关键词: 佛波酯, 细胞凋亡, 蛋白激酶B, 免疫印迹法, 激光扫描共焦显微镜, BGC-823细胞

Abstract: Objective To study the role of protein kinase B (PKB) in gastric cancer BGC-823 cells treated by TPA. Methods Flow cytomety(FAC) and fluorescence microscope were used to detect the effect of TPA on gastric cancer BGC-823 cells, after cells were treated by BrdU and stained by DAPI, respectively. The effect of TPA on the expression level of PKB protein and its phosphorylation of Ser 473 and Thr 308 were investigated by Western blotting. When nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation, the effect of TPA on the expression level of PKB protein and its phosphorylation of Ser 473 in cytoplasm and nuclei were detected by Western blotting. Meanwhile, the effect of TPA on the distribution of PKB was observed under laserscanning confocal microscope with immunofluorescence technique. Results TPA induced the apoptosis of gastric cancer BGC-823 cells. It also induced the inhibition of PKB protein expression in a TPA concentrationdependent and timedependent manner in gastric cancer BGC-823 cells, regardless of dephosphorylation of PP2A.TPA inhibited the phosphorylation of PKB at Ser 473, but it did not affect its phosphorylation at Thr308. TPA only attenuated the expression level of PKB and its phosphorylation of Ser 473 in nuclei, whereas it did not change the distribution of PKB in BGC-823 cells. Conclusion These data suggest that the inhibition of PKB plays an important role in mediating the effect of TPA in gastric cell line. Apoptosis induced by TPA is partly due to the inhibition of PKB and its phosphorylation of Ser 47

Key words: TPA, Cell apoptosis, Protein kinase B (PKB), Western blotting, Laserscanning confocal microscope, BGC-823 cells