Keizo Kagawa

Side effects of high-dose interferon therapy for chronic hepatitis C

BACKGROUND/AIMSnVarious side effects have been reported in patients treated with alpha interferon, but their incidence and prognosis remain unknown.nnnMETHODSnNine hundred and eighty-seven patients with chronic active hepatitis C received 6 to 10 MU of alpha interferon per day for 2 weeks and 3 times per week for 22 weeks. Autoantibodies, thyroid function tests, and fasting plasma glucose concentrations were evaluated prior to alpha interferon therapy.nnnRESULTSnOf the 987 patients, 310 were required reduction in the dose of alpha interferon to 3 MU/day or cessation of alpha interferon therapy because of adverse reactions such as flu-like symptoms, leukopenia, and thrombocytopenia. Of the remaining 677, five developed diabetes mellitus, 12 had hyperthyroidism, and six acquired hypothyroidism. Of the 18 with thyroid disorders, five demonstrated antimicrosomal antibodies before therapy. Forty-four patients revealed high or low concentrations of thyroid stimulating hormone at the end of alpha interferon therapy. Three patients developed interstitial pneumonia, one acquired systemic lupus erythematosus-like syndrome, two had autoimmune hepatitis, two developed rheumatoid arthritis, and one developed autoimmune thrombocytopenic purpura. No patients had a history of an autoimmune disorder. One patient experienced sudden hearing impairment and one had retinal detachment. Melena was seen in three patients; two of these cases were compatible with ischemic colitis. Symptoms of depression were seen in 23 patients, and one patient manifested memory loss.nnnCONCLUSIONnHigh-dose alpha interferon therapy induces various adverse effects. Most of the side effects cannot be predicted, but are reversible.

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.

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

Background:u2002 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.

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.

Oxidative stress may enhance the malignant potential of human hepatocellular carcinoma by telomerase activation

Background/Aims: Continuous oxidative stress (OS) plays an important role in the progression of chronic liver diseases and hepatocarcinogenesis through telomere shortening in hepatocytes. However, it has not been established how the OS influences the progression of human hepatocellular carcinomas (HCCs). We examined the correlations of OS with telomere length of cancer cells, telomerase activity and other clinicopathological factors in 68 HCCs.

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.

Oxidative stress is closely associated with tumor angiogenesis of hepatocellular carcinoma

BackgroundOxidative stress (OS) plays an important role in the progression of chronic liver disease and hepatocarcinogenesis. However, the role of OS in the progression of hepatocellular carcinoma (HCC) is unclear. The aim of this study was to assess whether OS promotes angiogenesis in HCC.MethodsThe expressions of vascular endothelial growth factor (VEGF), VEGF receptor2 (VEGFR2), and phosphorylated Akt were assessed, and microvessel density (MVD) and the cancer-associated fibroblast (CAF) population were examined by immunohistological staining in 55 HCC samples. The OS level in these tissues was assessed using 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) immunostaining, and an 8-OHdG enzyme-linked immunosorbent assay (ELISA). The expression and activation of angiogenic factors and the effect of growth stimulation of human umbilical vein endothelial cells (HUVECs) were also assessed in vitro, using HLE hepatoma-derived cells and conditioned medium with or without treatment with hydrogen peroxide (H2O2); a phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin; and an anti-oxidative agent, N-acetyl-l-cysteine (NAC).ResultsA higher OS grade was significantly associated with higher MVD, VEGF expression, Akt activity, and OS grade of CAFs, but not with the percentage of the CAFpopulation in HCC tissues. Additionally, cancer cells constituted a major population of OS marker-positive cells in HCC tissues. In vitro, H2O2 treatment induced up-regulation of VEGF at both the mRNA and protein levels, activated Akt, and resulted in the proliferation of HUVECs; the addition of wortmannin and NAC counteracted the effects of OS.ConclusionsOS enhances the malignant potential of HCC through the stimulation of angiogenesis by activation of the Akt-VEGF pathway.

Serum levels of macrophage colony stimulating factor (M-CSF) in liver disease.

We investigated the serum level of macrophage colony stimulating factor in acute and chronic liver disease. Levels of macrophage colony stimulating factor (mean +/- SD, ng/ml) were significantly higher in acute hepatitis (5.67 +/- 1.01, p < 0.01) and chronic active hepatitis (3.34 +/- 1.19, p < 0.01) than in healthy volunteers (1.90 +/- 0.25), asymptomatic hepatitis B virus carriers (1.98 +/- 0.40), and chronic persistent hepatitis (2.34 +/- 0.43). Levels of macrophage colony stimulating factor showed a highly significant correlation with the serum alanine aminotransferase levels in acute hepatitis (p < 0.01, rs = 0.903) and in chronic active hepatis (p < 0.01, rs = 0.672). Levels of macrophage colony stimulating factor in patients with cirrhosis (cirrhosis; 3.11 +/- 0.93 and hepatocellular carcinoma; 3.30 +/- 0.74) were significantly higher than in patients with chronic persistent hepatitis although the alanine aminotransferase levels were not significantly different. In cirrhosis, levels of macrophage colony stimulating factor correlated positively with the serum alanine aminotransferase levels (p < 0.05), total bilirubin levels (p < 0.05), and indocyanine green clearance (p < 0.05). An immunohistochemical study showed an increased number of macrophage colony stimulating factor positive mononuclear cells in portal areas in acute hepatitis. Our findings suggest that; (a) the serum levels of macrophage colony stimulating factor represent ongoing hepatocellular necrosis in acute and chronic liver disease, (b) the source of the increase in the serum macrophage colony stimulating factor levels in hepatic inflammation may be, in part, its production by infiltrating mononuclear cells in the liver, and (c) cirrhosis also causes elevated serum levels of macrophage colony stimulating factor.

Evaluation of hepatic proliferative activity in chronic liver diseases and hepatocellular carcinomas by proliferating cell nuclear antigen (PCNA) immunohistochemical staining of methanol-fixed tissues.

The proliferative activity of chronic liver diseases and hepatoellular carcinomas (HCCs) was studied by PCNA immunohistochemistry. Human liver tissues were obtained by surgical operation or needle biopsy, and PCNA was detected by immunohistochemistry. PCNA-labelling indices (PCNA-LIs) of methanol-fixed tissues corresponded with the incidence of S-phase cells previously reported, whereas paraformaldehyde-fixed tissues showed extremely high PCNA-LIs in all specimens. Therefore, methanol-fixed tissues were used for evaluation. The PCNA-LIs of the methanol-fixed tissues were: normal liver 0.78 ±0.38%, chronic persistent hepatitis 1.06±0.86%, chronic aggressive hepatitis 2A 1.01±0.50%, chronic aggressive hepatitis 2B 4.20±1.79%, inactive cirrhosis 0.81±0.49%, active cirrhosis 1.96±0.93%, HCC of Edmondsons type I 4.83±1.98%, type II 6.65±1.69%, and type III 38.7±30.6%. PCNA-positive cells showed little specific distribution; in periportal areas in chronic hepatitis, at the margins of pseudolobules in cirrhosis, and throughout the tumor in HCC. These findings indicated that proliferative activity increased during the progression of chronic hepatitis, but that it decreased at the stage of cirrhosis. In chronic liver diseases, the PCNA-LIs reflected hepatitis activity. HCC showed higher proliferative activity than liver cirrhosis, and the histological grade was correlated with the PCNA-LI.

Subcortical impairment in subclinical hepatic encephalopathy

We have used short latency somatosensory evoked potential (SEP) in 108 patients with liver cirrhosis by viral hepatitis to evaluate hepatic encephalopathy. Short-latency SEPs were recorded by the MEM-4104 apparatus (Nihon Kohden Inc., Tokyo) in response to median nerve stimulation. For a precise analysis of the early components, we averaged 1000 responses during a 30-msec period. Early SEP components were prolonged in patients with decompensated, but not in those with compensated, cirrhosis. We also examined the relationship between consciousness level and interpeak latency (IPL) N13-N20 of SEP and between consciousness level and electroencephalograph in 51 patients among 108 patients with liver cirrhosis. The IPL N13-N20 was prolonged in the decompensated stage with normal consciousness, but EEG findings had not deteriorated in this stage. EEG grade became worse in the stage of abnormal consciousness. The prolongation of the IPL N13-N20 was attributed to the central conduction impairment. We postulate that subcortical impairment may occur in patients with subclinical hepatic encephalopathy. when the cortex is little affected.