and thereby endogenous dynorphin from pronociceptive to antinociceptive. KOR/MOR could thus serve as a novel molecular target for pain management in women.

Estrogen and Its Receptor Enhance Mechanobiological Effects in Compressed Bone Mesenchymal Stem Cells

Mechanical stimulation and estrogen have been proven to be two important factors in promoting mesenchymal stem cell activity, which is closely associated with bone formation, mass maintenance and remodeling. However, the superposition effects of mechanical stimulation and estrogen on stem cells remain unknown. It is also unclear if the estrogen receptor (ER) plays only a key role in estrogen signaling or if it is also involved in the mechanotransduction of stem cells. To investigate the role of estrogen and its receptors in the mechanobiological effects in bone mesenchymal stem cells (BMSCs), isolated mesenchymal stem cells from bone marrow were exposed to mechanical pressure under additional estrogen treatment or ER blockade. Cell proliferation was examined using an MTT assay and alkaline phosphatase (ALP) activity was determined by a modified enzyme kinetic method. Alignment of the cytoskeleton was observed by Coomassie brilliant blue staining and F-actin fluorescent staining. Cellular ultrastructure was observed under transmission electron microscope. Expression of ERα was investigated using Western blot analysis. Results indicated that mechanical pressure promoted cell proliferation, ALP activity, ERα expression and F-actin stress fiber formation. Overall, this effect was enhanced by the addition of estrogen and inhibited by ER blockade. We concluded that pressure stimulated proliferation and differentiation capability via F-actin transduction in BMSCs. The effects were enhanced by the addition of estrogen, and the ER plays an important role in regulating mechanobiological effects and the mechanotransduction processes of BMSCs.

Effect of the simultaneous blockade of androgen and estrogen receptors on prostate cancer: Preliminary results.

168 Background: Androgens and estrogens have been shown to play an important role in normal prostate development and function as well as carcinogenesis and development of the castration resistant phenotype of disease. The aim of this study was to evaluate the effect of a simultaneous administration of an androgen receptor antagonist (bicalutamide) and a selective estrogen receptor modulator (raloxifene) on both androgen sensitive and androgen insensitive prostate cancer cell lines. Methods: Experiments were performed on LNCaP, PC3 and DU145 cell lines. Western blot was utilized for the identification of androgen and estrogen receptors (a andb) in the cell lines. Drug concentrations required to achieve IC 50 were obtained using the MTT assay; such concentrations were identified for the drugs individually and when used in combination. The effect of the drugs on apoptosis was assessed using flow cytometry. Results: Results of the IC 50 for the drugs alone and in combination by each cell line are shown in the table. An enhanced effect was observed when the drugs were used in combination in all the cell lines. It was evident that the combination of the drugs decreased the total drug required to achieve the IC50 decreases considerably. Apoptosis rates were also affected by the simultaneous administration of bicalutamide and raloxifene. The synergistic effect of the combination was reflected in the increase of the apoptosis rate in all cell lines. Conclusions: The simultaneous administration of bicalutamide and raloxifene has a synergistic effect on cell death and apoptosis of DU145, PC3 and LNCaP cell lines. The pathway(s) responsible for this observation may be independent of the androgen receptor as both AR negative cell lines were still affected by the combination over the SERM alone. [Table: see text] No significant financial relationships to disclose.

Estrogen dependence of the renal vasodilatory effect of nicotine in rats: Role of α 7 nicotinic cholinergic receptor/eNOS signaling

Aims We recently reported that acute exposure to nicotine vasodilates the renal vasculature of male rats via facilitation of endothelial nitric oxide synthase (eNOS). In this study, we investigated whether this effect of nicotine is sexually dimorphic and the role of estrogen in modulating the nicotine effect. Main methods Nicotine-evoked vasodilation was evaluated in phenylephrine-preconstricted perfused kidneys obtained from male, proestrus female, ovariectomized (OVX) and estrogen-replaced OVX (OVXE 2) rats. Key findings Nicotine infusion (5 × 10 − 5 , 1 × 10 − 4 , and 5 × 10 − 4 M) produced greater concentration-dependent reductions in the renal perfusion pressure (RPP) in an isolated kidney from proestrus females than from males. Inhibition of NOS by N G-nitro- l-arginine abolished the nicotine-evoked reduction in RPP and abolished the gender difference in the nicotine effect. Nicotine vasodilation was also attenuated in kidneys isolated from OVX and diestrus rats, models characterized by reduced estrogen levels. Further, estrogen or l-arginine supplementation in OVX rats largely restored the renal vasodilatory response to nicotine. Estrogen receptor blockade by tamoxifen abrogated the enhanced nicotine-evoked vasodilation elicited by E 2 in OVX rats. The nitrite/nitrate levels and protein expressions of eNOS and α 7 nicotinic cholinergic receptor (α 7 nAChRs) were significantly higher in renal tissues of OVXE 2 compared with OVX rats, suggesting a facilitatory effect for E 2 on α 7 nAChRs/eNOS signaling. Significance Estrogen-dependent facilitation of NOS signaling mediates the enhanced vasodilator capacity of nicotine in the renal vasculature of female rats. Preliminary evidence also suggests a potential role for α 7 nAChRs in this estrogen-dependent phenomenon.

Estradiol-17β-Induced Temporal and Spatial Partitioning of eNOS and Estrogen Receptors in Ovine Uterine Artery Endothelial Cells from Pregnant Sheep.

Pregnancy-induced elevations in uterine blood flow are closely correlated with concurrent increases in plasma estradiol-17β levels and the expression/activity of eNOS. Indeed during late ovine gestation a physiologic cause and effect relationship has been established in vivo in that unilateral blockade of estrogen receptors (w/ICI 182,780) or nitric oxide synthase (w/L-NAME) locally and by a similar magnitude reduces uteroplacental blood flow. Estradiol-17β binds to estrogen receptors alpha and beta (ERα/β) and binding to these receptors results in either rapid effects (non-classical, non-genomic) and/or chronic effects (classical-genomic). Interestingly, numerous proteins that are involved in the estradiol-17β-induced rapid eNOS activation and nitric oxide production are located in specialized membrane domains called the caveolae of uterine artery endothelial cells (UAECs). Furthermore, treatment with the calcium mobilizing agonist ATP induced rapid eNOS activation which is accompanied by eNOS repartitioning with alterations in its multi-site phosphorylation state. We hypothesized that estradiol-17β induces eNOS activation via the non-classical ER receptor and initiates the sequential and coordinated re-partitioning and activation of eNOS with altered multi-site phosphorylation. UAECs were isolated from uterine arteries of pregnant ewes (gestational day 120-130; term 147d). Confluent passage 4 UAECs were treated with vehicle (Control) or with estradiol-17β (10 nM) for 10 min. The caveolae were fractionated from UAECs using sucrose density gradient centrifugation, and immunoblotting was utilized to study the multi-site phosphorylation state of eNOS relative to the domain specific levels of ERs. In control UAECs, total eNOS was predominantly located in the caveolar domain, whereas it was detectable both in the caveolar and non-caveolar domains in estradiol-17β-treated cells. In control UAECs, stimulatory P635eNOS and P1177eNOS were both not detected in any of the fractions whereas inhibitory P114eNOS was detected strictly in the non-caveolar domain. In response to estradiol-17β, stimulatory P635eNOS was detected in all cellular domains whereas stimulatory P1177eNOS was detected mainly in the caveolar domain and barely visible in the non-caveolar domain. The inhibitory P114eNOS was decreased by estrogen to low levels in the non-caveolar domain and was not detected in the caveolar domain. In control UAECs, ERs were detected in a higher abundance in the non-caveolar domain compared to the caveolar domain. estradiol-17β did not alter the distribution of the ERs. Estradiol-17β produces temporal and spatial re-partitioning of eNOS from the caveolar to non-caveolar domains and alters its multi-site phosphorylation state and their distributions in UAECs. Furthermore, since estradiol-17β actions are not calcium mediated, the striking similarities of these results to the ATP-responses illustrate alternative activation pathways of calcium mobilizing agonists. These findings may be critical to understand estradiol-17β-induced gestational vascular adaptations. NIH HL49210, HD38843, HL87144, HL70562, R25GM083252. (platform)

In Vivo Treatments with Fulvestrant and Anastrozole Differentially Affect Gene Expression in the Rat Efferent Ductules1

Estrogen plays a key role in maintaining the morphology and function of the efferent ductules. We previously demonstrated that the antiestrogen fulvestrant markedly affected gene expression in the rat efferent ductules. The mechanism of fulvestrant action to modulate gene expression may involve not only the blockade of ESR1 and ESR2 estrogen receptors, but also the activation of ESR1 and ESR2 when the receptors are tethered to AP-1 or SP1 transcription factors, or the activation of the G protein-coupled estrogen receptor 1. We therefore compared the effects of two strategies to interfere with estrogen action in the rat efferent ductules: treatment with fulvestrant or with the aromatase inhibitor anastrozole. Whereas fulvestrant markedly increased Mmp7 and Spp1, and reduced Nptx1 mRNA levels, no changes were observed with anastrozole. Fulvestrant caused changes in epithelial morphology that were not seen with anastrozole. Fulvestrant shifted MMP7 immunolocalization in the epithelial cells from the supranuclear to the apical region; this effect was less pronounced with anastrozole. In vitro studies of (35)S-methionine incorporation showed that protein release was increased, whereas tissue protein content in the efferent ductules of fulvestrant-treated rats was decreased. Although fulvestrant markedly affected gene expression, no changes were observed on AP-1 and SP1 DNA-binding activity. The blockade of ESRs seems to be the major reason explaining the differences between both treatments. At least some of the effects of fulvestrant appear to result from compensatory mechanisms activated by the dramatic changes caused by ESR1 blockade.

Combined Src and ER blockade impairs human breast cancer proliferation in vitro and in vivo

Antiestrogen therapies arrest susceptible estrogen receptor (ER)-positive breast cancers by increasing p27. Since Src phosphorylates p27 to promote p27 proteolysis, Src activation observed in up to 40% of ER-positive cancers may contribute to antiestrogen resistance. In this article, we show that treatment with the Src-inhibitor saracatinib (AZD0530) together with ER-blocking drugs increased breast cancer cell cycle arrest via p27. Saracatinib and fulvestrant together more effectively increased p27, reduced Ki67, and impaired MDA-MB-361 xenograft tumor growth in vivo than either of the drugs alone. In contrast, saracatinib monotherapy rapidly gave rise to drug resistance. Since combined ER and Src inhibition delays development of resistance in vivo, these data support further clinical investigation of saracatinib in combination with fulvestrant for women with ER-positive breast cancer. Proteomic analysis revealed striking bypass activation of the mTOR pathway in saracatinib-resistant tumors. mTORC1 activation also arose following long-term culture of ER-positive breast cancer lines in the presence of saracatinib. These data indicate the utility of proteomic analysis of drug-resistant tumors to identify potential means of drug resistance. The use of mTOR kinase inhibitors with saracatinib may subvert drug resistance and prove to be more effective than saracatinib alone.