四嗪二甲酰胺抑制胰腺癌细胞AsPC-1增殖及其机制研究

Study of inhibitory effects of ZGDHu-1 on AsPC-1 pancreatic cancer cells and its molecular mechanism

目的观察四嗪二甲酰胺(ZGDHu-1)体外对AsPC-1细胞增殖、凋亡及细胞周期阻滞的影响。方法以不同浓度ZGDHu-1(0、0.025、0.125、0.25、0.5、1.25、2.5、12.5μmol·L^(-1))体外作用于AsPC-1细胞,MTT法检测细胞生长抑制率;瑞氏染色、Hoechst 33258荧光染色观察细胞形态;Annexin-V/PI双标记分析细胞凋亡率;流式细胞术分析细胞周期变化;Western blot分析凋亡相关蛋白的表达变化。结果 ZGDHu-1对AsPC-1细胞有明显的增殖抑制作用,呈现时间和浓度依赖性;ZGDHu-1能促进细胞凋亡,染色后出现典型的凋亡特征性改变,Annexin V^+/PI^-表达明显高于对照组(P<0.05);当ZGDHu-1浓度达到0.25μmol·L^(-1)时,开始阻滞AsPC-1细胞周期于G_2/M期,当浓度达1.25μmol·L^(-1)时,大部分细胞停留于G_2/M期;ZGDHu-1作用AsPC-1细胞48 h后,能明显激活caspase-3酶原;促凋亡蛋白Bax的表达水平较对照组升高。结论 ZGDHu-1在体外能够抑制AsPC-1的增殖,其主要通过激活caspase-3诱导细胞凋亡,同时ZGDHu-1能阻滞AsPC-1细胞周期于G_2/M期。

Aim To investigate the effect of ZGDHu-1 on inducing apoptosis of AsPC-1 cells in vitro and its mechanisms.Methods AsPC-1 cells were treated with different concentrations of ZGDHu-1(0,0.025,0.125,0.25,0.5,1.25,2.5,12.5μmol·L-1),and the inhibitory rate of AsPC-1 proliferation was measured by MTT assay.The morphology of apoptosis was observed by Wright’s staining and Hoechst 33258 fluorescence staining.Cell apoptotic rate was detected by Annexin-V/PI staining.Cell cycle was analyzed by flow cytometry.The expressions of apoptosis-related proteins were detected by Western blot.Results ZGDHu-1 significantly inhibited the proliferation of AsPC-1 cells in a time-and concentration-dependent manner.The morphological features of apoptosis were significantly changed and the apoptotic rates of AsPC-1 cells were elevated compared with control(P<0.05).ZGDHu-1 could induce the cell cycle of AsPC-1 arrest at G 2/M phase.Caspase-3 was greatly activated after treatment of ZGDHu-1 for 48 h.The expressions of Bax was up-regulated.Conclusions ZGDHu-1 could inhibit the proliferation and enhance apoptosis in AsPC-1 cells,as well as block the cell cycle at G 2/M phase.The activation of caspase-3 is of great importance in ZGDHu-1-induced apoptosis.