Mario Arciello

Effects of the Olive-Derived Polyphenol Oleuropein on Human Health

The use of the products derived from the olive tree on human health dates back centuries. In several civilizations, the olive tree had and still has a very strong cultural and religious symbolism. Notably, the official seal and emblem of the World Health Organization features the rod of Asclepius over a world map surrounded by olive tree branches, chosen as a symbol of peace and health. Recently, accumulating experimental, clinical and epidemiological data have provided support to the traditional beliefs of the beneficial effect provided by olive derivates. In particular, the polyphenols present in olive leaves, olives, virgin (unrefined) olive oil and olive mill waste are potent antioxidant and radical scavengers with anti-tumor and anti-inflammatory properties. Here, we review the positive impact on human health of oleuropein, the most prevalent polyphenol present in olives. In addition, we provide data collected in our laboratory on the role of oleuropein in counteracting lipid accumulation in a mouse model of non-alcoholic fatty liver disease.

Features of ceruloplasmin in the cerebrospinal fluid of Alzheimer’s disease patients

The level of the apo-form of the copper enzyme ceruloplasmin (CP) is an established peripheral marker in diseases associated with copper imbalance. In view of the proposal that disturbances of copper homeostasis may contribute to neurodegeneration associated with Alzheimer’s disease (AD), the present work investigates, by Western blot and non-reducing SDS-PAGE followed by activity staining, the features of CP protein, and the copper/CP relationship in cerebrospinal fluid (CSF) and serum of AD patients. Results show that only a fraction of total copper is associated with CP in the CSF, at variance with serum, both in affected and in healthy individuals. Furthermore, a conspicuous amount of apo-ceruloplasmin and a decrease of CP oxidase activity characterize the CSF of the affected individuals, and confirm that an impairment of copper metabolism occurs in their central nervous system. In the CSF of AD patients the decrease of active CP, associated with the increase in the pool of copper not sequestered by this protein, may play a role in the neurodegenerative process.

MicroRNAs in nonalcoholic fatty liver disease: novel biomarkers and prognostic tools during the transition from steatosis to hepatocarcinoma.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic-related disorder ranging from steatosis to steatohepatitis, which may progress to cirrhosis and hepatocellular carcinoma (HCC). The influence of NAFLD on HCC development has drawn attention in recent years. HCC is one of the most common malignant tumors and the third highest cause of cancer-related death. HCC is frequently diagnosed late in the disease course, and patients prognosis is usually poor. Early diagnosis and identification of the correct stage of liver damage during NAFLD progression can contribute to more effective therapeutic interventions, improving patient outcomes. Therefore, scientists are always searching for new sensitive and reliable markers that could be analysed through minimally invasive tests. MicroRNAs are short noncoding RNAs that act as posttranscriptional regulators of gene expression. Several studies identified specific miRNA expression profiles associated to different histological features of NAFLD. Thus, miRNAs are receiving growing attention as useful noninvasive diagnostic markers to follow the progression of NAFLD and to identify novel therapeutic targets. This review focuses on the current knowledge of the miRNAs involved in NAFLD and related HCC development, highlighting their diagnostic and prognostic value for the screening of NAFLD patients.

Inactivation of cytochrome c oxidase by mutant SOD1s in mouse motoneuronal NSC-34 cells is independent from copper availability but is because of nitric oxide

J. Neurochem. (2010) 112, 183–192.

Mitochondrial Dysfunctions and Altered Metals Homeostasis: New Weapons to Counteract HCV-Related Oxidative Stress

The hepatitis C virus (HCV) infection produces several pathological effects in host organism through a wide number of molecular/metabolic pathways. Today it is worldwide accepted that oxidative stress actively participates in HCV pathology, even if the antioxidant therapies adopted until now were scarcely effective. HCV causes oxidative stress by a variety of processes, such as activation of prooxidant enzymes, weakening of antioxidant defenses, organelle damage, and metals unbalance. A focal point, in HCV-related oxidative stress onset, is the mitochondrial failure. These organelles, known to be the “power plants” of cells, have a central role in energy production, metabolism, and metals homeostasis, mainly copper and iron. Furthermore, mitochondria are direct viral targets, because many HCV proteins associate with them. They are the main intracellular free radicals producers and targets. Mitochondrial dysfunctions play a key role in the metal imbalance. This event, today overlooked, is involved in oxidative stress exacerbation and may play a role in HCV life cycle. In this review, we summarize the role of mitochondria and metals in HCV-related oxidative stress, highlighting the need to consider their deregulation in the HCV-related liver damage and in the antiviral management of patients.

Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.

Environmental pollution: a tangible risk for NAFLD pathogenesis

The liver is crucial for human life, and the health of this organ often mirrors the health of the individual. The liver can be the target of several diseases, the most prevalent of which, as a consequence of development and changes in human lifestyles, is the nonalcoholic fatty liver disease (NAFLD). NAFLD is a multifactorial disease that embraces many histo-pathologic conditions and is highly linked to metabolic derangements. Technological progress and industrialization have also had the consequence of releasing pollutants in the environment, for instance pesticides or solvents, as well as by-products of discharge, such as the particulate matter. In the last decade, a growing body of evidence has emerged, shedding light on the potential impact of environmental pollutants on liver health and, in particular, on NAFLD occurrence. These contaminants have a great steatogenic potential and need to be considered as tangible NAFLD risk factors. There is an urgent need for a deeper comprehension of their molecular mechanisms of action, as well as for new lines of intervention to reduce their worldwide diffusion. This review wishes to sensitize the community to the effects of several environmental pollutants on liver health.

Copper depletion increases the mitochondrial-associated SOD1 in neuronal cells

The role of copper in the toxicity of mutant copper-dependent enzyme superoxide dismutase (SOD1) found in patients affected with the familial form of amyotrophic lateral sclerosis (fALS) is widely debated. Here we report that treatment of human neuroblastoma cells SH-SY5Y with a specific copper chelator, triethylene tetramine (Trien) induces the decrease of intracellular copper level, paralleled by decreased activity of SOD1. A comparable effect is observed in mouse NSC-34-derived cells, a motoneuronal model, transfected for the inducible expression of either wild-type or G93A mutant human SOD1, one of the mutations associated with fALS. In both cell types, the drop of SOD1 activity is not paralleled by the same extent of decrease in SOD1 protein content. This discrepancy can be explained by the occurrence of a fraction of copper-free SOD1 upon copper depletion, which is demonstrated by the partial recovery of the enzyme activity after the addition of copper sulphate to homogenates of SH-SY5Y cells. Furthermore, copper depletion produces the enrichment of the physiological mitochondrial fraction of SOD1 protein, in both cells models. However, increasing the fraction of mitochondrial, possibly copper-free, mutant human SOD1 does not further alter mitochondrial morphology in NSC-34-derived cells. Thus, copper deficiency is not a factor which may worsen mitochondrial damage, which is one of the earliest events in fALS associated with mutant SOD1.

Protective Effect of the Y220C Mutant p53 Against Steatosis: Good News?: ROLE OF THE Y220C MUTANT p53 IN NAFLD

Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, which may progress to fibrosis, and cirrhosis, leading eventually to hepatocarcinoma development. Recently, cases of hepatocarcinoma have been diagnosed in steatotic patients without nonalcoholic steatohepatitis (NASH) and cirrhosis. The p53 protein, besides its function as tumor suppressor, is emerging as an important regulator of cellular metabolism, but its role in steatosis remains unclear. We induced steatosis in HepG2 (wt‐p53) and Huh7.5.1 (Y220C‐mutant p53) cells using free fatty acids. We observed a different modulation of p53, different intracellular lipid content, and similar down‐regulation of the de novo lipid synthesis genes but opposite modulation of the fatty acid β‐oxidation pathway between HepG2 and Huh7.5.1. Accordingly, we found a diverse amount of apoptosis and reactive oxygen species between the two cell lines. Transfection of the wt‐p53 in Huh7.5.1 cells reverted the different lipid metabolism behavior observed in these cells. In conclusion, unlike the wt‐p53, the Y220C mutant provides a specific protection against steatosis and potentially against its progression. Our findings highlight for the first time an unknown role of a p53 mutant in the setting of steatosis. Being this mutation very frequent in human cancers, this study could be a breakthrough in explaining the occurrence of hepatocarcinoma in steatotic patients without NASH and cirrhosis. J. Cell. Physiol. 229: 1182–1192, 2014.

Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT?

To the Editor: We read with great interest the article by Akkari et al. [1], who evaluated the EMT induction and correlated oncogenic transformation induced by HCV protein NS5A in primary hepatocyte precursors. Epithelial–mesenchymal transition (EMT) is a biological process occurring during tissue development and regeneration, moreover, it is known to be involved in tumor progression and metastasis. The EMT is a phenomenon, which well represents the cellular plasticity, i.e., the ability of polarized epithelial cells, which through multiple biochemical changes acquire a mesenchymal cell phenotype. These cells exhibit loss of cell adhesion, enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a great ability to produce extracellular matrix (ECM) components. Several molecular processes are involved in EMT onset and progression. Among them we have to mention: activation of transcription factors (e.g., Snail, Twist, etc.), expression of specific cell-surface proteins, remodelling of cytoskeletal components, production of ECM-degrading enzymes and changes in the expression of specific microRNAs (e.g., microRNA 200 family). Most of them are commonly used as biomarkers to highlight a cellular EMT [2,3]. Akkari et al. have evaluated how a viral protein can alter the typical epithelial architecture of hepatic cells. In particular, they demonstrated that the ectopic expression of hepatitis C virus (HCV) non-structural protein 5a (NS5A) is able to induce EMT in bipotential mouse embryonic liver (BMEL) cells which acquire mesenchymal phenotype, increased motility and invasiveness, and are featured by the downregulation of epithelial markers (e.g., E-Cadherin) and the upregulation of mesenchymal markers (like Vimentin and Twist2). They also confirmed their results in an in vivo xenograft mouse model. To unravel the molecular mechanisms involved in EMT occurrence, they focused their experimental investigation on transforming growth factor (TGF)-b, a main cytokine involved in EMT induction. They have shown that NS5A is not responsible for TGF-b pathway activation but it acts in a synergistic manner with the cytokine in inducing EMT. Furthermore, they also screened, in their in vitro model, the modulation of expression of EMT biomarkers, such as Snail, Slug, Zeb and Twist 1/2. They demonstrated that only Twist2 expression was specifically increased by NS5A protein, and that, accordingly, through short hairpin RNA against Twist2, they were able to counteract EMT occurrence. Interestingly, the TGF-b treatment of cells lacking of Twist2 is able to promote EMT in any case, suggesting that NS5A and TGF-b trigger EMT by distinct pathways and thus identifying Twist2 as an NS5A EMT specific effector.