多西紫杉醇诱导人肺腺癌细胞A549及其多药耐药细胞A549/CDDP凋亡的研究

Docetacel-induced apoptosis of human lung adenocarcinoma cell line A549 and multidrug-resistant cell subline A549/CDDP

目的探讨凋亡在多西紫杉醇对人肺腺癌细胞A549及其多药耐药细胞A549/CDDP作用中的地位及其机制。方法应用透射电镜、流式细胞术、Annexin V/PI和免疫细胞化学等方法,观察多西紫杉醇对A549及A549/CDDP细胞形态学、细胞周期、凋亡以及Fas等蛋白表达的影响。结果多西紫杉醇作用后,A549及A549/CDDP细胞内空泡明显增多,可见少数凋亡小体和脱落的不含细胞器成分的细胞质,后者还出现大量多微核化细胞。多西紫杉醇具有显著的G2期阻滞作用,可同时诱导细胞凋亡、坏死和胞质自切,并以后两者为主。Fas蛋白在多西紫杉醇作用后表达明显增强。结论多西紫杉醇对A549及A549/CDDP细胞均具有显著的G2/M期阻滞作用,能同时诱导其凋亡、坏死和胞质自切,以后两者为主。Fas基因可能在多西紫杉醇抗肿瘤作用中具有重要作用。

Objective To investigate the apoptosis-inducing effect and its potential mechanisms of docetaxel on human lung adenocarcinoma cells A549 and multidrug resistant cell subline A549/CDDP. Methods Light microscope and TEM were used to observe cell morphological changes. FACs was used to detect the cell cycle arrest effect of docetaxel. TEM and Annexin V/PI staining were employed to measure apoptosis induced by docetaxel. Immuocytochemistry was carried out to confirm the expression of Bcl-2, Bax, Fas and other apoptosis related protein before and after treatment of A549 and A549/CDDP cells with docetaxel. Results After the treatment of docetaxel, vacuoles increased, microvilli decreased, and mitochondria swelled. A few apoptosis bodies and cytoplasm without organelles were observed both in A549/CDDP and A549 cells. Multimicronucleation appeared in A549/CDDP cells. FACs showed that docetaxel induced the arrest of A549 and A549/CDDP cells in G2 phase. Docetaxel could induce apoptosis, necrosis and autoschizis of A549 and A549/CDDP cells, with the latter two dominated. After treatment of docetaxel, Bax upregulated in A549/CDDP cells and Fas noticeably upregulated in both cells. The expression of other apoptosis related proteins had no visible changes. Conclusion Docetaxel could induce the arrest of A549 and A549/ CDDP cells in G2 phase and mainly results in cell necrosis and autuschizis; meanwhile, apoptosis also plays a role in it. The upregulation of Fas might be one of the mechanisms of docetaxel.