Okayama University Medical SchoolActa Medica Okayama0386-300X6942015Local Recurrence and Complications after Percutaneous Radiofrequency Ablation of Hepatocellular Carcinoma : A Retrospective Cohort Study Focused on Tumor Location219226ENJunichiToshimoriKazuhiroNousoShinichiroNakamuraNozomuWadaYukiMorimotoYasutoTakeuchiTetsuyaYasunakaKenjiKuwakiHidekiOhnishiFusaoIkedaHidenoriShirahaAkinobuTakakiKazuhideYamamotoOriginal Article10.18926/AMO/53558We conducted a retrospective cohort study to investigate the predisposing factors for local recurrence and complications after percutaneous radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC). HCC patients (n＝397) consecutively treated with RFA (256 males, 141 females, median age 69 years) were enrolled. In these patients, 1,455 nodules (median size 17mm) were ablated. Predisposing factors for overall recurrence and local recurrence in the context of tumor location and complications were examined. Local recurrence was observed for 113 of the 1,455 nodules. The 1-, 3- and 5-year local recurrence rates were 2.2ｵ, 7.4ｵ and 9.5ｵ, respectively. A multivariate Cox proportional hazard analysis revealed that large tumor size (＞2cm), tumor location (adjacent to the major portal branch or hepatic vein), and small ablated margin (＜3mm) were independent predisposing factors for local recurrence after RFA (HR＝1.70-2.81). Tumor location (adjacent to the major portal branch, hepatic vein, or diaphragm) was also revealed as a risk factor for liver damage due to RFA. HCC adjacent to the major portal vein or hepatic vein was associated with a higher risk for local recurrence and for complications;therefore, special precautions are necessary when applying RFA to HCC near vessels even when the tumors are located at an easy-to-puncture site.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0944-117445112010Laparoscopic findings of reddish markings predict hepatocellular carcinoma in patients with hepatitis B virus-related liver disease11721182ENBonShojiFusaoIkedaShin-ichiFujiokaHaruhikoKobashiTetsuyaYasunakaYasuhiroMiyakeHidenoriShirahaAkinobuTakakiKazuhiroNousoYoshiakiIwasakiKazuhideYamamotoFor patients with chronic hepatitis due to hepatitis B virus (HBV), factors predicting hepatocellular carcinoma (HCC) other than high levels of HBV-DNA and alanine aminotransferase (ALT) are needed to prevent HCC development, as many patients with chronic HBV infection fulfill these conditions. The purpose of this study was to clarify factors predictive of HCC development for those patients.
The study was a systematic cohort analysis of 303 consecutive patients with hepatitis B e-antigen, receiving laparoscopic examination for assessment of liver disease. Laparoscopic, histological, and clinical characteristics were investigated as related to HCC development.
HCC occurred in 27 patients during a mean follow-up of 8.0 +/- A 5.0 years, at the age of 37-72 years. Significant associations with HCC development were shown for liver cirrhosis, histological activity grade, reddish markings, and older age. Multivariate analysis revealed that HCC development was strongly associated with older age and male gender (P = 0.002 and P = 0.043, respectively). HCC occurred more frequently in patients of age a parts per thousand yen30 years even with early stage than in patients of age < 30 years (P = 0.031). Severe reddish markings, a laparoscopic finding of widespread parenchymal destruction, were highly associated with HCC development in patients of age a parts per thousand yen30 years at diagnosis (odds ratio = 1.67, P = 0.034), while histological activity grade and ALT level were not (P = 0.075 and P = 0.69, respectively).
HCC development is associated with older age, male gender, and liver cirrhosis. Reddish markings, rather than histological activity or ALT level, can be useful to predict HCC for HBV patients of age a parts per thousand yen30 years.No potential conflict of interest relevant to this article was reported.Public Library ScienceActa Medica Okayama1932-6203972014L-Carnitine Prevents Progression of Non-Alcoholic Steatohepatitis in a Mouse Model with Upregulation of Mitochondrial PathwayENHisashiIshikawaAkinobuTakakiRyuichiroTsuzakiTetsuyaYasunakaKazukoKoikeYasuyukiShimomuraHiroyukiSekiHiroshiMatsushitaYasuhiroMiyakeFusaoIkedaHidenoriShirahaKazuhiroNousoKazuhideYamamotoNon-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease characterized by lobular inflammation, hepatocellular ballooning, and fibrosis with an inherent risk for progression to cirrhosis and hepatocellular carcinoma (HCC). Mitochondrial dysfunction appears to play a role in the progression from simple steatosis to NASH. L-carnitine (L-b-hydroxy-g-N-trimethylaminobutyric acid), an essential nutrient that converts fat into energy in mitochondria, has been shown to ameliorate liver damage. The aim of the present study was to explore the preventive and therapeutic effect of L-carnitine in NASH model mice. Eight-week-old male STAM mice, a NASH-cirrhosis-hepatocarcinogenic model, were divided into 3 experimental groups and fed as follows: 1) high-fat diet (HFD) (control group); 2) HFD mixed with 0.28% L-carnitine (L-carnitine group); and 3) HFD mixed with 0.01% alpha-tocopherol (alpha-tocopherol group). After 4 or 8 weeks, mice were sacrificed. Blood samples and livers were collected, and hepatic tumors were counted and measured. Livers were subjected to histological study, immunohistochemical staining of 4-hydroxynonenal and ferritin, determination of 8-OHdG levels, mRNA and protein expressions for multiple genes, and metabolomic analysis. The intestinal microbiome was also analyzed. L-carnitine increased hepatic expression of genes related to long-chain fatty acid transport, mitochondrial beta-oxidation, and antioxidant enzymes following suppression of hepatic oxidative stress markers and inflammatory cytokines in NASH, and mice treated with L-carnitine developed fewer liver tumors. Although alpha-tocopherol resulted in NASH improvement in the same manner as L-carnitine, it increased periodontitis-related microbiotic changes and hepatic iron transport-related gene expression and led to less effective for anti-hepatocarcinogenesis. Conclusion: L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model by upregulating the mitochondrial beta-oxidation and redox system.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6862014Ultrastructural Analysis of an Enterolith Composed of Deoxycholic Acid369374ENMasayaIwamuroYuichiMiyashimaTakahiroYoshiokaToshihiroMurataYoshioMiyabeYoshinariKawaiHaruoUrataHidenoriShirahaHiroyukiOkadaKazuhideYamamotoCase Report10.18926/AMO/53026A 67-year-old Japanese man underwent enterotomy because of enterolith ileus. Component analysis by infrared spectroscopy revealed that the enterolith was composed of a high concentration of deoxycholic acid. We further analyzed and compared the ultrastructure of the enterolith and a commercially available powdered form of deoxycholic acid by means of scanning electron microscopy and energy dispersive X-ray spectroscopy. Energy dispersive X-ray spectroscopy analysis revealed that the ratios of carbon and oxygen in the enterolith were equal to those in the deoxycholic acid powder. Scanning electron microscopy analysis showed rectangular prism-shaped particles on the surface of the enterolith. This structure was similar to that of the deoxycholic acid powder. The surgically removed enterolith had a twisted and coiled appearance. Possible mechanisms underlying the formation of this unique form are discussed.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0815-93192952014Potential of adenovirus-mediated REIC/Dkk-3 gene therapy for use in the treatment of pancreatic cancer973983ENDaisukeUchidaHidenoriShirahaHironariKatoTeruyaNagaharaMasayaIwamuroJunroKataokaShigeruHoriguchiMasamiWatanabeAkinobuTakakiKazuhiroNousoYasutomoNasuTakahitoYagiHiromiKumonKazuhideYamamotoBackground and AimThe reduced expression in immortalized cells REIC/the dickkopf 3 (Dkk-3) gene, tumor suppressor gene, is downregulated in various malignant tumors. In a prostate cancer study, an adenovirus vector carrying the REIC/Dkk-3 gene (Ad-REIC) induces apoptosis. In the current study, we examined the effects of REIC/Dkk-3 gene therapy in pancreatic cancer.
MethodsREIC/Dkk-3 expression was assessed by immunoblotting and immunohistochemistry in the pancreatic cancer cell lines (ASPC1, MIAPaCa2, Panc1, BxPC3, SUIT-2, KLM1, and T3M4) and pancreatic cancer tissues. The Ad-REIC agent was used to investigate the apoptotic effect in vitro and antitumor effects in vivo. We also assessed the therapeutic effects of Ad-REIC therapy with gemcitabine.
ResultsThe REIC/Dkk-3 expression was lost in the pancreatic cancer cell lines and decreased in pancreatic cancer tissues. Ad-REIC induced apoptosis and inhibited cell growth in the ASPC1 and MIAPaCa2 lines in vitro, and Ad-REIC inhibited tumor growth in the mouse xenograft model using ASPC1 cells. The antitumor effect was further enhanced in combination with gemcitabine. This synergistic effect may be caused by the suppression of autophagy via the enhancement of mammalian target of rapamycin signaling.
ConclusionsAd-REIC induces apoptosis and inhibits tumor growth in pancreatic cancer cell lines. REIC/Dkk-3 gene therapy is an attractive therapeutic tool for pancreatic cancer.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0944-11744832013The impact of patatin-like phospholipase domain-containing protein 3 polymorphism on hepatocellular carcinoma prognosis405412ENYasutoTakeuchiFusaoIkedaYukiMoritouHiroakiHagiharaTetsuyaYasunakaKenjiKuwakiYasuhiroMiyakeHidekiOhnishiShinichiroNakamuraHidenoriShirahaAkinobuTakakiYoshiakiIwasakiKazuhiroNousoKazuhideYamamotoThe single nucleotide polymorphism (SNP) rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3) is associated with hepatic fat accumulation and disease progression in patients with non-alcoholic fatty liver disease and alcoholic liver disease (ALD). This study was conducted to determine whether PNPLA3 rs738409 SNPs affect development and prognosis of hepatocellular carcinoma (HCC) in patients with various liver diseases.
We enrolled 638 consecutive Japanese patients newly diagnosed with HCC between 2001 and 2010: 72 patients with hepatitis B virus (HBV), 462 with hepatitis C virus (HCV), and 104 with non-B non-C (NBNC).
NBNC patients exhibited large tumors of advanced TNM stages at HCC diagnosis, and had significantly poorer prognosis than HBV or HCV patients (P < 0.001 and < 0.001, respectively; log-rank test). The G/G genotype of PNPLA3 rs738409 SNP had significantly higher distribution in NBNC patients (P < 0.001) and was significantly associated with higher body mass index (BMI) and an increased aspartate aminotransferase to platelet ratio index. No significant differences were observed in survival with differences in PNPLA3 SNP genotypes among the patients, although ALD patients with the G/G genotype of PNPLA3 SNP and low BMI had significantly poorer survival than those with high BMI (P = 0.028).
The G/G genotype of PNPLA3 rs738409 SNP was more frequently distributed, and associated with BMI and fibrosis among NBNC-HCC patients but not among HBV or HCV patients. These genotypes might affect HCC prognosis in ALD patients, but not in HBV, HCV, or NAFLD patients.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6852014Hepatitis C Virus-specific T-cell Response Correlates with Hepatitis Activity and Donor IL28B Genotype Early after Liver Transplantation291302ENRyuichiroTsuzakiAkinobuTakakiTakahitoYagiFusaoIkedaKazukoKoikeYoshiakiIwasakiHidenoriShirahaYasuhiroMiyakeHiroshiSadamoriSusumuShinouraYuzoUmedaRyuichiYoshidaDaisukeNobuokaMasashiUtsumiEiichiNakayamaToshiyoshiFujiwaraKazuhideYamamotoOriginal Article10.18926/AMO/52898It is not known how the immune system targets hepatitis C virus (HCV)-infected HLA-mismatched hepatocytes under immune-suppressed conditions after orthotopic liver transplantation (OLT). In addition, the relationship between the HCV-specific immune response and IL28B variants as predictors of HCV clearance has not been well-characterized. We determined the IL28B polymorphisms for 57 post-OLT HCV carriers, and we assessed the HCV-specific immune responses by measuring the peripheral blood mononuclear cell-derived HCV-specific interferon-gamma (IFN-γ) response using an enzyme-linked immunospot assay. At 1-3 years after OLT, patients with no active hepatitis showed higher total spots on the immunospot assay. At＞3 years after OLT, patients with resolved HCV showed higher levels of core, NS3, NS5A, and total spots compared to the chronic hepatitis patients. The IL28B major genotype in the donors correlated with higher spot counts for NS5A and NS5B proteins at 1-3 years after OLT. In the post-OLT setting, the HCV-specific immune response could be strongly induced in patients with no active hepatitis with an IL28B major donor or sustained virological response. Strong immune responses in the patients with no active hepatitis could only be maintained for 3 years and diminished later. It may be beneficial to administer IFN treatment starting 3 years after OLT, to induce the maximum immunological effect.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6852014Nursing Support Increases the Efficacy of Interferon Therapy in Patients with Chronic Hepatitis C263268ENShihokoNambaKayokoMiyakeFusaoIkedaTomokoHazamaYuHitobeNorikoYamasakiHidenoriShirahaAkinobuTakakiKazuhiroNousoYoshiakiIwasakiKazuhideYamamotoOriginal Article10.18926/AMO/52894Nursing support might help patients with chronic hepatitis C (CHC) remain in good mental and physical condition during interferon (IFN) therapy. However, the effects of nursing support have not been studied adequately in this context. This case-control study evaluated the effects of nursing support during IFN therapy. Twenty-four CHC patients who received pegylated IFN and ribavirin were enrolled. Nurses advised patients on the maintenance of their mental and physical condition at weekly visits, based on the results of written questionnaires. An additional 24 patients who received IFN therapy without nursing support and who were matched for age, sex, platelet count, viral serogroup and IFN regimen were selected with propensity score matching as controls. The patients with nursing support during IFN therapy achieved higher sustained virological responses (79%) than those without nursing support (58%). Adherence to the IFN and ribavirin regimens at 24 weeks of therapy were slightly higher in the patients with nursing support than those without it, but these differences were not statistically significant. Adherence to ribavirin after 24 weeks of therapy was significantly higher in those with nursing support than those without it (93% and 66%, p＝0.045). These results suggested that nursing support services could contribute to the virological responses of CHC patients by promoting drug-regimen adherence.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0012-28238622012Serum Levels of Soluble Adhesion Molecules as Prognostic Factors for Acute Liver Failure122128ENAtsuyukiOhnishiYasuhiroMiyakeHiroshiMatsushitaKazuyukiMatsumotoAkinobuTakakiTetsuyaYasunakaKazukoKoikeFusaoIkedaHidenoriShirahaKazuhiroNousoKazuhideYamamotoBackground/Aims: In patients with septic shock, the degree of liver dysfunction is correlated with serum levels of soluble intercellular adhesion molecule (sICAM)-1. We aimed to assess the usefulness of serum levels of soluble adhesion molecules as prognostic factors for acute liver failure (ALF). Methods: Serum levels of soluble platelet endothelial cell adhesion molecule (sPECAM)-1, sICAM-3, soluble endothelial (sE) selectin, sICAM-1, soluble platelet selectin, and soluble vascular cell adhesion molecule-1 on admission were measured in 37 ALF patients and 34 healthy controls. Results: Twenty-two ALF patients (59%) reached to fatal outcomes. Serum levels of sPECAM-1, sICAM-3, sE-selectin and sICAM-1 were higher in ALF patients than healthy controls. In 37 ALF patients, by the multivariate logistic regression analysis, ratio of direct to total bilirubin (per 0.1 increase; OR 0.11, 95% CI 0.01-0.99), serum sPECAM-1 level (per 100 ng/ml increase; OR 4.37, 95% CI 1.23-15.5) and serum sICAM-1 level (per 100 ng/ml increase; OR 0.49, 95% CI 0.27-0.89) were associated with fatal outcomes. Using receiver operating characteristics curve, each area under the curve of serum sPECAM-1 and sICMA-1 levels as prognostic factors was 0.71 and 0.74, respectively. Conclusion: Serum sPECAM-1 and sICAM-1 levels may be useful for predicting the prognosis of ALF.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0815-93192612011Serum levels of platelet-derived growth factor-BB and vascular endothelial growth factor as prognostic factors for patients with fulminant hepatic failure116121ENHirokiTakayamaYasuhiroMiyakeKazuhiroNousoFusaoIkedaHidenoriShirahaAkinobuTakakiHaruhikoKobashiKazuhideYamamotoBackground and Aims:
In animal models for acute liver injury, the administration of some angiogenic factors such as vascular endothelial growth factor (VEGF) and granulocyte-colony stimulating factor (G-CSF) are shown to reduce liver injury and improve liver proliferative capacity. The aim of the present study was to assess the role of angiogenic factors in fulminant hepatic failure (FHF).
Methods:
Serum levels of nine angiogenic factors (angiopoietin-2, follistatin, G-CSF, hepatocyte growth factor [HGF], interleukin-8, leptin, platelet-derived growth factor [PDGF]-BB, platelet endothelial cell adhesion molecule-1 and VEGF) were measured using the Bio-Plex Protein Array System in 30 patients, 17 of whom were diagnosed with FHF, 13 with acute hepatitis (AH), and 20 controls.
Results:
Serum levels of PDGF-BB and VEGF were lower in FHF patients than AH patients and controls (PDGF-BB; 2050 +/- 1572 pg/mL vs 4521 +/- 2419 pg/mL vs 8506 +/- 5500 pg/mL, VEGF; 39 +/- 38 pg/mL vs 144 +/- 122 pg/mL vs 205 +/- 121 pg/mL). By using univariate logistic regression models, serum levels of PDGF-BB and VEGF were associated with poor outcomes. Serum PDGF-BB levels were strongly correlated with serum VEGF levels (r = 0.70). Furthermore, serum PDGF-BB levels were significantly correlated with platelet counts (r = 0.79), PT activity (r = 0.37) and D.Bil/T.Bil ratio (r = 0.50), while serum VEGF levels were significantly correlated with platelet counts (r = 0.68) and PT activity (r = 0.38).
Conclusions:
We consider that serum levels of PDGF-BB and VEGF are worth investigating as biomarkers for predicting outcomes of FHF patients.No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812622014柿胃石の成分分析における標準物質としての柿渋の有用性127131ENMasayaIwamuroYukoOkamotoToshihiroMurataYoshinariKawaiHidenoriShirahaHiroyukiOkadaKazuhideYamamotoThe definite diagnosis of persimmon phytobezoar (i.e., diospyrobezoar) is often accomplished by a component analysis using infrared spectroscopy. However, no studies have been conducted to investigate which substance is the best as a standard for the component analysis. Here we analyzed tannic acid, Japanese persimmon (kaki), fermented persimmon extract (kaki-shibu), conventional dried persimmon, and dried persimmon smoked in sulfur (ampo-kaki) by infrared spectroscopy to determine which would be optimal as a component analysis standard. The spectrum between 1,600 to 600cm−1 of a persimmon phytobezoar was quite similar to the spectrum of kaki-shibu rather than that of tannic acid. Consequently, we conclude that kaki-shibu should be used as a standard for infrared spectroscopy analyses of persimmon phytobezoars.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama1471-2407112011Hepatitis B virus core promoter mutations G1613A and C1653T are significantly associated with hepatocellular carcinoma in genotype C HBV-infected patientsENMasashiTatsukawaAkinobuTakakiHidenoriShirahaKazukoKoikeYoshiakiIwasakiHaruhikoKobashiShin-IchiFujiokaKohsakuSakaguchiKazuhideYamamotoBackground: Hepatitis B virus (HBV) is a major cause of hepatocarcinogenesis. To identify mutations relevant to hepatocellular carcinoma (HCC) development, we compared the full genome sequences of HBV from the sera of patients with and without HCC.
Methods: We compared the full genome sequences of HBV isolates from 37 HCC patients (HCC group 1) and 38 patients without HCC (non-HCC group 1). We also investigated part of the core promoter region sequences from 40 HCC patients (HCC group 2) and 68 patients without HCC. Of the 68 patients who initially did not have HCC, 52 patients remained HCC-free during the follow-up period (non-HCC group 2), and 16 patients eventually developed HCC (pre-HCC group 2). Serum samples collected from patients were subjected to PCR, and the HBV DNA was directly sequenced.
Results: All patients had genotype C. A comparison of the nucleotide sequences of the HBV genome between HCC group 1 and non-HCC group 1 revealed that the prevalence of G1613A and C1653T mutations in the core promoter region was significantly higher in the HCC group. These mutations tended to occur simultaneously in HCC patients. Multivariate analysis with group 2 revealed that the presence of HCC was associated with aging and the double mutation. Future emergence of HCC was associated with aging and the presence of a single G1613A mutation.
Conclusions: G1613A and C1653T double mutations were frequently found in patients with HCC. A single G1613A mutation was associated with future emergence of HCC. These mutations may serve as useful markers in predicting HCC development.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0944-11744742012Risk factors for recurrence after transarterial chemoembolization for early-stage hepatocellular carcinoma421426ENHideakiKinugasaKazuhiroNousoYasutoTakeuchiTetsuyaYasunakaHidekiOnishiShin-ichiroNakamuraHidenoriShirahaKenjiKuwakiHiroakiHagiharaFusaoIkedaYasuhiroMiyakeAkinobuTakakiKazuhideYamamotoRadiofrequency ablation (RFA) is a standard therapy for the treatment of hepatocellular carcinoma (HCC) with 3 or fewer tumors of up to 3 cm (early-stage HCC); when RFA is unsuccessful or unfeasible, transcatheter arterial chemoembolization (TACE) has often been performed. However, little information about the outcome of TACE for early-stage HCC has been reported and it is hard to decide whether to perform additional treatment following TACE in these difficult conditions. The aim of this study was to determine the risk factors for local or intrahepatic distant recurrence after TACE in early-stage HCC.
Among 1,560 newly diagnosed HCC patients who were admitted to Okayama University Hospital, 43 patients with early-stage HCC who received only TACE in at least one nodule were enrolled in this study. We analyzed the risk factors for local and distant recurrence by the Cox proportional hazard model.
The local recurrence rates and intrahepatic distant recurrence rates at 3 months, 6 months, and 1 year were 18.6, 33.4, and 61.8%, and 2.8, 2.8, and 10.2%, respectively. Among 12 parameters examined as possible risk factors for recurrence, heterogeneous Lipiodol uptake (risk ratio 3.38; 95% confidence interval 1.14-10.60) and high serum des-gamma-carboxy prothrombin (DCP) (2.58; 1.03-7.14) were significantly correlated with local recurrence, and the presence of multiple tumors (10.64; 1.76-93.75) was significantly correlated with intrahepatic distant recurrence.
Heterogeneous Lipiodol uptake, high serum DCP, and multiple tumors are risk factors for recurrence in patients with early-stage HCC who have undergone palliative TACE.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0815-931927102012Des-gamma-carboxyl prothrombin is associated with tumor angiogenesis in hepatocellular carcinoma16021608ENMinoruMatsubaraHidenoriShirahaJyunroKataokaMasayaIwamuroShigeruHoriguchiShin-ichiNishinaNobuyukiTakaokaMasayukiUemuraAkinobuTakakiShinichiroNakamuraYoshiyukiKobayashiKazuhiroNousoKazuhideYamamotoBackground and Aim: Hepatocellular carcinoma (HCC) is a hypervascular tumor, and angiogenesis plays an important role in its development. Previously, we demonstrated that des-gamma-carboxyl prothrombin (DCP) promotes both cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) by inducing the autophosphorylation of kinase insert domain receptor (KDR). In the present study, DCP-associated tumor angiogenesis was assessed by comparing hypovascular and common hypervascular HCC. Methods: The solitary HCCs of 827 patients were classified into two groups according to the tumor density at the arterial phase of a dynamic computed tomography scan; the initial clinical data of patients with the hyper- and hypovascular types were compared. The HCC tissues from 95 tumors were analyzed by immunohistochemical staining for DCP and phosphorylated KDR, and intratumoral microvessel density (MVD) was analyzed to evaluate microvessel angiogenesis. Results: The serum DCP levels (320 +/- 3532 mAU/mL) and tumor size (18.4 +/- 9.0 mm) of patients with hypervascular HCC were significantly greater than those with hypovascular HCC (38.7 +/- 80 mAU/mL and 14.6 +/- 5.2 mm, P < 0.001). Immunohistochemical analysis revealed that the expressions of DCP and phospho-KDR were significantly greater in hypervascular HCC (71.4% and 31.0%, respectively) than in hypovascular HCC (7.6% and 5.7%, respectively). Intratumoral MVD was significantly correlated with DCP (r = 0.48, P < 0.0001). Conclusions: des-gamma-carboxyl prothrombin production is associated with tumor angiogenesis in HCC.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama0020-7136131112012Runt-related transcription factor 3 reverses epithelial-mesenchymal transition in hepatocellular carcinoma25372546ENShigetomiTanakaHidenoriShirahaYutakaNakanishiShin-IchiNishinaMinoruMatsubaraShigeruHoriguchiNobuyukiTakaokaMasayaIwamuroJunroKataokaKenjiKuwakiHiroakiHagiharaJunichiToshimoriHidekiOhnishiAkinobuTakakiShinichiroNakamuraKazuhiroNousoTakahitoYagiKazuhideYamamotoLoss or decreased expression of runt-related transcription factor 3 (RUNX3), a tumor suppressor gene involved in gastric and other cancers, has been frequently observed in hepatocellular carcinoma (HCC). The objective of this study was to identify the regulatory mechanism of the epithelialmesenchymal transition (EMT) by RUNX3 in HCC. Human HCC cell lines, Hep3B, Huh7, HLF and SK-Hep1, were divided into low- and high-EMT lines, based on their expression of TWIST1 and SNAI2, and were used in this in vitro study. Ectopic RUNX3 expression had an anti-EMT effect in low-EMT HCC cell lines characterized by increased E-cadherin expression and decreased N-cadherin and vimentin expression. RUNX3 expression has previously been reported to reduce jagged-1 (JAG1) expression; therefore, JAG1 ligand peptide was used to reinduce EMT in RUNX3-expressing low-EMT HCC cells. Immunohistochemical analyses were performed for RUNX3, E-cadherin, N-cadherin and TWIST1 in 33 human HCC tissues, also divided into low- and high-EMT HCC, based on TWIST1 expression. E-cadherin expression was correlated positively and N-cadherin expression was correlated negatively with RUNX3 expression in low-EMT HCC tissues. Correlations between EMT markers and RUNX3 mRNA expression were analyzed using Oncomine datasets. Similarly, mRNA expression of E-cadherin was also significantly correlated with that of RUNX3 in low-EMT HCC, while mRNA expression of JAG1 was negatively correlated with that of RUNX3. These results suggest a novel mechanism by which loss or decreased expression of RUNX3 induces EMT via induction of JAG1 expression in low-EMT HCC.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6662012Eradication of Hepatitis C Virus Subgenomic Replicon by Interferon Results in Aberrant Retinol-Related Protein Expression461468ENKazukoKoikeAkinobuTakakiNobuyukiKatoMamoruOuchidaHirotakaKanzakiTetsuyaYasunakaHidenoriShirahaYasuhiroMiyakeKazuhideYamamotoOriginal Article10.18926/AMO/49042Hepatitis C virus (HCV) infection induces several changes in hepatocytes, such as oxidative stress, steatosis, and hepatocarcinogenesis. Although considerable progress has been made during recent years, the mechanisms underlying these functions remain unclear. We employed proteomic techniques in HCV replicon-harboring cells to determine the effects of HCV replication on host-cell protein expression. We examined two-dimensional electrophoresis (2-DE) and mass spectrometry to compare and identify differentially expressed proteins between HCV subgenomic replicon-harboring cells and their “cured” cells. One of the identified proteins was confirmed using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Full-length HCV genome RNA replicating and cured cells were also assessed using ELISA. Replicon-harboring cells showed higher expression of retinal dehydrogenase 1 (RALDH-1), which converts retinol to retinoic acid, and the cured cells showed higher expression of retinol-binding protein (RBP), which transports retinol from the liver to target tissues. The alteration in RBP expression was also confirmed by ELISA and Western blot analysis. We conclude that protein expression profiling demonstrated that HCV replicon eradication affected retinol-related protein expression.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama1471-2407112011Loss of runt-related transcription factor 3 expression leads hepatocellular carcinoma cells to escape apoptosisENYutakaNakanishiHidenoriShirahaShin-ichiNishinaShigetomiTanakaMinoruMatsubaraShigeruHoriguchiMasayaIwamuroNobuyukiTakaokaMasayukiUemuraKenjiKuwakiHiroakiHagiharaJunichiToshimoriHidekiOhnishiAkinobuTakakiShinichiroNakamuraYoshiyukiKobayashiKazuhiroNousoTakahitoYagiKazuhideYamamotoBackground: Runt-related transcription factor 3 (RUNX3) is known as a tumor suppressor gene for gastric cancer and other cancers, this gene may be involved in the development of hepatocellular carcinoma (HCC).
Methods: RUNX3 expression was analyzed by immunoblot and immunohistochemistry in HCC cells and tissues, respectively. Hep3B cells, lacking endogenous RUNX3, were introduced with RUNX3 constructs. Cell proliferation was measured using the MTT assay and apoptosis was evaluated using DAPI staining. Apoptosis signaling was assessed by immunoblot analysis.
Results: RUNX3 protein expression was frequently inactivated in the HCC cell lines (91%) and tissues (90%). RUNX3 expression inhibited 90 +/- 8% of cell growth at 72 h in serum starved Hep3B cells. Forty-eight hour serum starvation-induced apoptosis and the percentage of apoptotic cells reached 31 +/- 4% and 4 +/- 1% in RUNX3-expressing Hep3B and control cells, respectively. Apoptotic activity was increased by Bim expression and caspase-3 and caspase-9 activation.
Conclusion: RUNX3 expression enhanced serum starvation-induced apoptosis in HCC cell lines. RUNX3 is deleted or weakly expressed in HCC, which leads to tumorigenesis by escaping apoptosis.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama1471-240792009Twist expression promotes migration and invasion in hepatocellular carcinomaENNoriyukiMatsuoHidenoriShirahaTatsuyaFujikawaNobuyukiTakaokaNaokiUedaShigetomiTanakaShinichiNishinaYutakaNakanishiMasayukiUemuraAkinobuTakakiShinichiroNakamuraYoshiyukiKobayashiKazuhiroNousoTakahitoYagiKazuhideYamamotoBackground: Twist, a transcription factor of the basic helix-loop-helix class, is reported to regulate cancer metastasis. It is known to induce epithelial-mesenchymal transition (EMT). In this study, we evaluated the expression of twist and its effect on cell migration in hepatocellular carcinoma (HCC).
Methods: We examined twist expression using immunohistochemistry in 20 tissue samples of hepatocellular carcinoma, and assessed twist expression in HCC cell lines by RT-PCR and Western blot analysis. Ectopic twist expression was created by introducing a twist construct in the twist-negative HCC cell lines. Endogenous twist expression was blocked by twist siRNA in the twist-positive HCC cell lines. We studied EMT related markers, E-cadherin, Vimentin, and N-cadherin by Western blot analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. We used immunofluorescent vinculin staining to visualize focal adhesion.
Results: We detected strong and intermediate twist expression in 7 of 20 tumor samples, and no significant twist expression was found in the tumor-free resection margins. In addition, we detected twist expression in HLE, HLF, and SK-Hep1 cells, but not in PLC/RPF/5, HepG2, and Huh7 cells. Ectopic twist-expressing cells demonstrated enhanced cell motility, but twist expression did not affect cell proliferation. Twist expression induced epithelial-mesenchymal transition together with related morphologic changes. Focal adhesion contact was reduced significantly in ectopic twist-expressing cells. Twist-siRNA-treated HLE, HLF, and SK-Hep1 cells demonstrated a reduction in cell migration by 50, 40 and 18%, respectively.
Conclusion: Twist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X5832004Analysis of HCV genotypes from blood donors shows three new HCV type 6 subgroups exist in Myanmar.135142ENToshiyukiShinjiYi YiKyawKatsunoriGokanYasuhitoTanakaKojiOchiNobuchikaKusanoTakaakiMizushimaShin-ichiFujiokaHidenoriShirahaAye AyeLwinYasushiShiratoriMasashiMizokamiMyoKhinMasayukiMiyaharaShigeruOkadaNorioKoideArticle10.18926/AMO/32110The prevalence of hepatitis C virus (HCV) genotypes in Myanmar in comparison with the rest of Southeast Asia is not well known. Serum samples were obtained from 201 HCV antibody-positive volunteer blood donors in and around the Myanmar city of Yangon. Of these, the antibody titers of 101 samples were checked by serial dilution using HCV antibody PA test II and Terasaki microplate as a low-cost method. To compare antibody titers by this method and RNA identification, we also checked HCV-RNA using the Amplicor 2.0 test. Most high-titer groups were positive for HCV-RNA. Of the 201 samples, 110 were successfully polymerase chain reaction (PCR) amplified. Among them, 35 (31.8%) were of genotype 1, 52 (47.3%) were of genotype 3, and 23 (20.9%) were of type 6 variants, and phylogenetic analysis of these type 6 variants revealed that 3 new type 6 subgroups exist in Myanmar. We named the subgroups M6-1, M6-2, and M6-3. M6-1 and M6-2 were relatively close to types 8 and 9, respectively. M6-3, though only found in one sample, was a brand-new subgroup. These subtypes were not seen in Vietnam, where type 6 group variants are widely spread. These findings may be useful for analyzing how and when these subgroups were formed.No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X6362009Significance of Des-gamma-carboxy Prothrombin Production in Hepatocellular Carcinoma299304ENTatsuyaFujikawaHidenoriShirahaKazuhideYamamotoReview10.18926/AMO/31826<p>Serum des-gamma-carboxy prothrombin (DCP) is commonly used to detect hepatocellular carcinoma (HCC). This review focuses on the clinical features of DCP-positive HCC and the molecular function of DCP in HCC. DCP-positive HCC demonstrates more aggressive clinicopathological features than DCP-negative HCC. Analysis of the biological effects of DCP revealed that DCP acts as a growth factor in both an autocrine and paracrine manner. DCP stimulates HCC cell proliferation through the Met-Janus kinase 1-signal transducer and activator of transcription 3 signaling pathway, whereas for vascular endothelial cells, it stimulates cell proliferation and migration through the kinase insert domain receptor-phospholipase C-gamma-mitogen-activated protein kinase signaling pathway.</p>No potential conflict of interest relevant to this article was reported.Okayama University Medical SchoolActa Medica Okayama0386-300X4931995Gene expression of liver-specific proteins in hepatocyte spheroids in primary culture.161167ENTomoyukiTamuraNorioKoideHajimeHadaHidenoriShirahaTakaoTsujiArticle10.18926/AMO/30405Adult rat hepatocytes assemble to form multicellular spheroids under non-adherent environments such as immobilized chondroitin sulfate-proteoglycan in primary culture. Previously, we demonstrated that hepatocyte spheroids exhibited various differentiated structures as observed in the liver tissue. It was also shown that hepatocyte growth was highly suppressed and several differentiated functions, including albumin production and gluconeogenesis, were well preserved in spheroids. To investigate the differentiated functions of cultured hepatocytes in relation to cell morphology, we compared the expression of the albumin and transferrin genes in spheroids with those in monolayers by Northern blot analysis. Production of these proteins in the culture medium was simultaneously examined by ELISA. Gene expression and protein production of both albumin and transferrin were better preserved in spheroids. We also examined changes in the expression of liver-specific genes in response to IL-6. Reduced mRNA levels of both albumin and transferrin was only found in spheroids and no change was observed in monolayers. These results suggest that the regulation of tissue-specific gene expression is better preserved in spheroids, in which hepatocytes are in close contact with each other.No potential conflict of interest relevant to this article was reported.BioMed CentralActa Medica Okayama1471-240752005Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylationENTadashiHanafusaToshiyukiShinjiHidenoriShirahaKazuhiroNousoYoshiakiIwasakiEichiroYumotoToshiroOnoNorioKoide<p><b>Background:</b> Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulinlike
growth factors (IGF<sub>s</sub>) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in
human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered.<br />
<b>Methods:</b> In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation
constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity.<br />
Results: Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus
sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility
shift assay that p53 binding to the promoter region was diminished when methylated.<br />
<b>Conclusion:</b> From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of
IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells.</p>No potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812112009肝癌診療ガイドライン4145ENYoshiyukiKobayashiShinichiroNakamuraHidekiOhnishiJunichiToshimoriKenjiKuwakiHiroakiHagiharaYasuhiroMiyakeHidenoriShirahaKazuhiroNousoTakahitoYagiNoriakiTanakaKazuhideYamamotoNo potential conflict of interest relevant to this article was reported.岡山医学会Acta Medica Okayama0030-155812112009Des-γ-carboxy prothrombinは血管内皮細胞の増殖能と移動能を亢進させる18ENTatsuyaFujikawaHidenoriShirahaKazuhideYamamotoNo potential conflict of interest relevant to this article was reported.Acta Medica Okayama1996Improvement of serum amino acid profile in hepatic failure with the bio-artificial liver using multicellular hepatocyte spheroidsENNo potential conflict of interest relevant to this article was reported.