Shin Ohnishi

Identification of Soluble NH2-Terminal Fragment of Glypican-3 as a Serological Marker for Early-Stage Hepatocellular Carcinoma

For detection of hepatocellular carcinoma (HCC) in patients with liver cirrhosis, serum α-fetoprotein has been widely used, but its sensitivity has not been satisfactory, especially in small, well-differentiated HCC, and complementary serum marker has been clinically required. Glypican-3 (GPC3), a heparan sulfate proteoglycan anchored to the plasma membrane, is a good candidate marker of HCC because it is an oncofetal protein overexpressed in HCC at both the mRNA and protein levels. In this study, we demonstrated that its NH2-terminal portion [soluble GPC3 (sGPC3)] is cleaved between Arg358 and Ser359 of GPC3 and that sGPC3 can be specifically detected in the sera of patients with HCC. Serum levels of sGPC3 were 4.84 ± 8.91 ng/ml in HCC, significantly higher than the levels seen in liver cirrhosis (1.09 ± 0.74 ng/ml; P < 0.01) and healthy controls (0.65 ± 0.32 ng/ml; P < 0.001). In well- or moderately-differentiated HCC, sGPC3 was superior to α-fetoprotein in sensitivity, and a combination measurement of both markers improved overall sensitivity from 50% to 72%. These results indicate that sGPC3 is a novel serological marker essential for the early detection of HCC.

Dynamic Functional Relay between Insulin Receptor Substrate 1 and 2 in Hepatic Insulin Signaling during Fasting and Feeding

Insulin receptor substrate (Irs) mediates metabolic actions of insulin. Here, we show that hepatic Irs1 and Irs2 function in a distinct manner in the regulation of glucose homeostasis. The PI3K activity associated with Irs2 began to increase during fasting, reached its peak immediately after refeeding, and decreased rapidly thereafter. By contrast, the PI3K activity associated with Irs1 began to increase a few hours after refeeding and reached its peak thereafter. The data indicate that Irs2 mainly functions during fasting and immediately after refeeding, and Irs1 functions primarily after refeeding. In fact, liver-specific Irs1-knockout mice failed to exhibit insulin resistance during fasting, but showed insulin resistance after refeeding; conversely, liver-specific Irs2-knockout mice displayed insulin resistance during fasting but not after refeeding. We propose the concept of the existence of a dynamic relay between Irs1 and Irs2 in hepatic insulin signaling during fasting and feeding.

Increased expression of vascular endothelial growth factor in human hepatocellular carcinoma

BACKGROUND/AIMS Angiogenesis is critical for the development and progression of solid tumors. The purpose of this study was to evaluate the possible role of vascular endothelial growth factor (which is considered to be one of the most important factors involved in tumor-associated angiogenesis), in human hepatocellular carcinoma. METHODS Vascular endothelial growth factor gene and protein expression were analyzed by means of Northern hybridization and immunohistochemical methods in 5 hepatocellular carcinoma cell lines. Secretion of vascular endothelial growth factor was evaluated by immunoblotting of conditioned medium of these hepatocellular carcinoma cells. Further, we compared the level of vascular endothelial growth factor expression in hepatocellular carcinoma tissues along with that in surrounding tumor-free tissues obtained from 20 patients. We also analyzed mRNA expression of Flt-1, one of the vascular endothelial growth factor specific high-affinity receptors, in hepatocellular carcinoma cell lines. RESULTS Northern hybridization analysis and immunohistochemistry revealed that all cultured hepatocellular carcinoma cells exhibited a high level of vascular endothelial growth factor mRNA. In addition, vascular endothelial growth factor secretion by Hep G2, one of the hepatocellular carcinoma cell lines, was shown by Western blot. In vivo, we observed vascular endothelial growth factor expression in both hepatocellular carcinoma and non-hepatocellular carcinoma tissues. However, in 12 of 20 cases, vascular endothelial growth factor mRNA levels were significantly up-regulated in hepatocellular carcinoma tissues. In the majority of cases (10 out of 12 cases) with abundant tumor vascularity, vascular endothelial growth factor mRNA up-regulation in hepatocellular carcinoma tissues was observed. We failed to detect Flt-1 mRNA expression in hepatocellular carcinoma cells. CONCLUSIONS This study suggests that the possibility that hepatocellular carcinoma cells overexpress the vascular endothelial growth factor gene and protein. These findings support the hypothesis that vascular endothelial growth factor is one of the important factors involved in the angiogenesis of hepatocellular carcinoma, and may even be involved in the development and/or progression of hepatocellular carcinoma itself.

Adiponectin suppresses hepatic SREBP1c expression in an AdipoR1/LKB1/AMPK dependent pathway.

Adiponectin, one of the insulin-sensitizing adipokines, has been shown to activate fatty acid oxidation in liver and skeletal muscle, thus maintaining insulin sensitivity. However, the precise roles of adiponectin in fatty acid synthesis are poorly understood. Here we show that adiponectin administration acutely suppresses expression of sterol regulatory element-binding protein (SREBP) 1c, the master regulator which controls and upregulates the enzymes involved in fatty acid synthesis, in the liver of +Lepr(db)/+Lepr(db) (db/db) mouse as well as in cultured hepatocytes. We also show that adiponectin suppresses SREBP1c by AdipoR1, one of the functional receptors for adiponetin, and furthermore that suppressing either AMP-activated protein kinase (AMPK) via its upstream kinase LKB1 deletion cancels the negative effect of adiponectin on SREBP1c expression. These data show that adiponectin suppresses SREBP1c through the AdipoR1/LKB1/AMPK pathway, and suggest a possible role for adiponectin in the regulation of hepatic fatty acid synthesis.

Peroxisome Proliferator-Activated Receptor γ Inhibition Prevents Adhesion to the Extracellular Matrix and Induces Anoikis in Hepatocellular Carcinoma Cells

Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.

Adiponectin knockout mice on high fat diet develop fibrosing steatohepatitis

Background and Aim:  Low levels of serum adiponectin have been reported to be associated with obesity, diabetes, and non‐alcoholic steatohepatitis (NASH), as well as several malignancies. Adiponectin knockout (KO) mice have been reported to cause insulin resistance and neointimal formation of the artery. We used adiponectin KO mice fed a high fat (HF) diet, and investigated the effect of adiponectin on the progression of steatohepatitis and carcinogenesis in vivo.

Extensive mesenteric vein and portal vein thrombosis successfully treated by thrombolysis and anticoagulation

Abstract Mesenteric vein thrombosis is generally difficult to diagnose and can be fatal. A case of extensive thrombosis of the mesenteric and portal veins was diagnosed early and successfully treated in a 26‐year‐old man with Down syndrome who was admitted to hospital because of abdominal pain, severe nausea and high fever. Ultrasonography revealed moderate ascites, and there was minimal flow in the portal vein (PV) on the Doppler examination. Computed tomography (CT) showed remarkable thickening of the walls of the small intestine and extensive thrombosis of the mesenteric, portal and splenic veins. Because neither intestinal infarction nor peritonitis was seen, combined thrombolysis and anticoagulation therapy without surgical treatment was chosen. Urokinase was administered intravenously and later through a catheter in the superior mesenteric artery. Heparin and antibiotics were given concomitantly. The patients symptoms and clinical data improved gradually. After 10 days, CT revealed that collateral veins had developed and the thrombi in the distal portions of the mesenteric veins had dissolved, although the main trunk of the PV had not recanalized. The only risk factor of thrombosis that was detected was decreased protein S activity.

A minimal and optimal cytotoxic T cell epitope within hepatitis C virus nucleoprotein

Amino acid residues 81-100 of the hepatitis C virus (HCV) nucleoprotein contain a cytotoxic T cell epitope that is recognized by cytotoxic T lymphocytes (CTLs) in association with human leukocyte antigen B44. With panels of truncated and overlapping peptides, the minimal and optimal epitope recognized by CTLs was shown to be a 9-mer peptide (residues 88-96). The peptide can stimulate effectively CTLs that are able to recognize endogenously synthesized and processed HCV nucleoprotein.

Immunotherapy of hepatocellular carcinoma with autologous lymphokine-activated killer cells and/or recombinant interleukin-2

SummaryFive patients with hepatocellular carcinoma were subjected to immunotherapy: three patients were treated by adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), and two patients by systemic administration of rIL-2 alone. In one patient with diffuse-type hepatocellular carcinoma and portal vein thrombosis who was treated by infusion of LAK cells (a total number of 1.5x1010 cells/13 doses) and continuous rIL-2 administration (a total dose of 1.25x108 units) via a percultaneously placed hepatic arterial catheter, the size of the tumor reduced dramatically and the portal vein thrombosis retracted. In two patients who had LAK cells infused (totals of 6.6x109 cells/4 doses and 3.1x109 cells/2 doses, respectively) during hepatic angiogram followed by systemic administration of rIL-2 twice a day, no clinical improvement was noticed. In two patients who received rIL-2 alone systemically (total doses of 8.9x107 and 5.5x107 units, respectively), neither clinical improvement nor severe side effects were observed. The results suggest that adoptive immunotherapy combined with continuous local administration of rIL-2 via a percutaneously placed hepatic arterial catheter may be an effective therapy without apparent side effects for patients with hepatocellular carcinoma who cannot be treated by conventional cancer therapy.

Protein-losing enteropathy associated with hypocomplementemia and anti-nuclear antibodies.

Abstract: We report a case of protein-losing enteropathy associated with an autoimmune disorder, presumably systemic lupus erythematosus. Although typical manifestations of systemic lupus erythematosus were lacking, a high serum cholesterol level, a low serum complement level, positivity for anti-nuclear antibody, and positivity for anti-single-strand DNA antibody suggested an autoimmune mechanism as the cause of the condition. Although immunohistological examination of duodenal and ileal biopsy specimens failed to reveal deposits of immune complex or complement in the vessels, capillary hyperpermeability was suspected as the mechanism of the condition.