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目的:研究PYDDT诱导人结肠癌细胞HCT116凋亡的作用及分子机制。方法:以p53野生型人结肠癌细胞HCT116为研究对象,分别以MTT法测定PYDDT对细胞增殖的抑制作用,Annexin V/PI双染法测定细胞凋亡率,DCFH-DA法测定活性氧自由基(Reactive oxygen species,ROS),Western blot法检测p53和凋亡相关蛋白的表达。此外,采用抗氧化剂N-乙酰半胱氨酸(N-Acetylcysteine,NAc)预处理2 h,检测PYDDT对HCT116细胞凋亡率、p53及凋亡相关蛋白表达的影响。结果:与空白对照组相比,PYDDT呈剂量依赖抑制HCT116细胞的生长,5、10和20μM的PYDDT处理24 h后,细胞存活率分别为75.4%、59.4%和27.3%;凋亡率为20.2%、31.9%和76.3%;胞内ROS水平明显升高;Western blot结果表明,PYDDT处理24h后的HCT116细胞,p53及其下游靶基因Bax表达显著上调,而抗凋亡蛋白Bcl-2与空白对照组相比未明显改变,Caspase级联反应被激活。同时,5 mM的NAc能够逆转PYDDT(10μM)的促凋亡作用,PYDDT+NAc组p53、Bax的表达较空白对照组无差异,Caspase3未被激活。结论:PYDDT具有诱导HCT116细胞凋亡的作用,其机制是通过上调胞内ROS水平,继而上调p53及其下游靶基因Bax的表达,激活Caspase级联反应促发凋亡。
Objective: To study the effect and molecular mechanism of PYDDT on apoptosis of human colorectal cancer cell line HCT116. Methods: Human wild-type human colorectal cancer cell line HCT116 was used as the research object. The inhibitory effect of PYDDT on the cell proliferation was determined by MTT assay. The apoptotic rate was determined by Annexin V / PI double staining method. The activity of reactive oxygen species (Reactive oxygen species, ROS), Western blot detection of p53 and apoptosis-related protein expression. In addition, pretreatment with N-acetylcysteine (NAc) for 2 h was used to detect the effect of PYDDT on the apoptosis rate and the expression of p53 and apoptosis-related proteins in HCT116 cells. RESULTS: Compared with the blank control group, PYDDT inhibited the growth of HCT116 cells in a dose-dependent manner. After treated with 5, 10 and 20μM PYDDT for 24 h, the cell survival rates were 75.4%, 59.4% and 27.3%, respectively. The apoptosis rate was 20.2 %, 31.9%, and 76.3%, respectively. Western blot results showed that the expression of Bax in p53 and its downstream target gene was up-regulated in HCT116 cells treated with PYDDT for 24 h, while the expression of Bcl- The Caspase cascade was activated in the control group without significant change. At the same time, NAc at 5 mM reversed the pro-apoptotic effect of PYDDT (10μM). The expression of p53 and Bax in PYDDT + NAc group was not different from that in blank control group, while Caspase3 was not activated. CONCLUSION: PYDDT can induce the apoptosis of HCT116 cells by up-regulating the level of intracellular ROS and up-regulating the expression of p53 and its downstream target gene Bax, and activating Caspase cascade to induce apoptosis.