The in vivo antitumor effects of type I-interferon against hepatocellular carcinoma: the suppression of tumor cell growth and angiogenesis.

Type I-interferon (IFN) is considered to exert antitumor effects through the inhibition of cancer cell proliferation and angiogenesis. Based on the species-specific biological activity of IFN, we evaluated each antitumor mechanism separately. We further examined the antitumor effects of type I-IFN combined with sorafenib. Human IFN (hIFN) significantly inhibited the proliferation of human hepatocellular carcinoma (HCC) Hep3B cells and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Although mouse IFN (mIFN) did not inhibit the proliferation of Hep3B cells in vitro, mIFN, as well as hIFN, showed significant antitumor effects in mouse Hep3B cell-xenograft model. Furthermore, mIFN treatment amplified the antitumor effects of sorafenib in vivo with the suppression of angiogenesis. The DNA chip analysis showed that the mIFN treatment promoted the antitumor signal pathways of sorafenib, including anti-angiogenic effects. Unlike the effects observed in in vitro experiments, mIFN showed an antitumor effect in the mouse Hep3B cell-xenograft model, suggesting a role of the anti-angiogenic activity in the in vivo tumoricidal effects of type I-IFN. In addition, our findings suggested the clinical utility of combination therapy with type І-IFN and sorafenib for HCC.

Serum zinc value in patients with hepatitis virus-related chronic liver disease: association with the histological degree of liver fibrosis and with the severity of varices in compensated cirrhosis.

The relationships between the serum mineral concentrations and the endoscopic findings of esophageal varices have been poorly investigated. In this study, we investigated hepatitis virus-positive patients who had undergone a liver biopsy (n = 576) and 75 patients with compensated cirrhosis in order to evaluate the association of the zinc value with the severity of liver fibrosis and esophageal varices. The mean zinc values decreased with the progression of fibrosis (METAVIR score; F0-1: 71.3 ± 11.3, F2: 68.9 ± 11.7, F3: 66.3 ± 11.8, F4: 63.9 ± 15.0). In the hepatitis virus-related compensated cirrhosis, the mean zinc value decreased with the severity of varices (patients without varices: 66.3 ± 12.6, patients with low-risk varices: 62.5 ± 13.7, patients with high-risk varices: 55.6 ± 13.0). The zinc value was significantly lower in patients with varices than in those without varices (59.3 ± 13.6 vs 66.3 ± 12.6, p<0.05). The zinc value was also significantly lower in the patients with a high risk of bleeding than in those with a low risk (55.6 ± 13.0 vs 64.6 ± 13.1, p<0.01). These findings suggest that the zinc value is not only an indicator of an abnormal metal metabolism, but is also a simple parameter associated with hepatitis virus-related various conditions, including the degree of liver fibrosis and the severity of esophageal varices in compensated cirrhosis.

New malignant grading system for hepatocellular carcinoma using the Sonazoid contrast agent for ultrasonography.

BACKGROUND: The ultrasonography contrast agent Sonazoid provides parenchyma-specific contrast imaging (Kupffer imaging) based on its accumulation in Kupffer cells. This agent also facilitates imaging of the fine vascular architecture in tumors through maximum intensity projection (MIP). We examined the clinical utility of the malignancy grading system for hepatocellular carcinoma (HCC) using a combination of 2 different contrast-enhanced ultrasonography images. METHODS: We studied 121 histologically confirmed cases of HCC (well-differentiated, 45; moderately differentiated, 70; poorly differentiated, 6). The results of Kupffer imaging were classified as (1) iso-echoic pattern or (2) hypo-echoic pattern. The MIP patterns produced were classified into one of the following categories: fine, tumor vessels were not clearly visualized and only fine vessels were visualized; vascular, tumor vessels were visualized clearly; irregular, tumor vessels were thick and irregular. Based on the combined assessment of Kupffer imaging and the MIP pattern, the samples were classified into 4 grades: Grade 1 (iso-fine/vascular), Grade 2 (hypo-fine), Grade 3 (hypo-vascular), and Grade 4 (hypo-irregular). RESULTS: The distribution of moderately and poorly differentiated HCCs was as follows: Grade 1, 4 % (1/24); Grade 2, 52 % (15/29); Grade 3, 85 % (44/52); and Grade 4, 100 % (16/16). The grading system also predicted portal vein invasion in 72 resected HCCs: Grade 1, 0 % (0/4); Grade 2, 13 % (1/8); Grade 3, 23 % (11/48); and Grade 4, 67 % (8/12). CONCLUSIONS: This new malignant grading system is useful for estimation of histological differentiation and portal vein invasion of HCC.

Association of amino acid imbalance with the severity of liver fibrosis and esophageal varices.

BACKGROUND: The relationships between the metabolic parameters and the endoscopic findings of esophageal varices have been poorly investigated. We investigated the association of the branched-chain amino acids to tyrosine ratio (BTR) with the severity of liver fibrosis and esophageal varices. MATERIAL AND METHODS: We studied hepatitis C virus (HCV)-positive chronic liver disease patients who had undergone liver biopsy (n = 149). The relationship between the BTR values and the liver fibrotic stage was investigated. We also studied whether the BTR value was associated with the presence and bleeding risk of varices in patients with HCV-related compensated cirrhosis. RESULTS: The mean values of the BTR decreased with the progression of the fibrosis (METAVIR score: F0-1: 6.40 ± 1.19; F2: 5.85 ± 1.33; F3: 5.24 ± 0.97, F4: 4.78 ± 1.14). In the 58 patients with HCV-related compensated cirrhosis, the mean values of the BTR decreased with the severity of varices (patients without varices: 5.01 ± 1.15, patients with a low-risk varices: 4.42 ± 1.06, patients with a high-risk varices: 3.86 ± 1.02). The BTR value was significantly lower in the patients with varices than in those without varices (4.17 ± 1.07 vs. 5.01 ± 1.15, P < 0.01). The BTR value was also significantly lower in the patients with a high risk of hemorrhage than in those with a low risk (3.86 ± 1.02 vs. 4.78 ± 1.14, P < 0.01). Furthermore, the BTR value was the most significantly different parameter, with the smallest P-value among all the factors examined, including the platelet count and albumin level. CONCLUSION: A decreased BTR value was found to be associated with the progression of liver fibrosis and severity of varices.

Relationship between elevation of glycated albumin to glycated hemoglobin ratio in patients with a high bleeding risk of esophageal varices.

BACKGROUND/AIMS: Variceal hemorrhaging due to portal hypertension is a severe complication of liver cirrhosis. Although several biomarkers have been reported as predictors of the presence of varices, it is still difficult to assess the risk of variceal bleeding without esophagogastroduodenoscopy (EGD). The ratio of glycated albumin (GA) to glycated hemoglobin (HbA1c) was reported to increase with the progression of liver fibrosis. The aim of the study was to investigate whether the GA/HbA1c ratio is related to the severity and bleeding-risk of the varices. METHODOLOGY: We measured the GA/HbA1c ratio of HCV-related cirrhotic patients with Child-Pugh class A status and analyzed its relationship with the presence and bleeding risk of varices. RESULTS: The GA/HbA1c ratio was higher in the patients who had the varices with a high risk of hemorrhage than in the patients with a low risk of bleeding. In addition, the GA/HbA1c ratio was higher in patients with varices than that in patients without varices. Furthermore, the GA/HbA1c ratio was the most significantly different parameter of all the factors examined, including the platelet count, prothrombin activity and albumin level. CONCLUSIONS: The GA/HbA1c ratio is increased in patients with varices and with the bleeding risk of the varices.

Elevation of the glycated albumin to glycated hemoglobin ratio during the progression of hepatitis C virus related liver fibrosis.

AIM: To analyze the relationship between the glycated albumin (GA) to glycated hemoglobin (HbA1c) ratio and the histological grading of liver fibrosis. METHODS: The study retrospectively included consecutive hepatitis C virus positive chronic liver disease patients (n = 142) who had undergone percutaneous liver biopsy between January 2008 and March 2010 at our institution. The ratios of GA/HbA1c were calculated in all patients to investigate the relationship with the degree of the liver fibrosis. The values of the aspartate aminotransferase-to-platelet ratio index (APRI), an excellent marker for the evaluation of liver fibrosis, were also calculated. In addition, we combined the ratio of GA/HbA1c and the APRI in order to improve our ability to detect the presence of significant liver fibrosis. RESULTS: Sixty-one (43%) patients had either no fibrosis or minimal fibrosis (METAVIR score: F0-F1), while 25 (17%) had intermediate fibrosis (F2). Fifty-six (39%) patients had severe fibrosis (F3-F4) and 27 of them had cirrhosis (F4). The mean values of the GA/HbA1c increased with the progression of the fibrosis (F0-1: 2.83 ± 0.24, F2: 2.85 ± 0.24, F3: 2.92 ± 0.35, F4: 3.14 ± 0.54). There was a significant difference between the F0-F1 vs F4, F2 vs F4, and F3 vs F4 groups (P < 0.01, P < 0.01, P < 0.01 and P < 0.05, respectively). The GA/HbA1c ratio was significantly higher in the patients with cirrhosis (F4) than in those without cirrhosis (F0-F3) (3.14 ± 0.54 vs 2.85 ± 0.28, P < 0.0001). The GA/HbA1c ratio was also significantly higher in the patients with severe fibrosis (F3-F4) than in those without severe liver fibrosis (F0-F2) (3.03 ± 0.41 vs 2.84 ± 0.24, P < 0.001). Furthermore, the GA/HbA1c ratio was also significantly higher in the patients with significant fibrosis (F2-F4) than in those without significant liver fibrosis (F0-F1) (2.98 ± 0.41 vs 2.83 ± 0.24, P < 0.001). The diagnostic performance of the increased GA/HbA1c ratio (> 3.0) was as follows: its sensitivity and specificity for the detection of liver cirrhosis (F4) were 59.3% and 70.4%, respectively and its sensitivity and specificity for the detection of severe liver fibrosis (F3-F4) were 50.0% and 74.4%, respectively. With regard to the detection of significant fibrosis (F2-F4), its sensitivity was 44.4% and its specificity was 77.0%. Although even the excellent marker APRI shows low sensitivity (25.9%) for distinguishing patients with or without significant fibrosis, the combination of the APRI and GA/HbA1c ratio increased the sensitivity up to 42.0%, with only a modest decrease in the specificity (from 90.2% to 83.6%). CONCLUSION: The GA/HbA1c ratio increased in line with the histological severity of liver fibrosis, thus suggesting that this ratio is useful as a supportive index of liver fibrosis.

Cost-effectiveness analysis on the surveillance for hepatocellular carcinoma in liver cirrhosis patients using contrast-enhanced ultrasonography.

AIM: Sonazoid is a new contrast agent for ultrasonography (US). Contrast-enhanced ultrasonography (CEUS) using Sonazoid enables Kupffer imaging, which improves the sensitivity of hepatocellular carcinoma (HCC) detection. However, there are no studies on the cost-effectiveness of HCC surveillance using Sonazoid. METHODS: We constructed a Markov model simulating the natural history of HCV-related liver cirrhosis (LC) patients, and compared three strategies (no surveillance, US surveillance and CEUS surveillance). The transition probability and cost data were obtained from published data. The simulation and analysis were performed using TreeAge pro 2009 software. RESULTS: When compared to the no surveillance group, the US and CEUS surveillance groups increased the life expectancy by 1.67 and 1.99 quality-adjusted life-years (QALY), respectively, and the incremental cost effectiveness ratio (ICER) were 17 296 $US/QALY and 18 384 $US/QALY, respectively. These results were both less than the commonly-accepted threshold of $US 50 000/QALY. Even if the CEUS surveillance group was compared with the US surveillance group, the ICER was $US 24 250 and thus cost-effective. Sensitivity analysis showed that the annual incidence of HCC and CEUS sensitivity were two critical parameters. However, when the annual incidence of HCC is more than 2% and/or the CEUS sensitivity is more than 80%, the ICER was also cost-effective. CONCLUSIONS: Contrast-enhanced ultrasonography surveillance for HCC is a cost-effective strategy for LC patients and gains their longest additional life years, with similar degree of ICER in the US surveillance group. CEUS surveillance using Sonazoid is expected to be used not only in Japan, but also world-wide.

[A case of hepatocellular carcinoma with autoimmune hepatitis showing marked reduction of tumors by oral administration of UFT-E].

We report a case of hepatocellular carcinoma (HCC) in association with autoimmune hepatitis (AIH). In May 2003, a 66- year-old man was admitted to our hospital because of acute liver dysfunction. He was diagnosed with AIH, and his liver function was normalized by oral administration of the corticosteroid. In July 2007, when he was admitted for the treatment of bacterial pneumonia, two liver tumors (S4: ø4 cm and S2: ø1 cm) were revealed by abdominal CT scan, and the serum level of AFP was high. According to the findings of imaging diagnosis and laboratory data, the patient was diagnosed as having HCC. Since the standard invasive therapies of HCC were not accepted by the patient and his family, he was treated by oral administration of UFT-E (tegafur/uracil: 200 mg/day). Three months after the initiation of administration, CT scan showed a remarkable reduction of the tumors, and his serum AFP level was decreased to the normal range. This case shows that HCC develops in an AIH patient even if liver function is maintained in the normal range. It also suggests the clinical usefulness of UFT-E in the management of HCC given the difficulty of treatment by the standard therapies.

Long-term interferon therapy after radiofrequency ablation is effective in treating patients with HCV-associated hepatocellular carcinoma.

PURPOSE: This study investigates the usefulness of long-term interferon (IFN) therapy following radiofrequency ablation (RFA) for HCV-associated hepatocellular carcinoma (HCC). METHODS: This is a retrospective observational study. Patients underwent pegylated IFN-α/ribavirin combination therapy for 48 weeks and then were maintained on IFN-α administration on average for 68 weeks (mean total duration 116 weeks). Patients who underwent IFN monotherapy were maintained on IFN administration on average for 78 weeks. RESULTS: There were biases in the background factors between the IFN and non-IFN groups. Therefore, a covariate adjustment was performed using the propensity score. An analysis of 20-matched patients from each group showed the 5-year cumulative survival rate was higher in the IFN group than in the non-IFN group (100 and 76%, respectively), and the 3-year cumulative recurrence rate was significantly lower in the IFN group than in the non-IFN group (38.0 and 64.2%, respectively). In 14 patients (i.e., IFN responders), the serum alanine aminotransferase (ALT) level remained normalized at 30 IU/mL or lower, regardless of disappearance of serum HCV RNA. In these patients, the cumulative recurrence rate was low, the hazard ratio was 0.158 (95% confidence interval = 0.045-0.561, P = 0.004), and the serum albumin level was retained. CONCLUSION: These results show the importance of maintaining the liver function and suggest that long-term IFN administration after RFA inhibits recurrence and contributes to an improved outcome in patients (in particular, IFN responders) who initially develop HCC.

Vitamin K2 downregulates the expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma cells.

AIM: Vitamin K2 exerts an antitumor activity on human hepatocellular carcinoma (HCC), however, its inhibitory mechanism has not yet been clarified. This study was designed to identify the attractive target molecule of vitamin K2 and shed some light on its effects on fibroblast growth factor receptor (FGFR)3 in HCC cells. METHODS: The changes in the gene expression of HuH-7 after vitamin K2 treatment were evaluated by a DNA chip analysis. The mRNA and protein levels of FGFR were evaluated by semiquantitative reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and western blot analysis. The promoter activity of the FGFR3 gene was measured by a dual-luciferase assay. RESULTS: The DNA chip analysis revealed different inhibitory rates of gene expression of FGFR3 (60.6%) and FGFR1 (19.4%) after vitamin K2 treatment. Vitamin K2 suppresses the proliferation of HuH-7 in a dose-dependent manner and its inhibitory rate reached approximately 61.8% at the dose of 30 microM. FGFR3 mRNA was significantly reduced based on semiquantitative RT-PCR and decreased 61.5% by a real-time PCR method after vitamin K2 treatment, but FGFR1 mRNA was not. The level of FGFR3 protein was also reduced by vitamin K2 treatment. The luciferase assay demonstrated that vitamin K2 significantly suppressed the promoter activity of FGFR3. Furthermore, the FGFR3-ERK1/2 signaling pathway was suppressed by vitamin K2 treatment. CONCLUSION: These findings suggest that vitamin K2 may suppress the proliferation of HCC cells through the downregulation of the FGFR3 expression. The transcriptional suppression of FGFR3 may be a novel mechanism of the vitamin K2 action for HCC cells.

Hepatoma-derived growth factor is induced in liver regeneration.

AIM: Hepatoma-derived growth factor (HDGF) is a heparin-binding protein, which has been suggested to be involved in the development of kidneys, the cardiovascular system and the liver. We have shown that HDGF is highly expressed in parenchymal hepatocytes in the developing liver and promotes fetal hepatocyte proliferation. In the present study, we asked whether HDGF expression was related to liver regeneration. METHODS: We examined the mRNA and protein expressions of HDGF in two liver regeneration models. In addition, cellular distribution of HDGF in the regenerating liver was investigated by immunohistochemistry. RESULTS: In the carbon tetrachloride (CCl(4))-treated liver, HDGF expression was induced and the peak was detected at 24 h after the CCl(4 )injection. HDGF expression was also enhanced in the hepatectomy model and the peak was detected at 12 h after surgery. The increased expression of HDGF protein was also confirmed by western blotting. Expression of the HDGF gene in the regenerating liver was dominantly detected in parenchymal hepatocytes. CONCLUSION: These findings showed that HDGF expression was induced in parenchymal hepatocytes before the DNA synthesis in the regenerating liver, suggesting the possible involvement of HDGF in liver regeneration as an autocrine factor.

Involvement of hepatoma-derived growth factor in the growth inhibition of hepatocellular carcinoma cells by vitamin K(2).

BACKGROUND: Vitamin K(2) has been reported to suppress the growth of human hepatocellular carcinoma (HCC) in vitro and hepatocarcinogenesis in hepatitis C virus (HCV)-related cirrhosis in vivo. Hepatoma-derived growth factor (HDGF) is a unique nuclear targeting growth factor that is highly expressed in HCC cells and is a possible prognostic factor for patients with HCC. We investigated the regulation of HDGF expression by vitamin K(2). METHODS: Three HCC-derived cell lines, HepG2, HuH-7, and SK-Hep-1, were used. Cell number was determined with the MTT assay. The expression levels of HDGF mRNA and protein were measured by the real-time reverse transcriptase-polymerase chain reaction (PCR) method and ELISA and Western blot analysis, respectively. The HDGF promoter activity was measured by a dual luciferase-reporter assay. RESULTS: Vitamin K(2) suppressed the growth of the three HCC cell lines in a dose-dependent manner. Vitamin K(2) significantly suppressed the expression of the HDGF protein and mRNA in three cell lines. By a luciferase assay, vitamin K(2) significantly suppressed the promoter activity of the HDGF protein. Based on some luciferase-reporter plasmids containing truncated promoter regions, the possible responsive site of vitamin K(2) seems to reside in the region -1 to -150 bp of the HDGF gene. CONCLUSIONS: These findings suggested that regulation of the HDGF gene expression is one of the crucial mechanisms of vitamin K(2)-induced cell growth suppression for HCC.

Vitamin K2 inhibits the proliferation of HepG2 cells by up-regulating the transcription of p21 gene.

AIM: Vitamin K2 has been reported to inhibit the growth of human hepatocellular carcinoma (HCC) in vitro and suppress hepatocarcinogenesis in vivo. However, its inhibitory mechanism has not yet been clarified. METHODS: Different concentrations of vitamin K2 (30, 10, 1, 0.1 and 0.01 muM) were added to the HCC cell line HepG2 to assess effects on cell growth. The effect of vitamin K2 on cell cycle progression was determined by flow-cytometric analysis. The expression of cell cycle regulatory proteins p21 and p27 was then examined by Western blot. Whether vitamin K2 regulates the gene expression through action on the p21 promoter region was investigated by luciferase assay. RESULTS: Vitamin K2 inhibited the growth of HepG2 cells dose-dependently, and its inhibitory rate reached approximately 50% at the dose of 30 muM after 96 h treatment. After treatment with vitamin K2, the proportion of cells in G0-G1 phase increased, and in S phase decreased. Apoptotic cells were not detected. The expression of cell cycle regulatory protein p21 was induced by vitamin K2 treatment, but p27 was not. By the luciferase assay, vitamin K2 significantly activated the promoter of p21. Knock-down of p21 by siRNA reversed the growth inhibition of HepG2 cells by vitamin K2. CONCLUSIONS: The findings suggest that vitamin K2 suppresses the proliferation of HCC cells by blocking the cell cycle G1/S progression through the transcriptional induction of p21.

Chemoprevention of spontaneous development of hepatocellular carcinomas in fatty liver Shionogi mice by a cyclooxygenase-2 inhibitor.

Cyclooxygenase 2 (COX-2) and retinoid X receptor alpha (RXRalpha) are suggested to have roles in carcinogenesis. COX-2 inhibitors have been reported to suppress growth of hepatocellular carcinoma (HCC) cell lines in vitro. However, little is known about the preventive effect of these drugs on spontaneous hepatocarcinogenesis in vivo. Etodolac exists in a racemic mixture containing S- and R-etodolac. S-etodolac is responsible for COX-2 inhibitory activity and R-etodolac is related to the downregulation of RXRalpha. Here, the effect of etodolac on spontaneous development of HCC in fatty liver Shionogi mice is evaluated. Etodolac was administered at a low (2 mg/kg) or high (10 mg/kg) dose three times a week for 16 months starting at the age of 3 months. The development of HCC was suppressed slightly in the high-dose group, and suppressed markedly in the low-dose group, although the development of fatty liver was not inhibited in either group. Plasma prostaglandin E2 levels were also decreased significantly in the low-dose group, consistent with the suppression of HCC. The expression of RXRalpha and proliferating cell nuclear antigen in non-tumorous liver tissues was decreased significantly in both the low-dose and high-dose groups. These findings show that etodolac treatment at an optimum dose suppresses hepatocarcinogenesis in vivo, and may be useful for preventing the development of HCC in humans.

Hemochromatosis with mutation of the ferroportin 1 (IREG1) gene.

The HFE, H ferritin, TFR2, and ferroportin 1 genes of a Japanese patient diagnosed as having hemochromatosis were amplified by PCR and sequenced. A novel mutation in the ferroportin 1 was found in the patient. It was located in the noncoding region of the ferroportin 1; nucleotide 117 adenine was changed to guanine, 7 nucleotides downstream the iron responsive element (IRE) region. This mutation was not found in the patient's son or daughter, or in 50 healthy individuals. It was suggested that the mutation in the ferroportin 1 may be related to hemochromatosis of this patient.

Peroxisome proliferator-activated receptor gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2, and ciglitazone, induce growth inhibition and cell cycle arrest in hepatic oval cells.

There is growing evidence to show that hepatic oval cells contribute to liver regeneration, dysplastic nodule formation, and hepato-carcinogenesis. Peroxisome proliferator-activated receptors (PPARs) and their ligands play an important role in cell growth, inflammatory responses, and liver pathogenesis including fibrosis and cancer. However, little is known about the role of PPARgamma/its ligands in the growth and differentiation of hepatic oval cells. In this study, we found that OC15-5, a rat hepatic oval cell line, expressed PPARgamma at mRNA and protein levels, and a natural ligand for PPARgamma, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), and a synthetic ligand, ciglitazone, inhibited growth of OC15-5 cells by arresting at G1-S in a dose-dependent manner. Apoptosis was also induced in OC15-5 cells by 15d-PGJ2 treatment. In OC15-5 cells treated with 15d-PGJ2, the expression of CDK inhibitor, p27(Kip1), was up-regulated, while that of p21(WAF1/Cip1), p18(INK4C) CDK2, CDK4, and cyclin E was unchanged. In addition, delayed up-regulation of AFP expression was observed in OC15-5 cells after 15d-PGJ2 or ciglitazone treatment. This is the first report to show that the PPARgamma ligand was involved in the growth, cell cycle, and differentiation of hepatic oval cells, raising the possibility that the PPARgamma ligands may regulate liver regeneration and hepato-carcinogenesis.

Involvement of cell cycle regulatory proteins and MAP kinase signaling pathway in growth inhibition and cell cycle arrest by a selective cyclooxygenase 2 inhibitor, etodolac, in human hepatocellular carcinoma cell lines.

Recent studies have shown that selective cyclooxygenase-2 (COX-2) inhibitors induce growth inhibition and cell cycle arrest in hepatocellular carcinoma (HCC) cell lines. However, the mechanism by which COX-2 inhibitors regulate the cell cycle and whether or not growth signal pathways are involved in the growth inhibition remain unclear. In this study, we investigated the mechanisms of growth inhibition and cell cycle arrest by etodolac, a selective COX-2 inhibitor, in HCC cell lines, HepG2 and PLC/PRF/5, by studying cell cycle regulatory proteins, and the MAP kinase and PDK1-PKB/AKT signaling pathways. Etodolac inhibited growth and PCNA expression and induced cell cycle arrest in both HCC cell lines. Etodolac induced p21WAF1/Cip1 and p27Kip1 expression and inhibited CDK2, CDK4, CDC2, cyclin A and cyclin B1 expression, but did not affect cyclin D1 or cyclin E. HGF and 10% FBS induced ERK phosphorylation, but phosphorylation of p38, JNK and AKT was down-regulated by etodolac. PD98059, a selective inhibitor of ERK phosphorylation, induced growth inhibition, the expression of p27Kip1 and cell cycle arrest. In conclusion, p21WAF1/Cip1, p27Kip1, CDK2, CDK4, CDC2, cyclin A, cyclin B1 and the MAP kinase signaling pathway are involved in growth inhibition and cell cycle arrest by a selective COX-2 inhibitor in HCC cell lines.

Evaluation of interleukin-12-induced interferon-y production in vitro by peripheral blood mononuclear cells in patients with chronic liver disease.

BACKGROUND/AIMS: Interleukin-12 plays an important role in anti-tumor immune response by induction of interferon-gamma production by T cells and NK cells, and by activation of cytotoxic T cells and NK cells. We evaluated interleukin-12-induced interferon-gamma production as one of the immunological markers of patients with chronic liver diseases. METHODOLOGY: Interleukin-12-induced interferon-gamma production was measured in vitro in peripheral blood mononuclear cells from 28 hepatocellular carcinoma patients, 10 liver cirrhosis patients, 14 chronic hepatitis patients and 16 healthy individuals. RESULTS: The hepatocellular carcinoma patients exhibited a reduced interleukin-12 responsiveness for interferon-gamma production compared to the liver cirrhosis patients, the chronic hepatitis patients and the healthy individuals. The reduced interferon-gamma production seemed to roughly reflect clinical stage in the hepatocellular carcinoma patients. The interferon-gamma production correlated with neither alpha-fetoprotein nor protein induced by vitamin K absence II. CONCLUSIONS: The level of interleukin-12-induced interferon-gamma production by peripheral blood mononuclear cells in the patients with hepatocellular carcinoma was significantly lower than that in the patients with liver cirrhosis which is thought to be a premalignant state. The measurement of interferon-gamma production may be useful in evaluating severity of chronic liver disease from an immunological point of view.

Novel mutation and multiple mutations found in the human butyrylcholinesterase gene.

BACKGROUND: Mutations in human butyrylcholinesterase (BChE) are linked to low BChE activity and abnormal response to muscle relaxants. METHODS: Twenty Chinese patients with hepatic disease and low cholinesterase activity, and one Japanese patient and her mother were tested for BChE activity and BChE phenotype. The butyrylcholinesterase (BCHE gene) was amplified by polymerase chain reaction (PCR) and sequenced. Mutant BChE was expressed in 293 cells. RESULTS: A novel mutation was found in one Chinese patient at nucleotide 943, where A was changed to T (943 A-->T), causing substitution of threonine 315 by serine (T315S). The T315S mutant had half of the normal BChE activity. One Japanese patient with low BChE activity had three nucleotide substitutions, 355 C-->T, 988 T-->A, and 1615 G-->A. The amino acid substitutions were Q119stop, L330I, and A539T, respectively. The single mutant L330I had low BChE activity, but the double mutant L330I/A539T had normal activity. CONCLUSIONS: The L330I and the novel T315S mutation caused a decreased BChE activity. The T315S mutation is one of the first BChE mutations reported in the Chinese population. Multiple mutations in BChE may interact with each other in an intramolecular manner.

Inhibition of the expression of alpha-smooth muscle actin in human hepatic stellate cell line, LI90, by a selective cyclooxygenase 2 inhibitor, NS-398.

Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS-398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC.