Mechanism Of Apigenin Combined With Gefitinib In Inducing Apoptosis Of EGFR L858R+T790M Mutant H1975 Cells

Rationale:Cancer cells have high rates of glucose metabolism,which is primarily regulated by c-Myc,HIF-la and Glutl.c-Myc,HIF-la and Glutl over-expression is associated with drug resistance and poor prognosis in patients with NSCLC.Epidermal growth factor receptor inhibitors(EGFR-TKIs)inhibit glucose uptake in vitro and in vivo,but single drug do not inhibit the expression of c-Myc and HIF-la proteins in xengraft tumors.Phase ? clinical trials that blocked Myc amplification in SCLC,as well as phase ? clinical trials that blocked HIF-la in advanced NSCLC,did not benefit.Apigenin has an inhibitory effect on Glutl,but Glutl blockers do not inhibit xenograft growth.Gefitinib combined with apigenin to inhibit the glucose uptake of natural TKIs-resistant EGFR-T790M mutant NSCLC cells H1975,whether it affects H1975 cell apoptosis and its mechanism is unknown.Materials and Methods:1.Apigenin and gefitinib were incubated with H1975 cells at 1:1 ratio,grouped as:vehicle,apigenin,gefitinib,apigenin+gefitinib,detected at the end of time point.2.The cell viability of H1975 cells was measured by CCK-8,and the combination index was calculated by Chou-Talalay rule combined with CompuSyn software.3.Flow cytometry was used to measure cell cycle and cell apoptosis,Western Blot was used to measure the protein markers of cell cycle,apoptosis and migration,colony formation and cell scratch assay to evaluate the functional changes of H1975 cells.4.L-lactate,ATP kit was used to measure the intracellular lactate and ATP of H1975,Mitotracker green TM + Hoechst33342 double staining was used to evaluate mitochondrial mass,2-NBDG was used to measure H1975 cell glucose uptake,and Western Blot was used to measure glucose metabolism accosiated proteins.5.Western Blot was used to detect autophagy signaling pathway-associated proteins and HEF-1?,c-Myc,Kras and p-EGFR proteins.CCK-8 was used to measure the cytotoxicity of autophagy inhibitor chloroquine(CQ)and agonist(RAPA)of H1975 cells,and the apoptosis rate of apigenin + gefitinib + CQ was measured by flow cytometry.6.H1975 cells were incubated with c-Myc,Glutl and HIF-1? specific inhibitors 10058-F4,STF-31 and KC7F2,respectively.CCK-8 was used to detect the cytotoxicity,and Western Blot was used to detect c-Myc,HIF-1?,Glutl and p-EGFR protein expression.7.The relationship between the expression of c-Myc,HIF-la,Glutl,EGFR and the clinical stage and prognosis in Lung adenocarcinoma(LUAD)were performed by the common database GEPIA and Kaplan-Meier plotter.8.Statistical analysis:All data using mean ± standard deviation(mean±SD).Applied GraphPad Prism 7.0 unpaired student's t-test to calculate,ns means no statistically significant difference,*p<0.05,**p<0.01,***p<0.001,****p<0.0001.Chou-Talalay method combined with CompuSyn software to calculate the drug-drug combination index.CI>1 means antagonism,CI=1 means synergy,CI<1 means addition,CI<0.4 means strong addition.Results:1.0,20,30,40,50,60,70,80 ?M apigenin,cisplatin and gefitinib were incubated with 1975 cells for 72 h,respectively.All three drugs decreased the survival rate of H1975 cells in a concentration-dependent manner.Apigenin and gefitinib 1:1 combination showed a specific synergistic effect in inhibition EGFR L858R-T790M mutant H1975 cells,and synergistically enhanced with increasing concentration.2.Apigenin+gefitinib combined treatment induced G0/G1 cell cycle arrest in H1975 cells,decreased Cyclin D1,CDK4,E-cadherin,MMP2 and MMP9 expression,and inhibited H1975 cell proliferation,colony formation,and migration.3.Apigenin+gefitinib combined treatment significantly reduced Bcl-2 expression,promoted BIM,cleaved-caspase-3,cleaved-PARP-1 expression,and increased the apoptosis rate of H1975 cells.4.Apigenin+gefitinib combined treatment significantly reduced the intracellular L-lactic acid and ATP levels of H1975 cells,the cell morphology became thinner and the mitochondrial mass was reduced.Gefitinib and combination therapy both inhibited glucose uptake in 5,15 and 30 min,while the latter significantly inhibited Glutl,Glut3,Glut4,MCT1,and PDK1 protein expression.5.Apigenin+gefitinib combined treatment decreased c-Myc,HIF-1?,Kras,p-EGFR and p-AMPKa expression and increased p62,LC3-? protein accumulation,CQ enhanced combination therapy's cytotoxicity and apoptosis rate of H1975 cells.6.KC7F2 caused H1975 cells to form shuttle length,10058-F4 enhanced Glutl expression of the H1975 cells,inhibited HIF-la,p-EGFR expression.KC7F2 promoted c-Myc expression of the H1975 cells,did not change p-EGFR and Glutl expression.STF-31 inhibited p-EGFR expression of the H1975 cells,did not change c-Myc,HIF-la expression.The combination treatment inhibited c-Myc,HIF-la,p-EGFR,Glutl,MCT1,LDHA,GAPDH,and Bcl-2 expression in a concentration-and time-dependent manner,and increased Bax expression.7.Bioinformatics analysis showed that c-Myc expression was correlated with HIF-la and Glutl in lung adenocarcinoma,and HIF-la,EGFR,Glutl expression were related to each other.The expression of Glutl in adenocarcinoma was higher than that in matched adjacent tissues.The expression of c-Myc and Glutl was significantly increased with clinical stage,while Glutl,c-Myc,EGFR,and HIF-la high expression are associated with lung adenocarcinoma a poor prognosis.Conclusions:1.Apigenin combined with gefitinib shows a synergistic effect to increase EGFR T790M mutant H1975 cell inhibition.2.Apigenin cooperated with gefitinib induces the Go/Gi cell cycle arrest and cell migration in H1975 cells.3.Apigenin cooperated with gefitinib by inhibition Bcl-2 and inducing BIM,cleaved-caspase-3,and cleaved-PARP-1 to increase apoptosis in H1975 cells.4.Apigenin cooperated with gefitinib through inhibition the glucose transporter proteins,prevention glucose uptake,resulting in a significant reduction of intracellular lactate,ATP production and mitochondrial mass in H1975 cells.5.Apigenin cooperated with gefitinib via blocking autophagy flux and transcription factors c-Myc,HIF-1? and p-EGFR to increase H1975 cell apoptosis.6.Apigenin cooperated with gefitinib to inhibit transcription factors c-Myc,HIF-la and downstream p-EGFR and Glutl in a concentration and time-dependent manner,simultaneously reduce Bcl-2 and induce Bax protein expression.7.Glutl,c-Myc,EGFR,and HIF-la high expression are associated with lung adenocarcinoma a poor prognosis.