Research On The Proliferation,Invasion And Apoptosis Of T-Secretase Inhibitor In Rapamycin-Resistant Osteosarcoma Cells

Objective To observe the effects of the y-secretase inhibitor (DAPT) on the proliferation,invasion and apoptosis of rapamycin (RAPA)-resistant osteosarcoma cells, preliminary study the possibility of drugs reversing multidrug-resistance of osteosarcoma cells by DAPT,and probe into the connection between mTOR, Notch pathway and associated factors in multidrug resistance of osteosarcoma.Methods Established and cultured RAPA-resistant MG-63 cell line in vitro. The methyl thiazolyl tetrazolium (MTT) was used to detect resistance index (RI) of RAPA-resistant MG-63 cells,besides,to detect effects of different concentrations of RAPA and DAPT on the proliferation of MG63 cells and RAPA-resistant MG-63 cells. Scratch test was used to prove the migration of osteosarcoma cells. Using Transwell to detect invasion of MG-63 cell lines and compare mobility between MG-63 cells and RAPA-resistant MG-63 cells under different doses of rapamycin and DAPT.Annexin V-FITC/PI combined flow cytometry methods was used to explore the effects of different drugs on the apoptosis of MG-63 cells and RAPA-resistant MG-63 cells. The expression of p53 protein was detected in the two cell lines by Western blot.Results RAPA and DAPT could both inhibit the growth of two cells in vitro.Transwell showed the different migration rate between the MG-63 cell and RAPA-resistant MG-63 cell under effects of the two drugs. The flow cytometry result indicated that RAPA and DAPT could both increase the apoptosis rate of two cells,the early and late apoptosis rate of MG-63 cell were both proportional to the dose of two drugs,while the RAPA-resistant MG-63 cell presented significant difference in the early and late apoptosis rate compared to the blank group.Western blot found the discrepant p53 expression between the two cells under different dose of RAPA and DAPT, the further expression of p53 in group MG-63 was significantly increased in a dose-dependent manner with raised dose of RAPA and DAPT, yet the p53 expression in group RAPA-resistant MG-63 showed no significant difference with dose increasing.Conclusion RAPA and DAPT could both effectively inhibit proliferation and induce apoptosis of MG-63 cell and RAPA-resistant MG-63 cell. As the y-secretase inhibitors concentration increased,it showed obvious differences in apoptosis degree,apoptosis time and the p53 expression of group RAPA-resistant MG-63 compared to group MG-63. The above event might happened that blocking mTOR probably reduce the susceptibility of RAPA-resistant MG-63 cell to DAPT, and then inhibit the synergy between Notch pathway and p53.