Analysis of the anti-fibrotic effects of hop bitter acids on hepatic stellate cells and their anti-tumorigenic effects on hepatocellular carcinoma cells

Bitter acids (BA) from the hop plant Humulus lupulus L. exhibit multiple beneficial biological properties. However, no studies are available regarding the effects of hop bitter acids on liver cells or chronic liver diseases, respectively. Chronic liver diseases are accompanied by chronic hepatic inflammation which can lead to liver fibrosis. If left untreated, liver fibrosis can progress to cirrhosis, which can cause liver failure. Importantly, cirrhosis is also the main risk factor for the development of hepatocellular carcinoma (HCC). Both cirrhosis and HCC are associated with high morbidity and mortality, and up until now, no effective treatment options are available. The aim of this thesis was to assess the effects of BA on hepatic stellate cells, the central mediators of liver fibrosis, and on hepatocellular carcinoma cells. First, the effects of hop bitter acids on hepatic stellate cells (HSC) were analyzed. HSC were isolated from murine and human liver tissues and were incubated with a standardized hop bitter acid extract. At a concentration of 25 µg/ml hop bitter acids (BA) started to induce lactate dehydrogenase leakage. Already at lower concentrations BA led to a dose dependent inhibition of HSC proliferation and inhibited IkappaB-alpha-phosphorylation and nuclear p65 translocation in a dose dependent way. Accordingly, the same doses of BA inhibited the expression of pro-inflammatory and NFkappaB-regulated genes, but did not affect expression of genes not related to NFkappaB signaling. In addition to these effects on activated HSC, BA inhibited the in vitro activation of non-activated HSC as evidenced by delayed expression of collagen I and alpha-sma mRNA and protein. Together, these findings indicate that BA inhibit NFkappaB activation, and herewith, the activation and development of pro-fibrogenic phenotype of HSC in vitro. Next, we analyzed the effects of BA on hepatocellular carcinoma (HCC) cells. Here, we used two different standardized BA extracts enriched for either alpha-acids or beta-acids to get a first insight whether biological activity vary between these two groups of bitter acids. At a concentration of 25 µg/ml, only the beta-acid rich extract started to induce aspartate aminotransferase (AST) release as marker for cell injury, whereas at a dose of 50 µg/ml both extracts led to a significant increase of AST liberation. Already at lower concentrations both extracts dose-dependently inhibited proliferation and migration of HCC cells. Analysis of different signaling pathways revealed an inhibition of ERK1/2 phosphorylation, down-regulation of AP-1 activity and an alleviation of NFkappaB activity in HCC cells in vitro. Hereby, the beta-acid rich extract showed more pronounced effects. Furthermore, the stability of BA in a rodent chow supplemented with an alpha-acid rich extract was assessed applying high pressure liquid chromatography diode array detection technology. Mimicking feasible storage conditions we observed a very poor recovery, indicative of a strong degradation of BA. This prompted us not to proceed with application of this particular BA supplemented chow in rodent models, since in addition to imprecise BA-concentration also potential ill-defined degradation products would not have allowed a sound interpretation of such studies. In conclusion, hop bitter acids alleviate the pro-fibrogenic phenotype of hepatic stellate cells and inhibit central signaling pathways, which are known to play important roles in tumor development and progression, in hepatocellular carcinoma cells. These data suggest the potential use of hop bitter acids as functional nutrient or therapeutical agent for both prevention and treatment of chronic liver diseases. Further studies are needed to verify the beneficial effects in experimental models of liver injury in vivo. However, analysis of a newly designed BA-supplemented experimental chow for rodents revealed strong degradation processes even under optimal storage conditions. Therefore, special care has to be taken when planning long term BA-application experiments

Hop bitter acids inhibit tumorigenicity of hepatocellular carcinoma cells in vitro

Bitter acids (BAs) from the hop plant Humulus lupulus L. exhibit multiple beneficial biological properties with promising effects in cancer therapy and prevention, but information regarding the effects on hepatocellular carcinoma (HCC) is missing. Here, we used two different hop bitter acid extracts enriched for either α-acids or β-acids to obtain insight into whether biological activity varies between these two groups of BAs. At a concentration of 25 µg/ml, only the β-acid rich started to induce aspartate transaminase (AST) release, and a significant increase was detected with 50 µg/ml of both extracts. Already at lower concentrations both extracts led to a dose-dependent inhibition of proliferation, and migration was suppressed at a concentration as low as 5 µg/ml in HCC cells. The focus on different signaling pathways revealed an inhibition of ERK1/2 phosphorylation, downregulation of AP-1 activity and an alleviation of nuclear factor κB (NFκB) activity in HepG2 cells incubated with 5 µg/ml of both extracts, whereby the β-acid rich extract showed more pronounced effects. In conclusion, we identified ERK1/2, AP-1 and NFκB, which are important factors in tumor development and progression, as targets of hop BAs. Thus, these data suggest the potential use of BAs as functional nutrients for both prevention and treatment of HCC

Discrimination of steatosis and NASH in mice using nuclear magnetic resonance spectroscopy

Nonalcoholic fatty liver disease (NAFLD) is a common cause of hepatic dysfunction. The disease spectrum ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The aim of this study was to identify metabolic differences in murine models of simple hepatic steatosis and NASH for the distinction of these NAFLD stages. For 12 weeks, male BALB/c mice were fed either a control or two different high-fat diets leading to hepatic steatosis and NASH, respectively. Metabolic differences were determined by independent component analysis (ICA) of nuclear magnetic resonance (NMR) spectra of lipophilic and hydrophilic liver extracts, and urine specimens. The results from ICA clearly discriminated the three investigated groups. Discriminatory biomarkers in the lipophilic liver extracts were free cholesterol, cholesterol ester and lipid methylene. Discrimination of the hydrophilic liver extracts was mainly mediated by betaine, glucose, and lactate, whereas in urine taurine, trimethylamine-N-oxide, and trimethylamine were the most discriminatory biomarkers. In conclusion, NMR metabolite fingerprinting of spot urine specimens may allow the noninvasive distinction of steatosis and NASH

Increased expression of c-Jun in nonalcoholic fatty liver disease

Overnutrition is the major cause of nonalcoholic fatty liver disease (NAFLD) and its advanced form nonalcoholic steatohepatitis (NASH). We aimed to develop and characterize a murine model, which resembles both the pathology and nutritional situation, of NASH patients in Western societies. Mice were fed with a NASH-inducing diet (ND) containing sucrose, cholesterol and fats rich in saturated fatty acids in a composition, which mimics Western food. After 12 weeks, ND-fed mice revealed obesity and impaired glucose tolerance. In the liver, ND-feeding led to marked steatosis, hepatocellular damage, inflammation and beginning fibrosis. Transcriptome-wide gene expression analysis and search for over-represented transcription factor target sites among the differentially expressed genes identified activator protein-1 (AP-1) as the most likely factor to cause the transcriptional changes in ND livers. Combining differentially expressed gene and protein-protein interaction network analysis identified c-Jun as hub in the largest connected deregulated sub-network in ND livers. Accordingly, ND livers revealed c-Jun-phosphorylation and nuclear translocation. Moreover, hepatic c-Jun expression was enhanced in ND-fed mice. Combined tissue microarray technology and immunohistochemical analysis confirmed enhanced hepatic c-Jun levels in NAFLD patients, which correlated with inflammation, and notably, with the degree of hepatic steatosis. In summary, our new mouse model shows important pathological changes also found in human NASH and indicates c-Jun/AP-1 activation as critical regulator of hepatic alterations. Abundance of c-Jun in NAFLD likely facilitates development and progression of NASH

Enhanced expression of BMP6 inhibits hepatic fibrosis in non-alcoholic fatty liver disease

Objective: Bone morphogenetic protein 6 (BMP6) has been identified as crucial regulator of iron homeostasis. However, its further role in liver pathology including non-alcoholic fatty liver disease (NAFLD) and its advanced form non-alcoholic steatohepatitis (NASH) is elusive. The aim of this study was to investigate the expression and function of BMP6 in chronic liver disease. Design: BMP6 was analysed in hepatic samples from murine models of chronic liver injury and patients with chronic liver diseases. Furthermore, a tissue microarray comprising 110 human liver tissues with different degree of steatosis and inflammation was assessed. BMP6-deficient (BMP6−/−) and wild-type mice were compared in two dietary NASH-models, that is, methionine choline-deficient (MCD) and high-fat (HF) diets. Results: BMP6 was solely upregulated in NAFLD but not in other murine liver injury models or diseased human livers. In NAFLD, BMP6 expression correlated with hepatic steatosis but not with inflammation or hepatocellular damage. Also, in vitro cellular lipid accumulation in primary human hepatocytes induced increased BMP6 expression. MCD and HF diets caused more hepatic inflammation and fibrosis in BMP6−/− compared with wild-type mice. However, only in the MCD and not in the HF diet model BMP6−/− mice developed marked hepatic iron overload, suggesting that further mechanisms are responsible for protective BMP6 effect. In vitro analysis revealed that recombinant BMP6 inhibited the activation of hepatic stellate cells (HSCs) and reduced proinflammatory and profibrogenic gene expression in already activated HSCs. Conclusions: Steatosis-induced upregulation of BMP6 in NAFLD is hepatoprotective. Induction of BMP6-signalling may be a promising antifibrogenic strategy

Expression of fatty acid synthase in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD

Expression and function of methylthioadenosine phosphorylase in chronic liver disease

To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease. DESIGN: MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chrmatography-tandem mass spectrometry. RESULTS: MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs. CONCLUSION: MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis

ERK activation and autophagy impairment are central mediators of irinotecan-induced steatohepatitis

Objective Preoperative chemotherapy with irinotecan is associated with the development of steatohepatitis, which increases the risk of perioperative morbidity and mortality for liver surgery. The molecular mechanisms of this chemotherapeutic complication are widely unknown. Design Mechanisms of irinotecan-induced steatohepatitis were studied in primary human hepatocytes in vitro, in mice treated with irinotecan and in liver specimens from irinotecan-treated compared with control patients. Results Irinotecan dose-dependently induced lipid accumulation and pro-inflammatory gene expression in hepatocytes. This was accompanied by an impairment of mitochondrial function with reduced expression of carnitine palmitoyltransferase I and an induction of acylcoenzyme A oxidase-1 (ACOX1), oxidative stress and extracellular signal-regulated kinase (ERK) activation. ERK inhibition prevented irinotecan-induced pro-inflammatory gene expression but had only a slight effect on lipid accumulation. However, irinotecan also induced an impairment of the autophagic flux mediated by alkalisation of lysosomal pH. Re-acidification of lysosomal pH abolished irinotecan-induced autophagy impairment and lipid accumulation. Also in mice, irinotecan treatment induced hepatic ACOX1 expression, ERK phosphorylation and inflammation, as well as impairment of autophagy and significant steatosis. Furthermore, irinotecan-treated patients revealed higher hepatic ERK activity, expression of pro-inflammatory genes and markers indicative for a shift to peroxisomal fatty acid oxidation and an impaired autophagic flux. Pretreatment with the multityrosine kinase inhibitor sorafenib did not affect autophagy impairment and steatosis but significantly reduced ERK phosphorylation and inflammatory response in irinotecan-treated hepatocytes and murine livers. Conclusions Irinotecan induces hepatic steatosis via autophagy impairment and inflammation via ERK activation. Sorafenib appears as a novel therapeutic option for the prevention and treatment of irinotecan-induced inflammation

Suppression of MTAP in activated hepatic stellate cells with siRNA.

<p>Activated HSCs were transfected with siRNA against MTAP (siRNA1 and siRNA2) or control siRNA. (<b>A</b>,<b>B</b>) Analysis of MTAP expression by qRT-PCR and Western blotting. (<b>C</b>) Quantification of cellular MTA levels by means of LC-ESI-MS/MS. (<b>D</b>) Western blot analysis of phosphorylated p65. Actin staining was used to demonstrate equal protein loading. (<b>E</b>) NFκB reporter gene assay. (<b>F</b>) Analysis of caspase3/7 activity in HSCs with and without staurosporine (STS) treatment (500nM; 4h) to induce apoptosis. (<b>G</b>) Assessment of STS-induced apoptosis by flow cytometric analysis of annexin V-FITC / propidium iodide stained cells. Depicted are mean percentages of total apoptotic cells from 3 independent experiments. (*p<0.05 compared to ctrl. siRNA).</p