Afatinib Incombination With MEK Inhibitor On NCI-H522,A549,UM-SCC-74B And JHU-O28 Cell Lines

Part one: Afatinib incombination with GSK1120212 on non small cell lung cancer NCI-H522 and A549 cell lines[Objective]To explore how afatinib incombination with MEK inhibitor GSK1120212 influence NSCLC cell protein expression,proliferation and apotosis.[Materials and Methods]1.Fuction experiments: NCI-H522 and A549 cells were cultrued in proper medium and devided into three groups treat with afatinib,GSK1120212 and both drugs respectively.Each group contained four dishes,received final concentrations of 0,2.5,5.0,10?Mol/L for afatinib group,and 0,1.0,2.0,4.0?Mol/L for GSK1120212 group.Combination group was DMSO,5.0?Mol/L afatinib,2.0?Mol/L GSK1120212,5.0?Mol/L afatinib plus 2.0?Mol/L GSK1120212,respectively.Cells were cultrued for 24 hours and collected for western blot to detect the expression of p-EGFR,p-AKT,p-S6 and p-ERK1/2,etc.2.Proliferation experiments: Cells were cultrued in 96-well plates,withfinal concentrations of 0,0.125,0.25,0.5,1.0,2.0?Mol/L for afatinib and GSK1120212,and 1:1 combined for the third group.Every sigle dose for each group was designed for 3 wells,then cultrued for 72 hours to test proliferation rate.3.Apotosis experiments: Drug treatment was the same as combination group,cells were cultrued for 72 hours for flow cytometry.[Results]1.Afatinib alone inhibited PI3K/AKT/mTOR path way to suppress p-EGFR,p-AKT and p-S6 whereas induced p-ERK1/2 expression on NCI-H522 and A549 cell lines.2.GSK1120212 alone inhibited RAS/MEK/ERK path way to suppress p-ERK1/2 but induced p-AKT at the same time.3.Afatinib combine with GSK1120212 blocked both path ways thus decreased p-AKT,p-S6 and p-ERK1/2 preferably.4.Afatinib combine with GSK1120212 significantly lowered cell proliferation rate and promoted cell apotosis when compared to either drug(p<0.05).[Conclusion]Afatinib combine with GSK1120212 blocked both PI3K/AKT/mTOR and RAS/MEK/ERK path ways preferably,significantly inhibited cell proliferation and promoted apotosis.Part 2: Afatinib incombination with PD0325901 on squamous cell lines UM-SCC-74 B and JHU-O28[Objective]To explore how afatinib incombination with MEK inhibitor PD0325901 influence HNSCC cell protein expression,proliferation and apotosis.[Materials and Methods]1.Fuction experiments: UM-SCC-74 B and JHU-O28 cells were cultrued in proper medium and devided into three groups treat with afatinib,PD0325901 and both drugs respectively.Each group contained four dishes,received final concentrations of 0,2.5,5.0,10?Mol/L ?Mol/L for afatinib group,and 0,1.0,2.0,4.0 ?Mol/L for PD0325901 group.Combination group was DMSO,5.0?Mol/L afatinib,2.0?Mol/L PD0325901,5.0?Mol/L afatinib plus 2.0?Mol/L PD0325901,respectively.Cells were cultrued for 24 hours and collected for western blot to detect the expression of p-EGFR,p-AKT,p-S6 and p-ERK1/2,etc.2.Proliferation experiments: Cells were cultrued in 96-well plates,with final concentrations of 0,0.125,0.25,0.5,1.0,2.0?Mol/L for afatinib and PD0325901,and 1:1 combined for the third group.Every sigle dose for each group was designed for 3 wells,then cultrued for 72 hours to test proliferation rate.3.Apotosis experiments: Drug treatment was the same as combination group,cells were cultrued for 72 hours for flow cytometry.[Results]1.Afatinib alone inhibited PI3K/AKT/m TOR path way to suppress p-EGFR,p-AKT and p-S6 whereas induced p-ERK1/2 expression on UM-SCC-74 B and JHU-O28 cell lines.2.PD0325901 alone inhibited RAS/MEK/ERK path way to suppress p-ERK1/2 but induced p-AKT at the same time.3.Afatinib combine with PD0325901 blocked both path ways thus decreased p-AKT,p-S6 and p-ERK1/2 preferably.4.Afatinib combine with PD0325901 significantly lowered cell proliferation rate and promoted cell apotosis when compared to either drug(p<0.05).[Conclusion]Afatinib combine with PD0325901 blocked both PI3K/AKT/m TOR and RAS/MEK/ERK path ways preferably,significantly inhibited cell proliferation and promoted apotosis.