四嗪二甲酰胺对肺癌细胞株A549的体内外作用  

Effect of ZGDHu-1 on proliferation and apoptosis of A549 cells in vitro and antitumor activity in vivo

作　　者：周永列[1] 胡惟孝[2] 吕亚萍[2] 邱莲女[1] 王文松[1] 杨忠愚[2] 刘建栋[1] 饶国武[2] 

机构地区：[1]浙江省人民医院中心实验室,浙江杭州310014 [2]浙江工业大学药学院,浙江杭州310014

出　　处：《药学学报》2007年第1期26-34,共9页Acta Pharmaceutica Sinica

基　　金：浙江省科技厅资助项目(2003c33019).

摘　　要：为了观察四嗪二甲酰胺(ZGDHu-1)体外抑制肺癌细胞株A549增殖、诱导细胞凋亡和体内抗肿瘤活性的作用及其机制,将不同浓度的ZGDHu-1与A549细胞在体外培养,用台盼蓝染色、SRB法、5′-溴-2′脱氧尿苷-ELISA法,观察ZGDHu-1对A549细胞增殖的作用;用细胞形态学、DNA凝胶电泳、DNA含量及细胞周期分析、AnnexinV/PI双标记、Hoechst33258荧光染色等技术检测细胞凋亡。腹腔注射ZGDHu-1后观察其对裸鼠移植瘤生长的抑制作用。用RT-PCR和流式细胞术观察A549细胞bcl-2,bax,p53基因和蛋白质的表达改变。结果表明,ZGDHu-1能抑制A549细胞的增殖和活力,呈现作用时间和剂量的依赖关系。A549细胞经ZGDHu-1作用后,大部分细胞阻滞于G2-M期;出现DNA片段化,亚G1峰显著增加,AnnexinV+/PI-表达升高,Hoechst33258荧光染色后出现凋亡细胞的特征性改变等。ZGDHu-1以10,20及40mg.kg-1剂量给裸鼠体内用药14d后,移植瘤生长抑制率分别为43.7%,56.9%和60.0%。A549细胞经ZGDHu-1作用后,bcl-2基因和蛋白有所下调,但主要是上调bax基因和蛋白,导致bax/bcl-2比值明显增高,p53基因和蛋白表达也上调,均呈现剂量依赖性。ZGDHu-1在体内能明显抑制移植瘤的生长,体外通过诱导细胞凋亡抑制A549细胞增殖,其机制可能与上调bax和p53基因的表达有关。This study is to explore the mechanism and effect of N, N＇-di-（ m-methylphenyl）-3,6- dimethyl-1 ,4-dihydro-1 ,2,4,5-tetrazine-1, 4-dicarboamide （ZGDHu-1） on proliferation and apoptosis of A549 cells in vitro and on A549 xenograft tumor in nude mice. With different concentrations of ZGDHu-1 at different times were used to treat A549 cells in vitro. The proliferation was determined by living cell count, SRB assay and Brdu-ELISA. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin V/PI and Hoechst 33258 labeling method. The nude mice model of A549 xenograft tumor was established by subcutaneous inoculation. The suppression activity of ZGDHu- 1 by intraperitoneal injection on xenograft mice model was detected. The expressions of bcl-2, bax and p53 gene and protein were analyzed by RT-PCR and flow cytometry. ZGDHu-1 can inhibit A549 cell proliferation viability within a certain range of treating time and does, and a majority of A549 cells were arrested in Gz-M phase. The A549 cells apoptosis was confirmed by typical cell morphology, DNA fragment, Sub G1 phase, Hoechst 33258 and Annexin V/PI labeling method with a time and dose related manner. When the xenograft tumor mice model were treated with 10, 20 and 40 mg·kg^-1 ZGDHu-1 for 14 days, the tumor growth inhibition rate were 43.7% , 56.9% and 60.0% , respectively. The expression of bax, bax/bcl-2 and p53 gene and protein increased significantly and bcl-2 decreased slightly by the treatment of ZGDHu-1. ZGDHu-1 can significantly suppress the growth of A549 xenograft tumor in vivo and inhibited proliferation by inducing tumor cell apoptosis in vitro. The mechanism may associate with its upregulation of bax and p53 during the apoptosis process.

关 键 词：四嗪二甲酰胺 肺癌 细胞株A549 增殖 细胞凋亡 肿瘤移植 

分 类 号：R963[医药卫生—微生物与生化药学] R979.1[医药卫生—药理学]