Protein Kinase Inhibitors AT13148 Modulate Biological Activity Of Gastric Cancer Cells Via PI3K/Akt Signaling Pathway

Part I Protein kinase inhibitors AT13148 impact on the biological activity of gastric cancer cellsObjective To explore the proliferation and apoptosis suppression effect on gastric cancer cell of the AT13148 protein kinase inhibitors. Comparative analysis of different concentrations of protein kinase inhibitors AT13148 relevance to gastric cancer cell apoptosis. To futher confirm whether AT13148 inhibitory effect on the gastric cancer cells by promoting apoptosis.Methods MTT assay was determined in gastric cancer cells treated with AT13148 protein kinase inhibitors with the concentration of 0.1,1,5 or 10μmol/L. Brdu staining and trypan blue staining were used to analy the proliferation ability in gastric cancer cell lines HGC27, AGS, SNU-601, MKN-28, N87 or gastric epithelial cell GEC-1 treated with AT13148 protein kinase inhibitors. TUNEL, Annexin V and caspase 3 activity tests were utilized to confirm whether the inhibitory effect of AT13148 on the gastric cancer cells by promoting apoptosis.Results MTT, Brdu staining assays revealed that the proliferation ability of gastric cancer cell line, HGC27, AGS, SNU-601, MKN-28, N87 and gastric epithelial cell GEC-1 followed by AT13148 protein kinase inhibitors (0.1,1,5, 10μmol/L) treatment. Trypan blue dyeing experiments showed the dead cells after treatment increased significantly. And the most obvious impact was 48 h. At the same time, we also found that the proliferation suppression and effect on promoting death of AT13148 on gastric cancer cell was the concentration dependent.TUNEL, Annexin V and caspase 3 activity tests showed the numbers of apoptosis and the activity caspase-3 in HGC27 cell treated with AT13148 increased significantly; Additionally, TUNEL, determined by MTT and trypan blue dyeing experiment explored the result after the caspase and caspase 3 inhibitors blocking, and cell apoptosis and proliferation index are recovered.Conclusion Protein kinase inhibitor AT13148 can inhibit gastric cancer cell proliferation, its inhibition of tumor cell proliferation and the promotion of the tumor cell death is concentration dependent. Protein kinase inhibitor AT13148 inhibitory effect on the gastric cancer cells are realized by promoting apoptosis.Part II The mechanism of AT13148 regulating gastric cancer cell proliferation and apoptosis through PI3K/Akt signaling pathwaysObjective To investigate the mechanism of AT13148 regulating gastric cancer cell proliferation and apoptosis through PI3K/Akt signaling pathways.Methods Western blot assay was applied in HGC27 and AGS gastric cancer cells with the 1h treatment of protein kinase inhibitor AT 13148 at different concentrations (0.1,1,5, 10μmol/L) 1 h to determine the PI3K/Akt signal pathway, GSK3 beta, p70S6K and RSK phosphorylation level and the total protein level, and correlation analysis.Results Gastric carcinoma cell lines, HGC27 and AGS cells were treated with AT13148 (0.1,1,5,10μmol/L) by 1h, AKT, GSK3 beta, p70S6K and RSK phosphorylation levels were obviously drop and no significant changes in the total protein level, and 10μmol/L effect is most obvious.The results showed thatConclusion The inhibition of cells proliferation and the promote of apoptosis were associated with growth by inhibiting the activation of protein kinase, which is very important in the PI3K/Akt signaling pathway, such as Akt, GSK3β, p70S6K and RSK.Part III The regulation effect of Protein kinase inhibitors AT13148 on human gastric cancer xenografts tumor in nude mouseObjective To establishment the xenograft model in nude mice with human gastric cancer. To investigate regulation effect of Protein kinase inhibitors AT13148 on human gastric cancer xenografts tumor in nude mouse.Methods The nude mice xenograft tumor model was applied in HGC27 cells,30 nude mice were divided into three group, control group were treated with saline, AT13148 (15 mg/kg) and AT13148 (45 mg/kg) were used in tumor-bearing nude mice by oral administration. After treatment, recorded every three day, the the weight of mice and the volumes of HGC27 tumors in AT13148-treated mice of the groups of nude mice into tumors had product was measured; Three weeks later, all of the bearing-tumor nude mice were be executed. And Western blotting analysis was used to prove AT13148 can inhibit the activation of protein kinase in the body; IHC experiment was carried out to test the p-GSK3 beta expression level after AT13148 treatment in tumors in nude mice.Results After AT13148 HGC27 tumor-bearing nude mice (15 mg/kg) and AT13148 (45 mg/kg) treatment, compared with the control group, the volumes of HGC27 tumors in AT13148-treated mice were significantly lower than that of vehicle control mice, and the AT13148 (45 mg/kg) group reduced more than AT13148 (15 mg/kg) groups, but has no obvious effect on body weight of nude mice. The western blotting analysis proved that AT13148 can inhibit the activation of protein kinase in the nude mice; IHC experiments showed that p-GSK3 beta tumors in nude mice after AT13148 treatment was significantly lower than the control group, the expression of tan particles decreases.Conclusion AT13148 can inhibit the activation of protein kinase in vivo, such as Akt, GSK3β, p70S6K and so on. It also can block PI3K/Akt signaling pathway, inhibiting the malignant proliferation of tumor cells, but not much effect on the nude mice.