Yasuhide Mitsumoto

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.

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.

Rosuvastatin ameliorates high‐fat and high‐cholesterol diet‐induced nonalcoholic steatohepatitis in rats

Statins, which are inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and inhibit endogenous cholesterol synthesis, possess pleiotropic activities, such as anti‐inflammatory, anti‐oxidative and antifibrotic effects. Here, we investigated whether statins ameliorate steatohepatitis using a high‐fat and high‐cholesterol (HFHC) diet‐induced rat model.

Nuclear size measurement is a simple method for the assessment of hepatocellular aging in non-alcoholic fatty liver disease: Comparison with telomere-specific quantitative FISH and p21 immunohistochemistry

Telomere‐specific quantitative fluorescent in situ hybridization (Q‐FISH) accurately evaluates hepatocellular aging on histological sections, but it requires appropriate tissue processing. To establish a more simple method for the assessment of hepatocellular aging, the usefulness of nuclear size measurement was clarified using biopsy liver samples from 64 patients with non‐alcoholic fatty liver disease (NAFLD), a model for oxidative stress‐associated hepatocellular aging, and 11 control individuals. Relative telomere intensity (RTI) was measured on Q‐FISH, and the relative nuclear size (RNS) was calculated as the average nuclear size of the hepatocytes divided by that of lymphocytes. In normal individuals and NAFLD patients, the RTI and RNS were negatively correlated. The degree of nuclear enlargement in NAFLD patients was larger than that in normal individuals with the same telomere length, possibly reflecting telomere‐independent senescence. In NAFLD patients with RNS >2.0, the regenerative responses, indicated by the ratio of Ki‐67‐positive index to serum alanine aminotransferase level, were significantly reduced. The RNS positively correlated with the p21 expression, another marker of senescence. This all indicates that nuclear enlargement progresses in parallel with reduced regenerative responses, telomere shortening, and p21 upregulation. Nuclear size measurement is an effective method for estimation of hepatocellular aging.

Efficacy and safety of sorafenib in very elderly patients aged 80 years and older with advanced hepatocellular carcinoma

Sorafenib is the standard systemic therapy for patients with advanced hepatocellular carcinoma (HCC). We aimed to assess the efficacy and safety of sorafenib therapy in very elderly patients aged 80 years and older with advanced HCC.

Classification of patients with nonalcoholic fatty liver disease using rapid immunoassay of serum type IV collagen compared with that using liver histology and other fibrosis markers.

Non‐alcoholic fatty liver disease (NAFLD) can progress to non‐alcoholic fatty liver (NAFL) or non‐alcoholic steatohepatitis (NASH). We investigated the association among serum type IV collagen level, liver histology, and other fibrosis markers in NAFLD progression.

Risk estimation model for nonalcoholic fatty liver disease in the Japanese using multiple genetic markers

The genetic factors affecting the natural history of nonalcoholic fatty liver disease (NAFLD), including the development of nonalcoholic steatohepatitis (NASH) and NASH-derived hepatocellular carcinoma (NASH-HCC), are still unknown. In the current study, we sought to identify genetic factors related to the development of NAFLD, NASH, and NASH-HCC, and to establish risk-estimation models for them. For these purposes, 936 histologically proven NAFLD patients were recruited, and genome-wide association (GWA) studies were conducted for 902, including 476 NASH and 58 NASH-HCC patients, against 7,672 general-population controls. Risk estimations for NAFLD and NASH were then performed using the SNPs identified as having significant associations in the GWA studies. We found that rs2896019 in PNPLA3 [p = 2.3x10-31, OR (95%CI) = 1.85 (1.67–2.05)], rs1260326 in GCKR [p = 9.6x10-10, OR (95%CI) = 1.38(1.25–1.53)], and rs4808199 in GATAD2A [p = 2.3x10-8, OR (95%CI) = 1.37 (1.23–1.53)] were significantly associated with NAFLD. Notably, the number of risk alleles in PNPLA3 and GATAD2A was much higher in Matteoni type 4 (NASH) patients than in type 1, type 2, and type 3 NAFLD patients. In addition, we newly identified rs17007417 in DYSF [p = 5.2x10-7, OR (95%CI) = 2.74 (1.84–4.06)] as a SNP associated with NASH-HCC. Rs641738 in TMC4, which showed association with NAFLD in patients of European descent, was not replicated in our study (p = 0.73), although the complicated LD pattern in the region suggests the necessity for further investigation. The genetic variants of PNPLA3, GCKR, and GATAD2A were then used to estimate the risk for NAFLD. The obtained Polygenic Risk Scores showed that the risk for NAFLD increased with the accumulation of risk alleles [AUC (95%CI) = 0.65 (0.63–0.67)]. Conclusions: We demonstrated that NASH is genetically and clinically different from the other NAFLD subgroups. We also established risk-estimation models for NAFLD and NASH using multiple genetic markers. These models can be used to improve the accuracy of NAFLD diagnosis and to guide treatment decisions for patients.

Activation of apoptosis inhibitor of macrophage is a sensitive diagnostic marker for NASH-associated hepatocellular carcinoma

BackgroundA diagnostic marker is needed enabling early and specific diagnosis of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH). Our recent findings have indicated that circulating apoptosis inhibitor of macrophage (AIM), which usually associates with IgM pentamer in the blood, is activated by its dissociation from IgM. We investigated the serum levels of IgM-free AIM for AIM activation and its possible relationship with development of HCC in NASH.MethodsSerum levels of IgM-associated and IgM-free AIM were evaluated in patients with non-alcoholic fatty liver, NASH, and NASH-HCC using enzyme-linked immunosorbent assays and immunoblots. Liver biopsy specimens were graded and staged using Brunt’s classification.ResultsForty-two patients with fatty liver, 141 with NASH, and 26 with NASH-HCC were evaluated. Patients with stage 4 or grade 3 NASH (with or without HCC) exhibited significantly higher levels of both IgM-free and total AIM than those with fatty liver, whereas the ratio of IgM-free-to-total AIM was equivalent in these groups. Among patients with the same fibrosis stage of NASH, those with HCC had significantly higher IgM-free but not total AIM levels, resulting in a proportional increase in the IgM-free/total AIM ratio. Analysis of the areas under the receiver operating characteristic curves indicated the high sensitivity of the IgM-free AIM for NASH-HCC.ConclusionsOur observations suggest the activation of AIM in blood in the presence of NASH-HCC, with a significant increase in IgM-free AIM levels. IgM-free AIM serum levels appear to be a sensitive diagnostic marker for NASH-HCC.

Assessment of transient elastography in Japanese patients with non‐alcoholic fatty liver disease

Transient elastography (TE) is a non‐invasive method for predicting liver fibrosis. However, there are limited data regarding the performance of TE in Japanese patients with non‐alcoholic fatty liver disease (NAFLD). We aimed to evaluate the association between liver stiffness measurement (LSM) by TE and liver fibrosis stage, and define a cut‐off value for predicting liver fibrosis.

Development of a single expandable PEIT needle and its clinical application

The current mainstream percutaneous local treatment for HCC is RFA with supplementary PEIT. PEIT is sometimes employed for HCC in high-risk locations, such as HCC close to the heart. However, even with PEIT it is markedly difficult to treat HCC located at the back ofblood vessels deep in the liver. We developed a single expandable PEIT needle to treat difficult-to-puncture HCC. The tip of an inner needle can be expandable vertically to the long axis. We used this technique to successfully treat a case of HCC in S1 at the back of portal vein at the liver hilum. The needle is equipped with features absent in other devices and is expected to become a new option for percutaneous local therapy of HCC.