1,8-Cineole promotes G0/G1 cell cycle arrest and oxidative stress-induced senescence in HepG2 cells and sensitizes cells to anti-senescence drugs.

AIMS: 1,8-Cineole is a plant-derived monoterpene and a major constituent of Eucalyptus essential oil. Previously, we demonstrated that 1,8-cineole inhibited hepatocellular carcinoma (HCC) HepG2 cell growth. However, the underlying mechanisms remain unknown. Here, we evaluated the mechanisms of action of 1,8-cineole and the potential benefits of its combination with anticancer compounds harboring "anti-senescence" properties in HepG2 cells. MAIN METHODS: Cell viability was determined by the MTT assay. Cell cycle was assessed through flow cytometry (FC) and western blot (WB). Senescence was determined by the SA-ß-galactosidase assay, and apoptosis by caspase-3 activity, WB, and TUNEL. MAPKs (ERK, JNK, and p38), AMPK, and Akt/mTOR were analyzed by WB. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were evaluated by FC and fluorescence microscopy. KEY FINDINGS: 1,8-Cineole inhibited cell proliferation by promoting G0/G1 arrest. While 1,8-cineole was unable to trigger apoptosis, it induced cellular senescence. 1,8-Cineole promoted ROS production, ΔΨm depolarization, AMPK, ERK, and p38 activation and mTOR inhibition. Antioxidants, like N-acetyl-L-cysteine and vitamins, prevented HepG2 cell growth inhibition and senescence induced by 1,8-cineole. Pre-incubation with 1,8-cineole sensitized HepG2 cells to the anti-senescence compounds, quercetin, simvastatin, U0126, and SB202190. Combinations of 1,8-cineole and each compound synergistically inhibited cell viability, and combined treatment with 1,8-cineole and simvastatin induced apoptosis. SIGNIFICANCE: 1,8-Cineole induces G0/G1 arrest and senescence in HepG2 cells through oxidative stress and MAPK, AMPK, and Akt/mTOR pathways, and sensitizes cells to anti-senescence drugs, suggesting that 1,8-cineole has potential as an antineoplastic and adjuvant compound in combination with anti-senescence drugs in HCC therapy.

Serum and Fecal Oxylipins in Patients with Alcohol-Related Liver Disease.

BACKGROUND: Alcohol-related liver disease is one of the most prevalent chronic liver diseases worldwide. Mechanisms involved in the pathogenesis of alcohol-related liver disease are not well understood. Oxylipins play a crucial role in numerous biological processes and pathological conditions. Nevertheless, oxylipins are not well studied in alcohol-related liver disease. AIMS: (1) To characterize the patterns of bioactive ω-3 and ω-6 polyunsaturated fatty acid metabolites in alcohol use disorder and alcoholic hepatitis patients and (2) to identify associations of serum oxylipins with clinical parameters in patients with alcohol-related liver disease. METHODS: We performed a comprehensive liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of serum and fecal oxylipins derived from ω-6 arachidonic acid, ω-3 eicosapentaenoic acid, and docosahexaenoic acid in a patient cohort with alcohol-related liver disease. RESULTS: Our results show profound alterations in the serum oxylipin profile of patients with alcohol use disorder and alcoholic hepatitis compared to nonalcoholic controls. Spearman correlation of the oxylipins with clinical parameters shows a link between different serum oxylipins and intestinal permeability, aspartate aminotransferase, bilirubin, albumin, international normalized ratio, platelet count, steatosis, fibrosis and model for end-stage liver disease score. Especially, higher level of serum 20-HETE was significantly associated with decreased albumin, increased hepatic steatosis, polymorphonuclear infiltration, and 90-day mortality. CONCLUSIONS: Patients with alcohol-related liver disease have different oxylipin profiles. Future studies are required to confirm oxylipins as disease biomarker or to connect oxylipins to disease pathogenesis.

Intestinal dysbiosis and permeability: the yin and yang in alcohol dependence and alcoholic liver disease.

Alcohol dependence and alcoholic liver disease represent a major public health problem with substantial morbidity and mortality. By yet incompletely understood mechanisms, chronic alcohol abuse is associated with increased intestinal permeability and alterations of the gut microbiota composition, allowing bacterial components, bacteria, and metabolites to reach the portal and the systemic circulation. These gut-derived bacterial products are recognized by immune cells circulating in the blood or residing in remote organs such as the liver leading to the release of pro-inflammatory cytokines which are considered important mediators of the liver-gut-brain communication. Although circulating cytokines are likely not the sole factors involved, they can induce liver inflammation/damage and reach the central nervous system where they favor neuroinflammation which is associated with change in mood, cognition, and drinking behavior. In this review, the authors focus on the current evidence describing the changes that occur in the intestinal microbiota with chronic alcohol consumption in conjunction with intestinal barrier breakdown and inflammatory changes sustaining the concept of a gut-liver-brain axis in the pathophysiology of alcohol dependence and alcoholic liver disease.

Treatment of severe alcoholic hepatitis: past, present and future.

Alcoholic hepatitis (AH) manifests as a clinical syndrome characterized by recent jaundice and liver function deterioration in an actively drinking patient. The principal cause of AH is alcoholic steatohepatitis (ASH) defined histologically by the coexistence of steatosis, hepatocyte ballooning and satellitosis. While nonsevere AH usually responds to alcohol abstinence, severe AH, identified by Maddrey scoring ≥ 32, has a bad prognosis and is traditionally treated by a 28-day course of prednisone therapy. A recent trial, which showed no improvement of long-term survival but significant reduced mortality after 28 days of corticoid therapy compared to placebo, opens a debate on its efficacy. N-acetyl-cysteine supplementation combined with steroid therapy is also able to reduce the 28-day mortality compared to steroid alone. While guidelines recommend high-calorie intake and protein supplementation in decompensated liver diseases, intensive enteral nutrition together with corticoid treatment does not reduce mortality compared to corticoid alone in a recent study with ASH patients. Stimulation of liver regeneration through interleukin-22, granulocyte colony-stimulating factor or farnesoid X receptor agonists, inhibition of apoptosis, early liver transplantation and modulation of gut microbiota through antibiotic or faecal transplantation approaches constitute new therapeutic perspectives that are investigated in current clinical trials. Inhibition of oxidative stress, modulation of gut fungal populations and stimulation of progenitor cell proliferation and pro-regenerative inflammatory pathways constitute prospects for future human trials. For long-term survival, strategies for persistent alcohol abstinence remain the key of success, opening another large research field.

Intensive Enteral Nutrition Is Ineffective for Patients With Severe Alcoholic Hepatitis Treated With Corticosteroids.

BACKGROUND & AIMS: Severe alcoholic hepatitis (AH) is a life-threatening disease for which adequate oral nutritional support is recommended. We performed a randomized controlled trial to determine whether the combination of corticosteroid and intensive enteral nutrition therapy is more effective than corticosteroid therapy alone in patients with severe AH. METHODS: We enrolled 136 heavy consumers of alcohol (age, 18-75 y) with recent onset of jaundice and biopsy-proven severe AH in our study, performed at 18 hospitals in Belgium and 2 in France, from February 2010 through February 2013. Subjects were assigned randomly (1:1) to groups that received either intensive enteral nutrition plus methylprednisolone or conventional nutrition plus methylprednisolone (controls). In the intensive enteral nutrition group, enteral nutrition was given via feeding tube for 14 days. The primary end point was patient survival for 6 months. RESULTS: In an intention-to-treat analysis, we found no significant difference between groups in 6-month cumulative mortality: 44.4% of patients died in the intensive enteral nutrition group (95% confidence interval [CI], 32.2%-55.9%) and 52.1% of controls died (95% CI, 39.4%-63.4%) (P = .406). The enteral feeding tube was withdrawn prematurely from 48.5% of patients, and serious adverse events considered to be related to enteral nutrition occurred in 5 patients. Regardless of group, a greater proportion of patients with a daily calorie intake less than 21.5 kcal/kg/day died (65.8%; 95% CI, 48.8-78.4) than patients with a higher intake of calories (33.1%; 95% CI, 23.1%-43.4%) (P < .001). CONCLUSIONS: In a randomized trial of patients with severe AH treated with corticosteroids, we found that intensive enteral nutrition was difficult to implement and did not increase survival. However, low daily energy intake was associated with greater mortality, so adequate nutritional intake should be a main goal for treatment. ClinicalTrials.gov number: NCT01801332.

To chew or not to chew: that's the question.

A 25-year-old Somalian man was referred to the department of internal medicine because of nausea, vomiting and jaundice of recent onset. On physical examination, he was frankly icteric without clinical signs of chronic liver disease. Laboratory data showed evidence of acute hepatitis. Viral serologic tests for hepatitis A, B, C and E were negative. Antinuclear antibodies (ANA) were positive (titre 1 : 1280; speckled pattern, NV < 1 : 40) as well anti-actin antibodies (titre 75 UA, NV < 20). A liver biopsy was performed and showed a feature compatible with toxic hepatitis. On further questioning, the patient admitted to daily chew Khat when he was living in Somalia. We concluded to Khat-induced toxic hepatitis together with high-titre anti-nuclear antibody mimicking serologic patterns of auto-immune hepatitis.

Tetrahydro Iso-Alpha Acids and Hexahydro Iso-Alpha Acids from Hops Inhibit Proliferation of Human Hepatocarcinoma Cell Lines and Reduce Diethylnitrosamine Induced Liver Tumor Formation in Rats.

Chronic inflammation plays important role in the pathogenesis of hepatocellular carcinoma (HCC). To date, no antiinflammatory approach has shown its efficacy in preventing HCC occurrence in humans. Because tetra- and hexahydro isoalpha acids (THIAA and HHIAA) from hops elicit antiinflammatory properties, we evaluated these compounds for antitumor effects in vitro in human HCC cell lines (HepG2, Hep3B, Huh7) and in vivo in diethylnitrosamine (DEN)-induced animal model of HCC. In human HCC cell lines, THIAA and HHIAA reduced cell proliferation and viability which was associated with the inhibition of the NF-κB-DNA binding and tumor necrosis factor α mRNA expression. Both compounds also inhibited phosphorylation of the mTOR effector p70S6 kinase without affecting ERK, AKT, JNK, and GSK3ß phosphorylation or activator protein-1 activation. In DEN-treated rats, administration of THIAA and HHIAA in food reduced the tumor numbers and the expression of the cellular transformation marker glutathione-S-transferase in the liver. In conclusion, THIAA and HHIAA show antitumor properties in vitro in human HCC cell lines as well as in vivo in a chemically induced animal model of HCC.

The Ras/MAPK pathway and hepatocarcinoma: pathogenesis and therapeutic implications.

BACKGROUND: Hepatocellular carcinoma (HCC) is still a major health problem, often diagnosed at an advanced stage. The multikinase inhibitor sorafenib is to date the sole approved systemic therapy. Several signalling pathways are implicated in tumour development and progression. Among these pathways, the Ras/MAPK pathway is activated in 50-100% of human HCCs and is correlated with a poor prognosis. The aim of this work was to review the main intracellular mechanisms leading to aberrant Ras pathway activation in HCC and the potential therapeutic implications. MATERIALS AND METHODS: This review is based on the material found on PubMed up to December 2014. 'Ras signaling, Ras dysregulation, Ras inhibition, MAPK pathway, cancer, hepatocarcinoma and liver cancer' alone or in combination were the main terms used for online research. RESULTS: Multiple mechanisms lead to the deregulation of the Ras pathway in liver cancer. Ras and Raf gene mutations are rare events in human hepatocarcinogenesis in contrast to experimental models in rodents. Downregulation of several Ras/MAPK pathway inhibitors such as GAPs, RASSF proteins, DUSP1, Sprouty and Spred proteins is largely implicated in the aberrant activation of this pathway in the context of wild-type Ras and Raf genes. Epigenetic or post-transcriptional mechanisms lead to the downregulation of these tumour suppressor genes. CONCLUSION: Ras/MAPK pathway effectors may be considered as potential therapeutic targets in the field of HCC. In particular after the arrival of sorafenib, more Ras/MAPK inhibitors have emerged and are still in preclinical or clinical investigation for HCC therapy.

Supplementation of saturated long-chain fatty acids maintains intestinal eubiosis and reduces ethanol-induced liver injury in mice.

BACKGROUND & AIMS: Alcoholic liver disease is a leading cause of mortality. Chronic alcohol consumption is accompanied by intestinal dysbiosis, and development of alcoholic liver disease requires gut-derived bacterial products. However, little is known about how alterations to the microbiome contribute to pathogenesis of alcoholic liver disease. METHODS: We used the Tsukamoto-French mouse model, which involves continuous intragastric feeding of isocaloric diet or alcohol for 3 weeks. Bacterial DNA from the cecum was extracted for deep metagenomic sequencing. Targeted metabolomics assessed concentrations of saturated fatty acids in cecal contents. To maintain intestinal metabolic homeostasis, diets of ethanol-fed and control mice were supplemented with saturated long-chain fatty acids (LCFA). Bacterial genes involved in fatty acid biosynthesis, amounts of lactobacilli, and saturated LCFA were measured in fecal samples of nonalcoholic individuals and patients with active alcohol abuse. RESULTS: Analyses of intestinal contents from mice revealed alcohol-associated changes to the intestinal metagenome and metabolome, characterized by reduced synthesis of saturated LCFA. Maintaining intestinal levels of saturated fatty acids in mice resulted in eubiosis, stabilized the intestinal gut barrier, and reduced ethanol-induced liver injury. Saturated LCFA are metabolized by commensal Lactobacillus and promote their growth. Proportions of bacterial genes involved in fatty acid biosynthesis were lower in feces from patients with active alcohol abuse than controls. Total levels of LCFA correlated with those of lactobacilli in fecal samples from patients with active alcohol abuse but not in controls. CONCLUSIONS: In humans and mice, alcohol causes intestinal dysbiosis, reducing the capacity of the microbiome to synthesize saturated LCFA and the proportion of Lactobacillus species. Dietary approaches to restore levels of saturated fatty acids in the intestine might reduce ethanol-induced liver injury in patients with alcoholic liver disease.

Tumor reoxygenation following administration of Mitogen-Activated Protein Kinase inhibitors: a rationale for combination with radiation therapy.

BACKGROUND AND PURPOSE: The relevance of Mitogen Activated Protein Kinase (MAPK) inhibitors as co-treatments for radiation therapy is investigated, with special focus on a potential link between the MAPK pathway and tumor hypoxia, which is a critical determinant for response to therapy. MATERIALS AND METHODS: The effects of two MAPK inhibitors, Sorafenib and PD0325901, were monitored daily using in vivo EPR (Electron Paramagnetic Resonance) oximetry in FSaII and TLT tumor models in order to identify a window of reoxygenation, during which tumor blood flow, oxygen consumption and radiation sensitivity were assessed. RESULTS: Reoxygenation was shown after two days of treatments with Sorafenib or PD0325901 in two tumor models, which was further successfully exploited with Sorafenib for improving the radiation response of FSaII tumors by a factor of 1.5. The increase in tumor oxygenation was shown to be the result of two major factors: (i) an increase in blood flow for Sorafenib, that might be linked to its anti-angiogenic effect (vascular normalization), and (ii) a decrease in oxygen consumption for Sorafenib and PD0325901, due to an alteration of the mitochondrial activity. CONCLUSION: We evidenced tumor reoxygenation in vivo following MAPK inhibition and suggest a rationale for the combination of radiation therapy with Sorafenib.