Experimental Study On MTOR/P70S6K Signal Pathway Activity Related Proliferation And Apoptosis In A172 Human Glioblastoma Cell Line

Abstract:Objective:The purpose of this study was to investigate the significance of the mTOR/P70S6K signal pathway and the expression of Phospho-P70S6K on the proliferation and apoptosis in A172 human glioblastoma cells. Methods:Immunofluorescence and immunocytochemistry were employed to evaluate the expression of p-P70SK in A172 cells. The A172 cells proliferation was tested by MTT assay and cell apoptosis was tested by TUNEL, respectively. Firstly, the A 172 cells were randomly divided into group A1(Control), group B1(RPM,0.01μg/ml), groupC1(RPM,0.1μg/ml), group D1(RPM,1.0μg/ml) and group E1(RPM,10μg/ml). MTT assay was used to detect the A172 cells proliferation. Morphologic changes of the treated cells were then monitored by inverted microscope. Secondly, according to the MTT assay, the A172 cells were randomly divided into group A2(Control), group B2(RPM, 0.01μg/ml), group C2(RPM, 1.0μg/ml) again. Expression level of the p-P70S6K protein was followlly investigated among these groups. Thirdly, the cells were randomly divided into group A3(Control), group B3(RPM,1.0μg/ml). Cell apoptosis was tested by TUNEL and immunofluorescence labeling assay. Results:The MTT assay indicated that the proliferation of the A172 cells was different significantly between the RPM treated cells groups(group B1,C1,D1,E1) and the control group(group A1) (p<0.05). Significant morphologic changes occurred in the treated cells. Strongly positive expression of p-P70S6K in A172 cells was observed. Immunocytochemistry assay showed significant reduction of the expression of p-P70S6K protein in A172 treated cells(p<0.05). Apoptosis of the A172 cells was identified by TUNEL assay. And the apoptosis rate of the A172 cells was significantly different between the RPM treated group and the control group (p<0.05). Conclusion:The mTOR/P70S6K pathway played a very important role on the proliferation of the A172 cells. The expression level of the p-P70S6K protein could partially reflect the proliferative activity of the human glioblastoma A172 cells. Rapamycin, which could specifically inhibit the mTOR/P70S6K signal pathway, could significantly inhibit the proliferation of the A172 cells in dose-and time-dependent manner and induce the apoptosis in vitro. The profound effects of rapamycin in this study could be possibly because of its down-regulation effect on both the activity of the mTOR/P70S6K signal pathway and the expression of the p-P70S6K protein.