Hideki Horiuchi

Identification of genes differentially expressed in a newly isolated human metastasizing esophageal cancer cell line, T.Tn-AT1, by cDNA microarray

We isolated a metastasizing human esophageal squamous cell carcinoma (SCC) cell line, T.Tn‐AT1, from a parental non‐metastasizing cell line, T.Tn, by in vitro selection and by use of a nude mouse orthotopic inoculation model. Then, we compared the expression profiles of 9206 genes in T.Tn‐AT1 and T.Tn by cDNA microarray analysis. The gene expression profiles of T.Tn and T.Tn‐AT1 were very similar, and only 34 genes showed more than 3‐fold differential expression. Among the 34 genes, 29 genes were down‐regulated and only 5 genes were up‐regulated in T.Tn‐AT1 cells. Subsequently, we confirmed the expression levels of 14 of the 34 genes in T.Tn and T.Tn‐AT1 cells by means of reverse transcription‐polymerase chain reaction. The expression of 8 genes (KAL1, HPGD, NDN, REG1A, CXCR4, SPOCK, DIAPH2 and AIF1) was down‐regulated and that of one gene (VNN2) was up‐regulated in T.Tn‐AT1 cells. These 9 genes encoded proteins associated with metastatic processes, such as adhesion, migration, inflammation, proliferation, and differentiation. Thus, these genes might regulate the metastasis of esophageal SCC, and could be predictive markers for lymph node metastasis of esophageal SCC.

Frequent Occurrence of Abnormal E-Cadherin/β-Catenin Protein Expression in Advanced Gallbladder Cancers and Its Association with Decreased Apoptosis

Objective: Our aim is to assess the clinicopathological significance of E-cadherin and β-catenin expression, as well as their association with apoptosis in gallbladder cancers. Methods: The expression of E-cadherin and β-catenin proteins was examined in 4 biliary tract cancer cell lines and 49 gallbladder cancer specimens by immunofluorescent or immunohistochemical methods and Western blotting. The apoptotic status was evaluated in the cell lines by poly(ADP-ribose) polymerase Western blotting and in the tumors by the TdT-mediated dUTP nick end labeling assay. Results: Expression of poly(ADP-ribose) polymerase (apoptosis) was only seen in cell lines that expressed both E-cadherin and β-catenin. Reduced expression of E-cadherin and β-catenin was frequently seen in advanced gallbladder cancer cases (61 and 83%, respectively) relative to pT1 cases (25 and 63%, respectively). The 5-year survival rate in cases with reduced E-cadherin expression was 26%, significantly lower than in cases with preserved E-cadherin expression (70%; p = 0.017). Cases with reduced expression of both had lower apoptotic indices and showed a worse prognosis compared with cases with reduced expression of either E-cadherin or β-catenin (p = 0.04 and 0.049, respectively). Conclusions: The expression of E-cadherin or β-catenin frequently diminishes as the tumor progresses, and abnormalities of E-cadherin and β-catenin expression were associated with decreased apoptosis in gallbladder cancers. E-cadherin expression might be a useful prognostic marker in this tumor.

Frequent activation of mitogen-activated protein kinase relative to Akt in extrahepatic biliary tract cancer.

BackgroundLack of effective adjuvant therapy against advanced extrahepatic biliary tract carcinoma (BTC) requires that new therapeutic methods, such as molecular targeted therapy, be developed. The mitogen-activated protein kinase (MAPK) and Akt signaling pathways, which activate cell proliferation and suppress apoptosis, respectively, may function as important targets for such therapies. The aim of this study was to examine the expression patterns of phosphorylated MAPK (p-MAPK) and phosphorylated Akt (p-Akt) proteins in BTC cell lines and clinical specimens.MethodsExpression of p-MAPK and p-Akt proteins in four human BTC cell lines and in frozen sections of 20 advanced extrahepatic BTC specimens was analyzed by Western blotting. Thirty formalin-fixed BTC specimens were immunohistochemically stained for p-MAPK and p-Akt using labeled streptavidin–biotin conjugates.ResultsExpression of p-MAPK was observed in three of four (75%) BTC cell lines, whereas no expression of p-Akt was observed. Twenty-three of 30 formalin-fixed specimens stained positive for p-MAPK (77%), whereas only 47% stained positively for p-Akt. Expression of p-MAPK relative to that of p-Akt was also seen more frequently in the frozen specimens.ConclusionsThe results of this study suggest that MAPK is activated more frequently than Akt in extrahepatic biliary tract carcinoma.

Antitumor Effect of Gemcitabine on Orthotopically Inoculated Human Gallbladder Cancer Cells in Nude Mice

BackgroundThe prognosis of gallbladder carcinoma is poor; therefore, investigating the efficacy of new chemotherapy agents is essential for the treatments for this tumor. Recently, several studies have reported clinical trials using gemcitabine as treatment for advanced gallbladder cancers. However, the antitumor effects of gemcitabine on gallbladder carcinoma have not been examined in in vitro and in vivo model systems.MethodsWe examined the cytotoxicity of gemcitabine in four biliary tract cancer cell lines using the WST-1 assay. In addition, we examined the effect of gemcitabine on gallbladder cancers resulting from orthotopic inoculation of NOZ gallbladder tumor cells into nude mice. One week after transplantation, the mice were randomized into two groups: In Group A, the mice were treated by an intra-peritoneal injection of 0.9% sodium chloride for three weeks after inoculation (control). In Group B, the mice were treated by an intra-peritoneal injection of gemcitabine (125 mg / kg) for three weeks. All mice were sacrificed one week after the end of treatment, and macroscopic and histological findings were evaluated. The expression levels of proliferating-cell nuclear antigen (PCNA) were examined to investigate cellular proliferation activity, and Tunnel assays were performed to determine apoptotic status. Survival duration of the mice after gemcitabine treatment was compared to that of untreated mice.ResultsThe gemcitabine sensitivity of the four biliary tract cancer cell lines was similar in a dose dependent manner. In the in vivo models, the Group A mice showed huge tumors of the gallbladder, with liver invasion and lymph node metastases. However, there were no abdominal tumors in the Group B mice, and microscopic gallbladder cancer could only be detected from histological findings. The mean percent of PCNA-positive tumor cells was significantly higher in tumors from mice in Group A (71.9%) compared to those of Group B (34.7%). The mean percent of Tunnel-positive tumor cells was significantly lower in mice from Group A (2.0%) than those from Group B (5.7%). Survival duration was prolonged significantly in the gemcitabine-treated mice relative to untreated mice.ConclusionsGemcitabine treatment may inhibit tumor progression and prolong survival in gallbladder cancer by inhibiting cell proliferation and inducing apoptosis.

Overexpression of TSC-22 (transforming growth factor- β-stimulated clone-22) causes marked obesity, splenic abnormality and B cell lymphoma in transgenic mice

In this study, we generated transgenic (Tg) mice, which overexpressed transforming growth factor (TGF)-β stimulated clone-22 (TSC-22), and investigate the functional role of TSC-22 on their development and pathogenesis. We obtained 13 Tg-founders (two mice from C57BL6/J and 11 mice from BDF1). Three of 13 Tg-founders were sterile, and the remaining Tg-founders also could generate only a limited number of the F1 generation. We obtained 32 Tg-F1 mice. Most of the Tg-mice showed marked obesity. Histopathological examination could be performed on 31 Tg-mice; seventeen mice died by some disease in their entire life and 14 mice were killed for examination. Most of the Tg-mice examined showed splenic abnormality, in which marked increase of the megakaryocytes, unclearness of the margin of the red pulp and the white pulp, and the enlargement of the white pulp was observed. B cell lymphoma was developed in 10 (71%) of 14 disease-died F1 mice. These results indicate that constitutive over-expression of TSC-22 might disturb the normal embryogenesis and the normal lipid metabolism, and induce the oncogenic differentiation of hematopoietic cells.