| 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 H2228and EML4-ALK negative cell line A549(control) were treated with crizotinib at varying doses for different time periods (24h,48h,72h).Proliferation inhibition of cell lines was quantitated by MTT method.With PI staing in flowcytometry, crizotinib-induced apoptosis with concentration of300nmol/L of different time periods (24h,48h,72h) was measured.The expression of BIM as well as pro-apoptosis factor Bid and anti-apoptosis factor Bcl-2and Bcl-xL of cell lines before and after crizotinib treatment were examined by Western blot analsysis.siRNA technology was used to’silent’ the BIM gene expression.Proliferation inhibition of cell line after BIM gene was silented was quantitated by MTT method.Results:Crizotinib-induced proliferation inhibition of H2228cell line was observably higher than that of A549cell line (P<0.05).As concentration of crizotinib increased, proliferation inhibition of H2228cell lines was increased in a dose-dependent manner with IC50335nM.Apoptosis rate of H2228cell line was increased with the crizotinib’s concentration and exposure time.Up-regulated expression of BIM at protein levels together with down-regulated anti-apoptosis proteins Bcl-2and Bcl-xl and invariant pro-apoptosis factor Bid after exposure to crizotinib was confirmed by Western blot analsysis.Crizotinib-induced proliferation inhibition of H2228cell line after siRNA technology ’Silent’ BIM gene was found a significant decrease.Conclusions:Crizotinib inhibits proliferation in lung adenocarcinoma cell line H2228by induction of apoptosis with dose-and time-dependent manner, Crizotinib induces apoptosis of H2228cell line by down-regulated anti-apoptosis proteins Bcl-2and Bcl-xl and up-regulated expression of BIM at protein levels. |
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