Role Of Stat3/Survivin Signaling Pathway In The EML4-ALK Positive Lung Adenocarcinoma Cell-Line H2228 Before And After Crizotinib Induced Resistance

Objective:To investigate the role of the stat3/survivin 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:Proliferation inhibition of cell 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, p-stat3, stat3 and survivin of stat3/survivin signaling at crizotinib concentrations of 0,0.3, 1μM were determined at 72h using western blots. DNA sequencing was performed on H2228 cells and H2228 crizotinib resistance cells.Results:IC50 values for H2228 and H2228 crizotinib resistance cells at 72h were 334.5 nM and 3,418 nM, respectively. The resistance index of H2228 crizotinib resistance cells is 10.20. Crizotinib induces apoptosis in H2228 cells (P<0.05) and significantly inhibits the expression of p-ALK, p-stat3 and survivin. Compared with H2228 cells, expression of p-ALK, p-stat3 and survivin were not changed in H2228 crizotinib resistance cells.The 2067 G→A and 2182 G→C mutations of EML4-ALK V3b present in the H2228 crizotinib resistance cells.Conclusion:Crizotinib decreases the proliferation inhibition of H2228 cells in a time-dependent manner. Stat3/survivin may be involved in crizotinib-induced apoptosis of H2228 cells by down-regulating of p-ALK, p-stat3 and survivin. however, it has no effect on H2228 crizotinib resistance cells. H2228 cells acquired resistance may be related to change of amino acids of ALK gene.