Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK–positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mechanism of resistance was studied. Methods Cell viability was determined using the MTT assay. Crizotinib-induced apoptosis in H2228 and H2228 crizotinib-resistant cells treated with the indicated doses of crizotinib was measured at different times(24 h, 48 h, 72 h) using flow cytometry. The levels of p-ALK, ALK, p-STAT3, STAT3, and survivin after treatment of cells with 0, 0.3, and 1 μM crizotinib for 72 h were determined using Western blot analysis. DNA sequencing was used to identify mutations in H2228 crizotinib-resistant cells. Results The crizotinib IC50 values in H2228 and H2228 crizotinib-resistant cells at 72 h were 334.5 n M and 3418 n M, respectively. The resistance index of H2228 crizotinib-resistant cells was 10.20. Crizotinib induced apoptosis in H2228 cells and reduced the levels of p-ALK, p-STAT3, and survivin. In contrast, no changes in the levels of p-ALK, p-STAT3, and survivin were observed in H2228 crizotinib-resistant cells. The mutations 2067G→A and 2182G→C in EML4-ALK were present in the H2228 crizotinib-resistant cells. Conclusion Crizotinib decreased the viability of H2228 cells in a dose- and time-dependent manner. In the STAT3/survivin pathway, downregulation of p-ALK, p-STAT3, and survivin might contribute to crizotinib-induced apoptosis in H2228 cells. However, the STAT3/survivin pathway in H2228 crizotinib-resistant cells was unaffected by crizotinib treatment. Acquired resistance in H2228 cells might be related to ALK mutations.
Objective This study aimed to study the role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis of various lung adenocarcinoma cell lines and xenograft tumor models.Methods In vitro, H2228, H1993, and A549 cells were treated with crizotinib. The inhibition of proliferation was quantitated by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay. Apoptosis was quantified by flow cytometry. Expression of key proteins of the HGF/c-Met signaling pathway was examined by western blotting. In vivo, H1993 and A549 tumor cell xenograft models were established. Immunohistochemical analysis was used to determine protein expression of HGF and c-MET and the amount of phospho-c-MET(p-c-Met). Real-time quantitative polymerase chain reaction(PCR) was applied to examine the messenger RNA(m RNA) expression of c-MET and serine/threonine protein kinase(AKT). The expression and activation of the key proteins were evaluated by western blotting.Results In vitro, the growth of H1993, H2228, and A549 cells was inhibited after crizotinib treatment for 72 h. Apoptotic rates of H1993 and H2228 cells increased with the crizotinib concentration and exposure time. In vivo, the growth-inhibitory rate of crizotinib for H1993 xenografts was 72.3%. Positive expression rates of HGF and c-MET in H1993 xenografts were higher than those in A549 xenografts; the p-c-MET amount was the largest in H1993 xenograft control but the lowest in the H1993 xenograft with crizotinib treatment. The m RNA expression levels of c-MET and AKT in H1993 xenografts were higher than those of A549 xenografts. The protein levels of c-MET, AKT, and extracellular regulated protein kinases(ERK) in H1993 xenografts were higher than those in A549 xenografts; the p-AKT amount was higher in H1993 xenograft control than in A549 xenografts; the largest amount of p-c-MET was detected in H1993 xenograft control; the amount of p-ERK was the lowest in the H1993 xenograft with crizotinib treatment.Conclusion The HGF/c-Met signaling pathway may mediate
Shaozhang ZhouZhixin DongJinyi LvAiping ZengHuilin WangRuiling NingXiangqun Song
