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Biomarkers that enable an accurate diagnosis of hepatitis C virus (HCV)-induced liver diseases are necessary to prevent subsequent patient morbidity and suffering from the onset of hepatocellular carcinoma (HCC). In particular, the identification of novel biomarkers for liver cirrhosis (LC) will be an important new diagnostic tool since more than 70% of HCV-induced LCs are destined to develop into HCC. In our current study, we performed a search for new serological protein biomarkers of HCV-induced chronic hepatitis (CH), LC and HCC, using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The disease-affected spots were subsequently identified as isoforms of protein components of haptoglobin, transthyretin, the haptoglobin α-chain and apolipoprotein A-IV (apo A-IV), and in specific instances were significantly reduced in LC (p

Patterns of hypocholesterolemic lipid fractions in 295 patients with liver diseases, malignant tumors, arteriosclerotic and renal diseases with cholesterol (Chol) levels of

Interleukin (IL)-18 plays an important role in the pathogenesis of several liver diseases as well as Fas-mediated apoptosis. However, the effects of IL-18 on Fas-mediated liver injury have not been well elucidated. Therefore, we examined the effects of IL-18 on Fas-mediated apoptosis in in vitro and in vivo experiments. We found that recombinant IL-18 protected mouse hepatocellular carcinoma cell lines, BNL5, from Fas-mediated apoptosis in a dose-dependent manner with up-regulation of both nuclear factor (NF) kappaB and X-linked inhibitors of apoptosis (XIAP). IL-18 transgenic (Tg) mice were also protected from Fas-mediated liver injury and this was further confirmed by histological study and TUNEL staining. In IL-18 Tg mice, up-regulation of XIAP and down-regulation of caspase 3 were observed after injection of anti-Fas, which was consistent with the in vitro findings. These results suggest that IL-18 suppresses Fas-mediated apoptosis of hepatocytes by up-regulation of NFkappaB and XIAP, following inhibition of caspase-3 activity. This observation raises the possibility that IL-18 could be a therapeutic strategy for Fas-mediated liver injury as a negative regulator of XIAP.

CD40 is expressed primarily on B cells and plays an important role in antigen presentation, B cell proliferation, and T cell activation. It has been reported that the CD40 signal modulates apoptosis and has an anti-viral effect in certain cells. Therefore, we investigated the expression and the function of CD40 in HCV-associated chronic liver disease. The expression of CD40 on liver tissues was determined through immunohistochemistry on 50 liver specimens obtained from HCV-positive patients. The effect of CD40 signaling on apoptosis of HepG2 cells was assessed using the MTT assay. The effect of CD40 stimulation on NF-kappaB activation was determined in NF-kappaB reporter gene-transfected HepG2 cells with the Luciferase assay. CD40 positive hepatocytes were observed in both periportal and lobular areas, accompanied by inflammation. In both areas, CD40 staining intensity became significantly stronger, correlating with the histological grading. Similarly, it became stronger with the progression of the histological staging in F1, F2 and F3 cases; however, the expression level decreased in F4 cases. CD40 ligation induced apoptosis in HepG2 cells in the presence of 500 ng/ml of actinomycin D, while CD40 ligation alone could not. Anti-CD40 monoclonal antibody caused NF-kappaB activation in HepG2 cells in a dose-dependent manner. These results suggest that hepatocyte over-expression of CD40 might play an important role in regulating hepatocyte survival and death in HCV-associated chronic liver diseases.

Flavopiridol was one of the first cyclin-dependent kinase inhibitors demonstrated to have an antitumor effect in several cancer types. Here, we investigated the effects of flavopiridol on TNF-related apoptosis-inducing ligand (TRAIL) in the human hepatocellular carcinoma (HCC) cell lines HLE and HepG2, and evaluated the role of flavopiridol in apoptosis. To better understand the mechanism of increased TRAIL sensitivity in HCC cells, we determined the effect of flavopiridol on cell surface expression of TRAIL and TRAIL receptors using flow cytometry analysis. The levels of survivin, FLIP, Bcl-xL and X-chromosome-linked IAP (XIAP) in treated and untreated cells was also determined. Flavopiridol decreased cell viability in a dose-dependent manner in the two HCC cell lines tested. The pan-caspase inhibitor z-VAD-FMK did not inhibit the effect. However, subtoxic levels of flavopiridol dramatically enhanced TRAIL-induced apoptosis in both cells. Flavopiridol up-regulated TRAIL, TRAIL-R1 and TRAIL-R2 in both cell lines. In addition, flavopiridol down-regulated expression of survivin in both cell lines, and expression of FLIP and Bcl-xL were down-regulated in HLE cells. In summary, flavopiridol augmented TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of survivin, FLIP and Bcl-xL. Thus, combining flavopiridol with a TRAIL agonist may prove to be an effective new strategy for treatment of HCC.

The cellular apoptosis susceptibility protein (CAS) is the human homologue of the product of the essential yeast chromosome segregation gene, CSE1, and has important roles in tumor necrosis factor (TNF)-induced apoptosis and cell proliferation. In this study, we used immunoblotting and immunohistochemistry to look at CAS expression in human hepatocellular carcinoma (HCC) cells. We also studied the correlation between CAS expression and cell proliferation. To do this, we studied the expression of proliferating cell nuclear antigen (PCNA) by immunostaining and at apoptosis by in situ nick end-labeling (TUNEL), followed by calculation of the PCNA labeling index (PCNA LI) and TUNEL labeling index (TUNEL LI). CAS was constitutively expressed in human HCC cell lines and was primarily confined to the cytoplasm of the cells. PCNA LI and TUNEL LI were significantly higher in HCC than in non-tumor tissue (p

Cyclooxygenase (COX)-2 is upregulated in a variety of human cancers, including in hepatocellular carcinoma (HCC), whereas it is undetectable in most normal tissue. Evidence suggests that COX-2 is likely to be involved in hepatocarcinogenesis and, thus, COX-2 may be involved in an early process in carcinogenesis, dedifferentiation. To address this possibility, we investigated the effect of COX-2 inhibitors on TNF-related apoptosis, inducing ligand (TRAIL) sensitivity and its molecular mechanisms, with special attention to anti-apoptotic proteins. We used the highly selective COX-2 inhibitors, NS398 and CAY10404. We also used the MTT assay and cytological analysis of DAPI-stained DNA to assess viability and apoptosis in two HCC cells (SK-Hep1 and HLE). In order to ask what led to increased sensitivity to TRAIL in HCC cells, cell surface expression of TRAIL and TRAIL-receptors was investigated using flow cytometry analysis. Expression of survivin, X-chromosome-linked IAP (XIAP), Bcl-xL, AKT and phospho-AKT was also investigated using immunoblotting. COX-2 inhibitors resulted in a concentration-dependent decrease in cell viability in the two HCC cell lines tested. Subtoxic levels of COX-2 inhibitors did not significantly augment TNFalpha-induced apoptosis but did dramatically enhance TRAIL-induced apoptosis in both cell lines. TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5) expression was significantly up-regulated in SH-Hep1 and HLE cells. TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) expression was up-regulated only in SK-Hep1. Expression of survivin and Bcl-xL was down-regulated in SK-Hep1 and HLE cells in the presence of CAY10404 but XIAP was not affected. Expression of survivin, Bcl-xL and XIAP was down-regulated in SK-Hep1 cells in the presence of NS398. Survivin expression was also down-regulated in the presence of NS398 in HLE cells. Finally, NS398 also decreased phospho-AKT in SK-Hep1 cells. These results demonstrate that COX-2 inhibitors can induce apoptosis and augment TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of both survivin and AKT signaling.

The cellular apoptosis susceptibility protein (CAS) is the human homologue of the product of the essential yeast chromosome segregation gene, CSE1, and has important roles in tumor necrosis factor (TNF)-induced apoptosis and cell proliferation. In this study, we used immunoblotting and immunohistochemistry to look at CAS expression in human hepatocellular carcinoma (HCC) cells. We also studied the correlation between CAS expression and cell proliferation. To do this, we studied the expression of proliferating cell nuclear antigen (PCNA) by immunostaining and at apoptosis by in situ nick end-labeling (TUNEL), followed by calculation of the PCNA labeling index (PCNA LI) and TUNEL labeling index (TUNEL LI). CAS was constitutively expressed in human HCC cell lines and was primarily confined to the cytoplasm of the cells. PCNA LI and TUNEL LI were significantly higher in HCC than in non-tumor tissue (p0.01); however, the ratio of TUNEL LI/PCNA LI in HCC was significantly lower than that of non-tumor tissue. Immunohistochemistry revealed that the staining intensity score of CAS in HCC was significantly higher than that of non-tumor tissue (p0.05). These results indicate that there is an augmentation of pro-liferative activity and apoptosis in HCC tissue, as compared to non-tumor tissue. There was a significant positive correlation between CAS and PCNA LI (p0.05). In addition, we observed an inverse relationship between CAS expression and TUNEL LI, although the correlation did not reach statistical significance. These results suggest that CAS is expressed at higher levels in human HCC tissue than in non-tumor tissue. CAS may be associated with cell proliferation rather than apoptosis, and further, CAS might play an important role in the development of human HCCs.

Secondary bile acids have been implicated as an important etiological factor in colorectal cancer. We investigated the effects of ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) on the growth and cytotoxicity in HT29 human colonic adenocarcinoma cells. Proliferation assay, cell cycle analysis and cell death characterization by bile acids were performed. Both UDCA and DCA reduced their proliferation rate of HT29 over 48 h in a concentration- and time-dependent manner compared with control cultures. In terms of cell cycle effects, however, UDCA induced G2/M arrest, while DCA induced G1 arrest in a concentration- and time-dependent manner. As for the effects of each bile acid on cell toxicity, UDCA induced early apoptosis and DCA induced both early apoptosis and necrosis. Bile acids play an important role in regulating cell survival and cell death in colon adenocarcinoma cells.

It has been demonstrated that CD34-positive cells isolated from human peripheral blood differentiate into endothelial cells and contribute to neoangiogenesis in adults. We investigated the role of CD34-positive endothelial cells in liver samples from patients with hepatitis B virus (HBV)-associated chronic liver diseases. Tissue sections were obtained by liver biopsy from 25 patients with HBV-associated chronic liver diseases and were examined by immunohistochemistry using anti-CD34, anti-von Willebrand factor (vWF), and anti-vascular endothelial growth factor (VEGF) antibodies. CD34-positive, but vWF-negative endothelial cells were observed, particularly in the sinusoids and vascular endothelial cells. We counted these cells and expressed the results as a CD34-labeling index (LI). The CD34 LI did not correlate with VEGF expression and the CD34 LI of patients who progressed to hepatocellular carcinoma (HCC) tended to increase compared to those that did not progress to HCC. CD34 LI was an independent risk factor for development of HCC (relative risk, 35.689; P = 0.033). We conclude that CD34-positive endothelial cells in patients with HBV-associated chronic liver diseases might play a role in hepatocarcinogenesis.

PPARgamma is known to induce apoptosis in malignant tumor cells, but the mechanism of this induction is not well understood. We investigated induction of apoptosis with 15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), a PPARgamma ligand, in cholangio cell carcinoma (CCC) cells (RBE, ETK-1 or HuCCT-1). Apoptosis was induced in RBE and ETK-1 cells with 15d-PGJ2, but not in HuCCT-1 cells, although PPARgamma was expressed in all CCC cells. Apoptosis-related proteins were also expressed, including FLIP, bclx, Apaf-1 and XIAP, but expression levels differed among the three cell lines. RBE cells treated with 15d-PGJ2 showed caspase activation, and it appeared that PPARgamma-induced apoptosis was dependent on caspase activation. However, neither ETK-1 nor HuCCT-1 cells showed significant activation of caspase-8 or -3 with 15d-PGJ2 treatment, raising the possibility of a caspase-independent apoptosis induction pathway. XIAP was down-regulated by 15d-PGJ2 in all three CCC cell lines. Therefore, 15d-PGJ2 induces apoptosis in CCC cells via caspase-dependent or independent pathways. 15d-PGJ2 may also induce down-regulation of XIAP and may promote caspase cascade activation through TNF-family receptor signaling pathways.

Currently, pyogenic liver abscess is not frequent, but it is a severe infectious disease. However a strategy for the effective treatment of liver abscess is not established. We analyzed 75 cases of liver abscess over an eight year period and evaluated their prognosis, any associated underlying disease, or the effect of percutaneous transhepatic abscess drainage (PTAD). For all 75 cases, laboratory data were analyzed and imaging studies were performed. Next, PTAD and antibiotic administration were started on these cases as first choice treatments. These treatments were continued until the laboratory data of the patient were restored to within the normal range. Those cases that were PTAD non-effective or required operation for underlying diseases, underwent operations. Of the total 75 cases, 63 survived after treatment and 12 cases died. Bacteria were detected in 50 cases and Klebsiella pneumoniae was detected in 31 of these 50 cases, but 25 out of 75 cases were negative. The biliary system was the main route of infection. PTAD was effective, especially in cases that were complicated with disseminated intravascular coagulation (DIC) or acute renal failure (ARF). PTAD is an effective treatment for liver abscess, it is especially useful in the restoration of severe general conditions as indicated by this study.

Infection with Hepatitis B virus (HBV) genotype C predominates in Japan. We analyzed the efficacy of interferon (IFN) alpha or beta in the treatment of chronic hepatitis B patients with HBV genotype C and the clinical predictors for therapeutic response. Forty-three genotype C-infected, chronic hepatitis B e antigen (HBeAg)-positive patients (32 men and 11 women with a mean age of 35.6+/-10.1 years) who had been treated with IFN therapy were retrospectively studied. The patients were classified into two treatment groups. Short-term therapy group was administered a 5-6 MU dose three times weekly for 4 weeks, and the long-term therapy group for 24 weeks. At the end of the follow-up period, 4 (15%) of 27 short-term therapy group patients and 6 (38%) of 16 long-term therapy group patients had normalized serum ALT levels and seroconversion of HBeAg to anti-HBe (p=0.137). Multivariate analysis for parameters most important for the efficacy of IFN therapy was performed using Cox proportional hazard models in order to investigate the association between baseline characteristics of patients and the response to IFN treatment. As a result, the p-values of IFN treatment group and sex were

Infection with Hepatitis B virus (HBV) genotype C predominates in Japan. We analyzed the efficacy of interferon (IFN) alpha or beta in the treatment of chronic hepatitis B patients with HBV genotype C and the clinical predictors for therapeutic response. Forty-three genotype C-infected, chronic hepatitis B e antigen (HBeAg)-positive patients (32 men and 11 women with a mean age of 35.6+/-10.1 years) who had been treated with IFN therapy were retrospectively studied. The patients were classified into two treatment groups. Short-term therapy group was administered a 5-6 MU dose three times weekly for 4 weeks, and the long-term therapy group for 24 weeks. At the end of the follow-up period, 4 (15%) of 27 short-term therapy group patients and 6 (38%) of 16 long-term therapy group patients had normalized serum ALT levels and seroconversion of HBeAg to anti-HBe (p=0.137). Multivariate analysis for parameters most important for the efficacy of IFN therapy was performed using Cox proportional hazard models in order to investigate the association between baseline characteristics of patients and the response to IFN treatment. As a result, the p-values of IFN treatment group and sex were0.05, and both factors can be recognized as independent significant factors (relative risk, 2.93 and 2.53; p=0.027 and 0.040, respectively). Furthermore, the cumulative rates of seroconversion of HBeAg to anti-HBe analyzed by the Kaplan-Meier method was significantly higher in the female group (p=0.015) and in the long-term IFN therapy group (p=0.0046). In summary, long-term IFN therapy may be more effective than short-term IFN therapy for patients with chronic HBV genotype C infection.

Serum concentration levels of des-gamma-carboxy prothrombin (DCP), alpha-fetoprotein (AFP) and Lens culinaris agglutinin-reactive fraction (AFP-L3) are useful tumor markers for the diagnosis of hepatocellular carcinoma (HCC). Recently, a novel immunoassay using the electrochemiluminescence (ECLIA) was developed to enable measurement of low-concentration of DCP. This study investigated the usefulness of high-sensitive DCP for the early diagnosis of HCC. The subjects consisted of 90 patients with viral cirrhosis who could be followed for at least 24 months from 1992 to 1997. Fifty-six of these patients developed HCC and 34 patients had not by 1998. We measured the serum levels of high-sensitive DCP, AFP and %AFP-L3 every 3 months during 2 years before the detection of tumor in patients with HCC and during 2 years from 1995 to 97 in patients without HCC. Youden's index was calculated for evaluation of the ideal cut-off levels. The patterns of serial changes during 2 years were divided into two types: fluctuating type and non-fluctuating type. Cut-off levels of 40 mAU/ml for high-sensitive DCP (Youden's index = 0.435), 20 ng/ml for AFP (Youden's index = 0.442) and 10% for %AFP-L3 (Youden's index = 0.364) gave the highest index for each marker. When these markers were combined, the combination of high-sensitive DCP, AFP and %AFP-L3 gave the highest accuracy (sensitivity = 82.1%, specificity = 82.4%, accuracy = 82.2%). Fluctuating type of high-sensitive DCP, AFP and %AFP-L3 levels were found in 15 (17%), 29 (32%) and 11 (12%) patients, respectively. The rate of complication with HCC in the patients who showed the fluctuating type of high-sensitive DCP levels was significantly greater than that in the patients who showed non-fluctuating type (P

Serum concentration levels of des-gamma-carboxy prothrombin (DCP), alpha-fetoprotein (AFP) and Lens culinaris agglutinin-reactive fraction (AFP-L3) are useful tumor markers for the diagnosis of hepatocellular carcinoma (HCC). Recently, a novel immunoassay using the electrochemiluminescence (ECLIA) was developed to enable measurement of low-concentration of DCP. This study investigated the usefulness of high-sensitive DCP for the early diagnosis of HCC. The subjects consisted of 90 patients with viral cirrhosis who could be followed for at least 24 months from 1992 to 1997. Fifty-six of these patients developed HCC and 34 patients had not by 1998. We measured the serum levels of high-sensitive DCP, AFP and %AFP-L3 every 3 months during 2 years before the detection of tumor in patients with HCC and during 2 years from 1995 to 97 in patients without HCC. Youden's index was calculated for evaluation of the ideal cut-off levels. The patterns of serial changes during 2 years were divided into two types: fluctuating type and non-fluctuating type. Cut-off levels of 40 mAU/ml for high-sensitive DCP (Youden's index = 0.435), 20 ng/ml for AFP (Youden's index = 0.442) and 10% for %AFP-L3 (Youden's index = 0.364) gave the highest index for each marker. When these markers were combined, the combination of high-sensitive DCP, AFP and %AFP-L3 gave the highest accuracy (sensitivity = 82.1%, specificity = 82.4%, accuracy = 82.2%). Fluctuating type of high-sensitive DCP, AFP and %AFP-L3 levels were found in 15 (17%), 29 (32%) and 11 (12%) patients, respectively. The rate of complication with HCC in the patients who showed the fluctuating type of high-sensitive DCP levels was significantly greater than that in the patients who showed non-fluctuating type (P0.01). These results suggest that periodic measurement of serum DCP levels using ECLIA method is very useful for HCC screening and predicting the development of HCC.