Extrahepatic Cells Research Articles

A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components

Genotyping graft livers by short tandem repeats after human living-donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM-MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1-2% of BM-MSCs), called multilineage-differentiating stress-enduring (Muse) cells, for their ability to differentiate into liver-lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)-labeled human BM-MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species-specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP-positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM-MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM-MSCs that are capable of replacing major liver components during liver regeneration.

The Involving Roles of Intrahepatic and Extrahepatic Stem/Progenitor Cells (SPCs) to Liver Regeneration.

Liver regeneration is usually attributed to mature hepatocytes, which possess a remarkable potential to proliferate under mild to moderate injury. However, when the liver is severely damaged or hepatocyte proliferation is greatly inhibited, liver stem/progenitor cells (LSPCs) will contribute to the liver regeneration process. LSPCs in the developing liver have been extensively characterized, however, their contributing role to liver regeneration has not been completely understood. In addition to the restoration of the liver parenchymal tissue by hepatocytes or/and LSPCs, or in some cases bone marrow (BM) derived cells, such as hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), the wound healing after injury in terms of angiopoiesis by liver sinusoidal endothelial cells (LSECs) or/and sinusoidal endothelial progenitor cells (SEPCs) is another important aspect taking place during regeneration. To conclude, liver regeneration can be mainly divided into three distinct restoring levels according to the cause and severity of injury: hepatocyte dominant regeneration, LSPCs mediated regeneration, extrahepatic stem cells participative regeneration. In this review, we focus on the recent findings of liver regeneration, especially on those related to stem/progenitor cells (SPCs)-mediated regeneration and their potential clinical applications and challenges.

Specific extrahepatic cells, bone marrow-Muse cells, contribute to liver regeneration

Specific extrahepatic cells, bone marrow-Muse cells, contribute to liver regeneration

Simvastatin Induces Apoptosis and Suppresses Insulin-Like Growth Factor 1 Receptor in Bile Duct Cancer Cells.

Background/AimsStatins act as antineoplastic agents through the inhibition of cell proliferation. This study sought to demonstrate the effects of statins on extrahepatic bile duct cancer cell apoptosis and to document the changes in protein expression involved in tumor growth and suppression.MethodsHuman extrahepatic bile duct cancer cells were cultured. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine the effect of statins on cell proliferation. Apoptosis was measured by a cell death detection enzyme-linked immunosorbent assay and caspase-3 activity assay, and flow cytometry was used to determine the percentage of cells in each phase of the cell cycle. The protein expression of Bax, Bcl-2, insulin-like growth factor 1 (IGF-1) receptor, extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt was measured by Western blot analysis.ResultsSimvastatin suppressed cell proliferation by inducing G1 phase cell cycle arrest in bile duct cancer cells. Furthermore, it induced apoptosis via caspase-3 activation, downregulated the expression of the Bcl-2 protein, and enhanced the expression of the Bax protein. Moreover, simvastatin suppressed the expression of the IGF-1 receptor and IGF-1-induced ERK/Akt activation.ConclusionsSimvastatin induces apoptosis in bile duct cancer cells, which suggests that it could be an antineoplastic agent for bile duct cancer.

Novel surface markers directed against adult human gallbladder

Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1+SOX9+) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers.

The role of histone H3 lysine 9 trimethylation modification on cholangiocarcinoma moleculor mechanism

Objective To study the effect of histone H3 lysine 9 trimethylation(H3K9me3) modification on cholangiocarcinoma moleculor mechanism. Methods 9 cases cholangiocarcinoma tissue and 5 cases normal extrahepatic bile duct tissue were collected.Chromatin immunoprecipitation linked to microarrays(ChIP-chip) was adopted to profile the variations of histone H3K9me3 in genome-wide in cholangiocarcinoma cell and normal extrahepatic bile duct cell.CHIP-quantitative polymerase chain reaction(qPCR) was used to validate the microrray results.mRNA expression analysis of 6 selected genes by real-time quantitative reverse transcriptase-polymerase chain reaction(RT -qPCR) was performed to confirm the correlations between H3K9me3 and gene expression. Results 67(35 increased and 32 decreased) gene displayed significant histone H3K9me3 differences were found in tumor cell compared with normal extrahepatic bile duct cell.In bile duct cancer tissues, six selected oncogenes[calcium binding protein A2(S100A2), anaphase-promoting complex subunit 2 (ANAPC2), myeloid translocation gene on chromosome 16(CBFA2T3), Rho GTPase activating protein 4 isoform 2(ARHGAP),Rap guanine nucleotide exchange factor 3 isoform(RAPGEF3), protein kinase C alpha(PRKCA)]H3K9me3 modifications level were:(3. 51±3. 51)%,(8. 45± 0. 16)%,(0. 89±0. 27)%,(0. 66±0. 22)%,(0. 04±0. 01)%,(5. 21±1. 53)%, there was a statistically significant difference compared with normal bile duct cells.It was the Pearson correlation between selected genes expression and H3K9Me3 modification(r=-0. 539)in the turmor cell. Conclusion Compared with normal extrahepatic bile duct cell,there are significant changes in many genes of cholangiocarcinoma cell about histone H3K9Me3 profiling.And resulting in a number of related oncogenes and tumor suppressor genes expression change. Key words: Histone H3 lysine 9 trimethylation; Microarray; Chromatin immunoprecipitation; Cholangiocarcinoma

P113: Influence of liver normoxia on intra‐ and extra‐hepatic NK cell function in HCV infection

Journal of Viral HepatitisVolume 22, Issue S2 p. 77-78 Poster PresentationFree Access P113: Influence of liver normoxia on intra- and extra-hepatic NK cell function in HCV infection First published: 25 June 2015 https://doi.org/10.1111/jvh.107_12425AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume22, IssueS2Special Issue: 15th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 26th to 28th, 2015, in Berlin, GermanyJune 2015Pages 77-78 RelatedInformation

Salvianolic acid B accelerated ABCA1-dependent cholesterol efflux by targeting PPAR-γ and LXRα

ObjectivesCholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages. MethodsAfter PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [3H] cholesterol (1.0 μCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL2 or HDL3. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism. ResultsThe results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα. ConclusionThese results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway.

45. Adenovirus Variant With a Defined Set of Genetic Mutations in the Penton and Hexon Escapes Liver Sequestration After Intravascular Administration

Adenoviruses (Ad) are promising vectors for therapeutic interventions in humans. Despite significant knowledge regarding the biology of Ad interactions with cells in vitro, the molecular mechanisms governing in vivo Ad infectivity and bio-distribution remain poorly understood. Pharmacokinetic studies of Ad vectors after intravascular delivery demonstrate that the majority of an administered virus dose is rapidly sequestered from the circulation by the liver. Earlier we showed that Ad particles are distributed in liver tissue amongst three distinct cellular compartments, namely i) parenchymal liver cells – hepatocytes, ii) hepatic residential macrophages, Kupffer cells, and iii) hepatic sinusoid endothelial cells. Importantly, we found that the ablation of Ad interaction with only one of these cellular compartments cannot prevent virus trapping the liver shortly after intravascular virus injection and, likely, results in compensatory re-distribution of the virus among two remaining cellular compartments, thus functionally ensuring the quantitative removal of the virus from the blood. We speculated that to prevent Ad sequestration by the liver after intravascular administration, simultaneous ablation of virus interaction with all three cellular compartments of the liver is ultimately required. To evaluate this assumption experimentally, we introduced in a single human Ad5-based vector specific mutations that abrogate virus interactions with one, two, or all three hepatocellular compartments. To ablate Ad interaction with hepatocytes in vivo, we introduced T425A mutation in hexon hyper-variable loop HVR7 that completely abrogates virus interaction with blood coagulation FX. To prevent Ad interaction with liver sinusoid endothelial cells, we introduced a substitution of the RGD-motif-containing penton loop for an iso-functional integrin-interacting non-RGD-containing peptide. We also serendipitously found that the deletion of hexon HVR1 region, in addition to penton RGD-loop substitution and T425A HVR7 mutation, results in the reduction of virus interaction with Kupffer cells. Intravenous injection into mice of Ad variants containing individual mutations either in the penton or hexon or a vector with only two mutations in a single vector does not prevent sequestration of resultant Ad in the liver. However, Ad variant, containing all three mutations simultaneously, completely escaped being sequestered in the liver after intravascular injection and, instead, 20-fold higher amounts of the virus were recovered from the spleen, indicating the major change in virus bio-distribution, compared to unmodified vectors with a wild-type capsid, or vectors with individual mutations in either penton or hexon. To our knowledge this is the first demonstration of a feasibility of constructing Ad variants that would escape liver sequestration after intravascular administration. These variants may prove to be a useful platform for gene delivery to extra-hepatic cells and/or targeting disseminated metastatic cancers.

Analysis of the roles of glucose transporter 1 and hexokinase 2 in the metabolism of glucose by extrahepatic bile duct cancer cells.

Extrahepatic bile duct (EHD) cancer varies in uptake of FDG. The aim of the present study was to determine the role of glucose transporter (GLUT) 1 and hexokinase (HK) 2 in the glucose metabolism of EHD cancer cells using immunohistochemistry and 18F-FDG PET/CT. Twenty-six patients with EHD cancer who underwent baseline PET/CT and surgery were studied. Biopsies were immunohistochemically analyzed using antibodies against GLUT1 and HK2, and the expression was scored from 0 to 4 according to the percentage of stained cells. SUV and tumor-to-liver ratio (T/L ratio) were obtained from 18F-FDG PET/CT data. SUV and T/L ratio and GLUT1 and HK2 expression were compared with histological grades and tumor locations (proximal and distal EHD) to correlate glucose metabolism with the expression of GLUT1 and HK2. SUV, T/L ratio, and GLUT1 and HK2 expression did not differ as a function of histological grade and tumor location. GLUT1 and HK2 were expressed in 20 (76.9%) and 22 (84.6%) of 26 tumor biopsies, respectively. The GLUT1 score, SUV, and T/L ratio increased, and the GLUT1 score, but not the HK2 score, correlated significantly with SUV (ρ = 0.648) and T/L ratio (ρ = 0.703). There was no direct correlation between the expression of GLUT1 and that of HK2 (ρ = 0.2046, P = 0.3161). Although GLUT1 and HK2 regulate intracellular accumulation of FDG in many cancers, only GLUT1 expression was correlated with FDG uptake by EHD cancers.

Immunoregulation by lipids during the development of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) represents the most common liver disorder in western countries and it is commonly associated with obesity and progression of the metabolic syndrome. Comprehending a wide spectrum of pathologic features, it is currently well recognized that a key point for the integrity of hepatocyte functionality in NAFLD is the progression from simple steatosis to non-alcoholic steatohepatitis (NASH). Indeed, activation of the innate immune system in response to hepatic metabolic stresses represents a central process that determines the evolution and the reversibility of liver damage. Despite of the burden of studies published in recent years, it is still intriguingly unclear how accumulation of lipids in hepatocytes triggers the activation of the inflammatory response leading to the recruitment of infiltrating cells of extra-hepatic origins. In this review we offer a general view on recent advances pointing out how different classes of lipids are able to specifically affect hepatocytes functionality and survival, thus differently influencing the organization of the hepatic immune response. On the other hand, we gathered recent studies intending to illustrate the basic mechanisms through which several non-parenchymal hepatic and extra-hepatic cell populations get activated in response to lipids. Finally, we indicate latter findings proposing how the immune system majorly contributes to the progression of NASH.

255-POS

Objectives 27-hydroxycholesterol (27-OHC) is the hydroxylation product of cholesterol by the enzyme sterol 27-hydroxylase (CYP27A1). 27-OHC is a potent inhibitor of cholesterol synthesis and increases cholesterol efflux from extrahepatic cells in vitro. Its transport from peripheral cells to the liver contributes to the high-density lipoprotein HDL-dependent reverse-cholesterol-transport and represents a potentially anti-atherogenic mechanism. Our hypothesis was (1) that the rise in cholesterol concentration during pregnancy is attributed to a lower catabolism and excretion by a relative decrease in 27-OHC concentration during the course of pregnancy and (2) that 27-OHC plays a role in pregnancy related pathologies linked to a disturbed lipid metabolism. Methods 223 serum samples were analyzed. Group A: 33 patients with uncomplicated pregnancies were analyzed at three different time points (first to third trimester). Group B consists of 45 patients (24–42 weeks of gestation) with pregnancy pathologies (IUGR n = 14, preeclampsia n = 14, HELLP n = 10, intrahepatic cholestasis n = 7) and 45 gestational age matched controls. 27-OHC was measured by GC–MS and related to cholesterol concentrations. Statistics: Spearman’s rank coefficient and Kruskal–Wallis test with Dunn’s multiple comparisons. Results Group A: 27-OHC was positively correlated to total cholesterol concentration ( ρ = 0.518). The 27-OHC/cholesterol ratio dropped with the course of pregnancy with higher values in first and second trimester as compared to the non-pregnant state postpartum (mean (SD), 1st trimester: 1.367 (0.38) p p Conclusions Our analysis revealed an increased 27-OHC/cholesterol ratio in the first and second trimester, indicating a lower cholesterol catabolism accounting for increased cholesterol concentrations throughout gestation. However, it does not support a role of 27-OHC in the patho-mechanism of pregnancy pathologies. Disclosures B. Winkler: None. N. Kleine-Eggebrecht: None. N. Maass: None. M. Mohaupt: None. G. Escher: None. U. Pecks: None.

Plasma Exosomes and Drug Metabolic Cytochrome P450 Enzymes

The major drug metabolic enzymes, Cytochromes P450 (CYP), are abundantly present in hepatocytes, and to some extent in other extrahepatic cells such as monocytes. Since these enzymes metabolize the majority of xenobiotics leading to formation of Reactive Oxygen Species (ROS) and some toxic metabolites, hepatocytes and monocytes could produce extracellular vesicles such as exosomes. The exosomes produced by the liver and blood cells eventually secrete into the plasma, which may contain high levels of exosomes containing CYP enzymes. The level of plasma exosomal CYP enzymes may also be induced under certain conditions. Therefore, plasma exosomal CYP may be used as biological markers, as well as biocatalysts for various purposes in medicine, bioremediation, and industrial synthesis. This editorial briefly describes this novel and exciting area of research in the field of exosomes, and their potential as biomarkers and in therapy.

Abstract 4403: Preclinical evaluation of LY2801653, an orally bioavailable small molecule oncokinase inhibitor, in cholangiocarcinoma models

Abstract Cholangiocarcinoma (CCA) is an aggressive and locally invasive biliary tract malignancy with a poor prognosis and no approved drug treatment. LY2801653 is an orally bioavailable small molecule oncokinase inhibitor1 of MET, a receptor reported to be dysregulated and correlated with a poor outcome in CCA. We evaluated additional oncotargets of LY2801653 (MET/HGF, pMET, AXL/pAXL, the MKNK1/2 substrate p-eIF4E, and ROS1) in 7 CCA cell lines (EGI-1, TFK-1, SNU245, SNU478, SNU869, SNU1079, and SNU1196) and 5 CCA patient-derived mouse xenograft (PDX) tumors. Cell line expression of MET, EGFR and p-eIF4E were evaluated by western blot. Although HGF expression was not detected in any of the cell lines by ELISA, pMET was detected in 5 cell lines (not in TFK-1 or SNU1079). ROS1 fusion was not detected in the 7 cell lines using break-apart FISH probes. Very high AXL and pAXL were detected in the SNU1196 line by western blot. Despite the high levels of pAXL, further increases in pAXL were noted after addition of its ligand, Gas6. In the SNU1196 cell line, pAXL expression and cell proliferation were completely inhibited by LY2801653, but not by a MET-specific inhibitor PF4217903. HGF-induced SNU1196 cell migration was inhibited equally well by LY2801653 and PF4217903. Unlike CCA cell line derived mouse xenograft tumors, the 5 PDX tumors retained prominent desmoplastic stroma. As analyzed by immunohistochemistry (IHC), MET was highly expressed in 3 of the 5 PDX tumors, and high levels of p-eIF4E were expressed in all 5. Increased AXL expression (IHC) appeared to be associated with more poorly differentiated PDX tumors. LY2801653 demonstrated potent in vivo anti-tumor activity in several CCA xenograft models. In a xenograft mouse model with the extrahepatic CCA derived EGI-1 cell line, treatment with LY2801653 at 24 mg/kg dosed twice daily resulted anti-tumor effect of 38.5% (% treated/control). In the SNU869 extrahepatic CCA cell line-derived xenograft mouse model, combination treatment with LY2801653 (12 mg/kg twice daily) and either cisplatin (5 mg/kg once weekly) or gemcitabine (60 mg/kg once weekly) resulted in an additive anti-tumor effect as compared to LY2801653 alone. Moreover, the combination of LY2801653 with gemcitabine resulted in tumor regression in this model. In vivo studies are ongoing with LY2801653 in the extrahepatic SNU1196 CCA cell line-derived xenograft model as well as two of the PDX models. The preclinical data in this study support the ongoing phase 1 clinical evaluation of LY2801653 in cholangiocarcinoma patients (trial I3O-MC-JSBA, NCT01285037). (1 - Yan et al. Invest New Drugs 2013;31:833-44). Citation Format: Sau-Chi Betty Yan, Suzane L. Um, Victoria L. Peek, Megan N. Thobe, Kelly M. Credille, Jennifer R. Stephens, Jason R. Manro, Darryl W. Ballard, Jessica A. Baker, Joel D. Cook, Bruce W. Konicek, Jeremy R. Graff, Timothy R. Holzer, Richard A. Walgren. Preclinical evaluation of LY2801653, an orally bioavailable small molecule oncokinase inhibitor, in cholangiocarcinoma models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4403. doi:10.1158/1538-7445.AM2014-4403

In vivo and in vitro evaluation of effects of Mg-6Zn alloy on apoptosis of common bile duct epithelial cell.

Biodegradable magnesium alloy implants have attracted much attention because of their excellent biocompatibility and good mechanical properties. However, effects of Mg alloy on cell apoptosis remain unclear. The aim of this study was to investigate the effects of the Mg-6Zn alloy on the apoptosis and necrosis of common bile duct (CBD) epithelial cells. In the in vitro experiments, primary mouse extrahepatic bile epithelial cells (MEBECs) were exposed to Mg-6Zn alloy extracts with different concentrations (0, 40, 80, and 100%). Flow cytometry analysis indicated that low concentration Mg-6Zn extract can induce apoptosis of MEBECs, and high concentration Mg-6Zn extracts may relate to necrosis and/or 'apoptotic necrosis'. Real-time PCR results showed that when MEBECs were treated with 40% extracts for 3days, the relative apoptotic genes including Bax, Bax/Bcl-2 ratio, NF-κB and caspase-3 were higher than those in the control group. In the in vivo experiments, Mg-6Zn alloy stents were implanted into rabbits' CBD for 1, 2, 3weeks, respectively. Based on the hematoxylin and eosin (H&E) staining of peri-implant CBD tissue, no apoptotic bodies and necrotic cells were observed. Results of immunohistochemical staining also showed Mg-6Zn stents did not increase expression levels of apoptosis related gene such as Bax, Bcl-2, Bax/Bcl-2 ratio, TNF-α, NF-κB and caspase-3 in CBD, which indicating Mg-6Zn did not induce significant apoptosis in the in vivo experiments. The different results of in vitro and in vivo experiment may result from the low corrosion rate of Mg-6Zn alloy stents in vivo and local Mg(2+) ion concentration in CBD.

Isolation of neonatal extrahepatic cholangiocytes.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood. Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established(1-4). Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts. This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

646 Translational Control of the Circadian Rhythms by MicroRNA-34a in Human Cholangiocarcinoma

Background & aims: Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small noncoding RNAs that trigger mRNA translation, repression or degradation. Core clock genes are the molecular basis of circadian rhythms, which include BMAL1, CLOCK, Per1/2/3 and Cry1/2. We have previously demonstrated the downregulation of Per1 expression in human cholangiocarcinoma (CCA) that was predicted as the direct target of miR-34a. Thus, we aimed to evaluate the role of deregulated circadian rhythm and related microRNAs in CCA growth. Methods: Human intraand extrahepatic CCA cells and non-malignant (H69) human cholangiocytes were serum starved for 48 hr before stimulation with 50% serum for 2 hours. The 24-hour rhythmic expression of core clock genes, such as Per1,2/3, CLOCK, BMAL1 and Cry1/2 was evaluated in CCA and H69 cells by real-time PCR. To further evaluate the role of Per1, we overexpressed Per1 by transfecting the Mz-ChA-1 cells with Per1 cDNA and empty vector. In parallel, we also silenced miR-34a expression with lenti-miR-34a ZIP with relative controls. A stably overexpressing Per1 cell line and silencing of miR-34a were established, respectively. Then, wemeasured: (i) cell proliferation byMTS assays and PCNA immunoblots; (ii) cell cycle by BD Cycle test Plus reagent kit; (iii) apoptosis by Annexin V-PE apoptosis detection kit; and (iv) cell migration and invasion by commercially available kits. We utilized luciferase assay to demonstrate that Per1 act as a target of miR-34a. Results: The 24-hour rhythmical expression of Per1 was abolished in all CCA cell lines but not in H69 cells. The rhythmic expression of BMAL1, CLOCK, Per2/3, Cry1/2 was also lost in some of the CCA cell lines tested. After overexpression of Per1, the extrahepatic CCA cell line, Mz-ChA-1, showed: (i) reduced cell proliferation (by MTS assays and PCNA immunoblots); (ii) higher G0/G1 arrest and lower G2/M arrest; (iii) enhanced apoptosis; and (iv) decreased cell invasion and migration compared to control cells. Interestingly, miR-34a was rhythmically expressed in CCA cell lines and H69. The expression level of miR-34a was higher in CCA cell lines compared to H69. Moreover, the inhibition of miR-34a decreased proliferation, migration and invasion in CCA cell lines. Dual luciferase reporter assay demonstrated that miR-34a directly targets Per1. Summary and conclusions: Disruption of circadian rhythms, miR-34a and Per1 expression may contribute to the malignant phenotypes of human cholangiocarcinoma. Our findings suggest that modulation of miR-34a-dependent Per1 expression may represent a novel therapeutic approach for human CCA.

647 Sofosbuvir-Based Regimens Are Associated With High SVR Rates Across Genotypes and Among Patients With Multiple Negative Predictive Factors

Background & aims: Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small noncoding RNAs that trigger mRNA translation, repression or degradation. Core clock genes are the molecular basis of circadian rhythms, which include BMAL1, CLOCK, Per1/2/3 and Cry1/2. We have previously demonstrated the downregulation of Per1 expression in human cholangiocarcinoma (CCA) that was predicted as the direct target of miR-34a. Thus, we aimed to evaluate the role of deregulated circadian rhythm and related microRNAs in CCA growth. Methods: Human intraand extrahepatic CCA cells and non-malignant (H69) human cholangiocytes were serum starved for 48 hr before stimulation with 50% serum for 2 hours. The 24-hour rhythmic expression of core clock genes, such as Per1,2/3, CLOCK, BMAL1 and Cry1/2 was evaluated in CCA and H69 cells by real-time PCR. To further evaluate the role of Per1, we overexpressed Per1 by transfecting the Mz-ChA-1 cells with Per1 cDNA and empty vector. In parallel, we also silenced miR-34a expression with lenti-miR-34a ZIP with relative controls. A stably overexpressing Per1 cell line and silencing of miR-34a were established, respectively. Then, wemeasured: (i) cell proliferation byMTS assays and PCNA immunoblots; (ii) cell cycle by BD Cycle test Plus reagent kit; (iii) apoptosis by Annexin V-PE apoptosis detection kit; and (iv) cell migration and invasion by commercially available kits. We utilized luciferase assay to demonstrate that Per1 act as a target of miR-34a. Results: The 24-hour rhythmical expression of Per1 was abolished in all CCA cell lines but not in H69 cells. The rhythmic expression of BMAL1, CLOCK, Per2/3, Cry1/2 was also lost in some of the CCA cell lines tested. After overexpression of Per1, the extrahepatic CCA cell line, Mz-ChA-1, showed: (i) reduced cell proliferation (by MTS assays and PCNA immunoblots); (ii) higher G0/G1 arrest and lower G2/M arrest; (iii) enhanced apoptosis; and (iv) decreased cell invasion and migration compared to control cells. Interestingly, miR-34a was rhythmically expressed in CCA cell lines and H69. The expression level of miR-34a was higher in CCA cell lines compared to H69. Moreover, the inhibition of miR-34a decreased proliferation, migration and invasion in CCA cell lines. Dual luciferase reporter assay demonstrated that miR-34a directly targets Per1. Summary and conclusions: Disruption of circadian rhythms, miR-34a and Per1 expression may contribute to the malignant phenotypes of human cholangiocarcinoma. Our findings suggest that modulation of miR-34a-dependent Per1 expression may represent a novel therapeutic approach for human CCA.

Axitinib (AG-013736), an oral specific VEGFR TKI, shows potential therapeutic utility against cholangiocarcinoma.

Cholangiocarcinoma is a refractory cancer whose incidence has been increasing worldwide in recent years. Neoangiogenesis plays an important role in the growth of various solid cancers, including cholangiocarcinoma. Vascular endothelial growth factor plays an important role in tumor-induced angiogenesis and its expression is associated with the progression and prognosis of cholangiocarcinoma. This study examined whether axitinib (AG-013736, INLYTA(®)), a potent and selective second-generation inhibitor of vascular endothelial growth factor receptors 1, 2 and 3, could be a potentially useful therapeutic agent for cholangiocarcinoma. We performed expression profiling of angiogenesis-related molecules in eight cholangiocarcinoma cell lines and found that three of them showed high vascular endothelial growth factor expression. Among them, we examined the in vivo anti-tumor effect of axitinib on NCC-BD1 (a gemcitabine-sensitive extra-hepatic cholangiocarcinoma cell line) and TKKK (a gemcitabine-resistant intra-hepatic cholangiocarcinoma cell line) using subcutaneous xenograft models. Oral administration of axitinib significantly inhibited the growth of TKKK xenografts at a dose of 6 mg kg(-1) day(-1) (P<0.05), and the growth of NCC-BD1 xenografts at 30 mg kg(-1)day(-1) (P<0.05). Treated tumors showed a significant decrease of microvessel density and the tumor cell proliferation index and a mild but significant increase of the apoptotic index in comparison with untreated tumors. Our results suggest that axitinib should be a promising therapy for vascular endothelial growth factor-expressing cholangiocarcinoma, irrespective of tumor origin and gemcitabine sensitivity.

A high‐throughput, fluorescence‐based cholesterol efflux assay (LB44)

Numerous studies have established a negative correlation between cellular cholesterol efflux and cardiovascular disease.Cholesterol efflux from peripheral tissues and cells by a process called Reverse Cholesterol Transport (RCT). RCT allows extrahepatic cells, including macrophage‐derived foam cells in arterial atherosclerotic plaque, to transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Standard protocols for quantitation of cholesterol efflux involve labeling cells with tritiated cholesterol and measuring the release of labeled sterol in a 12 or 24 well plate. This protocol, however is not ideal for high‐throughput screening of large numbers of serum samples or screening compounds/small molecules for their effect on cholesterol efflux.We report here a simple, convenient and high‐throughput cell‐based assay using fluorescently‐labeled cholesterol in a 96‐well plate format. Our results demonstrate that the percentage cholesterol efflux elicited by 2% ApoB‐depleted normal human serum in J774 macrophage cells is comparable to those published in literature. Our assay method utilizes a positive control for this assay which induces efflux independent of any receptor, that is used as a measure of the reliability of the assay. This assay is highly reproducible with low inter‐assay variability (蠄 10%). We demonstrate that this is an efficient method of measuring cholesterol efflux in a cell‐based assay. The increased sensitivity of the assay coupled with its high‐throughput screening capability enables scientists to screen large libraries of reagents and serum samples and provides a valuable tool for drug discovery in the field of cardiovascular disease research.