M. Tarocchi

Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated mechanism

Experimental evidence indicates that reactive oxygen species (ROS) are involved in the development of hepatic fibrosis; they induce hepatic stellate cells (HSC) proliferation and collagen synthesis. To address the role of matrix metalloproteinase (MMP)‐2 in promoting HSC proliferation during hepatic injury, we investigated whether oxidative stress modulates the growth and invasiveness of HSC by influencing MMP‐2 activation. Cell invasiveness and proliferation, which were studied using Boyden chambers and by counting cells under a microscope, were evaluated after treatment with a superoxide‐producing system, xanthine plus xanthine oxidase (X/XO), in the presence or absence of antioxidants and MMP inhibitors. Expression and activation of MMP‐2 were evaluated via gel zymography, immunoassay, and ribonuclease protection assay. The addition of X/XO induced proliferation and invasiveness of human HSC in a dose‐dependent manner. The addition of antioxidants as well as MMP‐2–specific inhibitors impaired these phenomena. X/XO treatment increased MMP‐2 expression and secretion appreciably and significantly induced members of its activation complex, specifically membrane‐type 1 MMP and tissue inhibitor metalloproteinase 2. To study the intracellular signaling pathways involved in X/XO‐induced MMP‐2 expression, we evaluated the effects of different kinase inhibitors. The inhibition of extracellular signal‐regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3‐kinase (PI3K) abrogated X/XO‐elicited MMP‐2 upregulation and completely prevented X/XO‐induced growth and invasiveness of HSC. In conclusion, our findings suggest that MMP‐2 is required for the mitogenic and proinvasive effects of ROS on HSC and demonstrate that ERK1/2 and PI3K are the main signals involved in ROS‐mediated MMP‐2 expression. (HEPATOLOGY 2005;41:1074–1084.)

PPARγ and Oxidative Stress: Con(β) Catenating NRF2 and FOXO

Peroxisome-proliferator activator receptor γ (PPARγ) is a nuclear receptor of central importance in energy homeostasis and inflammation. Recent experimental pieces of evidence demonstrate that PPARγ is implicated in the oxidative stress response, an imbalance between antithetic prooxidation and antioxidation forces that may lead the cell to apoptotic or necrotic death. In this delicate and intricate game of equilibrium, PPARγ stands out as a central player devoted to the quenching and containment of the damage and to foster cell survival. However, PPARγ does not act alone: indeed the nuclear receptor is at the point of interconnection of various pathways, such as the nuclear factor erythroid 2-related factor 2 (NRF2), Wnt/β-catenin, and forkhead box proteins O (FOXO) pathways. Here we reviewed the role of PPARγ in response to oxidative stress and its interaction with other signaling pathways implicated in this process, an interaction that emerged as a potential new therapeutic target for several oxidative-related diseases.

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.

Molecular mechanism of hepatitis B virus-induced hepatocarcinogenesis

Hepatitis B virus (HBV) infection is a global public health problem with approximately 2 billion people that have been exposed to the virus. HBV is a member of a family of small, enveloped DNA viruses called hepadnaviruses, and has a preferential tropism for hepatocytes of mammals and birds. Epidemiological studies have proved a strong correlation between chronic hepatitis B virus infection and the development of hepatocellular carcinoma (HCC). HCC is the fifth most common malignancy with about 700000 new cases each year, and more than 50% of them arise in HBV carriers. A large number of studies describe the way in which HBV can contribute to HCC development. Multiple mechanisms have been proposed, including the accumulation of genetic damage due to immune-mediated hepatic inflammation and the induction of oxidative stress. There is evidence of the direct effects of the viral proteins HBx and HBs on the cell biology. Integration of HBV-DNA into the human genome is considered an early event in the carcinogenic process and can induce, through insertional mutagenesis, the alteration of gene expression and chromosomal instability. HBV has also epigenetic effects through the modification of the genomic methylation status. Furthermore, the virus plays an important role in the regulation of microRNA expression. This review will summarize the many mechanisms involved in HBV-related liver carcinogenesis.

Thiazolidinediones Inhibit Hepatocarcinogenesis in Hepatitis B Virus―Transgenic Mice by Peroxisome Proliferator-Activated Receptor γ―Independent Regulation of Nucleophosmin

Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effect in epithelial cancers, including hepatocellular carcinoma (HCC). The effective anticancer properties and the underlying molecular mechanisms of these drugs in vivo remain unclear. In addition, the primary biological target of TZD, the ligand‐dependent transcription factor peroxisome proliferator‐activated receptor γ (PPARγ), is up‐regulated in HCC and seems to provide tumor‐promoting responses. The aim of our study was to evaluate whether chronic administration of TZD may affect hepatic carcinogenesis in vivo in relation to PPARγ expression and activity. The effect of TZD oral administration for 26 weeks was tested on tumor formation in PPARγ‐expressing and PPARγ‐deficient mouse models of hepatic carcinogenesis. Proteomic analysis was performed in freshly isolated hepatocytes by differential in gel electrophoresis and mass spectrometry analysis. Identified TZD targets were confirmed in cultured PPARγ‐deficient hepatocytes. TZD administration in hepatitis B virus (HBV)–transgenic mice (TgN[Alb1HBV]44Bri) reduced tumor incidence in the liver, inhibiting hepatocyte proliferation and increasing apoptosis. PPARγ deletion in hepatocytes of HBV‐transgenic mice (Tg[HBV]CreKOγ) did not modify hepatic carcinogenesis but increased the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as a TZD target in PPARγ‐deficient hepatocytes. TZD inhibited NPM expression at protein and messenger RNA levels and decreased NPM promoter activity. TZD inhibition of NPM was associated with the induction of p53 phosphorylation and p21 expression. Conclusion: These findings suggest that chronic administration of TZD has anticancer activity in the liver via inhibition of NPM expression and indicate that these drugs might be useful for HCC chemoprevention and treatment. HEPATOLOGY 2010

Peroxisome proliferator activated receptors at the crossroad of obesity, diabetes, and pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth cause of cancer death with an overall survival of 5% at five years. The development of PDAC is characteristically associated to the accumulation of distinctive genetic mutations and is preceded by the exposure to several risk factors. Epidemiology has demonstrated that PDAC risk factors may be non-modifiable risks (sex, age, presence of genetic mutations, ethnicity) and modifiable and co-morbidity factors related to the specific habits and lifestyle. Recently it has become evident that obesity and diabetes are two important modifiable risk factors for PDAC. Obesity and diabetes are complex systemic and intertwined diseases and, over the years, experimental evidence indicate that insulin-resistance, alteration of adipokines, especially leptin and adiponectin, oxidative stress and inflammation may play a role in PDAC. Peroxisome proliferator activated receptor-γ (PPARγ) is a nuclear receptor transcription factor that is implicated in the regulation of metabolism, differentiation and inflammation. PPARγ is a key regulator of adipocytes differentiation, regulates insulin and adipokines production and secretion, may modulate inflammation, and it is implicated in PDAC. PPARγ agonists are used in the treatment of diabetes and oxidative stress-associated diseases and have been evaluated for the treatment of PDAC. PPARγ is at the cross-road of diabetes, obesity, and PDAC and it is an interesting target to pharmacologically prevent PDAC in obese and diabetic patients.

Effect of a counseling-supported treatment with the Mediterranean diet and physical activity on the severity of the non-alcoholic fatty liver disease

AIM To determine the clinical effectiveness of nutritional counseling on reduction of non-alcoholic fatty liver disease (NAFLD) severity, weight loss, metabolic and anthropometric indexes and liver enzymes. METHODS Forty-six adults with NAFLD received a 6-mo clinical and a dietary intervention (based on Mediterranean diet) carried out respectively by a gastroenterologist and a nutritionist with counseling license. The counseling process consisted of monthly meeting (about 45 min each). The effect of the treatment was evaluated monitoring liver enzymes, metabolic parameters, cardiovascular risk indexes, NAFLD severity [assessed by ultrasound (US)] and related indexes. All parameters were assessed at baseline. Biochemistry was also assessed at mid- and end-interventions and US was repeated at end-intervention. RESULTS The percentage of patients with steatosis grade equal or higher than 2 was reduced from 93% to 48% and steatosis regressed in 9 patients (20%). At the end of the treatment the end-point concerning the weight (i.e., a 7% weight reduction or achievement/maintenance of normal weight) was accomplished by 25 out of 46 patients (i.e., 54.3%). As far as the liver enzymes is concerned, all three liver enzymes significantly decrease during the treatment the normalization was particularly evident for the ALT enzyme (altered values reduced from 67% down to 11%). Several parameters, i.e., BMI, waist circumference, waist-to-hip ratio, AST, ALT, GGT, HDL, serum glucose, Tot-Chol/HDL, LDL/HDL, TG/HDL, AIP, HOMA, FLI, Kotronen index, VAI, NAFLD liver fat score and LAP, showed a significant improvement (P < 0.01) between baseline and end-treatment. CONCLUSION Outcomes of this study further strengthen the hypothesis that MedDiet and more active lifestyle can be considered a safe therapeutic approach for reducing risk and severity of NAFLD and related disease states. The proposed approach may be proposed as a valid and recommended approach for improving the clinical profile of NAFLD patients.

Nuclear receptors and pathogenesis of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a median overall survival time of 5 mo and the five years survival less than 5%, a rate essentially unchanged over the course of the years. A well defined progression model of accumulation of genetic alterations ranging from single point mutations to gross chromosomal abnormalities has been introduced to describe the origin of this disease. However, due to the its subtle nature and concurring events PDAC cure remains elusive. Nuclear receptors (NR) are members of a large superfamily of evolutionarily conserved ligand-regulated DNA-binding transcription factors functionally involved in important cellular functions ranging from regulation of metabolism, to growth and development. Given the nature of their ligands, NR are very tempting drug targets and their pharmacological modulation has been widely exploited for the treatment of metabolic and inflammatory diseases. There are now clear evidences that both classical ligand-activated and orphan NR are involved in the pathogenesis of PDAC from its very early stages; nonetheless many aspects of their role are not fully understood. The purpose of this review is to highlight the striking connections that link peroxisome proliferator activated receptors, retinoic acid receptors, retinoid X receptor, androgen receptor, estrogen receptors and the orphan NR Nur, chicken ovalbumin upstream promoter transcription factor II and the liver receptor homologue-1 receptor to PDAC development, connections that could lead to the identification of novel therapies for this disease.

COUP-TFII in pancreatic adenocarcinoma: clinical implication for patient survival and tumor progression.

Despite the accumulating knowledge of alterations in pancreatic cancer molecular pathways, no substantial improvements in the clinical prognosis have been made and this malignancy continues to be a leading cause of cancer death in the Western World. The orphan nuclear receptor COUP‐TFII is a regulator of a wide range of biological processes and it may exert a pro‐oncogenic role in cancer cells; interestingly, indirect evidences suggest that the receptor could be involved in pancreatic cancer. The aim of this study was to evaluate the expression of COUP‐TFII in human pancreatic tumors and to unveil its role in the regulation of pancreatic tumor growth. We evaluated COUP‐TFII expression by immunohistochemistry on primary samples. We analyzed the effect of the nuclear receptor silencing in human pancreatic cancer cells by means of shRNA expressing cell lines. We finally confirmed the in vitro results by in vivo experiments on nude mice. COUP‐TFII is expressed in 69% of tested primary samples and correlates with the N1 and M1 status and clinical stage; Kaplan–Meier and Cox regression analysis show that it may be an independent prognostic factor of worst outcome. In vitro silencing of COUP‐TFII reduces the cell growth and invasiveness and it strongly inhibits angiogenesis, an effect mediated by the regulation of VEGF‐C. In nude mice, COUP‐TFII silencing reduces tumor growth by 40%. Our results suggest that COUP‐TFII might be an important regulator of the behavior of pancreatic adenocarcinoma, thus representing a possible new target for pancreatic cancer therapy.

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.