Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition

<p>Abstract</p> <p>Background</p> <p>Dysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated <it>in vitro </it>the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its <it>in vivo </it>effect in a subcutaneous xenograft model with HepG2 cells.</p> <p>Results</p> <p>Salirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC₅₀ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. <it>In vivo</it>, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.</p> <p>Conclusions</p> <p>Our results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model.</p

A POSSIBLE ROLE OF GUT MICROBIOTA IN THE BEHAVORIAL CONTROL OF ALCOHOL-DEPENDENT SUBJECTS

These observations suggest that alterations at the level of the gut microbiota influence the gut permeability and activate specific inflammation pathways that are related to psychological symptoms of alcoholdependence. Altogether these observations are consistent with a role of inflammation as one mediator of a gut-brain communication in AD patients

Linalool induces cell cycle arrest and apoptosis in HepG2 cells through oxidative stress generation and modulation of Ras/MAPK and Akt/mTOR pathways

Aims Linalool is a plant-derived monoterpene with anticancer activity, however its mechanisms of action remain poorly understood. The aim of this work was to elucidate the anticancer mechanisms of action of linalool in hepatocellular carcinoma (HCC) HepG2 cells. Main methods Cell viability and proliferation were determined by WST-1 assay and BrdU incorporation, respectively. Cell cycle analysis was assessed through flow cytometry (FC) and western blot (WB). Apoptosis was determined by caspase-3 activity, TUNEL assay and WB. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by FC and fluorescence microscopy. Expression of Ras, MAPKs (ERK, JNK and p38) and Akt/mTOR pathways were evaluated by WB. Key findings Linalool (0–2.5 mM) dose-dependently inhibited cell proliferation by inducing G0/G1 cell cycle arrest, through Cdk4 and cyclin A downregulation, p21 and p27 upregulation, and apoptosis, characterized by MMP loss, caspase-3 activation, PARP cleavage and DNA fragmentation. Low concentrations of linalool (1.0 mM) reduced membrane-bound Ras and Akt activity whereas higher amounts (2.0 mM) triggered mTOR inhibition and ROS generation, in correlation with MAPKs activation and Akt phosphorylation. ROS scavenger N-acetyl-L-cysteine partially rescued HepG2 cell growth and prevented MPP depolarization, ERK and JNK activation. Moreover, specific ERK and Akt phosphorylation inhibitors potentiated linalool anti-cancer activity, pointing Akt and ERK activation as pro-survival mechanisms in response to higher concentrations of linalool. Significance This report reveals that linalool induces G0/G1 arrest and apoptosis in HepG2 cells involving Ras, MAPKs and Akt/mTOR pathways and suggests that linalool is a promising anticancer agent for HCC therapy.Instituto de Investigaciones Bioquímicas de La Plat

Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital

Background: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. Methods: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. Results: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5 UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. Conclusions: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular

Intestinal fungi contribute to development of alcoholic liver disease

This study was supported in part by NIH grants R01 AA020703, U01 AA021856 and by Award Number I01BX002213 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development (to B.S.). K.H. was supported by a DFG (Deutsche Forschungsgemeinschaft) fellowship (HO/ 5690/1-1). S.B. was supported by a grant from the Swiss National Science Foundation (P2SKP3_158649). G.G. received funding from the Yale Liver Center NIH P30 DK34989 and R.B. from NIAAA grant U01 AA021908. A.K. received support from NIH grants RC2 AA019405, R01 AA020216 and R01 AA023417. G.D.B. is supported by funds from the Wellcome Trust. We acknowledge the Human Tissue and Cell Research (HTCR) Foundation for making human tissue available for research and Hepacult GmbH (Munich, Germany) for providing primary human hepatocytes for in vitro analyses. We thank Dr. Chien-Yu Lin Department of Medicine, Fu-Jen Catholic University, Taiwan for statistical analysis.Peer reviewedPublisher PD

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat

<p>Abstract</p> <p>Background</p> <p>There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model.</p> <p>Results</p> <p>Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.</p> <p>In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. <b>Conclusions</b>: We found an increase in core-α-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.</p

Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFβ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFβ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH

Global prevalence and genotype distribution of hepatitis C virus infection in 2015 : A modelling study

Publisher Copyright: © 2017 Elsevier LtdBackground The 69th World Health Assembly approved the Global Health Sector Strategy to eliminate hepatitis C virus (HCV) infection by 2030, which can become a reality with the recent launch of direct acting antiviral therapies. Reliable disease burden estimates are required for national strategies. This analysis estimates the global prevalence of viraemic HCV at the end of 2015, an update of—and expansion on—the 2014 analysis, which reported 80 million (95% CI 64–103) viraemic infections in 2013. Methods We developed country-level disease burden models following a systematic review of HCV prevalence (number of studies, n=6754) and genotype (n=11 342) studies published after 2013. A Delphi process was used to gain country expert consensus and validate inputs. Published estimates alone were used for countries where expert panel meetings could not be scheduled. Global prevalence was estimated using regional averages for countries without data. Findings Models were built for 100 countries, 59 of which were approved by country experts, with the remaining 41 estimated using published data alone. The remaining countries had insufficient data to create a model. The global prevalence of viraemic HCV is estimated to be 1·0% (95% uncertainty interval 0·8–1·1) in 2015, corresponding to 71·1 million (62·5–79·4) viraemic infections. Genotypes 1 and 3 were the most common cause of infections (44% and 25%, respectively). Interpretation The global estimate of viraemic infections is lower than previous estimates, largely due to more recent (lower) prevalence estimates in Africa. Additionally, increased mortality due to liver-related causes and an ageing population may have contributed to a reduction in infections. Funding John C Martin Foundation.publishersversionPeer reviewe

Muscle mass depletion in chronic liver diseases: An accelerated model of aging or a distinct entity?

Sarcopenia was initially defined as loss of muscle mass, strength and function related to aging. This phenomenon is a multifactorial process. The evaluation of the geriatric population in which sarcopenia has extensively been studied opens the field for other chronic diseases. Cirrhosis is one of them and the term ‘‘sarcopenia’’ is now also used in this pathological situation. It must be emphasized that the pathophysiology of sarcopenia in cirrhosis is likely different from the pathogenesis in geriatric patients. Furthermore, cirrhosis has heterogeneous causes. Therefore, we need a better understanding of the changes in muscle physiology specifically in chronic liver diseases as well as easy, accurate, reproducible and validated tools, taking into consideration etiology specific aspects to identify sarcopenia in every cirrhotic patient