四溴苯三唑对人结肠癌SW480细胞凋亡的诱导作用及其机制

Induction effect of tetrabromobenzotriazole on apoptosis of human colon cancer SW480 cells and its mechanism

目的:探讨4,5,6,7-四溴苯三唑(TBB)对人结肠癌SW480细胞的促凋亡作用,并初步探讨其可能的作用机制。方法:选取处于对数生长期的人结肠癌SW480细胞,将其分为对照组(给予0μmol·L^(-1)TBB)和实验组(给予1、3、10、30和100μmol·L^(-1) TBB)。采用MTT比色法检测SW480细胞存活率,采用AnnexinⅤ-FITC/PI双染法及流式细胞术检测SW480细胞凋亡率及细胞中活性氧(ROS)水平,采用Western blotting法检测细胞中抗凋亡蛋白p-Akt、Bcl-2和促凋亡蛋白Bad、pro-caspase-9及cleaved-caspase-3表达水平。结果:MTT法检测,与对照组比较,实验组SW480细胞存活率明显降低(P<0.05),且呈浓度依赖性;AnnexinⅤ-FITC/PI双染法及流式细胞术检测,随着处理时间的增加,与对照组比较,TBB作用3、6、12和24h时SW480细胞凋亡率明显高于作用0h时(P<0.05);流式细胞术检测,TBB作用3、6、12和24h时结肠癌SW480细胞中ROS水平高于作用0h时(P<0.05);Western blotting法检测,SW480细胞中抗凋亡蛋白p-Akt和Bcl-2表达水平逐渐降低,促凋亡蛋白Bad和cleaved-caspase-3表达水平逐渐升高,pro-caspase-9蛋白表达水平逐渐降低,与对照组(0h)比较差异有统计学意义(P<0.05)。结论:TBB对体外培养的人结肠癌SW480细胞有明显的杀伤作用,可诱导细胞凋亡,其机制可能与抑制Akt活性或促进细胞中ROS产生有关。

Objective:To investigate the promotion effect of4,5,6,7tetrabromobenzotriazole(TBB)on the apoptosis of human colon cancer SW480cells,and to explore its possible mechanism.Methods:The human colon cancer SW480cells at logarithmic growth phase were divided into control group(0μmol·L-1TBB)and experiment group(1,3,10,30,and100μmol·L-1TBB).The viability of cells was measured by MTT assay;the apoptotic rate of the SW480cells and the level of intracellular reactive oxygen species(ROS)were analyzed by AnnexinⅤ-FITC/PI double staining and flow cytometry.The expression levels of anti apoptotic proteins p-Akt and Bcl-2,and pro apoptotic proteins Bad,pro caspase9and cleaved caspase3were detected by Western blotting method.Results:The MTT results showed that the viabilities of SW480cells in experiment group were decreased in a dose dependent manner,which were lower than that in control group(P<0.05).The AnnexinⅤ-FITC/PI double staining and flow cytometry results showed that the apoptotic rates of SW480cells in experiment group(3,6,12,and24h)were significantly higher than that in control group(0h)(P<0.05).The flow cytometry results showed the levels of ROS in SW480cells after treated with TBB for3,6,12and24h were higher than that in0h group(P<0.05).The Western blotting results showed that the expression levels of anti apoptotic proteins p-Akt and Bcl-2in SW480cells in experiment group(3,6,12,and24h)were decreased obviously,whereas the expression levels of the pro apoptotic proteins Bad and cleaved caspase3were increased and the expression level of pro caspase9was decreased.compared with those in control group(0h)(P<0.05).Conclusion:TBB could inhibit the cell proliferation and induce the apoptosis of human colon cancer SW480cells,and its mechanism may be related to the inhibition of the activity of Akt and the promotion of the level of intracellular ROS.