AD-ING4对MCF-7/ADR乳腺癌细胞抑制及其对化疗药物增敏机制探讨  

Growth inhibition and chemotherapy sensitization effect and its molecular mechanism of human breast cancer multi-drug resistant MCF-7/ADR cells by adenovirus-mediated ING4gene in vitro

作　　者：王新昇[1] 张冠新[1] 董晓强[2] 杨吉成[3] 盛伟华[3] 

机构地区：[1]青海省人民医院普通外科,青海西宁810007 [2]苏州大学附属第一医院普通外科,江苏苏州215006 [3]苏州大学医学部细胞与分子生物教研室,江苏苏州215123

出　　处：《中华肿瘤防治杂志》2014年第22期1794-1799,1806,共7页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的探讨重组腺病毒介导的人生长抑制因子4(inhibitor of growth 4,ING4)基因对人乳腺癌多药耐药MCF-7/ADR细胞体外抑制和化疗增敏作用及相关分子机制。方法用携带ING4基因的重组腺病毒感染MCF-7/ADR细胞,本实验分为胎牛血清(PBS)阴性对照组、Ad-GFP空载体腺病毒组、Ad-ING4重组腺病毒组、3.0μg/mL ADR组和Ad-ING4+3.0μg/mL ADR组5组,应用半定量反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法检测各组ING4基因及蛋白质在MCF-7/ADR细胞中的表达情况;用CCK-8法检测各组MCF-7/ADR细胞生长情况;分别用流式细胞术(FCM)PI单染法和AnnexinⅤ-PE/7-AAD双荧光标记法检测各组细胞周期和细胞凋亡情况;并使用RT-PCR和蛋白质印迹法检测各组MCF-7/ADR细胞中Bax、Bcl-2和Survivin等相关基因的表达情况。结果腺病毒介导的ING4基因在MCF-7/ADR细胞中成功表达;Ad-ING4和ADR对MCF-7/ADR细胞生长均有明显的生长抑制和促凋亡作用,Ad-ING4使MCF-7/ADR细胞周期阻滞在G2/M期,F=129.294,P<0.05。Ad-ING4联合ADR对细胞的生长抑制和促凋亡作用更明显,培养至第4天,其生长抑制率可达(73.13±3.78)%,显著高于Ad-ING4组(19.29±1.53)%和ADR组(48.39±2.96)%,F=257.397,P<0.05;其凋亡率可达(26.48±3.13)%,显著高于Ad-ING4组(11.57±1.26)%和ADR组(17.21±2.34)%,F=30.253,P=0.001。蛋白质印迹法结果分析显示,导入ING4基因和ADR处理使Bax的表达增强,Bcl-2和Survivin的表达下降。与其他组相比较,Ad-ING4联合ADR使上述效应更加显著,差异有统计学意义,P值均<0.05。结论 Ad-ING4在体外可抑制MCF-7/ADR细胞的生长,并诱导其凋亡,对ADR的细胞毒作用具有协同增敏效应,其机制可能与ING4降低Bcl-2/Bax比值和抑制Survivin的表达有关。OBJECTIVE To explore the anti-cancer effect and chemotherapy sensitization of adenovirus-mediated ING4 gene on the human breast cancer multidrug resistant cell line MCF-7/ADR and its possible mechanisms in vitro. METHODS The human breast cancer multidrug resistant ceils MCF 7/ADR were infected by Ad-ING4 combined with doxorubicin （ADR）in vitro and were randomly divided into five groups： the PBS negative control group （PBS group）, the idling adenovirus group （Ad-GFP group）, the adenovirus mediated ING4 group （Ad-ING4 group）, the ADR positive control group with the final concentration 3.0μg/mL （ADR group）, the group of Ad-ING4 combined with ADR finalconcentration 3.0 μg/mL （referred to as the joint group）. The methods of reverse transcription-polymerase chain reaction （RT-PCR） and Western blot were used to detect the expression of ING4 in breast cancer MCF-7/ADR cells. The influ- ence of Ad-ING4 transfection on cell proliferation was evaluated by CCK-8 assay, The cell cycle of MCF-7/ADR cells in- fected with Ad-ING4 and doxorubicin was detected by flow cytometry（FCM）. The influence of Ad-ING4 and doxorubicin on MCF-7/ADR cell apoptosis was detected by double fluorescent labeling with Annexin V PE/7-AAD. The expression of apoptosis-related genes （Bax, Bcl-2, Survivin） was detected by RT PCR and Western blot. RESULTS The adenovi rus-mediated ING4 was successfully transcribed in MCF-7/ADR cells. The adenovirus mediated ING4 and doxorubicin significantly inhibited MCF-7/ADR ceil growth, induced cell apoptosis and made the cell cycle partly arrested in G2/M phase（F= 129. 294,P〈0.05）. The Ad-ING4 combined with Doxoruhicin could acquire higher rate of growth inhibition and apoptosis. After cultured for four days, the growth inhibition rate of the joint group was significantly higher than Ad- ING4 group and ADR group, （73.13±3.78）% vs （19. 29±1. 53）% and （48. 39±2. 96）%, F=257. 397,P〈0. 05）. The apoptosis rate of the joint group was significantly higher than

关 键 词：ING4基因 乳腺肿瘤 化疗增敏 细胞凋亡 

分 类 号：R737.9[医药卫生—肿瘤]