Thiazolidinediones inhibit hepatocarcinogenesis in hepatitis B virus-transgenic mice by peroxisome proliferator-activated receptor gamma-independent regulation of nucleophosmin.

UNLABELLED: 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 gamma (PPARgamma), 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 PPARgamma expression and activity. The effect of TZD oral administration for 26 weeks was tested on tumor formation in PPARgamma-expressing and PPARgamma-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 PPARgamma-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. PPARgamma deletion in hepatocytes of HBV-transgenic mice (Tg[HBV]CreKOgamma) did not modify hepatic carcinogenesis but increased the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as a TZD target in PPARgamma-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.

Alcohol induced hepatic fibrosis: role of acetaldehyde.

Alcohol abuse is one of the major causes of liver fibrosis worldwide. Although the pathogenesis of liver fibrosis is a very complex phenomenon involving different molecular and biological mechanisms, several lines of evidence established that the first ethanol metabolite, acetaldehyde, plays a key role in the onset and maintenance of the fibrogenetic process. This review briefly summarizes the molecular mechanisms underlying acetaldehyde pro-fibrogenic effects. Liver fibrosis represents a general wound-healing response to a variety of insults. Although mortality due to alcohol abuse has been constantly decreasing in the past 20 years in Southern Europe and North America, in several Eastern-European countries and Great Britain Alcoholic Liver Disease (ALD) shows a sharply increasing trend [Bosetti, C., Levi, F., Lucchini, F., Zatonski, W.A., Negri, E., La, V.C., 2007. Worldwide mortality from cirrhosis: an update to 2002. J. Hepatol. 46, 827-839]. ALD has a complex pathogenesis, in which acetaldehyde (AcCHO), the major ethanol metabolite, plays a central role. Ethanol is mainly metabolized in the liver by two oxidative pathways. In the first one ethanol is oxidized to acetaldehyde by the cytoplasmic alcohol dehydrogenase enzyme (ADH), acetaldehyde is then oxidized to acetic acid by the mitochondrial acetaldehyde dehydrogenase (ALDH). The second pathway is inducible and involves the microsomal ethanol-oxidizing system (MEOS), in which the oxidation of ethanol to acetaldehyde and acetic acid also leads to generation of reactive oxygen species (ROS). Chronic ethanol consumption significantly inhibits mitochondrial ALDH activity while the rate of ethanol oxidation to acetaldehyde is even enhanced, resulting in a striking increase of tissue and plasma acetaldehyde levels [Lieber, C.S., 1997. Ethanol metabolism, cirrhosis and alcoholism. Clin. Chim. Acta 257, 59-84]. This review will focus on the molecular mechanisms by which acetaldehyde promote liver fibrosis.

Fatigue in primary biliary cirrhosis: a possible role of comorbidities.

BACKGROUND AND AIMS: Fatigue is considered to be a specific manifestation of primary biliary cirrhosis (PBC). Recent reports have, however, questioned these findings. Considering the high rate of comorbidities in PBC patients and the fact that fatigue is a multifactorial symptom, we hypothesized that it might also be due to nonhepatic causes. Our aim was to evaluate fatigue in PBC patients and its relationship with comorbidities and depression. METHODS: We enroled 49 Italian PBC patients (44 women; mean age: 58.9 years, range: 21-73 years) and 30 matched healthy controls, who completed the Fatigue Impact Scale (FIS), Modified FIS (MFIS), Fatigue Severity Score (FSS) and Rand Medical Outcomes Study Depression Screener. Comorbidities and several clinical and biochemical data were investigated. Linear regression, analysis of variance and post-hoc analysis were applied. RESULTS: Fatigue was higher in patients than in controls (FIS: 33 vs. 24; MFIS: 24 vs. 14; FSS: 3.3 vs. 1.9). Physical domain was significantly different in all the three questionnaires (FIS: P=0.05; MFIS: P=0.002; FSS: P=0.0002). Comorbidities (38% of patients) were independently associated with higher fatigue scores (FIS: 45; MFIS: 32; FSS: 3.3). Depressed patients (30%) were more fatigued, even if not always significantly (FIS: 43; MFIS: 29; FSS: 3.5), than controls and patients with no depression. Patients without comorbidities or depression (51%) did not have higher fatigue than controls (FIS: 20; MFIS: 17; FSS: 2.4). CONCLUSIONS: Fatigue in patients with PBC was higher, but not always significantly, than in healthy controls. Comorbidities and depression might have played a role in its pathogenesis. Our data arouse doubts about the specificity of fatigue in PBC and the pathogenetic role of liver impairment.

Adherence to a healthful life attenuates lipid parameters among a healthy Italian population.

BACKGROUND AND AIM: During the last 5 years, an increasing body of evidence on the association between adherence to the Mediterranean diet (MD), calculated through specific diet scores, and health status have been accumulated, but limited data are available regarding the association between MD scores and biomarkers. Similarly, many studies have demonstrated a significant protection against chronic diseases from a global healthy lifestyle (HL) pattern which includes not only dietary habits but also physical activity and abstinence from smoking, whereas few data regarding the influence of a HL pattern on circulating biomarkers are available. Using the framework of an epidemiological study conducted in Florence, Italy between 2002 and 2004 we evaluated the association between two different scores (a score of adherence to the MD and a score of adherence to a healthful life which includes abstinence from smoking and a moderate-to-high physical activity level) and some circulating parameters linked to chronic diseases. METHODS AND RESULTS: Dietary habits and anthropometric and biochemical profiles were studied in 932 individuals (365 M; 567 F) with a median age of 47.5 years. Subjects who reported a greater adherence to the MD were found more frequently to be male, married and over 45 years of age. A general linear model dividing the study population into quartiles of scores was used. After adjustment for age, gender, educational status, body mass index and total energy intake, we observed no influence of adherence to the MD on circulating levels of biomarkers. On the other hand, an inverse association between circulating levels of lipid parameters (namely total cholesterol, LDL-cholesterol and triglycerides) and higher scores of adherence to a HL, was reported. In addition, a significant difference between the highest and the lowest quartiles of HL scores for homocysteine plasma levels was observed (p=0.04). CONCLUSION: A high adherence to a HL, which includes not only a high adherence to the MD but also to other lifestyle factors (i.e. abstinence from smoking, and increasing physical activity during leisure time), is able to lower lipid parameters and homocysteine in a clinically healthy Italian population.

Hyperhomocysteinemia and hypercoagulability in primary biliary cirrhosis.

AIM: To assess the hypercoagulability in PBC and its relationship with homocysteine (HCY) and various components of the haemostatic system. METHODS: We investigated 51 PBC patients (43F/8M; mean age: 63+/-13.9 yr) and 102 healthy subjects (86 women/16 men; 63+/-13 yr), and evaluated the haemostatic process in whole blood by the Sonoclot analysis and the platelet function by PFA-100 device. We then measured HCY (fasting and after methionine loading), tissue factor (TF), thrombin-antithrombin complexes (TAT), D-dimer (D-D), thrombomodulin (TM), folic acid, vitamin B6 and B12 plasma levels. C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism was analyzed. RESULTS: Sonoclot RATE values of patients were significantly (P<0.001) higher than those of controls. Sonoclot time to peak values and PFA-100 closure times were comparable in patients and controls. TAT, TF and HCY levels, both in the fasting and post-methionine loading, were significantly (P<0.001) higher in patients than in controls. Vitamin deficiencies were detected in 45/51 patients (88.2%). The prevalence of the homozygous TT677 MTHFR genotype was significantly higher in patients (31.4%) than in controls (17.5%) (P<0.05). Sonoclot RATE values correlated significantly with HCY levels and TF. CONCLUSION: In PBC, hyper-HCY is related to hypovitaminosis and genetic predisposing factors. Increased TF and HCY levels and signs of endothelial activation are associated with hypercoagulability and may have an important role in blood clotting activation.

Low adherence of a clinically healthy Italian population to nutritional recommendations for primary prevention of chronic diseases.

BACKGROUND AND AIM: Several data demonstrated that dietary habits significantly affect the health state of the population. During recent years all the major scientific associations have provided nutritional recommendations for primary prevention of chronic diseases but few data are available about prevalence of adherence to these recommendations in an otherwise healthy population. The aims of this study were to evaluate dietary habits, and to assess the adherence of the general population to the recommendations for correct nutritional behaviour. METHODS AND RESULTS: Dietary habits, anthropometric and biochemical parameters were evaluated in a population of 932 (367 M; 565 F) clinically healthy subjects living in Florence, enrolled in an epidemiologic study conducted between 2002 and 2004. By comparing the dietary pattern with the nutritional guidelines, the study population reported a hyperproteic and hyperlipidic nutritional pattern, with a considerably low contribution from polyunsaturated fats (PUFA). A low fibre intake is shown in both genders. In addition, food consumption pattern showed an increased consumption of some foods such as meat, both fresh and processed, and a low intake of some "healthy" foods like fruit and vegetables. CONCLUSIONS: We found several nutritional flaws in the dietary habits of a clinically healthy Italian population. In particular, we reported a high intake of animal protein and total fats with a very low contribution from PUFA.

Antidiabetic thiazolidinediones induce ductal differentiation but not apoptosis in pancreatic cancer cells.

AIM: Thiazolidinediones (TZD) are a new class of oral antidiabetic drugs that have been shown to inhibit growth of same epithelial cancer cells. Although TZD were found to be ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), the mechanism by which TZD exert their anticancer effect is presently unclear. In this study, we analyzed the mechanism by which TZD inhibit growth of human pancreatic carcinoma cell lines in order to evaluate the potential therapeutic use of these drugs in pancreatic adenocarcinoma. METHODS: The effects of TZD in pancreatic cancer cells were assessed in anchorage-independent growth assay. Expression of PPARgamma was measured by reverse-transcription polymerase chain reaction and confirmed by Western blot analysis. PPARgamma activity was evaluated by transient reporter gene assay. Flow cytometry and DNA fragmentation assay were used to determine the effect of TZD on cell cycle progression and apoptosis respectively. The effect of TZD on ductal differentiation markers was performed by Western blot. RESULTS: Exposure to TZD inhibited colony formation in a PPARgamma-dependent manner. Growth inhibition was linked to G1 phase cell cycle arrest through induction of the ductal differentiation program without any increase of the apoptotic rate. CONCLUSION: TZD treatment in pancreatic cancer cells has potent inhibitory effects on growth by a PPAR-dependent induction of pancreatic ductal differentiation.

The potential of antidiabetic thiazolidinediones for anticancer therapy.

The thiazolidinediones (TZDs) are a class of synthetic compounds for treatment of insulin-resistant Type 2 diabetes mellitus. TZDs are known activators of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and exert their antidiabetic action largely through this nuclear receptor family. Moreover, increasing experimental evidences of PPAR-gamma-independent effects are accumulating. Apart from the established metabolic actions, TZD treatment exerts additional biological effect such as control of cell growth, differentiation, motility and programmed cell death. In this context, considerable interest has focused on TZDs as potential chemopreventive agents in oncology; however, despite encouraging observation on the potential anticancer effect of these drugs in several in vitro experimental models, controversial results have been obtained with animal models and in pilot clinical trials. This review summarises the molecular mechanisms of the antineoplastic actions of TZDs and the relevance of these findings in human pathology and therapy.

Acetaldehyde inhibits PPARgamma via H2O2-mediated c-Abl activation in human hepatic stellate cells.

BACKGROUND & AIMS: Accumulating evidence indicates that acetaldehyde (AcCHO) is one of the main mediators of fibrogenesis in alcoholic liver disease. AcCHO stimulates synthesis of fibrillar collagens in hepatic stellate cells, but the molecular events directly involved in the activation of collagen genes are debatable. METHODS: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that is expressed in stellate cells, and its activation by specific ligands inhibits collagen synthesis. In this study, we evaluated the effects of AcCHO on PPARgamma transcriptional activity and its correlation with the AcCHO-induced collagen synthesis in hepatic stellate cells. RESULTS: AcCHO treatment inhibited ligand-dependent and -independent PPARgamma transcriptional activity, and this effect was correlated with an increased phosphorylation of a mitogen-activated protein kinase site at serine 84 of the human PPARgamma. Transfection of the PPARgammaSer84Ala mutant completely prevented the effect of AcCHO on PPARgamma activity and in parallel abrogated the induction of collagen gene expression by AcCHO. The effect of AcCHO on PPARgamma activity and phosphorylation was blocked by extracellular signal-regulated kinase (ERK) 1/2 and protein kinase C (PKC)delta inhibitors as well as by catalase, suggesting that hydrogen peroxide is involved in the molecular cascade responsible for PPARgamma phosphorylation via activation of the PKCdelta/ERK pathway. Furthermore, inhibition of c-Abl completely abrogated the effect of AcCHO on either PPARgamma function or collagen synthesis; in addition, expression of the PPARgammaSer84Ala mutant prevented the profibrogenic signals mediated by c-Abl activation. CONCLUSIONS: Our results showed that the induction of collagen expression by AcCHO in stellate cells is dependent on PPARgamma phosphorylation induced by a hydrogen peroxide-mediated activation of the profibrogenic c-Abl signaling pathway.

Endothelial nitric oxide synthase -786T>C, but not 894G>T and 4a4b, polymorphism influences plasma homocysteine concentrations in persons with normal vitamin status.

BACKGROUND: Nitric oxide (NO) plays a relevant role in various events during atherogenesis. In vitro data suggest that NO may modulate homocysteine (Hcy) concentrations. The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) -786T>C, 894G>T, and 4a4b polymorphisms in influencing Hcy concentrations. METHODS: Blood samples were obtained from 1287 unrelated persons. Plasma Hcy was measured by fluorescence polarization immunoassay, folate and vitamin B(12) by RIA, vitamin B(6) by HPLC, and eNOS and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms by PCR with restriction fragment length polymorphism analysis. RESULTS: MTHFR 677C>T polymorphism significantly influenced Hcy concentrations after adjustment for all confounding variables (P <0.0001 for trend). Univariate analysis showed that the eNOS -786T>C polymorphism, but not 894G>T and 4a4b, was significantly associated with the risk of having Hcy in the third tertile [>13.4 micromol/L; odds ratio (OR) = 1.2; 95% confidence interval (CI), 1.02-1.5; P = 0.03]. After adjustment for all variables known to influence Hcy, the -786T>C polymorphism still influenced Hcy concentrations (OR = 1.9; 95% CI, 1.1-3.2; P = 0.01). By analyzing the influence of eNOS polymorphisms on plasma Hcy concentrations according to vitamin concentrations (folate, vitamin B(6), and vitamin B(12)), age, and smoking habits, we found a significant association between the eNOS -786T>C polymorphism and Hcy in nonsmokers, in persons with normal vitamin status, and in persons <60 years. CONCLUSION: The eNOS -786T>C polymorphism, but not 894G>T and 4a4b, influences plasma Hcy concentrations mildly but significantly and independently.

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.

Helicobacter pylori cag pathogenicity island is associated with reduced expression of interleukin-4 (IL-4) mRNA and modulation of the IL-4delta2 mRNA isoform in human gastric mucosa.

Interleukin-4 (IL-4) and IL-4delta2 mRNA gastric expression was evaluated in healthy subjects and patients who did not have ulcers but were infected with Helicobacter pylori with or without the cag pathogenicity island (cag PAI). IL-4 mRNA was physiologically expressed by gastric epithelium and negatively influenced by H. pylori. Also, nonepithelial cells in the lamina propria of H. pylori-infected patients expressed IL-4 mRNA, whereas IL-4delta2 mRNA was found only in cag PAI-negative patients. Thus, gastric IL-4 takes part in the local immune response to H. pylori.

Antidiabetic thiazolidinediones inhibit collagen synthesis and hepatic stellate cell activation in vivo and in vitro.

BACKGROUND & AIMS: The ligand-dependent transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in hepatic stellate cells (HSC), and its transcriptional activity is reduced during cell transdifferentiation in culture. PPARgamma transcriptional activation decreases platelet-derived growth factor-induced proliferation and inhibits alpha-smooth muscle actin expression in cultured HSC. The aim of our study was to evaluate whether oral administration of synthetic PPARgamma ligands, thiazolidinediones (TZD), might affect collagen deposition in animal models of liver fibrosis. METHODS: The effect of 2 TZD (pioglitazone or rosiglitazone) was tested on liver fibrosis induced in rats by either toxin administration (dimethylnitrosamine or carbon tetrachloride) or bile duct ligation. In vivo PPARgamma activation was evaluated by gel shift assay using nuclear extracts from HSC isolated from control and treated rats. RESULTS: Oral administration of TZD reduced extracellular matrix deposition and HSC activation in both toxic and cholestatic models of liver fibrosis. PPARgamma-specific DNA binding was significantly impaired in nuclear extracts of HSC isolated from fibrotic rats compared with HSC from control rats. TZD administration restored PPARgamma DNA binding in HSC nuclei. In vitro, TZD-induced PPARgamma activation inhibited collagen and fibronectin synthesis induced by transforming growth factor (TGF)-beta1 in human HSC, as measured by enzyme-linked immunosorbent assay and Northen blotting. TZD also reduced the TGF-beta1-induced activity of a 3.5-kilobase procollagen type I promoter transfected in human HSC. CONCLUSIONS: These findings indicate that PPARgamma activation in HSC retards fibrosis in vivo and suggest the use of TZD for the treatment of liver fibrosis.