Tomoki Nakajima

Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, reduces hepatic steatosis and lipid peroxidation in fatty liver Shionogi mice with hereditary fatty liver.

Abstract: Background and aims: The fatty liver Shionogi (FLS) mouse, a unique model for nonalcoholic fatty liver disease (NAFLD), is an inbred strain that develops spontaneous hepatic steatosis without obesity or diabetes mellitus. Peroxisome proliferator‐activated receptor (PPAR) α controls fatty acid metabolism. In the present study, we investigated the effect of fenofibrate, a PPARα agonist, on hepatic steatosis in FLS mice.

Hepatitis B virus-related insertional mutagenesis in chronic hepatitis B patients as an early drastic genetic change leading to hepatocarcinogenesis

Growing evidence demonstrates that hepatitis B virus (HBV) integration and resulting insertional mutagenesis play an important role in cell growth or maintenance in hepatocellular carcinomas (HCCs). To determine if HBV integration occurs and affects cellular genes at such a stage of infection, we analysed viral–host junctions in chronic hepatitis tissues without HCC using PCR amplification with primers specific to human Alu-repeat and HBV. We obtained 42 independent viral–host junctions from six patients examined and identified chromosomal locations for 20 of the 42 junctions. In six clones, each integration apparently affected a single gene. These six candidate genes included one known tumor suppressor gene, three human homologs of drosophila genes that are critical for organ development, one putative oncogene and one recently found chemokine. Our data, together with previously reported HBV integrants in HCCs, suggested preferential HBV integration into chromosome 3 (P=0.022). Our virus-tagging approach provided (a) firm evidence of HBV integration in hepatocytes at an early stage of chronic infection and (b) revealed cellular genes possibly affected by HBV integration and potentially involved in early steps of the process leading to carcinogenesis.

Premature telomere shortening and impaired regenerative response in hepatocytes of individuals with NAFLD

Abstract: Aims: The risk factors associated with poor prognosis of nonalcoholic fatty liver disease (NAFLD) are not fully understood. Our aim was to assess the role of progressive hepatocellular telomere shortening in the clinical course of NAFLD.

The copper chelator trientine has an antiangiogenic effect against hepatocellular carcinoma, possibly through inhibition of interleukin‐8 production

Recent studies have revealed that copper is an important cofactor for several angiogenic agents. We examined the antiangiogenic effect against hepatocellular carcinoma (HCC) of the copper chelator trientine, especially focusing on the relationship between copper and interleukin‐8 (IL‐8), a potent angiogenic factor produced by hepatoma cells. HuH‐7 hepatoma cells were transplanted into nude mice and the growth of xenograft tumors was compared to and without administration of trientine. Using the resected tumor, microvessel density, apoptotic potential and proliferative activity were analyzed histologically and IL‐8 mRNA was semiquantified by RT‐PCR. In addition, HuH‐7 cells were cultured in control medium, medium supplemented with copper, medium supplemented with trientine and medium supplemented with both copper and trientine. Human IL‐8 levels were measured in the supernatants by ELISA. Using the extracts from cultured cells, IL‐8 mRNA was semiquantified by RT‐PCR. Trientine suppressed the growth of xenograft tumors significantly. Histologically, apoptotic potential was increased significantly and microvessel density, decreased. The production of IL‐8 from the tumor was suppressed by trientine. In vitro, IL‐8 production by HuH‐7 cells in copper‐containing medium was significantly greater than that in copper‐free medium, and this effect was weakened when trientine was added. However, no significant change was apparent when trientine was added to the medium alone. In conclusion, the chelating effect of trientine prevented copper from functioning as a cofactor in angiogenesis, which resulted in reduced IL‐8 production from HuH‐7 cells.

Association of gankyrin protein expression with early clinical stages and insulin-like growth factor-binding protein 5 expression in human hepatocellular carcinoma†

Gankyrin (also known as PSMD10) is a liver oncoprotein that interacts with multiple proteins including MDM2 and accelerates degradation of the tumor suppressors p53 and Rb. We produced a monoclonal anti‐gankyrin antibody and immunohistochemically assessed the clinicopathological significance of gankyrin overexpression in 43 specimens of human hepatocellular carcinoma (HCC). Specific cytoplasmic staining for gankyrin was observed in 62.8% (27/43) of HCCs, which was significantly associated with low TNM stage (P = 0.004), no capsular invasion (P = 0.018), no portal venous invasion (P = 0.008), and no intrahepatic metastasis (P = 0.012). The cumulative survival rate of patients with gankyrin‐positive HCC was significantly higher than that with gankyrin‐negative HCC (P = 0.037). p53 and MDM2 were positively stained by antibodies in 30.2% and 23.3%, respectively, of HCCs, but neither was inversely associated with gankyrin expression. In the Huh‐7 human HCC cell line, overexpression of gankyrin up‐regulated expression of insulin‐like growth factor binding protein 5 (IGFBP‐5), whereas suppression of gankyrin expression by siRNA down‐regulated it. Supression of IGFBP‐5 expression inhibited proliferation of Huh‐7 cells as well as U‐2 OS osteosarcoma cells. In HCC specimens, positive staining for IGFBP‐5 was observed by immunohistochemistry in 41.9% (18/43), and the level of expression was significantly correlated with that of gankyrin (rho = 0.629, P < 0.001). Conclusion: These results suggest that gankyrin plays an oncogenic role(s) mainly at the early stages of human hepatocarcinogenesis, and that IGFBP‐5 inducible by gankyrin overexpression may be involved in it. (HEPATOLOGY 2008.)

Transient biochemical response in interferon therapy decreases the development of hepatocellular carcinoma for five years and improves the long-term survival of chronic hepatitis C patients

The development of hepatocellular carcinoma (HCC) was significantly reduced in both sustained responders (SR) and transient biochemical responders (TR) in chronic hepatitis C (CH-C) patients who received interferon (IFN) therapy. However, the long-term clinical outcome of TR remains unclear. One thousand three hundred and seventy CH-C Japanese patients who received IFN therapy and 54 control cirrhotic patients were enrolled. TR were defined as those patients who showed a normal serum alanine aminotransferase level (<==30 IU/l) at the end of therapy and then relapsed. Mean follow-up period was 5.6 years (6.1 years in 48 cirrhotic patients) in the IFN group and 8.3 years in the 54 control cirrhotic patients. HCC was detected in 114 patients in the IFN group among whom 4 were in the 425 SR, 21 were in the 359 TR and 89 were in the 586 non-responders (NR). The cumulative incidence of HCC was significantly (P=0.0001) inhibited in both SR and TR compared with NR. Its inhibitory effect in TR was within 5 years. Platelet count did not significantly decrease for 2-4 years after IFN therapy in TR, but it significantly decreased in NR 2 years after IFN therapy. The cumulative survival in both SR and TR was significantly higher than NR (SR vs NR; P=0.0001, TR vs NR; P=0.0305). These results indicate that IFN therapy lowers the rate of the progression of HCC and improves the long-term survival even in CH-C patients who transiently respond to IFN therapy.

Role of cell-cycle turnover and oxidative stress in telomere shortening and cellular senescence in patients with chronic hepatitis C.

Background:  In addition to the telomere shortening that occurs with cell division, oxidative stress can damage or shorten telomeres and induce a condition termed premature senescence, possibly before telomeres become critically short. We investigated the effects of cell‐cycle turnover and oxidative stress on cellular senescence in hepatitis C virus (HCV)‐related chronic liver injury.

High expression of p300 in HCC predicts shortened overall survival in association with enhanced epithelial mesenchymal transition of HCC cells

P300 impacts the transcription of several genes involved in biological behavior of human malignancies including hepatocellular carcinomas (HCC). We found p300 is highly expressed in 47% of surgically resected HCC specimens by immunohistochemistry, which correlated with advanced TNM staging (P = 0.034), vascular invasion (P = 0.036), intrahepatic metastasis (P = 0.001) and shortened overall survival (P = 0.028). In vitro study, knocking down of p300 expression in hepatoma cells recovered E-cadherin expression, inhibited the translocation of beta (β)-catenin into the nuclei, decreased cyclin D1 activity and suppressed the migration/invasion of HCC cells. Furthermore, suppression of p300 led to down-regulation of epithelial-mesenchymal transition (EMT)-related molecules such as Snail, Twist and HIF-1 alpha. These observations suggest that p300 contributes to the EMT-related progression of HCCs.

Centrosome aberration accompanied with p53 mutation can induce genetic instability in hepatocellular carcinoma.

Centrosome duplication is controlled in a cell cycle-specific manner and occurs once every cell cycle, thereby ensuring the balanced segregation of chromosomes during the mitotic phase. Numerical or structural abnormalities can arise in the centrosomes of malignant cells. Under defective cell cycle checkpoint systems, cancer cells with abnormal centrosomes can survive and re-enter the cell cycle, promoting unbalanced chromosome segregation and genetic instability. We investigated the centrosome aberrations in 33 patients diagnosed with hepatocellular carcinoma (HCC), using fluorescent pericentrin immunostaining. We also studied the p53 mutation, proliferative activity, and DNA ploidy in these cases. In normal hepatocytes, one centrosome was identified per cell as a round dot, usually in the vicinity of the nuclear membrane. However, in cancer cells from HCC tissue, several patterns of centrosome abnormalities occurred, including supernumerary centrosomes and centrosomes with an abnormal shape and size. Although the frequency of abnormal centrosomes in each tissue was relatively low compared with previous reports in other cancers, nevertheless, centrosome aberration was found in 30 out of 33 HCC tissues. The percentage of tumor cells with abnormal centrosomes was significantly higher in the nondiploid tumors (15.8±15.9‰) than in the diploid tumors (5.4±5.1‰) (P<0.05), and tended to be higher in the tumors with p53 mutation (11.6±13.1‰) than in those with wild-type p53 (5.6±6.8‰). Furthermore, 82% of nondiploid tumors exhibited p53 mutation, whereas only 41% of diploid tumors showed p53 mutation. The percentage of tumor cells with centrosome abnormalities were not related to tumor stage, size or proliferative activity. Therefore, our results indicate that hepatic cancer cells, under centrosome aberration and a defective checkpoint system possibly caused by p53 mutation, have the potential for genetic instability and aggressive behavior. This potential effect occurs irrespective of the tumor size or stage.

In vivo expression patterns of survivin and its splicing variants in chronic liver disease and hepatocellular carcinoma.

Abstract: Aim: Our aim was to clarify the significance of expression levels and post‐transcriptional splicing patterns of survivin during multistep hepatocarcinogenesis and tumor progression.