Bim在克唑替尼诱导EML4-ALK融合基因阳性肺腺癌细胞株H2228凋亡中的作用机制

摘要

目的：探讨Bim在克唑替尼诱导EML4-ALK融合基因阳性肺腺癌细胞株H2228凋亡过程中的作用机制。方法在不同时间点（24、48、72h）用不同浓度克唑替尼分别处理EML4-ALK阳性肺腺癌H2228细胞株和EML4-ALK阴性肺癌A549细胞株，采用MTT法检测克唑替尼作用72h的细胞增殖抑制情况；PI单染流式细胞仪检测经300nmol/L克唑替尼作用24、48和72h的细胞凋亡率；采用Western blotting法检测克唑替尼诱导前后Bim蛋白（ Bim-EL、Bim-L和Bim-S）的表达水平，同时对促凋亡分子Bid和抗凋亡分子Bcl-2、Bcl-xL的蛋白水平进行联合检测；采用siRNA技术“沉默”Bim基因的表达，用流式细胞仪检测 siRNA“沉默”Bim基因后克唑替尼诱导H2228细胞株的凋亡率。结果克唑替尼作用72h后，肺腺癌H2228细胞株和A549细胞株的细胞增殖抑制率均随着克唑替尼药物浓度的增加而逐渐升高，呈剂量依赖性。克唑替尼作用于H2228细胞72h 的 IC50值为335nmol/L。300nmol/L 克唑替尼作用 H2228细胞株24、48、72h 后的凋亡率分别为（19�19±0�61）％、（35�47±1�17）％、（43�58±4�84）％，作用A549细胞株的凋亡率分别为（12�71±0�1）％、（18�22±0�13）％、（19�36±0�45）％。随着克唑替尼（300nmol/L）作用时间的延长，细胞凋亡率亦随之增加，并呈时间依赖性（ P＜0�05）。在凋亡细胞中发现Bim蛋白水平升高，以及抗凋亡蛋白 Bcl-2、Bcl-xL表达降低，但促凋亡蛋白 Bid 在不同药物浓度及时间点的表达水平无明显变化。此外，经siRNA 技术“沉默”Bim 基因后，克唑替尼诱导的H2228细胞株凋亡率明显降低。结论克唑替尼通过下调抗凋亡蛋白 Bcl-2、Bcl-xL及上调Bim的表达水平来发挥其诱导细胞凋亡作用，该过程可被Bim siRNA抑制。%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-apop-tosis 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. Ap-optosis rate of H2228 cell line increased with the concentration and exposure time of crizotinib. Up-regulated expression of Bim at pro- tein 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 af-ter Bim gene was silent by siRNA technology. Conclusion Crizotinib 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.