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1.  Naringenin modulates skeletal muscle differentiation via estrogen receptor α and β signal pathway regulation 
Genes & Nutrition  2014;9(5):425.
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 β)-dependent signals important for 17β-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 ERβ, 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 ERβ could balance this negative effect avoiding the toxic effects produced by oxidative stress .
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-014-0425-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s12263-014-0425-3
PMCID: PMC4172642  PMID: 25156241
Estrogen; Estrogen receptors; Naringenin; Signal transduction; Skeletal muscle differentiation
2.  Lysosomal Function Is Involved in 17β-Estradiol-Induced Estrogen Receptor α Degradation and Cell Proliferation 
PLoS ONE  2014;9(4):e94880.
The homeostatic control of the cellular proteome steady-state is dependent either on the 26S proteasome activity or on the lysosome function. The sex hormone 17β-estradiol (E2) controls a plethora of biological functions by binding to the estrogen receptor α (ERα), which is both a nuclear ligand-activated transcription factor and also an extrinsic plasma membrane receptor. Regulation of E2-induced physiological functions (e.g., cell proliferation) requires the synergistic activation of both transcription of estrogen responsive element (ERE)-containing genes and rapid extra-nuclear phosphorylation of many different signalling kinases (e.g., ERK/MAPK; PI3K/AKT). Although E2 controls ERα intracellular content and activity via the 26S proteasome-mediated degradation, biochemical and microscopy-based evidence suggests a possible cross-talk among lysosomes and ERα activities. Here, we studied the putative localization of endogenous ERα to lysosomes and the role played by lysosomal function in ERα signalling. By using confocal microscopy and biochemical assays, we report that ERα localizes to lysosomes and to endosomes in an E2-dependent manner. Moreover, the inhibition of lysosomal function obtained by chloroquine demonstrates that, in addition to 26S proteasome-mediated receptor elimination, lysosome-based degradation also contributes to the E2-dependent ERα breakdown. Remarkably, the lysosome function is further involved in those ERα activities required for E2-dependent cell proliferation while it is dispensable for ERα-mediated ERE-containing gene transcription. Our discoveries reveal a novel lysosome-dependent degradation pathway for ERα and show a novel biological mechanism by which E2 regulates ERα cellular content and, as a consequence, cellular functions.
doi:10.1371/journal.pone.0094880
PMCID: PMC3988130  PMID: 24736371
3.  Xenoestrogens challenge 17β-estradiol protective effects in colon cancer 
Several epidemiological, cellular, and molecular studies demonstrate the role of environmental chemicals with endocrine disrupting activities, typical of Westernized societies, in the pathogenesis of numerous diseases including cancer. Nonetheless this information, the design and execution of studies on endocrine disruptors are not yet cognizant that the specific actions of individual hormones often change with development and ageing, they may be different in males and females and may be mediated by different receptors isoforms expressed in different tissues or at different life stages. These statements are particularly true when assessing the hazard of endocrine disruptors against 17β-estradiol (E2) actions in that this hormone is crucial determinant of sex-related differences in anatomical, physiological, and behavioral traits which characterize male and female physiology. Moreover, E2 is also involved in carcinogenesis. The oncogenic effects of E2 have been investigated extensively in breast and ovarian cancers where hormone-receptor modulators are now an integral part of targeted treatment. Little is known about the E2 preventive signalling in colorectal cancer, although this disease is more common in men than women, the difference being more striking amongst pre-menopausal women and age-matched men. This review aims to dissect the role and action mechanisms of E2 in colorectal cancer evaluating the ability of estrogen disruptors (i.e., xenoestrogens) in impair these E2 actions. Data discussed here lead to define the possible role of xenoestrogens in the impairment and/or activation of E2 signals important for colorectal cancer prevention.
doi:10.4251/wjgo.v6.i3.67
PMCID: PMC3955780  PMID: 24653796
17β-Estradiol; Estrogen receptors; Xenoestrogens; Bisphenol A; Flavonoids; Colorectal cancer
4.  Xenoestrogens Alter Estrogen Receptor (ER) α Intracellular Levels 
PLoS ONE  2014;9(2):e88961.
17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.
doi:10.1371/journal.pone.0088961
PMCID: PMC3930606  PMID: 24586459
5.  Differences between conventional and non-conventional MRI techniques in Parkinson’s disease 
Functional Neurology  2013;28(2):73-82.
Summary
Magnetic resonance imaging (MRI) provides an in vivo assessment of cortical and subcortical regions affected in Parkinson’s disease (PD). This review summarizes the most important conventional and non-conventional MRI techniques applied in this field.
Standard neuroimaging techniques have played a marginal role in the diagnosis and follow-up of PD, essentially being used only to discriminate atypical syndromes from PD, to exclude secondary causes such as vascular lesions, and to confirm the absence of specific imaging features found in atypical parkinsonisms. However, non-conventional MRI techniques, i.e. new neuroimaging approaches such as magnetic resonance spectroscopy, diffusion tensor imaging, and functional MRI, may allow the detection of structural, functional and metabolic changes useful not only for differential diagnosis, but also for early diagnosis and outcome and treatment monitoring in PD. In addition, we illustrate the advantages of high-field MRI over lower magnetic fields, highlighting the great potential of advanced neuroimaging techniques.
PMCID: PMC3812728  PMID: 24125556
diffusion tensor imaging; functional magnetic resonance imaging; high-field MRI; magnetic resonance spectroscopy; Parkinson’s disease; volumetric MRI
6.  The effect of physician’s recommendation on seasonal influenza immunization in children with chronic diseases 
BMC Public Health  2012;12:984.
Background
Despite recommendations by Health Authorities, influenza immunization coverage remains low in children with chronic diseases. Different medical providers involved in the management of children with chronic conditions may affect the pattern of influenza vaccine recommendations and coverage. The likelihood of vaccination by type of provider in children with chronic conditions is poorly understood. Therefore, the objectives of this study were to analyze the pattern and the effect of recommendations for seasonal influenza immunization provided by different physician profiles to families of children with chronic diseases and to measure the frequency of immunization in the study population.
Methods
We recruited children with chronic diseases aged 6 months–18 years who subsequently presented to specialty clinics for routine follow-up visits, during spring 2009, in three Italian Regions Families of children with chronic diseases were interviewed during routine visits at reference centers through a face-to-face interview. We analyzed the following immunization predictors: having received a recommendation toward influenza immunization by a health provider; child’s sex and age; mothers and fathers’ age; parental education and employment; underlying child’s disease; number of contacts with health providers in the previous year. Influenza immunization coverage was calculated as the proportion of children who received at least one dose of seasonal influenza vaccine in the previous season. We calculated prevalence ratios and we used a generalized linear model with Poisson family, log link and robust error variance to assess the effect of socio-demographic variables, underlying diseases, and recommendations provided by physicians on influenza immunization.
Results
We enrolled 275 families of children with chronic diseases. Overall influenza coverage was 57.5%, with a low of 25% in children with neurological diseases and a high of 91.2% in those with cystic fibrosis. While 10.6% of children who did not receive any recommendation toward influenza immunization were immunized, among those who received a recommendation 87.5-94.7% did, depending on the health professional providing the recommendation. Receiving a recommendation by any provider is a strong predictor of immunization (PR = 8.5 95% CI 4.6;15.6) Most children received an immunization recommendation by a specialty (25.8%) or a family pediatrician (23.3%) and were immunized by a family pediatrician (58.7%) or a community vaccinator (55.2%).
Conclusions
Receiving a specific recommendation by a physician is a strong determinant of being immunized against seasonal influenza in children with chronic diseases independently of other factors. Heterogeneity exists among children with different chronic diseases regarding influenza recommendation despite international guidelines. Increasing the frequency of appropriate recommendations toward influenza immunization by physicians is a single powerful intervention that may increase coverage in children with chronic conditions.
doi:10.1186/1471-2458-12-984
PMCID: PMC3585468  PMID: 23153092
Influenza; Immunization; Chronic disease; Children; Physicians; Recommendations
7.  Neuroprotective Effects of 17β-Estradiol Rely on Estrogen Receptor Membrane Initiated Signals 
Besides its crucial role in many physiological events, 17β-estradiol (E2) exerts protective effects in the central nervous system. The E2 effects are not restricted to the brain areas related with the control of reproductive function, but rather are widespread throughout the developing and the adult brain. E2 actions are mediated through estrogen receptors (i.e., ERα and ERβ) belonging to the nuclear receptor super-family. As members of the ligand-regulated transcription factor family, classically, the actions of ERs in the brain were thought to mediate only the E2 long-term transcriptional effects. However, a growing body of evidence highlighted rapid, membrane initiated E2 effects in the brain that are independent of ER transcriptional activities and are involved in E2-induced neuroprotection. The aim of this review is to focus on the rapid effects of E2 in the brain highlighting the specific role of the signaling pathway(s) of the ERβ subtype in the neuroprotective actions of E2.
doi:10.3389/fphys.2012.00073
PMCID: PMC3319910  PMID: 22493583
estrogen receptor α; estrogen receptor β; 17β-estradiol; neuroprotective effects; membrane initiated signals
8.  Hepatitis B: Epidemiology and prevention in developing countries 
World Journal of Hepatology  2012;4(3):74-80.
Hepatitis B virus (HBV) infection is a serious global public health problem. The infection may be transmitted through sexual intercourse, parenteral contact or from an infected mother to the baby at birth and, if contracted early in life, may lead to chronic liver disease, including cirrhosis and hepatocellular carcinoma. On the basis of the HBV carrier rate, the world can be divided in 3 regions of high, medium and low endemicity. The major concern is about high endemicity countries, where the most common route of infection remains vertical transmission from mother to child. Screening of all pregnant women and passive immunization with human hepatitis B immunoglobulin are not affordable for many developing countries. The infection rate can be reduced by modifying behavior, improving individual education, testing all blood donations, assuring asepsis in clinical practice and screening all pregnant women. However, availability of a safe and efficacious vaccine and adoption of appropriate immunization strategies are the most effective means to prevent HBV infection and its consequences. The unsolved problem for poorest countries, where the number of people currently infected is high, is the cost of the vaccine. A future challenge is to overcome the social and economic hurdles of maintaining and improving a prevention policy worldwide to reduce the global burden of the disease.
doi:10.4254/wjh.v4.i3.74
PMCID: PMC3321493  PMID: 22489259
Hepatitis B; Developing countries; Endemicity; Seroprevalence; Vaccine
9.  Prelamin A mediated recruitment of SUN1 to the nuclear envelope directs nuclear positioning in human muscle 
Cell death and differentiation  2011;18(8):1305-1315.
Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among nuclear envelope partners of lamin A are SUN1 and SUN2, which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.
doi:10.1038/cdd.2010.183
PMCID: PMC3097169  PMID: 21311568
10.  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 β, more considerably in FOCA-. Importantly, the activation state of estrogen receptor β 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.
doi:10.1186/2042-6410-3-4
PMCID: PMC3298494  PMID: 22284681
androgenic and non-androgenic progestin; combined oral contraceptive; estrogen receptors; global DNA methylation; monocyte-derived macrophages; TNFα
11.  The Effects of 17β-estradiol in Cancer are Mediated by Estrogen Receptor Signaling at the Plasma Membrane 
Two different isoforms of the estrogen receptors (i.e., ERα and ERβ) mediate pleiotropic 17β-estradiol (E2)-induced cellular effects. The ERs are principally localized in the nucleus where they act by globally modifying the expression of the E2-target genes. The premise that E2 effects are exclusively mediated through the nuclear localized ERs has been rendered obsolete by research over the last 15 years demonstrating that ERα and ERβ proteins are also localized at the plasma membranes and in other extra-nuclear organelles. The E2 modulation of cancer cell proliferation represents a good example of the impact of membrane-initiated signals on E2 effects. In fact, E2 via ERα elicits rapid signals driving cancer cells to proliferation (e.g., in breast cancer cells), while E2-induced ERβ rapid signaling inhibits proliferation (e.g., in colon cancer cells). In this review we provide with an overview of the complex system of E2-induced signal transduction pathways, their impact on E2-induced cancer cell proliferation, and the participation of E2-induced membrane-initiated signals in tumor environment.
doi:10.3389/fphys.2011.00030
PMCID: PMC3129035  PMID: 21747767
estrogens; estrogen receptors; membrane-initiated signals; cell proliferation; cell apoptosis
12.  Epidemiologic Determinants Affecting Cigarette Smoking Cessation: A Retrospective Study in a National Health System (SSN) Treatment Service in Rome (Italy) 
This retrospective study aims to evaluate epidemiologic characteristics of patients attending stop smoking courses, based on group therapy, testing their influence on smoking cessation in univariate and multivariate model. A total of 123 patients were included in this study. Mean age was 53 (±11). Sixty-seven percent were women. At the end of the courses 66% of patients stopped smoking, after 12 months only 39% remained abstinent. Patients younger than 50 years statistically tended to continue smoking 6 months (P = .02–R.R. = 1.49, C.I. 95%: 1.06–2.44) and 12 months (P = .03–R.R. = 1.37, C.I. 95%: 1.02–2.52) after the end of the courses. A low self-confidence in quitting smoking was significantly related to continuing tobacco consumption after 6 months (P = .016–R.R. = 1.84, C.I. 95%: 1.14–2.99). Low adherence to therapeutic program was statistically associated to maintenance of tobacco use at 6 months (P = .006–R.R. = 1.76, C.I. 95%: 1.32–2.35) and 12 months (P = .050–R.R. = 1.45, C.I. 95%: 1.11–1.88). This association was confirmed at 6 months in the analysis performed on logistic regression model (P = .013).
doi:10.1155/2010/183206
PMCID: PMC2853854  PMID: 20396671
13.  Molecular analysis of the apoptotic effects of BPA in acute myeloid leukemia cells 
Background:
BPA (bisphenol A or 2,2-bis(4-hydroxy-phenol)propane) is present in the manufacture of polycarbonate plastic and epoxy resins, which can be used in impact-resistant safety equipment and baby bottles, as protective coatings inside metal food containers, and as composites and sealants in dentistry. Recently, attention has focused on the estrogen-like and carcinogenic adverse effects of BPA. Thus, it is necessary to investigate the cytotoxicity and apoptosis-inducing activity of this compound.
Methods:
Cell cycle, apoptosis and differentiation analyses; western blots.
Results:
BPA is able to induce cell cycle arrest and apoptosis in three different acute myeloid leukemias. Although some granulocytic differentiation concomitantly occurred in NB4 cells upon BPA treatment, the major action was the induction of apoptosis. BPA mediated apoptosis was caspase dependent and occurred by activation of extrinsic and intrinsic cell death pathways modulating both FAS and TRAIL and by inducing BAD phosphorylation in NB4 cells. Finally, also non genomic actions such as the early decrease of both ERK and AKT phosphorylation were induced by BPA thus indicating that a complex intersection of regulations occur for the apoptotic action of BPA.
Conclusion:
BPA is able to induce apoptosis in leukemia cells via caspase activation and involvement of both intrinsic and extrinsic pathways of apoptosis.
doi:10.1186/1479-5876-7-48
PMCID: PMC2717713  PMID: 19538739
14.  High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients 
PLoS ONE  2009;4(4):e5219.
Background
Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up.
Methodology/Principal Findings
We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group.
Conclusions/Significance
We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.
doi:10.1371/journal.pone.0005219
PMCID: PMC2667632  PMID: 19381331
15.  CagA and VacA Helicobacter pylori antibodies in gastric cancer 
BACKGROUND:
Infection with different genotypes of virulent Helicobacter pylori strains (cytotoxin-associated gene A [CagA]-and/or vacuolating cytotoxin A [VacA]-positive) can play a role in the development of atrophic gastritis, duodenal ulcer (DU) and gastric cancer (GC).
OBJECTIVE:
To determine whether patients with GC and H pylori-negative histological staining had previously been infected with H pylori CagA- and/or VacA-positive virulent strains.
METHODS:
Twenty-three GC patients with a mean (± SD) age of 68.14±9.8 years who tested H pylori-negative on histological staining took part in the study. Three control groups were included. The first group comprised 19 patients with past H pylori infection and DUs eradicated 10 years earlier, with a mean age of 58±18.2 years. H pylori-negative status for this group was determined every year with Giemsa staining, and follow-up testing occured 120±32 months (mean ± SD) after therapy. The subsequent control groups included 20 asymptomatic children, with a mean age of 7±4.47 years, and with H pylori-negative fecal tests; the final group contained 30 patients without clinical symptoms of H pylori infection, with a mean age of 68±11.6 years, who tested H pylori-negative by histological staining.
RESULTS:
Prevalence of CagA and VacA seropositivity, respectively was 82.6% and 73.91% in GC patients; 84.2% and 84.2% in H pylori-negative DU patients; 25% and 5% in H pylori-negative children; and 36.6% and 16.6% in the patients without clinical symptoms on histological staining. CagA and VacA antibody positivity was not significantly different between GC patients and patients with DUs that had been eradicated 10 years earlier. Significant positivity was found between the children’s group and the H pylori-negative (with past DUs) group (P<0.001). A statistically significant difference was found in age between groups (P<0.03).
CONCLUSIONS:
Patients with GC, even when H pylori-negative at the time of the present study, may have been infected by H pylori before the onset of the disease, as confirmed by CagA and VacA seropositivity. These data reinforce the hypothesis that H pylori may be a direct carcinogenic agent of GC.
PMCID: PMC2662200  PMID: 18354754
CagA; Gastric cancer; Helicobacter pylori; VacA
16.  Estrogen Signaling Multiple Pathways to Impact Gene Transcription 
Current Genomics  2006;7(8):497-508.
Steroid hormones exert profound effects on cell growth, development, differentiation, and homeostasis. Their effects are mediated through specific intracellular steroid receptors that act via multiple mechanisms. Among others, the action mechanism starting upon 17β-estradiol (E2) binds to its receptors (ER) is considered a paradigmatic example of how steroid hormones function. Ligand-activated ER dimerizes and translocates in the nucleus where it recognizes specific hormone response elements located in or near promoter DNA regions of target genes. Behind the classical genomic mechanism shared with other steroid hormones, E2 also modulates gene expression by a second indirect mechanism that involves the interaction of ER with other transcription factors which, in turn, bind their cognate DNA elements. In this case, ER modulates the activities of transcription factors such as the activator protein (AP)-1, nuclear factor-κB (NF-κB) and stimulating protein-1 (Sp-1), by stabilizing DNA-protein complexes and/or recruiting co-activators. In addition, E2 binding to ER may also exert rapid actions that start with the activation of a variety of signal transduction pathways (e.g. ERK/MAPK, p38/MAPK, PI3K/AKT, PLC/PKC). The debate about the contribution of different ER-mediated signaling pathways to coordinate the expression of specific sets of genes is still open. This review will focus on the recent knowledge about the mechanism by which ERs regulate the expression of target genes and the emerging field of integration of membrane and nuclear receptor signaling, giving examples of the ways by which the genomic and non-genomic actions of ERs on target genes converge.
PMCID: PMC2269003  PMID: 18369406
Estrogen; estrogen receptors; genomic and non-genomic action mechanism; gene transcription
17.  Nutritional flavonoids impact on nuclear and extranuclear estrogen receptor activities 
Genes & Nutrition  2006;1(3-4):161-176.
Flavonoids are a large group of nonnutrient compounds naturally produced from plants as part of their defence mechanisms against stresses of different origins. They emerged from being considered an agricultural oddity only after it was observed that these compounds possess a potential protective function against several human degenerative diseases. This has led to recommending the consumption of food containing high concentrations of flavonoids, which at present, especially as soy isoflavones, are even available as overthecounter nutraceuticals. The increased use of flavonoids has occurred even though their mechanisms are not completely understood, in particular those involving the flavonoid impact on estrogen signals. In fact, most of the human health protective effects of flavonoids are described either as estrogenmimetic, or as antiestrogenic, while others do not involve estrogen signaling at all. Thus, the same molecule is reported as an endocrine disruptor, an estrogen mimetic or as an antioxidant without estrogenic effects. This is due in part to the complexity of the estrogen mechanism, which is conducted by different pathways and involves two different receptor isoforms. These pathways can be modulated by flavonoids and should be considered for a reliable evaluation of flavonoid, both estrogenicity and antiestrogenicity, and for a correct prediction of their effects on human health.
doi:10.1007/BF02829966
PMCID: PMC3454832  PMID: 18850212
17β-Estradiol; Estrogen Receptor-α; Estrogen Receptor-β; Flavonoids; Gene Transcription; Signal Transduction Cascade
18.  Palmitoylation-dependent Estrogen Receptor α Membrane Localization: Regulation by 17β-Estradiol 
Molecular Biology of the Cell  2005;16(1):231-237.
A fraction of the nuclear estrogen receptor α (ERα) is localized to the plasma membrane region of 17β-estradiol (E2) target cells. We previously reported that ERα is a palmitoylated protein. To gain insight into the molecular mechanism of ERα residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ERα membrane localization. The cancer cell lines expressing transfected or endogenous human ERα (HeLa and HepG2, respectively) or the ERα nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ERα enacts ERα association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D1 promoter activity, DNA synthesis). Moreover, E2 reduces both ERα palmitoylation and its interaction with caveolin-1, in a time- and dose-dependent manner. These data point to the physiological role of ERα palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation.
doi:10.1091/mbc.E04-07-0547
PMCID: PMC539167  PMID: 15496458
19.  Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells 
Molecular Biology of the Cell  2003;14(6):2583-2591.
We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1–S transition by the parallel stimulation of both PKC-α and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17β-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1–S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1–S phase transition.
doi:10.1091/mbc.E02-09-0621
PMCID: PMC194905  PMID: 12808053
20.  Distinct Nongenomic Signal Transduction Pathways Controlled by 17β-Estradiol Regulate DNA Synthesis and Cyclin D1 Gene Transcription in HepG2 Cells 
Molecular Biology of the Cell  2002;13(10):3720-3729.
Estrogens induce cell proliferation in target tissues by stimulating progression through the G1 phase of the cell cycle. Activation of cyclin D1 gene expression is a critical feature of this hormonal action. The existence of rapid/nongenomic estradiol-regulated protein kinase C (PKC-α) and extracellular signal-regulated kinase (ERK) signal transduction pathways, their cross talk, and role played in DNA synthesis and cyclin D1 gene transcription have been studied herein in human hepatoma HepG2 cells. 17β-Estradiol was found to rapidly activate PKC-α translocation and ERK-2/mitogen-activated protein kinase phosphorylation in this cell line. These actions were independent of each other, preceding the increase of thymidine incorporation into DNA and cyclin D1 expression, and did not involve DNA binding by estrogen receptor. The results obtained with specific inhibitors indicated that PKC-α pathway is necessary to mediate the estradiol-induced G1-S progression of HepG2 cells, but it does not exert any effect(s) on cyclin D1 gene expression. On the contrary, ERK-2 cascade was strongly involved in both G1-S progression and cyclin D1 gene transcription. Deletion of its activating protein-1 responsive element motif resulted in attenuation of cyclin D1 promoter responsiveness to estrogen. These results indicate that estrogen-induced cyclin D1 transcription can occur in HepG2 cells independently of the transcriptional activity of estrogen receptor, sustaining the pivotal role played by nongenomic pathways of estrogen action in hormone-induced proliferation.
doi:10.1091/mbc.E02-03-0153
PMCID: PMC129978  PMID: 12388769
21.  CD99 Drives Terminal Differentiation of Osteosarcoma Cells by Acting as a Spatial Regulator of ERK 1/2† 
Journal of Bone and Mineral Research  2013;29(5):1295-1309.
Differentiation therapy is an attractive treatment for osteosarcoma (OS). CD99 is a cell surface molecule expressed in mesenchymal stem cells and osteoblasts that is maintained during osteoblast differentiation while lost in OS. Herein, we show that whenever OS cells regain CD99, they become prone to reactivate the terminal differentiation program. In differentiating conditions, CD99-transfected OS cells express osteocyte markers, halt proliferation, and largely die by apoptosis, resembling the fate of mature osteoblasts. CD99 induces ERK activation, increasing its membrane-bound/cytoplasmic form rather than affecting its nuclear localization. Through cytoplasmic ERK, CD99 promotes activity of the main osteogenic transcriptional factors AP1 and RUNX2, which in turn enhance osteocalcin and p21WAF1/CIP1, leading to G0/G1 arrest. These data underscore the alternative positions of active ERK into distinct subcellular compartments as key events for determining OS fate. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
doi:10.1002/jbmr.2141
PMCID: PMC4255300  PMID: 24677094
CD99; OSTEOSARCOMA; OSTEOBLAST DIFFERENTIATION; MAPK SIGNALING; RUNX2

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