Ethanol sensitizes hepatocytes for TGF-ß-triggered apoptosis.

Alcohol abuse is a global health problem causing a substantial fraction of chronic liver diseases. Abundant TGF-ß-a potent pro-fibrogenic cytokine-leads to disease progression. Our aim was to elucidate the crosstalk of TGF-ß and alcohol on hepatocytes. Primary murine hepatocytes were challenged with ethanol and TGF-ß and cell fate was determined. Fluidigm RNA analyses revealed transcriptional effects that regulate survival and apoptosis. Mechanistic insights were derived from enzyme/pathway inhibition experiments and modulation of oxidative stress levels. To substantiate findings, animal model specimens and human liver tissue cultures were investigated. RESULTS: On its own, ethanol had no effect on hepatocyte apoptosis, whereas TGF-ß increased cell death. Combined treatment led to massive hepatocyte apoptosis, which could also be recapitulated in human HCC liver tissue treated ex vivo. Alcohol boosted the TGF-ß pro-apoptotic gene signature. The underlying mechanism of pathway crosstalk involves SMAD and non-SMAD/AKT signaling. Blunting CYP2E1 and ADH activities did not prevent this effect, implying that it was not a consequence of alcohol metabolism. In line with this, the ethanol metabolite acetaldehyde did not mimic the effect and glutathione supplementation did not prevent the super-induction of cell death. In contrast, blocking GSK-3ß activity, a downstream mediator of AKT signaling, rescued the strong apoptotic response triggered by ethanol and TGF-ß. This study provides novel information on the crosstalk between ethanol and TGF-ß. We give evidence that ethanol directly leads to a boost of TGF-ß's pro-apoptotic function in hepatocytes, which may have implications for patients with chronic alcoholic liver disease.

MicroRNAs play a role in spontaneous recovery from acute liver failure.

UNLABELLED: Acute liver failure (ALF) represents a life-threatening situation characterized by sudden and massive liver cell death in the absence of preexisting liver disease. Although most patients require liver transplantation to prevent mortality, some recover spontaneously and show complete liver regeneration. Because of the rarity of this disease, the molecular mechanisms regulating liver regeneration in ALF patients remain largely unknown. In this study, we investigated the role of microRNAs (miRs) that have been implicated in liver injury and regeneration in sera from ALF patients (n = 63). Patients with spontaneous recovery from ALF showed significantly higher serum levels of miR-122, miR-21, and miR-221, compared to nonrecovered patients. In liver biopsies, miR-21 and miR-221 displayed a reciprocal expression pattern and were found at lower levels in the spontaneous survivors, whereas miR-122 was elevated in both serum and liver tissue of those patients. As compared to nonrecovered patients, liver tissue of spontaneous survivors revealed not only increased hepatocyte proliferation, but also a strong down-regulation of miRNA target genes that impair liver regeneration, including heme oxygenase-1, programmed cell death 4, and the cyclin-dependent kinase inhibitors p21, p27, and p57. CONCLUSION: Our data suggest that miR-122, miR-21, and miR-221 are involved in liver regeneration and might contribute to spontaneous recovery from ALF. Prospective studies will show whether serological detection of those miRNAs might be of prognostic value to predict ALF outcome.

BRaf and MEK inhibitors differentially regulate cell fate and microenvironment in human hepatocellular carcinoma.

PURPOSE: Small molecule inhibitors of the mitogen-activated protein kinase (MAPK) pathway, such as sorafenib, represent novel treatment options for advanced hepatocellular carcinoma. The aim of our study was to identify downstream targets as biomarker candidates that are directly linked to the oncogenic MAPK pathway in hepatocellular carcinoma and correlate with inhibition of this pathway by multikinase inhibitors. EXPERIMENTAL DESIGN: Hepatocellular carcinoma cell lines and fresh tumor and tumor-free liver tissues from patients with hepatocellular carcinoma were incubated with different BRaf or MEK inhibitors and analyzed for kinase phosphorylation, proliferation, induction of apoptosis, and chemokine secretion. RESULTS: Hepatocellular carcinoma cell lines responded differentially to these inhibitors in a dose-dependent manner, even those targeting the same kinase. Sorafenib inhibited both MEK1 and ERK1/2 phosphorylation at high but increased signaling at low concentrations. Similarly, PLX4720 increased MEK/ERK signaling independently from mutations in BRaf or NRas. MEK inhibitors decreased ERK1/2 phosphorylation in a dose-dependent manner. These signaling characteristics correlated with inhibition of proliferation, induction of apoptosis, and chemokine secretion. Fresh tissues derived from patients diagnosed with primary hepatocellular carcinoma responded to these inhibitors with changes in their microenvironment following the patterns observed in hepatocellular carcinoma cells. CONCLUSIONS: Oncogenic signaling of the MAPK pathway influences hepatocellular carcinoma sensitivity to treatment with BRaf and MEK inhibitors about cell fate independently from mutations in BRaf and NRas. MAPK inhibitors have a strong impact on chemokine secretion as a consequence of interference with oncogenic signaling. Therefore, novel biomarker candidates associated with the hepatocellular carcinoma microenvironment may be developed for prediction and monitoring of treatment response to small molecule inhibitors.

Increased epithelial permeability is the primary cause for bicarbonate loss in inflamed murine colon.

BACKGROUND: Bicarbonate loss into the lumen occurs during intestinal inflammation in different species. However, candidate pathways like CFTR or DRA are inhibited in the inflamed gut. This study addressed the question whether and how inflammation-associated increased intestinal permeability may result in epithelial HCO(3)(-) loss. METHODS: Murine proximal colon was studied because it does not express functional DRA but is inflamed in the tumor necrosis factor α overexpressing mouse model (TNF(ΔARE)). Luminal alkalization, (3)H-mannitol fluxes, impedance spectroscopy, and dilution potentials were measured in Ussing chambers, whereas expression and localization of tight junction-associated proteins were analyzed by Western blots and immunohistochemistry. RESULTS: Luminal alkalization rates and (3)H-mannitol fluxes were increased in TNF(+/ΔARE) proximal colon, whereas forskolin-stimulated I(sc) was not altered. Epithelial resistance was reduced, but subepithelial resistance increased. The epithelial lining was intact, and enterocyte apoptosis rate was not increased despite massively increased Th1 cytokine levels and lymphoplasmacellular infiltration. Measurement of dilution potentials suggested a loss of cation selectivity with increased anion permeability. Western analysis revealed a downregulation of occludin expression and an upregulation of both claudin-2 and claudin-5, with no change in ZO-1, E-cadherin, claudin-4, and claudin-8. Immunohistochemistry suggested correct occludin localization but reduced tight junction density in TNF(+/ΔARE) surface epithelium. CONCLUSIONS: Inflammation during TNF-α overexpression leads to increased epithelial permeability in murine proximal colon, decreased tight junctional cation selectivity, and increased HCO(3)(-) loss into the lumen. Inflammation-associated colonic HCO(3)(-) loss may occur through leaky tight junctions rather than through HCO(3)(-) secreting ion transporters.

Increased apoptosis induction in hepatocellular carcinoma by a novel tumor-targeted TRAIL fusion protein combined with bortezomib.

UNLABELLED: As the result of an increasing incidence and a prevalent therapy resistance of hepatocellular carcinoma (HCC), there is a strong need for novel strategies to enhance treatment responses in HCC. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising anticancer drug because it can selectively induce apoptosis in cancer cells, but not in healthy cells. Nevertheless, most tumor cells show TRAIL resistance, emphasizing the requirement for apoptosis-sensitizing agents and TRAIL molecules with improved tumor specificity. In this study, we employed a recombinant TRAIL molecule, in which three TRAIL protomers were expressed as a single polypeptide chain (scTRAIL), and a novel TRAIL variant, in which scTRAIL was additionally fused to an antibody fragment recognizing epidermal growth factor receptor (EGFR) to improve its HCC-targeting properties. We analyzed the proapoptotic effects of both TRAIL versions in combination with the proteasome inhibitor bortezomib (BZB) in hepatoma cells and primary human hepatocytes as well as in intact explants from HCC and healthy liver tissue. We demonstrate that EGFR-targeted TRAIL in combination with BZB induced significantly higher caspase activation and cell death in hepatoma cells, but not in primary hepatocytes. Importantly, when incubated with fresh liver explants, the combination of EGFR-targeted TRAIL and BZB displayed selective cytotoxicity for HCC, but not for tumor-free liver tissue, which could even be verified in liver explants from the same individuals. Unlike nontargeted TRAIL, EGFR-targeted TRAIL combined with BZB exerted no toxicity in liver tissues from nonalcoholic fatty liver disease patients. CONCLUSION: EGFR-targeted TRAIL reveals increased antitumor activity toward HCC without inducing toxicity to tumor-free liver tissue and might therefore represent a promising novel strategy for HCC treatment.

Biopsy-controlled liver fibrosis staging using the enhanced liver fibrosis (ELF) score compared to transient elastography.

BACKGROUND AND AIMS: Chronic liver diseases are characterized by inflammatory and fibrotic liver injuries that often result in liver cirrhosis with its associated complications such as portal hypertension and hepatocellular carcinoma. Liver biopsy still represents the reference standard for fibrosis staging, although transient elastography is increasingly used for non-invasive monitoring of fibrosis progression. However, this method is not generally available and is associated with technical limitations emphasizing the need for serological biomarkers staging of liver fibrosis. The enhanced liver fibrosis (ELF) score was shown to accurately predict significant liver fibrosis in different liver diseases, although extracellular matrix components detected by this score may not only mirror the extent of liver fibrosis but also inflammatory processes. METHODS: In this prospective biopsy-controlled study we evaluated the utility of the ELF score in comparison to transient elastography to predict different stages of fibrosis in 102 patients with chronic liver diseases. RESULTS: Both techniques revealed similar area under receiver operating characteristic curve values for prediction of advanced fibrosis stages. Compared to transient elastography, the ELF score showed a broader overlap between low and moderate fibrosis stages and a stronger correlation with inflammatory liver injury. CONCLUSIONS: Both the ELF score as well as transient elastography allowed for high quality fibrosis staging. However, the ELF score was less discriminative in low and moderate fibrosis stages and appeared more strongly influenced by inflammatory liver injury. This should be considered when making clinical interpretations on the basis of ELF score values.

Prospective biopsy-controlled evaluation of cell death biomarkers for prediction of liver fibrosis and nonalcoholic steatohepatitis.

UNLABELLED: Fibrosis and steatosis are major histopathological alterations in chronic liver diseases. Despite various shortcomings, disease severity is generally determined by liver biopsy, emphasizing the need for simple noninvasive methods for assessing disease activity. Because hepatocyte cell death is considered a crucial pathogenic factor, we prospectively evaluated the utility of serum biomarkers of cell death to predict different stages of fibrosis and steatosis in 121 patients with chronic liver disease. We compared the M30 enzyme-linked immunosorbent assay (ELISA), which detects a caspase-cleaved cytokeratin-18 (CK-18) fragment and thereby apoptotic cell death, with the M65 ELISA, which detects both caspase-cleaved and uncleaved CK-18 and thereby overall cell death. Both biomarkers significantly discriminated patients with different fibrosis stages from healthy controls. However, whereas both markers differentiated low or moderate from advanced fibrosis, only the M65 antigen could discriminate even lower stages of fibrosis. The M65 assay also performed better in distinguishing low (≤10%) and higher (>10%) grades of steatosis. In a subgroup of patients, we evaluated the biomarkers for their power to predict nonalcoholic steatohepatitis (NASH). Importantly, both markers accurately differentiated healthy controls or simple steatosis from NASH. However, only serum levels of M65 antigen could differentiate simple steatosis from healthy controls. CONCLUSION: Cell death biomarkers are potentially useful to predict fibrosis, steatosis, or NASH. Compared with the widely used apoptosis marker M30, the M65 assay had a better diagnostic performance and even differentiated between lower fibrosis stages as well as between healthy individuals and patients with simple steatosis.

Apoptosis of regulatory T lymphocytes is increased in chronic inflammatory bowel disease and reversed by anti-TNFα treatment.

BACKGROUND AND AIMS: Inappropriate immune responses contribute to the continuous stimulation of the intestinal immune system in chronic inflammatory bowel disease (IBD). Among several pathogenic factors, a numerical deficiency of regulatory T (Treg) cells has been suggested to lead to an insufficient compensation of chronically activated T lymphocytes. This study was conducted to investigate whether increased apoptosis contributes to Treg cell deficiency in IBD and whether successful treatment with antitumour necrosis factor α (TNFα) is achieved by reducing of Treg cell apoptosis. METHODS: Apoptosis of CD4(+)Foxp3(+) Treg cells in tissue sections of patients with active IBD was analysed by immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) staining. Apoptosis of peripheral blood CD4(+)CD25(+)Foxp3(+) Treg cells was investigated by flow cytometry and annexin-V staining. In addition, caspase activity and apoptosis were measured in sera of patients with IBD treated with anti-TNFα by a luminometric caspase enzyme assay. RESULTS: It is demonstrated that patients with active IBD revealed increased apoptosis of local CD4(+)Foxp3(+) Treg cells in the inflamed mucosa compared with non-inflamed control colon tissue. Moreover, in peripheral blood a reduced frequency and increased apoptosis of Treg cells were found and accompanied by elevated caspase activity in the serum. During anti-TNFα treatment, Treg cell apoptosis declined in close correlation with elevated peripheral Treg cell numbers and a decrease of caspase activation and disease activity. CONCLUSIONS: These data suggest that increased apoptosis of Treg cells plays a potentially important role in the pathogenesis of IBD and can be reversed by anti-TNFα treatment. Measurement of Treg cell apoptosis and serum caspase activity might therefore represent promising tools for monitoring disease activity and treatment response in patients with IBD.