Weeks Of E2 Treatment Research Articles

Estradiol treatment increases M2-like visceral adipose tissue macrophages in obese ovariectomized mice regardless of its anorectic action

ABSTRACT Estradiol (E2) treatment has been known to induce changes in food intake, energy expenditure, and weight gain. However, its direct effects on adipose tissue macrophages (ATM) in vivo are not fully understood. Thus, we aimed to explore this aspect at cellular and molecular levels in ovariectomized obese mice. We examined the changes in ATMs after eight weeks of a high-fat diet (HFD) in male, female, and ovariectomized (OVX) mice. After eight weeks, osmotic pumps were inserted into OVX mice to provide two weeks of E2 treatment. We additionally set up a vehicle Pair-Fed (PF) control group that supplied the same amount of HFD consumed by the E2-treated group. We then investigated the in vivo phenotypic changes of visceral adipose tissue (VAT) macrophages. The percentage of M1-like ATMs decreased by the anorectic effect of E2, while M2-like ATMs increased regardless of the anorexia. E2 treatment increased the expression of anti-inflammatory genes but decreased pro-inflammatory genes in VAT. Monocyte recruitment and local proliferation contributed to M2-like ATMs. Furthermore, M2-like phenotypes were induced by E2 treatment in human macrophages. E2 treatment increases M2-like macrophages and improves the tissue milieu of VAT regardless of the anorectic reaction of E2.

Abstract 828: Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cell lines.

Abstract Models of long-term estrogen-deprived breast cancer cells are utilized in the laboratory to mimic clinical aromatase inhibitor-resistant breast cancer and serve as a tool to discover new therapeutic strategies. The MCF-7:5C and MCF-7:2A subclones are derived from the parental MCF-7 cell line through long-term estrogen deprivation (Sweeney, et al. HMBCI 2012). In addition to the different responses to antiestrogens observed between MCF-7:5C and MCF-7:2A cells, they also have different apoptotic responses to estrogen (E2); the MCF-7:5C cells undergo apoptosis and die during the first week of E2 treatment, whereas the “late-apoptosis” MCF-7:2A cells die after two weeks of E2 treatment. To discover the mechanisms by which MCF-7:2A cells survive two weeks of E2 treatment, systematic experiments were performed in this study. E2 increased the cleavages of poly (ADP-ribose) polymerase (PARP) and Caspase 9 in both cell lines within 72 hours. RT-PCR microarrays of apoptosis-related genes also showed the gene expression pattern in MCF-7:2A cells was similar to that of MCF-7:5C cells after E2 treatment. Importantly, E2 caused apoptosis in both cell lines after 72 hours of E2 treatment, detected by Annexin V binding assay through flow cytometry, but to varying degrees. The MCF-7:5C cells showed approximately double the amount of Annexin V binding (and thereby apoptosis) compared to the MCF-7:2A cells. Further experiments showed that E2 induced similar responses of the unfolded protein response (UPR), demonstrated by the markers eukaryotic initiation factor subunit alpha (eIF2α) and inositol-requiring enzyme 1 alpha (IRE1α), although MCF-7:2A cells have higher basal levels of phosphorylated eIF2α compared with MCF-7:5C. The question arises, then, of why E2 does not kill the 2A cells until the second week of treatment, when their molecular responses closely mirror the 5C cells. Our microarray database demonstrates increased superoxide dismutase 2 (SOD2) in response to E2 in MCF-7:2A but not in MCF-7:5C cells, as well as higher basal levels of glutathione. These could represent protective mechanisms in the MCF-7:2A cells to prevent E2-induced cell death. We are currently investigating whether MCF-7:2A cells are resistant to the oxidative stress induced by E2. All of these preliminary data imply that the antioxidant system may play a critical role to defend MCF-7:2A cells from apoptosis induced by E2. VCJ is supported by the Department of Defense Breast Program under Award number W81XWH-06-1-0590 Center of Excellence; subcontract under the SU2C (AACR) Grant number SU2C-AACR-DT0409; the Susan G Komen For The Cure Foundation under Award number SAC100009; GHUCCTS CTSA (Grant # UL1RR031975) and the Lombardi Comprehensive Cancer Center Support Grant (CCSG) Core Grant NIH P30 CA051008. The views and opinions of the author(s) do not reflect those of the US Army or the Department of Defense. Citation Format: Elizabeth E. Sweeney, Ping Fan, V. Craig Jordan. Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cell lines. [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 828. doi:10.1158/1538-7445.AM2013-828

The AF-1 Activation Function of Estrogen Receptor α Is Necessary and Sufficient for Uterine Epithelial Cell Proliferation In Vivo

Estrogen receptor-α (ERα) regulates gene transcription through the 2 activation functions (AFs) AF-1 and AF-2. The crucial role of ERαAF-2 was previously demonstrated for endometrial proliferative action of 17β-estradiol (E2). Here, we investigated the role of ERαAF-1 in the regulation of gene transcription and cell proliferation in the uterus. We show that acute treatment with E2 or tamoxifen, which selectively activates ERαAF-1, similarly regulate the expression of a uterine set of estrogen-dependent genes as well as epithelial cell proliferation in the uterus of wild-type mice. These effects were abrogated in mice lacking ERαAF-1 (ERαAF-1(0)). Four weeks of E2 treatment led to uterine hypertrophy and sustained luminal epithelial and stromal cell proliferation in wild-type mice, but not in ERαAF-1(0) mice. However, ERαAF-1(0) mice still presented a moderate uterine hypertrophy essentially due to a stromal edema, potentially due to the persistence of Vegf-a induction. Epithelial apoptosis is largely decreased in these ERαAF-1(0) uteri, and response to progesterone is also altered. Finally, E2-induced proliferation of an ERα-positive epithelial cancer cell line was also inhibited by overexpression of an inducible ERα isoform lacking AF-1. Altogether, these data highlight the crucial role of ERαAF-1 in the E2-induced proliferative response in vitro and in vivo. Because ERαAF-1 was previously reported to be dispensable for several E2 extrareproductive protective effects, an optimal ERα modulation could be obtained using molecules activating ERα with a minimal ERαAF-1 action.

Milk Fat Globule-EGF Factor 8 is Required for Recovery of Pancreas From Cerulein-Induced Pancreatitis

Gastric cancer (GC) is a male-predominant cancer associated with Helicobacter pylori (HP). Epidemiological studies suggest that female hormones reduce GC risk and our previous work demonstrated that ovariectomized female mice have increased GC risk. We examined the effect of 17 beta-estradiol (E2) on HP-induced gastric cancer in hypergastrinemic 8 week-old male and female INS-GAS mice inoculated with HP SS1 or vehicle-only. At 16 weeks post-infection (WPI), mice were treated with E2, Tamoxifen, E2 and Tamoxifen or placebo pellets for 12 weeks. Gastric histopathology was evaluated before and after treatment. At 28 WPI, stomachs were evaluated by HP quantitative culture, promoter CpG methylation of 24 tumor suppressors/oncogenes, and gene expression, and serum inflammatory cytokines measured by Luminex. At 16 WPI, HP infection induced robust gastric pathology in mice of both genders (P<0.001). After 12 weeks of E2 treatment, gastric pathology was significantly reduced in infected E2 males compared to infected placebo males (P<0.001). Tamoxifentreated and dual-treated infected males also showed reduction in gastric pathology compared to infected placebo males (both P<0.01) indicating that Tamoxifen may act agonistically in the stomach. In contrast, Tamoxifen treatment in females did not alter pathology in females. By microarray assessment, infected E2 males differentially expressed 412 probes compared to infected placebo males (Q<0.05). Cancer (115/412 probes) and inflammatory response (38/412) were among the top five differentially expressed biological functions using Ingenuity pathway analysis. Serum keratinocyte chemoattractant (KC) was decreased in infected E2 males and infected placebo females (P<0.05, P<0.001 respectively) compared to infected placebo males. Infected E2 males had significantly higher amounts of IL-6 and IL-10 compared to infected placebo males (P<0.01, P<0.05, respectively). MIP1a and MIP1b was significantly lower in infected placebo males compared to infected placebo females (both P<0.01) but these cytokines in infected E2 males did not differ from females. Both KC and MIP1a/b are strong neutrophil activators that were deregulated by E2 in infected males and trended toward the levels in infected females. IL-6 and IL-10 increased in infected E2 males to levels similar to that noted in infected females. Initial CpG methylation results indicate increased promoter methylation in 4 of 24 genes caused by HP infection in male mice. Studies are underway to elucidate the effect of E2 on methylation status. Our results demonstrate that E2 treatment can prevent the progression of HP-induced pathology by shifting the inflammatory response in males to one that resembles a female inflammatory response.

The effect of short-term estradiol therapy on clotting and inflammatory markers in older men receiving hormonal suppression therapy for prostate cancer

The cardio protective effect of estrogen in women has come under scrutiny as recent evidence from long-term trials has demonstrated negative findings. In contrast, the effect of endogenous sex hormones, specifically estrogen, on cardiovascular disease, inflammation and clotting parameters in men has not been well-studied. Men receiving androgen deprivation therapy for prostate cancer provide a unique model to study the effect of estrogen alone on inflammation and clotting factors. In a short-term randomized controlled trial of 17-β estradiol (E2) versus placebo, we measured sex hormones, markers of inflammation including homocysteine (HC), C-reactive protein (CRP), interleukin-6 (IL-6) and coagulation factors including fibrinogen, plasminogen activator-inhibitor-1 (PAI-1) and anti-thrombin-III (AT-III) in 27 older men without bone metastases receiving androgen deprivation therapy or neoadjuvant treatment for prostate cancer. After 9 weeks of E2 treatment, there was no difference in inflammation or clotting parameters between groups, but after 9 weeks of treatment AT-III increased in the E2 treated group and decreased in the placebo group. CRP, homocysteine and IL-6 did not show any significant differences. We also evaluated the above parameters in 12 men 3 weeks after acute steroid withdrawal with androgen deprivation therapy and found no significant changes.We found an increase in AT-III in men receiving E2 which may be related to gonadal steroid withdrawal, but no significant differences in other inflammatory or clotting factor parameters. While the current report is very preliminary in a small group of subjects, further studies are needed to determine the long-term effects of E2 in this population of hypogonadal men.

Estrogen-Induced Rat Breast Carcinogenesis is Characterized by Alterations in DNA Methylation, Histone Modifications, and Aberrant microRNA Expression

Breast cancer is the most common malignancy in women continuing to rise worldwide. Breast cancer emerges through a multi-step process, encompassing progressive changes from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma, and metastasis. In the current study, we analyzed the morphological changes and alterations of DNA methylation, histone methylation and microRNA expression during estradiol-17β (E2)-induced mammary carcinogenesis in female August Copenhagen Irish (ACI) rats. E2-induced breast carcinogenesis in ACI rats provides a physiologically relevant and genetically defined animal model for studying human sporadic breast cancer. The pattern of morphological changes in mammary glands during E2-induced carcinogenesis was characterized by transition from normal appearing alveolar and ductular hyperplasia to focal hyperplastic areas of atypical glands and ducts accompanied by a rapid and sustained loss of global DNA methylation, LINE-1 hypomethylation, loss of histone H3 lysine 9 and histone H4 lysine 20 trimethylation, and altered microRNAs expression. More importantly, these alterations in the mammary tissue occurred after 6 weeks of E2-treatment, whereas the atypical hyperplasia, which represents a putative precursor lesion to mammary carcinoma in this model, was detected only after 12 weeks of exposure, demonstrating clearly that these events are directly associated with the effects of E2 and are not a consequence of the preexisting preneoplastic lesions. The results of this study show that deregulation of cellular epigenetic processes plays a crucial role in the mechanism of E2-induced mammary carcinogenesis in ACI rats, especially in the tumor initiation process.

The regulatory effect of tamoxifen on fibronectin expression in estrogen-dependent MCF-7 breast carcinoma cells

We investigated the regulatory effect of tamoxifen (TAM) on fibronectin (FN) expression in estrogen-dependent MCF-7 breast carcinoma cells both in vitro and in vivo. in vitro, MCF-7 cells were cultured with 17beta-estradiol (E2) and/or TAM. In the animal experiment in vivo, MCF-7 tumors were grown in ovariectomized athymic mice by implanting a sustained release E2 pellet. The E2 pellets were removed after 3 weeks of E2 treatment. Animals were then divided into four groups: 1) an E2 (0.72 mg/pellet) pellet [E2(+)]; 2) an E2 and a TAM (5 mg/pellet) pellet [E2(+)TAM]; 3) no treatment [E2(-)] and 4) a TAM pellet [E2(-)TAM]. Following each treatment for 4 weeks, all animals were sacrificed and tumors were removed. Specimens, cells (in vitro) or tumors (in vivo), were homogenized and assayed for FN by Western blots. In the in vitro experiment, FN expression in MCF-7 cells decreased by incubating with 10(-9) M E2 and increased with 10(-6) M TAM. The effect of TAM increasing FN expression was inhibited by incubation accompanied with 10(-9) M E2 or 1 microg/ml transforming growth factor-beta (TGF-beta) neutralizing antibody. In the in vivo animal experiment FN expression in the tumors of E2(+) mice was lower than that of E2(-) mice. However, TAM increased FN expression in the tumors regardless of E2 pellet. These results suggest that TAM increases FN expression of MCF-7 breast carcinoma cells and that these regulatory effects of TAM on FN expression are partly mediated by TAM-induced TGF-beta.

Regulation of estrogen receptor and epidermal growth factor receptor by tamoxifen under high and low estrogen environments in MCF-7 cells grown in athymic mice.

The purpose of this study was to investigate whether tamoxifen (TAM) treatment causes a downregulation of estrogen receptor (ER) and whether TAM induces epidermal growth factor receptor-1 (EGFR). We investigated the expression of ER and EGFR after the treatment of TAM in MCF-7 tumors grown in athymic mice under high and low estrogen environments. MCF-7 tumors were grown in ovariectomized athymic mice by implanting a sustained release 17beta-estradiol (E2) pellet. The E2 pellets were removed after 3 weeks of E2 treatment. Animals were then divided into the following 4 groups: i) an E2 (0. 72 mg/pellet) pellet [E2(+)]; ii) an E2 and a TAM (5 mg/pellet) pellets [E2(+)TAM]; iii) no treatment [E2(-)]; iv) a TAM pellet [E2(-)TAM]. A significant reduction in tumor size was observed in the estrogen-depleted group [E2(-) and E2(-)TAM] compared with the estrogen-completed group [E2(+) and E2(+)TAM]. TAM inhibited estrogen-stimulated growth in the estrogen-completed mice. No additional reduction of the tumor by TAM was observed in the estrogen-depleted mice. Both ER and EGFR protein levels in the tumors of the estrogen-depleted mice were higher than in the estrogen-completed mice. Expression of ER and EGFR protein was increased by TAM in the estrogen-completed mice, however it was decreased by TAM in the estrogen-depleted mice. Changes of ER and EGFR protein levels were similar in all treatments. Transforming growth factor-alpha (TGF-alpha) in tumors, which is known as a ligand of EGFR and as an estrogen-inducible protein in ER positive MCF-7 cells, was decreased by TAM in the estrogen-completed mice, by contrast, it was increased by TAM in the estrogen-depleted mice. Downregulation of ER was observed in TAM-treated mice in an estrogen-depleted environment, this action of TAM was similar to E2. These results suggest that increase of EGFR expression does not lead to a loss of ER after short-term TAM treatment in MCF-7 tumors.

The Effect of Short-Term Treatment With Micronized Estradiol on Bone Turnover and Gonadotrophins in Older Men

Evidence for the role of estrogen in male bone metabolism has been confirmed by studies on a man with a genetic defect in the estrogen receptor as well as men with aromatase defects. All exhibited tall stature, delayed epiphysial closure, decreased bone density and increased bone turnover. Estrogen is likely to affect bone turnover in men throughout life; therefore, we hypothesized that older men would show decreased bone resorption in response to estrogen therapy. To test our hypothesis, fourteen community-dwelling men with osteopenia of the femoral neck were treated for 9 weeks with micronized estradiol, 1 mg/d, a dose which is effective in postmenopausal women. Each subject served as his own control. Markers of bone resorption, N-terminal collagen crosslinks (NTX) and C-terminal collagen crosslinks (CTX) and markers of bone formation, osteocalcin (OC) and bone specific alkaline phosphatase (BSAP) were measured every 3 weeks during a 9-week treatment period and 9 weeks post-treatment. Sex hormones, gonadotrophins and calciotropic hormones were measured at baseline, 9 weeks on treatment and 9 weeks post- treatment. After 9 weeks of treatment, estradiol and estrone levels increased significantly by greater than 6-fold and 15-fold, respectively. SHBG levels increased significantly by 17%. Testosterone and free testosterone levels decreased significantly by 27% and 34%, respectively. Markers of bone resorption showed wide variation at baseline and while on treatment. There was no correlation between changes in bone markers and changes in estrogen levels. During treatment, 11 patients showed a decrease of NTX or CTX, but three showed an increase. These three and one other subject had high initial levels of FSH and LH, suggesting some degree of primary gonadal failure, which decreased during estrogen therapy. Thus, the change in NTX (and CTX) after 9 weeks of E2 treatment was correlated with initial FSH (r= -.66, p= .01) and LH (r= -.73, p= .003) values. In addition, the largest decrease in free testosterone at 9 weeks was correlated with the higher values for NTX, CTX and BAP (r=-0.66, -0.68, -0.70 respectively; p≤.01 for each of the markers). Treatment was generally well tolerated. Side effects of treatment were minimal, and included breast tenderness and decreased libido which reversed after treatment. We conclude that it is feasible to give low dose estrogen to healthy older men, but that the effects on bone turnover are not consistent. Changes in central feedback and in endogenous sex hormone production may alter the response of bone turnover to exogenous estrogen in this population.

Estradiol regulation of proopiomelanocortin gene expression and peptide content in the hypothalamus.

Previous studies have shown that the hypothalamic concentrations of beta-endorphin (beta-EP) and other proopiomelanocortin (POMC)-derived peptides change in the female rat following castration and gonadal steroid replacement. In this study we have measured POMC mRNA by solution hybridization assay in the medial basal hypothalamus (MBH) of ovariectomized rats treated with a regimen of estradiol (E2) that we have previously shown alters brain beta-EP peptide content. In addition the effect of progesterone (P) was also studied. In the first experiment the concentration of beta-EP and alpha-melanocyte-stimulating hormone (alpha-MSH) in the MBH of castrated rats decreased significantly after 3 weeks of E2 treatment compared to castrated unreplaced rats: beta-EP decreased from 6.00 +/- 0.46 to 4.32 +/- 0.38 ng/mg protein and alpha-MSH decreased from 3.00 +/- 0.23 to 2.35 +/- 0.15 ng/mg protein (p less than 0.05). A similar decrease in peptide content was noted in the anterior hypothalamus/preoptic area. A parallel reduction in the concentration of POMC mRNA was measured in the MBH of the E2-replaced animals: 1.17 +/- 0.14 vs. 0.72 +/- 0.08 pg/microgram RNA (p less than 0.02). In a second study castrated rats were studied after 2 weeks of E2 or E2 plus P treatment. After 2 weeks, POMC peptide levels did not change significantly in the MBH of either the E2- or E2 plus P-treated rats. POMC mRNA, however, was significantly reduced from 1.10 +/- 0.10 pg/micrograms RNA in the unreplaced rats to 0.58 +/- 0.05 and 0.61 +/- 0.06 pg/microgram RNA after E2 or E2 plus P, respectively (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of estrogen receptor mRNA in normal and neoplastic rat pituitary tissues.

The effects of estradiol 17β (E2) on the regulation of estrogen receptor (ER) mRNA amounts in normal and neoplastic rat pituitary tissues were analyzed by in situ hybridization with an oligonucleotide probe. E2 treatment produced a significant increase in ER mRNA amounts in two transplantable pituitary tumors (MtT/Wl5 and MtT/F4) and in the GH3 cell line. ER mRNA amounts were also increased In normal pituitaries after 6 weeks of E2 treatment, but the differences were not significant. Biochemical assay of ER proteins confirmed the presence of ER protein in MtT/Wl 5 and MtT/F4 tumors. "Cytoplasmic" ER proteins were decreased by E2 in the MtT/Wl5 tumor.These results indicate that ER mRNA is present in normal pituitaries and in pituitary tumors and can be regulated by estrogen treatment in vivo and in vitro. The inhibitory effects of high estrogen concentration on proliferation of transplantable pituitary tumors in vivo and GH3 cells in vitro is not explained by the absence of ER mRNAs in these tumors.

Persistence of low hypothalamic dopaminergic activity after removal of chronic estrogen treatment.

The purpose of this study was to determine whether inhibition of tuberoinfundibular dopaminergic (TIDA) neuron function which occurs during chronic estrogen administration persists after removal of the estrogen. Ovariectomized (OVX) Fischer 344 (F344) rats were implanted for 4 weeks with a Silastic capsule containing estradiol-17 beta (E2) and controls with an empty capsule for 4 weeks. Other rats which received E2 for 4 weeks had the capsule removed and experiments performed 4 weeks later. At the end of 4 weeks of E2 treatment, anterior pituitary (AP) weight was increased sixfold, serum prolactin (PRL) 65-fold, and AP DNA content fivefold over OVX control rats. Four weeks after removal of E2, AP weight, serum PRL, and AP DNA content declined, but remained significantly above OVX control values. At the end of 4 weeks of E2 treatment and after E2 withdrawal, release of [3H]dopamine (DA) from median eminence (ME) tissue superfused in vitro was lower than from ME of OVX control rats although [3H]DA accumulation was not significantly different among the treatment groups. Administration of apomorphine (APO), a dopamine agonist, significantly reduced plasma prolactin levels in OVX control rats, in rats at the end of 4 weeks E2 treatment, and in rats after 4 weeks of E2 withdrawal. Injection of haloperidol (HALO) produced similar increases in plasma PRL/estimated PRL-cell DNA in OVX controls, at the end of E2 treatment or after E2 withdrawal. However, injection of morphine (MOR), a drug which increases the release of PRL by inhibiting hypothalamic dopaminergic activity, resulted in a rise in plasma PRL/estimated PRL-cell DNA in OVX control rats that was significantly greater compared to rats at the end of E2 treatment or after E2 withdrawal. Since rats treated with E2 released less [3H]DA from ME tissue in vitro, and were less responsive to MOR, it can be that animals treated for 4 weeks with E2 show a decreased ability to release DA from TIDA neurons which persists even after termination of E2 treatment. These results suggest that chronic high circulating E2 levels result in a depression of TIDA neuronal activity which is sustained after E2 is removed.