Comparison study of the expressions of myristoylated alanine-rich C kinase substrate in hepatocellular carcinoma, liver cirrhosis, chronic hepatitis, and normal liver.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent substrate for protein kinase C (PKC) in a variety of cells. The aim of this study was not only to evaluate the expression and localization of MARCKS in various pathological liver tissues, including HCC, but also to analyze the difference in MARCKS expression between hepatitis virus-induced HCC and cirrhosis. The level of MARCKS and its phosphorylated proteins, as well as its localization, were determined using Western blot and/or immunohistochemistry in HCC and other pathological liver tissues. We also analyzed the change of MARCKS localization on the influence of MARCKS phosphorylation in the HLF cancer cell line by phosphorylation study. In addition, the relationship between MARCKS expression and proliferative activity was studied in HCC. In the immunohistochemical study, a very small amount of MARCKS protein was found along the contour of the hepatocellular membrane in normal liver and in cases of chronic hepatitis. MARCKS was up-regulated in liver cirrhosis tissue and was localized in the cytoplasm of hepatocytes. The expression of MARCKS was down-regulated in HCC tissues, as compared with non-tumorous liver cirrhosis tissues from the same patients. Furthermore, MARCKS was serine-phosphorylated in liver cirrhosis and HCC, and phosphorylated MARCKS was detected in a cytosolic fraction of these tissues. In a phosphorylation study using the HLF HCC cell line, MARCKS was displaced from the plasma membrane to the cytosol following the activation of protein kinase C (PKC) by phorbol 12-myristrate 13-acetate (PMA). Furthermore, the activity of cyclin D1 and cyclin E kinases was found to be higher in HCCs with low MARCKS expression than in HCCs with high MARCKS expression. These results suggest that up-regulation of MARCKS might be essential in the generation of cirrhotic nodules through chronic hepatitis from normal liver, and that the phosphorylation and/or down-regulation of MARCKS might play an important role in the development and progression of HCC from liver cirrhosis.

Enhanced expression of adaptor molecule p46 Shc in nuclei of hepatocellular carcinoma cells: study of LEC rats.

Shc protein is known to be related to cell proliferation and carcinogenesis. However, the involvement of Shc in hepatocellular carcinoma (HCC) remains unknown. In the present study, we report that p46 Shc is probably expressed in the nuclei of hepatocytes and/or cancer cells during the development of HCC in Long-Evans Cinnamon (LEC) rats. The expression and localization of Shc in various pathological liver tissues obtained from LEC rats were analyzed by immunohistochemical study and Western blotting. Furthermore, tyrosine phosphorylation of Shc in various pathological liver tissues of LEC rats was studied by immunoprecipitation using a monoclonal anti-phosphotyrosine antibody. Although p66 Shc was detected in none of the liver tissues, regardless of pathological status, the expression of p46 Shc and that of p52 Shc increased proportionately with the development of HCC in LEC rats. Furthermore, although p52 Shc was localized only in the cytoplasm of hepatocytes and/or cancer cells, p46 Shc was found to express in both the nuclei and the cytoplasm of hepatocytes and/or cancer cells in precancerous and cancerous tissues of LEC rat liver. Tyrosine phosphorylation of p46 Shc and p52 Shc was detected only in cancer cells, and p46 Shc in such cells was much more heavily phosphorylated than p52 Shc. These results suggest that enhanced expression of p46 Shc and p52 Shc, as well as p46 Shc tyrosine phosphorylation, was involved not only in the process from normal liver to chronic hepatitis, but also in the transition from chronic hepatitis into HCC in LEC rats. Furthermore, unlike p52 Shc, p46 Shc was detected not only in the cytoplasm but also in the nuclei of hepatocytes (especially in transformed hepatocytes), and p46 Shc expressed in the nuclei may be closely related to hepatocarcinogenesis in LEC rats.

Enlargement of thermal ablation zone by the combination of ethanol injection and radiofrequency ablation in excised bovine liver.

The efficacy of radiofrequency ablation (RFA) and RFA with concurrent ethanol injection (EI-RFA) was compared. RFA (3-cm-electrode) was applied to bovine liver using three types of RFA equipment; Radionics, RITA and Radio Therapeutics Corporation (RTC). For EI-RFA, 5 ml of 99.5% ethanol was injected around the electrode. A total number of 40 RFA and EI-RFA treatments were performed. We compared RFA with EI-RFA by examining the size, shape of ablation zone, treatment time, power, and needle tip temperature. Liver specimens were examined for pathological changes. EI-RFA produced a larger zone of ablation than RFA alone using Radionics and RITA (Radionics, 35.3+/-7.4 cm(3) vs 23.2+/-7.7 cm(3), p<0.05; RITA, 30.7+/-10.3 cm(3) vs 19.7+/-4.7 cm(3), p<0.05), corresponding to shortest diameters of coagulation zone (Radionics, 3.7+/-0.4 cm vs 3.0+/-0.4 cm, p<0.05; RITA, 3.8+/-0.4 cm vs 3.1+/-0.3 cm, p<0.01). However, a larger ablation zone was not seen with the RTC device. The ablated volume per energy and the ablated volume per current density administered were greater with EI-RFA than with RFA using Radionics (p<0.05). The shape of the ablated zone changed from ellipsoid to spherical with EI-RFA using Radionics. No pathological differences between RFA and EI-RFA samples were detected. For a given amount of energy and current administered, ethanol injection caused a better ablation effect, in terms of the size and shape of the ablated zone, than RFA with Radionics and RITA equipment.

Percutaneous ethanol and lipiodol injection therapy for hepatocellular carcinoma.

Radiofrequency ablation (RFA) therapy is of great significance in the treatment of hepatocellular carcinoma (HCC) or metastatic liver tumors. RFA is able to achieve widely coagulated necrosis in a few sessions without major complications. However, HCC cases exist that are resistant to RFA therapy for several reasons. In the present study, we performed injection of the mixture of ethanol and lipiodol (percutaneous ethanol-lipiodol injection therapy: PELIT) for HCCs that lacked clear visuality of the entire shape of the tumor by ultrasonography (US) or computed tomography (CT), or that were difficult to treat with RFA alone due to their locations in the liver or due to severe liver dysfunction of the patients. Local recurrence rates of HCC treated with PELIT were shown to be low in patients followed up for at least 4 months. In all patients treated with PELIT, lipiodol was accumulated in the entire region of the tumor after several trials of PELIT and the accumulation was kept for many months. The biopsy examination from the tumor treated with PELIT showed that HCC cells were totally destroyed by the PELIT. Although RFA therapy serves as a central role for the treatment of HCCs, PELIT, considered to be milder therapy, is likely to be important as a supportive treatment for HCCs and useful for the treatment of HCCs that are difficult to treat with RFA.

Clinical significance of lens culinaris agglutinin-reactive fraction of serum alpha-fetoprotein in patients with hepatocellular carcinoma.

alpha-fetoprotein (AFP) is an important marker for the diagnosis of hepatocellular carcinoma (HCC) and has been widely used in clinical settings. Recently, the importance of lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) has been indicated. However, the clinical significance of the level of AFP-L3 protein in relation to the characteristics of HCC has not been fully evaluated. In the present study, both the ratio of AFP-L3 (AFP-L3%) and the absolute value of AFP-L3 (AFP-L3-AV) were examined in 80 patients with HCC, and evaluated with respect to characteristics of HCC such as grade of differentiation, size of tumor and morphological findings. Among HCC-specific tumor markers, AFP, AFP-L3% and protein induced in vitamin K absence (PIVKA-II), AFP showed the highest positive rate in patients with HCC, while AFP-L3% showed the lowest rate. AFP-L3% and AFP-L3-AV were, however, most significantly correlated with the grade of HCC differentiation, while AFP showed the least significant correlation. Furthermore, AFP-L3% was most significantly correlated with the size of HCC in patients with solitary HCC. Conversely, neither AFP-L3-AV nor PIVKA-II showed a significant correlation with the size of HCC. In relation to morphological differences of HCC, although AFP-L3%, AFP-L3-AV and PIVKA-II were significantly higher in the diffuse type of HCC than in the nodular type of HCC, AFP was most significantly correlated with the morphological differences of HCC. These results indicate that tumor markers for HCC, such as AFP, AFP-L3%, AFP-L3-AV and PIVKA-II, may play different roles in predicting the characteristics of HCC.

Combination therapy of percutaneous ethanol injection and radiofrequency ablation against hepatocellular carcinomas difficult to treat.

Radiofrequency ablation (RFA) is an effective modality for the treatment of hepatocellular carcinoma (HCC), because it can induce large coagulated necrosis in a few sessions. We have recently reported that the combination therapy of percutaneous ethanol injection (PEI) with RFA (PEI-RFA) created enhancement of coagulated necrosis compared with RFA alone. In the present study, we adopted PEI-RFA for the treatment of HCCs located in the regions that are difficult to treat with RFA alone. Five patients with biopsy-proven HCC and liver cirrhosis underwent PEI-RFA therapy. In these patients, HCCs were located beside the gallbladder, inferior vena cava or portal vein or kidney, or immediately under the diaphragm. Prior to RFA, 99.5% ethanol was injected into the region of HCC located in the regions where RFA energy appears to be difficult to reach. In all cases, HCC was totally coagulated by PEI-RFA. Injecting ethanol prior to RFA therapy caused no major side effects. These results indicate that PEI-RFA may be effective for the treatment of HCCs located in the regions that are difficult to treat with RFA alone as well as large-sized HCCs.

CD28-negative CD8-positive cytotoxic T lymphocytes mediate hepatocellular damage in hepatitis C virus infection.

The pathogenic mechanism for hepatocellular damage in hepatitis C virus (HCV) infection has not been clearly understood. Analysis of costimulatory molecules on lymphocytes may give us insight into the pathogenic mechanism of hepatocellular damage in HCV infection. Peripheral blood mononuclear cells (PBMCs) and liver infiltrating mononuclear cells (LIMCs) isolated from the HCV-infected patients were analyzed with antibodies directed against a variety of costimulatory molecules by flow cytometry. Blocking experiment against HLA-A24-restricted HCV-specific CTLs and immunohistochemical analysis were also performed. PBMCs expressing CD8, CD28, CD80, or CD154 were significantly reduced in HCV-infected patients compared with the healthy controls. CD28(+)CD8(+) PBMCs in the patients inversely correlated with ALT levels. Conversely, levels of CD28(-)CD8(+) LIMCs correlated with ALT levels. HCV-specific CTL activity was blocked by the treatment with anti-CD8 antibody, but not with anti-CD4 or anti-CD28 antibody. Immunohistochemical analysis revealed the accumulation of CD28(+) cells around the portal area in the liver of a patient with chronic active hepatitis C. These results suggest that CD28(+)CD8(+) T cells leave the circulation, move to the livers, and are activated in the portal area in proportion to the extent of liver diseases. CD28(-)CD8(+) T cells may finally function as effector T cells causing the hepatocellular damage in HCV infection.

Reduced expression of cell cycle regulator p18(INK4C) in human hepatocellular carcinoma.

Cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (CdkIs) are frequently altered in human cancer. p18INK4C, a member of the INK4 family of CdkIs, is a potential tumor-suppressor gene product. However, the expression of p18INK4C in hepatocellular carcinoma (HCC) remains unknown. The aim of this study was to examine the expression of p18INK4C in various liver diseases including HCC and to assess its clinical significance in HCC. To that end, we examined the expression of p18INK4C by immunohistochemistry in various liver diseases, including 51 HCCs, and also studied the relationship between p18INK4C expression, the phosphorylation of retinoblastoma protein (pRb), and the activity level of Cdk4 and Cdk6. Immunohistochemical analysis revealed the frequent loss of p18INK4C expression in HCC, especially in poorly differentiated HCC. The loss of p18INK4C expression was shown to be associated with a poor prognosis compared with that associated with p18INK4C- positivity. Further, the kinase activity of Cdk4 was found to be higher in p18INK4C-negative HCCs than in p18INK4C- positive HCCs. However, the level of Cdk6 activity was similar in the 2 groups of HCCs. In p18INK4C- positive HCCs, p18INK4C dominantly interacted with Cdk4 rather than with Cdk6. pRb phosphorylated at serine(Ser) 780 was detected more frequently in p18INK4C - negative than in p18INK4C - positive HCCs. In conclusion, the loss of p18INK4C expression may play a role in the differentiation and development of HCC through the up-regulation of Cdk4 activity.