EML4-ALK融合基因阳性表达的人肺腺癌H2228细胞对克唑替尼耐药后BIM信号通路的影响

Role of BIM signaling in crizotinib-induced apoptosis in the EML4-ALK-positive lung adenocarcinoma cell line H2228

目的建立人肺腺癌H2228克唑替尼耐药细胞株,探讨克唑替尼(crizotinib)诱导EML4-ALK融合基因阳性表达的人肺腺癌细胞H2228在BIM信号通路中的作用。方法克唑替尼按50 nmol/L、100 nmol/L、200 nmol/L、500 nmol/L、1 000 nmol/L的浓度逐步递增法诱导人肺腺癌H2228细胞株获得性耐药;分别采用MTT法和流式细胞术检测H2228和H2228/CR细胞在不同浓度克唑替尼作用后的IC50和细胞凋亡率;采用Western blot法检测H2228和H2228/CR细胞在BIM信号通路关键分子ALK、p-ALK、ERK、p-ERK及BIM蛋白表达的变化。结果成功诱导克唑替尼耐药细胞株H2228/CR;MTT法检测H2228/CR细胞和H2228细胞的IC50分别为3 418 nmol/L、335 nmol/L,H2228/CR细胞的耐药指数为10.20,H2228/CR细胞在不同浓度克唑替尼作用下的增殖抑制率低于H2228细胞(P<0.05);流式细胞术检测显示克唑替尼对H2228细胞有促凋亡作用,且呈时间依赖性(P<0.05),H2228/CR细胞的凋亡率明显低于H2228细胞的凋亡率(P<0.05);Western blot法检测显示H2228/CR细胞的p-ALK、p-ERK的表达不受抑制,BIM蛋白表达无上调趋势。结论实验表明H2228/CR耐药细胞中的BIM蛋白的表达与其耐药有关。初步阐明EML4-ALK阳性表达的非小细胞肺癌克唑替尼耐药产生的可能机制,BIM在诱导EML4-ALK阳性表达的人肺腺癌细胞株H2228对克唑替尼产生耐药发挥重要作用,提示BIM可作为克服克唑替尼耐药的一个靶点。

Objective To establish a crizotinib-resistant line of the EML4-ALK-positive lung adenocarcinoma cell line H2228 and compare it with the crizotinib-sensitive parental line to examine the possible role of BIM in crizotinib-induced apoptosis. Methods H2228 human lung cells were exposed to gradually increasing doses of crizotinib（50,100,200,500,1000 nmol/L）to create a crizotinibresistant line（H2228/CR）. Then H2228/CR and parental H2228 cells were exposed to different doses of crizotinib,and their growth was compared using the MTT assay,while levels of apoptosis were compared using flow cytometry. Western blotting was used to compare levels of ALK,p-ALK,ERK,p-ERK and BIM in the two cell lines. Results After 8 months of crizotinib selection,the resistant line H2228/CR showed an IC50 of 3,418 nmol/L compared to 335 nmol/L for the parental H2228 line. The RI for H2228/CR cells was10.20. Crizotinib inhibited the growth of H2228/CR cells to a much smaller extent than it inhibited H2228,and it led to a much smaller proportion of apoptotic cells in H2228/CR cultures. Levels of phospho-ERK were higher in H2228/CR cells,implying downregulation of BIM. Conclusion BIM may help mediate crizotinib-induced apoptosis in lung cancer,and its down-regulation may contribute to crizotinib resistance.