Endocrine disruptors differently influence estrogen receptor ß and androgen receptor in male and female rat VSMC.

Sex steroid hormones differently control the major physiological processes in male and female organisms. In particular, their effects on vascular smooth muscle cells (VSMCs) migration are at the root of sex/gender-related differences reported in the cardiovascular system. Several exogenous substances, defined endocrine disruptor chemicals (EDCs), could interfere with these androgen and estrogen effects; however, the sex/gender-related susceptibility of VSMC motility to EDCs is completely unknown. Here, the effect of naturally occurring (naringenin, Nar) and synthetic (bisphenol A, BPA) EDCs on male and female VSMC motility has been evaluated. 17ß-estradiol (E2, 0.1 nM-1 µM) induced a dose-dependent inhibition of motility in female-derived VSMC. In contrast, neither dihydrotestosterone (DHT, 0.01-100 nM) nor the common precursor of sex steroid hormones, testosterone (Tes, 0.01-100 nM) modified male-derived VSMC motility. Estrogen receptor (ER) ß subtype-dependent activation of p38 was necessary for the E2 effect on cell motility. High BPA concentration prevented E2 effects in female-derived cells being without any effect in male-derived cells. Nar mimicked E2 effects on female-derived cells even in the presence of E2 or BPA. Intriguingly, Nar also inhibited the male-derived VSMC mobility. This latter effect was prevented by ERß inhibitor, but not by the androgen receptor (AR) inhibitor. As a whole, ERß-dependent signals in VSMC results more susceptible to the impact of EDCs than AR signals suggesting a possible high and overall susceptibility of female to EDCs. However, several male-derived cells, including VSMC, express ERß, which could also serve as target of EDC disruption in male organisms.

Cleome arabica leaf extract has anticancer properties in human cancer cells.

CONTEXT: Cleome arabica L. (Capparidaceae) is a desert plant widely distributed in the North part of Africa whose leaves are used in traditional medicine as a sedative for abdominal and rheumatic pains. OBJECTIVES: The anticancer activity of methanol Cleome arabica leaf extracts (CALE) is investigated in different human cancer cell lines. MATERIALS AND METHODS: Five different human cancer cell lines, representative of the most common cancers in Western countries (i.e., breast adenocarcinoma, colon carcinoma, neuroblastoma, hepatoma, cervix carcinoma) were treated with different concentrations of CALE (i.e., 1, 5, 10, 25, 50, 100 and 200 µg/ml). Cell viability and cell cycle were measured by using a hemocytometer chamber and a cytofluorimeter, respectively. Epidermal growth factor (EGF) was used as a positive control. Western blots were performed to evaluate the CALE effects on pathways involved in cell growth regulation and on apoptotic cascade activation. RESULTS AND CONCLUSION: Our results confirm that CALE has a high content of polyphenolic compounds (i.e., 32.21 ± 3.44%), mainly as flavonoids (24.56 ± 4.67%). In all tested cell lines CALE treatment reduces cell number in a dose-dependent manner (ED50 = 175 ± 30 µg/ml). CALE (100 and 200 µg/ml) increases by three-fold the activation of the apoptotic cascade involving caspase-3 activation and the cleavage of its substrate poly(ADP-ribose) polymerase (PARP). Intriguingly, CALE treatment (200 µg/ml) also blocks EGF-induced cell growth by preventing the growth factor-triggered AKT and ERK phosphorylation. As a whole, these data strongly suggest that CALE possesses anticancer effects in all tested cancer cell lines.

The pro-apoptotic effect of quercetin in cancer cell lines requires ERß-dependent signals.

Quercetin has potentially beneficial effects on disease prevention, including cancer. An intriguing issue regarding the mechanisms of action of quercetin is the ability of this drug to modulate estrogen receptor (ER) activities. In a previous study, we demonstrated that quercetin elicited apoptosis through an ERα-dependent mechanism. However, the contribution of ERß in quercetin-induced apoptosis remains elusive. Here, we report that quercetin, at nutritionally relevant concentrations, mimicked the 17ß-estradiol (E2)-induced apoptotic effect in both ERß1-transfected HeLa and in ERß1-containing DLD-1 colon cancer cell lines by inducing the activation of p38. p38 activation is responsible for pro-apoptotic activation of caspase-3 and the cleavage of poly(ADP-ribose) polymerase. Notably, no inactivation or downregulation of the survival kinases (i.e., AKT and ERK1/2) or the antiapoptotic protein Bcl-2 was observed after quercetin stimulation. On the contrary, quercetin acted similarly to E2 by increasing the levels of the oncosuppressor protein PTEN and by impeding ERß-dependent cyclin D1 promoter activity, which subsequently resulted in the transcription of the estrogen-responsive element remaining unchanged. As a whole, these data indicate that quercetin mimics the E2 effects in the presence of ERß1, thus maintaining its anti-carcinogenic potential. In addition, the quercetin pro-apoptotic action in the presence of ERα may render it as a dual-sided protective agent against E2-related cancer in the reduction of tumour growth in organs that express ERα and/or ERß.

Embryologic origin of endometriosis: analysis of 101 human female fetuses.

The etiology of endometriosis, a gynecological disease characterized by the presence of endometrial glands and stroma outside the uterine cavity, is still unknown. Our research group has recently demonstrated the presence of ectopic endometrium in human female fetuses at different gestational ages. In this manuscript we describe four new cases of fetal endometriosis found among a series of 52 female fetuses analyzed at autopsy. The anatomical localization of this ectopic endometrium, and its histological and immunohistochemical characteristics are depicted. We suggest that endometriosis is caused by dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis. The clinical and pathological implications of these findings are discussed.

The naringenin-induced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background.

Fruit and vegetable consumption has generally been associated with the prevention or suppression of cancer. However, food could contain a multitude of chemicals (e.g., bisphenol A; BPA) that could synergize or antagonize the effects of diet-derived compounds. Remarkably, food containers (e.g., water and infant bottles) are the largest source of exposure to BPA for human beings. Here, the effects of the coexposure of naringenin (Nar, 1.0 × 10(-9) M to 1.0 × 10(-4) M) and BPA (1.0 × 10(-5) M) in estrogen-dependent breast cancer cell lines expressing (i.e., MCF-7 and T47D) or not expressing (i.e., MDA-MB-231) estrogen receptor α (ERα) are reported. Although both Nar and BPA bind to ERα, they induce opposite effects on breast cancer cell growth. BPA induces cell proliferation, whereas Nar only decreases the number of ERα-positive cells (i.e., MCF-7 and T47D). Notably, even in the presence of BPA, Nar impairs breast cancer cell proliferation by activating caspase-3. The molecular pathways involved require p38 activation, whereas, the BPA-induced AKT activation is completely prevented by the Nar treatment. As a whole, Nar maintains its proapoptotic effects even in the presence of the food contaminant BPA, thus, enlarging the chemopreventive potential of this flavanone.

Bisphenol A impairs estradiol-induced protective effects against DLD-1 colon cancer cell growth.

Bisphenol A (BPA), a prototype of endocrine disruptors, mimics 17ß-estradiol (E2)-induced proliferation in several cancer cells by binding to estrogen receptor α (ERα). However, scarce and conflicting data are available concerning the effect of BPA on estrogen receptor ß (ERß)-mediated functions. Here, the detailed analysis of the effect of BPA, alone or in combination with E2, on ERß-mediated cellular functions is reported in ERß-expressing colon cancer cell line. BPA binds to ERß without activating any receptor activities. On the other hand, BPA inhibits E2-induced genomic activity of ERß as well as ERß extra-nuclear activities (i.e., ERß:p38 association and p38 activation). As a consequence, BPA impairs the E2-induced activation of the apoptotic cascade which is at the root of the protective role played by the hormone against colon cancer growth. Thus, women may be considered a highly susceptible population with an increased risk of colon cancers after BPA exposures.

Naringenin and 17beta-estradiol coadministration prevents hormone-induced human cancer cell growth.

Flavonoids have been described as health-promoting, disease-preventing dietary components. In vivo and in vitro experiments also support a protective effect of flavonoids to reduce the incidence of certain hormone-responsive cancers. In particular, our previous results indicate that the flavanone naringenin (Nar), decoupling estrogen receptor alpha (ERalpha) action mechanisms, drives cancer cells to apoptosis. Because these studies were conducted in the absence of the endogenous hormone 17beta-estradiol (E2), the physiological relevance of these findings is not clear. We investigate whether the antiproliferative Nar effect persists in the presence of physiological E2 concentration (i.e. 10 nM), using both ERalpha-transfected (HeLa cells) and ERalpha-containing (HepG2 cells) cancer cell lines. Ligand saturation experiments indicate that Nar decreases the binding of E2 to ERalpha without impairing the estrogen response element (ERE)-driven reporter plasmid activity. In contrast, Nar stimulation prevents E2-induced extracellular regulated kinases (ERK1/2) and AKT activation and still induces the activation of p38, the proapoptotic member of mitogen-activating protein kinase (MAPK) family. As a consequence, Nar stimulation impedes the E2-induced transcription of cyclin D1 promoter and reverts the E2-induced cell proliferation, driving cancer cell to apoptosis. Thus, these results suggest that coexposure to this low-affinity, low-potency ligand for ERalpha specifically antagonizes the E2-induced ERalpha-dependent rapid signals by reducing the effect of the endogenous hormone in promoting cellular proliferation. As a whole, these data indicate that Nar is an excellent candidate as a chemopreventive agent in E2-dependent cancers.

17ß-estradiol--a new modulator of neuroglobin levels in neurons: role in neuroprotection against H2O2-induced toxicity.

Although discovered in 2000, neuroglobin (Ngb) functions are still uncertain. A contribution to the role played by Ngb in neurons could certainly derive from the identification of Ngb endogenous modulators. Here, we evaluate the possibility that Ngb could be regulated by 17ß-estradiol (E2) signaling in both SK-N-BE human neuroblastoma cell line and mouse hippocampal neurons. 1 nM E2 rapidly induced a 300% increase in Ngb levels in both models. The E2 effect was specific, being not induced by testosterone or dihydrotestosterone. The E2-induced Ngb increase requires estrogen receptor (ER) ß, but not ERα, as evaluated by the mimetic effect of ERß-specific agonist DPN and by the blockage of E2 effect in ERß-silenced SK-N-BE cells. Furthermore, both rapid (15 min) ERß-dependent activation of p38/MAPK and transcriptional ERß activity were required for the estrogenic regulation of Ngb. Finally, E2 exerted a protective effect against H2O2-induced neuroblastoma cell death which was completely prevented in Ngb-silenced cells. Overall, these data suggest that Ngb is part of the E2 signaling mechanism that is activated to exert protective effects against H2O2-induced neurotoxicity.

17beta-Estradiol regulates the first steps of skeletal muscle cell differentiation via ER-alpha-mediated signals.

17beta-Estradiol (E(2)) mediates a wide variety of complex biological processes determining the growth and development of reproductive tract as well as nonreproductive tissues of male and female individuals. While E(2) effects on the reproductive system, bone, and cardiovascular system are quite well established, less is known about how it affects the physiology of other tissues. Skeletal muscle is a tissue that is expected to be E(2) responsive since both isoforms of estrogen receptor (ER-alpha and ER-beta) are expressed. Significant sex-related differences have been described in skeletal muscle, although the role played by E(2) and the mechanisms underlying it remain to be determined. Here, we demonstrate that E(2) increases the glucose transporter type 4 translocation at membranes as well as the expression of well-known differentiation markers of myogenesis (i.e., myogenin and myosin heavy chain) in rat myoblast cells (L6). These E(2)-induced effects require rapid extranuclear signals and the presence of ER-alpha, whereas no contribution of IGF-I receptor has been observed. In particular, ER-alpha-dependent Akt activation participates in regulating the first step of myogenic differentiation. Moreover, both receptors mediate the E(2)-induced activation of p38, which, in turn, affects the expression of myogenin and myosin heavy chain. All together, these data indicate that E(2) should be included in the list of skeletal muscle trophic factors.

The nutritional flavanone naringenin triggers antiestrogenic effects by regulating estrogen receptor alpha-palmitoylation.

Naringenin (Nar) is a component of fruits and vegetables associated with healthful benefits, such as in osteoporosis, cancer, and cardiovascular diseases. These protective effects have been linked with Nar antiestrogenic as well as estrogenic activities. Previous studies indicate that Nar impaired estrogen receptor (ER) alpha signaling by interfering with ERalpha-mediated activation of ERK and phosphoinositide 3-kinase signaling pathways in the absence of effects at the transcriptional level. The present studies evaluated the hypothesis that these Nar antagonistic effects occur at the level of the plasma membrane. Our results indicate that Nar induces ERalpha depalmitoylation faster than 17beta-estradiol, which results in receptor rapid dissociation from caveolin-1. Furthermore, Nar impedes ERalpha to bind adaptor (modulator of nongenomic actions of the ER) and signaling (c-Src) proteins involved in the activation of the mitogenic signaling cascades (i.e. ERK and phosphoinositide 3-kinase). On the other hand, Nar induces the ER-dependent, but palmitoylation-independent, activation of p38 kinase, which in turn is responsible for Nar-mediated antiproliferative effects in cancer cells. Altogether, these data highlight new ER-dependent mechanisms on the root of antiproliferative and antiestrogenic effects of Nar. Moreover, the different modulation of ERalpha palmitoylation exerted by different ligands represents a pivotal mechanism that drives cancer cell to proliferation or apoptosis.

Naringenin modulates skeletal muscle differentiation via estrogen receptor α and ß signal pathway regulation.

Several experiments sustain healthful benefits of the flavanone naringenin (Nar) against chronic diseases including its protective effects against estrogen-related cancers. These experiments encourage Nar use in replacing estrogen treatment in post-menopausal women avoiding the serious side effects ascribed to this hormone. However, at the present, scarce data are available on the impact of Nar on E2-regulated cell functions. This study was aimed at determining the impact of Nar on the estrogen receptor (ERα and ß)-dependent signals important for 17ß-estradiol (E2) effect in muscle cells (rat L6 myoblasts, mouse C2C12 myoblasts, and mouse skeletal muscle satellite cells). Dietary relevant concentration of Nar delays the appearance of skeletal muscle differentiation markers (i.e., GLUT4 translocation, myogenin, and both fetal and slow MHC isoforms) and impairs E2 effects specifically hampering ERα ability to activate AKT. Intriguingly, Nar effects are specific for E2-initiating signals because IGF-I-induced AKT activation, and myoblast differentiation markers were not affected by Nar treatment. Only 7 days after Nar stimulation, early myoblast differentiation markers (i.e., myogenin, and fetal MHC) start to be accumulated in myoblasts. On the other hand, Nar stimulation activates, via ERß, the phosphorylation of p38/MAPK involved in reducing the reactive oxygen species formation in skeletal muscle cells. As a whole, data reported here strongly sustain that although Nar action mechanisms include the impairment of ERα signals which drive muscle cells to differentiation, the effects triggered by Nar in the presence of ERß could balance this negative effect avoiding the toxic effects produced by oxidative stress .

Oral contraceptives modify DNA methylation and monocyte-derived macrophage function.

BACKGROUND: Fertile women may be encouraged to use contraception during clinical trials to avoid potential drug effects on fetuses. However, hormonal contraception interferes with pharmacokinetics and pharmacodynamics and modifies internal milieus. Macrophages depend on the milieu to which they are exposed. Therefore, we assessed whether macrophage function would be affected by the use of combined oral contraceptives (OCs) and if this influence depended on the androgenic or non-androgenic properties of progestin. METHODS: Healthy adult women were enrolled and stratified into two groups: women who did not use OCs (Fs) and women treated with OCs (FOCs). FOCs were further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins. Routine hematological, biochemical, inflammatory and endothelial dysfunction parameters were measured. Monocyte-derived macrophages (MDMs) were evaluated for the expression and activity of estrogen receptors and androgen receptors, and release of tumor necrosis factor α (TNFα) was measured from unstimulated and lipopolysaccharide-stimulated cells. RESULTS: As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased. Hormonal levels also varied: cortisol was higher in FOCs, while luteinizing hormone, follicle-stimulating hormone, and testosterone were lower in FOCs. Asymmetric dimethylarginine, an index of endothelial function, was lower in FOC than in Fs, as were cysteine and bilirubin. The androgenic properties of progestins affected the activity of OCs: in particular, white blood cell count, hemoglobin, high-density lipoprotein and calcium were higher in FOCA- than in FOCA+, whereas percentage oxygen saturation and γ-glutamyl transpeptidase were lower in FOCA- than in FOCA+. Importantly, FOCs had a lower global DNA methylation, indicating that OC may have epigenetic effects on gene expression. OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor ß, more considerably in FOCA-. Importantly, the activation state of estrogen receptor ß in FOCs was decreased, while estrogen receptor α was not active in either Fs or FOCs. Unstimulated MDMs obtained from FOCs showed higher release of TNFα in comparison with Fs. After lipopolysaccharide stimulation, the release of TNFα was significantly higher in Fs than in FOCs. CONCLUSIONS: OC use induced many changes in hematological and plasmatic markers, modifying hormonal levels, endothelial function, inflammation index and some redox state parameters, producing a perturbation of the internal milieu that impacted macrophagic function. In fact, different levels of estrogen receptor expression and release of TNFα were observed in macrophages derived from OC users. Some of the above activities were linked to the androgenic properties of progestin. Even though it is not known whether these effects are reversible, the results indicate that to avoid potential skewing of results only a single type of OC should be used during a single clinical trial.

Environmental endocrine disruptors: does a sex-related susceptibility exist?

Several substances present in the environment, now classified as endocrine disruptors (EDs), strongly interfere with both androgen and oestrogen actions in reproductive tissues. However, nowadays it is well recognized that these sex steroid hormones are more than regulators of gonadal functions. In fact, they, in synergy with genes, are responsible of sex-related differences in anatomical, physiological, and behavioral traits which characterize males and females of many vertebrate species, including humans. Thus, even if EDs are present in minute amount (part for trillion) in environment, their effects in male and female physiology could be greater than before expected also prejudicing the sex-steroid hormone-induced integrated physiological responses in women and men. In addition, differences in male and female susceptibility to EDs could be present even if scarce information on this aspect is still available. Here we have reviewed the state of the art on the sex-related susceptibility to EDs underlying the mechanism at the root of these effects.

Nutrition and human health from a sex-gender perspective.

Nutrition exerts a life-long impact on human health, and the interaction between nutrition and health has been known for centuries. The recent literature has suggested that nutrition could differently influence the health of male and female individuals. Until the last decade of the 20th century, research on women has been neglected, and the results obtained in men have been directly translated to women in both the medicine and nutrition fields. Consequently, most modern guidelines are based on studies predominantly conducted on men. However, there are many sex-gender differences that are the result of multifactorial inputs, including gene repertoires, sex steroid hormones, and environmental factors (e.g., food components). The effects of these different inputs in male and female physiology will be different in different periods of ontogenetic development as well as during pregnancy and the ovarian cycle in females, which are also age dependent. As a result, different strategies have evolved to maintain male and female body homeostasis, which, in turn, implies that there are important differences in the bioavailability, metabolism, distribution, and elimination of foods and beverages in males and females. This article will review some of these differences underlying the impact of food components on the risk of developing diseases from a sex-gender perspective.

Quercetin-induced apoptotic cascade in cancer cells: antioxidant versus estrogen receptor alpha-dependent mechanisms.

The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti-proliferative effects in many cancer cell lines. Antioxidant or pro-oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin-induced anti-proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor alpha (ERalpha; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H(2)O(2)-induced ROS production both in the presence and in the absence of ERalpha. However, this flavonoid induces the activation of p38/MAPK, leading to the pro-apoptotic caspase-3 activation and to the poly(ADP-ribose) polymerase cleavage only in the presence of ERalpha. Notably, no down-regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERalpha-dependent mechanism involving caspase- and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.