Masaru Moriyama

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.)

MDM2 Promoter SNP309 Is Associated with the Risk of Hepatocellular Carcinoma in Patients with Chronic Hepatitis C

Purpose: A single nucleotide polymorphism (SNP) in the promoter region of MDM2 gene, SNP309, has recently been shown to be associated with accelerated tumor formation in both hereditary and sporadic cancers in humans. However, the association of SNP309 with hepatocellular carcinoma is unknown. We evaluated the association of SNP309 with the risk of hepatocellular carcinoma development among Japanese patients with chronic hepatitis C virus infection. Experimental Design: We genotyped the SNP309 at the MDM2 promoter in 435 Japanese patients with chronic hepatitis C virus infection, including 187 patients with hepatocellular carcinoma and 48 healthy subjects, using a fluorogenic PCR. Presence of SNP was also confirmed by direct sequencing of the MDM2 promoter region. Results: The proportion of G/G genotype of the SNP309 in patients with hepatocellular carcinoma (33%) was significantly higher than that in patients without hepatocellular carcinoma (23%), with an odds ratio (95% confidence interval) of 2.28 (1.30-3.98). A multivariate analysis revealed that MDM2 SNP309 (G/G versus T/T), age >60 years, male gender, presence of cirrhosis, serum α-fetoprotein >20 μg/L, and serum albumin <3.2 g/dL were independently associated with the hepatocellular carcinoma development at odds ratio of 2.27, 2.46, 3.08, 4.15, 4.87, and 6.33, respectively. Conclusions: The MDM2 promoter SNP309 is associated with the presence of hepatocellular carcinoma in Japanese patients with chronic hepatitis C.

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.)

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.

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.

Absence of tyrosine kinase mutations in Japanese colorectal cancer patients

Tyrosine kinases, which are important regulators of intracellular signal-transduction pathways, have mutated forms that are often associated with oncogenesis and are attractive targets for therapeutic intervention. Recently, systematic mutational analyses of tyrosine kinases revealed that a minimum of 30% of colorectal cancer contain at least one mutation in the tyrosine kinases. To further explore these mutations, we examined all reported mutations of NTRK3, FES, KDR, EPHA3, NTRK2, JAK1, PDGFRA, EPHA7, EPHA8, ERBB4, FGFR1, MLK4 and GUCY2F genes in the 24 colorectal cancer cell lines. Unexpectedly, among 24 colorectal cancer cell lines, only two cell lines (LoVo and CaR1) harbored mutation C1408T (R470C) in MLK4 gene. The mutation rate was extremely low compared to that previously reported. Therefore, we analyzed mutations in 46 colorectal cancer samples resected from the same number of Japanese patients. Surprisingly, none of the 46 samples contained any of the mutations reported. Based on our study, we advise that a more comprehensive tyrosine kinase gene mutation assay is necessary in the future.

Identification of genes associated with sensitivity to 5-fluorouracil and cisplatin in hepatoma cells

Background: The prognosis of patients with advanced hepatoma is grim. Although chemotherapy is adapted to such patients, the efficacy is low and the outcome cannot be predicted before therapy. In this study, we aimed to identify genes associated with sensitivity to 5-fluorouracil and cisplatin, drugs widely used in treatment, using gene expression profiles. Methods: Gene expression was evaluated in eight human hepatoma cell lines using an in-house cDNA microarray including 2300 known genes. The 50% growth inhibitory concentrations (GI50) of 5-fluorouracil and cisplatin were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and designated as chemosensitivity. Genes with expression ratios associated with GI50 were selected using the permutation test. Results: For 5-fluorouracil and cisplatin, 21 and 40 genes, respectively, were selected. From among the genes associated with 5-fluorouracil and cisplatin, several encoding metabolic enzymes were selected. In addition, several genes involved in the cell cycle and transcription were identified. Conclusions: We identified genes that may be associated with sensitivity to 5-fluorouracil and cisplatin. A list of these genes may be useful to elucidate how these drugs work on human hepatoma.