Plasma proteome plus site-specific N-glycoprofiling for hepatobiliary carcinomas.

Hepatobiliary cancer is the third leading cause of cancer death worldwide. Appropriate markers for early diagnosis, monitoring of disease progression, and prediction of postsurgical outcome are still lacking. As the majority of circulating N-glycoproteins are originated from the hepatobiliary system, we sought to explore new markers by assessing the dynamics of N-glycoproteome in plasma samples from patients with hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), or combined HCC and CCA (cHCC-CCA). Using a mass spectrometry-based quantitative proteomic approach, we found that 57 of 5358 identified plasma proteins were differentially expressed in hepatobiliary cancers. The levels of four essential proteins, including complement C3 and apolipoprotein C-III in HCC, galectin-3-binding protein in CCA, and 72 kDa inositol polyphosphate 5-phosphatase in cHCC-CCA, were highly correlated with tumor stage, tumor grade, recurrence-free survival, and overall survival. Postproteomic site-specific N-glycan analyses showed that human complement C3 bears high-mannose and hybrid glycoforms rather than complex glycoforms at Asn85. The abundance of complement C3 with mannose-5 or mannose-6 glycoform at Asn85 was associated with HCC tumor grade. Furthermore, stepwise Cox regression analyses revealed that HCC patients with a hybrid glycoform at Asn85 of complement C3 had a lower postsurgery tumor recurrence rate or mortality rate than those with a low amount of complement C3 protein. In conclusion, our data show that particular plasma N-glycoproteins with specific N-glycan compositions could be potential noninvasive markers to evaluate oncological status and prognosis of hepatobiliary cancers.

Etifoxine, a TSPO Ligand, Worsens Hepatitis C-Related Insulin Resistance but Relieves Lipid Accumulation.

Etifoxine, an 18 kDa translocator protein (TSPO) agonist for the treatment of anxiety disorders in clinic, may be able to cause acute liver injury or cytolytic hepatitis. TSPO has been demonstrated to participate in inflammatory responses in infective diseases as well as to modulate glucose and lipid homeostasis. Hepatitis C virus (HCV) infection disrupts glucose and lipid homoeostasis, leading to insulin resistance (IR). Whether TSPO affects the HCV-induced IR remains unclear. Here, we found that the administration of etifoxine increased the TSPO protein expression and recovered the HCV-mediated lower mitochondrial membrane potential (MMP) without affecting HCV infection. Moreover, etifoxine reversed the HCV-induced lipid accumulation by modulating the expressions of sterol regulatory element-binding protein-1 and apolipoprotein J. On the other hand, in infected cells pretreated with etifoxine, the insulin-mediated insulin receptor substrate-1/Akt signals, forkhead box protein O1 translocation, and glucose uptake were blocked. Taken together, our results pointed out that etifoxine relieved the HCV-retarded MMP and reduced the lipid accumulation but deteriorated the HCV-induced IR by interfering with insulin signal molecules.

Calcitriol Inhibits HCV Infection via Blockade of Activation of PPAR and Interference with Endoplasmic Reticulum-Associated Degradation.

Vitamin D has been identified as an innate anti-hepatitis C virus (HCV) agent but the possible mechanisms for this issue remain unclear. Here, we clarified the mechanisms of calcitriol-mediated inhibition of HCV infection. Calcitriol partially inhibited HCV infection, nitric oxide (NO) release and lipid accumulation in Huh7.5 human hepatoma cells via the activation of vitamin D receptor (VDR). When cells were pretreated with the activators of peroxisome proliferator-activated receptor (PPAR)-α (Wy14643) and -γ (Ly171883), the calcitriol-mediated HCV suppression was reversed. Otherwise, three individual stimulators of PPAR-α/ß/γ blocked the activation of VDR. PPAR-ß (linoleic acid) reversed the inhibition of NO release, whereas PPAR-γ (Ly171883) reversed the inhibitions of NO release and lipid accumulation in the presence of calcitriol. The calcitriol-mediated viral suppression, inhibition of NO release and activation of VDR were partially blocked by an inhibitor of endoplasmic reticulum-associated degradation (ERAD), kifunensine. Furthermore, calcitriol blocked the HCV-induced expressions of apolipoprotein J and 78 kDa glucose-regulated protein, which was restored by pretreatment of kifunensine. These results indicated that the calcitriol-mediated HCV suppression was associated with the activation of VDR, interference with ERAD process, as well as blockades of PPAR, lipid accumulation and nitrative stress.

Celastrol inhibits hepatitis C virus replication by upregulating heme oxygenase-1 via the JNK MAPK/Nrf2 pathway in human hepatoma cells.

BACKGROUND AND PURPOSE: Celastrol, a quinone methide triterpene isolated from the root extracts of Tripterygium wilfordii, can greatly induce the gene expression activity of heme oxygenase-1 (HO-1) to achieve disease prevention and control. HO-1 induction was recently shown to result in anti-HCV activity by inducing type I interferon and inhibiting hepatitis C virus (HCV) NS3/4A protease activity. The aim of the present study is to evaluate the anti-HCV activity of celastrol and characterize its mechanism of inhibition. METHODS: The anti-HCV activity of celastrol was evaluated using the HCV subgenomic replicon and HCVcc infection systems. The anti-HCV mechanism of celastrol targeting HO-1 expression was clarified using specific inhibitors against several signaling pathways. The transcriptional regulation of celastrol on target gene expression was determined using promoter-based reporter activity assay. The synergistic effect of celastrol and a numbers of clinically used anti-HCV drugs was determined via a drug combination assay. RESULTS: Celastrol inhibited HCV replication in both the HCV subgenomic and HCVcc infection systems with EC50 values of 0.37 ± 0.022 and 0.43 ± 0.019 µM, respectively. Celastrol-induced heme oxygenase 1 (HO-1) expression promoted antiviral interferon responses and inhibition of NS3/4A protease activity, thereby blocking HCV replication. These antiviral effects were abrogated by treatment with the HO-1-specific inhibitor SnMP or silencing of HO-1 expression by transfection of shRNA, which indicates that HO-1 induction contributes to the anti-HCV activity of celastrol. JNK mitogen-activated protein kinase and nuclear factor erythroid 2-related factor 2 (Nrf2) were confirmed to be involved in the inductive effect of celastrol on HO-1 expression. Celastrol exhibited synergistic effects in combination with interferon-alpha, the NS5A inhibitor daclatasvir, and the NS5B inhibitor sofosbuvir. CONCLUSION: Celastrol can serve as a potential supplement for blocking HCV replication. Targeting the JNK/Nrf2/HO-1 axis presents a promising strategy against HCV infection.

Lipoprotein lipase liberates free fatty acids to inhibit HCV infection and prevent hepatic lipid accumulation.

Lipoprotein lipase (LPL) has been identified as an anti-hepatitis C virus (HCV) host factor, but the cellular mechanism remains elusive. Here, we investigated the cellular mechanism of LPL involving in anti-HCV. The functional activation of peroxisome proliferator-activated receptor (PPAR) α signal by LPL transducing into hepatocytes was investigated in HCV-infected cells, primary human hepatocytes, and in HCV-core transgenic mice. The result showed that the levels of transcriptional transactivity and nuclear translocation of PPARα in Huh7 cells and primary human hepatocytes were elevated by physiologically ranged LPL treatment of either very-low density lipoprotein or HCV particles. The LPL-induced hepatic PPARα activation was weakened by blocking the LPL enzymatic activity, and by preventing the cellular uptake of free unsaturated fatty acids with either albumin chelator or silencing of CD36 translocase. The knockdowns of PPARα and CD36 reversed the LPL-mediated suppression of HCV infection. Furthermore, treatment with LPL, like the direct activation of PPARα, not only reduced the levels of apolipoproteins B, E, and J, which are involved in assembly and release of HCV virions, but also alleviated hepatic lipid accumulation induced by core protein. HCV-core transgenic mice exhibited more hepatic miR-27b, which negatively regulates PPARα expression, than did the wild-type controls. The induction of LPL activity by fasting in the core transgenic mice activated PPARα downstream target genes that are involved in fatty acid ß-oxidation. Taken together, our study reveals dual beneficial outcomes of LPL in anti-HCV and anti-steatosis and shed light on the control of chronic hepatitis C in relation to LPL modulators.

Fluoxetine regulates cell growth inhibition of interferon-α.

Fluoxetine, a well-known anti-depression agent, may act as a chemosensitizer to assist and promote cancer therapy. However, how fluoxetine regulates cellular signaling to enhance cellular responses against tumor cell growth remains unclear. In the present study, addition of fluoxetine promoted growth inhibition of interferon-alpha (IFN-α) in human bladder carcinoma cells but not in normal uroepithelial cells through lessening the IFN-α-induced apoptosis but switching to cause G1 arrest, and maintaining the IFN-α-mediated reduction in G2/M phase. Activations and signal transducer and transactivator (STAT)-1 and peroxisome proliferator-activated receptor alpha (PPAR-α) were involved in this process. Chemical inhibitions of STAT-1 or PPAR-α partially rescued bladder carcinoma cells from IFN-α-mediated growth inhibition via blockades of G1 arrest, cyclin D1 reduction, p53 downregulation and p27 upregulation in the presence of fluoxetine. However, the functions of both proteins were not involved in the control of fluoxetine over apoptosis and maintained the declined G2/M phase of IFN-α. These results indicated that activation of PPAR-α and STAT-1 participated, at least in part, in growth inhibition of IFN-α in the presence of fluoxetine.

Prediction of early hepatocellular carcinoma recurrence using germinal center kinase-like kinase.

Germinal center kinase-like kinase (GLK) is a key controller of autoimmunity. In this study, we assessed the clinical relevance and tumorigenic effects of GLK in hepatocellular carcinoma (HCC). Using immunohistochemistry, we showed that the GLK proportion score increased in both cancerous and adjacent non-cancerous liver tissue from patients with HCC recurrence. A Kaplan-Meier analysis revealed that patients with a wide distribution of GLK in non-cancerous liver tissue had a higher rate of HCC recurrence than those with very low or no GLK expression. Multivariate Cox regression analyses indicated that a high GLK proportion score in non-cancerous liver tissue was an independent predictor of early HCC recurrence after resection. Lentiviral vector-mediated overexpression of GLK activated the nuclear factor kappa B (NFκB) signaling cascade and accelerated cell cycle progression in primary human hepatocytes, thereby promoting proliferation. An increase in GLK expression coincided with NFκB activation and enhanced expression of proliferating cell nuclear antigen in HCC tissue. Our findings demonstrate a potential hepatocarcinogenic effect of GLK and the feasibility of using GLK to predict early HCC recurrence.

Apolipoprotein J, a glucose-upregulated molecular chaperone, stabilizes core and NS5A to promote infectious hepatitis C virus virion production.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to glucose abnormality. HCV depends on lipid droplets (LDs) and very-low density lipoproteins for assembly/releasing; however, the components and locations for this process remain unidentified. Apolipoprotein J (ApoJ), upregulated by glucose, functions as Golgi chaperone of secreted proteins and resides abundantly in very-low density lipoproteins. This study investigates the interplay between glucose, ApoJ and HCV virion production. METHODS: The effects of high glucose on ApoJ expression and HCV production were evaluated with cultivated HuH7.5, primary human hepatocytes, and in treatment naive chronic hepatitis C patients. How ApoJ affects HCV lifecycle was assessed using siRNA knockdown strategy in JFH1 infected and subgenomic replicon cells. The interactions and locations of ApoJ with viral and host components were examined by immunoprecipitation, immunofluorescence and subcellular fractionation experiments. RESULTS: HCV infection increased ApoJ expression, which in parallel with HCV infectivity was additionally elevated with high glucose treatment. Serum ApoJ correlated positively with fasting blood glucose concentration and HCV-RNA titre in patients. ApoJ silencing reduced intracellular and extracellular HCV infectivity and extracellular HCV-RNA, but accumulated intracellular HCV-RNA in HCV-infected cells. ApoJ interacted with HCV core and NS5A and stabilized the dual protein complex. HCV infection dispersed cytoplasmic ApoJ from the compact zones of the Golgi to encircle LDs, where co-localization of the core, NS5A, HCV-RNA, subcellular markers for LDs, endoplasmic reticulum (ER), Golgi, and membrane contact sites occurred. CONCLUSIONS: ApoJ facilitates infectious HCV particle production via stabilization of core/NS5A, which might surround LDs at the ER-Golgi membrane contact site.

Rosiglitazone regulates anti-inflammation and growth inhibition via PTEN.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonist has anti-inflammatory and anticancer properties. However, the mechanisms by which PPARγ agonist rosiglitazone interferes with inflammation and cancer via phosphatase and tensin homolog-(PTEN)-dependent pathway remain unclear. We found that lower doses (<25 µ M) of rosiglitazone significantly inhibited lipopolysaccharide-(LPS)-induced nitric oxide (NO) release (via inducible nitric oxide synthase, iNOS), prostaglandin E2 (PGE2) production (via cyclooxygenase-2, COX-2), and activation of Akt in RAW 264.7 murine macrophages. However, rosiglitazone did not inhibit the production of reactive oxygen species (ROS). In PTEN knockdown (shPTEN) cells exposed to LPS, rosiglitazone did not inhibit NO release, PGE2 production, and activation of Akt. These cells had elevated basal levels of iNOS, COX-2, and ROS. However, higher doses (25-100 µ M) of rosiglitazone, without LPS stimulation, did not block NO release and PGE2 productions, but they inhibited p38 MAPK phosphorylation and blocked ROS generation in shPTEN cells. In addition, rosiglitazone caused G1 arrest and reduced the number of cells in S + G2/M phase, leading to growth inhibition. These results indicate that the anti-inflammatory property of rosiglitazone is related to regulation of PTEN independent of inhibition on ROS production. However, rosiglitazone affected the dependence of PTEN-deficient cell growth on ROS.

Andrographolide exerts anti-hepatitis C virus activity by up-regulating haeme oxygenase-1 via the p38 MAPK/Nrf2 pathway in human hepatoma cells.

BACKGROUND AND PURPOSE: This study aimed to evaluate the anti-hepatitis C virus (HCV) activity of andrographolide, a diterpenoid lactone extracted from Andrographis paniculata, and to identify the signalling pathway involved in its antiviral action. EXPERIMENTAL APPROACH: Using HCV replicon and HCVcc infectious systems, we identified anti-HCV activity of andrographolide by measuring protein and RNA levels. A reporter activity assay was used to determine transcriptional regulation of anti-HCV agents. A specific inhibitor and short hairpin RNAs were used to investigate the mechanism responsible for the effect of andrographolide on HCV replication. KEY RESULTS: In HCV replicon and HCVcc infectious systems, andrographolide time- and dose-dependently suppressed HCV replication. When combined with IFN-α, an inhibitor targeting HCV NS3/4A protease (telaprevir), or NS5B polymerase (PSI-7977), andrographolide exhibited a significant synergistic effect. Andrographolide up-regulated the expression of haeme oxygenase-1 (HO-1), leading to increased amounts of its metabolite biliverdin, which was found to suppress HCV replication by promoting the antiviral IFN responses and inhibiting NS3/4A protease activity. Significantly, these antiviral effects were attenuated by an HO-1-specific inhibitor or HO-1 gene knockdown, indicating that HO-1 contributed to the anti-HCV activity of andrographolide. Andrographolide activated p38 MAPK phosphorylation, which stimulated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated HO-1 expression, and this was found to be associated with its anti-HCV activity. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that andrographolide has the potential to control HCV replication and suggest that targeting the Nrf2-HO-1 signalling pathway might be a promising strategy for drug development.

Blockade of reactive oxygen species and Akt activation is critical for anti-inflammation and growth inhibition of metformin in phosphatase and tensin homolog-deficient RAW264.7 cells.

CONTEXT: Metformin is widely used for treatment of type 2 diabetes and has a potential application on the treatment of inflammation and cancer. Phosphatase and tensin homolog (PTEN) plays a critical role in cancer cell growth and inflammation; however, precise mechanisms remain unclear. OBJECTIVE: We aimed to investigate the possible mechanisms of how PTEN regulates metformin against cell growth and inflammation. MATERIALS AND METHODS: We established PTEN knockdown in RAW264.7 murine macrophages (shPTEN cells) to detect inflammatory mediators using commercial kits, production of reactive oxygen species (ROS) by flow cytometry, cell growth by MTT assay and phosphorylated levels of signal molecules by western blot. RESULTS: The shPTEN cells had a significant large amount of inflammatory mediators, such as inducible nitric oxide synthase (iNOS)/nitric oxide (NO) and cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)); and also elevated the production of ROS and increased cell proliferation. These effects were accompanied with the activation of Akt and p38 mitogen-activated protein kinase (MAPK), and the inactivation of an AMP-activated protein kinase (AMPK) activator and extracellular signal-regulated kinase 1/2. Pretreatment with metformin not only blocked these inflammatory mediators, but also caused growth inhibition induced by significant apoptosis. Furthermore, inactivation of Akt, blockade of ROS generation and independence of activations of AMPK and MAPK by metformin were also observed. CONCLUSION: Macrophages with PTEN deficiency developed a continuous inflammatory microenvironment, which further aggravated tumor cell growth. Moreover, metformin affected PTEN-deficient cells dependent of inhibition of ROS production and Akt activation against enlarged inflammatory mediators and/or cell growth in shPTEN cells.

The dyslipidemia-associated SNP on the APOA1/C3/A5 gene cluster predicts post-surgery poor outcome in Taiwanese breast cancer patients: a 10-year follow-up study.

BACKGROUND: Post-surgery therapies are given to early-stage breast cancer patients due to the possibility of residual micrometastasis, and optimized by clincopathological parameters such as tumor stage, and hormone receptor/lymph node status. However, current efficacy of post-surgery therapies is unsatisfactory, and may be varied according to unidentified patient genetic factors. Increases of breast cancer occurrence and recurrence have been associated with dyslipidemia, which can attribute to other known risk factors of breast cancer including obesity, diabetes and metabolic syndrome. Thus we reasoned that dyslipidemia-associated nucleotide polymorphisms (SNPs) on the APOA1/C3/A5 gene cluster may predict breast cancer risk and tumor progression. METHODS: We analyzed the distribution of 5 selected APOA1/C3/A5 SNPs in recruited Taiwanese breast cancer patients (n=223) and healthy controls (n=162). The association of SNP (APOA1 rs670) showing correlation with breast cancer with baseline and follow-up parameters was further examined. RESULTS: APOA1 rs670 A allele carriage was higher in breast cancer patients than controls (59.64% vs. 48.77%, p=0.038). The rs670 A allele carrying patients showed less favorable baseline phenotype with positive lymph nodes (G/A: OR=3.32, 95% CI=1.77-6.20, p<0.001; A/A: OR=2.58, 95% CI=1.05-6.32, p=0.039) and negative hormone receptor expression (A/A: OR=4.85, 95%CI=1.83-12.83, p=0.001) in comparison to G/G carriers. Moreover, rs670 A/A carrying patients had higher risks in both tumor recurrence (HR=3.12, 95% CI=1.29-7.56, p=0.012) and mortality (HR=4.36, 95% CI=1.52-12.47, p=0.006) than patients with no A alleles after adjustments for associated baseline parameters. Furthermore, the prognostic effect of rs670 A/A carriage was most evident in lymph node-negative patients, conferring to the highest risks of recurrence (HR=4.98, 95% CI=1.40-17.70, p=0.013) and mortality (HR=9.87, 95%CI=1.60-60.81, p=0.014) than patients with no A alleles. CONCLUSIONS: APOA1 rs670 A/A carriage showed poor post-surgery prognosis in Taiwanese lymph node-negative breast cancer patients, whose prognosis were considered better and adjuvant treatment might be less stringent according to currently available assessment protocols. Our findings suggest that APOA1 rs670 indicate a post-surgery risk of breast cancer disease progression, and that carriers of this SNP may benefit from more advanced disease monitoring and therapy regimens than the current regular standards. Furthermore, control of lipid homeostasis might protect APOA1 rs670 minor allele carriers from breast cancer occurrence and progression.

Glycogen synthase kinase-3ß regulates anti-inflammatory property of fluoxetine.

A selective serotonin reuptake inhibitor fluoxetine not only is widely used in the treatment of depression but also has an anti-inflammatory property. Glycogen synthase kinase-3beta (GSK-3ß) is a vital factor in the inflammation process. How fluoxetine interferes with inflammation via a GSK-3ß-dependent pathway remains unclear. The aim of this study is to investigate the effects of fluoxetine on lipopolysaccharide (LPS)-induced inflammation. Results showed that fluoxetine decreased mortality rate of the mice. It also inhibited LPS-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) in serum and RAW264.7 murine macrophages and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Upon LPS stimulation, fluoxetine caused a delay but increased in the phosphorylated levels of GSK-3ß (ser9), whereas it did not affect LPS-induced activation of mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS). Fluoxetine in combination with phosphatidylinositol 3-kinases/Akt inhibitors (LY294002 and Wortmannin) did not have a synergistic inhibition on LPS-induced NO release and PGE2 production. In addition, peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9622 showed no reverse effects of this inhibition of fluoxetine. GSK-3ß knockdown blocked the inhibitory effects of fluoxetine on LPS-induced iNOS/NO release and COX-2/PGE2 production. These results indicated that GSK-3ß regulated anti-inflammatory property of fluoxetine. However, Akt activation, ROS generation, and altered PPARγ activity were not involved in this inhibition of fluoxetine.

Expression of T-cell lymphoma invasion and metastasis 2 (TIAM2) promotes proliferation and invasion of liver cancer.

The T-cell lymphoma invasion and metastasis 2 (TIAM2) gene is the homolog of human TIAM1, a Rac-specific guanine nucleotide exchange factor that plays important roles in neuron development and human malignancies. Although the role of TIAM1 is well characterized, the physiological and pathological functions of TIAM2 remain unknown. In our study, human cDNA and protein panels were evaluated for endogenous expression of TIAM2. Four hepatocellular carcinoma (HCC) cell lines and 91 HCC samples were used to demonstrate expression of TIAM2S (the short form of TIAM2) in cancer cells. In addition, HepG2 cells stably expressing TIAM2S were used for tumorigenic assays in both cellular and mouse models. We demonstrate that endogenous TIAM2S was induced in several human cancers including HCC. TIAM2S expression was undetectable in normal human liver but was induced in all HCC cell lines and in 86% (78/91) of HCC biopsies. TIAM2S expression was positively associated with TIAM1 expression, hepatitis B virus (HBV) infection and metastatic phenotype. Expression of recombinant TIAM2S in HepG2 cells promoted growth and invasiveness. In vivo study using a xenografted mouse model demonstrated that induced endogenous expression of TIAM2S converted non-invasive human HCC cells into highly aggressive vascular tumors. Further examination revealed that TIAM2S expression resulted in up-regulation of N-cadherin and vimentin, and in redistribution of E-cadherin. These findings show, for the first time, that human TIAM2S is involved in HCC pathogenesis, and that increased expression of TIAM2S promotes epithelial-to-mesenchymal transition and results in proliferation and invasion in liver cancer cells.

Novel nucleotide and amino acid covariation between the 5'UTR and the NS2/NS3 proteins of hepatitis C virus: bioinformatic and functional analyses.

Molecular covariation of highly polymorphic viruses is thought to have crucial effects on viral replication and fitness. This study employs association rule data mining of hepatitis C virus (HCV) sequences to search for specific evolutionary covariation and then tests functional relevance on HCV replication. Data mining is performed between nucleotides in the untranslated regions 5' and 3'UTR, and the amino acid residues in the non-structural proteins NS2, NS3 and NS5B. Results indicate covariance of the 243(rd) nucleotide of the 5'UTR with the 14(th), 41(st), 76(th), 110(th), 211(th) and 212(th) residues of NS2 and with the 71(st), 175(th) and 621(st) residues of NS3. Real-time experiments using an HCV subgenomic system to quantify viral replication confirm replication regulation for each covariant pair between 5'UTR243 and NS2-41, -76, -110, -211, and NS3-71, -175. The HCV subgenomic system with/without the NS2 region shows that regulatory effects vanish without NS2, so replicative modulation mediated by HCV 5'UTR243 depends on NS2. Strong binding of the NS2 variants to HCV RNA correlates with reduced HCV replication whereas weak binding correlates with restoration of HCV replication efficiency, as determined by RNA-protein immunoprecipitation assay band intensity. The dominant haplotype 5'UTR243-NS2-41-76-110-211-NS3-71-175 differs according to the HCV genotype: G-Ile-Ile-Ile-Gly-Ile-Met for genotype 1b and A-Leu-Val-Leu-Ser-Val-Leu for genotypes 1a, 2a and 2b. In conclusion, 5'UTR243 co-varies with specific NS2/3 protein amino acid residues, which may have significant structural and functional consequences for HCV replication. This unreported mechanism involving HCV replication possibly can be exploited in the development of advanced anti-HCV medication.

Identification of hnRNPH1, NF45, and C14orf166 as novel host interacting partners of the mature hepatitis C virus core protein.

The hepatitis C virus core protein (HCVc) forms the viral nucleocapsid and is involved in viral persistence and pathogenesis, possibly by interacting with host factors to modulate viral replication and cellular functions. Here, we identified 36 cellular protein candidates by one-dimensional SDS-PAGE and LC-MS/MS-based proteomics after affinity purification with HCVc174, a matured form of HCVc from HCV-1b genotype, tagged with biotin and calmodulin-binding peptide/protein A at N- and C-termini, respectively. By pull-down and confocal imaging techniques, we confirmed that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1), nuclear factor 45 (NF45), and C14orf166 are novel HCVc174-interacting host proteins, known to participate in mRNA metabolism, gene regulation, and microtubule organization, respectively. Unlike the other 2 proteins, NF45 interacted with HCVc174 in an RNA-dependent manner. These 3 proteins colocalized with ectopic HCVc-1b in both the cytoplasm and nucleus, which demonstrated their spatial interaction with naturally translocated HCVc174 after HCVc biogenesis. Such colocalization, however, shifted to the cytoplasm in cells with replicating virus of 1b or 2a genotype, indicating that active viral replication confined these interacting proteins in the cytoplasm. Collectively, our findings suggest that spatial interactions of hnRNPH1, NF45, and C14orf166 with HCVc174 likely modulate HCV or cellular functions during acute and chronic HCV infection.

ACSL3 and GSK-3ß are essential for lipid upregulation induced by endoplasmic reticulum stress in liver cells.

The endoplasmic reticulum (ER) is essential for lipid biosynthesis, and stress signals in this organelle are thought to alter lipid metabolism. Elucidating the mechanisms that underlie the dysregulation of lipid metabolism in hepatocytes may lead to novel therapeutic approaches for the treatment of lipid accumulation. We first tested the effects of several inhibitors on lipid dysregulation induced by tunicamycin, an ER stress inducer. Triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL) 1, 3, and 4, was the most potent among these inhibitors. We then analyzed the expression of the ACSL family during ER stress. The expression of ACSL3 was induced by ER stress in HuH-7 cells and in mice livers. ACSL3 shRNA, but not ACSL1 shRNA, inhibited the induction of lipid accumulation. GSK-3ß inhibitors attenuated ACSL3 expression and the lipid accumulation induced by ER stress in HuH-7 cells. shRNA that target GSK-3ß also inhibited the upregulation of ACSL3 and lipid accumulation in HuH-7 and HepG2 cells. The hepatitis B virus mutant large surface protein, which is known to induce ER stress, increased the lipid content of cells. Similarly, Triacsin C, and GSK-3ß inhibitors abrogated the lipid dysregulation caused by the hepatitis B virus mutant large surface protein. Altogether, ACSL3 and GSK-3ß represent novel therapeutic targets for lipid dysregulation by ER stress.

A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide. The pre-S1 and -S2 mutant large HBV surface antigen (LHBS), in which the pre-S1 and -S2 regions of the LHBS gene are partially deleted, are highly associated with HBV-related HCC. METHODS: The pre-S region of the LHBS gene in two hundred and one HBV-positive serum samples was PCR-amplified and sequenced. A pre-S oligonucleotide gene chip was developed to efficiently detect pre-S deletions in chronic HBV carriers. Twenty serum samples from chronic HBV carriers were analyzed using the chip. RESULTS: The pre-S deletion rates were relatively low (7%) in the sera of patients with acute HBV infection. They gradually increased in periods of persistent HBV infection: pre-S mutation rates were 37% in chronic HBV carriers, and as high as 60% in HCC patients. The Pre-S Gene Chip offers a highly sensitive and specific method for pre-S deletion detection and is less expensive and more efficient (turnaround time 3 days) than DNA sequencing analysis. CONCLUSION: The pre-S1/2 mutants may emerge during the long-term persistence of the HBV genome in carriers and facilitate HCC development. Combined detection of pre-S mutations, other markers of HBV replication, and viral titers, offers a reliable predictive method for HCC risks in chronic HBV carriers.

CD81 down-regulation on B cells is associated with the response to interferon-alpha-based treatment for chronic hepatitis C virus infection.

The lymphocytic CD81 molecule, capable of modulating type-1/-2 T-helper responses and serving as a putative receptor for hepatitis C virus (HCV), might influence the outcome of anti-HCV treatment. This study characterized the interferon-alpha-induced alteration of lymphocytic CD81. The CD81 levels in healthy subjects and naïve chronic HCV patients were compared, with the results showing that the two groups had comparable surface CD81 levels for total peripheral blood lymphocytes, subpopulation-B, -T, and -NK cells. In vitro interferon-alpha treatment could suppress the CD81 expression from both groups. Subsequently, we compared the in vitro interferon-alpha modulatory effects on lymphocytic CD81 from patients having received anti-HCV therapy with either sustained virological response (SVR) or without SVR. There was a significant down-regulation of the B-cell's CD81 only in the SVR group. The CD81 modulation was further investigated using Daudi lymphoid cell line, showing declined surface CD81 levels following treatment with interferon-alpha, interferon-beta or polyI:C. Thus, interferons could directly decrease CD81 expression. The interferon-alpha effect could be restored by 2-aminopurine, suggesting that double-stranded RNA activated kinase might be involved in the suppression of CD81. In conclusion, CD81 down-regulation is a primary host response to interferon-alpha-based therapy and an immunophenotype associated with anti-HCV SVR.