Coumestrol induces oxidative stress and impairs migration and embryonic growth.

In brief: Healthy development of the placenta is dependent on trophoblast cell migration and reduced oxidative stress presence. This article describes how a phytoestrogen found in spinach and soy causes impaired placental development during pregnancy. Abstract: Although vegetarianism has grown in popularity, especially among pregnant women, the effects of phytoestrogens in placentation lack understanding. Factors such as cellular oxidative stress and hypoxia and external factors including cigarette smoke, phytoestrogens, and dietary supplements can regulate placental development. The isoflavone phytoestrogen coumestrol was identified in spinach and soy and was found to not cross the fetal-placental barrier. Since coumestrol could be a valuable supplement or potent toxin during pregnancy, we sought to examine its role in trophoblast cell function and placentation in murine pregnancy. After treating trophoblast cells (HTR8/SVneo) with coumestrol and performing an RNA microarray, we determined 3079 genes were significantly changed with the top differentially changed pathways related to the oxidative stress response, cell cycle regulation, cell migration, and angiogenesis. Upon treatment with coumestrol, trophoblast cells exhibited reduced migration and proliferation. Additionally, we observed increased reactive oxygen species accumulation with coumestrol administration. We then examined the role of coumestrol within an in vivo pregnancy by treating wildtype pregnant mice with coumestrol or vehicle from day 0 to 12.5 of gestation. Upon euthanasia, fetal and placental weights were significantly decreased in coumestrol-treated animals with the placenta exhibiting a proportional decrease with no obvious changes in morphology. Therefore, we conclude that coumestrol impairs trophoblast cell migration and proliferation, causes accumulation of reactive oxygen species, and reduces fetal and placental weights in murine pregnancy.

Genistein and coumestrol reduce MCF-7 breast cancer cell viability and inhibit markers of preferential metastasis, bone matrix attachment and tumor-induced osteoclastogenesis.

The propensity of breast cancer to preferentially metastasize to the skeleton is well known. Once established in bone metastatic breast cancers have a poor prognosis due to their ability to promote extensive bone loss which augments tumor burden. Unfortunately, current anti-resorptive therapies for skeletal metastasis are typically prescribed after secondary tumors have formed and are palliative in nature. One group of compounds with the potential to reduce both tumor burden and osteolysis are phytoestrogens (PE), but the mechanisms mediating a beneficial effect are unclear. Therefore, the current study examined the effect of genistein and coumestrol alone or in combination on breast cancer cell number, expression of mediators of preferential skeletal metastasis, bone matrix attachment and tumor-induced osteoclast formation. Results showed that genistein and coumestrol significantly reduced viable cell number in an estrogen receptor dependent manner (p < 0.05), whereas combinations of PE had no effect. In addition, genistein and coumestrol significantly reduced expression of genes driving epithelial to mesenchymal transition (snail), bone attachment (CXCR4 and integrin αV) and osteolysis (PTHrP and TNF-α). In keeping with this genistein and coumestrol significantly suppressed attachment of breast cancer cells to bone matrix and inhibited tumor and RANKL-induced osteoclast formation. Our data suggests that phytoestrogens not only decrease breast cancer cell viability but also antagonize essential tumor bone interactions that establish and drive the progression of skeletal metastasis.

Protective Effects of Coumestrol on Metabolic Dysfunction and Its Estrogen Receptor-Mediated Action in Ovariectomized Mice.

Coumestrol, a phytoestrogen compound found in various plants, has been shown to act as a potent estrogen receptor (ER) agonist, with a higher binding affinity for ERß than for ERα. However, there is currently limited information regarding its beneficial effects in postmenopausal disorders and its ER-mediated mechanisms. Herein, we investigated the effects of coumestrol (subcutaneous or oral treatment) on metabolic dysfunction in ovariectomized (OVX) mice fed a high-fat diet, in comparison with the effects of 17ß-estradiol (E2) replacement. Coumestrol was administered daily at a dose of 5 mg/kg for 10 weeks. Coumestrol treatment through the subcutaneous route stimulated uterine growth in OVX mice at a level lower than that of E2. E2 and coumestrol prevented body fat accumulation, adipocyte hypertrophy, and hepatic steatosis, and enhanced voluntary physical activity. Coumestrol showed estrogen-mimetic effects in the regulation of the protein expressions involved in browning of white fat and insulin signaling, including increased hepatic expression of fibroblast growth factor 21. Importantly, the metabolic effects of coumestrol (oral administration at 10 mg/kg for 7 weeks) were mostly abolished following co-treatment with an ERß-selective antagonist but not with an ERα-selective antagonist, indicating that the metabolic actions of coumestrol in OVX mice are primarily mediated by ERß. These findings provide important insights into the beneficial effects of coumestrol as a phytoestrogen supplement for the prevention and treatment of postmenopausal symptoms.

Phytoestrogen Coumestrol Selectively Inhibits Monoamine Oxidase-A and Amyloid ß Self-Aggregation.

Pueraria lobata leaves contain a variety of phytoestrogens, including flavonoids, isoflavonoids, and coumestan derivatives. In this study, we aimed to identify the active ingredients of P. lobata leaves and to elucidate their function in monoamine oxidase (MAO) activation and Aß self-aggregation using in vitro and in silico approaches. To the best of our knowledge, this is the first study to elucidate coumestrol as a selective and competitive MAO-A inhibitor. We identified that coumestrol, a coumestan-derivative, exhibited a selective inhibitory effect against MAO-A (IC50 = 1.99 ± 0.68 µM), a key target protein for depression. In a kinetics analysis with 0.5 µg MAO-A, 40-160 µM substrate, and 25 °C reaction conditions, coumestrol acts as a competitive MAO-A inhibitor with an inhibition constant of 1.32 µM. During an in silico molecular docking analysis, coumestrol formed hydrogen bonds with FAD and pi-pi bonds with hydrophobic residues at the active site of the enzyme. Moreover, based on thioflavin-T-based fluorometric assays, we elucidated that coumestrol effectively prevented self-aggregation of amyloid beta (Aß), which induces an inflammatory response in the central nervous system (CNS) and is a major cause of Alzheimer's disease (AD). Therefore, coumestrol could be used as a CNS drug to prevent diseases such as depression and AD by the inhibition of MAO-A and Aß self-aggregation.

Coumestrol mitigates retinal cell inflammation, apoptosis, and oxidative stress in a rat model of diabetic retinopathy via activation of SIRT1.

Diabetes-induced oxidative stress is vital in initiating neuronal damage in the diabetic retina, leading to diabetic retinopathy (DR). This study investigates the possible effects of coumestrol (CMS) on streptozotocin (STZ)-induced DR. First, we established a rat model of DR by STZ injection and a cell model involving high-glucose (HG) exposure of human retinal microvascular endothelial cells (hRMECs). We characterized the expression patterns of oxidative stress indicators, pro-inflammatory cytokines, and pro-apoptotic proteins in hRMECs. Polymerase chain reaction showed sirtuin 1 (SIRT1) to be poorly expressed in the retinal tissues of STZ-treated rats and HG-exposed hRMECs, but its expression was upregulated upon treatment with CMS treatment. Furthermore, CMS treatment attenuated the STZ-induced pathologies such as oxidative stress, inflammation, and cell apoptosis. Consistent with the in vivo results, CMS activated the expression of SIRT1, thereby inhibiting oxidative stress, inflammation, and apoptosis of HG-treated hRMECs. From these findings, we concluded that CMS ameliorated DR by inhibiting inflammation, apoptosis and oxidative stress through activation of SIRT1.

Coumestrol ameliorates doxorubicin-induced cardiotoxicity via activating AMPKα.

Doxorubicin (DOX) acts as the cornerstone in multiple tumour chemotherapy regimens, however, its clinical application is often impeded due to the induction of a severe cardiotoxicity that eventually provokes left ventricular dysfunction and congestive heart failure. Coumestrol (CMT) is a common dietary phytoestrogen with pleiotropic pharmacological effects. The present study aims to investigate the role and mechanism of CMT on DOX-induced cardiotoxicity. Mice were intragastrically administrated with CMT (5 mg/kg/day) for consecutive 2 weeks and then received a single intraperitoneal injection of DOX (15 mg/kg) to mimic the clinical toxic effects after 8-day additional feeding. To verify the role of 5' AMP-activated protein kinase alpha (AMPKα), AMPKα2 global knockout mice were used. H9C2 cells were cultured to further validate the beneficial role of CMT in vitro. CMT administration notably ameliorated oxidative damage, cell apoptosis and cardiac dysfunction in DOX-treated mice. Besides, we observed that DOX-induced reactive oxygen species overproduction and cardiomyocyte apoptosis were also reduced by CMT incubation in H9C2 cells. Mechanistically, CMT activated AMPKα and Ampkα deficiency abolished the beneficial effects of CMT in vivo and in vitro. Finally, we proved that protein kinase A (PKA) was required for CMT-mediated AMPKα activation and cardioprotective effects. CMT activated PKA/AMPKα pathway to alleviate DOX-induced oxidative damage, cell apoptosis and cardiac dysfunction. Our findings provide a promising therapeutic agent for cancer patients receiving anthracycline chemotherapy.

Evaluation of phytoestrogens in inducing cell death mediated by decreasing Annexin A1 in Annexin A1-knockdown leukemia cells.

BACKGROUND: Phytoestrogens are plant compounds that are structurally similar to estrogen and that possess anti-cancer properties. Previous studies have reported that coumestrol, daidzein and genistein could induce cell death by reducing Annexin A1 protein in leukemic cell lines. Annexin A1 (ANXA1) is involved in cell progression, metastasis, and apoptosis in several types of cancer cells. The present study sought to investigate if the effects of phytoestrogens on apoptosis, cell cycle arrest and phagocytosis in ANXA1-knockdown leukemic cells are mediated through ANXA1 or occurred independently. METHODS: Transfection of ANXA1 siRNA was conducted to downregulate ANXA1 expression in Jurkat, K562 and U937 cells. Apoptosis and cell cycle assays were conducted using flow cytometry. Western blot was performed to evaluate ANXA1, caspases and Bcl-2 proteins expression. Phagocytosis was determined using hematoxylin and eosin staining. RESULTS: The expression of ANXA1 after the knockdown was significantly downregulated in all cell lines. Genistein significantly induced apoptosis associated with an upregulation of procaspase-3, -9, and - 1 in Jurkat cells. The Bcl-2 expression showed no significant difference in Jurkat, K562 and U937 cells. Treatment with phytoestrogens increased procaspase-1 expression in Jurkat and U937 cells while no changes were detected in K562 cells. Flow cytometry analysis demonstrated that after ANXA1 knockdown, coumestrol and genistein caused cell cycle arrest at G2/M phase in selected type of cells. The percentage of phagocytosis and phagocytosis index increased after the treatment with phytoestrogens in all cell lines. CONCLUSION: Phytoestrogens induced cell death in ANXA1-knockdown leukemia cells, mediated by Annexin A1 proteins. Graphical abstract.

Antiobesity effects of coumestrol through expansion and activation of brown adipose tissue metabolism.

Coumestrol is a dietary phytoestrogen with estrogen-mimicking characteristics. This study investigated the molecular mechanisms of antiobesity effects of coumestrol. Two weeks of coumestrol treatment reduced body weight and improved glucose tolerance of high-fat diet (HFD)-fed mice. Notably, coumestrol treatment reduced adiposity but expanded brown adipose tissue mass. In addition, coumestrol treatment induced up-regulation of brown adipocyte markers and lipolytic gene expression in adipose tissue. Mechanistically, coumestrol induced an increase in mitochondrial contents of brown adipose tissue, which was associated with up-regulation of adenosine monophosphate-activated protein kinase and sirtuin 1. In vitro knockdown of estrogen receptor 1 inhibited the effect of coumestrol on brown adipogenic marker expression, increase in mitochondrial contents and oxygen consumption rate in brown adipocytes. Furthermore, lineage tracing of platelet-derived growth factor receptor A-positive (PDGFRA+) adipocyte progenitors confirmed increased levels of de novo brown adipogenesis from PDGFRA+ cells by coumestrol treatment. In conclusion, our results indicate that coumestrol has antiobesity effects through the expansion and activation of brown adipose tissue metabolism.

Phytoestrogen coumestrol attenuates brain mitochondrial dysfunction and long-term cognitive deficits following neonatal hypoxia-ischemia.

INTRODUCTION: Neonatal Hypoxia-Ischemia (HI) is a major cause of morbidity and mortality, and is frequently associated with short and long-term neurologic and cognitive impairments. The HI injury causes mitochondrial damage leading to increased production of reactive oxygen species (ROS). Phytoestrogens are non-steroidal plant substances structurally and functionally similar to estrogen. Coumestrol is a potent isoflavonoid with a protective effect against ischemic brain damage in adult rats. Our aim was to determine if coumestrol treatment following neonatal HI attenuates the long-term cognitive deficits induced by neonatal HI, as well as to investigate one possible mechanism underlying its potential effect. METHODS: On the 7th postnatal day, male Wistar rats were submitted to the Levine-Rice HI model. Intraperitoneal injections of 20 mg/kg of coumestrol, or vehicle, were administered immediately pre-hypoxia or 3 h post-hypoxia. At 12 h after HI the mitochondrial status and ROS levels were determined. At 60th postnatal day the cognitive deficits were revealed in the Morris water maze reference and working spatial memories. Following behavioral analysis, histological assessment was performed and reactive astrogliosis was measured by GFAP expression. RESULTS: Results demonstrate that both pre- and post-HI administration of coumestrol were able to counteract the long-term cognitive and morphological impairments caused by HI, as well as to block the late reactive astrogliosis. The pre-HI administration of coumestrol was able to prevent the early mitochondrial dysfunction in the hippocampus of injured rat pups. CONCLUSION: Present data suggest that coumestrol exerts protection against experimental neonatal brain hypoxia-ischemia through, at least in part, early modulation of mitochondrial function.

Phytoestrogens Weaken the Blood-Milk Barrier in Lactating Mammary Epithelial Cells by Affecting Tight Junctions and Cell Viability.

During lactation, mammary epithelial cells (MECs) form the blood-milk barrier by less-permeable tight junctions (TJs) to prevent the leakage of milk components. Phytoestrogens affect the proliferation, differentiation, and apoptosis of MECs. However, it remains unclear whether phytoestrogens are involved in the blood-milk barrier. Therefore, we investigated the influence of phytoestrogens (coumestrol, genistein, and daidzein) by using an in vitro mouse-MEC-culture model. The results showed that coumestrol and genistein changed the expression of TJ proteins (claudins-3 and -4 and occludin), weakened barrier function, and reduced ß-casein production. Daidzein also weakened barrier function without inhibiting ß-casein production. Additionally, coumestrol and genistein induced apoptosis in MECs. These results indicate that phytoestrogens weaken the blood-milk barrier by directly affecting TJs and the cellular viability of lactating MECs in different ways.

Coumestrol induces mitochondrial dysfunction by stimulating ROS production and calcium ion influx into mitochondria in human placental choriocarcinoma cells.

STUDY QUESTION: Does coumestrol inhibit proliferation of human placental choriocarcinoma cells? SUMMARY ANSWER: Coumestrol promotes cell death in the choriocarcinoma cells by regulating ERK1/2 MAPK and JNK MAPK signaling pathways and through disruption of Ca2+ and ROS homeostasis. WHAT IS KNOWN ALREADY: A number of patients who suffer from choriocarcinomas fail to survive due to delayed diagnosis or a recurrent tumor and resistance to traditional chemotherapy using platinum-based agents and methotrexate. To overcome these limitations, it is important to discover novel compounds which have no adverse effects yet can inhibit the expression of a target molecule to develop, as a novel therapeutic for prevention and/or treatment of choriocarcinomas. STUDY DESIGN, SIZE, DURATION: Effects of coumestrol on human placental choriocarcinoma cell lines, JAR and JEG3, were assessed in diverse assays in a dose- and time-dependent manner. PARTICIPCANTS/MATERIALS, SETTING, METHODS: Effects of coumestrol on cell proliferation, apoptosis (annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of reactive oxygen species (ROS), lipid peroxidation, glutathione levels and endoplasmic reticulum (ER) stress proteins in JAR and JEG3 cells were determined. Signal transduction pathways in JAR and JEG3 cells in response to coumestrol were determined by western blot analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Results of the present study indicated that coumestrol suppressed proliferation and increased apoptosis in JAR and JEG3 cells by inducing pro-apoptotic proteins, Bax and Bak. In addition, coumestrol increased ROS production, as well as lipid peroxidation and glutathione levels in JAR and JEG3 cells. Moreover, coumestrol-induced depolarization of mitochondrial membrane potential (MMP) and increased cytosolic and mitochondrial Ca2+ levels in JAR and JEG3 cells. Consistent with those results, treatment of JAR and JEG3 cells with a Ca2+ chelator and an inhibitor of IP3 receptor decreased coumestrol-induced depolarization of MMP and increased proliferation in JAR and JEG3 cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: A lack of in vivo animal studies is a major limitation of this research. The effectiveness of coumestrol to induce apoptosis of human placental choriocarcinoma cells requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that coumestrol induces apoptotic effects on placental choriocarcinoma cells by regulating cell signaling and mitochondrial-mediated functions, with a potential to impair progression of the cancer. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.).

Antioxidant dietary approach in treatment of fatty liver: New insights and updates.

Non-alcoholic fatty liver disease (NAFLD) is a common clinicopathological condition, encompassing a range of conditions caused by lipid deposition within liver cells. To date, no approved drugs are available for the treatment of NAFLD, despite the fact that it represents a serious and growing clinical problem in the Western world. Identification of the molecular mechanisms leading to NAFLD-related fat accumulation, mitochondrial dysfunction and oxidative balance impairment facilitates the development of specific interventions aimed at preventing the progression of hepatic steatosis. In this review, we focus our attention on the role of dysfunctions in mitochondrial bioenergetics in the pathogenesis of fatty liver. Major data from the literature about the mitochondrial targeting of some antioxidant molecules as a potential treatment for hepatic steatosis are described and critically analysed. There is ample evidence of the positive effects of several classes of antioxidants, such as polyphenols (i.e., resveratrol, quercetin, coumestrol, anthocyanins, epigallocatechin gallate and curcumin), carotenoids (i.e., lycopene, astaxanthin and fucoxanthin) and glucosinolates (i.e., glucoraphanin, sulforaphane, sinigrin and allyl-isothiocyanate), on the reversion of fatty liver. Although the mechanism of action is not yet fully elucidated, in some cases an indirect interaction with mitochondrial metabolism is expected. We believe that such knowledge will eventually translate into the development of novel therapeutic approaches for fatty liver.

Isoflavones and their metabolites influence the milk component synthesis ability of mammary epithelial cells through prolactin/STAT5 signaling.

SCOPE: Isoflavones are a class of polyphonic compounds present in legumes and are called phytoestrogens because of their estrogen-like activity. Estrogen influences the behavior of mammary epithelial cells (MECs) during pregnancy and lactation. In this study, we investigated the direct influences of isoflavones and their metabolites in milk production ability of MECs. METHODS AND RESULTS: Mouse MECs were cultured with prolactin and dexamethasone (glucocorticoid analog) to induce milk production ability. Subsequently, lactating MECs were treated with each isoflavone. Coumestrol, biochanin A, genistein, and formononetin decreased the intracellular and secreted ß-casein. On the other hand, p-ethylphenol, daidzein, and equol did not significantly influence ß-casein production at any concentration. Coumestrol, biochanin A and genistein down-regulated the mRNA expression of whey acidic protein (WAP), lactoferrin and α-lactalbumin. In contrast, p-ethylphenol, daidzein and equol up-regulated ß-casein and/or WAP with α-lactalbumin. Furthermore, coumestrol and genistein down-regulated the expression of prolactin receptor and signal transducer and activator of transcription 5 (STAT5) accompanied by a decrease in STAT5 phosphorylation. CONCLUSION: Isoflavones and their metabolites influence the milk production ability of MECs through different interactions with prolactin/STAT5 signaling. Simultaneous intake of multiple isoflavones by consumption of legumes may induce promotive or adverse effects on lactating MECs.

Coumestrol Down-Regulates Melanin Production in Melan-a Murine Melanocytes through Degradation of Tyrosinase.

Pigmentation reflects skin darkening caused by melanin production, but excessive melanin synthesis may cause problems, such as melasma, solar lentigo, dark spots, and freckles. Considerable effort has been devoted to alleviating these undesired symptoms through the development of safe and effective depigmenting agents. Coumestrol, a plant-derived natural isoflavone with an estrogen-like structure and actions, is known to have anti-aging ability, but its potential depigmenting efficacy has not been evaluated. In the present study, we investigated the effects of coumestrol on melanin synthesis in normal melan-a murine melanocytes. Coumestrol significantly reduced melanin synthesis in a concentration-dependent manner up to a concentration of 25 µM without causing cytotoxicity. It also brightened tissue in an artificial skin model (MelanoDerm) that incorporates both human keratinocytes and melanocytes. Interestingly, although coumestrol did not inhibit tyrosinase activity or transcript level in melan-a cells, it clearly decreased the expression level of tyrosinase protein at a concentration of 25 µM. This coumestrol-induced reduction in tyrosinase protein levels was prevented by pretreatment with the proteasome inhibitor MG-132 or the lysosomal proteolysis inhibitor chloroquine. Collectively, our findings indicate that coumestrol exerts an inhibitory effect on melanin synthesis in melan-a cells, at least in part, through degradation of tyrosinase. These findings suggest that coumestrol is a good candidate for use in depigmentary reagents from a cosmetic and clinical perspective.

Coumestrol Counteracts Interleukin-1ß-Induced Catabolic Effects by Suppressing Inflammation in Primary Rat Chondrocytes.

In the present study, we investigated the anti-catabolic effects of coumestrol, a phytoestrogen derived from herbal plants, against interleukin-1ß-induced cartilage degeneration in primary rat chondrocytes and articular cartilage. Coumestrol did not affect the viability of human normal oral keratinocytes and primary rat chondrocytes treated for 24 h and 21 days, respectively. Although coumestrol did not significantly increase the proteoglycan contents in long-term culture, it abolished the interleukin-1ß-induced loss of proteoglycans in primary rat chondrocytes and knee articular cartilage. Furthermore, coumestrol suppressed the expression of matrix-degrading enzymes such as matrix metalloproteinase-13, -3, and -1 in primary rat chondrocytes stimulated with interleukin-1ß. Moreover, the expression of catabolic factors such as nitric oxide synthase, cyclooxygenase-2, prostaglandin E2, and inflammatory cytokines in interleukin-1ß-stimulated primary rat chondrocytes was suppressed by coumestrol. In summary, these results indicate that coumestrol counteracts the catabolic effects induced by interleukin-1ß through the suppression of inflammation. Therefore, based on its biological activity and safety profile, coumestrol could be used as a potential anti-catabolic biomaterial for osteoarthritis.

Cytotoxic activity of soy phytoestrogen coumestrol against human breast cancer MCF-7 cells: Insights into the molecular mechanism.

Coumestrol is a phytoestrogen present in soybean products and recognized as potential cancer therapeutic agent against breast cancer. However, the clear molecular mechanism of anticancer-activity of coumestrol in breast carcinoma has not been reported. It is well established that copper levels are elevated in different malignancies. Therefore, the objective of this study was to investigate the copper-dependent cytotoxic action of coumestrol in human breast cancer MCF-7 cells. Results showed that coumestrol inhibited proliferation and induced apoptosis in MCF-7 cells, which was prevented by copper chelator neocuproine and ROS scavengers. Coumestrol treatment induced ROS generation coupled to DNA fragmentation, up-regulation of p53/p21, cell cycle arrest at G1/S phase, mitochondrial membrane depolarization and caspases 9/3 activation. All these effects were suppressed by ROS scavengers and neocuproine. These results suggest that coumestrol targets elevated copper for redox cycling to generate ROS leading to DNA fragmentation. DNA damage leads to p53 up-regulation which directs the cell cycle arrest at G1/S phase and promotes caspase-dependent apoptosis of MCF-7 cells. In conclusion, copper targeted ROS-mediated p53-dependent mechanism better explains the cytotoxic action of coumestrol in MCF-7 cells. Thus, targeting elevated copper levels might be a potential therapeutic strategy for selective cytotoxic action against malignant cells.

Effects of neonatal treatment with two phytoestrogens on male rat sexual behavior and partner preference.

The aim of this work was to compare the effect of neonatal treatment with the phytoestrogens coumestrol (COU) and genistein (GEN), administered in equimolecular doses, on the sexual behavior and partner preference of male rats. Four groups of male rats were injected daily from day 1 to 5 with 150 µg of GEN, an equivalent amount of COU, 1 µg of ß-estradiol 3-benzoato (EB), or olive oil (VEH) (control). A fifth group remained intact. In the GEN group, intromission and ejaculation latencies decreased, whereas ejaculatory frequency increased. Contrasting results were observed in COU males. EB males could not ejaculate and their mount and intromission latencies increased significantly. To determine sexual-partner preferences, a multiple partner preference arena was used and two types of tests were performed, the first one without allowing contact test (CT) with the stimulus animals, followed by a CT. COU and GEN groups did not show preference for any stimulus animal, whereas the EB males preferred the expert male. When CT with the stimulus animals was allowed, GEN-males preferred the receptive female, unlike the COU and EB groups. It is concluded that neonatal treatment with COU and GEN induced opposite effects, the effects of COU being more estrogenic.

Coumestrol inhibits autoantibody production through modulating Th1 response in experimental autoimmune thyroiditis.

Coumestrol is a common phytoestrogen found in plants and Chinese medicinal herbs. Its influences on experimental autoimmune thyroiditis (EAT) were investigated in this study. Female adult CBA/J mice were fed with drinking water containing 1% Tween80 only (Control group), 0.8 mg/l (L group) and 8 mg/l coumestrol (H group) from 6 to 15 weeks of age, respectively. Their serum coumestrol concentrations were determined by high performance liquid chromatography, which were undetectable, 43.70 ± 21.74 ng/ml and 135.07 ± 70.40 ng/ml, respectively. In addition, the mice (n = 14-16/group) were immunized twice with thyroglobulin (Tg) and Freund's adjuvant to induce EAT during the meantime. Although no overt changes in the extent of intrathyroidal mononuclear cell infiltration were shown in the two coumestrol-treated groups as compared with the controls, serum anti-Tg IgG2a, IgG3 and IgG1 titers, ratio of IgG2a to IgG1 and the percentage of T helper (Th)1 cells in the splenocytes were significantly reduced in the L group. Another consistent change was the significantly decreased expression of splenic IFN-γ mRNA after low dose of coumestrol exposure. Uterine weight was also markedly reduced in the mice of L group. These findings suggest that coumestrol treatment may have some beneficial actions against thyroid-specific autoantibody production in the development of autoimmune thyroiditis through suppression of Th1 response due to its anti-estrogenic activity.

Coumestrol suppresses proliferation of ES2 human epithelial ovarian cancer cells.

Coumestrol, which is predominantly found in soybean products as a phytoestrogen, has cancer preventive activities in estrogen-responsive carcinomas. However, effects and molecular targets of coumestrol have not been reported for epithelial ovarian cancer (EOC). In the present study, we demonstrated that coumestrol inhibited viability and invasion and induced apoptosis of ES2 (clear cell-/serous carcinoma origin) cells. In addition, immunoreactive PCNA and ERBB2, markers of proliferation of ovarian carcinoma, were attenuated in their expression in coumestrol-induced death of ES2 cells. Phosphorylation of AKT, p70S6K, ERK1/2, JNK1/2, and p90RSK was inactivated by coumestrol treatment in a dose- and time-dependent manner as determined in western blot analyses. Moreover, PI3K inhibitors enhanced effects of coumestrol to decrease phosphorylation of AKT, p70S6K, S6, and ERK1/2. Furthermore, coumestrol has strong cancer preventive effects as compared to other conventional chemotherapeutics on proliferation of ES2 cells. In conclusion, coumestrol exerts chemotherapeutic effects via PI3K and ERK1/2 MAPK pathways and is a potentially novel treatment regimen with enhanced chemoprevention activities against progression of EOC.

Multiple phytoestrogens inhibit cell growth and confer cytoprotection by inducing manganese superoxide dismutase expression.

Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. As data on phytoestrogens continues to accumulate, it is clear that there is significant overlap in the cellular effects elicited by these various compounds. Here, we show that one mechanism by which a number of phytoestrogens achieve their growth inhibitory and cytoprotective effects is via induction of the mitochondrial manganese superoxide dismutase (MnSOD). Eight phytoestrogens, including resveratrol, coumestrol, kaempferol, genistein, daidzein, apigenin, isoliquirtigenin and glycitin, were tested for their ability to induce MnSOD expression in mouse C2C12 and primary myoblasts. Five of these, resveratrol, coumestrol, kaempferol, genistein and daidzein, significantly increased MnSOD expression, slowed proliferative growth and enhanced stress resistance (hydrogen peroxide LD50) . When siRNA was used to prevent the MnSOD induction by genistein, coumestrol or daidzein, none of these compounds exerted any effect on proliferative growth, and only the effect of coumestrol on stress resistance persisted. The estrogen antagonist ICI182780 prevented the increased MnSOD expression and also the changes in cell growth and stress resistance, indicating that these effects are mediated by estrogen receptors (ER). The absence of effects of resveratrol or coumestrol, but not genistein, in ERß-null cells further indicated that this ER in particular is important in mediating these effects. Thus, an ER-mediated induction of MnSOD expression appears to underlie the growth inhibitory and cytoprotective activities of multiple phytoestrogens.