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Janda E.,University of Catanzaro | Janda E.,Interregional Research Center for Food Safety and Health | Lascala A.,University of Catanzaro | Carresi C.,University of Catanzaro | And 11 more authors.
Autophagy | Year: 2015

Oxidative stress (OS) stimulates autophagy in different cellular systems, but it remains controversial if this rule can be generalized. We have analyzed the effect of chronic OS induced by the parkinsonian toxin paraquat (PQ) on autophagy in astrocytoma cells and primary astrocytes, which represent the first cellular target of neurotoxins in the brain. PQ decreased the basal levels of LC3-II and LC3-positive vesicles, and its colocalization with lysosomal markers, both in the absence and presence of chloroquine. This was paralleled by increased number and size of SQSTM1/p62 aggregates. Downregulation of autophagy was also observed in cells chronically exposed to hydrogen peroxide or nonlethal concentrations of PQ, and it was associated with a reduced astrocyte capability to protect dopaminergic cells from OS in co-cultures. Surprisingly, PQ treatment led to inhibition of MTOR, activation of MAPK8/JNK1 and MAPK1/ERK2- MAPK3/ERK1 and upregulation of BECN1/Beclin 1 expression, all signals typically correlating with induction of autophagy. Reduction of OS by NMDPEF, a specific NQO2 inhibitor, but not by N-acetylcysteine, abrogated the inhibitory effect of PQ and restored autophagic flux. Activation of NQO2 by PQ or menadione and genetic manipulation of its expression confirmed the role of this enzyme in the inhibitory action of PQ on autophagy. PQ did not induce NFE2L2/NRF2, but when it was co-administered with NMDPEF NFE2L2 activity was enhanced in a SQSTM1-independent fashion. Thus, a prolonged OS in astrocytes inhibits LC3 lipidation and impairs autophagosome formation and autophagic flux, in spite of concomitant activation of several pro-autophagic signals. These findings outline an unanticipated neuroprotective role of astrocyte autophagy and identify in NQO2 a novel pharmacological target for its positive modulation. © 2015, Taylor and Francis Group, LLC.


Parafati M.,University of Catanzaro | Parafati M.,University of Piemonte Orientale | Lascala A.,University of Catanzaro | Morittu V.M.,University of Catanzaro | And 12 more authors.
Journal of Nutritional Biochemistry | Year: 2015

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries. Defective autophagy of lipid droplets (LDs) in hepatocytes, also known as lipophagy, has recently been identified as a possible pathophysiological mechanism of NAFLD. Experimental and epidemiological evidence suggests that dietary polyphenols may prevent NAFLD. To address this hypothesis and analyze the underlying mechanisms, we supplemented bergamot polyphenol fraction (BPF) to cafeteria (CAF) diet-fed rats, a good model for pediatric metabolic syndrome and NAFLD. BPF treatment (50 mg/kg/day supplemented with drinking water, 3 months) potently counteracted the pathogenic increase of serum triglycerides and had moderate effects on blood glucose and obesity in this animal model. Importantly, BPF strongly reduced hepatic steatosis as documented by a significant decrease in total lipid content (-41.3%±12% S.E.M.), ultrasound examination and histological analysis of liver sections. The morphometric analysis of oil-red stained sections confirmed a dramatic reduction in LDs parameters such as total LD area (48.5%±15% S.E.M.) in hepatocytes from CAF+BPF rats. BPF-treated livers showed increased levels of LC3 and Beclin 1 and reduction of SQSTM1/p62, suggesting autophagy stimulation. Consistent with BPF stimulation of lipophagy, higher levels of LC3II were found in the LD subcellular fractions of BPF-expose livers. This study demonstrates that the liver and its lipid metabolism are the main targets of bergamot flavonoids, supporting the concept that supplementation of BPF is an effective strategy to prevent NAFLD. © 2015 Elsevier Inc..


Malara N.M.,Italian Institute of Technology | Givigliano F.,University of Catanzaro | Trunzo V.,University of Catanzaro | Macrina L.,University of Milan | And 12 more authors.
Journal of biological regulators and homeostatic agents | Year: 2014

The clinical development of locally and advanced non-small cell lung cancer (NSCLC) suffers from a lack of biomarkers as a guide in the selection of optimal prognostic prediction. Circulating Tumour Cells (CTCs) are correlated to prognosis and show efficacy in cancer monitoring in patients. However, their enumeration alone might be inadequate; it might also be critical to understand the viability, the apoptotic state and the kinetics of these cells. Here, we report what we believe to be a new and selective approach to visually detect tumour specific CTCs. Firstly, using labelled human lung cancer cells, we detected a specific density interval in which NSCL-CTCs were concentrated. Secondly, to better characterize CTCs in respect to their heterogeneous composition and tumour reference, blood and tumour biopsy were performed on specimens taken from the same patient. The approach consisted in comparing phenotype profile of CTCs, and their progenitor Tumour Stem Cells, (TSCs). Moreover, NSCL-CTCs were cultivated in short-time human cultures to provide response to drug sensitivity. Our bimodal approach allowed to reveal two items. Firstly, that one part of a tumour, proximal to the bronchial structure, displays a predominance of CD133+. Secondly, specific NSCL-CTCs Epithelial Cell Adhesion Molecule (EpCAM)+CD29+ can be used as a negative prognostic factor as well the high expression of CTCs EpCAM+. These data were confirmed by drug-sensitivity tests, in vitro, and by the survival curves, in vivo.


Muscoli C.,University of Catanzaro | Muscoli C.,Interregional Research Center for Food Safety and Health | Muscoli C.,Drug Center | Dagostino C.,Drug Center | And 16 more authors.
Mediators of Inflammation | Year: 2013

Activation of the N-methyl-D-aspartate receptor (NMDAR) is fundamental in the development of hyperalgesia. Overactivation of this receptor releases superoxide and nitric oxide that, in turn, forms peroxynitrite (PN). All of these events have been linked to neurotoxicity. The receptors and enzymes involved in the handling of glutamate pathway - specifically NMDARs, glutamate transporter, and glutamine synthase (GS) - have key tyrosine residues which are targets of the nitration process causing subsequent function modification. Our results demonstrate that the thermal hyperalgesia induced by intrathecal administration of NMDA is associated with spinal nitration of GluN1 and GluN2B receptor subunits, GS, that normally convert glutamate into nontoxic glutamine, and glutamate transporter GLT1. Intrathecal injection of PN decomposition catalyst FeTM-4-PyP5+ prevents nitration and overall inhibits NMDA-mediated thermal hyperalgesia. Our study supports the hypothesis that nitration of key proteins involved in the regulation of glutamate transmission is a crucial pathway used by PN to mediate the development and maintenance of NMDA-mediated thermal hyperalgesia. The broader implication of our findings reinforces the notion that free radicals may contribute to various forms of pain events and the importance of the development of new pharmacological tool that can modulate the glutamate transmission without blocking its actions directly. © 2013 Carolina Muscoli et al.


Muscoli C.,University of Catanzaro | Muscoli C.,Interregional Research Center for Food Safety and Health | Muscoli C.,Drug Center | Lauro F.,University of Catanzaro | And 25 more authors.
Journal of Biological Regulators and Homeostatic Agents | Year: 2014

Morphine and related opioid drugs are currently the major drugs for severe pain. Their clinical utility is limited in the management of severe cancer pain due to the rapid development of tolerance. Restoring opioid efficacy is therefore of great clinical importance. A great body of evidence suggests the key role of free radicals and posttranslational modulation in the development of tolerance to the analgesic activity of morphine. Epidemiological studies have shown a relationship between the Mediterranean diet and a reduced incidence of pathologies such as coronary heart disease and cancer. A central hallmark of this diet is the high consumption of virgin olive oil as the main source of fat which contains antioxidant components in the non-saponifiable fraction, including phenolic compounds absent in seed oils. Here, we show that in a rodent model of opiate tolerance, removal of the free radicals with phenolic compounds of olive oil such as hydroxytyrosol and oleuropein re-instates the analgesic action of morphine. Chronic injection of morphine in mice led to the development of tolerance and this was associated with increased nitrotyrosin and malonildialdeide (MDA) formation together with nitration and deactivation of MnSOD in the spinal cord. Removal of free radicals by hydroxytyrosol and oleuropein blocked morphine tolerance by inhibiting nitration and MDA formation and replacing the MnSOD activity. The phenolic fraction of virgin olive oil exerts antioxidant activities in vivo and free radicals generation occurring during chronic morphine administration play a crucial role in the development of opioid tolerance. Our data suggest novel therapeutic approach in the management of chronic cancer pain, in particular for those patients who require long-term opioid treatment for pain relief without development of tolerance. Copyright © by BIOLIFE, s.a.s.


Tassone E.J.,University of Catanzaro | Perticone M.,University of Catanzaro | Sciacqua A.,University of Catanzaro | Mafrici S.F.,University of Catanzaro | And 7 more authors.
Acta Diabetologica | Year: 2015

Current guidelines suggest the use of low doses of acetylsalicylic acid (ASA) for patients with diabetes mellitus (DM) in primary prevention. However, the evidences demonstrating the beneficial effect of ASA in primary prevention are conflicting. In this pilot study, we evaluated in a group of diabetic patients, in primary prevention, the impact of ASA treatment on oxidative stress and vascular function. We enrolled 22 newly diagnosed diabetic patients, without any previous clinical evidence of cardiovascular disease, to receive, in primary prevention, ASA (100 mg/daily). We tested, in basal condition, after 4 weeks of ASA administration and after 4 weeks of pharmacological washout, the impact of ASA treatment on endothelial function, assessed by a semipletysmographic method, measuring the main oxidative stress parameters related to it. As expected, after 4 weeks of treatment, ASA induced a significant reduction of plasma thromboxane-A2, as a consequence of cyclooxygenase-1 inhibition. By contrast, ASA significantly increased the plasma and urine 8-iso-PGF2α, a well-known prothrombotic molecule, parallel to an increase of plasma NOX2 levels. The enhancement of this oxidative pathway is associated with a significant impairment of endothelial vasodilation, assessed by reactive hyperemia index (RHI). The pharmacological washout reverted all parameters to basal condition. Our findings suggest that ASA utilization for primary prevention in diabetic patients causes a significant increase of oxidative stress burden impairing the vascular function. Present data, if confirmed on a larger population, could permanently discourage the use of the ASA for the primary prevention in patients with DM. © 2014, The Author(s).

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