Takuya Tsutsumi

Reciprocal regulation of α-fetoprotein and albumin gene expression by butyrate in human hepatoma cells

Abstract Background/Aims: Butyrate, a product of colonic bacterial flora, functions as an antiproliferative agent and induces cell differentiation in a variety of cell types. In the present study, the effects of butyrate on cell growth and expression of α-fetoprotein (AFP) and albumin genes in HuH-7 human hepatoma cells were investigated. Methods: The HuH-7 cells were treated with sodium butyrate (0-1 mmol/L), and numbers of viable cells were counted at 24, 48, and 72 hours after treatment. To elucidate the effects of sodium butyrate on AFP and albumin gene expression, Northern blotting and transient chloramphenicol acetyltransferase plasmid transfection experiments were performed. Results: Cell growth was dose dependently inhibited by sodium butyrate. By Northern blot analysis, the level of AFP messenger RNA was reduced by treatment with sodium butyrate, whereas the level of albumin messenger RNA was elevated by this treatment. In transient chloramphenicol acetyltransferase plasmid transfection experiments, sodium butyrate repressed the AFP promoter activity but did not change the AFP enhancer or silencer activities. In contrast, the albumin promoter activity was stimulated by sodium butyrate. Conclusions: These results suggest that butyrate leads to the reciprocal differentiating regulation of AFP and albumin gene expression at the transcriptional level in human 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.

Interaction of interferon-α with interleukin-1β or tumor necrosis factor-α on hepatitis B virus enhancer activity

Abstract The interaction of IFN-α with IL-1β or TNF-α on hepatitis B surface antigen (HBsAg) expression was analysed in hepatitis B virus (HBV)-DNA integrated PLC/PRF/5 and non-integrated HuH-7 human hepatoma cells. Secretion of HBsAg in PLC/PRF/5 cells was reduced by IFN-α, IL-1β or TNF-α, and synergistically depressed when -α was used in combination with IL-1β or TNF-α. By Northern blot analysis, the levels of HBsAg mRNA were suppressed by IFN-α in combination with IL-1β or TNF-α. In the chloramphenicol acetyltransferase plasmid transfection assay, IFN-α in combination with IL-1β or TNF-α caused a much greater suppression of HBV enhancer activity than IFN-α, IL-1β or TNF-α alone in both hepatoma cells. These findings suggest that the interaction of IFN-α with IL-1β or TNF-α synergistically represses HBV enhancer activity, resulting in depressed expression of HBsAg.

Regulation of albumin and α-fetoprotein gene expression by colloid osmotic pressure in human hepatoma cells

BACKGROUND Colloid osmotic pressure has been thought to regulate albumin synthesis; however, the exact mechanism remains obscure. In the present study, the effect of colloid osmotic pressure on the albumin and alpha-fetoprotein gene expression in HuH-7 human hepatoma cells was analyzed. METHODS HuH-7 cells were treated with albumin or dextran (mean mol wt, 70,000), and changes in the levels of albumin and alpha-fetoprotein messenger RNA (mRNA) were analyzed by Northern blotting. Furthermore, in transient chloramphenicol acetyltransferase (CAT) plasmid transfection experiments, effects of colloid osmotic pressure on CAT activities were studied. RESULTS By Northern blot analysis, the levels of both albumin and alpha-fetoprotein mRNA were dose-dependently suppressed by the elevation of colloid osmotic pressure and returned to pretreatment levels 48 hours after the culture medium containing dextran was replaced with a dextran-free fresh medium. In transient CAT plasmid transfection experiments, the increased level of colloid osmotic pressure resulted in the repression of both albumin and alpha-fetoprotein promoter activities. In contrast, alpha-fetoprotein enhancer activity, which possibly regulates not only alpha-fetoprotein but also albumin gene expression, was not affected by changes in colloid osmotic pressure. CONCLUSIONS These results suggest that colloid osmotic pressure regulates both albumin and alpha-fetoprotein gene transcription through the modulation of their promoter activities.

Regulation of alkaline phosphatase gene expression in human hepatoma cells by bile acids

Bile acid‐dependent secretion and the translationally regulated synthesis of alkaline phosphatase (ALP) in rat liver cell culture and by bile duct ligation has already been demonstrated. With the advent of ALP cDNA cloned sequences, the mechanism of the effect of bile acids on ALP activity and the expression of the ALP gene in different hepatoma cells was investigated. The HuH7 and HepG2 cells were treated with taurine‐conjugated cholic acid (CA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) at 0–1mmol/L and the numbers of viable cells were counted at 24, 48 and 72h after treatment. A [3H]‐thymidine incorporation study was performed with different bile acids at a concentration of 1 mmol/L for 48h. The cellular activity of ALP in HuH7 and HepG2 cells by bile acids was measured by a substrate‐specific enzymatic assay. To elucidate the effects of bile acids on ALP gene expression, a northern blotting experiment using hybridization with mouse placental ALP cDNA was performed. Cellular ALP activity was time‐ and dose‐dependently increased in both HuH7 and HepG2 cells treated by CA and CDCA; however, no change in ALP activity was observed following treatment with UDCA compared with controls. Induction of ALP activity was dominant in HepG2 cells and independent of cell growth and proliferation. The addition of UDCA synergistically reduced the increased activity of ALP produced by CA and CDCA in both HuH7 and HepG2 cells. By northern blot analysis, the level of ALP mRNA was elevated by CA and CDCA; however, levels of ALP mRNA were suppressed by UDCA. In conclusion, CA and CDCA cause up‐regulation of ALP mRNA and UDCA leads to down‐regulation of ALP mRNA by its interaction with either CA or CDCA. We assume that increased ALP synthesis in hepatoma cells after bile acid treatment results from an enhanced rate of transcription rather than translation of mRNA.

Hepatocyte growth factor down-regulates the α-fetoprotein gene expression in PLC/PRF/5 human hepatoma cells

Abstract Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the α-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of β-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.

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.

Sodium butyrate induces alkaline phosphatase gene expression in human hepatoma cells

Background and Aims: Butyrate, a natural product of colonic bacterial flora, has been reported to increase the activities of a number of enzymes, including alkaline phosphatase, (ALP) in several cancer cell lines. However, butyrate‐induced ALP gene expression in human hepatoma cells has not been previously demonstrated. In the present study, the effects of sodium butyrate on cell growth and proliferation, cellular activity and expression of ALP gene in human hepatoblastoma‐derived HepG2 cells were investigated.

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.