Abstract: Objective To explore the role of BH3-only sub-family member Bim in crizotinib-induced apoptosis of EML4ALK fusion gene positive lung adenocarcinoma cell line H2228. Methods EML4-ALK positive cell line H2228 and EML4-ALK negative cell line A549 were treated with crizotinib at various concentrations for different time（24h，48h，72h）. Proliferation inhibition rate of cell lines were determined by MTT method. With PI staing in flowcytometry，crizotinibinduced apoptosis with concentration of 300nmol／L of different time（24h，48h，72h） were measured. The expression of Bim （Bim-EL， Bim-L and Bim-S） as well as pro-apoptosis factor Bid and anti-apoptosis factor Bcl-2 and Bcl-xL of cell lines before and after crizotinib administration were examined by Western blotting. SiRNA technology was used to ‘silent’ the Bim gene expression. Proliferation inhibition rate of cell lines after silent Bim gene were determined by MTT method. Results As concentration of crizotinib increased，crizotinibinduced proliferation inhibition rates of H2228 and A549 cell lines for 72h were increased in a dose-dependent manner. IC50 of H2228 cell line treated by crizotinib for 72h was 335nmol／L. After treated by 300nmol／L crizotinib for 24h，48h，72h， the apoptosis rates of H2228 and A549 cell lines were（19.19±0.61）％，（35.47±1.17）％，（43.58±4.84）％ and （12.71±0.1）％，（18.22±0.13）％，（19.36±0.45）％， respectively. Apoptosis rate of H2228 cell line increased with the concentration and exposure time of crizotinib. Up-regulated expression of Bim at protein levels together with down-regulated antiapoptosis proteins Bcl-2 and Bcl-xL and in variant proapoptosis factor Bid after exposure to crizotinib was confirmed by Western blotting. Crizotinib-induced proliferation inhibition of H2228 cell line significantly decreased after Bim gene was silent by siRNA technology.
ConclusionCrizotinib inhibits proliferation in lung adenocarcinoma cell line H2228 with dose and time-dependent manner. Crizotinib induces apoptosis of H2228 cell line by downregulating anti-apoptosis proteins Bcl-2 and Bcl-xL as well as up-regulating expression of Bim. These pro-apoptosis processes can be blocked by Bim siRNA.