Nobuko Ishii

Early recognition of hepatocellular carcinoma based on altered profiles of alpha-fetoprotein.

BACKGROUND The sugar-chain structures of circulating alpha-fetoprotein in patients with hepatocellular carcinomas differ from those in patients with cirrhosis. We studied the reactivity of alpha-fetoprotein with two lectins, Lens culinaris agglutinin A and erythroagglutinating phytohemagglutinin, to monitor the evolution of hepatocellular carcinoma in patients with cirrhosis. METHODS Among 361 patients with cirrhosis caused mainly by chronic hepatitis B or hepatitis C virus infection, 33 with base-line serum alpha-fetoprotein concentrations > or = 30 ng per milliliter or more were found to have hepatocellular carcinomas during a mean follow-up of 35 months. The lectin-reactive profiles of the alpha-fetoprotein in the serum of these 33 patients were analyzed and compared with those in the serum of 32 patients with cirrhosis who had increased base-line serum alpha-fetoprotein concentrations and were followed for at least 24 months but in whom hepatocellular carcinoma did not develop. RESULTS At the time of tumor detection, 24 (73 percent) of the 33 patients with cirrhosis and hepatocellular carcinoma had higher percentages of L. culinaris agglutinin A-reactive alpha-fetoprotein (alpha-fetoprotein L3), erythroagglutinating phytohemagglutinin-reactive alpha-fetoprotein (alpha-fetoprotein P4+P5), or both than the 32 patients with cirrhosis but no hepatocellular carcinoma. Among the 24 patients, one or both of the markers were first elevated 3 to 18 months before the hepatocellular carcinoma was detected by imaging techniques. CONCLUSIONS Measurements of the alpha-fetoprotein L3 and alpha-fetoprotein P4+P5 fractions of serum alpha-fetoprotein allow the differentiation of hepatocellular carcinoma from cirrhosis in some cases and serve as predictive markers for the development of hepatocellular carcinoma during the follow-up of patients with cirrhosis.

Interferon-|[alpha]| sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-|[kappa]|B inactivation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in a variety of cancer cells with little or no effect on normal cells. Human hepatoma cells, however, are resistant to TRAIL-induced apoptosis. Since interferon-α (IFN-α) is capable of enhancing TNF-α-induced apoptosis in certain cancer cells, we evaluated the effect of IFN-α on TRAIL-induced apoptosis of human hepatoma cells. IFN-α pretreatment enhanced TRAIL-induced apoptosis of HuH-7 and Hep3B cells, in which IFN-α upregulated the expression of DR5, a death receptor of TRAIL, and downregulated the expression of survivin, which has an antiapoptotic function. In contrast, IFN-α did not enhance TRAIL-induced apoptosis of HepG2 cells, in which expression of DR5 and survivin was not affected by IFN-α. On the other hand, TRAIL activated NF-κB composed of RelA-p50 heterodimer, a key transcription factor regulating cell survival, in HuH-7 and HepG2 cells. However, IFN-α pretreatment repressed the TRAIL-mediated activation of NF-κB and decreased its transcriptional activity in HuH-7 but not in HepG2 cells. Moreover, IFN-α pretreatment clearly augmented TRAIL-mediated caspase-8 activation in HuH-7 cells. Our results suggest that IFN-α could sensitize certain human hepatoma cells to TRAIL-induced apoptosis by stimulating its death signaling and by repressing the survival function in these cells.

Association between nonalcoholic fatty liver, markers of obesity, and serum leptin level in young adults.

OBJECTIVES:The aim of the present study was to clarify the risk factors for nonalcoholic fatty liver in young adults.METHODS:One thousand two hundred two students, aged 18–21 yr, received matriculation health examinations, including measurements of body mass index and percent body fat and determination of serum levels of ALT at Nagasaki University in 1998. One hundred twenty-nine were found to have borderline or elevated levels of serum ALT, and 105 of the 129 students (75 men and 30 women) were subjected to further analysis for the presence of fatty liver using ultrasonography, by which both the degree of steatosis and the abdominal wall fat index (AFI) corresponding to the ratio of visceral to s.c. adipose tissue (V/S ratio) were evaluated, in addition to determination of the serum level of leptin.RESULTS:Of 105 students, 74 (70%) had fatty liver. The incidence of moderately to severely fatty liver was significantly higher in men than in women. In parameters related to obesity, the close correlation between body mass index and percent body fat was observed in both sexes. The serum level of leptin correlated well with percent body fat and AFI (V/S ratio) in women, whereas it did not correlate with AFI (V/S ratio) in men. Multiple logistic regression analysis indicated that AFI (V/S ratio) was the only independent risk factor for fatty liver in both sexes.CONCLUSIONS:These results suggest that visceral fat distribution is a key risk factor for nonalcoholic fatty liver in young adults.

Evaluation of Nontumorous Tissue Damage by Transcatheter Arterial Embolization for Hepatocellular Carcinoma

The serial changes in serum hepatic enzyme activities by transcatheter arterial embolization (TAE) were analyzed in 17 patients with hepatocellular carcinoma to estimate the contribution to the value by the damage of tumor or nontumorous hepatic cells. The serum levels of relatively tumor-specific fructose 1,6-diphosphate (FDP) aldolase were elevated after TAE in the cases of both superselective and nonsuperselective TAE that were performed from the segmental and the nonsegmental hepatic artery, respectively, but we found the marked elevation of FDP aldolase in the cases of the superselective TAE. In contrast, the non-tumor-specific fructose 1-phosphate (F1P) aldolase was markedly elevated only in the cases of nonsuperselective TAE. The total amount of FDP aldolase released by TAE correlated significantly with the integrated tumor tissue volume (P less than 0.005), whereas the total amount of F1P aldolase output correlated significantly with the integrated nontumorous tissue volume (P less than 0.005) as defined by lipiodol accumulation on computerized tomography scan. The consequent changes in the total nontumorous liver volumes after TAE were also analyzed by the follow-up computerized tomography scan. The nonsuperselective TAE caused the significant total nontumorous liver atrophy when compared with the superselective TAE. The progression of the total nontumorous liver atrophy correlated significantly with F1P aldolase output by TAE (P less than 0.001) but not with FDP aldolase output. These results suggest that the outputs of FDP and F1P aldolase are useful to estimate the degree of the tumorous and nontumorous tissue damage by TAE, respectively, and F1P aldolase output can be used to predict the progression of liver atrophy caused by TAE.

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.

Differential effects of calcineurin inhibitors, tacrolimus and cyclosporin a, on interferon-induced antiviral protein in human hepatocyte cells†

The premise of our study is that selective inhibition of interferon (IFN) by calcineurin inhibitors contribute to the increased severity of hepatitis C virus (HCV) posttransplantation. Therefore, we examined the influence of calcineurin inhibitors in the human hepatocyte cell line on IFN‐α‐induced phosphorylation of Janus kinase (Jak) and signal transducers and activators of transcription (STAT), nuclear translocation of IFN‐stimulated gene factor 3 (ISGF‐3), IFN‐stimulated regulatory element (ISRE)‐contained promoter activity, and the expressions of antiviral proteins. Tacrolimus (Tac), but not cyclosporin A (CyA), had an inhibitory effect on IFN‐α‐induced double‐stranded ribonucleic acid (RNA)‐dependent protein kinase (PKR) in a dose‐dependent manner. STAT‐1 also acted in a similar fashion to PKR. IFN‐α combined with Tac attenuated the ISRE‐containing promoter gene activity as compared with IFN‐α alone. In contrast, its expression in pretreated CyA was slightly attenuated. In pretreated Tac, but not CyA, the levels of IFN‐α‐induced tyrosine phosphorylated STAT‐1 and ‐2 were clearly lower than those induced by IFN‐α alone. Tac and CyA did not decrease the IFN‐α‐induced JAK‐1 phosphorylation. The nuclear translocation rate of tyrosine phosphorylated STAT‐1 was inhibited by pretreatment of both Tac and CyA by western blotting and immunohistochemistry. In an HCV replicon system, pretreated Tac diminished the replication inhibitory effect of IFN‐α. In this study, we show that calcineurin inhibitors, especially Tac, are the negative regulators of IFN signaling in the hepatocyte; the greatest cause of such inhibition is the phosphorylation disturbance of STAT‐1, next to inhibition of the nuclear translocation of STAT‐1. In conclusion, disturbance of tyrosine phosphorylation of STAT‐1 resulted in diminished ISRE‐containing promoter activity and a decline in antiviral protein expression. Moreover, the replication of HCV was activated. This phenomenon is detrimental to IFN therapy after liver transplantation, and the selection of calcineurin inhibitors may warrant further discussion depending on the transplant situation. Liver Transpl 14:292–298, 2008.

Relationship between sustained elevation of serum alanine aminotransferase and progression from cirrhosis to hepatocellular carcinoma: comparison in patients with hepatitis B virus- and hepatitis C virus-associated cirrhosis.

Abstract  Most patients with hepatocellular carcinoma (HCC) in Japan have hepatitis B virus (HBV)‐or hepatitis C virus (HCV)‐associated cirrhosis. In the present study, the risk of HCC in patients with cirrhosis was analysed by the levels of serum alanine aminotransferase (ALT). One hundred and one (78%) of 129 patients with cirrhosis registered from April 1979 were followed at monthly intervals with the measurement of serum ALT. Of 101 patients, 38 tested positive for hepatitis B surface antigen (HBsAg) but negative for antibody to HCV (anti‐HCV; HBV group), 47 tested negative for HBsAg but positive for anti‐HCV (HCV group) and nine tested positive and seven tested negative for both. Mean serum ALT during follow‐up was calculated on the basis of monthly values during the observation period that started at enrolment and ended with the detection of HCC or at the end of March 1994. By the end of March 1994, 37 (37%) patients developed HCC; 12 were in the HBV group, 21 in the HCV group and four were in the group positive for both. Mean serum ALT during the observation period was similar in patients who developed HCC and those who did not develop HCC in the HBV group. In contrast, the value was significantly higher in patients who developed HCC than in patients who did not develop HCC in the HCV group (P < 0.05).

Changes in HBsAg carrier rate in Goto Islands, Nagasaki Prefecture, Japan

Annual mass examinations in an area where hepatitis B virus (HBV) infection is very prevalent revealed that 12.1% of inhabitants born during 1946-50 were positive for hepatitis B surface antigen (HBsAg), compared with only 0.6% of those born during 1971-75. To find out why the HBV carrier rate has fallen, changes in the modes of HBV infection were examined. The HBsAg positivity rate among mothers who gave birth to HBV carrier children in 1965 and before was 26.8% and that for such mothers whose babies were born in 1966 and after was 66.7%, whereas the HBsAg positivity rate among children born to HBV carrier mothers in 1965 and before and in 1966 and after were 30.8% and 28.3%, respectively. None of the 503 inhabitants who had no HBV markers in 1976 had become carriers by 1981. These findings indicate that the decrease in the prevalence of HBV carriage is caused mainly by the reduction in occurrence of horizontal transmission of HBV in infancy.

p48 (ISGF-3γ) Is Involved in Interferon-α-induced Suppression of Hepatitis B Virus Enhancer-1 Activity

Interferon-α (IFN-α) suppresses hepatitis B virus (HBV) gene expression by reducing its enhancer-1 activity. IFN-α induces transcription factors, interferon-stimulated gene factor 3 (ISGF3), and interferon regulatory factor-1 (IRF-1), which activate interferon-inducible gene expression through binding to the interferon-stimulated regulatory element (ISRE) “AGTTTCNNTTTCNC” in the gene promoters. We found the ISRE-like sequence “AGGCTTTCACTTTCTC” in the HBV enhancer-1 region and elucidated the role of this sequence. Gel mobility shift assay showed binding ofin vitro translated IRF-1 and in vitrotranslated p48 (ISGF3-γ), which is a component of ISGF3 to this sequence. However, nuclear extracts binding to this sequence from human hepatoma cells (HuH-7) treated with IFN-α contained only the protein consisted of p48. In transfection experiments, IFN-α suppressed the HBV enhancer-1 activity, and overexpression of p48 enhanced this inhibitory effect. Both mutation and deletion of the ISRE-like sequence in the HBV enhancer-1 region reduced the suppressive effect of IFN-α. Our results suggest that the ISRE-like sequence in the HBV enhancer-1 can interact with the protein containing p48 and mediate the IFN-α-induced suppression of the enhancer activity.

Receptor-independent augmentation of adenovirus-mediated gene transfer with chitosan in vitro

Recombinant adenovirus is one of the most widely used viral vectors for gene delivery. This study was designed to evaluate the ability of chitosan, a cationic, linear polysaccharide composed of beta(1,4) linked glucosamine partly containing N-acetyl-glucosamine, to enhance the in vitro infectivity of adenovirus to mammalian cells. Wild type and a fiber-mutant replication-defective recombinant adenoviruses expressing beta-galactosidase were used. In the latter, an RGD peptide, the binding site for alpha(v)beta3 and alpha(v)beta5 integrin, was introduced in the fiber knob enabling adenovirus receptor-independent viral infection. Enhanced effect of chitosan on the infectivity of both adenoviruses was observed in Chinese hamster ovary cells that do not express the receptor for adenovirus with beta-galactosidase activity assay and x-gal staining. These data indicate the receptor-independent mechanism(s) for this enhancement effect. In addition, we found that pH of the culture medium, and molecular mass and concentration of chitosan are also critical factors. Thus, the highest effect was obtained with 0.1-1 microg/ml of chitosan with molecular mass of 19K and 40K in the culture medium of pH 6.4; on the other hand, the effect was negligible with the higher chitosan concentrations (10 microg/ml or more), lower or higher molecular mass (11K and 110K) of chitosan, or at pH of 7.4. Studies using several cell lines with variable levels of adenoviral infectivity revealed that this enhanced effect is evident in the cells with poor infectivity to adenovirus. Since chitosan is biocompatible and inexpensive, these data indicate that chitosan may be a potential candidate for a non-viral vector to safely increase adenoviral infectivity to mammalian cells, particularly those with poor susceptibility to adenoviral infection.