邻香草醛基席夫碱配体衍生物VALD-3对结直肠癌细胞增殖、迁移及凋亡的影响及其作用机制

Effects of VALD-3,a derivative of o-vanilla Schiff base ligand,on proliferation,migration and apoptosis of colorectal cancer cells and its mechanism

目的评价邻香草醛基席夫碱配体衍生物VALD-3对结直肠癌细胞增殖、迁移及凋亡的影响,并探讨其作用机制。方法体外培养HT-29和HCT116细胞,MTT法检测VALD-3(5、10、20、40 mg/L)对两种细胞增殖的抑制作用;倒置显微镜观察VALD-3(10、20、40 mg/L)对两种细胞形态变化的影响;细胞划痕试验检测VALD-3(10、20、40 mg/L)对HT-29细胞迁移能力的影响;流式细胞术和Hoechst 33258荧光染色法检测VALD-3(10、20、40 mg/L)对HT-29细胞凋亡的影响;Western blot法检测VALD-3(5、10、20、40 mg/L)对HT-29细胞中凋亡相关蛋白表达水平的影响。实验均设阴性对照组(不加VALD-3)。结果与阴性对照组比较,10、20、40 mg/L VALD-3组HT-29和HCT116细胞存活率明显下降(t=7.717~2006.148,P均<0.05);10、20和40 mg/L VALD-3组HT-29和HCT116细胞随着VALD-3浓度增加,细胞数目明显减少,且细胞出现不规则形态,逐渐变圆、变小,细胞碎片增多;10、20和40 mg/L VALD-3组HT-29细胞划痕迁移率均明显降低(t=7.596~73.780,P均<0.01),凋亡率明显升高(t=7.092~8.057,P均<0.01),且凋亡细胞明显增多,产生强烈的亮蓝色荧光,染色质浓缩,细胞核碎裂;5、10、20、40 mg/L VALD-3组HT-29细胞中cleaved caspase-3和Bax蛋白水平明显升高(t=2.998~24.901,P均<0.05),40 mg/L VALD-3组Bcl-2蛋白水平明显降低(t=10.035,P<0.05),20、40 mg/L VALD-3组cleaved caspase-8明显升高(t分别为12.630和8.064,P均<0.01)。结论VALD-3可抑制结直肠癌细胞的增殖和迁移,诱导细胞凋亡,这些作用是通过调节cleaved caspase-3、cleaved caspase-8、Bax和Bcl-2蛋白表达实现的。

Objective To the effects of VALD-3,a derivative of o-vanilla Schiff base ligand,on proliferation,migration and apoptosis of colorectal cancer cells and evaluate its mechanism.Methods HT-29 and HCT116 cells were cultured in vitro,and the inhibitory effects of VALD-3(5,10,20 and 40 mg/L)on proliferation of the two kinds of cells were detected by MTT assay;The effects of VALD-3(10,20 and 40 mg/L)on the morphological changes of the cells were observed by inverted microscope,while the effects on the migration ability of HT-29 cells were detected by cell scratch test,and the effects on the apoptosis of HT-29 cells were detected by flow cytometry and Hoechst 33258 fluorescence staining;The effects of VALD-3(5,10,20 and 40 mg/L)on the expression of apoptosis-related proteins in HT-29 cells were detected by Western blot.Negative control groups were set up(with no VALD-3).Results Compared with the negative control group,the survival rates of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3 treated groups significantly decreased(t=7.717~2006.148,each P<0.05);the number of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3 groups decreased significantly with the increase of VALD-3 concentration,the cells appeared irregular morphology and gradually became round and smaller,and cellfragments increased;In 10,20 and 40 mg/L VALD-3 treated groups,the migration rate of HT-29 cell scratches decreased significantly(t=7.596~73.780,each P<0.01),the apoptosis rate increased significantly(t=7.092~8.057,each P<0.01),and the number of apoptotic cells increased significantly with strong bright blue fluorescence,chromatin concentration and nuclear fragmentation;The levels of cleaved caspase-3 and Bax protein in HT-29 cells treated with 5,10,20 and 40 mg/L VALD-3 significantly increased(t=2.998~24.901,each P<0.05),the level of Bcl-2 protein in 40 mg/L VALD-3 group decreased significantly(t=10.035,P<0.05),and the levels of cleaved caspase-8 in 20 and 40 mg/L VALD-3 group significantly increased(t=12.630 and 8.064 respectively,each P<0.O1).Conclusion VALD-3 inhibited the proliferation and migration of colorectal cancer cells and induced apoptosis by regulating the expression of cleaved caspase-3,cleaved caspase-8,Bax and Bcl-2 proteins.