Tripchlorolide Induces Autophagy In Lung Cancer Cells Via Inhibiting The PI3K Signaling Pathway And Improves Cisplatin Sensitivity Of A549/DDP Cells

Object:As a most prevalent disease,lung cancer has been ranked among the leading causes of death worldwide.A new drug was important for the treatment of lung cancer.Tripchlorolide(T4),an attenuated monomer,has been extracted from tripterygium or obtained from the hydroxyl acylation and chlorination of triptolide and presents higher activity and lower toxicity than triptolide.Studies have found Tripchlorolide can induce autophagy in Lung Cancer Cells,but its mechanism has not yet been elucidated.In our study,we discussed whether Tripchlorolide induced Autophagy in A549 and A549/DDP Lung Cancer Cells via inhibiting the PI3K/Akt/mTOR Signaling Pathway and improved Cisplatin Sensitivity of A549/DDP Cells.This study aims to investigate the role the role of T4 in lung cancer treatment and the underlying molecular mechanisms.Methods:1.A549 cells were cultured in DMEM-high glucose supplemented with 10% fetal bovine serum(FBS)in a humidified incubator under 5% CO2 at 37℃.A549/DDP cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum(FBS)and in a humidified incubator under 5% CO2 at 37℃.We first determined the optimum concentration of Tripchlorolide via CCK8,then the cells were treated with the optimum concentration of Tripchlorolide.After 24 h,we detected the changes of LC3 II and the changes of PI3K/Akt/mTOR Signaling Pathway by western blotting.2.A549 and A549/DDP lung cancer cells were seeded in 6-well plates,and the cells were with a pretreatment with wortmannin(inhibitor of PI3K),perifosine(inhibitor of AKT)or rapamycin(inhibitor of mTOR)combined with a subsequent T4 treatment.After 24 h,we also observed the changes of LC3 II and the changes of PI3K/Akt/mTOR Signaling Pathway by western blotting.3.A549 and A549/DDP lung cancer cells were seeded in 96-well plates,from CCK8,we detected the activity of PI3 K and AKT.4.A549 and A549/DDP lung cancer cells were seeded in 6-well plates,then we used overexpressed AKT to transfect cells and observe the changes of LC3 II.5.A549 and A549/DDP lung cancer cells were seeded in 6-well plates or 96-well plates,then we detected the Cisplatin Sensitivity of A549/DDP Cells via CCK8,qPCR and western blotting.Results:1.We confirmed the optimum concentration of Tripchlorolide is 200 nM,24 h via CCK8.From western blotting,the protein level of PI3-K,PI3-P,Akt,TSC2,m TOR,p70S6 K and 4E-BP1 had little change,but their protein phosphorylation changed a lot.2.After a pretreatment with rapamycin(inhibitor of PI3K),wortmannin(inhibitor of AKT)or perifosine(inhibitor of mTOR)combined with a subsequent T4 treatment,the expression levels of PI3-K,PI3-P,Akt,TSC2,m TOR,p70S6 K and 4E-BP1 were minimally affected by wortmannin,perifosine,or rapamycin plus T4 treatment,but their phosphorylated products were greatly affected in A549 lung cancer cells and slightly affected in A549/DDP lung cancer cells.3.The expression of LC3 II paralleled the increase of T4 concentration both in A549 and A549/DDP cells,which was repressed by the overexpressed AKT.4.After T4 treatment with cells,cell killing effect of Cisplatin increased significantly by CCK8.And T4 decreased the expression of MDR1 to improve the cisplatin sensitivity of A549/DDP cells via qPCR and western blotting.Conclusions:1.Tripchlorolide can induces cell death in both A549 and A549/DDP cells by autophagy.2.Tripchlorolide induces autophagy mainly via PI3K/AKT/mTOR Signaling Pathway.3.Tripchlorolide effectively improves the sensitivity of A549/DDP cells to cisplatin.