Hop-derived Humulinones Reveal Protective Effects in in vitro Models of Hepatic Steatosis, Inflammation and Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is emerging as leading cause of liver disease worldwide. Specific pharmacologic therapy for NAFLD is a major unmet medical need. Recently, iso-alpha acids, hop-derived bitter compounds in beer, have been shown to beneficially affect NAFLD pathology. Humulinones are further hop derived bitter acids particularly found in modern styles of beer. So far, biological effects of humulinones have been unknown. Here, we investigated the effect of humulinones in in vitro models for hepatic steatosis, inflammation and fibrosis. Humulinones dose-dependently inhibited fatty acid induced lipid accumulation in primary human hepatocytes. Humulinones reduced the expression of fatty acid uptake transporter CD36 and key enzymes of (de novo) lipid synthesis. Conversely, humulinones increased the expression of FABP1, CPT1 and ACOX1, indicative for increased lipid combustion. Furthermore, humulinones ameliorated steatosis induced pro-inflammatory gene expression. Furthermore, humulinones significantly reduced the expression of pro-inflammatory and pro-fibrogenic factors in control as well as lipopolysaccharide treated activated hepatic stellate cells, which play a key role in hepatic fibrosis. In conclusion, humulinones beneficially affect different pathophysiological steps of NAFLD. Our data suggest humulinones as promising therapeutic agents for the prevention and treatment of NAFLD.

Regional hyperthermia with cisplatin added to gemcitabine versus gemcitabine in patients with resected pancreatic ductal adenocarcinoma: The HEAT randomised clinical trial.

BACKGROUND: Regional hyperthermia (RHT) with cisplatin added to gemcitabine showed efficacy in gemcitabine-pre-treated patients with advanced pancreatic ductal adenocarcinoma. We conducted a randomised clinical trial to investigate RHT with cisplatin added to gemcitabine (GPH) compared with gemcitabine (G) in the adjuvant setting of resected pancreatic ductal adenocarcinoma. METHODS: This randomised, multicentre, open-label trial randomly assigned patients to either GPH (gemcitabine 1000 mg/m2 on day 1, 15 and cisplatin 25 mg/m2 with RHT on day 2, 3 and 15,16) or to G (gemcitabine 1000 mg/m2 on day 1,8,15), four-weekly over six cycles. Disease-free survival (DFS) was the primary end-point. Secondary end-points included overall survival (OS) and safety. RESULTS: A total of 117 eligible patients (median age, 63 years) were randomly allocated to treatment (57 GPH; 60 G). With a follow-up time of 56.6 months, the median DFS was 12.7 compared to 11.2 months for GPH and G, respectively (p = 0.394). Median post-recurrence survival was significantly prolonged in the GPH-group (15.3 versus 9.8 months; p = 0.031). Median OS reached 33.2 versus 25.2 months (p = 0.099) with 5-year survival rates of 28.4% versus 18.7%. Excluding eight patients who received additional capecitabine in the G-arm (investigators choice), median OS favoured GPH (p = 0.052). Adverse events CTCAE (Common Terminology Criteria for Adverse Events) grade ≥3 occurred in 61.5% (GPH) versus 63.6% (G) of patients. Two patients in the G-group died because of treatment-related toxic effects. CONCLUSIONS: The randomised controlled Hyperthermia European Adjuvant Trial study failed to demonstrate a significant difference in DFS. However, it suggests a difference in post-recurrence survival and a trend for improved OS. CLINICALTRIALS: gov, number NCT01077427.

Frequency and clinical correlates of somatic Ying Yang 1 mutations in sporadic insulinomas.

CONTEXT: Insulinomas represent pancreatic neuroendocrine neoplasms that cause severe morbidity attributed to their often pronounced endocrine activity. Apart from hereditary forms such as multiple endocrine neoplasia type 1 (MEN-1), genetic causes for sporadic insulinoma development had remained obscure until recently. Applying next-generation sequencing methods, disease-causing genetic alterations have been identified in various endocrine tumors. OBJECTIVE AND DESIGN: Paired tumor and blood DNA from eight patients with sporadic insulinomas (five females and two malignant tumors) were analyzed by whole-exome sequencing. After this initial analysis, Ying Yang 1 (YY1) mutation status was assessed in a larger cohort of 39 additional insulinomas (including eight malignant and one liver metastasis) from three German hospitals by targeted sequencing. The mutation status was correlated with various clinical parameters. RESULTS: A range of one to 12 somatic genetic variants were identified by exome sequencing. A recurrent somatic Thr372Arg YY1 point mutation was detected in two patients of the initial cohort and four patients of the second cohort (total, six of 47; 13%). The presence of the mutation was associated with a trend toward higher age (63.5 y; IQR, 48.0-74.0 vs 45.0 y; IQR, 33.0-63.0; P = .05), and all affected patients were females (six of six; P = .04). All other clinical parameters, including the presence of malignancy and metastatic spread, tumor localization, and hypoglycemic episodes were not different between YY1-mutated and nonmutated tumor carriers. CONCLUSIONS: The somatic Thr372Arg YY1 mutation is a relevant finding in female patients with sporadic insulinomas. The prevalence of this mutation in this Caucasian population is considerably lower compared to that of a recently described Asian cohort.

Hop bitter acids exhibit anti-fibrogenic effects on hepatic stellate cells in vitro.

Female inflorescences of the hop plant Humulus lupulus L. contain a variety of secondary metabolites with bitter acids (BA) as quantitatively dominating secondary metabolites. The use of hops in beer brewing has a long history due to the antibacterial effects of the BA and their typical bitter taste. Furthermore, hop cones are used in traditional medicine and for pharmaceutical purposes. Recent studies indicate that BA may affect activity of the transcription factor NFκB. NFκB plays a key role in the activation process of hepatic stellate cells (HSC), which is the key event of hepatic fibrosis. The aim of this study was to investigate the effect of BA on HSC (activation) and their potential to inhibit molecular processes involved in the pathogenesis of hepatic fibrosis. HSC were isolated from murine and human liver tissue and incubated with a characterized fraction of bitter acids purified from a CO(2) hop extract. At a concentration of 25µg/ml BA started to induce LDH leakage. Already at lower concentrations BA lead to a dose dependent inhibition of HSC proliferation and inhibited IκB-α-phosphorylation, nuclear p65 translocation and binding activity in a dose dependent way (up to 10µg/ml). Accordingly, the same BA-doses inhibited the expression of pro-inflammatory and NFκB regulated genes as MCP-1 and RANTES, but did not affect expression of genes not related to NFκB signaling. In addition to the effect on activated HSC, BA inhibited the in vitro activation process of freshly isolated HSC as evidenced by delayed expression of collagen I and α-SMA mRNA and protein. Together, these findings indicate that BA inhibit NFκB activation, and herewith the activation and development of profibrogenic phenotype of HSC. Thus, bitter acids appear as potential functional nutrients for the prevention or treatment hepatic fibrosis in chronic liver disease.

Inferring statin-induced gene regulatory relationships in primary human hepatocytes.

MOTIVATION: Statins are the most widely used cholesterol-lowering drugs. The primary target of statins is HMG-CoA reductase, a key enzyme in cholesterol synthesis. However, statins elicit pleitropic responses including beneficial as well as adverse effects in the liver or other organs. Today, the regulatory mechanisms that cause these pleiotropic effects are not sufficiently understood. RESULTS: In this work, genome-wide RNA expression changes in primary human hepatocytes of six individuals were measured at up to six time points upon atorvastatin treatment. A computational analysis workflow was applied to reconstruct regulatory mechanisms based on these drug-response data and available knowledge about transcription factor (TF) binding specificities and protein-drug interactions. Several previously unknown TFs were predicted to be involved in atorvastatin-responsive gene expression. The novel relationships of nuclear receptors NR2C2 and PPARA on CYP3A4 were successfully validated in wet-lab experiments. AVAILABILITY: Microarray data are available at the Gene Expression Omnibus (GEO) database at www.ncbi.nlm.nih.gov/geo/, under accession number GSE29868. CONTACT: andreas.zell@uni-tuebingen.de; adrian.schroeder@uni-tuebingen.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cellular damage to human hepatocytes through repeated application of 5-aminolevulinic acid.

BACKGROUND/AIMS: 5-Aminolevulinic acid (ALA), a precursor of porphyrins is used for photodynamic diagnosis and therapy within topical or systemic applications. A potential toxic effect on the human liver is of major interest and therefore we investigated the impact of a repeated application of ALA without illumination on cultures of human hepatocytes. METHODS: After ALA treatment of hepatocytes in vitro the porphyrin synthesis, albumin secretion, liver-specific enzyme release, and malondialdehyde levels were determined. In order to reduce levels of reactive oxygen substances, mannitol and the antioxidant enzymes superoxide dismutase and catalase were supplemented. RESULTS: Porphyrin biosynthesis by human hepatocytes in vitro was repeatedly stimulated by ALA (0.001-1.0 mM), which was indicated by an accumulation of protoporphyrin IX. A repetitive treatment (up to four times) of hepatocytes with ALA resulted in an impairment of the hepatic function and viability, depending on the ALA concentration (0.1-1.0 mM) and frequency of application (2-3 times). This was also accompanied by increased malondialdehyde levels indicating enhanced lipid peroxidation. Only superoxide dismutase was able to reduce cellular damage and prevent specific function. CONCLUSIONS: Repeated, not single, ALA treatment without illumination may cause deleterious effects to the liver, which are mediated by oxygen radicals and inhibited by an antioxidant.