The β-Amyloid (βA) peptide is the major component of senile plaques that are one of the hallmarks of Alzheimer’s Disease (AD). It is well recognized that Aβ exists in multiple assembly states, such as soluble oligomers or insoluble fibrils, which affect neuronal viability and may contribute to disease progression. In particular, common βA-neurotoxic mechanisms are Ca2+ dyshomeostasis, reactive oxygen species (ROS) formation, altered signaling, mitochondrial dysfunction and neuronal death such as necrosis and apoptosis. Recent study shows that the ubiquitin-proteasome pathway play a crucial role in the degradation of short-lived and regulatory proteins that are important in a variety of basic and pathological cellular processes including apoptosis. Guanosine (Guo) is a purine nucleoside present extracellularly in brain that shows a spectrum of biological activities, both under physiological and pathological conditions. Recently it has become recognized that both neurons and glia also release guanine-based purines. However, the role of Guo in AD is still not well established. In this study, we investigated the machanism basis of neuroprotective effects of GUO against Aβ peptide-induced toxicity in neuronal (SH-SY5Y), in terms of mitochondrial dysfunction and translocation of phosphatidylserine (PS), a marker of apoptosis, using MTT and Annexin-V assay, respectively. In particular, treatment of SH-SY5Y cells with GUO (12,5-75 μM) in presence of monomeric βA25-35 (neurotoxic core of Aβ), oligomeric and fibrillar βA1-42 peptides showed a strong dose-dependent inhibitory effects on βA-induced toxic events. The maximum inhibition of mitochondrial function loss and PS translocation was observed with 75 μM of Guo. Subsequently, to investigate whether neuroprotection of Guo can be ascribed to its ability to modulate proteasome activity levels, we used lactacystin, a specific inhibitor of proteasome. We found that the antiapoptotic effects of Guo were completely abolished by lactacystin. To rule out the possibility that this effects resulted from an increase in proteasome activity by Guo, the chymotrypsin-like activity was assessed employing the fluorogenic substrate Z-LLL-AMC. The treatment of SH-SY5Y with Guo (75 μM for 0-6 h) induced a strong increase, in a time-dependent manner, of proteasome activity. In parallel, no increase of ubiquitinated protein levels was observed at similar experimental conditions adopted. We then evaluated an involvement of anti and pro-apoptotic proteins such as Bcl-2, Bad and Bax by western blot analysis. Interestingly, Bax levels decreased after 2 h treatment of SH-SY5Y with Guo. Taken together, these results demonstrate that Guo neuroprotective effects against βA-induced apoptosis are mediated, at least partly, via proteasome activation. In particular, these findings suggest a novel neuroprotective pathway mediated by Guo, which involves a rapid degradation of pro-apoptotic proteins by the proteasome. In conclusion, the present data, raise the possibility that Guo could be used as an agent for the treatment of AD.

High serum levels of Interleukin-6 (IL-6) correlate with poor outcome in breast cancer patients. However no data are available on the relationship between IL-6 and stem/progenitor cells which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in mammospheres (MS), multi-cellular structures enriched in stem/progenitor cells of the mammary gland, and also in MCF-7 breast cancer cells. We show that MS from node invasive breast carcinoma tissues express IL-6 mRNA at higher levels than MS from matched non-neoplastic mammary glands. We find that IL-6 mRNA is detectable only in basal-like breast carcinoma tissues, an aggressive variant showing stem cell features. Our results reveal that IL-6 triggers a Notch-3-dependent up-regulation of the Notch ligand Jagged-1, whose interaction with Notch-3 promotes the growth of MS and MCF-7 derived spheroids. Moreover, IL-6 induces a Notch-3-dependent up-regulation of the carbonic anhydrase IX gene, which promotes a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, an autocrine IL-6 loop relies upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, our data support the hypothesis that IL-6 induces malignant features in Notch-3 expressing, stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

A reduced cancer risk associated with fruit and vegetable phytochemicals initially dictated chemopreventive approaches focused on specific green variety consumption or even single nutrient supplementations. However, these strategies not only failed to provide any health benefits but gave rise to detrimental effects. In parallel, public-health chemoprevention programmes were developed in the USA and Europe to increase whole vegetable consumption. Among these, the National Cancer Institute (NCI) sponsored plan “5 to 9 a day for a better health” was one of the most popular. This campaign promoted wide food choice through the consumption of at least 5 to 9 servings a day of colourful fruits and vegetables. In this study the effects of the diet suggested by NCI on transcription, translation and catalytic activity of both xenobiotic metabolizing (XME) and antioxidant enzymes were studied in the animal model. In fact, the boost of both antioxidant defences and “good” phase-II together with down-regulation of “bad” phase-I XMEs is still considered one of the most widely-used strategies of cancer control. Six male Sprague Dawley rats for each treatment group were used. According to the Italian Society of Human Nutrition, a serving of fruit, vegetables and leafy greens corresponds to 150, 250 and 50 g, respectively, in a 70 kg man. Proportionally, rats received one or five servings of lyophilized onion, tomato, peach, black grape or lettuce – for white, red, yellow, violet or green diet, respectively - or five servings of each green (“5 a day” diet) by oral gavage daily for 10 consecutive days. Liver subcellular fractions were tested for various cytochrome P450 (CYP) linked-monooxygenases, phase-II supported XMEs such as glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UDPGT) as well as for some antioxidant enzymes. Hepatic transcriptional and translational effects were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. dROMs test was used to measure plasmatic oxidative stress. Routine haematochemical parameters were also monitored. While the five servings administration didn’t significantly vary XME catalytic activity, the lower dose caused a complex pattern of CYP inactivation with lettuce exerting particularly strong effects (a loss of up to 43% and 45% for CYP content and CYP2B1/2-linked XME, respectively; P<0.01). “5 a day” supplementation produced the most pronounced modulations (a loss of up to 60% for CYP2E1-linked XME and a reduction of CYP content of 54%; P<0.01). Testosterone hydroxylase activity confirmed these results. RT-PCR and Western blot analysis revealed that the “5 a day” diet XMEs inactivations were a result of both a transcriptional and a translational effect while lettuce didn’t exert such effects. All administrations brought out none or fewer modulation of phase-II supported XMEs. Apart from “5 a day” supplementation and the single serving of lettuce, which strongly induced DT- diaphorase (an increase of up to 141 and 171%, respectively; P<0.01), antioxidant enzymes were not significantly changed. RT-PCR analysis confirmed DT-diaphorase induction brought about by the administration of both “5 a day” diet and a single serving of lettuce. Furthermore, it unmasked a similar result for heme-oxygenase. dROMs test provided insight into a condition of high systemic oxidative stress as a consequence of animal diet supplementation with “5 a day” diet and a single serving of lettuce (an increase of up to 600% and 900%, respectively; P<0.01). Haematochemical parameters were mildly affected by such dietary manipulations. According to the classical chemopreventive theory, these results could be of particular relevance. In fact, even if antioxidant enzymes were only mildly affected, the phase-I inactivating ability of these vegetables would be a worthy strategy to cancer control. However, the recorded systemic considerable amount of reactive oxygen species and the complexity of these enzymes and their functions suggest caution in the widespread use of vegan/vegetarian diets as human chemopreventive strategies. In fact, recent literature rather suggests that only diets rich in fruits and vegetables and poor in certain types of fat, together with moderate caloric intake, could be associated with reduced cancer risk.

No description available.

No description available.

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).

Alzheimer's disease (AD) is probably caused by both genetic and environmental risk factors. The major genetic risk factor is the E4 variant of apolipoprotein E gene called apoE4. Several risk factors for developing AD have been identified including lifestyle, such as dietary habits. The mechanisms behind the AD pathogenesis and the onset of cognitive decline in the AD brain are presently unknown. In this study we wanted to characterize the effects of the interaction between environmental risk factors and apoE genotype on neurodegeneration processes, with particular focus on behavioural studies and neurodegenerative processes at molecular level. Towards this aim, we used 6 months-old apoE4 and apoE3 Target Replacement (TR) mice fed on different diets (high intake of cholesterol and high intake of carbohydrates). These mice were evaluated for learning and memory deficits in spatial reference (Morris Water Maze (MWM)) and contextual learning (Passive Avoidance) tasks, which involve the hippocampus and the amygdala, respectively. From these behavioural studies we found that the initial cognitive impairments manifested as a retention deficit in apoE4 mice fed on high carbohydrate diet. Thus, the genetic risk factor apoE4 genotype associated with a high carbohydrate diet seems to affect cognitive functions in young mice, corroborating the theory that the combination of genetic and environmental risk factors greatly increases the risk of developing AD and leads to an earlier onset of cognitive deficits. The cellular and molecular bases of the cognitive decline in AD are largely unknown. In order to determine the molecular changes for the onset of the early cognitive impairment observed in the behavioural studies, we performed molecular studies, with particular focus on synaptic integrity and Tau phosphorylation. The most relevant finding of our molecular studies showed a significant decrease of Brain-derived Neurotrophic Factor (BDNF) in apoE4 mice fed on high carbohydrate diet. Our results may suggest that BDNF decrease found in apoE4 HS mice could be involved in the earliest impairment in long-term reference memory observed in behavioural studies. The second aim of this thesis was to study possible involvement of leptin in AD. There is growing evidence that leptin has neuroprotective properties in the Central Nervous System (CNS). Recent evidence has shown that leptin and its receptors are widespread in the CNS and may provide neuronal survival signals. However, there are still numerous questions, regarding the molecular mechanism by which leptin acts, that remain unanswered. Thus, given to the importance of the involvement of leptin in AD, we wanted to clarify the function of leptin in the pathogenesis of AD and to investigate if apoE genotype affect leptin levels through studies in vitro, in mice and in human. Our findings suggest that apoE4 TR mice showed an increase of leptin in the brain. Leptin levels are also increased in the cerebral spinal fluid of AD patients and apoE4 carriers with AD have higher levels of leptin than apoE3 carriers. Moreover, leptin seems to be expressed by reactive glial cells in AD brains. In vitro, ApoE4 together with Amyloid beta increases leptin production by microglia and astrocytes. Taken together, all these findings suggest that leptin replacement might not be a good strategy for AD therapy. Our results show that high leptin levels were found in AD brains. These findings suggest that, as high leptin levels do not promote satiety in obese individuals, it might be possible that they do not promote neuroprotection in AD patients. Therefore, we hypothesized that AD brain could suffer from leptin resistance. Further studies will be critical to determine whether or not the central leptin resistance in SNC could affect its potential neuroprotective effects.

Introduction – Although imatinib (IM) is a recognized gold standard in chronic myeloid leukemia (CML) therapy, resistance has emerged in a significant proportion of patients. Aim – The aim of this study was: (1) to investigate the role of genetic variants in genes encoding for IM transporters, as candidate of IM responsiveness and (2) to test the influence of miRNAs on IM response, focusing on efflux transporters. Methods – As a first step, a panel of polymorphisms (SNPs) was genotyped in a subgroup population of 189 patients enrolled in the Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial. The association with cytogenetic response and molecular response (MR) was assessed for each SNP. As a second step, an in vitro IM-resistant model (K-562 CML cell line) was established. miRNAs profiles were analyzed using Taqman arrays and in silico search was performed for miRNAs deregulated after IM treatment. mRNA and protein expression were quantified using TaqMan realtime PCR and Western blotting, respectively. Results – (1) Among Caucasian patients, ABCB1 rs60023214 significantly correlated with complete MR (P = 0.005). Concerning SNPs combination in IM uptake transporters, the associations with treatment outcomes were statistically significant for both major and complete MR (P = 0.005 and P = 0.01, respectively). (2) ABCB1 protein was not expressed under any conditions of treatment, differently from ABCG2. Two deregulated miRNAs, namely miR-212 and miR-328, were identified to be inversely correlated with ABCG2 (r2= 0.57; p=0.03 and r2=0.47; p=0.06, respectively). Experiments of loss and gain of function confirmed the functional influence of these miRNAs on ABCG2. Conclusion – The multiple candidate gene approach identified single and combination of SNPs that can be proposed as predictor of IM response. The in vitro study suggested that IM resistance could be mediated by miRNA-dependent mechanism. Further studies are needed to validate these preliminary findings.

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The detection of Colorectal Cancer (CRC), at early stages, is one of the proven strategies resulting in a higher cure rate. In recent years, several studies have appeared identifying potential cancer markers in serum, plasma and stool in an attempt to improve actual screening procedures. Thus, the aim of the study was (1) Evaluate MN frequency, (2) Evaluate plasma ultrafiltrate capacity to induce MN formation, (3) Evaluate SEPT9 and NOTCH3 promoter methylation profile in peripheral blood lymphocytes from subjects resulted positive to fecal occult blood test and examined by colonoscopy. MN frequency was significantly higher in subjects with histological diagnosis of CRC and adenoma than control (p ≤ 0.001 and p ≤ 0.01, respectively). About, CF-MN analysis, a statistically significant difference was observed between CRC and control (p ≤ 0.05). On the other hand, SEPT9 and NOTCH3 promoter methylation status was significantly lower in CRC subjects than controls; additionally, NOTCH3 promoter methylation status was significantly lower in CRC subjects than adenoma subjects (p ≤ 0.01). The results obtained allow conclude that MN frequency varies according CRC pathologic status and, together with other variables, is a valid biomarker for adenoma and CRC risk. Additionally, the plasma of patients affected with CRC not only serve as a biomarker for oxidative stress but also as biomarker of genetic damage correlated with the carcinogenic process that verifies in colon-rectum. SEPT9 and NOTCH3 promoter methylation status, at peripheral blood level, varies according hystopathological changes observed in colon-rectum, suggesting that promoter methylation profile of these genes could be a reliable biomarker for CRC risk.