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Katsuya Shiraki

Publications and source records attributed to Katsuya Shiraki.

At least 19 recordsLinked to original sources
PubMed2006

Identification of novel hepatitis C virus-specific cytotoxic T lymphocyte epiotpe in NS3 region.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are thought to be effective in limiting viral spread and in clearing virus during infection. Therefore, we attempted to establish HCV-specific CTL and identify novel HCV-specific CTL epitopes in a patient with acute hepatitis C by a novel screening method using recombinant vaccinia viruses (rVV) and synthetic peptides. CD8(+)CD45RA(-) T cells (memory T cells) were isolated from peripheral blood mononuclear cells (PBMC) of a patient with acute hepatitis C. HCV-specific CTL were cloned at limited dilutions and tested for HCV-specific CTL activity using a standard (51)Cr release assay. CTL assay was performed using rVV expressing regions of HCV-J, and overlapping and truncated synthetic peptides from HCV-J. CTL recognizing the NS3 region were isolated by (51)Cr release assay with rVV-HCV. Isolated CTL were restricted by HLA class I molecules B(*)5603. We confirmed that isolated CTL recognized 8-mer amino acids in the NS3 region of HCV-J by (51)Cr release assay with overlapping and truncated synthetic peptides. In conclusion, we isolated HCV-specific CTL restricted by HLA-B(*)5603 and identified a novel HCV-specific CTL epitope (IPFYGKAI, amino acids 1373-1380) in the NS3 region. The identified HCV-specific CTL epitope might be useful for HCV therapy.

Journal Article↗
PubMed2006

Adenovirus-mediated transfection of caspase-8 sensitizes hepatocellular carcinoma to TRAIL- and chemotherapeutic agent-induced cell death.

Caspase-8 belongs to the cysteine protease family and is known to be activated at the initial step in the cascade of TRAIL-induced apoptosis. The activation of procaspase-8 can be blocked by a relatively large amount of c-FLIP, which renders resistance to death receptor-mediated apoptosis in many types of cancer cells. To ask if extrinsic over-expression of caspase-8 contributes to the induction of apoptosis, we introduced the caspase-8 gene into HCC cells using an adenoviral (Adv) vector (Adv-Casp8). We demonstrated that Adv-Casp8 increased expression of active forms of caspase-8 in MOI-dependent manner. A large amount of Adv-Casp8 (MOI of 50) induced apoptosis significantly in HCC cells and resulted in downregulation of c-FLIP (in SK-Hep1, HLE, and HepG2 cells), XIAP, survivin, and Bcl-xL (in HLE cells) and dynamic release of cytochrome c and Smac from the mitochondria into the cytosol. On the other hand, a small amount of Adv-Casp8 (MOI of 10) causes a slight but detectable increase in the level of apoptosis with only a small effect on anti-apoptotic proteins and mitochondrial activation. However, small amounts of Adv-Casp8 augmented TRAIL- or chemotherapeutic agent-induced cell death (with an MOI of 10 or 20, respectively). These results suggest both that exogenous over-expression of caspase-8 by Adv-Casp8 may be essential for induction of HCC cell death and that the combination of Adv-Casp8 and TRAIL or chemotherapeutic agents could provide a useful strategy for treatment of HCC.

Adenoviridae↗
PubMed2006

Proteasome inhibition sensitizes hepatocellular carcinoma cells to TRAIL by suppressing caspase inhibitors and AKT pathway.

The ubiquitin-proteasome pathway is responsible for regulating cell cycle proteins, tumor-suppressor molecules, oncogenes, transcription factors, and pro- and anti-apoptotic proteins. The aim of this study is to evaluate the effects of proteasome inhibitors on human hepatocellular carcinoma (HCC) cells. HCC cells SK-Hep1, HLE and HepG2 were treated with the proteasome inhibitors MG132 and MG115. Our data showed that both inhibitors induce apoptosis in the three cell types tested in a dose-dependent manner. Moreover, subtoxic levels of MG132 and MG115 sensitized HCC cells to TRAIL-induced apoptosis. To investigate the mechanism of increased TRAIL sensitivity in HCC cells, we first examined surface expression of TRAIL and its receptors. MG132 upregulated TRAIL and its receptors (TRAIL-R1 and -R2) in SK-Hep1 and HLE, whereas MG115 upregulated them in SK-Hep1. MG132 downregulated expression of X-linked inhibitor of apoptosis protein (XIAP) in SK-Hep1 and HLE, and of survivin in all three cell-types. MG115 downregulated expression of XIAP in SK-Hep1, and survivin in SK-Hep1 and HepG2. Furthermore, MG132 downregulated phospho-AKT and its downstream target phospho-BAD, indicating that MG132 activated the mitochondrial apoptosis pathway by inhibiting phosphorylation of AKT and BAD. In conclusion, proteasome inhibitors induced apoptosis and augmented TRAIL sensitivity via both the IAP family and AKT pathways. Thus, combining proteasome inhibitors with a TRAIL agonist may provide a new therapeutic strategy for HCC.

Animals↗
PubMed2006

Corona enhancement in ultrasonographical post-vascular phase images with microbubble contrast agent: a novel specific sign for hepatocellular carcinomas.

In 1.5 Harmonic Imaging ultrasonography (1.5 HI US), images are obtained in a band intermediate between the fundamental and 2nd harmonic components, resulting in stronger contrast enhancement than in conventional harmonic imaging. We attempted to assess the hemodynamics of hepatocellular carcinomas (HCC) with special attention to blood drainage using 1.5 HI US. Forty-two HCC nodules, metastatic liver tumors and hepatic hemangiomas were studied. In contrast studies, intermittent ultrasound transmission was performed for a period of up to 45 sec after the injection of contrast agent, which was regarded as the vascular phase. The time point of 5 min later was specified as the post-vascular phase, and images were obtained by single manual transmission for comparison of contrast enhancement with surrounding hepatic parenchyma. In addition, histological examination was performed. 1.5 HI US clearly demonstrated the strong tumor vessels in most HCCs. Corona enhancement, in which the areas surrounding the tumor are enhanced, was observed in 71.4% (30/42) of HCC nodules during the post-vascular phase. This sign was not observed in any other tumors. Histological findings revealed that CD34-positive-endothelial cells were prominent in the surrounding area of HCC. In conclusion, 1.5 HI US is an effective tool for evaluating hemodynamics in both early- and post-vascular phase. Corona enhancement may be due to the trapping of contrast agent in the endothelial cells in the surround of HCC nodules and be a novel specific sign for HCC.

Adult↗
PubMed2006

COX-2 inhibitors sensitize human hepatocellular carcinoma cells to TRAIL-induced apoptosis.

Cyclooxygenase (COX)-2 is upregulated in a variety of human cancers, including in hepatocellular carcinoma (HCC), whereas it is undetectable in most normal tissue. Evidence suggests that COX-2 is likely to be involved in hepatocarcinogenesis and, thus, COX-2 may be involved in an early process in carcinogenesis, dedifferentiation. To address this possibility, we investigated the effect of COX-2 inhibitors on TNF-related apoptosis, inducing ligand (TRAIL) sensitivity and its molecular mechanisms, with special attention to anti-apoptotic proteins. We used the highly selective COX-2 inhibitors, NS398 and CAY10404. We also used the MTT assay and cytological analysis of DAPI-stained DNA to assess viability and apoptosis in two HCC cells (SK-Hep1 and HLE). In order to ask what led to increased sensitivity to TRAIL in HCC cells, cell surface expression of TRAIL and TRAIL-receptors was investigated using flow cytometry analysis. Expression of survivin, X-chromosome-linked IAP (XIAP), Bcl-xL, AKT and phospho-AKT was also investigated using immunoblotting. COX-2 inhibitors resulted in a concentration-dependent decrease in cell viability in the two HCC cell lines tested. Subtoxic levels of COX-2 inhibitors did not significantly augment TNFalpha-induced apoptosis but did dramatically enhance TRAIL-induced apoptosis in both cell lines. TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5) expression was significantly up-regulated in SH-Hep1 and HLE cells. TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) expression was up-regulated only in SK-Hep1. Expression of survivin and Bcl-xL was down-regulated in SK-Hep1 and HLE cells in the presence of CAY10404 but XIAP was not affected. Expression of survivin, Bcl-xL and XIAP was down-regulated in SK-Hep1 cells in the presence of NS398. Survivin expression was also down-regulated in the presence of NS398 in HLE cells. Finally, NS398 also decreased phospho-AKT in SK-Hep1 cells. These results demonstrate that COX-2 inhibitors can induce apoptosis and augment TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of both survivin and AKT signaling.

Apoptosis↗
PubMed2006

Cellular apoptosis susceptibility protein and proliferation in human hepatocellular carcinoma.

The cellular apoptosis susceptibility protein (CAS) is the human homologue of the product of the essential yeast chromosome segregation gene, CSE1, and has important roles in tumor necrosis factor (TNF)-induced apoptosis and cell proliferation. In this study, we used immunoblotting and immunohistochemistry to look at CAS expression in human hepatocellular carcinoma (HCC) cells. We also studied the correlation between CAS expression and cell proliferation. To do this, we studied the expression of proliferating cell nuclear antigen (PCNA) by immunostaining and at apoptosis by in situ nick end-labeling (TUNEL), followed by calculation of the PCNA labeling index (PCNA LI) and TUNEL labeling index (TUNEL LI). CAS was constitutively expressed in human HCC cell lines and was primarily confined to the cytoplasm of the cells. PCNA LI and TUNEL LI were significantly higher in HCC than in non-tumor tissue (p<0.01); however, the ratio of TUNEL LI/PCNA LI in HCC was significantly lower than that of non-tumor tissue. Immunohistochemistry revealed that the staining intensity score of CAS in HCC was significantly higher than that of non-tumor tissue (p<0.05). These results indicate that there is an augmentation of pro-liferative activity and apoptosis in HCC tissue, as compared to non-tumor tissue. There was a significant positive correlation between CAS and PCNA LI (p<0.05). In addition, we observed an inverse relationship between CAS expression and TUNEL LI, although the correlation did not reach statistical significance. These results suggest that CAS is expressed at higher levels in human HCC tissue than in non-tumor tissue. CAS may be associated with cell proliferation rather than apoptosis, and further, CAS might play an important role in the development of human HCCs.

Apoptosis↗
PubMed2006

The cyclin-dependent kinase inhibitor flavopiridol sensitizes human hepatocellular carcinoma cells to TRAIL-induced apoptosis.

Flavopiridol was one of the first cyclin-dependent kinase inhibitors demonstrated to have an antitumor effect in several cancer types. Here, we investigated the effects of flavopiridol on TNF-related apoptosis-inducing ligand (TRAIL) in the human hepatocellular carcinoma (HCC) cell lines HLE and HepG2, and evaluated the role of flavopiridol in apoptosis. To better understand the mechanism of increased TRAIL sensitivity in HCC cells, we determined the effect of flavopiridol on cell surface expression of TRAIL and TRAIL receptors using flow cytometry analysis. The levels of survivin, FLIP, Bcl-xL and X-chromosome-linked IAP (XIAP) in treated and untreated cells was also determined. Flavopiridol decreased cell viability in a dose-dependent manner in the two HCC cell lines tested. The pan-caspase inhibitor z-VAD-FMK did not inhibit the effect. However, subtoxic levels of flavopiridol dramatically enhanced TRAIL-induced apoptosis in both cells. Flavopiridol up-regulated TRAIL, TRAIL-R1 and TRAIL-R2 in both cell lines. In addition, flavopiridol down-regulated expression of survivin in both cell lines, and expression of FLIP and Bcl-xL were down-regulated in HLE cells. In summary, flavopiridol augmented TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of survivin, FLIP and Bcl-xL. Thus, combining flavopiridol with a TRAIL agonist may prove to be an effective new strategy for treatment of HCC.

Antineoplastic Agents↗
PubMed2006

CD40 expression in HCV-associated chronic liver diseases.

CD40 is expressed primarily on B cells and plays an important role in antigen presentation, B cell proliferation, and T cell activation. It has been reported that the CD40 signal modulates apoptosis and has an anti-viral effect in certain cells. Therefore, we investigated the expression and the function of CD40 in HCV-associated chronic liver disease. The expression of CD40 on liver tissues was determined through immunohistochemistry on 50 liver specimens obtained from HCV-positive patients. The effect of CD40 signaling on apoptosis of HepG2 cells was assessed using the MTT assay. The effect of CD40 stimulation on NF-kappaB activation was determined in NF-kappaB reporter gene-transfected HepG2 cells with the Luciferase assay. CD40 positive hepatocytes were observed in both periportal and lobular areas, accompanied by inflammation. In both areas, CD40 staining intensity became significantly stronger, correlating with the histological grading. Similarly, it became stronger with the progression of the histological staging in F1, F2 and F3 cases; however, the expression level decreased in F4 cases. CD40 ligation induced apoptosis in HepG2 cells in the presence of 500 ng/ml of actinomycin D, while CD40 ligation alone could not. Anti-CD40 monoclonal antibody caused NF-kappaB activation in HepG2 cells in a dose-dependent manner. These results suggest that hepatocyte over-expression of CD40 might play an important role in regulating hepatocyte survival and death in HCV-associated chronic liver diseases.

Antibodies, Monoclonal↗
PubMed2005

Relative predictive factors for hepatocellular carcinoma after HBeAg seroconversion in HBV infection.

AIM: To determine the predictive factors for hepatocellular carcinoma (HCC) development in patients after spontaneous or therapeutic HBeAg seroconversion. METHODS: In 48 patients who seroconverted to anti-HBe positive during follow-up, the background factors for HCC development were analyzed. RESULTS: HCC was developed in six patients during follow-up (average follow-up after HBeAg seroconversion: 10.9+/-5.4 years). The incidence of HCC evaluated by Kaplan-Meier analysis was significantly higher in patients with abnormal aspartate aminotransferase (AST>40 IU/L) level, lower platelet counts (PLT< 10 x 10(4)/microL), lower albumin level (Alb<30 g/L), positive HBV-DNA or older age at seroconversion (>40 years). However, lower platelet count was the only predictive factor for HCC development shown by multivariate proportional-hazard analysis. CONCLUSION: Active hepatitis or advanced hepatitis at HBeAg seroconversion or progressive hepatitis even after HBeAg seroconversion would be the risk factors for HCC development. These predictive factors should be taken into account in determining the frequency of biochemical study or imaging studies for HCC surveillance.

Adolescent↗
PubMed2005

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NF kappa B.

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor kappa B (NF kappa B). METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNF alpha and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NF kappa B. RESULTS: E3330 decreased NF kappa B levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNF alpha, Fas ligand, and E3330 increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability. CONCLUSION: Inhibition of NF kappa B sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.

Apoptosis↗
PubMed2005

Primary biliary cirrhosis after aortoiliac reconstruction surgery using a Y-graft.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by anti-mitochondrial antibodies and destruction of intra-hepatic bile ducts. Though little is known about the etiology of PBC, some reports suggest that xenobiotics and viral infections may induce PBC. We describe a case of PBC after the aortoiliac reconstruction surgery using a Y-graft.

Aorta, Abdominal↗
PubMed2005

1.5 Harmonic Imaging Sonography with microbubble contrast agent improves characterization of hepatocellular carcinoma.

AIM: To investigate the usefulness of 1.5 Harmonic Imaging Sonography with the use of the contrast agent Levovist for the diagnosis of hepatocellular carcinoma (HCC) and for the evaluation of therapeutic response. METHODS: Phantom experiments were performed to compare the contrast effects of 2(nd) harmonic imaging and 1.5 Harmonic Imaging Sonography. 1.5 Harmonic Imaging Sonography was employed to examine 36 patients with HCC (42 nodules) before and after the treatment and to compare against the findings obtained using other diagnostic imaging modalities. RESULTS: In 1.5 Harmonic Imaging Sonography, the tumor vessels of HCCs were clearly identified during the early phase, and late-phase images clearly demonstrated the differences in contrast enhancement between the tumor and surrounding hepatic parenchyma. Blood flow within the tumor was detected in 36 nodules (85.7%) during the early phase and in all 42 nodules (100%) during the late phase using 1.5 Harmonic Imaging Sonography, in 38 nodules (90.5%) using contrast-enhanced CT, in 34 nodules (81.0%) using digital subtraction angiography (DSA), and in 42 nodules (100%) using US CO(2) angiography. Following transcatheter arterial embolization, 1.5 Harmonic Imaging Sonography detected blood flow and contrast enhancement within the tumors that were judged to contain viable tissue in 20 of 42 nodules (47.6%). However, 6 of these 10 cases were not judged in contrast-enhanced CT. 1.5 Harmonic Imaging Sonography was compared with the US CO(2) angiography findings as the gold standard, and the sensitivity and specificity of these images for discerning viable and nonviable HCC after transcatheter arterial embolization were 100% and 100%, respectively. CONCLUSION: 1.5 Harmonic Imaging Sonography permits the vascular structures of HCCs to be identified and blood flow within the tumor to be clearly demonstrated. Furthermore, 1.5 Harmonic Imaging Sonography is potentially useful for evaluating the therapeutic effects of transcatheter arterial embolization on HCC.

Adult↗
PubMed2005

Basket pattern blood flow signals discovered in a case of splenic hamartoma by power Doppler ultrasonography.

We present the gray-scale ultrasonography (GSUS), power Doppler ultrasonography (PDUS), abdominal computed tomography (CT), and magnetic resonance imaging (MRI) findings for a case of splenic hamartoma in a 27-year-old man, showing a phi 50 mm homogeneous, iso- and hypo-echoic splenic mass with evidence of a small plural cystic lesion. This splenic hamartoma showed increased vascularity on power Doppler sonograms. PDUS showed multiple circular blood flow signals inside the mass (i.e. a basket pattern), which was consistent with the small plural cystic lesion shown by GSUS. Spectral analysis also confirmed arterial and venous flow. CT scans showed that the mass had low-density relative to the normal spleen and MRI showed that the mass was isodense, relative to the normal spleen. Therefore, CT and MRI are not useful for the diagnosis of splenic hamartoma. Ultrasonography can be used to diagnose splenic hamartoma without administration of a contrast material and therefore is an indispensable method for the diagnosis of splenic hamartoma.

Adult↗