Development of a Prediction Model for Short-Term Remission of Patients with Crohn's Disease Treated with Anti-TNF Drugs.

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.

Evolution after switching to biosimilar infliximab in inflammatory bowel disease patients in clinical remission. / Evolución tras el intercambio a infliximab biosimilar en pacientes con enfermedad inflamatoria intestinal en remisión clínica.

BACKGROUND AND AIM: The biosimilar of infliximab (CT-P13) has been approved for the same indications held by the infliximab reference product (Remicade®); however, there are few clinical data on switching in inflammatory bowel disease (IBD). The aim of this study was to assess the efficacy, safety, bioavailability profile and factors associated with relapse after switching to biosimilar infliximab in IBD patients in clinical remission. MATERIAL AND METHOD: Observational study with IBD patients treated with Remicade® for at least 6 months and in clinical remission for at least 3 months who switched to infliximab biosimilar. The incidence of relapse, adverse effects and possible changes in drug bioavailability (trough level and antidrug antibodies) were evaluated. RESULTS: Thirty six patients were included (63.9% CD) with a mean follow-up of 8.4 months (SD±3.5). The 13.9% had clinical relapse. The longer clinical remission time before switching (HR=0.54, 95% CI=0.29-0.98, P=.04) and detectable infliximab levels at the time of switching (HR=0.03, 95% CI=0.001-0.89, P=.04) were associated with a lower risk of relapse. No differences were found between infliximab levels at the time of switching and at weeks 8 and 16 (P=.94); 8.3% of the patients had some adverse event, requiring the suspension of biosimilar in one patient for severe pneumonia. CONCLUSION: Switching to biosimilar infliximab in a real-life cohort of IBD patients in clinical remission did not have a significant impact on short-term clinical outcomes. The factors associated with relapse were similar to those expected in patients continuing with Remicade®.

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.

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.

Cardiotrophin-1 reduces ischemia/reperfusion injury during liver transplant.

BACKGROUND: Orthotopic liver transplantation (OLT) is currently the elective treatment for advanced liver cirrhosis and acute liver failure. Ischemia/reperfusion damage may jeopardize graft function during the postoperative period. Cardiotrophin-1 (CT-1) has demonstrated cytoprotective properties in different experimental models of liver injury. There is no evidence to demonstrate its potential use in the prevention of the ischemia/reperfusion injury that occurs during OLT. The present study is the first report to show that the administration of CT-1 to donors would benefit the outcome of OLT. MATERIALS AND METHODS: We tested the cytoprotective effect of CT-1 administered to the donor prior to OLT in an experimental pig model. Hemodynamic changes, hepatic histology, cell death parameters, activation of cell signaling pathways, oxidative and nitrosative stress, and animal survival were analyzed. RESULTS: Our data showed that CT-1 administration to donors increased animal survival, improved cardiac and respiratory functions, and reduced hepatocellular injury as well as oxidative and nitrosative stress. These beneficial effects, related to the activation of AKT, ERK, and STAT3, reduced caspase-3 activity and diminished IL-1ß and TNF-α expression together with IL-6 upregulation in liver tissue. CONCLUSIONS: The administration of CT-1 to donors reduced ischemia/reperfusion injury and improved survival in an experimental pig model of OLT.

Impact of age on liver regeneration response to injury after partial hepatectomy in a rat model.

BACKGROUND: Liver resection is a feasible treatment for multiple liver diseases. There is no evidence about the impact of age on liver regeneration. OBJECTIVE: To assess the effect of age on liver regeneration in an experimental in vivo animal model of 70%-partial hepatectomy. METHODS: Forty young (Y) and old (O) Wistar male rats (n = 80) were distributed into four groups [controls (C), sham operated (SO), hepatectomy 6 h (H6), and 48 h (H48)]. Different morphometric and biochemical factors, oxidative and nitrosative stress, lipid peroxidation, cytokines kinetics, and histopathologic tissular parameters were determined. RESULTS: Early postoperative mortality was higher in aged rats (P = 0.049). Morphometric determinations, liver regeneration index, and total volume weight were favorable to young rats. Serum transaminase levels were higher in aged rats. Parameters of necrosis (measured by histopathologic injury [HI: 0-I-II-III]), regeneration (measured by bromodeoxyuridine-BrdU incorporation) and apoptosis (determined by the TDT-mediated dUTP nick end labeling-TUNEL) were well-synchronized in young rats. Parameters of oxidative stress such as reduced (GSH), oxidized (GSSG) glutathione and lipid peroxidation (measured by hepatic malondialdehyde -MDA-) were lower in young animals throughout the studied period. Nitrosative stress measured by nitric oxide (NO) end-products was higher in late stages in resected old rats. Pro-inflammatory cytokines (TNF- α) reached higher and earlier levels in aged rats while pro-regenerative cytokines (IL-6) were significantly higher in early stages for young rats and in late stages for aged rats. The levels of TGF-ß were higher in young rats. CONCLUSION: Liver regeneration is delayed and reduced in aged animals submitted to liver resection.

Calcium-dependent nitric oxide production is involved in the cytoprotective properties of n-acetylcysteine in glycochenodeoxycholic acid-induced cell death in hepatocytes.

The intracellular oxidative stress has been involved in bile acid-induced cell death in hepatocytes. Nitric oxide (NO) exerts cytoprotective properties in glycochenodeoxycholic acid (GCDCA)-treated hepatocytes. The study evaluated the involvement of Ca2+ on the regulation of NO synthase (NOS)-3 expression during N-acetylcysteine (NAC) cytoprotection against GCDCA-induced cell death in hepatocytes. The regulation of Ca2+ pools (EGTA or BAPTA-AM) and NO (L-NAME or NO donor) production was assessed during NAC cytoprotection in GCDCA-treated HepG2 cells. The stimulation of Ca2+ entrance was induced by A23187 in HepG2. Cell death, Ca2+ mobilization, NOS-1, -2 and -3 expression, AP-1 activation, and NO production were evaluated. GCDCA reduced intracellular Ca2+ concentration and NOS-3 expression, and enhanced cell death in HepG2. NO donor prevented, and l-NAME enhanced, GCDCA-induced cell death. The reduction of Ca2+ entry by EGTA, but not its release from intracellular stores by BAPTA-AM, enhanced cell death in GCDCA-treated cells. The stimulation of Ca2+ entrance by A23187 reduced cell death and enhanced NOS-3 expression in GCDCA-treated HepG2 cells. The cytoprotective properties of NAC were related to the recovery of intracellular Ca2+ concentration, NOS-3 expression and NO production induced by GCDCA-treated HepG2 cells. The increase of NO production by Ca2+-dependent NOS-3 expression during NAC administration reduces cell death in GCDCA-treated hepatocytes.

Usefulness of monitoring antitumor necrosis factor serum levels during the induction phase in patients with Crohn's disease.

AIMS: The aims of this study were (a) to know the kinetics of antitumor necrosis factor (TNF) drug serum levels during the induction phase in patients with Crohn's disease; (b) to identify variables associated with these levels; and (c) to assess the relation between these levels and short-term effectiveness in Crohn's disease patients. METHODS: Patients with Crohn's disease naïve to anti-TNF treatment were prospectively included. Remission was defined as a Crohn's disease activity index (CDAI) score <150 after 14 weeks of treatment. Blood samples were obtained at baseline and at weeks 4, 8, and 14. Adalimumab and infliximab levels were measured, receiver operating characteristic (ROC) curves were constructed, and the area under the ROC curve was calculated. RESULTS: One-hundred fifty patients with Crohn's disease were included, 79 (53%) received infliximab and 71 (47%) had CDAI > 150 at study entry. At week 14, 52 out of 71 patients with CDAI > 150 at baseline (73%) had clinical remission. There were no differences in infliximab levels between patients with and without remission (8 vs. 9.1 µg/mL, P > 0.05) or with and without response (7 vs. 11 µg/mL, P > 0.05) at week 14. There was a trend to higher levels of adalimumab concentration in responders in comparison with nonresponders (13 vs. 6.7 µg/mL, P = 0.05) and in patients who achieved remission in comparison with nonremitters (13.5 vs. 8.4 µg/mL, P = 0.06). In the multivariate analysis, no variable was predictive of short-term remission, including infliximab and adalimumab serum levels. CONCLUSION: Determining anti-TNF serum levels during the induction phase is not useful for predicting short-term remission in patients with Crohn's disease.

The reduction of cell death and proliferation by p27(Kip1) minimizes DNA damage in an experimental model of genotoxicity.

Hepatocellular carcinoma (HCC) is the fifth most commonly occurring cancer worldwide. The expression of p27 has been related to reduced severity of tumor grade and recurrence of HCC. The study assessed the role of p27 on the cell proliferation and death, and DNA mutagenesis in experimental genotoxicity induced by aflatoxin B1 (AFB(1)) in cultured hepatocytes obtained from control and p27(Kip1) deficient mice. The overexpression of p27 was assessed with wild type p27(Kip1) expression vector in HepG2 cells. The expression of p27, p21 and p53 was assessed in well and poorly-differentiated liver tumors. DNA damage and cell death induced by AFB(1) were related to a reduction of p27 and p21 expression in cultured hepatocytes. AFB(1)-induced nuclear phosphorylated (Ser 10) p27 degradation was related to a rise of nuclear KIST, Rsk-1 and Rsk-2 expression and cytoplasm phosphorylated (Thr 198) p27 expression. The overexpression of p27 reduced cell proliferation, cell death and DNA damage in AFB(1)-treated hepatocytes. The enhanced survival of patients with well differentiated compared to poorly-differentiated tumors was related to high expression of p27, p21 and p53 in liver sections. The study showed that the p27 reduced cell proliferation and death, as well as the accumulation of DNA damage in hepatocarcinogenesis.

Mitochondrial-driven ubiquinone enhances extracellular calcium-dependent nitric oxide production and reduces glycochenodeoxycholic acid-induced cell death in hepatocytes.

Ca(2+) mobilization, nitric oxide (NO), and oxidative stress have been involved in cell death induced by hydrophobic bile acid in hepatocytes. The aim of the study was the elucidation of the effect of the antioxidant mitochondrial-driven ubiquinone (Mito Q) on the intracellular Ca(2+) concentration, NO production, and cell death in glycochenodeoxycholic acid (GCDCA)-treated HepG2 cells. The role of the regulation of the intracellular Ca(2+) concentration by Ca(2+) chelators (EGTA or BAPTA-AM), agonist of Ca(2+) entrance (A23187) or NO (L-NAME or NO donor), was assessed during Mito Q cytoprotection in GCDCA-treated HepG2 cells. Cell death, NO synthase (NOS)-1, -2, and -3 expression, Ca(2+) mobilization, and NO production were evaluated. GCDCA reduced the intracellular Ca(2+) concentration and NOS-3 expression and enhanced cell death in HepG2. NO donor prevented and L-NAME enhanced GCDCA-induced cell death. The reduction of Ca(2+) entry by EGTA, but not its release from intracellular stores by BAPTA-AM, reduced the expression of NOS-3 and enhanced cell death in control and GCDCA-treated cells. Mito Q prevented the reduction of intracellular Ca(2+) concentration, NOS-3 expression, NO production, and cell death in GCDCA-treated HepG2 cells. The conclusion is that the recovery of Ca(2+)-dependent NOS-3 expression by Mito Q may be considered an additional cytoprotective property of an antioxidant.

Bevacizumab Allows Preservation of Liver Function and its Regenerative Capacity after Major Hepatectomy.

BACKGROUND: Parallel to the safety of liver resections, new chemotherapy drugs have emerged for the control of liver metastases. However, there is unclear evidence about the combination of intensive BVZ-therapy and extended resections. The main aim was to analyse the impact of Bevacizumab (BVZ) in terms of liver safety and tolerability in two experimental models: a basal-toxicity situation and after major hepatectomy. METHODS: Eighty male-Wistar rats were grouped as toxicity analysis (sham-operated rats-OS-) and regeneration after- surgery analysis (hepatectomy rats-H-). Eight further subgroups were created according to sacrifice (6- hours-6h- or 24-hours-24h-) and dose (µg) of BVZ (none, 100, 200, 400). Several measurements were performed, including biochemical serum samples, histopathological analysis, cytokines (IL-6, TNF-α, TGF-ß), oxidative-stress (GSH/GSSG, ATP), lipid-peroxidation (TBARS) and epidermal and vascular endothelium growth-factors (EGF and VEGF). RESULTS: In the toxicity analysis, safe results with BVZ were observed, with no significant differences among the groups. A trend towards a lower oxidative status was observed in the OS 6 h-100, -200 and -400 versus the OS 6 h-none group. Similar results were observed in the hepatectomy model, with stable oxidative-stress-index and IL-6, TNF- α, and TGF- ß levels. Despite higher lipid peroxidation status, overall regeneration was preserved. As expected, VEGF was almost undetectable in BVZ-treated groups after resection, but not in the non-resection group. CONCLUSION: It was concluded that liver status was not impaired by BVZ even at the high-dose. Similarly, liver regeneration after extended hepatectomy in BVZ-treated animals was well-preserved. Extended liver resections may be encouraged in BVZ-treated patients due to its excellent tolerability and good liver regeneration status.

Functional rare variants influence the clinical response to anti-TNF therapy in Crohn's disease.

BACKGROUND: The effect of low-frequency functional variation on anti-tumor necrosis factor alpha (TNF) response in Crohn's disease (CD) patients remains unexplored. The objective of this study was to investigate the impact of functional rare variants in clinical response to anti-TNF therapy in CD. METHODS: CD anti-TNF naïve patients starting anti-TNF treatment due to active disease [Crohn's Disease Activity Index (CDAI > 150)] were included. The whole genome was sequenced using the Illumina Hiseq4000 platform. Clinical response was defined as a CDAI score <150 at week 14 of anti-TNF treatment. Low-frequency variants were annotated and classified according to their damaging potential. The whole genome of CD patients was screened to identify homozygous loss-of-function (LoF) variants. The TNF signaling pathway was tested for overabundance of damaging variants using the SKAT-O method. Functional implication of the associated rare variation was evaluated using cell-type epigenetic enrichment analyses. RESULTS: A total of 41 consecutive CD patients were included; 3250 functional rare variants were identified (2682 damaging and 568 LoF variants). Two homozygous LoF mutations were found in HLA-B and HLA-DRB1 genes associated with lack of response and remission, respectively. Genome-wide LoF variants were enriched in epigenetic marks specific for the gastrointestinal tissue (colon, p = 4.11e-4; duodenum, p = 0.011). The burden of damaging variation in the TNF signaling pathway was associated with response to anti-TNF therapy (p = 0.016); damaging variants were enriched in epigenetic marks from CD8+ (p = 6.01e-4) and CD4+ (p = 0.032) T cells. CONCLUSIONS: Functional rare variants are involved in the response to anti-TNF therapy in CD. Cell-type enrichment analysis suggests that the gut mucosa and CD8+ T cells are the main mediators of this response.

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.

Biomarkers for hepatocellular carcinoma: diagnostic and therapeutic utility.

Because of the high prevalence and associated-mortality of hepatocellular carcinoma (HCC), early diagnosis of the disease is vital for patient survival. In this regard, tumor size is one of the two main prognostic factors for surgical resection, which constitutes the only curative treatment for HCC along with liver transplantation. However, techniques for HCC surveillance and diagnosis that are currently used in clinical practice have certain limitations that may be inherent to the tumor development. Thus, it is important to continue efforts in the search for biomarkers that increase diagnostic accuracy for HCC. In this review, we focus on different biological sources of candidate biomarkers for HCC diagnosis. Although those biomarkers identified from biological samples obtained by noninvasive methods have greater diagnostic value, we have also considered those obtained from liver tissue because of their potential therapeutic value. To date, sorafenib is the only US Food and Drug Administration-approved antineoplastic for HCC. However, this therapeutic agent shows very low tumor response rates and frequently causes acquired resistance in HCC patients. We discuss the use of HCC biomarkers as therapeutic targets themselves, or as targets to increase sensitivity to sorafenib treatment.

Plasma protein biomarkers of hepatocellular carcinoma in HCV-infected alcoholic patients with cirrhosis.

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers in the world, with limited options for treatment unless timely diagnosed. Chronic hepatitis C virus (HCV) infection and persistent heavy alcohol consumption are independent risk factors for HCC development, which may induce a specific protein expression pattern different from those caused separately. The aim of the study was to identify protein biomarkers for the detection of HCC in HCV-infected alcoholic patients with cirrhosis in order to improve survival. We compared protein expression profiles of plasma samples from 52 HCV-infected alcoholic patients with and without HCC, using 2-D DIGE coupled with MALDI-TOF/TOF mass spectrometry. The 2-D DIGE results were analyzed statistically using Decyder software, and verified by western-blot and ELISA. In plasma samples from HCV-infected alcoholic patients, we found significantly differential expression profiles of carboxypeptidase-N, ceruloplasmin (CP), complement component 4a (C4a), fibrinogen-alpha (FGA), immunoglobulin mu chain C region, serum albumin, and serum paraoxonase/arylesterase 1 (PON1). Deregulation of plasma/serum levels of the identified proteins was associated to HCV, ethanol consumption, and/or HCC progression. In the validation through ELISA, C4a serum concentration was increased in HCC patients (2.4±1 ng/mg vs 1.8±0.6 ng/mg; p = 0.029), being the only independent predictor of HCC in the multivariate analysis (OR = 2.15; p = 0.015), with an AUROC = 0.70. The combination of C4a, FGA, CP and PON1 improved slightly the predictive ability of C4a alone (AUROC 0.81). In conclusion, we identified proteins related to acute-phase response, oxidative stress, or immune response, whose differential expression in plasma may be attributed to the presence of HCC. Among them, C4a, and its combination with CP, FGA and PON1, could be considered as potentially reliable biomarkers for the detection of HCC in HCV-infected alcoholic patients.

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.

Proteomics in HepG2 hepatocarcinoma cells with stably silenced expression of PRDX1.

Peroxiredoxin 1 (PRDX1) is a member of the peroxiredoxin family. Aberrant expression of PRDX1 has been described in various cancers. We investigated the significance of this up-regulation in non-challenged hepatocellularcarcinoma (HCC) cells by establishing a HepG2 cell line stably expressing a Prdx1 shRNA. Prdx1 silencing reversed, at least partially, the tumoural phenotype of HepG2 cells, resulting in morphological changes, delayed cell growth, down-regulation of transcripts for AFP, osteopontin and ß-catenin and decreased γ-glutamyl transpeptidase activity, and oppositely up-regulation of transcripts for E-cadherin and proapoptotic proteins (BAX, CASP3) and increased alkaline phosphatase and CASP3 activities. Proteomic profiling identified 16 spots differentially expressed in Prdx1-silenced cells. Most of the variations involved the down-regulation of proteins with pivotal roles in cell proliferation and differentiation, in agreement with the observed phenotypic changes. We also investigated the effect of Prdx1 silencing on thiol protein oxidation. Proteins prone to reversible cysteine oxidation play major physiological functions. Notably, the down-regulation and altered redox status of key enzymes of carbohydrate and amino acid metabolism suggested a disturbance of the Warburg effect and glutamine utilization, two major pathways in the proliferation of tumour cells. Overall, these observations suggest that PRDX1 acts as a pro-cancer protein in HCC HepG2 cells.

Targeting hepatoma using nitric oxide donor strategies.

AIMS: The study evaluated the role of increased intracellular nitric oxide (NO) concentration using NO donors or stably NO synthase-3 (NOS-3) overexpression during CD95-dependent cell death in hepatoma cells. The expression of cell death receptors and caspase activation, RhoA kinase activity, NOS-3 expression/activity, oxidative/nitrosative stress, and p53 expression were analyzed. The antitumoral activity of NO was also evaluated in the subcutaneous implantation of NOS-3-overexpressing hepatoma cells, as well NO donor injection into wild-type hepatoma-derived tumors implanted in xenograft mouse models. RESULTS: NO donor increased CD95 expression and activation of caspase-8 and 3 in HepG2, Huh7, and Hep3B cells. NOS-3 overexpression increased oxidative/nitrosative stress, p53 and CD95 expression, cellular Fas-associated death domain (FADD)-like IL-1beta converting enzyme (FLICE) inhibitory protein long (cFLIP(L)) and its short isoform (cFLIP(S)) shift, and cell death in HepG2 (4TO-NOS) cells. The inhibition of RhoA kinase and p53 knockdown using RNA interference reduced cell death in 4TO-NOS cells. The supplementation with hydrogen peroxide (H(2)O(2)) increased NOS-3 activity and cell death in 4TO-NOS cells. NOS-3 overexpression or NO donor injection into hepatoma-derived tumors reduced the size and increased p53 and cell death receptor expression in nude mice. INNOVATION AND CONCLUSIONS: The increase of intracellular NO concentration promoted oxidative and nitrosative stress, Rho kinase activity, p53 and CD95 expression, and cell death in cultured hepatoma cells. NOS-3-overexpressed HepG2 cells or intratumoral NO donor administration reduced tumor cell growth and increased the expression of p53 and cell death receptors in tumors developed in a xenograft mouse model.