A 75-year-old female was admitted to our hospital and diagnosed with hepatocellular carcinoma (HCC). Computed tomography (CT) revealed a liver tumor with tumor thrombi in the portal trunk and main hepatic vein, as well as small lung metastases. The patient had good liver function with no sign of hepatitis B or C infection. She underwent right trisectionectomy of the liver with tumor thrombectomy. Intrahepatic recurrence and progression of lung metastases were observed 4 months later. Intrahepatic recurrent tumors were treated with transcatheter arterial chemoembolization (TACE), and lung metastases were treated with systemic combination chemotherapy of 5-fluorouracil (5FU) and interferon-α (IFN-α). Computed tomography showed no viable lesions in the liver and lung 6 months after these treatments. The patient has been disease free for 18 months. Prognosis is poor for patients with hepatocellular carcinoma with portal vein tumor thrombus (PVVT) or extrahepatic metastasis. This systemic combination chemotherapy with 5-fluorouracil and interferon-α might be effective for patients with good liver function when intrahepatic lesions are well controlled by multidisciplinary treatments, including hepatic resection with tumor thrombectomy.

Hepatocellular carcinoma (HCC) is a common malignancy and often resistant to chemotherapy. Many chemotherapy regimens have been tried to control advanced HCC, but have produced a low response rate and no clear impact. CPT-11, a derivative of camptothecin, works as type-I DNA topoisomerase inhibitor and showed a major objective response rate in patients with metastatic colorectal cancer. In this study, the mechanism underlying chemo-resistance to SN-38, an active form of CPT-11, in HCC was investigated in relation to anti-apoptotic pathways NF-kappaB and PI3K/Akt. Hep3B was the most resistant to SN-38 among three hepatoma cell lines. NF-kappaB was constitutively activated in Hep3B, and SN-38 further enhanced the nuclear translocation of NF-kappaB. However, inactivation of NF-kappaB by adenovirus expressing IkappaB super-repressor or MG-132, proteasome inhibitor, did not sensitize Hep3B to SN-38-induced apoptosis. On the other hand, SN-38 phosphorylated Akt and pretreatment with PI3K inhibitors increased SN-38-induced apoptosis, indicating that resistance to SN-38 in Hep3B occurs partly through the PI3K/Akt not the NF-kappaB pathway. Blocking of PI3K/Akt may thus be helpful for overcoming chemo-resistance of HCC.

Background/Aims: Thioredoxin is a small redox-active protein with anti-oxidant and anti-apoptotic effects. We have previously reported that thioacetamide-induced acute hepatitis was attenuated in thioredoxin transgenic mice. The aim of the present study was to investigate the protective effect of thioredoxin for hepatic fibrosis.Methods: We subjected thioredoxin transgenic mice to thioacetamide-induced hepatic fibrosis. We also studied the effect of thioredoxin on the activation process of primary-cultured hepatic stellate cell.Results: The expression of endogenous thioredoxin was induced in hepatocytes of thioacetamide-induced murine and rat fibrotic livers. Overexpression of thioredoxin inhibited tumor necrosis factor-alpha-induced apoptosis of HepG2 cells. T'hioacetamide-induced fibrosis and accumulation of malondialdehyde were suppressed in transgenic mice as compared with wild type mice. Hepatic stellate cells isolated from transgenic mice were less proliferative than those isolated from wild type mice. Recombinant thioredoxin significantly inhibited DNA synthesis of primary-cultured stellate cells under serum or platelet-derived growth factor stimulation.Conclusions: Thioredoxin has a potential to attenuate hepatic fibrosis via suppressing oxidative stress and inhibiting proliferation of stellate cells. (C) 2004 Published by Elsevier B.V. on behalf of the European Association for the Study of the Liver.

In gastric cancer, lymph node metastasis is one of the major prognostic factors and forms the basis for surgical removal of local lymph nodes. Recently, several studies have demonstrated that overexpression of lymphangiogenic growth factor VEGF‐C or VEGF‐D induces tumor lymphangiogenesis and promotes lymphatic metastasis in mouse tumor models. We examined whether these processes could be inhibited in naturally metastatic tumors by blocking of their cognate receptor VEGFR‐3 signaling pathway. Using a mouse orthotopic gastric cancer model which has a high frequency of lymph node metastasis, we estimated lymphatic vessels in gastric cancers by immunostaining for VEGFR‐3 and other specific lymphatic markers, LYVE‐1 and prox‐1. Then we systemically administered anti‐VEGFR‐3 blocking antibodies. This treatment resulted in the inhibition of regional lymph node metastasis and reduction of lymphatic vessel density in the primary tumors. In addition, increased density of LYVE‐1‐positive lymphatic vessels of primary tumors was closely correlated with lymph node metastasis in human samples of gastric cancer. Antilymphangiogenesis by inhibiting VEGFR‐3 signaling could provide a potential strategy for the prevention of lymph node metastasis in gastric cancer.

Background/Aims: This study using primary-cultured rat hepatic stellate cells (HSCs) was aimed to reveal the effect of carbenoxolone and the other gap-junction blockers on the proliferation and activation of HSCs.Methods: HSC morphology was microscopically evaluated. DNA synthesis was determined by [H-3]thymidine incorporation. Expression of HSC activation markers and cell cycle-related proteins was evaluated by Western blot. Collagen alpha1(I) mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction.Results: Carbenoxolone triggered the morphological change of activated HSCs without inducing apoptosis. Culture-induced DNA synthesis was suppressed to 22.6 and 8.51%, respectively, by 40 and 80 muM carbenoxolone. The other gap-junction blockers failed to affect the morphology and the DNA synthesis of activated HSCs. Carbenoxolone decreased the expression of cyclins D1/2 and cyclin-dependent kinases 4/6. Platelet-derived growth factor (PDGF)-BB-elicited DNA synthesis was reduced to 45.6 and 3.27%, respectively, by 40 and 80 muM carbenoxolone. Phosphorylation of c-Raf, MEK and mitogen-activated protein kinase, but not PDGF receptor beta, under PDGF-BB stimulation was attenuated by carbenoxolone. Collagen alpha1(I) mRNA expression was significantly reduced. In addition, carbenoxolone suppressed the activation process of quiescent HSCs.Conclusions: Carbenoxolone reduced the DNA synthesis and the expression of collagen alpha1(I) mRNA in activated HSCs independently of its pharmacological action as gap-junction blocker. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

BACKGROUND:The low-molecular-weight GTPase Rho is known to act as a molecular switch by activating several downstream effectors, one of which is Rho-associated coiled-coil forming protein kinase (ROCK). ROCK/Rho kinase mediates cytoskeleton-dependent cell functions, such as actomyosin-based smooth muscle contraction and integrin-mediated cell adhesion. A specific inhibitor of ROCK/Rho kinase, Y-27632, was recently developed. The present study examined whether Y-27632 could provide a beneficial effect on hepatic ischemia/reperfusion (I/R)-induced injury through the attenuation of microcirculatory disturbance.MATERIALS AND METHODS:In male Sprague-Dawley rats, normothermic partial ischemia was induced by clamping the hepatic pedicle to the left and median lobes for 90 min, followed by 2 h of reperfusion. In the treatment group, Y-27632 was intravenously administered prior to ischemic insult. Hepatic microcirculation was investigated by using intravital fluorescence microscopy. Liver enzyme release and histological changes of the liver tissue were also evaluated.RESULTS:Y-27632 significantly improved sinusoidal perfusion and reduced the number of leukocytes sticking in hepatic sinusoids and adhering in postsinusoidal venules. The postischemic narrowing of both sinusoids and postsinusoidal venules was also markedly suppressed. Consequently, liver enzyme release was reduced and postischemic histological damage was suppressed.CONCLUSIONS:A specific ROCK/Rho kinase inhibitor, Y-27632, was useful to alleviate hepatic I/R-induced injury through ameliorating postischemic microcirculation. The administration of Y-27632 may be a novel strategy for conquering hepatic I/R-induced injury.

Thioredoxin (Trx) is a small redox-active protein with antioxidant and antiapoptotic effects. Trx transgenic (Tg) mice are more resistant to cerebral infarction and survive longer than wild-type (WT) C57BL/6 mice. The aim of the present study was to investigate the protective role of Trx in acute hepatitis models. The expression of endogenous Trx was decreased in thioacetamide (TAA)-induced acute hepatitis. TAA (100 mug/g) was injected intraperitoneally in WT and Tg mice. Survival rate after TAA injection was higher in Tg mice than in WT mice. The level of oxidative stress was significantly less in Tg mice than in WT mice, as shown by the protein carbonylation assay and lipid peroxidation assay. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells were less in Tg mice than in WT mice, which was consistent with DNA laddering assay. Caspase-3 and caspase-9 activities and cytochrome c release were significantly inhibited in Tg mice compared with those in WT mice. In addition, lipopolysaccharide (LPS) plus D-galactosamine (GalN, or anti-Fas antibody Jo2) were injected. Survival rate after LPS plus GalN injection was much higher in Tg mice than in WT mice. In contrast, there was no difference in survival rate after Jo2 injection between WT and Tg mice. In conclusion, transgene of Trx attenuated TAA- or LPS-induced acute lethal hepatitis. In addition to an antioxidant effect, Trx has the potential to protect acute liver injury via an antiapoptotic effect, which mainly inhibits mitochondria-mediated apoptosis signaling.

The protective effect of a specific ROCK inhibitor, Y-27632, on endotoxin-induced liver inJury was investigated by intravital fluorescence microscopy. In Sprague-Dawley rats, lipopolysaccharide (LPS) combined with D-galactosamine (Ga1N) was administered intravenously and Y-27632 was given orally before LPS/Ga1N injection. Y-27632 significantly improved sinusoidal perfusion and reduced the number of leukocytes sticking in hepatic sinusoids and adhering in postsinusoidal venules. Serum liver enzyme release and 48 hr survival rate were also improved significantly.Our results indicate Y-27632 may be a novel therapeutic strategy for endotoxin-induccd liver injury.

The aim of this study was to target Kupffer cells (KCs) selectively and efficiently by the intraportal injection of fusigenic cationic liposomes with hemagglutinating virus of Japan components (HVJ cationic liposomes). Phosphorothioate FITC-oligodeoxynucleotides (FITC-ODNs) encapsulated in either HVJ cationic liposomes, HVJ anionic liposomes or conventional cationic liposomes without HVJ were transferred to the rat. FITC-ODNs in HVJ cationic liposomes administered via portal vein were selectively transfected to KCs for up to 24 h with no apparent cytotoxicity at higher transfection efficiency than FITC-ODNs in conventional cationic liposomes without HVJ administered via portal vein or tail vein. On the other hand, FITC-ODNs in HVJ anionic liposomes were observed mainly in hepatocytes, not KCs. This new method will be useful for the modulation of KCs activity in both basic research and clinical applications.

The protective effect of a specific ROCK/Rho kinase inhibitor, Y-27632, on hepatic microcirculatory disturbance was investigated by using intravital microscopy. In Sprague-Dawley rats, normothermic ischemia was induced by clamping the hepatic pedicle to the left and median lobe for 90 min, followed by 120 min of reperfusion. Y-27632 significantly improved sinusoidal perfusion rate and reduced the number of leukocytes sticking in hepatic sinusoids and adhering in postsinusoidal venules. The postischemic decrease in the diameter of postsinusoidal venules and sinusoids was also significantly suppressed. The improvement of hepatic microcirculation led to a reduction of postischemic serum liver enzyme release. Our results indicate Y-27632 may be a novel therapeutic strategy for hepatic ischemia/reperfusion-induced injury.

The draft human genome sequence was published on February 15, 2001, which will provide a huge amount of information on human genetics, human disease, and human cell biology. Now, medical scientists and cell biologists are turning their attention to illustrating gene expression pattern using gene microarray and to identifying the functions and the expression patterns of proteins encoded by the genes. Hepatic stellate cell is one of the sinusoid-constituent cells that play multiple roles in the liver pathophysiology. Transformation of stellate cells from the vitamin A-storing phenotype to the "myofibroblastic" one closely correlates to hepatic fibrosis during chronic liver trauma. Analyses of the molecular mechanisms of stellate cell activation have made a great progress, in particular, in the field of intracellular signal transduction of transforming growth factor-beta and platelet-derived growth factor, integrin signaling related to cell-adhesion, and cell motility-associated Rho and focal-adhesion kinase. Accumulation of the information on the stellate cell activation would shed light on the establishment of a novel therapeutic strategy against fibrosis of human liver disease.

BACKGROUND/AIMS:This study using primary culture models was aimed to reveal the stellate cell-derived factors that regulate hepatocyte proliferation. METHODS:Rat hepatocytes and stellate cells were cultured in serum-free Williams-E medium. We prepared hepatocyte mono-culture and two different co-cultures of hepatocytes and stellate cells; (1) co-culture on the same surface (Co-mix.) and (2) co-culture without contact between hepatocytes and stellate cells using a culture insert (Co-sep.). The change in the number and the DNA synthesis of hepatocytes was evaluated. RESULTS:The number of hepatocytes decreased to 76% of the original number after 48 h of starting mono-culture, while it remained at 106% in mixed co-culture (Co-mix.) and increased to 135% in separated co-culture (Co-sep.). The hepatocyte DNA synthesis was enhanced by carbenoxolone in Co-mix. and reduced by NK1 in each co-culture. PD153035 had no effect. Heparitinase-I (20 mU/ml) and sodium chrolate (25 mM) reduced the hepatocyte DNA synthesis in Co-sep. to 71.8 and 61.6%, respectively. Activation of mitogen-activated protein kinase was induced in hepatocytes stimulated by conditioned mediums. CONCLUSIONS:Hepatocyte proliferation was stimulated in the presence of stellate cells through hepatocyte growth factor, extracellular heparan sulfate (HS), and HS proteoglycan, and might be negatively regulated by gap junction-dependent mechanism.

BACKGROUND/AIMS:Intrahepatic cholangiocarcinoma (ICC) arises from intrahepatic bile duct epithelium and is the second most prevalent among primary liver cancers. The aim of this study was to clarify the mechanism of cholangiocarcinogenesis. METHODS:We studied the incidence of microsatellite instability (MSI) involving eight highly polymorphic microsatellite markers and alternations of the K-ras, p53 and mdm-2 genes in human ICC tissues. Overexpression of mdm-2 oncoprotein was also immunohistochemically studied. RESULTS:Of all 65 cases examined, K-ras gene mutation was found in three cases (4.6%) at codon 12. Analysis of p53 alterations was performed in 28 cases including 22 frozen samples and mutations were found in three cases (10.7%). Overexpression of mdm-2 protein was observed in 25 (41.7%) out of 60 cases analyzed. In 22 frozen samples, seven (31.8%) cases showed mdm-2 amplification and four (18.2%) cases revealed MSI-positive phenotype. Among the cases analyzed, all the tumors with mdm-2 amplification/overexpression harbored the wild-type p53 gene and all the microsatellite instability-positive cases were from mass-forming (MF) + periductal-infiltrating (PI) subtype. CONCLUSIONS:These results suggest that mdm-2 plays a role, which might be partially through inhibiting p53 activity, in cholangiocarcinogenesis and that M