阿帕替尼通过ABCB1逆转非小细胞肺癌紫杉醇耐药的机制  

Mechanism of ABCB1 reversing paclitaxel resistance in treatment of non-small cell lung cancer with Apatinib

作　　者：张勇 黄念[1] 李秋波 许瑞琪 ZHANG Yong;HUANG Nian;LI Qiu-bo;XU Rui-qi(Department of Oncology,First People's Hospital of Jingmen City,Jingmen 448000,China)

机构地区：[1]荆门市第一人民医院肿瘤科,湖北荆门448000

出　　处：《中华肿瘤防治杂志》2021年第19期1448-1455,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：荆门市科学技术研究与开发计划引导项目(2018YDKY032)。

摘　　要：目的探讨阿帕替尼通过调控ABCB1逆转非小细胞肺癌(NSCLC)紫杉醇(PTX)耐药性的相关机制。方法肺腺癌细胞株A549购自中国科学院生物化学与细胞生物学研究所。用PTX诱导NSCLC细胞株A549转化为PTX耐药细胞株A549/PTX,通过阿帕替尼培养将A549/PTX细胞株分为PTX组和阿帕替尼剂量组。MTT法测定药物对肺癌细胞抑制率,分析阿帕替尼逆转A549/PTX细胞的PTX耐药性。通过对PTX积累实验、Rh123蓄积实验、碘化丙锭染色法测定A549/PTX细胞周期以及AnnexinⅤ-FITC/PI双染法对A549/PTX细胞凋亡的分析。结果A549/PTX细胞中PTX的平均IC_(50)值为(1.40±0.15)nmol/L较A549细胞的(0.03±0.01)nmol/L增大,差异有统计学意义,t=22.302,P<0.05,证明成功建立NSCLC PTX耐药细胞株A549/PTX。MTT法证明阿帕替尼对A549/PTX抑制率较PTX提升,且呈阿帕替尼浓度依赖性,F=21.600,P<0.05。PTX在A549/PTX细胞中的积累(0.31±0.03)少于A549细胞(1.64±0.11),差异有统计学意义,t=29.416,P<0.05。PTX在细胞里蓄积量减少可能是A549/PTX细胞耐药的主要原因之一。随着阿帕替尼浓度的增加,细胞内Rh123蓄积量增加,证明阿帕替尼是通过对ABCB1蛋白的抑制来实现对PTX治疗NSCLC耐药性的逆转作用。当PTX联合阿帕替尼用药时,A549/PTX细胞的G_(2)/M期细胞受到阻滞,细胞凋亡率增加,2种药物存在拮抗作用,F=236.496,P<0.05。[^(125)I]-IAAP标记ABCB1底物,通过UV-Vis定量分析得出,阿帕替尼能有效抑制ABCB1的底物,并且呈浓度依赖性。结论阿帕替尼通过抑制ABCB1的表达逆转PTX治疗NSCLC的耐药性,抑制癌细胞增殖且促进癌细胞凋亡。Objective To investigate the mechanism of Apatinib reversing paclitaxel resistance in non-small cell lung cancer(NSCLC)by regulating ABCB1.Methods Lung adenocarcinoma cell line A549cells were purchased from the Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences.The NSCLC cell line A549was induced to transform into paclitaxel-resistant cell line A549/PTX by paclitaxel,and the A549/PTX cell line was divided into paclitaxel group and Apatinib dose group by Apatinib culture.MTT assay was used to determine the inhibition rate of the drug on lung cancer cells and to analyze the reversal of paclitaxel resistance in A549/PTX cells by Apatinib.A549/PTX cell cycle was determined by paclitaxel accumulation assay,Rh123accumulation assay,propidium iodide staining,and AnnexinⅤ-FITC/PI doublestaining method for the analysis of apoptosis in A549/PTX cells.Results The mean IC_(50) value of paclitaxel in A549/PTX cells(1.40±0.15)nmol/L was increased compared with A549cells(0.03±0.01)nmol/L,and the difference was statistically significant(t=22.302,P<0.05),demonstrating the successful establishment of NSCLC paclitaxel-resistant cell line A549/PTX.MTT assay demonstrated that the inhibition rate of Apatinib on A549/PTX inhibition was elevated compared to paclitaxel in an Apatinib concentration-dependent manner(F=21.600,P<0.05).The accumulation of paclitaxel in A549/PTX cells(0.31±0.03)was less than that in A549cells(1.64±0.11),and the difference was statistically significant,t=29.416,P<0.05.The reduced accumulation of paclitaxel in the cells may be one of the main reasons for drug resistance in A549/PTX cells.As the concentration of Apatinib increased,the intracellular Rh123accumulation increased,demonstrating that the reversal of resistance to paclitaxel treatment of NSCLC was achieved by the inhibition of ABCB1protein by Apatinib.When paclitaxel was administered in combination with Apatinib,the G_(2)/M phase cells of A549/PTX cells were blocked and the apoptosis rate increased,and there was an antagonistic

关 键 词：阿帕替尼 ABCB1 逆转 紫杉醇耐药性 非小细胞肺癌 

分 类 号：R734.2[医药卫生—肿瘤]