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Yoichiro Takami,
Hirofumi Uto,
Tsutomu Tamai,
Yuko Sato,
Yo-Ichi Ishida,
Hiroyuki Morinaga,
Yoichi Sakakibara,
Akihiro Moriuchi,
Makoto Oketani,
Akio Ido, Tomoaki Nakajima,
Takeshi Okanoue,
Hirohito Tsubouchi
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ABSTRACT: Aim: Oxidative stress is involved in the progression of non-alcoholic steatohepatitis (NASH). However, there are few biomarkers that are easily measured and accurately reflect the disease states. The aim of this study was to identify novel oxidative stress markers using the 2-nitrobenzenesulfenyl (NBS) stable isotope labeling method and to examine the clinical utility of these diagnostic markers for NASH. Methods: Proteins extracted from phosphate buffered saline- and hydrogen peroxide-loaded human primary hepatocyte were labeled with the [(12)C]- and [(13)C]-NBS reagents, respectively. Pairs of peaks with 6-Da differences in which the [(13)C]-NBS labeling was more intense than the [(12)C]-NBS labeling were detected by MALDI-TOF/MS and identified by MS/MS ion searching. Results: Four pairs of peaks, m/z 1705-1711, m/z 1783-1789, m/z 1902-1908 and m/z 2790-2796, were identified as cytochrome c oxidase VIb (COX6B), liver carboxylesterase 1 (CES1), carbamoyl-phosphate synthase 1 (CPS1) and superoxide dismutase (MnSOD), respectively. Furthermore, serum MnSOD protein levels were significantly higher in NASH patients than in simple steatosis (SS) patients. The serum MnSOD levels tended to increase in parallel with the stage of fibrosis. Conclusion: The NBS labeling technique was useful to identify biomarkers. Serum MnSOD may be a useful biomarker that can distinguish between SS and NASH.
Hepatology Research 03/2010; 40(4):438-45. · 2.20 Impact Factor
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Mio Endo,
Kohichiroh Yasui, Tomoaki Nakajima,
Yasuyuki Gen,
Kazuhiro Tsuji,
Osamu Dohi,
Keika Zen,
Hironori Mitsuyoshi,
Masahito Minami,
Yoshito Itoh,
Masafumi Taniwaki,
Shinji Tanaka,
Shigeki Arii,
Takeshi Okanoue,
Toshikazu Yoshikawa
[show abstract]
[hide abstract]
ABSTRACT: To determine whether JUN (the oncogene encoding c-Jun protein) is amplified and overexpressed in hepatocellular carcinoma (HCC).
DNA copy number aberrations were investigated using a high-density oligonucleotide microarray. DNA copy numbers were determined by fluorescence in situ hybridization. Genomic DNA and mRNA were quantified using real-time quantitative PCR.
A novel amplification was found at the chromosomal region 1p32-31 in a JHH-2 HCC cell line within which JUN is amplified and overexpressed. However, no copy number gain of JUN (>2-fold) was observed in 34 primary HCC tumors. Rather, a loss of JUN (<0.5-fold) was seen in 13 (38%) out of the 34 tumors and expression of JUN was significantly lower in 26 (70%) out of the 37 HCC tumors compared with their nontumorous counterparts.
Although JUN was amplified and overexpressed in JHH-2 HCC cells, amplification and overexpression of JUN may be rare in primary HCCs.
Anticancer research 12/2009; 29(12):4989-94. · 1.73 Impact Factor
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Kohichiroh Yasui,
Yuichi Harano,
Hironori Mitsuyoshi,
Kazuhiro Tsuji,
Mio Endo, Tomoaki Nakajima,
Masahito Minami,
Yoshito Itoh,
Yoh Zen,
Yasuni Nakanuma,
Toshikazu Yoshikawa,
Takeshi Okanoue
[show abstract]
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ABSTRACT: Steatosis is a histological finding associated with the progression of chronic hepatitis C. The aims of this study were to elucidate risk factors associated with steatosis and to evaluate the association between steatosis and hepatic expression of genes regulating lipid metabolism.
We analyzed 297 Japanese patients infected with hepatitis C virus and a subgroup of 100 patients who lack metabolic factors for steatosis. We determined intrahepatic mRNA levels of 18 genes regulating lipid metabolism in these 100 patients using real-time reverse transcription-polymerase chain reaction. Levels of peroxisome proliferator-activated receptor alpha and sterol regulatory element-binding protein 1 proteins were assessed by immunohistochemistry.
Steatosis was present in 171 (57%) of 297 patients. The presence of steatosis was independently associated with a higher body mass index, higher levels of gamma-glutamyl transpeptidase and triglyceride, and a higher fibrosis stage. Steatosis was present in 43 (43%) of 100 patients lacking metabolic factors. Levels of mRNA and protein of peroxisome proliferator-activated receptor alpha, which regulates beta-oxidation of fatty acid, were lower in patients with steatosis than in patients without steatosis.
These findings indicate that impaired degradation of lipid may contribute to the development of hepatitis C virus-related steatosis.
Journal of Gastroenterology 09/2009; 45(1):95-104. · 4.16 Impact Factor
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Masami Miyagawa,
Masahito Minami,
Kota Fujii,
Rei Sendo,
Kojiro Mori,
Daisuke Shimizu, Tomoaki Nakajima,
Kohichiroh Yasui,
Yoshito Itoh,
Masafumi Taniwaki,
Takeshi Okanoue,
Toshikazu Yoshikawa
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ABSTRACT: Hepatitis B virus (HBV) reactivation in hepatitis B surface antigen (HBsAg)-negative patients following treatment with rituximab has been reported increasingly. The aim of this study was to investigate the molecular mechanisms underlying HBV reactivation in an HBsAg-negative patient. HBV was reactivated in a 75-year-old man following chemotherapy with rituximab, without elevation of HBsAg. The patient's full-length HBV genome was cloned and the entire sequence was determined. Transfection studies were performed in vitro using recombinant wild-type HBV (wild-type), the patient's HBV (patient), and two chimeric HBV constructs, in which the preS/S region of the patient and wild-type virus had been exchanged with one another. Secreted HBsAg and intra- and extra-cellular HBV DNA were measured. The number of amino acid substitutions in HBV from this patient was much higher than in previous reports of HBV mutants, such as occult HBV and vaccine escape HBV mutants. Levels of HBsAg and HBV DNA production in vitro were significantly lower in the patient compared to wild-type transfections. From analyses of the chimeric constructs, the altered preS/S region was responsible mainly for this impairment. These results show that highly mutated HBV can reactivate after chemotherapy with rituximab, despite an unusually large number of mutations, resulting in impaired viral replication in vitro. Severe immune suppression, probably caused by rituximab, may permit reactivation of highly mutated HBV. These findings have important clinical implications for the prevention and management of HBV reactivation and may explain partially the mechanism of recent, unusual cases of HBV reactivation.
Journal of Medical Virology 01/2009; 80(12):2069-78. · 2.82 Impact Factor
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Yasuyuki Gen,
Kohichiroh Yasui,
Keika Zen, Tomoaki Nakajima,
Kazuhiro Tsuji,
Mio Endo,
Hironori Mitsuyoshi,
Masahito Minami,
Yoshito Itoh,
Shinji Tanaka,
Masafumi Taniwaki,
Shigeki Arii,
Takeshi Okanoue,
Toshikazu Yoshikawa
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ABSTRACT: RhoA, a member of the Rho family of small GTPases, directs the organization of the actin cytoskeleton and is involved in regulating cell shape and movement. Its activity is negatively regulated by p190-B RhoGAP (GTPase-activating protein). We investigated DNA copy number aberrations in human hepatocellular carcinoma and esophageal squamous cell carcinoma cell lines using a high-density oligonucleotide microarray and found a novel amplification at chromosomal region 14q12. We identified ARHGAP5 (the gene encoding p190-B RhoGAP) as a probable target for the amplification at 14q12, and our results showed that p190-B RhoGAP promotes cells spreading and migration by negatively regulating RhoA activity in Huh-7 hepatocellular carcinoma cells.
Cancer letters 12/2008; 275(1):27-34. · 4.86 Impact Factor
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Keika Zen,
Kohichiroh Yasui, Tomoaki Nakajima,
Yoh Zen,
Kan Zen,
Yasuyuki Gen,
Hironori Mitsuyoshi,
Masahito Minami,
Shoji Mitsufuji,
Shinji Tanaka,
Yoshito Itoh,
Yasuni Nakanuma,
Masafumi Taniwaki,
Shigeki Arii,
Takeshi Okanoue,
Toshikazu Yoshikawa
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[hide abstract]
ABSTRACT: Using high-density oligonucleotide microarrays, we investigated DNA copy-number aberrations in cell lines derived from hepatocellular carcinomas (HCCs) and detected a novel amplification at 17p11. To identify the target of amplification at 17p11, we defined the extent of the amplicon and examined HCC cell lines for expression of all seven genes in the 750-kb commonly amplified region. Mitogen-activated protein kinase (MAPK) 7, which encodes extracellular-regulated protein kinase (ERK) 5, was overexpressed in cell lines in which the gene was amplified. An increase in MAPK7 copy number was detected in 35 of 66 primary HCC tumors. Downregulation of MAPK7 by small interfering RNA suppressed the growth of SNU449 cells, the HCC cell line with the greatest amplification and overexpression of MAPK7. ERK5, phosphorylated during the G2/M phases of the cell cycle, regulated entry into mitosis in SNU449 cells. In conclusion, our results suggest that MAPK7 is likely the target of 17p11 amplification and that the ERK5 protein product of MAPK7 promotes the growth of HCC cells by regulating mitotic entry.
Genes Chromosomes and Cancer 11/2008; 48(2):109-20. · 3.31 Impact Factor
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ABSTRACT: Aim: 2',5' oligoadenylate synthetase (2-5AS), an enzyme induced by interferon, is an accurate indicator of the antiviral effect of interferon. We measured it during pegylated interferon based therapies in patients with chronic hepatitis C virus (HCV) in order to determine the dynamics of antiviral status in vivo and the relationship between the response to exogenous interferon and the outcome of therapy. Methods: A total of 160 patients with chronic HCV were treated with pegylated interferon alfa 2a or 2b or non-pegylated interferon, with or without ribavirin. Serum 2-5AS activity was measured by radioimmunoassay assay kits every 2 weeks. Results: In 60 patients treated with pegylated interferon alfa 2a or 2b, 2-5AS levels increased to 7-40 times (average 235 pmol/dL) above the pretreatment levels (30 pmol/dL), which were significantly higher than the levels during non-pegylated interferon therapy. Ribavirin did not enhance 2-5AS levels. 2-5AS levels between sustained virological response (SVR) and non-SVR, including null responders to pegylated interferon plus ribavirin therapy were not significantly different. Conclusion: 2-5AS levels were significantly higher in patients treated with pegylated interferon than in those treated with non-pegylated interferon, suggesting that pegylated interferon is more potent at inducing interferon response genes resulting in an improved antiviral effect. Ribavirin did not appear to be related to interferon response gene induction.
Hepatology Research 09/2008; 38(12):1213-20. · 2.20 Impact Factor
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Tomoaki Nakajima,
Kohichiroh Yasui,
Keika Zen,
Yoshikazu Inagaki,
Hideki Fujii,
Masahito Minami,
Shinji Tanaka,
Masafumi Taniwaki,
Yoshito Itoh,
Shigeki Arii,
Johji Inazawa,
Takeshi Okanoue
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ABSTRACT: Aim: Deregulation of E2F1 transcriptional activity is observed in a variety of cancers, including hepatocellular carcinoma (HCC). The aim of the present study is to identify transcriptional target genes of E2F1 in HCC. Methods: We determined expression levels for E2F1 and ten candidate genes thought to be targets of E2F1 in primary HCCs using a real-time quantitative reverse transcription-PCR assay. Following small interfering RNA (siRNA)-mediated knockdown of E2F1 in HCC cell lines, we quantified mRNA levels of the candidate E2F1 target genes. Results: E2F1 was significantly over-expressed in 41 primary HCCs as compared to non-tumorous liver tissues. Among the candidates, MYBL2, whose product is the transcriptional factor B-Myb, which is involved in controlling cell-cycle progression and apoptosis, was significantly over-expressed in primary HCCs. Additionally, expression levels of MYBL2 correlated with those of E2F1. Knockdown of E2F1 resulted in a decrease in expression of MYBL2. A copy-number gain for MYBL2 was observed in 36 of 66 primary HCCs, suggesting that MYBL2 expression is up-regulated by amplification in addition to being regulated by E2F1. Moreover, siRNA-mediated knockdown of MYBL2 led to reduced expression of CDC2 (which encodes CDC2), cyclin A2 (CCNA2), and topoisomerase II alpha (TOP2A), implicating these genes in the cell cycle and suggesting that they may be downstream targets of B-Myb. Conclusion: MYBL2 is a probable transcriptional target of E2F1 in HCC and may therefore be a useful biomarker for diagnosis and an attractive target for molecular therapies useful to treat HCC.
Hepatology Research 07/2008; 38(9):886-95. · 2.20 Impact Factor
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Yoshikazu Inagaki,
Kohichiroh Yasui,
Mio Endo, Tomoaki Nakajima,
Keika Zen,
Kazuhiro Tsuji,
Masahito Minami,
Shinji Tanaka,
Masafumi Taniwaki,
Yoshito Itoh,
Shigeki Arii,
Takeshi Okanoue
[show abstract]
[hide abstract]
ABSTRACT: High-density single nucleotide polymorphism (SNP) array analysis revealed novel amplification at 1q21 in cell lines derived from hepatocellular carcinomas (HCCs). Fluorescence in situ hybridization and real-time quantitative polymerase chain reaction studies verified amplification at 1q21. An increase in copy number at the region was detected in 32 of the 36 primary HCC tumors (89%). To identify the targets for amplification, we examined 19 HCC cell lines for expression levels of all 26 genes located within the 700-kb amplified region. Five genes were overexpressed in cell lines with amplification at 1q21. Among these, CREB3L4 (cAMP responsive element binding protein 3-like 4), INTS3 (integrator complex subunit 3), and SNAPAP (SNAP-associated protein) were significantly overexpressed in tumors from 18 HCC patients, compared with counterpart nontumorous tissues. The findings suggest that CREB3L4, INTS3, and SNAPAP are probable targets for the amplification mechanism and may therefore be involved, together or separately, in the development or progression of HCCs.
Cancer Genetics and Cytogenetics 02/2008; 180(1):30-6. · 1.39 Impact Factor
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Masahide Hamaguchi,
Takao Kojima,
Yoshito Itoh,
Yuichi Harano,
Kota Fujii, Tomoaki Nakajima,
Takahiro Kato,
Noriyuki Takeda,
Junichi Okuda,
Kazunori Ida,
Yutaka Kawahito,
Toshikazu Yoshikawa,
Takeshi Okanoue
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ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) is closely associated with the metabolic syndrome.
We evaluated the association among the metabolic syndrome, visceral fat accumulation, and the severity of fatty liver with a new scoring system of ultrasonographic findings in apparently healthy Japanese adults.
Subjects consisted of 94 patients who received liver biopsy and 4,826 participants who were selected from the general population. Two hepatologists scored the ultrasonographic findings from 0 to 6 points. We calculated Cohen's kappa of within-observer reliability and between-observer reliability. We evaluated the predictive value of the score by the area under a conventional receiver operating characteristic curve (AUC).
Within-observer reliability was 0.95 (95% CI 0.93-0.97, P<0.001) and between-observer reliability was 0.95 (95% CI 0.93-0.97, P<0.001). The AUC to diagnose NAFLD was 0.980. The sensitivity was 91.7% (95% CI 87.0-95.1, P<0.001) and the specificity was 100% (95% CI 95.4-100.0, P<0.001). The AUC to diagnose visceral obesity was 0.821. The sensitivity was 68.3% (95% CI 51.9-81.9, P=0.028) and the specificity was 95.1% (95% CI 86.3-99.0, P<0.001). Adjusted odds ratio of the score for the metabolic syndrome was 1.37 (95% CI 1.26-1.49, P<0.001).
The scoring system with abdominal ultrasonography could provide accurate information about hepatic steatosis, visceral obesity, and the metabolic syndrome in apparently healthy people who do not consume alcohol.
The American Journal of Gastroenterology 12/2007; 102(12):2708-15. · 7.28 Impact Factor
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Masahide Hamaguchi,
Takao Kojima,
Yoshito Itoh,
Yuichi Harano,
Kota Fujii, Tomoaki Nakajima,
Takahiro Kato,
Noriyuki Takeda,
Junichi Okuda,
Kazunori Ida,
Yutaka Kawahito,
Toshikazu Yoshikawa,
Takeshi Okanoue
The American Journal of Gastroenterology 11/2007; 102(12):2708-2715. · 7.28 Impact Factor
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Masahide Hamaguchi,
Takao Kojima,
Noriyuki Takeda,
Takayuki Nakagawa,
Hiroya Taniguchi,
Kota Fujii,
Tatsushi Omatsu, Tomoaki Nakajima,
Hiroshi Sarui,
Makoto Shimazaki,
Takahiro Kato,
Junichi Okuda,
Kazunori Ida
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ABSTRACT: The frequent association of nonalcoholic fatty liver disease with components of the metabolic syndrome such as obesity, hyperglycemia, dyslipidemia, and hypertension is well known. However, no prospective study has examined the role of the metabolic syndrome in the development of this disease.
To characterize the longitudinal relationship between the metabolic syndrome and nonalcoholic fatty liver disease.
A prospective observational study.
A medical health checkup program in a general hospital.
4401 apparently healthy Japanese men and women, 21 to 80 years of age, with a mean body mass index (BMI) of 22.6 kg/m2 (SD, 3.0).
Alcohol intake was assessed by using a questionnaire. Biochemical tests for liver and metabolic function and abdominal ultrasonography were done. Modified criteria of the National Cholesterol Education Program Adult Treatment Panel III were used to characterize the metabolic syndrome.
At baseline, 812 of 4401 (18%) participants had nonalcoholic fatty liver disease. During the mean follow-up period of 414 days (SD, 128), the authors observed 308 new cases (10%) of nonalcoholic fatty liver disease among 3147 participants who were disease-free at baseline and who completed a second examination. Regression of nonalcoholic fatty liver disease was found in 113 (16%) of 704 participants who had the disease at baseline and who completed a second examination. Men and women who met the criteria for the metabolic syndrome at baseline were more likely to develop the disease during follow-up (adjusted odds ratio, 4.00 [95% CI, 2.63 to 6.08] and 11.20 [CI, 4.85 to 25.87], respectively). Nonalcoholic fatty liver disease was less likely to regress in those participants with the metabolic syndrome at baseline.
Ultrasonography may lead to an incorrect diagnosis of nonalcoholic fatty liver disease in 10% to 30% of cases and cannot distinguish steatohepatitis from simple steatosis. Self-reported alcohol intake may cause bias. Because all of the participants were Japanese, generalizability to non-Japanese populations is uncertain.
The metabolic syndrome is a strong predictor of nonalcoholic fatty liver disease.
Annals of internal medicine 12/2005; 143(10):722-8. · 16.73 Impact Factor
