Objective To transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing chol-angiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis. Method...Objective To transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing chol-angiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis. Methods QBC939 cells were transfected with antisense vector of human COX-2 gene using LipoVecTM transfecting te-chnique. Transfected cells were selected with G418; COX-2 mRNA was examined using reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein expression was detected by immunocytochemistry using isozyme selective anti-bodies. The proliferative status of transfected cells was measured by using methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry. Results RT-PCR showed a lower COX-2 mRNA level in antisense vector transfected cells and immunocytochemistry showed a weaker COX-2 protein expression in antisense vector transfected cells. The antisense vector transfected cells proli-ferative index decreased significantly (P< 0.01), the percentage of S phase decreased remarkably (P< 0.05) in antisense vec-tor transfected cells (9.27% ±1.91%) compared with that in QBC939 cells without transfection(16.35% ±2.87%), and the percentage of G0/G1 phase increased remarkably (P< 0.05) in antisense vector transfected cells (75.16%±4.13%) compared with that in QBC939 cells without transfection (57.31% ±10.16%). Transfection with antisense vector of human COX-2 gene had no significant influence on the apoptosis in QBC939 cells (P> 0.05). Conclusion Transfection with antisense vector of human COX-2 gene could inhibit the proliferation of human cholan-giocarcinoma QBC939 cells.展开更多
AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells ...AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.展开更多
文摘Objective To transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing chol-angiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis. Methods QBC939 cells were transfected with antisense vector of human COX-2 gene using LipoVecTM transfecting te-chnique. Transfected cells were selected with G418; COX-2 mRNA was examined using reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein expression was detected by immunocytochemistry using isozyme selective anti-bodies. The proliferative status of transfected cells was measured by using methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry. Results RT-PCR showed a lower COX-2 mRNA level in antisense vector transfected cells and immunocytochemistry showed a weaker COX-2 protein expression in antisense vector transfected cells. The antisense vector transfected cells proli-ferative index decreased significantly (P< 0.01), the percentage of S phase decreased remarkably (P< 0.05) in antisense vec-tor transfected cells (9.27% ±1.91%) compared with that in QBC939 cells without transfection(16.35% ±2.87%), and the percentage of G0/G1 phase increased remarkably (P< 0.05) in antisense vector transfected cells (75.16%±4.13%) compared with that in QBC939 cells without transfection (57.31% ±10.16%). Transfection with antisense vector of human COX-2 gene had no significant influence on the apoptosis in QBC939 cells (P> 0.05). Conclusion Transfection with antisense vector of human COX-2 gene could inhibit the proliferation of human cholan-giocarcinoma QBC939 cells.
基金Supported by the Royal Golden Jubilee PhD Program of the Thailand Research Fund (RGJ/PHD/0112/2542)
文摘AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.
