Hiroyoshi Taniguchi

Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses

The persistent nature of hepatitis C virus (HCV) infection suggests that HCV encodes proteins that enable it to overcome host antiviral responses. Toll‐like receptor 3 (TLR3)‐mediated signaling, which recognizes the double‐stranded RNA that is produced during viral replication and induces type I interferons, including interferon β (IFN‐β), is crucial to the host defense against viruses. Recent studies suggest that a TIR domain–containing adaptor protein, TRIF, and two protein kinases, TANK‐binding kinase‐1 (TBK1) and IκB kinase‐ϵ (IKKϵ), play essential roles in TLR3‐mediated IFN‐β production through the activation of the transcriptional factor interferon regulatory factor 3 (IRF‐3). We report that the HCV NS3 protein interacts directly with TBK1, and that this binding results in the inhibition of the association between TBK1 and IRF‐3, which leads to the inhibition of IRF‐3 activation. In conclusion, these results suggest the mechanisms of the inhibition of the innate immune responses of HCV infection by NS3 protein. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:1004–1012.)

Vitamin K2 inhibits the growth and invasiveness of hepatocellular carcinoma cells via protein kinase A activation.

Heatocellular carcinoma (HCC) is a common human malignancy. Its high mortality rate is mainly a result of high intrahepatic recurrence and portal venous invasion (PVI). We previously reported that the development of PVI is related to levels of des‐gamma‐carboxy prothrombin (DCP), a serum protein that increases at a notably higher rate in patients with HCC. Because DCP is produced by a vitamin K shortage, we examined the biological effects of extrinsic supplementation of vitamin K2 in HCC cells in vitro and in vivo. Consequently, vitamin K2 inhibits the growth and invasion of HCC cells through the activation of protein kinase A, which modulates the activities of several transcriptional factors and inhibits the small GTPase Rho, independent of suppression of DCP. In addition, administration of vitamin K2 to nude mice inoculated with liver tumor cells reduced both tumor growth and body weight loss. In conclusion, similar to an acyclic retinoid—which was previously reported to prevent the recurrence of HCC—vitamin K2, another lipid‐soluble vitamin, may be a promising therapeutic means for the management of HCC. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:243–251.)

UDP-Glucuronosyltransferase 1A7 Genetic Polymorphisms Are Associated with Hepatocellular Carcinoma in Japanese Patients with Hepatitis C Virus Infection

Purpose: Genetic polymorphisms of UDP-glucuronosyltransferase 1A7 (UGT1A7), which detoxifies endogenous and environmental carcinogens, have been reported to be associated with hepatocellular carcinoma (HCC) in German populations. On the other hand, we reported that interleukin-1β (IL-1β) gene polymorphisms were associated with hepatitis C virus (HCV)-related HCC. In this study, we evaluated the association of both genes with the risk of HCC in Japanese HCV-infected patients. Experimental Design: Genetic polymorphisms of UGT1A7 and IL-1β were investigated in 280 Japanese patients (122 with HCC and 158 without HCC) with chronic HCV infections, by use of standard PCR-based genotyping techniques. Results: We designated the UGT1A7*1 allele (a haplotype conferring higher activity) as H and the *2, *3, and *4 alleles (haplotypes conferring lower activity) as L. The proportions of UGT1A7 L/L and H/L alleles (genotypes) in patients with HCC (25% and 45%, respectively) were higher than those in patients without HCC (15% and 39%, respectively) with odds ratios of 2.73 (95% confidence interval, 1.40–5.35) and 1.80 (95% confidence interval, 1.05–3.09), respectively, compared with the UGT1A7 H/H alleles. Multivariate analyses revealed that UGT1A7 L/L and IL-1β/−31T/T−511C/C genotypes, the presence of cirrhosis, age >60 years, male sex, and α-fetoprotein >20 μg/ml were associated with the presence of HCC (odds ratios, 2.33, 2.67, 4.20, 3.12, 3.09, and 2.90, respectively). Conclusion: The UGT1A7 polymorphisms together with IL-1β were associated with the presence of HCC in Japanese HCV-infected patients.

Large‐scale search of single nucleotide polymorphisms for hepatocellular carcinoma susceptibility genes in patients with hepatitis C

Hepatitis C virus (HCV) infection is a major risk factor for developing hepatocellular carcinoma (HCC). The host genetic factors that are involved in the development of HCC in patients with HCV infection remain to be investigated. To search for single nucleotide polymorphisms (SNPs) in HCC susceptibility genes, 393 SNPs in 171 candidate genes were examined in 188 Japanese patients with chronic HCV infection, including 77 patients with HCC. HCC‐related SNPs were then examined in another 188 patients (including 93 patients with HCC) with chronic HCV infection. Haplotype analyses of HCC‐related genes were performed in a total of 376 patients. Of the 393 SNPs, 31 SNPs in 29 genes were significantly associated with HCC based on an initial screening (P < .05). Of these 31 SNPs, 3 SNPs of 3 genes (SCYB14, GFRA1, and CRHR2) were significantly associated with HCC in a secondary screening. Haplotype analyses of these 3 genes identified 2 haplotype blocks associated with HCC. In conclusion, these SNPs and haplotypes located in the SCBY14, CRHR2, and GFRA1 genes will be used as markers to identify a subgroup of Japanese patients with chronic HCV infection who are at high risk of developing HCC. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:846–853.)

Potential contribution of tumor suppressor p53 in the host defense against hepatitis C virus.

Infection by hepatitis C virus (HCV) usually results into chronic hepatitis that can ultimately lead to cirrhosis and hepatocellular carcinoma. Type 1 interferons (IFN‐α/β) constitute the primary cellular defense against viral infection including HCV. IFN binding to their receptors activates associated Jak1 and Tyk2 kinases, which ultimately leads to phosphorylation and assembly of a signal transducer and activator of transcription protein (STAT)1‐STAT2‐interferon regulatory factor (IRF)9 trimetric complex called interferon‐stimulated gene factor 3 that translocates into the nucleus and binds to the interferon‐ stimulated response elements (ISRE), leading to transcriptional induction of several antiviral genes, including double‐stranded RNA‐activated protein kinase (PKR), 2′,5′‐ oligoadenylate synthetase (OAS), and myxovirus resistance protein A (MxA). Understanding the mechanisms of how the virus evades this cellular innate defense and establishes a chronic infection is the key for the development of better therapeutics against HCV infection. Here, we demonstrate that p53 could have a crucial role in the cellular innate defense against HCV. We observed significantly higher levels of HCV RNA replication and viral protein expression in the Huh7 cells when their p53 expressions were knocked down. Moreover, IFN treatment was less effective in inhibiting the HCV RNA replication in the p53–knocked‐down (p53kd) Huh7 cells. In fact, the activation of the ISRE and the induction of ISGs were significantly attenuated in the p53kd Huh7 cells and p53 was found to directly interact with IRF9. Conclusion: These observations underscore the potential contributions of the tumor suppressor p53 in cellular antiviral immunity against HCV with possible therapeutic implications. (HEPATOLOGY 2008.)

Interferons Specifically Suppress the Translation from the Internal Ribosome Entry Site of Hepatitis C Virus through a Double-Stranded RNA–Activated Protein Kinase–Independent Pathway

Interferon (IFN) therapy is used worldwide as the best available treatment for hepatitis C virus (HCV) infection; however, little is known about how IFN or other drugs work against liver diseases. The effect of 6 drugs (glycyrrhizin, ursodeoxycholic acid, ribavirin, methylprednisolone, IFN-alpha, and IFN-beta) on HCV RNA translation from the HCV internal ribosome entry site (IRES) was investigated, using a bicistronic reporter containing the HCV IRES. IFNs suppressed both cap-dependent and HCV IRES-dependent translation, with HCV IRES-dependent translation being more significantly suppressed. In contrast to HCV IRES, IFN did not suppress either foot-and-mouth disease virus IRES-dependent or encephalomyocarditis virus IRES-dependent translation more than it suppressed cap-dependent translation. Moreover, dominant inhibition of HCV IRES-dependent over cap-dependent translation depended neither on the double-stranded RNA-activated protein kinase activation nor on La protein function. These results indicate a novel antiviral effect of IFNs against HCV.

Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A

Innate immunity is part of the antiviral response. Interferon (IFN)-beta plays a leading role in this system. To investigate the influence of hepatitis C virus (HCV) on innate immunity, we examined the effect of viral proteins on IFN-beta induction. HepG2 cells were co-transfected with plasmids for seven HCV proteins (core protein, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and the IFN-beta promoter luciferase. Toll-like receptor (TLR) 3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) play key roles in dsRNA-mediated activation of interferon regulatory factor (IRF)-3 and IFN-beta; therefore, the participation of TLR3/TRIF in NS5B-mediated IFN induction was examined. Among seven HCV proteins, only NS5B, a viral RNA-dependent RNA polymerase (RdRp), activated the IFN-beta promoter. However, mutant NS5B without RdRp activity or template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5B required the positive regulatory domain III, a binding sequence for IRF-3. Moreover, IRF-3 was phosphorylated by NS5B. Both inhibition of TLR3 expression by small interfering RNA and expression of the dominant negative form of TRIF significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA. Meanwhile, NS4A, NS4B, and NS5A block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.

Clinical features associated with radiological response to sorafenib in unresectable hepatocellular carcinoma: a large multicenter study in Japan

There have been no established predictive factors of responders to sorafenib in patients with unresectable hepatocellular carcinoma (HCC). This study aimed to investigate the factors predicting a good response to sorafenib in Japanese patients with HCC.

Hepatitis C Virus Core Protein and Hepatitis Activity Are Associated through Transactivation of Interleukin-8

BACKGROUND We evaluated the association between variations in hepatitis C virus (HCV) core protein and hepatitis severity in patients with chronic HCV infection who achieved remission without viral eradication and had a biochemical response to interferon (IFN) therapy, to evaluate the effect of HCV core sequence in the absence of the influence of host factors. METHODS. Using serum from 10 patients with a biochemical response and 10 patients with no response, we measured serum levels of interleukin (IL)-1 beta , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN- gamma , and tumor necrosis factor- alpha before and after IFN therapy. Expression vectors with the core region were transfected into Huh7 cells, and cytokine induction was evaluated by reporter assay. RESULTS In biochemical responders, only IL-8 levels decreased after IFN therapy (P=.04). Changes in the C-terminal hydrophobic region were observed more frequently in biochemical responders. Activation of the IL-8 promoter by HCV core protein was significantly decreased in biochemical responders after IFN therapy (P=.04). When 69 C-terminal amino acids from before IFN therapy were replaced with those from after IFN therapy in 3 biochemical responders, their ability to transactivate IL-8 decreased. CONCLUSIONS Differences in amino acids in the HCV core protein correlates with hepatitis activity through the modulation of IL-8 induction in HCV-infected patients.

Sorafenib Therapy for BCLC Stage B/C Hepatocellular Carcinoma; Clinical Outcome and Safety in Aged Patients: A Multicenter Study in Japan

Background and aims: We aimed to compare clinical outcomes and safety after sorafenib therapy between patients with Barcelona Clinic Liver Cancer (BCLC) stage B or C hepatocellular carcinoma (HCC) aged ≥75 years (aged group, n=179) and those with BCLC stage B or C HCC aged <75 years (control group, n=279). Patients and methods: We compared overall survival (OS), progression free survival (PFS), best treatment response and sorafenib related serious adverse events (SAEs) of grade 3 or more in the two groups. Furthermore, for reducing the selection bias, we compared clinical outcome of these two groups using propensity score matching analysis. Results: The median OS and PFS intervals were 9.7 and 3.8 months in the aged group and 8.2 and 3.3 months in the control group (P=0.641 for OS and P=0.068 for PFS). Disease control rates were 49.2% (88/179) in the aged group and 49.1% (137/279) in the control group (P>0.999). Objective response rates were 15.1% (27/179) in the aged group and 14.3% (40/279) in the control group (P=0.892). Treatment related SAEs of grade 3 or more were observed in 51 patients (28.5%) in the aged group and in 69 patients (24.7%) in the control group (P=0.385). In the propensity score matched cohort (132 pairs), no significant difference in the two groups was observed in terms of OS (P=0.898) and PFS (P=0.407). Conclusion: In BCLC stage B or C HCC patients treated with sorafenib, life expectancy, disease progression, treatment efficacy and SAEs are unaffected by age over 75 years.