Search

Introduction The aim of this study was to present two outbreaks of bovine abortion due to Leptospira infection in cattle herds located in the northern part of Sicily (Italy). The animals were positive for Leptospira interrogans serogroup Sejroe serovar Hardjo in a microscopic agglutination test (MAT). Material and Methods A total of 23 Charolaise cows (farm A) and 75 Limousine bulls and Cinisara and Modicana cows (farm B) were enrolled in this study. The blood samples were collected from all subjects at the following time points: before a cycle of intramuscular treatment with oxytetracycline dihydrate (T0), after 5–6 weeks from the treatment (T1), and every 10 weeks until seronegativisation (T2 in Farm A and T3 in Farm B). A serological test (MAT) was used for the diagnosis of leptospirosis. Results Two samples from farm A (2/23) and 29 samples from farm B (29/75) were positive to Leptospira interrogans, serogroup Sejroe, serovar Hardjo in the MAT. Leptospira spp. DNA was detected by real-time PCR in the urine sample of one positive cow on farm A, and in placenta and brain samples belonging to one aborted foetus on farm B. Conclusion It is important to use serological and molecular diagnostic techniques complementarily to identify infected individuals.

Aim of this study is to report a laboratory investigation performed following the isolation of Brucella ovis, causing ovine epididymitis, in a traditional sheep farm in Sicily (South Italy). This disease represents a newly emerging risk for Italian livestock and is listed among diseases of EU priority (EU Reg 2016/429). Blood samples from 56 rams and 143 ewes were analyzed by both Enzyme-Linked Immunosorbent Assay (ELISA) and Complement Fixation Test (CFT). Genital swabs from all rams and 15 lactating ewes were collected to perform real-time PCR. Eighteen serologically positive rams were slaughtered and postmortem-inspected. Samples of testicle, epididymis, lymph nodes, and urine were also collected in order to perform microbiological, molecular, and histopathological analysis. Twelve slaughtered rams showed anatomo-pathological lesions. Real-time PCR for B. ovis BOV_A0504 gene was positive for 13 testicles and epididymis and 11 urine while B. ovis was isolated from epididymis and testicles of 7 slaughtered rams. This is the first exhaustive laboratory report of a microbiological, molecular, and serological pattern of the disease in sheep in Italy. Despite the impact on health and animal welfare, the epidemiology of B. ovis infection is still unknown, particularly in our country where the disease is considered endemic.

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system, caused by a combination of genetic and environmental factors. In recent years, a role in MS pathogenesis was assigned to the gut microbiota. However, different signatures of gut dysbiosis have been shown to depend on environmental factors, like diet and lifestyle. In this study, we compared the gut microbiome in MS patients and their household healthy relatives sharing lifestyle and environmental factors. Faecal metagenomic DNA was extracted and the V3–V4 regions of the conserved bacterial 16S ribosomal RNA gene were amplified and sequenced. While overall bacterial communities were similar, specific families differed between healthy and MS subjects. We observed an increase in Ruminococcaceae, Christensenellaceae, Desulfovibrionaceae, Clostridiales, and Family XIII in MS patients, while Bacteroidaceae, Tannerellaceae, Veillonellaceae, and Burkholderiaceae were more abundant in healthy controls. In addition, principle coordinate analysis showed that the gut microbiome of all MS patients formed a cluster being less diverse than the household relatives and that gut microbiota of MS patients with EDSS 4.5–7 formed a distinct cluster in respect to their controls. Overall, our study is consistent with the hypothesis that MS patients have gut microbial dysbiosis and evidenced the importance of environmental factors in shaping the gut microbiome.

Sanitary management and population control of feral pigs remains a major problem in public health, particularly in natural parks where hunting is prohibited and the extensive farming of livestock is common. Macracanthorhynchus hirudinaceus is a zoonotic parasite species with a worldwide distribution of which the natural definitive hosts are primarily pigs and wild boars (Sus scrofa). The present study describes the main anatomo-pathological and parasitological findings in the first case of M. hirudinaceus in feral pigs in the Madonie park in Sicily (Southern Italy). Overall, 52 acanthocephalans were collected from the small intestine of four infected feral pigs. The prevalence among the 36 examined animals was 11.1% with a mean Abundance (mA) and mean Intensity (mI) of 1.4 and 13, respectively. Pathological examination revealed grossly visible nodules on the external surface of the intestines, corresponding to the proboscis of M. hirudinaceus attached deeply into the intestinal wall. In these sites, severe inflammatory reactions in the tissue involved and the destruction of normal intestinal architecture, as well as necrosis and ulceration in the mucosa, submucosa, and part of the muscolaris mucosae were described. This is the first official report of this neglected zoonosis in Italy, in particular in a natural park where the extensive farming of domestic pigs is practiced. This could favor the spread of this parasite in domestic animals and the environment, increasing the accidental risk of infection in human residents of these areas.

Borrelia burgdorferi is a bacterial pathogen transmitted by Ixodes ticks and is responsible for Lyme disease in both humans and dogs. The aim of this work was to evaluate B. burgdorferi diffusion among stray dogs in Palermo (Sicily, Italy) by serological methods in order to study the risk factors associated with the infection. Serum and blood samples of 316 dogs were collected from a shelter in Palermo, and were analyzed for the presence of antibodies against B. burgdorferi by indirect immunofluorescence assay (IFA), and of the ospA gene by real-time PCR, respectively. Seventeen sera (5.4%) were positive for the antibodies via IFA and one blood (0.3%) for ospA via real time PCR. On the basis of serological results, the evaluation of the potential risk factors (sex, age, breed and coat color) was carried out. The multivariate analysis indicated that male sex is a factor significantly associated with B. burgdorferi seropositivity. This study confirms that male dogs have a higher risk of developing the disease than females, and represents the first investigation on the spread of B. burgdorferi among stray dogs in Sicily.

Q fever is an important and widespread zoonotic disease caused by the bacterium Coxiella burnetii, which has been recently reclassified as belonging to the order Legionellales. Q fever in humans is considered an endemic, mostly occupational, disease in several Mediterranean Countries. The infection is often asymptomatic in ruminants but infected animals excrete the bacteria into the environment, mainly during and after parturition or abortion. The aim of this study was to estimate the seroprevalence of C. burnetii infection and related risk factors in Sicilian ovine farms. A total of 6641 serum samples, from sheep belonging to 205 flocks, were examined by Enzyme Linked ImmunoSorbent Assay (ELISA). The seroprevalence at the animal level was 18% (95% CI), while at farm level it was 73.6% (95% CI). Moreover, an evaluation of potential risk factors was carried out on farms, including: farm size, contact with other animals, production type, farm localization and presence of water sources. The multivariate analysis indicated only two factors significantly associated with C. burnetii seropositivity: flock size (χ2 = 10.82; p These findings represent a first piece of information on Coxiella burnetii circulation in the examined areas. There is actually no specific vaccination plan to reduce the infection prevalence in animals; therefore, a better knowledge on the pathogen spreading could be helpful to take adequate preventive measures, especially considering the potential consequences and risks for human public health too.

INTRODUCTION AND OBJECTIVE Little is known about the development of chronic Q fever caused by Coxiella burnetii in occupational risk groups and in the general population in Italy, as well as in many countries in the world. The aim of this study was to highlight the presence of the infection in a sample of workers operating outdoors (but not directly in contact with animals), in three provinces of western Sicily, in order to detect the human seroprevalence and compare the obtained data with those found in animals raised in the same territory. MATERIALS AND METHODS The study included 126 generic seasonal agricultural workers (labourers), 84 male and 42 female; none of whom were aware of any previous contact with Coxiella burnetii. Their immunologic status against Coxiella burnetii was tested through research and titration of both phases I and II specific antibodies (IgG) with an indirect immunofluorescence assay, using anti-antibodies labeled with fluorescein isothiocyanate. All data were statistically analyzed, comparing all positive results from the three provinces through variance analysis, and then comparing human results with those obtained from animals raised in the considered areas, specifically, 1,511 cows, 46 of which were found positive (3.04%), and 3,391 sheep, 548 of which were found positive (16.16%). RESULTS Anti-Coxiella antibodies were found in nine of 42 females sampled (21.4%; 95%CI=[9.0-33.8]) and 21 of 84 males sampled (25.0%; 95%CI=[15.7-34.3]). 60% (18 of 30; 95%CI=[42.5-77.5]) of seropositive samples were positive either for Phase I antigen or for both Phase I and II antigens, representing cases of chronic infection. Applying Spearman's rank correlation, the percentage of seropositive humans was significantly correlated with that observed for sheep (r=1.00; p

Staphylococcus aureus is the major cause of foodborne diseases worldwide. In this retrospective study, 84 S. aureus strains were characterized. The collection comprises 78 strains isolated during 1998 and 2014 from dairy products and tissue samples from livestock bred for dairy production in Sicily. One isolate was obtained from a pet (dog), one from an exotic animal (a circus elephant), and four human isolates were obtained during a severe food poisoning outbreak that occurred in Sicily in 2015. All the strains were characterized by pulsed-field gel electrophoresis (PFGE), for antibiotic resistance and presence of toxin genes. PFGE results showed 10 different pulsotypes, with three relatively frequent and three unique. The antibiotic resistance profiling showed that penicillin G (35.7%) and tetracycline (20.2%) resistance is largely spread. Most isolates contained at least one toxin gene making them a potential threat for public health. Enterotoxin sec gene was observed in 28.6% and seg in 23.8% of the strains, respectively; the human isolates were the only ones to concurrently harbor both seg and sei genes. In addition, 24 isolates were randomly selected and analyzed by multilocus sequence typing. Interestingly, the analysis showed the presence of 12 sequence types (STs), of which 6 were novel. One of them, ST700, was detected in 29% of the isolates and was found to be spread throughout Sicily. ST700 has been present in the island for almost 16 years (1998–2014) and it shows no host preference since it was isolated from different ruminant species. Four human isolates shared both the pulsotype (PT10) and the sequence type (ST9), as well as the virulence genes (seg-sei); this observation suggests that the isolates originated from a single clone, although they were obtained from two different individuals.

We recently reported that Amyloid Precursor Protein (APP) regulates global protein synthesis in a variety of human dividing cells, including non-small cell lung cancer (NSCLC) cells. More specifically, APP depletion causes an increase of both cap- and IRES-dependent translation. Since growth and proliferation are tightly coupled processes, here, we asked what effects artificial downregulation of APP could have elicited in NSCLC cells proliferation. APP depletion caused a G0/G1 arrest through destabilization of the cyclin-C protein and reduced pRb phosphorylation at residues Ser802/811. siRNA to cyclin-C mirrored the cell cycle distribution observed when silencing APP. Cells arrested in G0/G1 (and with augmented global protein synthesis) increased their size and underwent a necrotic cell death due to cell membrane permeabilization. These phenotypes were reversed by overexpression of the APP C-terminal domain, indicating a novel role for APP in regulating early cell cycle entry decisions. It is seems that APP moderates the rate of protein synthesis before the cell clears growth factors- and nutrients-dependent checkpoint in mid G1. Our results raise questions on how such processes interact in the context of (at least) dividing NSCLC cells. The data presented here suggest that APP, although required for G0/G1 transitions, moderates the rate of protein synthesis before the cell fully commits to cell cycle progression following mechanisms, which seem additional to concurrent signals deriving from the PI3-K/Akt/mTORC-1 axis. APP appears to play a central role in regulating cell cycle entry with the rate of protein synthesis; and its loss-of-function causes cell size abnormalities and death.

Background: The diffusion of antimicrobial resistance is a significant concern for public health worldwide. Staphylococcus aureus represents a paradigm microorganism for antibiotic resistance in that resistant strains appear within a decade after the introduction of new antibiotics. Methods: Fourteen S. aureus isolates from human specimens and twenty-one from samples of animal origin, were compared for their antimicrobial resistance and biofilm capability. In addition, they were characterized at the molecular level to detect the antimicrobial resistance mecA gene and genes related with enterotoxin, toxin, and biofilm production. Results: Both phenotypic and molecular analysis showed main differences among human- and animal-derived isolates. Among the human-derived isolates, more multidrug-resistant isolates were detected and mecA gene, enterotoxin, and toxin genes were more prevalent. Different genes involved in biofilm production were detected with bap present only in animal-derived isolates and sasC present in both isolates, however, with a higher prevalence in the human-derived isolates. Biofilm capability was higher in human-derived isolates mainly associated to the sasC gene. Conclusions: The overall results indicate that human S. aureus isolates are more virulent and resistant than the isolates of animal origin randomly selected with no infection anamnesis. This study confirms that selection for more virulent and resistant S. aureus strains is related to the clinical practice.

Brucellosis is a worldwide zoonotic disease. The role of sheep and goats is particularly important as they are considered reservoir animals. Although it has been eradicated in some countries, it continues to be an important disease in many farming areas of the Mediterranean region. The study reports the results of field and microbiological examinations carried out at the Istituto Zooprofilattico Sperimentale della Sicilia over the three year period 2007–2009.

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.

17β-Estradiol (E2) 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 E2 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 E2 responsive since both isoforms of estrogen receptor (ER-α and ER-β) are expressed. Significant sex-related differences have been described in skeletal muscle, although the role played by E2 and the mechanisms underlying it remain to be determined. Here, we demonstrate that E2 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 E2-induced effects require rapid extranuclear signals and the presence of ER-α, whereas no contribution of IGF-I receptor has been observed. In particular, ER-α-dependent Akt activation participates in regulating the first step of myogenic differentiation. Moreover, both receptors mediate the E2-induced activation of p38, which, in turn, affects the expression of myogenin and myosin heavy chain. All together, these data indicate that E2 should be included in the list of skeletal muscle trophic factors.

Estrogen receptors (ERalpha and ERbeta) mediate opposite functions on cancer growth induced by 17beta-estradiol (E2). E2 binding to ERalpha induces a cancer promoting response, whereas E2 binding to ERbeta exerts a protective action against cancer growth. Moreover, E2 can diversely modulate the ERalpha and ERbeta levels intensifying or decreasing their actions in target tissues. Only molecular mechanisms at the root of E2 ability to down-regulate the ERalpha levels are known. Here, we report the first molecular mechanism underlying E2-induced ERbeta up-regulation in DLD-1 colon cancer cells. E2 induces a short term (2 and 4h after stimulation) translation of ERbeta mRNA followed by a late (24h after stimulation) enhanced transcription. Both processes required the E2-induced persistent and palmitoylation-dependent p38/MAPK activation. Overall, our data suggest a finely tuned control exerted by rapid signals on different cellular molecular events important for the protective effects of E2 against colon cancer growth.

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.

This study investigated the prevalence of feline haemoplasma infections in a number of stray cat colonies in Milan, Northern Italy. Blood samples from 260 stray cats were evaluated, with conventional PCR, for the presence of DNA associated with Mycoplasma haemofelis (Mhf) and “Candidatus Mycoplasma haemominutum” (CMhm). Odd ratios (OR) were calculated to identify risk factors for haemoplasma infections. PCR was positive in 86 out of 260 subjects (33.1%), with a prevalence of 10.8% (28/260 cats) for Mhf and 22.3% (58/260 cats) for CMhm. No coinfections were registered. There were significant associations between infections and season of sampling, that is, a negative association between winter sampling and a haemoplasma positive status (OR=0.29, P=0.001), or CMhm positive status (OR=0.29, P=0.01). Haemoplasma infections are common in stray cats in Milan. Thus, domestic cats with outdoor access should be routinely monitored and treated for ectoparasites to minimize risks of disease acquisition. Moreover, as these infections are transmitted via blood, feline blood donors from this area should be screened by PCR and preferably be drawn from a population of indoor cats regularly treated for fleas.

Emerging diseases caused by piroplasms pose a health risk for man and other animals, and domestic cats have been proposed as potential reservoirs for some piroplasm infections. The aim of this study was to identify the frequency of the piroplasmsBabesia microtiandCytauxzoon felisin stray cats from northern Italy and to identify possible risk factors associated with these infections. Blood samples from 260 stray cats enrolled in a trap-neuter-release (TNR) program in northern Italy were examined with conventional PCR for the presence ofBabesia microtiandCytauxzoon felisDNA. No sample (0.0%) tested positive forC. felis, whilstB. microtiDNA was detected in two samples (0.8%). Both infected cats were in good clinical condition and recovered well from the neutering surgery. One of these two cats had a triple coinfection withBabesia microti,CandidatusMycoplasma haemominutum, andAnaplasma phagocytophilum. Evidence presented in this study indicates that the blood borne protozoansBabesia microtiandCytauxzoon felisare not widely distributed in stray cat populations in Milan, northern Italy, and that the significance of cats as a reservoir host forB. microtiin this area is limited.

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 .

Feline vector-borne diseases can be caused by a range of pathogens transmitted by arthropods. Many of these infections have zoonotic implications, and stray cats are potential sentinels for human and pet health. This study investigated the prevalence of selected vector-borne infections in stray colony cats in Milan. Blood samples from 260 stray cats were evaluated, using conventional polymerase chain reaction tests (cPCRs), for the presence of DNA associated with Rickettsia species, Anaplasma phagocytophilum and Ehrlichia species. Positive cPCR results occurred in 127/260 subjects (48.9%; 95% confidence interval [CI] = 40.7–58.1), with a prevalence of 31.9% (83/260, 95% CI = 25.4–39.6) for Rickettsia species, 17.7% (46/260, 95% CI= 13.0–23.6) for A phagocytophilum, and 5.4% (14/260, 95% CI = 2.9–9.0) for Ehrlichia species. There was no statistical association between a positive PCR test for vector-borne infections surveyed and colony location, age, gender, body condition score or complete blood count abnormalities, nor feline immunodeficiency virus, feline leukaemia virus or Toxoplasma gondii status. The only variable linked to positive PCR results was detection of signs of ocular infection and PCR positivity for Rickettsia species ( P = 0.04, odds ratio [OR] = 2.2, 95% CI = 1.1–4.4, P = 0.02). There is a significant prevalence of vector-borne infections with zoonotic potential in urban stray cats in Milan. Thus, dogs and pet cats with outdoor access should be monitored and treated for ectoparasites on a regular basis to minimise risks of disease and the potential transmission of zoonotic agents to people.

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.

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

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.

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