Uterine Weight Gain Research Articles

THE CHEMICAL COMPOSITION OF PARABEN AND ITS EFFECTS ON HEALTH

Parabens are a group of alkyl esters of p-hydroxybenzoic acid and usually include methyl paraben, ethyl paraben, propyl paraben, butyl paraben, isobutyl paraben, isopropyl paraben and benzyl paraben. Parabens (or their salts) are widely used as preservatives in cosmetics and medicine due to their relatively low toxicity and long history of safe use. Testing of parabens has shown, to varying degrees, that individual paraben compounds have weak estrogenic activity in some in vitro screening tests, such as estrogen receptor ligand binding, regulation of CAT gene expression, and proliferation of MCF-7 cells. Reported in vivo effects include uterine weight gain (eg, butyl, isobutyl, and benzyl parabens) and male reproductive tract effects (eg, butyl and propyl parabens). However, in relation to estrogen as a control during in vivo studies, parabens are much less active than estrogen. While exposure to sufficient doses of exogenous estrogens can increase the risk of certain adverse effects, it is hypothesized that similar risks may arise from exposure to much weaker endocrine-active chemicals (EACs) is still speculation. In evaluating the possibility that exposure to weakly active EACs may be associated with adverse effects due to their endocrine mode of action, it is important to consider both the dose and potency of these compounds compared to estrogen. In this review, a comparative approach involving dose and potency is used to assess whether in utero or adult exposure to parabens may be associated with adverse effects due to their estrogen-modulating mode of action. Keywords: parabens, p-Hydroxybenzoates, PHBA, paraben applications, Methyl paraben.

Protective Effects of Resveratrol Against Adenomyosis in a Mouse Model.

Adenomyosis is a uterine condition in which endometrial glands and stroma are commonly pathologically observed in the myometrium. In this study, we sought to determine the effect of resveratrol on the progression of adenomyosis. Adenomyosis was induced in mice given tamoxifen neonatally. All mice were subjected to body weight measurement and hotplate testing every four weeks beginning four weeks after birth. All mice with adenomyosis were randomly separated into 3 groups at 16weeks: untreated, low-dose resveratrol (25mg/kg), and high-dose resveratrol (50mg/kg). After 3weeks of treatment, final hotplate test and body weight measurement were performed, and the uterine horn blood samples were collected. Adenomyosis in mice caused body weight loss and uterine weight gain, reduced hotplate latency, and progression of endometrial fibrosis. The underlying biological process could be coupled with the overexpression of many cells' proliferation and immune-regulation-related genes. Resveratrol treatment could slow the progression of adenomyosis by enhancing hotplate latency, lowering endometrial fibrosis, and restoring cell proliferation- and immune-regulation-associated gene expression levels in endometrium and plasma. However, resveratrol treatment also reduced the body weight and uterine weight. In conclusion, Resveratrol might be a potential compound for treating patients with adenomyosis.

The Ethanolic Extract of Gomphrena celosioides Mart. Does Not Alter Reproductive Performance or Embryo-Fetal Development, nor Does It Cause Chromosomal Damage.

Gomphrena celosioides is a native Brazilian plant found in the State of Mato Grosso do Sul. It is used in folk medicine to treat kidney diseases, skin diseases, infections, rheumatism, gastrointestinal diseases, and respiratory diseases. It is also used as an abortifacient. To evaluate the effects of the ethanolic extract of Gomphrena celosioides (EEGc) on reproductive performance, embryo development, and chromosome stability, Swiss mice were randomly divided into experimental groups (n = 10). The animals in the control group received the vehicle Tween 80-1% in the proportion of 0.1 mL/10 g of body weight orally, from the first to the 18th gestational day. The animals in the treatment groups received the EEGc (100, 1000, and 2000 mg/kg) from the first to the 18th gestational day. The animals underwent evaluations of their reproductive performance and embryofetal development. The results showed that the EEGc did not change the animals' final weight, weight gain, uterine weight, or net weight gain. The evaluation showed that the absolute and relative organs' weights did not vary between the different experimental groups. In addition, the EEGc did not change the numbers of implants, live fetuses, dead fetuses, or fetal resorptions. There were no differences in post-operative loss rates, implantations, or resorptions, nor were there differences in fetal viability or sex ratio. The use of the EEGc did not result in different frequencies of malformations. In addition, the EEGc did not alter the frequency of chromosomal damage or frequency of micronuclei. Based on our findings, we considered the extract of Gomphrena celosioides to be safe for use during pregnancy, although some parameters indicated caution in its use.

Two Synthetic Peptides Corresponding to the Human Follicle-Stimulating Hormone β-Subunit Promoted Reproductive Functions in Mice.

A follicle stimulating hormone (FSH) is widely used in the assisted reproduction and a synthetic peptide corresponding to a receptor binding region of the human (h) FSH-β-(34–37) (TRDL) modulated reproduction. Furthermore, a 13-amino acid sequence corresponding to hFSH-β-(37–49) (LVYKDPARPKIQK) was recently identified as the receptor binding site. We hypothesized that the synthetic peptides corresponding to hFSH-β-(37–49) and hFSH-β-(34–49), created by merging hFSH-β-(34–37) and hFSH-β-(37–49), modulate the reproductive functions, with the longer peptide being more biologically active. In male or female prepubertal mice, a single injection of 200 μg/g BW ip of hFSH-β-(37–49) or hFSH-β-(34–49) hastened (p < 0.05) puberty, whereas the same treatments given daily for 4 d promoted (p < 0.05) the gonadal steroidogenesis and gamete formation. In addition of either peptide to the in vitro cell cultures, promoted (p < 0.05) the proliferation of primary murine granulosa cells and the estradiol production by upregulating the expression of Ccnd2 and Cyp19a1, respectively. In adult female mice, 200 μg/g BW ip of either peptide during diestrus antagonized the FSH-stimulated estradiol increase and uterine weight gain during proestrus. Furthermore, hFSH-β-(34–49) was a more potent (p < 0.05) reproductive modulator than hFSH-β-(37–49), both in vivo and in vitro. We concluded that hFSH-β-(37–49) and especially hFSH-β-(34–49), have the potential for reproductive modulation.

Ameliorating Effect of the Edible Mushroom Hericium erinaceus on Depressive-Like Behavior in Ovariectomized Rats.

Estrogen deficiency during menopause causes a variety of neurological symptoms, including depression. The edible Lion's Mane mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (HE), is a medicinal mushroom that has the potential for a neuroprotective effect and ameliorating neurological diseases, such as depression, anxiety, and neurodegenerative diseases. HE contains phytoestrogens, including daidzein and genistein. However, the ameliorating effect of HE on menopausal symptoms is not well understood. Here we investigated the impact of methanol extract of the HE fruiting body on depressive-like behavior in postmenopausal model rats. The activation of estrogen receptor alpha (ERα) causes body weight loss and uterine weight gain. Body weight gain and uterine weight loss by estrogen deficiency in ovariectomized (OVX) rats were reversed with 17β-estradiol (E2) but not with HE. Thus, the phytoestrogens in HE may hardly activate ERα. Estrogen receptor beta (ERβ) is expressed in the brain, and activation of ERβ ameliorates menopausal depressive symptoms. Notably, depressive-like behavior in OVX rats evaluated in forced swim test was reduced by administration of not only E2 but also HE for 92 d. Long-term activation of ERα increases the risk of breast and uterine cancers. HE, therefore, may be effective in treating menopausal depression without the risk of carcinogenesis caused by ERα activation.

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17β-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

Acacetin improves endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats by estrogen receptors

The aim of the work was to investigate the effects of acacetin on endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats and explore its mechanism. Seven-week-old male spontaneously hypertensive rats (SHR) were selected to establish a rat model of hypertension with insulin resistance induced by 10% fructose. The nuclear factor kappa B p65 (NF-κB p65) and Collagen I were observed by Immunohistochemistry. Immunofluorescence was used to observe estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ), and G protein-coupled receptor 30 (GPR30). Western blotting was used to detect interleukin (IL-1β), Arginase 2 (ARG2), Nostrin, endothelial nitric oxide synthase (eNOS), TGF-β, Smad3, ERK pathway proteins such as p-c-Raf, p-MEK1/2, p-ERK, ERK, p-P90RSK and p-MSK1. We found that acacetin did have an improvement on endothelial dysfunction and fibrosis. Meanwhile, it was also found to have a significant effect on the level of estrogen in this model by accident. Then, the experiment of uterine weight gain in mice confirmed that acacetin had a certain estrogen-like effect in vivo and played its role through the estrogen receptors pathway. In vitro experience HUVEC cells were stimulated with 30 mM/L glucose and 100 mM/L NaCl for 24 h to establish the endothelial cell injury model. HUVEC cells were treated with 1 μM/L estrogen receptors antagonist (ICI 182780) for 30 min before administration. Cell experiments showed that acacetin could reduce the apoptosis of HUVEC cells, the levels of inflammatory cytokines and the expression of TGF-β, Collagen I and Smad3 in endothelial cell injury model. After treatment with ICI 182780, the improvement of acacetin was significantly reversed. The results showed that acacetin relieved endothelial dysfunction and reduced the aortic fibrosis in insulin-resistant SHR rats by reducing the release of inflammatory factors and improving vasodilatory function through estrogen signaling pathway.

Abstract 4376: OP-1250, a complete estrogen receptor antagonist (CERAN) that shrinks estrogen receptor positive tumors and exhibits favorable pharmacokinetics

Abstract Fulvestrant (FASLODEX), a Complete Estrogen Receptor ANtagonist (CERAN) is recognized as the most effective endocrine therapy for estrogen receptor positive (ER+) metastatic breast cancer. Unfortunately, fulvestrant must be injected i.m. and has poor drug exposure. Thus, an oral compound that shares fulvestrant's drug virtues with improved drug exposure might be both more effective and more convenient than fulvestrant. This realization has sparked intensive efforts to develop such a compound, however most efforts have focused on discovering novel selective estrogen receptor degraders (SERDS) most of which have also displayed agonist activities on gene expression and uterus, and are therefore are also selective estrogen receptor modulators (SERMs). We predict that CERANs that fully block the activities of estrogen receptor alpha (ERalpha) will have a clinical advantage over SERM/SERDs in shrinking breast tumors as well as preventing and treating endocrine resistance. We have developed a small molecule, OP-1250 that, like fulvestrant, is a CERAN that completely turns off both transcriptional activation functions of ERalpha, as can be seen in a uterine cell assay, and also prevents uterine weight gain in ovariectomized mice. In addition, it blocks degrades ERalpha and blocks the proliferation of a large variety of ER+ breast cells. Using RNAseq, we found many estrogen-regulated biomarkers that are also regulated by SERM/SERDs, however CERANS such as OP-1250 and fulvestrant have no, or even an inverse effect on many such genes. Unlike fulvestrant, OP-1250 gives high and stable oral drug exposure in multiple species. Consequentially, OP-1250 effectively shrinks ER+ tumors in multiple human tumor xenografts in mice at very low daily doses of 3 mg/kg, even in models containing activating ERalpha mutant receptors. Importantly, the pharmacokinetics and biodistribution of OP-1250 has a favorable profile that results in unusual potency of OP-1250 in vivo. Citation Format: Leslie Hodges-Gallagher, Cyrus L. Harmon, Richard Sun, David C. Myles, Peter Kushner. OP-1250, a complete estrogen receptor antagonist (CERAN) that shrinks estrogen receptor positive tumors and exhibits favorable pharmacokinetics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4376.

Raw and salt-processed Achyranthes bidentata attenuate LPS-induced acute kidney injury by inhibiting ROS and apoptosis via an estrogen-like pathway

BackgroundTraditional Chinese medicine suggests that Radix Achyranthis Bidentatae nourishes and protects the kidneys, the effect of which is enhanced following a salt treatment. Raw and salt-processed Achyranthes bidentata are produced via different processing techniques from the same crude Achyranthes root. The anti-inflammatory and immunomodulatory properties of this plant have been verified earlier. However, there is a scarcity of experimental evidence for the renal-protective effects. AimThe purpose of present study is to compare the protective effects of raw and salt-processed Achyranthes on lipopolysaccharide (LPS) - induced acute kidney injury in mice and chemically characterize their extracts. MethodThe monomer components of raw and salt-processed Achyranthes extracts were analyzed using high performance liquid chromatography (HPLC). The aggregation and distribution of 2-Deoxy-D-glucose (2-DG) near infrared fluorescence probe in mice was examined with a small animal imaging systems. The pathological and morphological changes of kidneys were observed by H&E staining, and the serum urea nitrogen (BUN) and serum creatinine (Scr) levels were used to evaluate the renal function. The levels of cytokines in serum were detected by cytometric bead array. Flow cytometry assay was performed to assess the apoptosis and reactive oxygen species (ROS) in the kidney cells, and cell surface marker expression including CD45+, F4/80+, and Ly-6G+. The estrogenic activities of the raw and salt-processed Achyranthes were observed by uterine weight gain test in sexually immature mice. Western blot was used to detect the protein expression levels in the kidney. ResultsChemical analysis showed that the salt-processed Achyranthes contained more ginsenoside Ro and chikusetsusaponin Ⅳa than the raw Achyranthes, but there was no difference in the contents of β-ecdysterone, 25R-inokosterone, and 25S-inokosterone.in vivo near-infrared fluorescence imaging showed a significant reduced inflammation in the AKI mice. Histological studies showed that the raw and salt-processed Achyranthes markedly decreased the inflammatory infiltration, swelling and vacuolar degeneration in renal tissues and the Scr and BUN. Importantly, the raw and salt-processed Achyranthes extracts demonstrated different degrees of inhibition on the LPS-induced AKI, with salt-processed Achyranthes showing better inhibition. Results of flow cytometry showed a significant inhibition of IFN-γ, TNF-α, and IL-2, and promoted IL-10, along with reduced macrophages (CD45 + F4/80+), neutrophils (CD45+ Ly-6G+) and phagocytes. Furthermore, the extracts reduced the accumulation of ROS and apoptosis in the kidney, and also regulated the expression of apoptosis marker proteins TLR4, Bcl-2, Bax, cleaved caspase 3 and cleaved caspase 9 levels. Notably, they increased ERα, ERβ, and GPR30 in the renal tissues of AKI mice and LPS non-treated mice. In the subsequent experiments, it was found that the raw and salt-processed Achyranthes extracts increased the uterine coefficient in sexually immature mice, improved the LPS-induced decrease in NRK52e cell viability, and reduced the apoptosis, which could be antagonized by ICI182, 780 (estrogen receptor-unspecific antagonist, Faslodex). ConclusionsThe renal-protective effect of raw and salt-processed Achyranthes was exhibited through antiapoptotic and antioxidant mechanisms via an estrogen-like pathway, along with a modulation of the inflammatory response by regulating immune cells. Ginsenoside Ro and Chikusetsu saponin IVa were found to be the key factors to enhance the protective effect of salt-processed Achyranthes.

Uterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein.

Background:Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects.Objectives:Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects.Methods:Female mice were exposed to GEN on postnatal days (PND)1–5 and uterine tissues collected on PND5, PND22–26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators.Results:GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including Foxa2, Wnt4, and Sox17. These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of Foxa2, Wnt4, and Sox17, as well as genes important for neonatal uterine differentiation (Wnt7a, Hoxa10, and Msx2), were severely disrupted on PND5 in GEN-exposed mice.Discussion:Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants. https://doi.org/10.1289/EHP6336

Abstract P5-05-02: Preclinical development of OP-1250, an oral complete estrogen receptor antagonist (CERAN) that shrinks ER-positive breast tumors in xenograft models

Abstract Fulvestrant (FASLODEX) is the most effective endocrine therapy for estrogen receptor positive (ER+) metastatic breast cancer (MBC). Despite this superiority, fulvestrant must be delivered by intramuscular injection and its efficacy appears limited by poor drug exposure. Thus, an oral compound that shares fulvestrant’s pharmacodynamic virtues with improved drug exposure might be both more convenient and more effective than fulvestrant. Fulvestrant development was aimed at creating a Complete Estrogen Receptor ANtagonist (CERAN) using the rodent uterine weight gain assay. It is now understood that the ability of fulvestrant to shut off both the AF1 and AF2 transcriptional activation functions of the ER underlies this activity. In contrast to CERANs, selective estrogen receptor modulators (SERMs) such as tamoxifen efficiently shut off AF2 signaling (turned on by estradiol), but incompletely antagonize AF1, a function that is turned on by multiple cell signaling pathways and shown to play a role in the development of endocrine resistance. We have developed a sensitive cell culture assay that distinguish between CERANs and SERMs. Fulvestrant is also a Selective Estrogen Receptor Degrader (SERD), and it has become widely but mistakenly assumed that such ability makes a compound a CERAN. In our assay several SERDs under clinical investigation, including AZD9496 and GDC-0810, were identified as SERMs that exhibited ER-mediated agonism on genes dependent on AF1 activity. Further, these SERM/SERDs stimulate cell proliferation of CAMA-1 and HCC-1500 breast cancer cells even in the absence of estrogen. These proliferation assays may model clinical breast cancer progression in which tumors become less dependent on estrogen and more sensitive to AF1 signaling mediated by SRC family kinases, IGFs, and FGFs. We thus predict that SERM/SERDs will be inferior in preventing and treating resistance compared to CERAN/SERDs. Guided by our cell culture assay, we have developed the CERAN/SERD OP-1250. OP-1250 completely blocks both AF2 and AF1 activity as seen in cell culture and the ovex mouse uterine weight gain assay. Unlike SERM/SERDs, OP-1250 is a potent inhibitor of CAMA1 and HCC-1500 breast cancer cell proliferation. OP-1250 also is a SERD in a large variety of cell lines including those in which some SERM/SERDs have little activity. OP-1250 achieves high and stable drug exposure in multiple animal species unlike fulvestrant and many SERMs. As might be expected for a compound with this spectrum of activities, OP-1250 shrinks tumors in xenograft models in which fulvestrant (because of limited drug exposure) and SERM/SERDs (because of residual estrogen-like action) fail to shrink the tumors. These models include HCC-1500 xenografts with a wild type ER and HCI-013 patient-derived xenografts with ESR1Y537S. These results suggest that OP-1250 has potential to be a superior compound to treat ER+ MBC, especially in patients whose tumors allow high activity of AF1 and including those with activating mutations ins ESR1. We expect to bring OP-1250 into the clinic shortly. Citation Format: Leslie Hodges-Gallagher, Richard Sun, David C Myles, Cyrus L Harmon, Peter J Kushner. Preclinical development of OP-1250, an oral complete estrogen receptor antagonist (CERAN) that shrinks ER-positive breast tumors in xenograft models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-02.

N-acetylcysteine and alpha-lipoic acid improve antioxidant defenses and decrease oxidative stress, inflammation and serum lipid levels in ovariectomized rats via estrogen-independent mechanisms

Sexual hormone deficiency has been associated with metabolic changes, oxidative stress and subclinical inflammation in postmenopausal women. Hormone replacement therapies are effective in many instances, even though some patients either do not respond or are not eligible. The aim of this study was to evaluate the impact of short- (15 days) versus long-term (60 days) sexual hormone depletion and whether antioxidant supplementation with N-acetylcysteine (NAC) and alpha-lipoic acid (LA) improves oxidative stress, metabolic, and inflammatory parameters in ovariectomized (OVX) rats. Short-term OVX rapidly depleted circulating estrogen, causing uterine atrophy and body weight gain without affecting oxidative damage, inflammatory and lipid metabolism markers. In contrast, long-term OVX augmented oxidative damage in serum and peripheral tissues as well as increased serum total cholesterol, TNF-α and IL6 levels. Triglycerides, glucose and HDL cholesterol were not altered. Long-term OVX-induced oxidative stress was associated with depletion of GSH and total non-enzymatic antioxidants as well as decreased activity of Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), but not Superoxide Dismutase (SOD) and Catalase (CAT). NAC and LA supplementation prevented GSH and total non-enzymatic antioxidants depletion as well as restored GPx and GR activities, TNF-α, IL6 and cholesterol in OVX rats. NAC and LA effects appear to be independent on NRF2 activation and estrogen-like activity, since NAC/LA did not promote NRF2 activation and were not able to emulate estrogen effects in OVX rats and estrogen-receptor-positive cells. The herein presented data suggest that NAC and LA may improve some deleterious effects of sexual hormone depletion via estrogen-independent mechanisms.

Estrogenic Effects of the Extracts from the Chinese Yam (Dioscorea opposite Thunb.) and Its Effective Compounds in Vitro and in Vivo.

Background: The aim of this study was to explore the estrogenic effects of the extracts from Chinese yam and its effective compounds. Methods: The activity of the yam was investigated by the uterine weight gain of mice and a proliferation assay of breast cancer cell lines (MCF-7 cell); the estrogenic activity was comprehensively evaluated by a serum pharmacology experiment. The levels of estradiol (E2), follicle stimulating hormone (FSH), and luteinizing hormone (LH) were also measured. Western blot analysis and antagonist assays with faslodex (ICI182,780), methylpiperidino-pyrazole (MPP), Delta (9) –tetrahydrocannabinol (THC), and G-15 were used to explore the mechanism of the effects of the yam. To find the effective compounds of the yam which play a role in its estrogen-like effects, we used the same methods to study the effects of adenosine and arbutin. Results: The Chinese yam and two main compounds, adenosine and arbutin, have estrogen-like effects. The mechanism of the yam which plays a role in its estrogen-like effects was mainly mediated by the estrogen receptors ERα, ERβ, and GPR30; that of adenosine was mainly mediated by estrogen receptors ERα and ERβ, and that of arbutin was mainly mediated by estrogen receptors ERβ and GPR30. Conclusions: The Chinese yam has estrogen-like effects; adenosine and arbutin are two of the effective compounds in the yam which play a role in its estrogen-like effects.

Novel oestrogen receptor \u03b2-selective ligand reduces obesity and depressive-like behaviour in ovariectomized mice

Hormonal changes due to menopause can cause various health problems including weight gain and depressive symptoms. Multiple lines of evidence indicate that oestrogen receptors (ERs) play a major role in postmenopausal obesity and depression. However, little is known regarding the ER subtype-specific effects on obesity and depressive symptoms. To delineate potential effects of ERβ activation in postmenopausal women, we investigated the effects of a novel oestrogen receptor β-selective ligand (C-1) in ovariectomized mice. Uterine weight, depressive behaviour, and weight gain were examined in sham-operated control mice and ovariectomized mice administered placebo, C-1, or 17β-oestradiol (E2). Administration of C-1 or E2 reduced body weight gain and depressive-like behaviour in ovariectomized mice, as assessed by the forced swim test. In addition, administration of E2 to ovariectomized mice increased uterine weight, but administration of C-1 did not result in a significant increase in uterine weight. These results suggest that the selective activation of ERβ in ovariectomized mice may have protective effects against obesity and depressive-like behaviour without causing an increase in uterine weight. The present findings raise the possibility of the application of ERβ-ligands such as C-1 as a novel treatment for obesity and depression in postmenopausal women.

Novel selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer.

Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17β-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2.

Steroid hormone intervenes in the endometrial tumorigenesis of pten ablation.

Background:Endometrial cancer, the most common gynecological cancer, is closely associated with endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations. Phosphatase and tensin homologue (PTEN) is a tumor suppressor genes completely lost or mutated in >50% of primary endometrioid endometrial cancers. Estrogen-dependent endometrioid carcinoma is the most common type of endometrial cancer. Progesterone is a hormone that antagonizes the growth-promoting properties of estrogen in the uterus. Progestin is used as a conservative endocrine treatment of early endometrial cancer in order to preserve fertility as well as a palliative measure for advanced-stage patients. Progesterone therapy has been shown to be effective in preventing endometrial cancer as well as controlling growth of the endometrium. However, the effectiveness of progestin for women with endometrial cancer is less clear.Methods:In order to understand the effect of steroid hormone on endometrial cancer progression, we used a mouse endometrial cancer model with conditional loss of Pten in the mouse uterus (PRcre/+ Ptenf/f, Ptend/d). To assess the effect of steroid hormones, ovariectomized Ptenf/f and Ptend/d mice were treated with estrogen or progesterone over a period of three month.Results:Uterine weight gain was significantly decreased in ovariectomized PRcre/+ Ptenf/f mice compared to intact PRcre/+ Ptenf/f mice. Ovariectomized PRcre/+ Ptenf/f mice treated with P4 or vehicle also exhibited decreased uterine cancer size compared with intact PRcre/+ Ptenf/f mice. Proliferation of ovariectomized PRcre/+ Ptenf/f mice treated with P4 is highly decreased compared to other groups. The levels of stromal progesterone receptor were highly increased in ovariectomized PRcre/+ Ptenf/f mice treated with P4 which resulted in decreased epithelial proliferation.Conclusions:These results suggest that P4 treatment significantly reduces tumor mass but does not affect cancer progression in PRcre/+ Ptenf/f mice.

Agouti-related peptide plays a critical role in leptin's effects on female puberty and reproduction

Deficient leptin signaling causes infertility via reduced activity of GnRH neurons, causing a hypogonadal state in both rodents and humans. Because GnRH neurons do not express leptin receptors, leptin's effect on GnRH neurons must be indirect. Neurons within the hypothalamic arcuate nucleus that coexpress AGRP and NPY are considered to be important intermediate neurons involved in leptin regulation of GnRH neurons. Previously, we reported that the absence of AGRP and haploinsufficiency of MC4R in leptin receptor mutant (Lepr(db/db)) females result in restoration of fertility and lactation despite the persistence of obesity and insulin resistance. The overarching hypothesis in the present study is that the absence or reduction of leptin's inhibition of AGRP/NPY neurons leads to suppression of GnRH release in cases of leptin signaling deficiency. Since TAC2 (NKB)-TAC3R signaling plays a role in puberty maturation and is modulated by metabolic status, the other aim of this study is to test whether TAC2/NKB neurons in ARC regulated by melanocortinergic signals herein affect leptin's action on puberty and reproduction. Our data showed that AGRP deficiency in Lepr(db/db) females restores normal timing of vaginal opening and estrous cycling, although uterine weight gain and mammary gland development are morphologically delayed. Nonetheless, Agrp(-/-) Lepr(db/db) females are fertile and sustain adequate nutrition of pups with lactation to weaning age. AGRP deficiency results in advanced vaginal opening in wild-type female mice. The postpubertal increase in hypothalamic TAC2 mRNA was not observed in Lepr(db/db) females, whereas AGRP deficiency restored it in Lepr(db/db) females. Additionally, MC4R activation with MTII induced FOS expression in TAC2 neurons, supporting the concept of melanocortinergic regulation of TAC2 neurons. These studies suggest that AGRP imposes an inhibitory effect on puberty and that TAC2 neurons may transmit melanocortinergic inhibition of GnRH neurons.

Abstract 197: Reduction in lobular but not ductal hyperplasia by the PGR inhibitor ORG33628 in a CYP19A1 overexpressing mouse model of breast cancer risk.

Abstract Background: The Women's Health Initiative demonstrated increased breast cancer risk in women exposed to estrogen with progesterone. Genetically engineered mice with conditional mammary epithelial cell targeted Cytochrome P45019A1 (CYP19A1/aromatase) over-expression (tet-op-CYP19A1/MMTV-rtTA mice) tested the impact of a selective progesterone receptor (PGR) inhibitor, ORG33628, on mammary hyperplasia and cancer development. This model demonstrates increased percentages of PGR+ mammary epithelial cells (MECs) cells at the hyperplastic stage and triple negative (Estrogen Receptor (ER)-, PGR-, Receptor tyrosine-protein kinase erbB-2 (HER2/neu)-) adenocarcinomas. Purpose: Test if ORG33628 reduces mammary hyperplasia and blocks cancer formation in tet-op-CYP19A1/MMTV-rtTA mice. Methods: Female tet-op-CYP19A1/MMTV-rtTA mice were implanted subcutaneously with a pellet containing either ORG33628 7.5mg (60-day release) or placebo at age 10m. A smaller study tested ORG33628 in CYP19A1/MMTV-rtTA/trp53+/- mice. Mice were necropsied at age 12m and mammary tissue examined to determine if ORG33628 altered lobular/ductal hyperplasia prevalence or blocked progression to hyperplastic alveolar nodules (HANs) or cancer. Immunohistochemistry (IHC) was employed to compare MEC proliferative index (PI) (Ki-67) and percentage of MECs expressing nuclear-localized ER/PGR. An uterotropic assay tested if ORG33628 blocked estrogen-induced changes in the uterus/cervix. Ovariectomized 6w old mice were implanted subcutaneously with pellets of 0.72g 17β-estradiol and either ORG33628 or placebo, necropsied 3 days later, and uterine weight, PI and ER/PGR expression compared in uterine and cervical epithelium and stroma. Results: ORG33628 treatment reduced lobular hyperplasia prevalence (Fisher's exact, p&amp;lt;0.05) and percentages of PGR+ MEC (9.3% to 5.2%, t-test, p&amp;lt;0.05), but did not alter ductal hyperplasia or HAN prevalence or block cancer formation. ORG33628 reduced estrogen-induced uterine weight gain (t-test, p&amp;lt;0.05) and PI in cervical epithelium (38.6% to 15%, t-test, p&amp;lt;0.05) in the uterotropic assay. Conclusions: ORG33628 reduced lobular but not ductal hyperplasia and did not prevent progression to HANs or cancer. In normal development, estrogen plays the more important role in ductal development and progesterone in lobular development paralleling the isolated impact of ORG33628 on lobular disease here. These results suggest that reducing ductal hyperplasia may be a prerequisite for cancer prevention or that reducing lobular hyperplasia at 10m in this model is too late due to the presence of triple negative cancer progenitor cells. Support: P30CA051008, RO1CA112176, T32CA009686-15, KG080359. Content solely responsibility of authors and does not necessarily represent official NCI/NIH views. Citation Format: Sarah A. Dabydeen, Weisheng Wang, Edgar S. Diaz-Cruz, Levy Kopelovich, Robert I. Glazer, Priscilla A. Furth. Reduction in lobular but not ductal hyperplasia by the PGR inhibitor ORG33628 in a CYP19A1 overexpressing mouse model of breast cancer risk. [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 197. doi:10.1158/1538-7445.AM2013-197

The effects of Sutaehwan-Gami on menopausal symptoms induced by ovariectomy in rats

BackgroundThis study was undertaken to evaluate the beneficial effects of a modified prescription of Sutaehwan named Sutaehwan-Gami (SG), created by adding Rhizoma dioscoreae and Carthami semen to Sutaehwan, on menopausal symptoms.MethodsTo evaluate the estrogenic effect of SG, we first examined estrogen receptor (ER) activation by SG treatment in breast adenocarcinoma cells and confirmed the estrogenic effect of SG in vivo ovariectomized rats. The animals were randomized into four groups: Sham operated group (Sham), saline treated ovariectomized group (OVX), SG treated group (SG) and raloxifene treated group (RLX). Animals were provided with SG at a dose of 500 mg/kg bw/day and RLX at a dose of 5.4 mg/kg bw/day with standard rat pellets for 3 months.ResultsSG significantly increased ERα phosphorylation, and its downstream effectors, extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) phosphorylation in breast adenocarcinoma cells. Treatment with SG reversed ovariectomy-induced uterine weight reduction and weight gain. Decreases in the levels of GOT and GPT were observed in the SG group. The significantly reduced E2β level in OVX rats was raised by treatment with SG. Moreover, SG significantly increased the phosphorylation levels of ERK and Akt in the uterus.ConclusionTaken together, these data indicate that SG has phytoestrogen-like properties through ERK and Akt activation, implying that it could be protective and beneficial for the management of menopausal symptoms.

Conditional Ablation of the Progesterone Receptor Specifically in the Uterine Epithelium Demonstrates Its Necessary Role in the Regulation of Uterine Function.

Uterine progesterone receptor (PR), expressed in both the epithelium and stroma, mediates epithelial-stromal crosstalk during embryo implantation and pregnancy. Ex vivo tissue reconstitution studies have pointed to stromal PR as being critical for the regulation of uterine epithelial gene expression (i.e., Indian hedgehog (Ihh)) and inhibition of mitogenic estrogen (E2) action whereas the role of epithelial PR has been largely unexplored. Here, we investigated the vivo role of epithelial PR during pregnancy and in the regulation of epithelial gene expression using compartment-specific ablation of PR. Using the Wnt7a-Cre mouse model, PR was ablated specifically in the uterine epithelium. This ablation rendered female mice infertile, demonstrating that epithelial PR is critical for female fertility. We examined uterine function by assessing the ability of these mice to undergo the decidual response. The epithelial PR conditional knockout mice failed to display a decidual response indicating that, despite the presence of PR in the stroma, decidualization is unable to occur without epithelial PR. We next wanted to determine if epithelial PR may regulate the expression of progesterone (P4)-target genes. Epithelial PR is critical for P4-induction of epithelial P4-target genes (Ihh, Areg, Gata2, Cyp26a1), but not stromal P4-target genes (Il13ra2). Transfection analysis revealed that epithelial PR can transactivate Ihh gene expression in Hec1a cells further confirming its critical role in P4-target gene expression. Therefore, epithelial PR plays a critical role in epithelial P4-target gene expression which differs from previous observations using tissue reconstitution experiments. In the uterus, P4 inhibits E2 actions such as uterine weight gain, epithelial proliferation, and E2-target gene expression. Normal P4-inhibition of estrogen-induced weight gain was observed in these mice. However, examination of P4-inhibition of specific E2-target genes demonstrated that epithelial PR plays a gene-specific role in P4-inhibition exhibiting inhibition of Ltf and Clca3 expression, but not Lif expression. We are currently studying its potential role in P4-inhibition of E2-induced epithelial proliferation. In summary, using an in vivo mouse model, we have shown that epithelial PR is necessary for female fertility as a mediator of decidualization, P4-target gene expression, and the P4-inhibition of E2 action. This work was supported by NIH Grant RO1CA077530 (to J.P.L.) and R01HD042311 (to F.J.D.). (platform)