Glaucocalyxin A Induces Apoptosis Of NSCLC By Inhibiting The PI3K/Akt/GSK3β Pathway

Objective:To investigate the anti-lung cancer effects of Glaucocalyxin A(GLA)in vivo and in vitro and its potential molecular mechanism,and to provide experimental evidence for further clinical application of GLA.Methods:1.Normal lung epithelial cells(BEAS-2B)and non-small cell lung cancer cells(A549and H460)were treated with GLA at different concentrations(0,2.5,5,10,20,40μM)for0-72 h.The effect of GLA on cell viability was detected by CCK-8 method,and OD values of each group were calculated.Subsequently,the mother liquor of bluecycalycline was diluted successively to(0,2.5,5,10μM),and the effect of GLA on cell clonogenesis was detected by plate cloning assay.In addition,we also used Ed U method to analyze cell proliferation;2.A549 and H460 cells were treated with different concentrations of GLA(0,5,10,20μM)for 24 h,and the effect of GLA on cell apoptosis was detected by flow cytometry.The expression changes of apoptosis-related proteins(Bax,Bcl-2,Caspase3,C-Caspase3,PARP,C-PARP)were detected by Western blot.3.A549 cells were treated with different concentrations of GLA(0,5,10,20μM)for24 h,and the effects of GLA on mitochondrial membrane potential were detected by flow cytometry.4.A549 cells and H460 cells were treated with different concentrations of GLA(0,5,10,20μM)for 24 h.Western blot was used to detect the expression changes of PI3K/Akt/GSK3βrelated proteins(PI3K,PTEN,p-PDK1,PDK1,Akt,p-Akt,GSK3β,p-GSK3β).5.A549 cells were injected subcutaneously into the left back of nude mice to establish a tumor bearing mouse model of A549.24 h later,20 mg/kg GLA was intraperitoneally injected into the experimental group,while the control group was injected with equal volume PBS.The drug was adistered every 48 hours thereafter,and the tumor size was measured with vernier-caliper every three days.The tumor volume(mm~3)=width~2(mm~2)×length(mm)×0.5 was calculated,and the weight of mice was weighed by an electronic balance.The mice were sacrificed on the 28th day,the tumors were extracted and weighed,and the apoptosis of tumor tissues was detected by TUNEL and Ki67immunohistochemistry.The expressions of apoptosis-related proteins and PI3K/Akt/GSK3β-related proteins were detected by Western blot.Results:1.GLA inhibited the cell viability and colony formation of human NSCLC cells in a concentration-dependent and time-dependent manner.No statistical changes were observed in BEAS-2B cells.2.GLA induced apoptosis of A549 and H460 cells in a concentration-dependent manner,up-regulated Bax protein,down-regulated Bcl-2 expression,regulated the decline of Caspase3 and PARP protein,and promoted the increase of C-Caspase3 and C-PARP protein.3.GLA decreased the mitochondrial membrane potential of A549 cells in a concentration-dependent manner.4.GLA inhibited PI3K/Akt/GSK3βpathway in NSCLC cells in a concentration-dependent manner.The protein expressions of PDK1,Akt and GSK3βremained unchanged after GLA treatment.At the same time,PTEN increased gradually,while PI3K levels,PDK1 phosphorylated(Ser241),Akt(Thr308)and(Ser473)and phosphorylated GSK3β(Ser9)decreased.5.In vivo studies showed that 20 mg/kg GLA could significantly inhibit tumor growth,induce cell apoptosis in tumor tissues,up-regulate Bax protein,down-regulate Bcl-2expression,and enhance Caspase3 and PARP cleavage.These data show that changes in the PI3K/Akt/GSK3βpathway and apoptosis pathway are consistent with those found in vitro.Conclusions:In conclusion,the results of this study in vitro and in vivo suggest that the effect of GLA on NSCLC is mediated by inhibiting PI3K/Akt/GSK3βpathway and inducing cell apoptosis.