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Toxoplasma gondii is a major food and waterborne transmitted parasite world-wide. The tissues and meat samples of many warm blooded animals can contain tissues cysts from chronic toxoplasmosis. Water and vegetable can be contaminated by the parasitic oocysts shed through the feces of infected cats, representing the definitive host of the parasite. A sensitive PCR for Toxoplasma gondii detection is described. The first step amplified the region between the 28S and 18S rDNA in the closely related T. gondii and Neospora caninum; RFLP analysis distinguished the DNA from the two morphologically identical parasites. Although N. caninum is not involved in human transmission, so far, it is important for animal health since is a major responsible for abortion in cattle. The nested PCR was used in a dilution assay in pork sausage samples spiked with T. gondii parasitic DNA. The analysis showed that up to 200fg equivalent to two single parasites only, could be detected. Similar detection limit for T. gondii can be obtained with real-time PCRs, but real time methods need special consumables and expensive equipment.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Because of their relative simplicity and the barriers to gene flow, islands are ideal systems to study the distribution of biodiversity. However, the knowledge that can be extracted from this peculiar ecosystem regarding epidemiology of economically relevant diseases has not been widely addressed. We used information available in the scientific literature for 10 old world islands or archipelagos and original data on Sicily to gain new insights into the epidemiology of the Mycobacterium tuberculosis complex (MTC). We explored three nonexclusive working hypotheses on the processes modulating bovine tuberculosis (bTB) herd prevalence in cattle and MTC strain diversity: insularity, hosts and trade. Results suggest that bTB herd prevalence was positively correlated with island size, the presence of wild hosts, and the number of imported cattle, but neither with isolation nor with cattle density. MTC strain diversity was positively related with cattle bTB prevalence, presence of wild hosts and the number of imported cattle, but not with island size, isolation, and cattle density. The three most common spoligotype patterns coincided between Sicily and mainland Italy. However in Sicily, these common patterns showed a clearer dominance than on the Italian mainland, and seven of 19 patterns (37%) found in Sicily had not been reported from continental Italy. Strain patterns were not spatially clustered in Sicily. We were able to infer several aspects of MTC epidemiology and control in islands and thus in fragmented host and pathogen populations. Our results point out the relevance of the intensity of the cattle commercial networks in the epidemiology of MTC, and suggest that eradication will prove more difficult with increasing size of the island and its environmental complexity, mainly in terms of the diversity of suitable domestic and wild MTC hosts., This is a contribution to the EU FP7 grant number 278976 ANTIGONE and the research project FAU2008-00004-C03. PA was supported by a Beatriu de Pinós fellowship funded by “Comissionat per a Universitats i Recerca del Departament d'Innovació, Universitats i Empresa” of the Generalitat de Catalunya, and the COFUND Programme–Marie Curie Actions under 7th Framework Programme of the European Community. PA is currently funded from the SFRH/BPD/90320/2012 post-doctoral grant by Portuguese Fundação para a Ciência e a Tecnologia and European Social Fund.

We found both in vitro and in vivo that survival of NSCLC cells in a hypoxic microenvironment requires Notch-1 signaling. A hypoxic tumor environment represents a problem for NSCLC treatment because it plays a critical role in cancer resistance to chemotherapy, tumor recurrence, and metastasis. Here we targeted hypoxic tumor tissue in an orthotopic NSCLC model. We inhibited the Notch-1/IGF-1R/Akt-1 axis using 3 agents: a γ-secretase inhibitor or GSI (MRK-003), a fully humanized antibody against the human IGF-1R (MK-0646), and a pan-Akt inhibitor (MK-2206), alone or in various combinations including therapeutics currently in clinical use. All treatments but Akt inhibition significantly prolonged the median survival of mice compared with controls. GSI treatment caused specific cell death of hypoxic tumors. Tumors excised from mice displayed a significant reduction of markers of hypoxia. Moreover, GSI treatment caused reduced metastasis to the liver and brain. MK-0646 was not specific to a hypoxic tumor environment but substantially increased the median survival of treated mice compared with controls. NSCLC cells evaded MK-0646 treatment by specifically overactivating EGF-R both in vivo and in 5 cell lines in vitro. This phenomenon is achieved at the level of protein stability. MK-0646 treatment caused increased erlotinib sensitivity in NSCLC cells poorly responsive to it. Sequential treatment with MK-0646 followed by erlotinib prolonged median survival of mice significantly. When the 2 drugs were administered simultaneously, no survival benefit was observed, and this combination therapy proved less effective than MK-0646 used as single agent. Our data offer novel information that may provide insights for the planning of clinical trials in humans, likely for maintenance therapy of NSCLC patients.

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. © 2012 IUBMB IUBMB Life, 64(8): 690–696, 2012

Lung cancer is the leading cause of cancer-related deaths in the Western world. The lungs can be affected by a number of histologically diverse malignancies. Nonetheless, the vast majority of lung cancers are classified as non-small-cell lung cancer (NSCLC). Despite extensive research on different therapeutic regimens, the overall 5-year survival of patients diagnosed with NSCLC (all stages) is a dismal 15%. Although strongly correlated with tobacco smoke, there is an increasing NSCLC morbidity in individuals who have never smoked. The pattern of genetic lesions found in NSCLC derived from smokers and never-smokers appears to be different. This fact led to the hypothesis that different, still unidentified carcinogens are responsible for lung cancer onset in never-smokers. All the aforementioned considerations compel the scientific community to find novel therapeutic targets to fight such a deadly disease. In recent years critical pathways governing embryonic development have been increasingly linked to cancer. Here we will focus on the role of Notch signaling in lung cancer. Notch receptors' activity can be blocked through the use of different strategies, thus representing a promising alternative/complement to the arsenal of therapeutic strategies currently used to treat lung cancer.

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 17β-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) β, but not ERα, as evaluated by the mimetic effect of ERβ-specific agonist DPN and by the blockage of E₂ effect in ERβ-silenced SK-N-BE cells. Furthermore, both rapid (15 min) ERβ-dependent activation of p38/MAPK and transcriptional ERβ 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.

Bisphenol A (BPA), a prototype of endocrine disruptors, mimics 17β-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 β (ERβ)-mediated functions. Here, the detailed analysis of the effect of BPA, alone or in combination with E2, on ERβ-mediated cellular functions is reported in ERβ-expressing colon cancer cell line. BPA binds to ERβ without activating any receptor activities. On the other hand, BPA inhibits E2-induced genomic activity of ERβ as well as ERβ extra-nuclear activities (i.e., ERβ: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. © 2010 IUBMB IUBMB Life, 62(9): 684–687, 2010

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 α (ERα) action mechanisms, drives cancer cells to apoptosis. Because these studies were conducted in the absence of the endogenous hormone 17β-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 ERα-transfected (HeLa cells) and ERα-containing (HepG2 cells) cancer cell lines. Ligand saturation experiments indicate that Nar decreases the binding of E2 to ERα 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 ERα specifically antagonizes the E2-induced ERα-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. © 2009 IUBMB IUBMB Life, 62(1):51–60, 2010

Recently, aqueous solutions polluted by BPA have been bioremediated by us using laccase immobilized on hydrophobic membranes in non-isothermal bioreactors. BPA degradation was checked using analytical methods. To assess in vitro the occurred bioremediation, the proliferation and viability indexes of MCF-7 cells incubated in the presence of aqueous solutions of BPA, or of enzyme-treated BPA solutions, have been measured as a function of the initial BPA concentration.The results demonstrated that: i) at each initial BPA concentration used, both the proliferation and viability indexes are a function of the duration of enzyme treatment; ii) proliferation and viability are uncoupled biological processes with respect to BPA enzyme treatment.Non-isothermal bioreactors are a useful tool for the bioremediation of aqueous solutions polluted by BPA, which is an example of an endocrine disruptor that belongs to the alkyl phenol family. Keywords: MCF-7, BPA, Bioremediation, Proliferation index, Viability index

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.

A wide variety of environmental contaminants exert estrogenic actions in wildlife, laboratory animals, and in human beings through binding to nuclear estrogen receptors (ERs). Here, the mechanism(s) of bisphenol A (BPA) to induce cell proliferation and the occurrence of its bioremediation by treatment with laccase are reported. BPA, highly present in natural world and considered as a model of environmental estrogen action complexity, promotes human cancer cell proliferation via ER alpha-dependent signal transduction pathways. Similar to 17 beta-estradiol, BPA increases the phosphorylation of both extracellular regulated kinase and AKT. Specific inhibitors of these kinase completely, block the BPA effect on cancer cell proliferation. Notably, high BPA concentrations (i.e., 0.1 and 1 mM) are cytotoxic even in ER alpha-devoid cancer cells, indicating that an ER alpha-independent mechanism participates to BPA-induced cytotoxicity. On the other hand, BPA oxidation by laccase impairs the binding of this environmental estrogen to ER alpha loosing at all ERa-dependent effect on cancer cell proliferation. Moreover, the laccase-catalyzed oxidation of BPA reduces the BPA cytotoxic effect. Thus, laccase appears to impair BPA action(s), representing an invaluable bioremediation enzyme.

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.

Adipose tissue has recently been described as one of the major endocrine gland that plays a role in energy homeostasis, lipid metabolism, immune response, and reproduction. An excess of white adipose tissue, caused by a complex interaction between genetic, hormonal, behavioral, and environmental factors, results in obesity: a heterogeneous disorder that predisposes humans to a variety of diseases. Among several hormones, estrogens promote, maintain, and control the typical distribution of body fat and adipose tissue metabolism through still unknown mechanisms. These steroids are known to regulate fat mass, adipose deposition and differentiation, and adipocyte metabolism. Moreover, estrogen deficiency results in increases in adipose tissue, preferentially in visceral fat, which would link obesity to the susceptibility of related disorders. In this review the role of estrogens in adipose tissue differentiation and in the protection against the onset of obesity will be discussed with particular attention being drawn to the underlying molecular mechanisms mediated by estrogen receptor isoforms ERalpha and ERbeta.

Vascular smooth muscle cells (VSMC) have been isolated from male and female rat aorta and studied to assess their susceptibility to ultraviolet radiation-induced oxidative stress. Interestingly, a gender difference, in terms of reactive oxygen species production, was detected in both basal and irradiated VSMC. Namely, VSMC from male rats were more susceptible to radiation-induced stress and easier underwent apoptosis in comparison to cells from female rats. Conversely, the latter, in the same experimental conditions, clearly displayed signs of premature senescence. These results indicate that a sort of “gender memory” can be conserved in VMSC in primary culture.

The cellular functions regulated by 17beta-estradiol (E2) start after the hormone binds to its receptors (i.e., ERalpha and ERbeta). These act as ligand-dependent transcription factor transactivating target genes. In addition, E2 induces non-genomic actions, whose activation is triggered by a fraction of the ERs localized at the plasma membrane. Palmitoylation allows ERalpha to localize at the plasma membrane, to associate with caveolin-1, and, upon E2 stimulation, to activate rapid signals relevant for cell proliferation. The existence of a mechanism, which allows ERbeta localization at the plasma membrane and its putative role in anti-proliferative E2 effects is completely unknown. Here, the susceptibility of ERbeta to undergo palmitoylation and the role played by this process has been analyzed in DLD-1 containing endogenous ERbeta or in HeLa cells transiently transfected with ERbeta or ERalpha expression vectors. As for ERalpha, palmitoylation is necessary for ERbeta localization at the plasma membrane and its association with caveolin-1 but, in contrast to ERalpha, the E2 binding increases ERbeta association with caveolin-1 and the p38 member of MAPK family. Moreover, the palmitoyl acyl transferase (PAT) inhibitor blocks the ability of ERbeta-E2 complex to activate p38 impairing the receptor-dependent activation of downstream proapoptotic cascade (i.e., caspase-3 activation and poly(ADP-ribose)polymerase (PARP) cleavage). Consequently, palmitoylation must be considered to be a molecular device for ERbeta, which allows these receptors to interact with the plasma membrane and to regulate E2-induced non-genomic functions relevant to the anti-proliferative effect of this hormone.

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 17beta-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-kappaB (NF-kappaB) 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.

In previous studies we targeted hypoxic Non-Small Cell Lung Cancer (NSCLC) tumor tissue using gamma-secretase inhibitors (GSI) in a preclinical model of advanced NSCLC. GSI treatment of mice suggested reactivation of mTORC-1 in otherwise quiescent hypoxic NSCLC tissue. Methods included immunohistochemistry and cytochemistry, immunoblotting, immunoprecipitation, siRNA-mediated gene knockdown, nucleotide and amino acid analogs incorporation, FACS analysis, various imaging techniques, Q-PCR, gene transfection, cap binding assays, RNA and antibodies arrays. GSI treatment of mice showed a re-induced phosphorylation of 4E-BP1 at T37/46 in hypoxic tumor masses. In vitro studies indicated a role for APP in this phenomenon. APP downregulation caused a rearrangement in 4E-BP1's phosphorylation pattern at residues T37/46, S65 and T70, reversed by overexpression of the APP C-terminal domain. Increased recruitment of eIF4A on the mRNA cap was observed. These events coincided with a drastic increase in the rate of global protein synthesis, an event that appeared to be mTOR-independent. Meanwhile, NSCLC cells with depleted APP were arrested in G0/G1 through a cyclin C-mediated mechanism. Cells arrested in G0/G1 with augmented rate of global protein synthesis (both cap- and IRES-dependent) increased their size and underwent a substantial necrotic cell death due to cell membrane permeabilization. These results indicate a novel role for APP in synchronizing the rate of protein synthesis and cell cycle progression, raising questions on how these processes interact in malignant transformation. The data presented here suggest that cells of different origin, including NSCLC cells, have developed some mechanisms to couple cell growth with cell cycle progression independent of overlapping signals which derive, among others, from the PI3-K pathway. APP seems to play a central role in this synchronization, and its loss of function causes cell size abnormalities and death. Citation Format: Anna Sobol, Paola Galluzzo, Shuang Liang, Brittany Rambo, Sylvia Skucha, Megan Weber, Maurizio Bocchetta. Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C40.

In a previous study we found both in vitro and in an orthotropic NSCLC model in SCID mice that survival of NSCLC cells in hypoxic microenvironment is dependent upon Notch-1 signaling. Active Notch-1 was expressed in hypoxic tumor microenvironment only. Notch-1 provides survival stimuli through exacerbated Akt-1 activation due to suppression of PTEN transcription and positive regulation of the transcription of IGF-1 and its receptor. Hypoxic tumor environment is a problem for NSCLC treatment because it is responsible for cancer resistance to chemotherapy, tumor recurrence and metastasization. Also, hypoxic tumor microenvironment is quiescent and represses mTORC-1 activity. We show a number of experiments aimed at targeting hypoxic tumor tissue in the orthotropic NSCLC model. We attempted to target the Notch-1/IGF-1R/Akt-1 axis using three agents (a γ-secretase inhibitor, MRK-003; a fully humanized antibody against the human IGF-1R that promotes the receptor degradation, MK-0646; a pan-Akt inhibitor, MK-2206, all drugs provided by Merck Inc.) alone or in various combinations. We also used in combination studies drugs that are used for the treatment of advanced NSCLC, namely cisplatin and Erlotinib. The orthotropic model was established using two cell lines non-dependent on Notch-3 signaling. siRNA to Notch-3 in A549 and H1437 affects neither the growth rate nor the survival of these cells. All treatments (besides MK-2206) significantly prolonged the median survival of mice compared to controls (16 mice/experimental arm). MRK-003 treatment yielded specific death of hypoxic tumor areas. The tumors excised from the mice reaching an end-point displayed a significant reduction of markers of hypoxia as assessed by RT-qPCR using human specific primers. Moreover, qPCR measurements of the ratio between human/mouse GAPDH in DNA extracted from the whole brains and livers showed a significant reduction in metastasization in MRK-003 treated mice. MK-0646 was not specific to hypoxic tumor environment, but substantially increased the median survival of treated mice compared to controls. NSCLC cells evaded MK-0646 treatment by activating EGF-R and ErbB3 exclusively both in vivo and in five cell lines in vitro. This phenomenon is achieved at the level of protein stability. One outcome of MK-0646 treatment is that cells poorly or not responsive to Erlotinib become more responsive to it. Sequential treatment with MK-0646 (three weeks), then combination of MK-0646 plus Erlotinib prolonged median survival of mice dramatically. If the two drugs where administered in combination from day one no survival advantage was observed in median survival compared to mice treated with Erlotinib alone and was actually less effective than MK-0646 used as single agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5227. doi:1538-7445.AM2012-5227