Shotaro Tsuruta

Utilization of variant-type of human α-fetoprotein promoter in gene therapy targeting for hepatocellular carcinoma

We previously reported that the retroviral vector (LNAFW0.3TK) expressing the herpes simplex thymidine kinase (HSVtk) gene under the control of the 0.3 kb human α-fetoprotein (AFP) promoter provided the ganciclovir (GCV)-mediated cytotoxicity in the high AFP-producing (HuH-7) but not in the low AFP-producing (huH-1/cl.2) human hepatoma cells. In the present study, we constructed the retroviral vector (LNAFM0.3TK) in which the HSVtk gene expression is regulated by the variant-type of the 0.3 kb human AFP promoter with a G-to-A substitution at nucleotide −119, a point mutation responsible for hereditary persistence of human AFP and the vector was applied to three human hepatoma cell lines, HuH-7, huH-1/cl.2 and intermediate AFP-producing cells (PLC/PRF/5). By the reporter gene transfection assay, the activity of the variant-type of the promoter was much higher than that of the wild-type of the promoter in both HuH-7 and huH-1/cl.2 cells. Consistent with this, LNAFM0.3TK infection could sensitize huH-1/cl.2 cells, as well as HuH-7 and PLC/PRF/5 cells to GCV, but did not affect cell growth of nonhepatoma cells (HeLa). In addition, the bystander effect was achieved more efficiently by LNAFM0.3TK infection than LNAFW0.3TK infection in HuH-7 cells. These results suggest that the variant-type of the human AFP promoter ensures the therapeutic gene expression in gene therapy particularly for the low AFP-producing hepatoma cells.

Baseline Serum Cholesterol Is Associated with a Response to Pegylated Interferon Alfa-2b and Ribavirin Therapy for Chronic Hepatitis C Genotype 2

Background. HCV infection is associated with lipid disorders because this virus utilizes the host lipid metabolism to sustain its life cycle. Several studies have indicated that higher concentrations of serum cholesterol and LDL before treatment are important predictors of higher rates of sustained virological response (SVR). However, most of these studies involved patients infected with HCV genotype 1. Thus, we performed a multi-institutional clinical study to evaluate the impact of lipid profiles on SVR rates in patients with HCV genotype 2. Methods. A total of 100 chronic hepatitis C patients with HCV genotype 2 who received peg-IFN alfa-2b and ribavirin therapy were consecutively enrolled. The significance of age, sex, BMI, AST level, ALT level, WBC, hemoglobin, platelet count, gamma-glutamyltransferase, total cholesterol level (TC), LDL level, HCV RNA, and histological evaluation was examined for SVR using logistic regression analysis. Results. The 100 patients infected with HCV genotype 2 were divided into 2 groups, an SVR group and a non-SVR group. Characteristics of each group were subsequently compared. There was no significant difference in the level of HCV RNA, BMI, platelet, TG, or stage of fibrosis between the groups. However, there were significant differences in the levels of TC and LDL-C. In multivariate logistic regression analysis using baseline characteristics, high TC level was an independent and significant risk factor (relative risk 18.59, P = 0.015) for SVR. Conclusion. Baseline serum total cholesterol levels should be considered when assessing the likelihood of sustained treatment response following the course of peg-IFN and ribavirin therapy in patients with chronic HCV genotype 2 infection.

Frequency of elevated biomarkers in patients with cryptogenic hepatocellular carcinoma

Background The incidence of hepatocellular carcinoma (HCC) continues to increase in Japan, but the clinical characteristics of Japanese patients with HCC have not been well described. The aim of this study was to determine the frequencies and utilities of elevated α-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) levels as biomarkers in cryptogenic HCC. Material/Methods A total of 2638 patients with HCC diagnosed between 1999 and 2010 in the Nagasaki Association Study of Liver (NASLD) were recruited for this study. The cause of HCC was categorized into 4 groups; HCC-B, HCC-C, HCC-BC, and HCC-nonBC. The significance of factors was examined for HCC-nonBC using logistic regression analysis in all patients. Results Multivariate analysis identified age, sex, BMI, alcohol consumption, platelet count, AST, ALT, AFP, DCP, and TNM stage as independent and significant risk factors for HCC-nonBC. According to TNM stage, the median AFP levels in HCC-nonBC with TNM stages I, II, and III were significantly lower than in either HCC-B or HCC-C. In TNM stage IV, the median AFP level in HCC-nonBC was significantly lower than in either HCC-B or HCC-BC. The median DCP levels in HCC-nonBC with TNM stages I and II were significantly higher than those in either HCC-B or HCC-C. In TNM stage III, the median DCP level in HCC-nonBC was significantly higher than that in HCC-C. Conclusions DCP was more sensitive than AFP for the diagnosis of early stage cryptogenic HCC. DCP should be used as the main serum test for cryptogenic HCC detection.

Inhibitory effect of prostaglandin Δ12-PGJ2 on cell proliferation and α-fetoprotein expression in HuH-7 human hepatoma cells

9-deoxy-Δ9, Δ12-13,14-dihydro-prostaglandin D2 (Δ12-PGJ2) is a potent inhibitor of proliferation of tumor cells. In the present study, the effect of Δ12-PGJ2 on the α-fetoprotein (AFP) and the albumin gene expression was analyzed in HuH-7 human hepatoma cells. Δ12-PGJ2 inhibited the cell growth and reduced the medium AFP concentrations dose-dependently. To determine whether this decline of AFP depends only on the relative decrease in cell numbers by Δ12-PGJ2, or is in part, due to the decrease in the cellular AFP synthesis by Δ12-PGJ2, Northern blot analysis was performed in this study. By Northern blotting, it was shown that Δ12-PGJ2 caused a marked reduction in the levels of the AFP mRNA and the albumin mRNA. In contrast, the level of the β-actin mRNA was not changed by Δ12-PGJ2. In the transient chloramphnicol acetyltransferase plasmid transfection experiments, Δ12-PGJ2 did not suppress the AFP enhancer activity, which possibly regulates both the AFP and the albumin gene expression in HuH-7 hepatoma cells, but resulted in the selective repression of the AFP and the albumin promoter activity. These results suggest that Δ12-PGJ2 suppresses not only cell growth but also expression of the AFP gene and the albumin gene at the transcriptional level in human hepatoma cells.

Differential Regulation of Albumin Gene Expression by Heparin-Binding Epidermal Growth Factor-Like Growth Factor in α-Fetoprotein-Producing and -Nonproducing Human Hepatoma Cells

The effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on albumin gene expression were analyzed in α-fetoprotein (AFP)-producing and AFP-nonproducing human hepatoma cells, HuH-7 and huH-1/cl.2, in which the AFP silencer is inactive and active, respectively. HB-EGF selectively suppressed the AFP enhancer activity, resulting in decreased levels of both albumin and AFP mRNA in HuH-7 cells. In contrast, HB-EGF did not influence the albumin transcripts in huH-1/cl.2 cells or primary culture of rat hepatocytes, although the AFP enhancer activity was reduced by HB-EGF in huH-1/cl.2 cells as well as in HuH-7 cells. These results indicate that the AFP silencer interacts with the AFP enhancer to block its activity regulating both the albumin and AFP promoters, and that HB-EGF downregulates the albumin gene expression only in the absence of the AFP silencer through the repression of the AFP enhancer activity.

Relationship of α-fetoprotein levels and development of hepatocellular carcinoma in hepatitis C patients with liver cirrhosis

α-fetoprotein (AFP) is a tumor marker of hepatocellular carcinoma (HCC) and has also been reported to reflect the effectiveness of long-term low-dose interferon (IFN) therapy in hepatitis C virus (HCV)-infected patients with chronic liver disease. The correlation between AFP levels and the incidence of HCC has been discussed over a long period. We investigated whether high levels of AFP at the time of diagnosis were associated with an increased incidence of HCC in patients with HCV. A total of 107 HCV patients with liver cirrhosis without other risks were evaluated for the predictive value of non-invasive risk factors for HCC, including age, gender, alcohol intake, aspartate and alanine aminotransferase levels, bilirubin, albumin, platelet count and AFP levels at study entry, as well as the IFN therapy received. During the follow-up period, HCC developed in 68 (63.6%) patients. Kaplan-Meier estimates were made to assess the cumulative risk of HCC. The 10-year cumulative incidence rate of HCC was 80%. Cox regression analysis was performed on several variables, including age, gender, alcohol consumption, experience of IFN therapy and biochemical parameters. The following factors were identified as exhibiting an increased risk of HCC by univariate analysis: aspartate transaminase (AST) ≥71 IU/l, alanine transaminase (ALT) ≥60 IU/l, AFP ≥6 ng/ml and IFN therapy. Multivariate analysis identified that the AFP level [6–19 ng/ml: hazard ratio (HR), 2.22; P=0.006 and ≥20 ng/ml: HR, 2.09; P=0.003] was an independent and significant risk factor for the development of HCC. A slightly elevated (6–19 ng/ml) AFP level may be a risk factor for HCC in certain cases. By contrast, AFP levels <6 ng/ml indicate a low risk of HCC development in HCV patients with liver cirrhosis.

Effect of simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on α-fetoprotein gene expression through interaction with the ras-mediated pathway

BACKGROUND/AIMS The ras proto-oncogene encodes a small GTP-binding protein (Ras) which regulates cell growth and differentiation by relaying signals from the cell surface to the nucleus. In the present study, the role of Ras signal transduction pathway in alpha-fetoprotein (AFP) gene expression was evaluated in HuH-7 human hepatoma cells using simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, which blocks Ras function through inhibition of farnesylation, and the ras(val-12) expression vector. METHODS The HuH-7 cells were treated with simvastatin (10 micromol/l), or both simvastatin and mevalonate (300 micromol/l), and numbers of viable cells were counted after treatment. To elucidate the effects of simvastatin on AFP gene expression and the interactive effect of simvastatin on Ras signal transduction pathway, Northern blotting and transient chloramphenicol acetyltransferase plasmid transfection assays were performed. RESULTS Cell growth was inhibited by simvastatin, and this growth inhibition was restored by addition of mevalonate. Levels of AFP mRNA but not albumin mRNA were elevated by simvastatin in a dose-dependent manner (1-10 micromol/l). AFP promoter and enhancer activities were stimulated by simvastatin. In contrast, both activities were repressed by transfection with the ras(val-12) expression vector. The ras(val-12)-mediated repression was restored by simvastatin and returned to the repressed level by simvastatin plus mevalonate. CONCLUSIONS These results indicate that the Ras signal transduction pathway functions to down-regulate the AFP gene transcription in human hepatoma cells.

Gene Targeting to Hepatomas (AFP)

Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis worldwide, especially in eastern Asia and Africa (1). Recent advances in delivering genes to mammalian cells stimulate the possibility of gene therapy for human diseases, including cancer gene therapy (2). One approach of gene therapy for cancers is the transduction of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells, because the killing effect of the HSV-tk product on the virus-infected cells is seen in only proliferating cells (3). HSV-tk can efficiently phosphorylate nucleoside analogs, and the phosphorylated products act as a chain terminator of DNA synthesis, leading to cell death (4). In addition, successful application of suicide gene therapy for cancer, in part, relies on the bystander effect, where the active chemotherapeutic agent produced in target cells diffuses from cells to neighboring malignant cells in sufficient concentrations to induce growth inhibition (5).

Analysis of interferon regulatory factor 1 and 2 gene expression in human hepatoma cell lines

Abstract The transcription activator, Interferon regulatory factor-1 (IRF-1), has been shown to function as a tumor suppressor and the structurally related interferon regulatory factor-2 (IRF-2) as a counterpart. The loss of IRF-1 function or overexpression of IRF-2 are possible mechanisms of cancer development. In the present study, IRF-1 and IRF-2 gene expression was analyzed in human hepatoma cell lines (HuH7, huH 1, HepG2 and PLC/PRF/5) and the normal liver. We found a small amount of exon-skipped IRF-1 mRNA in HuH7 and huH 1, and the low level of IRF-1 mRNA in PLC/PRF/5, but we could not find the overexpression of IRF-2 mRNA in any hepatoma cells lines. Thus, it is possible that hepatocarcinogenesis involves some alterations in expression of IRF-1 gene.