AP-1 Inhibition by SR 11302 Protects Human Hepatoma HepG2 Cells from Bile Acid-Induced Cytotoxicity by Restoring the NOS-3 Expression.

The harmful effects of bile acid accumulation occurring during cholestatic liver diseases have been associated with oxidative stress increase and endothelial nitric oxide synthase (NOS-3) expression decrease in liver cells. We have previously reported that glycochenodeoxycholic acid (GCDCA) down-regulates gene expression by increasing SP1 binding to the NOS-3 promoter in an oxidative stress dependent manner. In the present study, we aimed to investigate the role of transcription factor (TF) AP-1 on the NOS-3 deregulation during GCDCA-induced cholestasis. The cytotoxic response to GCDCA was characterized by 1) the increased expression and activation of TFs cJun and c-Fos; 2) a higher binding capability of these at position -666 of the NOS-3 promoter; 3) a decrease of the transcriptional activity of the promoter and the expression and activity of NOS-3; and 4) the expression increase of cyclin D1. Specific inhibition of AP-1 by the retinoid SR 11302 counteracted the cytotoxic effects induced by GCDCA while promoting NOS-3 expression recovery and cyclin D1 reduction. NOS activity inhibition by L-NAME inhibited the protective effect of SR 11302. Inducible NOS isoform was no detected in this experimental model of cholestasis. Our data provide direct evidence for the involvement of AP-1 in the NOS-3 expression regulation during cholestasis and define a critical role for NOS-3 in regulating the expression of cyclin D1 during the cell damage induced by bile acids. AP-1 appears as a potential therapeutic target in cholestatic liver diseases given its role as a transcriptional repressor of NOS-3.

GCDCA down-regulates gene expression by increasing Sp1 binding to the NOS-3 promoter in an oxidative stress dependent manner.

During the course of cholestatic liver diseases, the toxic effect of bile acids accumulation has been related to the decreased expression of endothelial nitric oxide synthase (NOS-3) and cellular oxidative stress increase. In the present study, we have investigated the relationship between these two biological events. In the human hepatocarcinoma cell line HepG2, cytotoxic response to GCDCA was characterized by the reduced activity of the respiratory complexes II+III, the increased expression and activation of the transcription factor Sp1, and a higher binding capacity of this at positions -1386, -632 and -104 of the NOS-3 promoter (pNOS-3). This was associated with a decreased promoter activity and a consequent reduction of NOS-3 expression. The use of antioxidants in GCDCA-treated cells caused a lower activation of Sp1 and the recovery of the pNOS-3 activity and NOS-3 expression and activity. Similarly, the specific inhibition of Sp1 resulted in the improvement of NOS-3 expression. Both, antioxidant treatment and Sp1 inhibition were associated with the reduction of cell death-related parameters. Bile duct ligation in rats confirmed in vitro results concerning the activation of Sp1 and the reduction of NOS-3 expression. Our results provide direct evidence for the involvement of Sp1 in the regulation of NOS-3 expression during cholestasis. Thus, the identification of Sp1 as a potential negative regulator of NOS-3 expression represents a new mechanism by which the accumulation of bile acids causes a cytotoxic effect through the oxidative stress increase, and provides a new potential target in cholestatic liver diseases.

Sensitivity to anti-Fas is independent of increased cathepsin D activity and adrenodoxin reductase expression occurring in NOS-3 overexpressing HepG2 cells.

Stable overexpression of endothelial nitric oxide synthase (NOS-3) in HepG2 cells (4TO-NOS) leads to increased nitro-oxidative stress and upregulation of the cell death mediators p53 and Fas. Thus, NOS-3 overexpression has been suggested as a useful antiproliferative mechanism in hepatocarcinoma cells. We aimed to identify the underlying mechanism of cell death induced by NOS-3 overexpression at basal conditions and with anti-Fas treatment. The intracellular localization of NOS-3, the nitro-oxidative stress and the mitochondrial activity were analysed. In addition, the protein expression profile in 4TO-NOS was screened for differentially expressed proteins potentially involved in the induction of apoptosis. NOS-3 localization in the mitochondrial outer membrane was not associated with changes in the respiratory cellular capacity, but was related to the mitochondrial biogenesis increase and with a higher protein expression of mitochondrial complex IV. Nitro-oxidative stress and cell death in NOS-3 overexpressing cells occurred with the expression increase of pro-apoptotic genes and a higher expression/activity of the enzymes adrenodoxin reductase mitochondrial (AR) and cathepsin D (CatD). CatD overexpression in 4TO-NOS was related to the apoptosis induction independently of its catalytic activity. In addition, CatD activity inhibition by pepstatin A was not effective in blocking apoptosis induced by anti-Fas. In summary, NOS-3 overexpression resulted in an increased sensitivity to anti-Fas induced cell death, independently of AR expression and CatD activity.

Lack of agreement for defining 'clinical suspicion of rejection' in liver transplantation: a model to select candidates for liver biopsy.

The gold standard to diagnose acute cellular rejection (ACR) after liver transplantation (LT) is histological evaluation, but there is no consensus to select patients for liver biopsy. We aimed to evaluate the agreement among clinicians to select candidates for liver biopsy early after LT. From a protocol biopsy population (n = 690), we randomly selected 100 LT patients in whom the biopsy was taken 7-10 days after LT. The clinical information between LT and protocol biopsy was given to nine clinicians from three transplant centres who decided whether a liver biopsy was needed. The agreement among clinicians to select candidates for liver biopsy was poor: κ = 0.06-0.62, being κ < 0.40 in 76% of comparisons. The concordance between indication for liver biopsy and moderate-severe ACR in the protocol biopsy was κ < 0.30 in all cases. A multivariate model based on the product age-by-MELD (OR = 0.81; P = 0.013), delta eosinophils (OR = 1.5; P = 0.002) and mean tacrolimus trough concentrations <6 ng/ml within the prior 4 days (OR = 11.4; P = 0.047) had an AUROC = 0.84 to diagnose moderate-severe histological ACR. In conclusion, the agreement among clinicians to select patients for liver biopsy is very poor. If further validated the proposed model would provide an objective method to select candidates for liver biopsy after LT.

Identification of candidate biomarkers for hepatocellular carcinoma in plasma of HCV-infected cirrhotic patients by 2-D DIGE.

BACKGROUND: The current methods available for screening and detecting hepatocellular carcinoma (HCC) have insufficient sensitivity and specificity, and only a low percentage of diagnosis of small tumours is based on these assays. Because HCC is usually asymptomatic at potentially curative stages, identification of biomarkers for the early detection of HCC is essential to improve patient survival. AIM: The aim of this study was to identify candidate markers for HCC development in the plasma from hepatitis C virus (HCV)-infected cirrhotic patients. METHODS: We compared protein expression profiles of plasma samples from HCV-infected cirrhotic patients with and without HCC, using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) coupled with MALDI-TOF/TOF mass spectrometry. The 2-D DIGE results were analysed statistically using Decyder™ software, and verified by western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: In the plasma of HCV-infected HCC patients, we observed decreased expression of complement component 9, ficolin-3 (FCN3), serum amyloid P component (SAP), fibrinogen-gamma and immunoglobulin gamma-1 chain, and increased expression of vitronectin (VTN) and galectin-3 binding protein (G3BP) by DIGE analysis. ELISA confirmed DIGE results for VTN and G3BP but not for SAP or FCN3 in a larger patient population. CONCLUSIONS: The proteins VTN and SAP are candidate biomarkers for HCC development in HCV-infected cirrhotic patients.