Effect Of Type Ⅱ CGMP-Dependent Protein Kinase On Biology Activity Of Gastric Cancer Cell Line

ObjectiveTo investigate the effects of cGMP dependent protein kinaseⅡon biology activity of human gastric cancer cell line BGC-823.Methods(1) The remcombinant adenovirus vector encoding the PKGⅡgene (Ad-PKGⅡ) and empty vector LacZ(Ad-LacZ) was amplified in 293 A cells. Several gastric cancer cell lines including AGS,SGC-7901 and BGC-823 were infected with Ad-LacZ and Ad-PKGⅡ.X-Gal staining and Western blotting were used to detect the expression of target protein and the multiplicity of infection(MOI).The most sensitive cell line was chosen as an ideal model for studying the role of PKGⅡin tumorigenesis.(2) BGC-823 cells were infected with Ad-PKGⅡand the activity of the PKGⅡwas induced by cGMP analogue 8-pCPT-cGMP.The proliferation-inhibitory effect of PKGⅡwas analyzed by MTT assay,BrdU incorporation assay,and detection of proliferating cell nuclear antigen(PCNA) expression,and its effect on the cell cycle was analyzed by flow cytometry.(3) The effect of PKGⅡon apoptosis in BGC-823 cells that infection with Ad-PKGⅡwas evaluated by flow cytometry and DNA agarose gel electrophoresis.(4) The effect of activated PKGⅡon cell migration was examined by Boyden chamber migration assay and scrape-migration assay.Colony formation in soft agarose was performed to analyze the effect of PKGⅡon the anchorage-independent growth of the cells.The effect PKGⅡin vivo was investigated in an immunocompromised nude mice model.Results(1) Ad-PKGⅡand Ad-LacZ were successfully amplified.The results of β-Gal staining and Western blotting showed that BGC-823 was the most sensitive cells to adenoviral infection,with the infection rate of 94±18%at an MOI of 100,and the expression of PKGⅡwas maintained at a high level.On the other hand,the BGC-823 is a highly malignant cell line with strong proliferation capacity and migration activity.Therefore,BGC-823 cells were chosen as an ideal model for the following study.(2) PKGⅡactivation caused a significant decrease of live cell number and inhibited DNA synthesis in individual cells.PKGⅡactivation also inhibited the EGF-induced increase in PCNA expression.There was substantial cell arrest at the G1 phase and a decrease in the number of S phase cells in cells infected with PKGⅡand treated with 8-CPT-cGMP.These data indicated that the activation of PKGⅡinhibited the proliferation of human gastric cancer cells.(3) The activation of PKGⅡdid not cause apoptosis,since no change in the number of cells stained with Pi was observed.(4) The activation of PKGⅡcaused a significant inhibition on cell migration as well as a significant inhibition of colony formation in soft agarose, and significantly suppressed the in vivo growth of BGC-823 cells in immunocompromised nude mice.ConclusionThe expression and activity of PKGⅡin human Gastric cancer cell lines were significantly lower than that of normal cells.BGC-823 cells were infected with Ad-PKGⅡand increased the expression of PKGⅡ.Activation of PKGⅡby 8-pCPT-cGMP can significant inhibit biology activity of gastric cancer cells. All of these suggested that PKGⅡmight be a potential tumor suppressor.