Enumeration and Characterization of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Undergoing Transarterial Chemoembolization.

Circulating tumor cells (CTCs), and particularly circulating cancer stem cells (cCSC), are prognostic biomarkers for different malignancies and may be detected using liquid biopsies. The ex vivo culture of cCSCs would provide valuable information regarding biological aggressiveness and would allow monitoring the adaptive changes acquired by the tumor in real time. In this prospective pilot study, we analyzed the presence of EpCAM+ CTCs using the IsoFlux system in the peripheral blood of 37 patients with hepatocellular carcinoma undergoing transarterial chemoembolization (TACE). The average patient age was 63.5 ± 7.9 years and 91.9% of the patients were men. All patients had detectable CTCs at baseline and 20 patients (54.1%) showed CTC aggregates or clusters in their peripheral blood. The increased total tumor diameter (OR: 2.5 (95% CI: 1.3-4.8), p = 0.006) and the absence of clusters of CTCs at baseline (OR: 0.2 (95% CI: 0.0-1.0), p = 0.049) were independent predictors of a diminished response to TACE. Culture of cCSC was successful in five out of thirty-three patients, mostly using negative enrichment of CD45- cells, ultra-low adherence, high glucose, and a short period of hypoxia followed by normoxia. In conclusion, the identification of clusters of CTCs before TACE and the implementation of standardized approaches for cCSC culture could aid to predict outcomes and to define the optimal adjuvant therapeutic strategy for a true personalized medicine in hepatocellular carcinoma.

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.

Circulating Tumor Cells in Hepatocellular Carcinoma: A Comprehensive Review and Critical Appraisal.

Hepatocellular carcinoma (HCC) is the fifth most common neoplasm and a major cause of cancer-related death worldwide. There is no ideal biomarker allowing early diagnosis of HCC and tumor surveillance in patients receiving therapy. Liquid biopsy, and particularly circulating tumor cells (CTCs), have emerged as a useful tool for diagnosis and monitoring therapeutic responses in different tumors. In the present manuscript, we evaluate the current evidence supporting the quantitative and qualitative assessment of CTCs as potential biomarkers of HCC, as well as technical aspects related to isolation, identification, and classification of CTCs. Although the dynamic assessment of CTCs in patients with HCC may aid the decision-making process, there are still many uncertainties and technical caveats to be solved before this methodology has a true impact on clinical practice guidelines. More studies are needed to identify the optimal combination of surface markers, to increase the efficiency of ex-vivo expansion of CTCs, or even to target CTCs as a potential therapeutic strategy to prevent HCC recurrence after surgery or to hamper tumor progression and extrahepatic spreading.

Activation of mTOR Signaling Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and occurs mainly in patients with liver cirrhosis. The mammalian target of rapamycin (mTOR) signaling pathway is involved in many hallmarks of cancer including cell growth, metabolism re-programming, proliferation and inhibition of apoptosis. The mTOR pathway is upregulated in HCC tissue samples as compared with the surrounding liver cirrhotic tissue. In addition, the activation of mTOR is more intense in the tumor edge, thus reinforcing its role in HCC proliferation and spreading. The inhibition of the mTOR pathway by currently available pharmacological compounds (i.e., sirolimus or everolimus) is able to hamper tumor progression both in vitro and in animal models. The use of mTOR inhibitors alone or in combination with other therapies is a very attractive approach, which has been extensively investigated in humans. However, results are contradictory and there is no solid evidence suggesting a true benefit in clinical practice. As a result, neither sirolimus nor everolimus are currently approved to treat HCC or to prevent tumor recurrence after curative surgery. In the present comprehensive review, we analyzed the most recent scientific evidence while providing some insights to understand the gap between experimental and clinical studies.

mTOR Expression in Liver Transplant Candidates with Hepatocellular Carcinoma: Impact on Histological Features and Tumour Recurrence.

(1) Background: The mammalian target of rapamycin (mTOR) pathway activation is critical for hepatocellular carcinoma (HCC) progression. We aimed to evaluate the mTOR tissue expression in liver transplant (LT) patients and to analyse its influence on post-LT outcomes. (2) Methods: Prospective study including a cohort of HCC patients who underwent LT (2012⁻2015). MTOR pathway expression was evaluated in the explanted liver by using the "PathScan Intracellular Signalling Array Kit" (Cell Signalling). Kaplan-Meier and Cox regression analyses were performed to evaluate post-LT HCC recurrence. (3) Results: Forty-nine patients were included (average age 56.4 ± 6, 14.3% females). Phospho-mTOR (Ser2448) was over-expressed in peritumoral tissue as compared with tumoral tissue (ΔSignal 22.2%; p < 0.001). The mTOR activators were also increased in peritumoral tissue (phospho-Akt (Thr308) ΔSignal 18.2%, p = 0.004; phospho-AMPKa (Thr172) ΔSignal 56.3%, p < 0.001), as they were the downstream effectors responsible for cell growth/survival (phospho-p70S6K (Thr389) ΔSignal 33.3%, p < 0.001 and phospho-S6RP (Ser235/236) ΔSignal 54.6%, p < 0.001). MTOR expression was increased in patients with multinodular HCC (tumoral p = 0.01; peritumoral p = 0.001). Increased phospho-mTOR in tumoral tissue was associated with higher HCC recurrence rates after LT (23.8% vs. 5.9% at 24 months, p = 0.04). (4) Conclusion: mTOR pathway is over-expressed in patients with multinodular HCC and is it associated with increased post-LT tumour recurrence rates.

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.

Cardiotrophin-1 decreases liver apoptosis through calpastatin induction.

BACKGROUND: Cardiotrophin-1 (CT1) has been used to prevent cell death in different models of liver injury in rats. D-galactosamine induces cell death in culture rat and human hepatocytes. The present study evaluated the cytoprotective effects of CT1 in an experimental model of apoptosis induced by D-galactosamine in hepatocytes. METHODS: DNA fragmentation, calpain activity and Western blots of caspase-3, calpastatin and Stat3, and Akt phosphorylation were measured. Stat3 and Akt inhibitors were used to analyze the mechanisms of action of CT1. RESULTS: CT1 caused an increase in Stat3 and Akt phosphorylation and a decrease of DNA fragmentation, calpain activity, and caspase-3 induced by D-galactosamine. The reduction of calpain activity by CT1 was associated with an increase of calpastatin (its endogenous inhibitor). The effects of CT1 were also dependent on the activation of Sta3 or Akt. CONCLUSIONS: CT1 decreases cell death through a mechanism related to Stat3 and Akt phosphorylation and activation of calpastatin in D-galactosamine-treated hepatocytes.

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.

Evolution meets disease: penetrance and functional epistasis of mitochondrial tRNA mutations.

About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNA(Ile) that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both.

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.

Nitric oxide mimics transcriptional and post-translational regulation during α-tocopherol cytoprotection against glycochenodeoxycholate-induced cell death in hepatocytes.

BACKGROUND & AIMS: Reactive oxygen species (ROS) and nitric oxide (NO) exert a relevant role during bile acid-induced hepatotoxicity. Whether α-Tocopherol regulates oxidative and nitrosative stress, bile acid transporter expression and their NO-dependent post-translational modifications, and cell death were assessed in vitro and in vivo. METHODS: α-Tocopherol and/or NO donors (DETA-NONOate or CSNO, and V-PYRRO/NO) were administered to glycochenodeoxycholic acid (GCDCA)-treated cultured human hepatocytes or to bile duct obstructed rats. Cell injury, superoxide anion (O⁻2) production, as well as inducible nitric oxide synthase (NOS-2), cytochrome P4507A1 (CYP7A1), heme oxygenase-1, (HO-1) and bile acid transporter expression were determined. Cysteine S-nitrosylation and tyrosine nitration of Na(+)-taurocholate co-transporting polypeptide (NTCP), as well as taurocholic acid (TC) uptake were also evaluated. RESULTS: GCDCA-induced cell death was associated with increased (O⁻2) production, NTCP and HO-1 expression, and with a reduction of CYP7A1 and NOS-2 expression. α-Tocopherol reduced cell death, (O⁻2) production, CYP7A1, NTCP, and HO-1 expression, as well as increased NOS-2 expression and NO production in GCDCA-treated hepatocytes. α-Tocopherol and NO donors increased NTCP cysteine S-nitrosylation and tyrosine nitration, and reduced TC uptake in hepatocytes. α-Tocopherol and V-PYRRO/NO reduced liver injury and NTCP expression in obstructed rats. CONCLUSIONS: The regulation of CYP7A1, NTCP, and HO-1 expression may be relevant for the cytoprotective properties of α-Tocopherol and NO against mitochondrial dysfunction, oxidative stress and cell death in GCDCA-treated hepatocytes. The regulation of NO-dependent post-translational modifications of NTCP by α-Tocopherol and NO donors reduces the uptake of toxic bile acids by hepatocytes.

Clearance of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma Undergoing Surgical Resection or Liver Transplantation.

BACKGROUND: In patients with hepatocellular carcinoma (HCC), a complete clearance of circulating tumor cells (CTCs) early after liver transplantation (LT) or surgical resection (LR) could prevent tumor recurrence. METHODS: prospective pilot study including patients with HCC who underwent LR or LT from September 2017 to May 2020. Enumeration of CTCs was performed in peripheral blood samples (7 mL) using the Isoflux® system (Fluxion Biosciences) immediately before surgery, at post-operative day 5 and at day 30. A clinically relevant number of CTCs was defined as >30 CTCs/sample. RESULTS: 41 HCC patients were included (mean age 58.7 ± 6.3; 82.9% male). LR was performed in 10 patients (24.4%) and 31 patients (75.6%) underwent LT. The main etiology of liver disease was chronic hepatitis C (31.7%). Patients undergoing LR and LT were similar in terms of preoperative CTC count (p = 0.99), but clearance of CTCs within the first month was more pronounced in the LT group. Clusters of CTCs at baseline were associated with incomplete clearance of CTCs at day 30 (54.2% vs. 11.8%, p = 0.005), which in turn impacted negatively on survival (p = 0.038). CONCLUSION: Incomplete clearance of CTCs after surgery could be a surrogate marker of HCC aggressiveness.

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.

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.

Selecting patients with hepatocellular carcinoma for liver transplantation: incorporating tumor biology criteria.

Liver transplantation (LT) is the optimal therapeutic option for patients with liver cirrhosis and hepatocellular carcinoma (HCC). Due to universal donor shortage, only the patients with limited tumor burden (under the so-called Milan criteria) are considered as potential candidates for LT in most institutions. It is expected that in the near future, more liver grafts will be available for patients with HCC due to the implementation of new direct antivirals against hepatitis C, leaving a prone scenario to consider expanding Milan criteria. A moderate expansion of Milan criteria could be implemented without increasing the risk of tumor recurrence if patients with favorable biological behavior are carefully selected. Incorporating information regarding tumor biology in the decision-making algorithm would result in a more rational use of LT in patients with HCC. In the present review, surrogate markers of tumor biology are critically evaluated as potential tools to be combined with existing radiological criteria. In addition, the current state of liquid biopsy is discussed, as this cutting-edge technology may reshape the management of HCC in the upcoming years.

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.

Dysregulation of the Splicing Machinery Is Associated to the Development of Nonalcoholic Fatty Liver Disease.

CONTEXT: Nonalcoholic fatty liver disease (NAFLD) is a common obesity-associated pathology characterized by hepatic fat accumulation, which can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Obesity is associated with profound changes in gene-expression patterns of the liver, which could contribute to the onset of comorbidities. OBJECTIVE: As these alterations might be linked to a dysregulation of the splicing process, we aimed to determine whether the dysregulation in the expression of splicing machinery components could be associated with NAFLD. PARTICIPANTS: We collected 41 liver biopsies from nonalcoholic individuals with obesity, with or without hepatic steatosis, who underwent bariatric surgery. INTERVENTIONS: The expression pattern of splicing machinery components was determined using a microfluidic quantitative PCR-based array. An in vitro approximation to determine lipid accumulation using HepG2 cells was also implemented. RESULTS: The liver of patients with obesity and steatosis exhibited a severe dysregulation of certain splicing machinery components compared with patients with obesity without steatosis. Nonsupervised clustering analysis allowed the identification of three molecular phenotypes of NAFLD with a unique fingerprint of alterations in splicing machinery components, which also presented distinctive hepatic and clinical-metabolic alterations and a differential response to bariatric surgery after 1 year. In addition, in vitro silencing of certain splicing machinery components (i.e., PTBP1, RBM45, SND1) reduced fat accumulation and modulated the expression of key de novo lipogenesis enzymes, whereas conversely, fat accumulation did not alter spliceosome components expression. CONCLUSION: There is a close relationship between splicing machinery dysregulation and NAFLD development, which should be further investigated to identify alternative therapeutic targets.

Alteration of S-nitrosothiol homeostasis and targets for protein S-nitrosation in human hepatocytes.

The liver is one organ clearly influenced by nitric oxide (NO), and acute and chronic exposure to this substance has been associated with distinct patterns of liver disease. Disruption or deregulation of S-nitrosothiol (SNO) signalling leads to impairment of cellular function and disease, and this study was aimed to identify potential targets for protein S-nitrosation during alteration of SNO homeostasis in human hepatocytes. Cells were treated with S-nitroso-L-cysteine (CSNO), an effective physiological nitrosothiol for delivering NO bioactivity to cells. Treatment with CSNO augmented the levels of S-nitrosoproteins detected both by chemiluminescence and the biotin switch method. CSNO treatment also increased S-nitrosoglutathione reductase (GSNOR) activity that returned SNO content to basal levels. This increased enzymatic activity was related to augmented levels of ADH-5 mRNA, the gene encoding for GSNOR in humans. In addition, the treatment with the SNO also increased cell death. Twenty S-nitrosoproteins were identified in CSNO-treated hepatocytes, including mitochondrial aldehyde dehydrogenase, protein disulphide isomerase, Hsp60, GRP75 and Raf kinase inhibitor protein. The identification in the S-nitrosatable proteome of proteins involved in metabolism, maintenance of cellular homeostasis and signalling points to the relevance of protein S-nitrosation to the physiology and pathophysiology of human hepatocytes.

Impact of Early Initiated Everolimus on the Recurrence of Hepatocellular Carcinoma After Liver Transplantation.

BACKGROUND: Many centers implement everolimus-based immunosuppression in liver transplant patients with hepatocellular carcinoma. We aimed to explore the potential impact of early initiated everolimus on tumor recurrence after liver transplantation. METHODS: This study included 192 patients with hepatocellular carcinoma undergoing liver transplantation among who 64 individuals were prospectively enrolled (2012-2015) and received early initiated everolimus (ie, started between postoperative day 15 to 21), whereas the remaining 128 patients acted as historical controls without everolimus. Propensity score matching was performed to ensure comparability. Multivariate Cox regression and competing risks analysis were used to control for potential confounders. RESULTS: Patients with and without everolimus were comparable in terms of number of nodules (P = 0.37), total tumor diameter (P = 0.44), Milan criteria fulfillment (P = 0.56), and histological differentiation (P = 0.61), but there were increased microvascular invasion rates in the everolimus group (26.5% vs 13.3%; P = 0.026). Tumor recurrence rates were similar with and without everolimus (10.9% vs 9.9% at 36 months respectively; P = 0.18). After controlling for microvascular invasion among other potential confounders, everolimus had no significant impact on tumor recurrence, neither in the multivariate Cox regression (relative risk = 3.23; P = 0.09), nor in the competing risks analysis for tumor recurrence-death (relative risk = 1.02; P = 0.94). Patients receiving everolimus had reduced tacrolimus trough concentrations and lower serum creatinine within the first 18 months postliver transplantation. CONCLUSION: Everolimus may not be universally prescribed to prevent tumor recurrence in liver transplant patients with hepatocellular carcinoma. Future randomized trials should be focused on patients with histological features of increased tumor aggressiveness, in whom the potential benefit would be higher.