Shuichi Seki

Randomised trial of effects of interferon-α on incidence of hepatocellular carcinoma in chronic active hepatitis C with cirrhosis

Patients with chronic active hepatitis C and cirrhosis often develop hepatocellular carcinoma. Interferon (IFN) seems to be effective in some patients but whether it prevents carcinogenesis is unknown. In a prospective randomised controlled trial, we evaluated the effects of IFN-alpha in cirrhotic patients with HCV infection because of their high risk of hepatocellular carcinoma. 90 patients with compensated chronic active hepatitis C with cirrhosis were randomly allocated to receive IFN-alpha (6 MU three times weekly for 12-24 weeks) (45 patients) or symptomatic treatment (45 controls), and were followed up for 2-7 years. In nine controls, alanine aminotransferase (ALT) decreased to less than 80 IU/L but did not stay in the normal range. In 19 patients given IFN-alpha, ALT decreased to less than 80 IU/L (in seven patients, it became and stayed normal; p = 0.011, Wilcoxon rank-sum test). However, the mean change in ALT was not significantly different between the two groups. The mean change in peak alpha-fetoprotein values was smaller in patients given IFN-alpha than in controls (p = 0.021). The mean change in the serum albumin level was higher in the IFN-alpha group (p < 0.001). The histological activity index in the 12 IFN-alpha patients undergoing a second biopsy after therapy was improved (p = 0.031). Hepatitis C viral RNA disappeared in seven (16%) of the 45 IFN-alpha patients (95% CI, 7-29%) and in none of the 45 controls (0-8%; p = 0.018). Hepatocellular carcinoma was detected in two (4%, 1-15%) IFN-alpha patients and 17 (38%, 24-54%) controls (p = 0.002, Wilcoxon signed-rank test). The risk ratio of IFN-alpha treatment versus symptomatic treatment was 0.067 (0.009-0.530; p = 0.010 Coxs proportional hazards). IFN-alpha improved liver function in chronic active hepatitis C with cirrhosis, and its use was associated with a decreased incidence of hepatocellular carcinoma.

Characterization of a Stellate Cell Activation-associated Protein (STAP) with Peroxidase Activity Found in Rat Hepatic Stellate Cells

A proteome approach for the molecular analysis of the activation of rat stellate cell, a liver-specific pericyte, led to the discovery of a novel protein named STAP (stellate cellactivation-associated protein). We cloned STAP cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid sequence homology with myoglobin. STAP was dramatically induced in in vivo activated stellate cells isolated from fibrotic liver and in stellate cells undergoingin vitro activation during primary culture. This induction was seen together with that of other activation-associated molecules, such as smooth muscle α-actin, PDGF receptor-β, and neural cell adhesion molecule. The expression of STAP protein and mRNA was augmented time dependently in thioacetamide-induced fibrotic liver. Immunoelectron microscopy and proteome analysis detected STAP in stellate cells but not in other hepatic constituent cells. Biochemical characterization of recombinant rat STAP revealed that STAP is a heme protein exhibiting peroxidase activity toward hydrogen peroxide and linoleic acid hydroperoxide. These results indicate that STAP is a novel endogenous peroxidase catabolizing hydrogen peroxide and lipid hydroperoxides, both of which have been reported to trigger stellate cell activation and consequently promote progression of liver fibrosis. STAP could thus play a role as an antifibrotic scavenger of peroxides in the liver.

Identification of 27 5' CpG islands aberrantly methylated and 13 genes silenced in human pancreatic cancers.

Aberrantly methylated DNA fragments were searched for in human pancreatic cancers, using the genome scanning technique: methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 111 DNA fragments derived from CpG islands (CGIs), and 35 of them were from CGIs in the 5′ regions of known genes. Methylation-specific PCR (MSP) of the CGIs in seven pancreatic cancer cell lines and two pancreatic ductal epithelial cell lines showed that 27 CGIs in the 5′ regions were aberrantly methylated in at least one of the cancer cell lines. Quantitative reverse-transcription–PCR analysis showed that downstream genes of all the CGIs were either not expressed or only very weakly expressed in cancer cell lines with the aberrant methylation. In the pancreatic ductal epithelial cell lines, 18 genes were expressed at various levels, and nine genes were not expressed at all. Treatment of a cancer cell line with a demethylating agent, 5-aza-2′-deoxycytidine, restored the expression of 13 genes, RASGRF2, ADAM23, NEF3, NKX2-8, HAND1, EGR4, PRG2, FBN2, CDH2, TLL1, NPTX1, NTSR1 and THBD, showing their silencing by methylation of their 5′ CGIs. MSP of 24 primary pancreatic cancers showed that all these genes, except for THBD, were methylated in at least one cancer. Some of those were suggested to be potentially involved in pancreatic cancer development and progression.

In situ detection of oxidative DNA damage, 8-hydroxydeoxyguanosine, in chronic human liver disease

BACKGROUND/AIMS 8-Hydroxydeoxyguanosine (8-OHdG) is a promutagenic DNA lesion produced by oxygen radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress. METHODS Hepatic expression of 8-OHdG was immunohistochemically investigated in control and diseased human livers. RESULTS While no positive immunolabeling for 8-OHdG was observed in control livers, 8-OHdG was widely evident in diseased livers. Nuclear expression of 8-OHdG in the hepatocytes and bile duct cells were found in various forms of chronic hepatitis. 8-OHdG-positive hepatocytes were especially abundant in the periportal area with piecemeal necrosis and prominent cell infiltration. The number of positive hepatocytes significantly increased with the progression of severity of chronic hepatitis activity (r(s)=0.68, P<0.05). In alcoholic liver disease, nuclear expression of 8-OHdG was detected in the hepatocytes in the area of alcoholic hepatitis. Regarding primary biliary cirrhosis, 8-OHdG was preferentially detected in the nuclei of injured bile ducts (11 of 12 cases, 91.7%) and occasionally (2 of 12 cases, 16.7%) in the nuclei of hepatocytes around the bile duct lesions. CONCLUSIONS These results indicate that oxidative DNA damage is common in various forms of chronic liver disease suggesting a possible link between chronic inflammation and hepatocarcinogenesis.

Clinical usefulness of positron emission tomography with fluorine-18-fluorodeoxyglucose in the diagnosis of liver tumors

We studied various liver tumors by positron emission tomography with fluorine-18 fluorodeoxyglucose (FDG-PET) to examine the diagnostic usefulness of this technique. We also examined the relation between findings on FDG-PET and the characteristics of hepatocellular carcinoma.FDG-PET was performed in 78 patients with liver tumors, including 53 with primary liver cancer [48 hepatocellular carcinomas (HCC) and 5 cholangiocellular carcinomas (CCC)], 20 with metastatic liver cancer, 2 with liver hemangioma, and 3 with focal nodular hyperplasia. For quantitative evaluation, a region of interest (ROI) was placed over the entire tumor region, at the level of the maximum diameter of the tumor. A background ROI was then placed over the non-tumor region of the liver. The average activity within each ROI was subsequently corrected for radioactive decay, and the standardized uptake value (SUV) was calculated by dividing the tissue activity by the injected dose of radioactivity per unit body weight. SUV ratio was expressed as the tumor-to-non-tumor ratio of the SUV.The median SUV was significantly lower in HCC than in metastatic live cancer or CCC, and the median SUV ratio was significantly lower in HCC than in metastatic liver cancer or CCC. The median SUV was not higher in multiple HCC than in single HCC, but the median SUV ratio was significantly higher in multiple HCC than in single HCC. The median SUV and the median SUV ratio were significantly higher in the presence of portal vein thrombosis than in the absence of such thrombosis. The Cancer of the Liver Italian Program score and the α-fetoprotein value correlated significantly with both the SUV and SUV ratio. These results suggest that FDG-PET is clinically useful not only for the differential diagnosis of liver tumors but also for evaluation of the clinical characteristics of HCC.

Development of hepatocellular carcinoma in patients with chronic hepatitis C who had a sustained virological response to interferon therapy: a multicenter, retrospective cohort study of 1124 patients

Background: Interferon (IFN) improves hepatic inflammation/fibrosis and reduces the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C (CH‐C). However, HCC develops in some patients who have a sustained virological response (SVR) to IFN therapy. We designed this study to establish a follow‐up protocol for patients with CH‐C who have SVR to IFN therapy.

Inhibitory effect of a selective cyclooxygenase-2 inhibitor on liver metastasis of colon cancer.

COX‐2 overexpression is recognized in various cancers, but the role of COX‐2 in the progression of cancer, including the liver metastasis of colon cancer, is not clearly understood. We examined the role of COX‐2 in the mechanism of liver metastasis of colon cancer, using a highly metastasizable colon carcinoma cell line, LM‐H3. A COX‐2 inhibitor, JTE‐522, inhibited cell proliferation and invasion of LM‐H3 in vitro and clearly reduced the number of metastatic nodules on the surface of nude mouse livers in vivo. We also examined the effects of JTE‐522 on the production of growth factors and MMPs through the use of ELISA and gelatin zymography, respectively. JTE‐522 downregulated PDGF production by LM‐H3 but had no influence on VEGF production. JTE‐522 also inhibited MMP‐2 secretion by LM‐H3. JTE‐522 downregulated PGE2 production, but the associated changes in PGE2 did not affect PDGF and VEGF production by LM‐H3. We conclude that JTE‐522 downregulated the cell proliferation and invasive potential of LM‐H3 by reducing the production of PDGF and MMP‐2 and hypothesize that these inhibitory effects on the production of PDGF and MMP‐2 can lead to inhibition of liver metastasis of colon cancer. These data indicate that the COX‐2 inhibitor JTE‐522 has a high potential for use as a clinical agent for the treatment of liver metastasis of colon cancer.

Expression of antigens related to apoptosis and cell proliferation in chronic nonsuppurative destructive cholangitis in primary biliary cirrhosis

The initial injury in primary biliary cirrhosis (PBC) is the destruction of portal bile ducts. Little information is available on apoptosis and cell proliferation in such bile ducts, so we used immunohistochemical techniques to locate molecules related to apoptosis [Fas antigen, Lewis Y antigen (BM1/JIMRO), and bcl-2 protein] and to cell proliferation (proliferating cell nuclear antigen, PCNA) in 21 patients with PBC. In addition, nick-end labelling was done to locate DNA fragmentation. The expression of these molecules in chronic nonsuppurative destructive cholangitis (CNSDC) was examined. Cell death and PCNA expression were both found in portal bile ducts affected by CNSDC in 7 of the 13 CNSDC patients examined. Fas antigen was found on the plasma membrane and rough endoplasmic reticulum of bile-duct cells with CNSDC in the frozen sections of all 6 patients with CNSDC out of the 9 patients inspected, and this antigen was found also in bile-duct cells without CNSDC in 2 of these 9 patients. It was not found in anatomically normal liver (from 2 patients with Gilberts disease). The Lewis Y antigen was found in bile ducts with CNSDC and in proliferated ductules in all 16 patients examined. No bcl-2 protein was found in any bile-duct or ductule cells, but it was found in the cytoplasm of lymphocytes surrounding or invading CNSDC. DNA fragmentation was found in the nuclei of bile-duct cells with CNSDC by nick-end labelling. Our study indicated that Fas-mediated apoptosis might be involved in CNSDC, but that bcl-2 protein seems to participate less than Fas. Although the Lewis Y antigen was found in many bile ducts, the relationship between the antigen and apoptosis remains unknown because there was no evidence that this antigen mediates apoptosis.

Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver.

Abstract.Neural cell adhesion molecule (N-CAM) is distributed in most nerve cells and some non-neural tissues. The present immunohistochemical study has revealed, for the first time, the expression of N-CAM in perisinusoidal stellate cells of the human liver. Liver specimens were stained with monoclonal antibody against human Leu19 (N-CAM) by a streptoavidin-biotin-peroxidase-complex method. Light- and electron-microscopic analyses have shown that N-CAM-positive nerve fibers are distributed in the periportal and intermediate zones of the liver lobule. Perisinusoidal stellate cells in these zones are also positive for N-CAM. N-CAM is expressed on the surface of the cell, including cytoplasmic projections. Close contact of N-CAM-positive nerve endings with N-CAM-positive stellate cells has been observed. On the other hand, stellate cells in the centrilobular zone exhibit weak or no reaction for N-CAM. Perivascular smooth muscle cells and fibroblasts in the portal area and myofibroblasts around the central veins are negative for N-CAM. The present results indicate that the perisinusoidal stellate cells in the periportal and intermediate zones of the liver lobule characteristically express N-CAM, unlike other related mesenchymal cells, and suggest that the intralobular heterogeneity of N-CAM expression by stellate cells is related to the different maturational stages of these cells.

Clinicopathological significance of hypoxia‐inducible factor‐1α expression in human pancreatic carcinoma

Aims:  The transcription factor hypoxia‐inducible factor‐1α (HIF‐1α) plays a key role in the cellular adaptation to hypoxia and the activation of several genes that have been implicated in tumour growth. The aim of this study was to investigate the clinicopathological significance of HIF‐1α expression in pancreatic carcinoma.