The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Non-structural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70.Weconfirmed anNS5A/HSPinteraction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A.Atransient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion.
Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.
We have previously demonstrated that quercetin, a bioflavonoid, blocks hepatitis C virus (HCV) proliferation by inhibiting NS5A-driven internal ribosomal entry site (IRES)-mediated translation of the viral genome. Here, we investigate the mechanisms of antiviral activity of quercetin and six additional bioflavonoids. We demonstrate that catechin, naringenin, and quercetin possess significant antiviral activity, with no associated cytotoxicity. Infectious virion secretion was not significantly altered by these bioflavonoids. Catechin and naringenin demonstrated stronger inhibition of infectious virion assembly compared to quercetin. Quercetin markedly blocked viral translation whereas catechin and naringenin demonstrated mild activity. Similarly quercetin completely blocked NS5A-augmented IRES-mediated translation in an IRES reporter assay, whereas catechin and naringenin had only a mild effect. Moreover, quercetin differentially inhibited HSP70 induction compared to catechin and naringenin. Thus, the antiviral activity of these bioflavonoids is mediated through different mechanisms. Therefore combination of these bioflavonoids may act synergistically against HCV.
HSP70; NS5A; IRES; HCV; Bioflavonoid
This editorial reviews the recent evidence showing that Mallory-Denk bodies (MDBs) form in hepatocytes as the result of a drug-induced shift from the 26s proteasome formation to the immunoproteasome formation. The shift is the result of changes in gene expression induced in promoter activation, which is induced by the IFNγ and TNFα signaling pathway. This activates TLR 2 and 4 receptors. The TLR signaling pathway stimulates both the induction of a cytokine proinflammatory response and an up regulation of growth factors. The MDB- forming hepatocytes proliferate as a result of the increase in growth factor expression by the MDB- forming cells, which selectively proliferate in response to drug toxicity. All of these mechanisms are induced by drug toxicity, and are prevented by feeding the methyl donors SAMe and betaine, supporting the epigenetic response of MDB formation.
Toll-like receptor; Proinflammatory; Methyl donors; Epigenetic processes; Drug toxicity; 26s Proteasome; Immunoproteasome
There is clinical evidence that chronic liver diseases in which MDBs (Mallory Denk Bodies) form progress to hepatocellular carcinoma. The present study provides evidence that links MDB formation induced by chronic drug injury, with preneoplasia and later to the formation of tumors, which develop long after drug withdrawal. Evidence indicated that this link was due to an epigenetic cellular memory induced by chronic drug ingestion. Microarray analysis showed that the expressions of many markers of preneoplasia (UBD, Alpha Fetoprotein, KLF6 and Glutathione-S-Transferase mu2) were increased together when the drug DDC was refed. These changes were suppressed by S-adenosylmethionine feeding, indicating that the drug was affecting DNA and histones methylation in an epigenetic manner. The link between MDB formation and neoplasia formation was likely due to the over expression of UBD (also called FAT10), which is up regulated in 90% of human hepatocellular carcinomas. Immunohistochemical staining of drug primed mouse livers showed that FAT10 positive liver cells persisted up to 4 months after drug withdrawal and they were still found in the livers of mice, 14 months after drug withdrawal. The refeeding of DDC increased the percent of FAT10 hepatocytes.
Toll-like receptors (TLR) play a role in mediating the proinflammatory response, fibrogenesis and carcinogenesis in chronic liver diseases such as alcoholic liver disease, non-alcoholic liver disease, hepatitis C and hepatocellular carcinoma. This is true in experimental models of these diseases. For this reason, we investigated the TLR proinflammatory response in the chronic intragastric tube feeding rat model of alcohol liver disease. The methyl donor S-adenosylmethionine was also fed to prevent the gene expression changes induced by ethanol. Ethanol feeding tended to increase the up regulation of the gene expression of TLR2 and TLR4. SAMe feeding prevented this. TLR4 and MyD88 protein levels were significantly increased by ethanol and this was prevented by SAMe. This is the first report where ethanol feeding induced TLR2 and SAMe prevented the induction by ethanol. CD34, FOS, interferon responsive factor 1 (IRF-1), Jun, TLR 1,2,3,4,6 and 7 and Traf-6 were found to be up regulated as seen by microarray analysis where rats were sacrified at high blood alcohol levels compared to pair fed controls. Il-6, IL-10 and IFNγ were also up regulated by high blood levels of ethanol. The gene expression of CD14, MyD88 and TNFR1SF1 were not up regulated by ethanol but were down regulated by SAMe. The gene expression of IL-1R1 and IRF1 tended to be up regulated by ethanol and this was prevented by feeding SAMe. The results suggest that SAMe, fed chronically prevents activation of TLR pathways caused by ethanol. In this way the proinflammatory response, fibrogenesis, cirrhosis and hepatocellular carcinoma formation due to alcohol liver disease could be prevented by SAMe.
Toll-like receptor (TLR); S-adenosylmethionine (SAMe); alcoholic liver disease (ASH)
There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells over expressing CYP2E1 were subjected to arachidonic acid, iron and 100 mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up regulated by ethanol alone and betaine tended to prevent this. All 4 agents down regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol.
Catechin; EGCG; Quercetin; Betaine; Oxidative Stress
An alcohol bolus causes the blood alcohol level (BAL) to peak at 1-2 hours post ingestion. The ethanol elimination rate is regulated by alcohol metabolizing enzymes, primarily alcohol dehydrogenase (ADH1), acetaldehyde dehydrogenase (ALDH), and cytochrome P450 (CYP2E1). Recently, S-adenosylmethionine (SAMe) was found to reduce acute BALs 3h after an alcohol bolus. The question, then, was: what is the mechanism involved in this reduction of BAL by feeding SAMe? To answer this question, we investigated the changes in ethanol metabolizing enzymes and the epigenetic changes that regulate the expression of these enzymes during acute binge drinking and chronic drinking.
Rats were fed a bolus of ethanol with or without SAMe, and were sacrificed at 3h or 12 h after the bolus.
RT-PCR and Western blot analyses showed that SAMe significantly induced ADH1 levels in the 3h liver samples. However, SAMe did not affect the changes in ADH1 protein levels 12h post bolus. Since SAMe is a methyl donor, it was postulated that the ADH1 gene expression up regulation at 3h was due to a histone modification induced by methylation from methyl transferases. Dimethylated histone 3 lysine 4 (H3K4me2), a modification responsible for gene expression activation, was found to be significantly increased by SAMe at 3h post bolus.
These results correlated with the low BAL found at 3h post bolus, and support the concept that SAMe increased the gene expression to increase the elimination rate of ethanol in binge drinking by increasing H3K4me2.
Over expression of FAT10 is characteristic of numerous types of carinoma including liver, gastric and colon carcinomas. In the case of colon carcinoma it is possible to determine at the point in the progression from the benign to the malignant process of colon cancer development by determining which stage in the neoplastic process FAT10 overexpression occurs. This stage was determined by measuring the intensity of fluoresence of immunohistochemically stained normal mucosa, tubular adenomas, hyperplastic polyps, serrated adenomas, villotubular, villous adenomas and invasive adenocarcinoma stages. Using this approach it was found the overexpression of FAT10 began at the serrated adenoma stage and continued to include the villous and villotubular stages and the invasive adenocaricnoma stage. The FAT10 overexpression by invasive adenocarcinoma was accompanied by the expression of the catalytic subunits of the immunoproteasome which is functionally tied to overexpression of FAT10, Toll-like receptor activation and the proinflammatory response.
FAT10; immunoproteasome; Toll-like receptors; interferon; proinflammatory response
Mallory-Denk Bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway’s (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFα and IFNγ receptors. SAMe prevented the increase in the TNFα and IFNγ receptors, supporting the idea that TNFα and IFNγ were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome.
26S proteasome; Immunoproteasome; TNF alpha; Interferon gamma; inflammatory response; Mallory-Denk Body
Microarrays were done on the livers of mice fed DDC for 10 weeks, withdrawn 1 month (DDC primed livers) and refed 6 days,and compared with mice fed the control diet. The expression of a large number of genes changed when DDC was fed or refed. A Venn diagram analysis identified 649 genes where gene expression was changed in the same direction. The epigenetic memory of the DDC primed liver involved an increase in the expression of ubiquitin D, alpha fetoprotein, connective tissue growth factor, integrin beta 2, DNA methyl transferase 3a and DNA damage –inducible 45 gamma. DNA methyl transferase 3b was down regulated as was Cbp/p300. When DDC was refed, DNA methyltransferase and histone deacetylase were up regulated as shown by microarray analysis. Histone3 lysine9 acetylation was increased by DDC and DDC refeeding and DNA methyltransferases were not changed as shown by Western blot analysis. The data suggests the concept that the epigenetic memory that explains why DDC primed hepatocytes form MBs in 7 days of DDC refeeding is primarily the result of epigenetic modifications of gene expression through changes in histone acetylation and methylation, as well as DNA methylation.
epigenetics; Mallory bodies; phenotypic change; genetic memory
Mallory-Denk bodies (MDBs) are found in chronic liver diseases. Previous studies showed that Diethyl-1, 4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) induced formation of MDBs and the up regulation of UbD expression in mouse liver. UbD is a protein over expressed in hepatocellular carcinomas. It is a potential preneoplastic marker in the mouse. It is hypothesized that inflammatory cytokines play a critical role in UbD up regulation and MDB formation. TNFa and IFNg treatment of HCC cell line Hepa 1–6, induced the expression of UbD and the expression of genes coding for the immunoproteasome (LMP2, LMP7, and MECL-1 subunits). TNFa and IFNg induced the activity of the UbD promoter, using a luciferase assay. The co-treatment with TNFa and IFNg induced the activity of the UbD promoter through an Interferon Sequence Responsive Element (ISRE). In addition, long term treatment with TNFa and IFNg induced the formation of MDB-like aggresomes in Hepa 1–6 cells, which emphasizes the role of inflammation in the formation of MDBs leading to the formation of liver tumors, in the mouse. Identifying the mechanism that regulates gene expression of UbD supports the hypothesis that down regulation of UbD and the proinflammatory gene expression would prevent MDBs and HCC formation. Previous studies indicate that S-adenosylmethionine or betaine prevented IFNg induced UbD and MDB formation.
MDB: Mallory-Denk Bodies; IFNg: Interferon gamma; TNFa: Tumor Necrosis Factor alpha; ISRE: Interferon stimulated response element; UbD: Di-Ubiquitin (Fat10); Ub: Ubiquitin
Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the down stream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-γ receptors by DDC feeding. INFγ stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described.
TLRs; 26s proteasome; immunoproteasome; interferon γ; proinflammatory cytokines
Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133+ TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG–dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2+/– mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.
NS5A is a key regulator of hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown NS5A to augment HCV IRES-mediated translation. Further, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also co-immunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) to be the interaction site. Further, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, while controls did not. Triple-alanine scan mutagenesis identified an exposed beta-sheet hairpin in C34 to be primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10 amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. Conclusion: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A-mediated IRES activity, sequence specific HSP recognition, and rational drug design.
HSP70; NS5A; IRES; HCV; Protein binding
Extremely drug-resistant (XDR; i.e. resistant to all antibiotics except colistin or tigecycline) Acinetobacter baumannii has emerged as one of the most common and highly antibiotic-resistant causes of infection. Diabetes is a risk factor for acquisition of and worse outcomes from A. baumannii infection. We sought to develop diabetic mouse models of A. baumannii bacteraemia and pneumonia and validate these models by comparing the efficacy of antibiotic treatment in these models with the established neutropenic mouse models.
Diabetic or neutropenic mice were infected via intravenous inoculation or inhalation in an aerosol chamber with an XDR A. baumannii. Treatment with colistin started 24 h after infection and continued daily for 7 days. Survival served as the primary endpoint while tissue bacterial burden and histopathological examination served as secondary endpoints.
Lethal infection was achieved for the neutropenic and diabetic mice when infected intravenously or via inhalation. Neutropenic mice were more susceptible to infection than diabetic mice in the pneumonia model and equally susceptible in the bacteraemia model. Both models of bacteraemia were sensitive enough to detect virulence differences among different clinical strains of A. baumannii. In the pneumonia model, colistin treatment was effective in improving survival, reducing lung bacterial burden and histologically resolving the infection compared with placebo only in diabetic mice.
We developed novel models of A. baumannii bacteraemia and pneumonia in diabetic mice. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies and evaluate drug efficacies against highly lethal A. baumannii infections.
bacteraemia; pneumonia; colistin; mice
The mechanism of Mallory Denk body formation is still not fully understood, but growing evidence implicates epigenetic mechanisms in MDB formation. In a previous study the epigenetic memory of MDB formation remained intact for at least four months after withdrawal from the DDC diet. In the present study, mice were fed a diet containing DDC or a diet containing DDC and S-adenosylmethionine (SAMe) to investigate the epigenetic memory of MDB formation. DDC feeding caused an increase in histone 3 acetylation, a decrease in histone 3 trimethylation, and an increase in histone ubiquitination. The addition of SAMe to the DDC diet prevented the DDC induced decrease of H3K4 and H3K9 trimethylation and the increase in histone ubiquitinylation. Changes in histone modifying enzymes, (HATs and HDACs) were also found in the liver nuclear extracts of the DDC/SAMe fed mice. Data mining of microarray analysis confirmed that gene expression changed with DDC refeeding, particularly the SAMe-metabolizing enzymes, Mat2a, AMD, AHCY and Mthfr. SAMe supplementation prevented the decrease of AHCY and GNMT, and prevented the increase in Mthfr, which provide a mechanism to explain how DDC inhibits methylation of histones. The results indicate that SAMe prevented the epigenetic cellular memory involved in the MDB formation
Sirt1, a deacetylase involved in regulating energy metabolism in response to calorie restriction, is up regulated after chronic ethanol feeding using the intragastric feeding model of alcohol liver disease. PGC1α is also up regulated in response to ethanol. These changes are consistent with activation of the Sirt1/PGC1α pathway of metabolism and aging, involved in alcohol liver disease including steatosis, necrosis and fibrosis of the liver. To test this hypothesis, male rats fed ethanol intragastrically for 1 month were compared with rats fed ethanol plus resveratrol or naringin. Liver histology showed macrovesicular steatosis caused by ethanol and this change was unchanged by resveratrol or naringin treatment. Necrosis occurred with ethanol alone but was accentuated by resveratrol treatment, as was fibrosis. The expression of Sirt1 and PGC1α was increased by ethanol but not when naringin or resveratrol was fed with ethanol. Sirt3 was also up regulated by ethanol but not when resveratrol was fed with ethanol. These results support the concept that ethanol induces the Sirt1/PGC1α pathway of gene regulation and both naringin and resveratrol prevent the activation of this pathway by ethanol. However, resveratrol did not reduce the liver pathology caused by chronic ethanol feeding.
This article reviews the evidence that ties the development of hepatocellular carcinoma (HCC) to the natural immune pro-inflammatory response to chronic liver disease, with a focus on the role of Toll-like receptor (TLR) signaling as the mechanism of liver stem cell/progenitor transformation to HCC. Two exemplary models of this phenomenon are reviewed in detail. One model applies chronic ethanol/lipopolysaccharide feeding to the activated TLR4 signaling pathway. The other applies chronic feeding of a carcinogenic drug, in which TLR2 and 4 signaling pathways are activated. In the drug-induced model, two major methyl donors, S-adenosylmethionine and betaine, prevent the upregulation of the TLR signaling pathways and abrogate the stem cell/progenitor proliferation response when fed with the carcinogenic drug. This observation supports a nutritional approach to liver cancer prevention and treatment. The observation that upregulation of the TLR signaling pathways leads to liver tumor formation gives evidence to the popular concept that the chronic pro-inflammatory response is an important mechanism of liver oncogenesis. It provides a nutritional approach, which could prevent HCC from developing in many chronic liver diseases.
Toll-like receptor; Hepatocellular carcinoma; Methyl donors, Epigenetic processes; Inflammation; Alcohol; Drug toxicity; Lipopolysaccharides
Liver stem cells are thought to preside in bile ducts and the canals of Hering. They extend into the liver parenchyma at a time when normal liver cell proliferation is suppressed and liver regeneration is stimulated. In the present study 69 liver biopsies and surgically excised liver tumors were studied for the presence of liver stem cells. It was found that human cirrhotic livers and hepatocellular carcinomas (HCC) frequently exhibited isolated single scattered hepatocyte stem cells within the liver parenchyma rather than in the portal tract, bile duct or the canal of Hering. These cells expressed liver stem cell markers. HCCs also contained isolated tumor cell which expressed the same stem cell markers. The markers used were GST-P, OV-6, CK-19, Oct-3/4 and FAT10. They were identified by immunofluorescent antibody staining. HGF, EGF, CK19, AIR, H19, Nanog, Oct-3/4 and FAT10 were identified by RNA-FISH. H19 is a non-coding RNA, which is expressed in most HCCs. Results: Immunohistochemistry and RNA-FISH performed on human livers identified isolated stem cells in liver parenchyma as follows: Stem cells identified by immunohistochemical markers (OV-6 and GST-P) and RNA-FISH markers (HGF, EGF, CK19 and H19) were found scattered in the liver parenchyma of cirrhotic livers and within hepatocellular carcinomas (HCCs). Precirrhotic ASH or NASH all stained negative for these stem cells. In HCCs, 13 out of 15 had stem cells located within the tumor (78%). In cirrhotic livers, 12 out of 28 (37%) had liver parenchymal stem cells present. In one case of stage 3 precirrhosis, stem cells were also found. Double staining for the markers showed colocalization of the markers in stem cells. Stem cells were found in 33% of HBV, 47% of HCV, 25% of alcoholic steatohepatitis (ASH) and 17% of non-alcoholic steatohepatitis (NASH). The frequency of stem cells found in the different disease categories correlates with the frequency of HCC occurring in these different diseases.
Epidermal growth factor (EGF); Hepatocyte growth factor (HGF); Glutathione S-transferase placental (GST-P); O. volvulus 6 (OV-6); AIR (antisense Igf2r)
Cancer stem cells (Tumor-initiating stem-like cells: TISCs) are resistant to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC), which is commonly observed in hepatitis C virus (HCV)-infected patients with obesity or alcohol abuse. However, it is unknown whether the TLR4-NANOG pathway serves as a universal oncogenic signaling in the genesis of TISCs and HCC. We aimed to determine whether Tlr4 is a putative proto-oncogene for TISCs in liver oncogenesis due to different etiologies and how Tlr4 is regulated at the transcriptional and epigenetic levels. CD133+/CD49f+ TISCs were isolated using FACS from HCC developed in HCV Core Tg mice fed alcohol, diethylnitrosamine-treated mice, and alcoholic patients with or without HCV infection. CD133+/CD49f+ cells isolated from the animal models and patients are tumorigenic both in vitro and in a xenograft model, and Tlr4 or Nanog silencing with shRNA attenuates their tumor initiating property. Functional oncogene screening of a cDNA library identified the organ size control pathway targets Yap1 and AKT activator Igf2bp3 as NANOG-dependent genes that inhibit TGF-β signaling in TISCs. Tlr4 expression is higher in TISCs compared to CD133−/CD49f+ cells, and DNA hypomethylation, histone acetylation and de-methylation drive Tlr4 induction through NOTCH signaling or Hif-1α. Taken together, Tlr4 is induced by hypoxia and NOTCH signaling and is a universal proto-oncogene responsible for the genesis of TLR4-NANOG dependent TISCs, and this pathway serves as a novel therapeutic target for HCC.
Alcohol; HCV; HCC; Tumor-initiating stem-like cells; Obesity
AIM: To examine the effects of ethanol-induced proteasome inhibition, and the effects of proteasome inhibition in the regulation of epigenetic mechanisms.
METHODS: Rats were fed ethanol for 1 mo using the Tsukamoto-French model and were compared to rats given the proteasome inhibitor PS-341 (Bortezomib, Velcade™) by intraperitoneal injection. Microarray analysis and real time PCR were performed and proteasome activity assays and Western blot analysis were performed using isolated nuclei.
RESULTS: Chronic ethanol feeding caused a significant inhibition of the ubiquitin proteasome pathway in the nucleus, which led to changes in the turnover of transcriptional factors, histone-modifying enzymes, and, therefore, affected epigenetic mechanisms. Chronic ethanol feeding was related to an increase in histone acetylation, and it is hypothesized that the proteasome proteolytic activity regulated histone modifications by controlling the stability of histone modifying enzymes, and, therefore, regulated the chromatin structure, allowing easy access to chromatin by RNA polymerase, and, thus, proper gene expression. Proteasome inhibition by PS-341 increased histone acetylation similar to chronic ethanol feeding. In addition, proteasome inhibition caused dramatic changes in hepatic remethylation reactions as there was a significant decrease in the enzymes responsible for the regeneration of S-adenosylmethionine, and, in particular, a significant decrease in the betaine-homocysteine methyltransferase enzyme. This suggested that hypomethylation was associated with proteasome inhibition, as indicated by the decrease in histone methylation.
CONCLUSION: The role of proteasome inhibition in regulating epigenetic mechanisms, and its link to liver injury in alcoholic liver disease, is thus a promising approach to study liver injury due to chronic ethanol consumption.
Alcohol liver injury; Betaine; Epigenetic mechanisms; Homocysteine methyltransferase; Proteasome inhibition; S-adenosylmethionine
M-30 and 4HNE adducts are two markers of active liver disease. M-30 is a serologic marker and 4HNE adducts are histologic markers. M-30 is a marker for apoptosis because it is a fragment of cytokeratin-18 left over from proteolysis by caspase 3. 4HNE is a marker of oxidative stress because it results from lipid peroxidation. Both markers are commonly found in nonalcoholic steatohepatitis and in alcoholic hepatitis. Liver biopsies from patients with steatohepatitis, 11 alcoholic and 11 non-alcoholics were stained for 4HNE and M-30. Almost all of the biopsies in both groups showed 4HNE and M-30 positive aggresomes in hepatocytes. Mallory Denk bodies (MDB) stained variably positive for M-30, whereas 4HNE was present in aggresomes independent of MDBs. However, they were sometimes located in hepatocytes which also contained MDBs as shown by confocal microscopy of double stained biopsies. The results indicate that the formation of M-30 and 4HNE aggresomes occurs through different pathways of liver cell injury in both types of steatohepatitis.
Caspase 3; oxidative stress; steatohepatitis; Mallory Denk bodies; aggresomes
In recent years, methyl one-carbon metabolism has received a great deal of attention because the disruption of methyl balance in a variety of genetically modified mice is associated with the development of various forms of liver injury, namely fatty liverdisease and hepatocellular carcinoma (HCC). In addition, patients with liver disease often have an abnormal expression of key genes involved in methionine metabolism as well as elevated serum levels of methionine and homocysteine (Hcy). S-adenosylmethionine (SAMe) has rapidly moved from being a methyl donor to a key metabolite that regulates hepatocyte proliferation, necrosis and differentiation. Biosynthesis of SAMe occurs in all mammalian cells as the first step in methionine catabolism in a reaction catalyzed by methionine adenosyltransferase (MAT). Decreased hepatic SAMe biosynthesis is a consequence of numerous forms of chronic liver injury. In an animal model of chronic liver SAMe deficiency, the liver is predisposed to further injury and develops spontaneous steatohepatitis and HCC. SAMe treatment in experimental animal models of liver injury shows that its hepatoprotective properties. Meta-analyses also showed that it is effective in the treatment of patients with cholestatic liver diseases. We studied the survival of liver cells treated with SAMe and betaine using Hepa 1–6 and E47/C34 cell lines. We showed that exogenous SAMe decreased the number of Hepa 1–6 and E47/C34 cells, and increased the number of dead cells in vitro. Betaine had no significant effect on the number of surviving cells and the number of dead cells. The combination of both methyl donors significantly increased the survival of liver cells and reduced necrosis, compare to SAMe alone. This study showed the inhibition of the proliferatino and increased necrosis in response to SAMe on liver cancer cell lines Hepa 1–6 and C34.
Liver; Methyl donors; SAMe; Betaine; cells death; necrosis