LINES OF RESEARCH PROF.SSA BALSANO
OLEUROPEIN OVERRIDES DAMAGE IN THE PRESENCE OF LIVER STEATOSIS
Non-alcoholic fatty liver disease (NAFLD), or the excessive accumulation of lipids in the liver not associated with alcohol abuse, is currently the most common liver disease in the world. In Western countries, the prevalence of NAFLD in the general population is 20-30%. NAFLD encompasses a large group of liver diseases, ranging from fatty liver (fatty liver) to NASH (nonalcoholic steatohepatitis) to cirrhosis, the most advanced stage of liver disease. Nonalcoholic steatohepatitis (NASH) is characterized by hepatocellular swelling, inflammation and oxidative stress, which are the main causes of NAFLD progression. These changes can stimulate the stellate cells of the liver causing fibrosis. In advanced stages, nonalcoholic steatohepatitis can cause cirrhosis and portal hypertension. Although it has been widely demonstrated that the progression of NAFLD is negatively influenced by poor lifestyles (high caloric intake, sedentary lifestyle), patients often do not change unhealthy behaviors or, in any case, take a long time to do so. Unfortunately, if the proper approach is not taken, NASH-related liver injury can progress, leading to liver cirrhosis and even hepatocellular carcinoma. In this context, it may be important to identify anti-inflammatory and antioxidant molecules capable of counteracting the progression of this disease.
Reactive oxygen species (ROS) are generated within cells by metabolic activities involved in the response to stress and inflammation. A compromised antioxidant defense system promotes the accumulation of oxidative stress by inducing tissue damage and promoting a pro-inflammatory state that leads to the secretion of inflammatory cytokines, such as: IL1-α, IL-6, IL-2, TNF-α and G -CSF.
Liver fat metabolism depends on mitochondrial activity and generates ATP but also reactive oxygen species (ROS). Considering the pivotal role of mitochondria in providing energy, it is not surprising that their function and behavior are crucial to the physiology of cellular health and that "mitochondrial dysfunction" has been implicated in a wide range of chronic inflammatory and related disorders as well as in NAFLD. Mitochondrial alterations negatively affect the balance between antioxidant and pro-oxidant systems, inducing the accumulation of ROS and consequently the progression of liver damage.
Sirtuins are a family of NAD + dependent deacetylases that mediate important metabolic pathways and regulate stress response mechanisms, including oxidative stress, especially during calorie restriction. It has been shown that, during the progression of NAFLD, two particular sirtuins: sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3) are related to metabolic changes, inflammatory signals and defense against oxidative stress. However, although both sirtuins have been associated with NAFLD / NASH progression, their specific roles still remain not fully defined.
Natural phytochemicals such as polyphenols are associated with the reduction of oxidative stress and the reduction of inflammation in chronic inflammatory diseases. Oleuropein (ole), a polyphenolic compound, is mainly present in the leaves and fruits of the olive tree and has both antioxidant and anti-inflammatory effects. Olive leaf extracts have therapeutic effects. The Ole has hepato / cardioprotective, antioxidant, anti-inflammatory, antihypertensive and anticancer properties.
However, the antioxidant properties of the Ole are still to be clarified, as they show conflicting mechanisms. In fact, in thyroid cancer cells, Ole is able to inhibit cell proliferation by reducing ROS levels, while in prostate cancer cells it shows pro-oxidant effects. Furthermore, it exhibits opposite behaviors under normal conditions compared to cancer cells. It is interesting to note that hydroxytyrosol, a metabolite of Ole, has an aromatic ring in its structure, which is a common feature of estrogens. In accordance with this aspect, the aforesaid phenolic compound has gender specific effects, probably at the level of binding with the estrogen receptor (ER).
According to literature data, it is clear that further studies are needed to determine the exact mechanism by which Ole exerts its antioxidant effects.
We wondered if, in the presence of hepatic steatosis, the improvement in liver damage related to oleuropein intake could be associated with an increase in antioxidant defenses, evaluating the activity of the enzymes of the SOD family and catalase (CAT) and there was a correlation with the enhancement of the autophagic response.
Porcu C, Sideri S, Martini M, Cocomazzi A, Galli A, Tarantino G, Balsano C.
Int J Mol Sci. 2018 Dec8;19(12):3948. doi: 10.3390/ijms19123948.
Balsano C, Porcu C, Sideri S.
Metallomics, 2018 Dec 12;10(12):1712-1722. doi: 10.1039/c8mt00219c
Barbaro B, Toietta G, Maggio R, Arciello M, Tarocchi M, Galli A, Balsano C.
Int J Mol Sci. 2014 Oct 14;15(10):18508-24. doi: 10.3390/ijms151018508.
INHIBITION OF FOCAL ADHESION KINASE (FAK) CONTROLS GROWTH OF PRIMITIVE LIVER CANCER (HCC)
In recent years, numerous studies have shown a substantial increase in the number of cases with NAFLD-related HCC, both in the presence and absence of liver cirrhosis.
Regardless of the etiology, during the hepatocarcinogenesis process, numerous signal transduction and gene expression control pathways are altered. However, the molecular mechanisms underlying the progression of NAFLD towards its pro-carcinogenic phenotype are not yet fully understood.
Among the numerous proteins involved in the onset and progression of HCC, Focal Adhesion Kinase (FAK) protein is often (26% of cases) found over-expressed or hyper-phosphorylated in cancer patients, suggesting a key role in this protein in the induction of the neoplastic phenotype. A recent study has shown a positive correlation between the over-expression of FAK, the function of methylase EZH2 and the aggressiveness of the tumor in patients with endometrial neoplasia, suggesting a potential link between the two proteins.
The study carried out has highlighted the correlations that exist between FAK and EZH2 proteins in in vitro and in vivo models of NAFLD or HCC.
The experiments conducted, both on in vitro models and on an in vivo model of NAFLD, showed a significant increase in the FAK protein, its phosphorylation in tyrosine 397 and its target protein Paxillin. However, the silencing of FAK in a human hepatoblastoma cell line was found to be associated with the activation of the de novo lipogenesis process and with a consequent increase in intra-cellular lipid accumulation. On the other hand, experiments conducted on a mouse model of progression of NAFLD towards HCC showed that levels of FAK and its phosphorylation in tyrosine 397 increased in parallel with the progression of liver damage up to the onset of tumor nodules. Finally, we highlighted the involvement of EZH2 in NAFLD, reporting an inverse correlation between EZH2 expression and the degree of severity of the disease.
Finally, a mouse model of hepatocarcinoma was used to characterize the role of FAK in hepatocarcinogenesis. In particular, our data demonstrate that silencing of FAK dramatically reduces tumor growth that also expresses lower levels of EZH2. Similarly, in vitro studies have highlighted the anti-proliferative / pro-apoptotic action of FAK silencing in HCC cells.
Our experimental approach has allowed us to demonstrate the key role of FAK in the development of NAFLD and their involvement in the progression of the latter in hepatocarcinoma. We have provided evidence of the existence of an inter-connection between the FAK and EZH2 proteins, suggesting p53 and E2F2 / 3 as possible mediators.
- Focal adhesion kinase depletion reduces human hepatocellular carcinoma growth by repressing enhancer of zeste homolog 2.
Gnani D, Romito I, Artuso S, Chierici M, De Stefanis C, Panera N, Crudele A, Ceccarelli S, Carcarino E, D'Oria V, Porru M, Giorda E, Ferrari K, Miele L, Villa E, Balsano C, Pasini D, Furlanello C, Locatelli F, Nobili V, Rota R, Leonetti C, Alisi A.
Cell Death Differ. 2017 May;24(5):889-902. doi: 10.1038/cdd.2017.34. Epub 2017 Mar 24.
- Focal adhesion kinase (FAK) mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV)-infected cells.
Alisi A, Arciello M, Petrini S, Conti B, Missale G, Balsano C.
PLoS One. 2012;7(8):e44147. doi: 10.1371/journal.pone.0044147. Epub 2012 Aug 28.
MOLECULAR MECHANISMS INDUCED BY HCV IN RELATION TO ITS PRO-FIBROTIC AND ONCOGENIC POTENTIAL.
Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and progression, which may lead to hepatocellular carcinoma (HCC) onset. The pivotal role of hepatic stellate cells (HSCs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the involvement of HCV in this pathologic process is not fully understood. However, the network of molecular interactions connecting HCV with several biological processes, including proliferation, inflammation, cytoskeleton and ECM remodelling is emerging as one of its master regulator. Through an “in vitro” model of HCV infection we have investigated the pro-invasive and pro-fibrogenic effects of HCV on both HCC and HCS cell lines. The HCV infection of HCC cell lines causes the occurrence of an aggressive phenotype characterized by a decreased adhesion, increased migration and invasiveness ability of these cells. The treatment of HSCs with a conditioned medium derived by HCV-infected cells causes an increase in cell proliferation and the occurrence of a fibrogenic profile, as highlighted by cytoskeletal remodeling and the increased release of hyaluronic acid (HA). Our data, in particular, show that FAK (Focal Adhesion Kinase) activation in infected hepatocytes is a key process in HCV-dependent hepatocarcinogenesis. In fact, through the up-regulation and the over-expression of this kinase, HCV promotes the production and release of TNF-alpha (Tumor Necrosis Factor α), a cytokine that is a main fibrogenesis mediator. This molecule is able to activate HSCs. Our findings demonstrated that HCV, through FAK activation, is able to increase the invasiveness of HCCs and, at the same time, to promote fibrogenic phenotype in HSCs.
- Conti B, Minutolo A, Arciello M, Balsano C. Are Hedgehog and Wnt/β-catenin pathways involved in hepatitis C virus-mediated EMT? J Hepatol. 2013 Mar;58(3):636-7.
- Alisi A, Arciello M, Petrini S, Conti B, Missale G, Balsano C. Focal adhesion kinase (FAK) mediates the induction of pro-oncogenic and fibrogenic phenotypes in hepatitis C virus (HCV)-infected cells. PLoS One. 2012; 7(8): 44147.
- Alisi A, Tomaselli S, Balsano C, Gallo A. Hepatitis C virus therapeutics: editing enzymes promising therapeutic targets? Hepatology. 2011 Aug;54(2):742
- Balsano C, Conti B, Arciello M. Regarding: epithelial-mesenchymal transition induced by hepatitis C virus core protein in cholangiocarcinoma. Ann SurgOncol. 2011 Mar;18(3):896.
ANALYSIS OF FATTY LIVER DISEASE DURING HCV INFECTION.
Hepatic steatosis is a common histological feature of patients infected by Hepatitis C virus (HCV). Steatosis occurs more frequently in HCV patients (72%) than in those affected by other common chronic liver diseases like Hepatitis B virus (HBV) or autoimmune hepatitis. Several data suggest a direct involvement of HCV in the pathogenesis of steatosis; this leads to a significant progression of fibrosis and to a reduced response to antiviral therapy. Furthermore, HCV-induced oxidative stress (OS) seems to play a key role in the alteration of lipid metabolism. Data obtained from our previous studies, performed on NAFLD and HCV patients, showed that infected patients are featured by an increased plasma levels of two oxysterols, 7β-hydroxycholesterol (7β-OH) and 7-ketocholesterol (7-K), two non-enzymatic products of cholesterol peroxidation. These molecules are ligands and agonists of Liver X Receptors (LXRs), nuclear receptors involved in several metabolic processes like glucose metabolism, lipid and cholesterol metabolism as well as in cholesterol transport. In contrast to what we have observed in the NAFLD cohort, HCV patients showed an inverse correlation between oxysterols and LDL-cholesterol. Our results not only demonstrate the impact of infection on lipid metabolism, but also highlight the significant metabolic differences that underlie the onset of HCV-related liver steatosis. Furthermore, by Real-Time PCR, we are analyzing the adipogenetic expression profile in HCC-infected cells. These investigations reveal a metabolic alteration induced by virus and its close connection with pathways involved in hepatic cell proliferation and transformation.
- Comparcola D, Nobili V, Alisi A, Balsano C. Effect of treatment with polyunsaturated fatty acids on HCV- or diet-induced fatty liver.J Hepatol. 2011 Jun;54(6):1325-6.
- Arciello M, Petta S, Leoni V, Iannucci G, Labbadia G, Cammà C, Craxì A, Balsano C. Inverse correlation between plasma oxysterol and LDL-cholesterol levels in hepatitis C virus-infected patients. Dig Liver Dis. 2012 Mar;44(3):245-50
ROLE OF CHOLESTEROL METABOLISM IN THE MODULATION OF THE IMMUNE RESPONSE IN THE COURSE OF CHRONIC HEPATITIS C VIRUS INFECTION
Chronic HCV infection is associated with both hepatic and extrahepatic manifestations, such as autoimmunity disorders. The progression of many autoimmune diseases is associated with the unbalance between Th17 cells and regulatory T (Treg) cells, a main immunoregulatory subsets involved in immune-mediated diseases. Several studies report that the Liver X Receptors (LXRs), involved in cholesterol homeostasis, influence Th17 cell differentiation and autoimmunity. Serum levels of oxysterols, specific ligands of LXRs and products of cholesterol oxidation, have been described to be higher in chronic hepatitis C (CHC) than Non-Alcoholic Fatty Liver Disease (NAFLD) patients. Furthermore, it has been reported a direct interaction between HCV-core protein and Retinoid X Receptor (RXR) alpha, the heterodimeric partner of LXRs, potentially deregulating the LXR activity during HCV infection.
On these bases, since cholesterol could represent a key element in regulating the Th17 cell differentiation and the onset of HCV-related autoimmune diseases, we will evaluate the impact of its reduced dietary intake on the immune system during this liver disease. To this aim we evaluated the impact of a Normocaloric Low Cholesterol Diet (NLCD) on modulation of peripheral Th17/Treg balance in adult CHC patients compared to control group (NAFLD/NASH patients). After 30 day diet, CHC patients showed a substantial reduction in the frequency of Th17 cells correlated with a significant decrease in serum levels of the cytokines Th17-related. At the same time, we have found an increase in the percentage of Treg cell, associated with an improvement of Treg/Th17 balance. We further observed increased gene expression of LXR target genes, i.e., SREBP1c (sterol regulatory element-binding protein-1c), and ABCA1 (ATP-Binding Cassette Transporter A1), after NLCD. These modulations were not observed in NAFLD patients.
In conclusion, this study suggests that a NLCD is able to regulate the Th17/Treg balance, fostering LXRs activation, and hampering the onset of HCV-related autoimmune diseases. Thus, our work could be a step forward in the management of chronic HCV-infected patients.
- MinutoloA, ContiB, GrelliS, ViscomiC, LabbadiaG, BalsanoC. Lymphocytes as liver damage mirror of HCV related adipogenesis deregulation. PloSOne 2014.
- MaggioR, ViscomiC, AndreozziP, D’Ettorre G, Viscogliosi G, BarbaroB,Gori M, Vullo V, Balsano C. Normocaloric low cholesterol diet modulates Th17/Treg balance in patients with chronic hepatitis C virus infection. PloSOne 2014.
ROLE OF ALTERATIONS IN INTESTINAL MICROFLORA IN THE PATHOGENESIS OF HEPATIC STEATOSIS
The liver damage can be triggered by diverse causes (as viral, toxic, metabolic, etc.), however the pathogenic mechanisms underlying the different types of damages (such as inflammation, steatosis, fibrosis and cirrhosis) are very similar. The necrosis of hepatocytes, followed by the recruitment of macrophages generally causes the induction and progression of chronic liver injury. The cells of the immune system and of inflammation (such as neutrophils, lymphocytes and Kupffer cells) can be selectively activated in relation to the type of liver damage.
The liver can react to the damage primarily through three mechanisms: apoptosis, regeneration of hepatocytes and fibrosis (via the activation of stellate cells).
In addition to the direct damage produced by various causative agents, it seems that alterations of the intestinal microflora play an important role in the induction of steatosis and in the progression of liver injury, such as in IBD (inflammatory bowel disease).
The main alterations of the intestinal microflora are bacterial overgrowth and the release into the blood of endotoxins (such as LPS, peptidoglycans, lipoproteins and various lipopeptides, etc.), well known PAMPs (Pathogen-Associated Molecular Pattern).
Endotoxemia was observed in various liver diseases and seems responsible of the induction of pathogenic processes which foster the insult to the liver. It is well known that bacterial products, through the circulation, can reach other body districts, including the liver, where they interact with specific recognition receptors, called TLRs (Toll Like Receptors), leading to the induction of NF-kB pathway.
In humans, the TLR receptor’s family is composed by 10 members; TLRs are involved in the innate immune response and act as pathogens sensors, but they are also implicated in many other processes, including the adaptive immune response, the regulation of the inflammatory response, the promotion of epithelial regeneration and the carcinogenesis. TLRs play an important role in the pathophysiology of liver diseases and represent the link between alterations in the intestinal bacterial flora, the endotoxemia and liver disease. Another link between fatty liver disease and IBD can be represented by oxidative stress (OS); it is well known, in fact, the link between the OS and hepatic steatosis. However, this may be relevant also in the bowel inflammation. Among the various immunoregulatory factors activated and / or produced during IBD, in fact, reactive oxygen species (ROS) are produced at exceptionally high levels.The devastating effects of ROS may contribute to both onset and progression of the disease.
Thus, would be of great interest to understand the relationship between hepatic steatosis and OS during IBD, also in children.
Therefore,our studies are direct to investigate the intricate network generated by IBD, the alterations of the intestinal microflora, the oxidative stress and the hepatic steatosis.
- Abenavoli L, Scarpellini E, Rouabhia S, Balsano C, Luzza F. Probiotics in non-alcoholicfattyliverdisease: which and when. Ann Hepatol. 2013 May-Jun;12(3):357-63.
- Alisi A, Panera N, Balsano C, Nobili V. Activation of the endotoxin/toll-like receptor 4 pathway: the way to go from nonalcoholicsteatohepatitis up to hepatocellular carcinoma. Hepatology. 2011 Mar;53(3):1069.
THE PATHOGENESIS OF NONALCOHOLIC FATTY LIVER DISEASE
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the western world and it is characterized by lipid accumulation in the liver of patients which do not consume alcohol and are not subjected to certain drugs or substances. NAFLD is a clinical-pathological condition which refers to a broad spectrum of conditions ranging from simple triglyceride accumulation in hepatocytes (the simple steatosis) to its form associated to inflammation (non-alcoholicsteatohepatitis-NASH), which may progress until to cirrhosis and hepatocarcinoma (HCC). NAFLD is frequently associated with the metabolic syndrome, and it is considered its hepatic manifestation. Today the NAFLD pathogenesis is explained by the “two-hit” theory. According to it, steatosis represents the ‘‘first hit’’ and makes the liver more susceptible to a ‘‘second hit’’, which in turn promotes inflammation, cell death and fibrosis, the histopathological features of NASH. Among the mechanisms underlying the intrahepatic lipid accumulation, the altered activities of proteins involved in modulation of lipogenesis, fatty acids beta-oxidation and cholesterol transport, known as nuclear receptors, such as Liver X Receptors (LXRs) and Peroxisome Proliferating Associated Receptors (PPARs), have a chief role. Several studies performed in rodents demonstrated that a high-fat diet (HFD) affects the transcriptional activity of these proteins. , Among the main factors involved in both onset and progression of NAFLD, without any doubt, oxidative stress (OS) and mitochondrial damages are key factors. OS, moreover, is strictly correlated to mitochondrial alterations, because these organelles are at the same time source of reactive oxygen species (ROS) and their main target. Mitonchondrial dysfunctions can cause a derangement of fatty acids beta-oxidation, which in turn can produce ROS overproduction and energy metabolism imbalance. The ROS overproduction associated to lipid accumulation promotes the onset of OS through a variety of mechanisms including lipid peroxidation, which is an extremely harmful event for the cellular physiological functions. In fact, the increase of ROS and lipid peroxidation products was observed both in animal models of hepatic steatosis and in NAFLD/NASH patients. Since at present there is not a pharmacological treatment which can effectively counteract the pathogenesis of NAFLD, several studies are aimed to identify molecules with therapeutic potential. In fact, numerous studies focused their interests on “natural” molecules, such as the polyphenols contained in numerous plants, because they are potentially able to counteract the onset and the progression of NAFLD.
On these bases we developed an in vitro model to study the effects of increased lipid content in hepatic cells. In particular, we investigated the genes involved in lipid and energy metabolism as well as in the pathways, known for their potential involvement in fibrosis or hepatocellular carcinoma, like Hedgehog (Hh) and p53, which have recently been associated to metabolic regulation. At the same time, we are performing our studies on a murine model of hepatic steatosis, C57BL/6 mice fed with a high fat diet (HFD), in order to analyse more in depth the effects on metabolism also in other body ditricts (eg. adipose tissue and heart). Finally, we aim to conduct studies on molecules easily assumable through diet, such as polyphenolic compounds of natural origin, in order to promote nutraceutical formulations that may have an important effect on human health.
- Peluso MME, Munnia A, Tarocchi M, Arciello M, Balsano C, Giese RW, Galli A. Exocycilic DNA adducts in a Murine Model of Non-AlcoholicSteatohepatitis. J Carcinog Mutagen. ISSN: 2157-2518. 2013.
- Gori M, Barbaro B, Arciello M, Maggio R, Viscomi C, Longo A, Balsano C. Protective effect of the Y220C mutant p53 against steatosis: good news? Journal Cell Physiol 2013.
- Gori M, Barbaro B, Arciello M, Balsano C. Paradoxical prosteatotic effect of hedgehog signaling pathway inhibition under conditions of steatosis. Hepatology. 2012 Oct;56(4):1587-8.
ROLE OF ENVIROMENTAL POLLUTION ON LIVER DISEASES
The liver is crucial for human life, and the health of this organ often mirrors the health of an 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, which is a multifactorial disease that embraces many histo-pathologic conditions. In the last decade, a considerable amount of experimental evidence revealed the pathological potential of a wide range of environmental pollutants on liver health and, in particular, on NAFLD occurrence. Technological progress and industrialization had the consequence of releasing pollutants in the environment, for instance pesticides or solvents, heavy metals, as well as by-products of discharge, such as the particulate matter. Several studies described and suggested that these contaminants have great steatogenic potential. Thus, also the enviromental pollutants must be considered as tangible risk factors for the onset of NAFLD and other metabolic diseases, whose spread is always greater and whose causes appear as yet little understood.
- Arciello M, Gori M, Maggio R, Barbaro B, Tarocchi M, Galli A, Balsano C. Environmental pollution: a tangible risk for NAFLD pathogenesis. Int J Mol Sci. 2013 Nov 7;14(11):22052-66. doi: 10.3390/ijms141122052.
POST-DOCTORAL VERONESI FELLOWSHIPS ROME 2014
Dr.ssa Roberta Maggio, collaborator, since 2012, of the Francesco Balsano Foundation at the Laboratory of Virology and Molecular Oncology, that is coordinated by Prof. Clara Balsano, partecipated to competition announcement “FUV Post-Doctoral Fellowship 2014” which permits the access to research grants funded by the Fondazione Umberto Veronesi, resulting the winner of the annual Fellowship Announcement "Post-doctoral Fellowships Rome - year 2014". This prestigious award was achieved through a research project, focused on the impact of the cholesterol modulation, through a normocaloric and hypocholesterolemic diet, on the immune response in patients with chronic hepatitis C, which display the possibility to develop HCV-related autoimmune diseases.