| Objectives: To determine the effects of Torin2 on cell proliferation and migration in human non-small lung cancer A549 cells and investigate its relative molecular mechanism,and provide theoretical basis for Torin2 used in the treatment of NSCLC in the future.Methods:1.A549 cells were divided into blank control,negative control and Torin2 treatment group.The inhibition rates of cell proliferation were measured by CCK-8 assay kit;2.flow cytometry was used to evaluate cell cycle and apoptosis;3.Cell migration was examined by Wound healing test and Transwell chamber assay.4.The expression levels of Akt,p-Akt473,4EBP1,p-4EBP137/46,Cyclin D1,Caspase 9 and E-cad were detected by Western blotting.Results:1.According to the results of CCK-8 assay,different concentrations of Torin2(0,50,100,200,400,800 nmol/L)treated with A549 cells for 48 hours,the inhibition rates of cell viability were(0.0+0.0)%,(22.7+6.2)%,(31.2+3.5)%,(38.8+4.2)%,(64.8+2.3)% and(85.0+1.3)%,respectively;with the increased concentration of Torin2,the cell proliferati ve activity was gradually decreased,which showed that Torin2 significantly inhibited the proliferation of A549 cells in a dose-dependent manner,and the half inhibitory concentration(IC50)was 214.96 nmol /L.2.Flow cytometry results showed that the apoptotic rates were(4.51±1.32)%,(9.56±2.34)%,and(16.40±3.57)%,respectively,in the control and the 200 and 400 nmol/L Torin2 treatment groups,with the treatment groups' significantly higher than the control's(P<0.05),indicated that Torin2 promoted the apoptosis of A549 cells.3.A549 cells were treated with Torin2 for 48 hours,and the cell proportion of G1 phase in the control group,200 nmol/L and 400 nmol/L Torin2 group were(58.15+1.71)%,(69.64+1.10)% and(69.27+0.70)%,respectively;while that of S phase were(33.06+2.29)%,(20.79+1.88)% and(22.62+1.23)%,respectively;compared with the control group,the percentage of the cells in G1 phase was increased(P<0.05)and that in S phase was decreased significantly(P < 0.05)in the Torin2 treatment groups,which elucidated that Torin2 induced G1/S phase arrest in A549 cells.4.Wound healing test showed that,the scratch width of the control group was significantly narrowed after 24 and 48 hours,whereas the change of that in Torin2 treatment group(200 and 400 nmol/L)is not obvious;meanwhile,Transwell chamber assay showed that the numbers of cell migration in group Torin2(200 and 400 nmol/L)treated with A549 cells for 48 hours were(107.2±18.9)and(13.4±3.0),respectively,which were decreased significantly compared with the control group,the migrated cell numbers of which were(190.2+14.2),the results above showed that Torin2 decreased the capacity of A549 cell migration.5.Compared with the control group,the expression levels of Cyclin D1,phosphorylated Akt473 and 4EBP137/46 were down-regulated and that of E-cad and Caspase 9 is up-regulated in Torin2 treatment(200 and 400 nmol/L)group.Conclusion:A novel mTOR inhibitor Torin2 inhibited the proliferation of A549 cells,decreased the cell migration ability,induced cell cycle arrest in G1/S stage and promoted cell apoptosis,which might be associated with its suppression on PI3K/Akt /mTOR signal pathway and the expression level of Cyclin D1 and E-Cad. |
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