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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。
ZGDHu-1 and A549 cells were cultured in vitro in order to observe the role of ZGDHu-1 in inhibiting the proliferation, inducing apoptosis and in vivo anti-tumor activity of lung cancer cell line A549. Trypan blue staining, SRB method and 5’-bromo-2’-deoxyuridine-ELISA method were used to observe the effect of ZGDHu-1 on the proliferation of A549 cells. Using cell morphology, DNA gel electrophoresis, DNA content and cell cycle analysis, AnnexinV / PI double labeling, Hoechst33258 fluorescence staining and other techniques to detect apoptosis. After intraperitoneal injection of ZGDHu-1, its inhibitory effect on the growth of xenografted tumor was observed. The expression of bcl-2, bax, p53 gene and protein in A549 cells was observed by RT-PCR and flow cytometry. The results showed that, ZGDHu-1 can inhibit the proliferation and viability of A549 cells, showing time-dependent and dose-dependent. The majority of A549 cells arrested in G2-M phase after ZGDHu-1 treatment. DNA fragmentation appeared, the sub-G1 peak increased and AnnexinV + / PI-expression increased. The characteristic of apoptotic cells after Hoechst33258 staining Change and so on. After treated with 10, 20 and 40 mg.kg-1 of ZGDHu-1 for 14 days in nude mice, the growth inhibition rates were 43.7%, 56.9% and 60.0%, respectively. Bcl-2 gene and protein were down-regulated in A549 cells after ZGDHu-1 treatment, but mainly up-regulated bax gene and protein, resulting in a significant increase of bax / bcl-2 ratio and upregulation of p53 gene and protein, Sex. ZGDHu-1 can significantly inhibit the growth of xenografts in vivo and inhibit the proliferation of A549 cells by inducing apoptosis in vitro. The mechanism may be related to the upregulation of bax and p53 gene expression.