Influence Of BIM Signaling Pathway In EML4-ALK Positive Lung Adenocarcinoma Cell-Line H2228 After Crizotinib Induced Resistance

OBJECTIVE:Establishing a resistant H2228 cell line of human lung adenocarcinoma by crizotinib inducing.To investigate the role of the BIM signaling in EML4-ALK positive lung adenocarcinoma cells H2228 before and after crizotinib-induced resistance and studies on the mechanism of resistance of H2228 cells.METHODS:Human adenocarcinoma lung cell H2228 were exposed to crizotinib in gradually increasing dose of 50nmol/L, 100nmol/L,200nmol/L,500nmol/L, 1000nmol/L, and establish acquired resistance in human lung adenocarcinoma cell line H2228 after eight months;Cell viability was determined by using MTT assay. Crizotinib-induced apoptosis of H2228 and H2228 crizotinib resistance cells at indicated doses of crizotinib was measured at different times points (24h, 48h,72h) using flow cytometry. The expressions and activity levels of p-ALK, ALK, and Bim-s、Bim-L、Bim-exL of BIM signaling at crizotinib concentrations of 0,0.3,1,10μM were determined at 72h using western blots.RESULTS:1、Crizotinib resistant cell line H2228/CR were successfully established by gradual increasing concentrations in 8 months;2、Crizotinib induces apoptosis in H2228 cells (P<0.05), comparing to H2228/CR cells, IC50 values for H2228 and H2228/CR were 335 nM and 3,418 nM, respectively. The resistance index of H2228 crizotinib resistance cells is 10.20;3、Crizotinib significantly inhibits the expression phospho-ALK and phospho-ERK of H2228, which increase expression of Bim-s、Bim-L、Bim-exL in H2228 cells; while phospho-ALK、phospho-ERK and Bim-s、Bim-L、 Bim-exL were not significant changes in the expression levels of H2228/CRCONCLUSION:Established resistant human lung adenocarcinoma cell line H2228/CR by in gradually increasing dose of crizotinib, detecting the inhibition rate, apoptosis and protein expression levels BIM confirmed that BIM is related to the in duction of EML4-ALK-positive lung cancer cell line H2228 for crizotinib-resistance and may play a regulated role in the resistanted process.