Soji Shimomura

Involvement of hepatoma-derived growth factor in the growth inhibition of hepatocellular carcinoma cells by vitamin K2

BackgroundVitamin K2 has been reported to suppress the growth of human hepatocellular carcinoma (HCC) in vitro and hepatocarcinogenesis in hepatitis C virus (HCV)-related cirrhosis in vivo. Hepatoma-derived growth factor (HDGF) is a unique nuclear targeting growth factor that is highly expressed in HCC cells and is a possible prognostic factor for patients with HCC. We investigated the regulation of HDGF expression by vitamin K2.MethodsThree HCC-derived cell lines, HepG2, HuH-7, and SK-Hep-1, were used. Cell number was determined with the MTT assay. The expression levels of HDGF mRNA and protein were measured by the real-time reverse transcriptase-polymerase chain reaction (PCR) method and ELISA and Western blot analysis, respectively. The HDGF promoter activity was measured by a dual luciferase-reporter assay.ResultsVitamin K2 suppressed the growth of the three HCC cell lines in a dose-dependent manner. Vitamin K2 significantly suppressed the expression of the HDGF protein and mRNA in three cell lines. By a luciferase assay, vitamin K2 significantly suppressed the promoter activity of the HDGF protein. Based on some luciferase-reporter plasmids containing truncated promoter regions, the possible responsive site of vitamin K2 seems to reside in the region −1 to −150 bp of the HDGF gene.ConclusionsThese findings suggested that regulation of the HDGF gene expression is one of the crucial mechanisms of vitamin K2-induced cell growth suppression for HCC.

Development of a new in situ hybridization method for the detection of global bacterial DNA to provide early evidence of a bacterial infection in spontaneous bacterial peritonitis

BACKGROUND & AIMS Despite the importance of identifying the causative pathogen(s), ascitic fluid cultures are occasionally negative in patients with spontaneous bacterial peritonitis (SBP). A novel strategy using the in situ hybridization (ISH) method was introduced to detect the bacterial genomic DNA phagocytized in the blood of patients with sepsis. In the present study, we developed a new ISH probe to detect global bacterial DNA (named as GB probe) and evaluated its utility for detecting the phagocytized bacterial DNA in SBP ascites. METHODS Hybridization of bacterial DNA with the GB probe was examined by dot-blot and ISH tests. In addition, the utility of the ISH method to detect the bacterial DNA in the leukocytes of SBP ascites was evaluated. RESULTS The GB probe hybridized with the genomic DNA of all 59 bacterial strains tested (59 species of 36 genus). Eleven of 51 patients with ascites (out of total 542 cirrhotic inpatients) were categorized as SBP. The ISH tests showed positive results in 10 of 11 SBP cases. However, the ISH tests all showed negative results in the 40 non-SBP ascitic samples. Therefore, the ISH tests yielded highly sensitive and specific results for detecting the phagocytized bacterial DNA in the leukocytes of SBP ascites. Moreover, all of the ISH test results were obtained within only one day. CONCLUSIONS Our newly established ISH method was found to provide both a rapid and sensitive detection of bacterial DNA in SBP ascites, thus suggesting its utility for providing early and direct evidence of bacterial infection in SBP ascites.

Anti‐interferon‐α neutralizing antibody is associated with nonresponse to pegylated interferon‐α plus ribavirin in chronic hepatitis C

Summary.  Pegylated interferon‐α (PEG‐IFN‐α) plus ribavirin (RBV) treatment fails to achieve a sustained virological response (SVR) in approximately 20–50% of patients with chronic hepatitis C virus (HCV) infection. We assessed the contribution of an anti‐IFN‐α neutralizing antibody (NAb) on the nonresponse to treatment. NAbs were detected using an antiviral assay that assessed the neutralizing effects of serum samples against IFN. Serum samples were obtained at the end of the treatment and evaluated for the presence of NAbs using recombinant IFN‐α as a standard. We studied 129 PEG‐IFN‐α/RBV‐treated patients. In the 82 end‐of‐treatment responders, no NAbs were detected. Of the 47 patients who did not respond, seven (15%) were positive for NAbs. We also examined an additional 83 patients who had not responded to PEG‐IFN‐α treatment, and detected 12 with NAbs. Patients with good IFN‐responsive characteristics, including HCV genotype 2/3 and major allele homozygotes for interleukin‐28B, were included in the 19 patients with NAbs. No NAbs interfered with the antiviral activity of natural human IFN‐β (nIFN‐β) and re‐treatement of patients with NAbs with nIFN‐β/RBV achieved SVR. Our analyses revealed that the emergence of anti‐IFN‐α NAbs was a candidate causal factor of PEG‐IFN‐α‐treatment failure. Therefore, these antibodies should be assayed in patients who do not respond to PEG‐IFN‐α therapy, and if detected, other effective treatments, i.e., medications that are not neutralized by anti‐IFN‐α NAbs, should be considered.

Elevation of the glycated albumin to glycated hemoglobin ratio during the progression of hepatitis C virus related liver fibrosis

AIM To analyze the relationship between the glycated albumin (GA) to glycated hemoglobin (HbA1c) ratio and the histological grading of liver fibrosis. METHODS The study retrospectively included consecutive hepatitis C virus positive chronic liver disease patients (n = 142) who had undergone percutaneous liver biopsy between January 2008 and March 2010 at our institution. The ratios of GA/HbA1c were calculated in all patients to investigate the relationship with the degree of the liver fibrosis. The values of the aspartate aminotransferase-to-platelet ratio index (APRI), an excellent marker for the evaluation of liver fibrosis, were also calculated. In addition, we combined the ratio of GA/HbA1c and the APRI in order to improve our ability to detect the presence of significant liver fibrosis. RESULTS Sixty-one (43%) patients had either no fibrosis or minimal fibrosis (METAVIR score: F0-F1), while 25 (17%) had intermediate fibrosis (F2). Fifty-six (39%) patients had severe fibrosis (F3-F4) and 27 of them had cirrhosis (F4). The mean values of the GA/HbA1c increased with the progression of the fibrosis (F0-1: 2.83 ± 0.24, F2: 2.85 ± 0.24, F3: 2.92 ± 0.35, F4: 3.14 ± 0.54). There was a significant difference between the F0-F1 vs F4, F2 vs F4, and F3 vs F4 groups (P < 0.01, P < 0.01, P < 0.01 and P < 0.05, respectively). The GA/HbA1c ratio was significantly higher in the patients with cirrhosis (F4) than in those without cirrhosis (F0-F3) (3.14 ± 0.54 vs 2.85 ± 0.28, P < 0.0001). The GA/HbA1c ratio was also significantly higher in the patients with severe fibrosis (F3-F4) than in those without severe liver fibrosis (F0-F2) (3.03 ± 0.41 vs 2.84 ± 0.24, P < 0.001). Furthermore, the GA/HbA1c ratio was also significantly higher in the patients with significant fibrosis (F2-F4) than in those without significant liver fibrosis (F0-F1) (2.98 ± 0.41 vs 2.83 ± 0.24, P < 0.001). The diagnostic performance of the increased GA/HbA1c ratio (> 3.0) was as follows: its sensitivity and specificity for the detection of liver cirrhosis (F4) were 59.3% and 70.4%, respectively and its sensitivity and specificity for the detection of severe liver fibrosis (F3-F4) were 50.0% and 74.4%, respectively. With regard to the detection of significant fibrosis (F2-F4), its sensitivity was 44.4% and its specificity was 77.0%. Although even the excellent marker APRI shows low sensitivity (25.9%) for distinguishing patients with or without significant fibrosis, the combination of the APRI and GA/HbA1c ratio increased the sensitivity up to 42.0%, with only a modest decrease in the specificity (from 90.2% to 83.6%). CONCLUSION The GA/HbA1c ratio increased in line with the histological severity of liver fibrosis, thus suggesting that this ratio is useful as a supportive index of liver fibrosis.

Vitamin K2 inhibits the proliferation of HepG2 cells by up‐regulating the transcription of p21 gene

Aim:  Vitamin K2 has been reported to inhibit the growth of human hepatocellular carcinoma (HCC) in vitro and suppress hepatocarcinogenesis in vivo. However, its inhibitory mechanism has not yet been clarified.

Hepatoma‐derived growth factor is induced in liver regeneration

Aim:  Hepatoma‐derived growth factor (HDGF) is a heparin‐binding protein, which has been suggested to be involved in the development of kidneys, the cardiovascular system and the liver. We have shown that HDGF is highly expressed in parenchymal hepatocytes in the developing liver and promotes fetal hepatocyte proliferation. In the present study, we asked whether HDGF expression was related to liver regeneration.

Chemoprevention of spontaneous development of hepatocellular carcinomas in fatty liver Shionogi mice by a cyclooxygenase‐2 inhibitor

Cyclooxygenase 2 (COX‐2) and retinoid X receptor α (RXRα) are suggested to have roles in carcinogenesis. COX‐2 inhibitors have been reported to suppress growth of hepatocellular carcinoma (HCC) cell lines in vitro. However, little is known about the preventive effect of these drugs on spontaneous hepatocarcinogenesis in vivo. Etodolac exists in a racemic mixture containing S‐ and R‐etodolac. S‐etodolac is responsible for COX‐2 inhibitory activity and R‐etodolac is related to the downregulation of RXRα. Here, the effect of etodolac on spontaneous development of HCC in fatty liver Shionogi mice is evaluated. Etodolac was administered at a low (2 mg/kg) or high (10 mg/kg) dose three times a week for 16 months starting at the age of 3 months. The development of HCC was suppressed slightly in the high‐dose group, and suppressed markedly in the low‐dose group, although the development of fatty liver was not inhibited in either group. Plasma prostaglandin E2 levels were also decreased significantly in the low‐dose group, consistent with the suppression of HCC. The expression of RXRα and proliferating cell nuclear antigen in non‐tumorous liver tissues was decreased significantly in both the low‐dose and high‐dose groups. These findings show that etodolac treatment at an optimum dose suppresses hepatocarcinogenesis in vivo, and may be useful for preventing the development of HCC in humans. (Cancer Sci 2006; 97: 768–773)

Investigation of TTV by in situ hybridization in patients with chronic hepatitis

To clarify whether TT virus (TTV) was present in liver tissues, 12 liver tissue samples from patients with chronic hepatitis positive for TTV in their serum and 11 samples from serum-negative patients were obtained by needle biopsies and investigated using in situ hybridization. Positive staining was observed in nine (75%) of 12 cases positive for TTV (serum-positive group) and three (27.3%) of 11 cases negative for TTV (serum-negative group) (P=0.061). Three kinds of staining patterns were observed: nuclear, cytoplasmic and both. In 58.3% (7/12) of the patients positive for TTV staining, the stained areas were found in both the nucleus and cytoplasm. Only cytoplasmic staining was observed in three cases from the serum-positive group. Only nuclear staining was observed in two cases from the serum-negative group. No significant differences were found in the clinical background between the in situ hybridization-positive and -negative groups, and between the serum-positive and -negative groups. The present study shows that TTV exists in the liver tissue, especially in hepatocytes, of chronic hepatitis patients and that the localization of TTV in the cell is different from case to case, although why this is so remains to be clarified.

Anticarcinogenic impact of interferon therapy on the progression of hepatocellular carcinoma in patients with chronic viral infection

Hepatocellular carcinoma (HCC) is mainly caused by a persistent infection due to the hepatitis B or hepatitis C virus. The number of HCC cases is increasing in Asian and African countries, as well as in European and American countries. Interferon (IFN) therapy, used for type B chronic liver diseases, inhibits hepatic carcinogenesis in patients with compensated cirrhosis. However, there is insufficient evidence that IFN therapy inhibits hepatic carcinogenesis in patients with chronic hepatitis B. There are few cases of HCC due to chronic hepatitis B, and long‐term follow‐up periods verifying the inhibitory effect of IFN on hepatic carcinogenesis have not been obtained. To improve the prognosis of type B chronic liver diseases, it is important that hepatitis treatment follows guidelines in which a patients age and the extent of hepatic fibrosis are taken into account. As for chronic hepatitis C, since a sustained virological response (SVR) in IFN therapy inhibits hepatic carcinogenesis and improves prognosis, treatment that aims for an SVR while taking into consideration host‐sided and virus‐sided factors is recommended for patients with type C chronic liver diseases. In areas with low incidence of HCC (e.g. USA), a large number of cases and a long‐term follow‐up period are needed before it can be accepted that IFN therapy inhibits hepatic carcinogenesis. After locally curative treatment of HCC, IFN therapy suppresses recurrence and improves survival rates.

Aspartate aminotransferase-linked immunoglobulin complexes in serum of a patient with primary biliary cirrhosis

A 51-year-old woman who had been treated for primary biliary cirrhosis (PBC) was admitted to our hospital for evaluation of unexplained, isolated, persistently increased aspartate aminotransferase (AST) activity. Results of laboratory tests on admission showed: AST 171 KU, alanine aminotransferase 28 KU, and anti-mitochondrial titer 1/1280. Results of hepatitis B surface antigen (HBs Ag) and hepatitis C virus antibody (HCV Ab; C100-3) assays were negative. Histology of a liver biopsy specimen was compatible with a diagnosis of PBC (stage III of Scheuers classificiation). The molecular size of serum AST was estimated to be more than 500 000 by high-performance size-exclusion liquid chromatography. Electrophoretic analysis showed an abnormal band of AST between supernatant AST (sAST) and mitochondrial AST (mAST), which band was characteristic of AST-immunoglobulin complexes (AST-Ig). Ouchterlony double-diffusion and immunoprecipitation tests identified the immunoglobulin component as IgM. The presence of AST-Ig appeared to be responsible for the elevated serum AST.