积雪草苷调节Wnt/β-catenin信号通路对结肠癌细胞恶性生物学行为的影响

Effect of Asiaticoside on the Malignant Biological Behavior of Colon Cancer Cells by Regulating the Wnt/β-Catenin Signaling Pathway

采用浓度2.5-100.0μmol/L的积雪草苷(ATS)处理人结肠癌细胞(HCT-116),CCK-8法检测细胞活性,筛选最佳药物浓度;将HCT-116细胞分为对照组(Control组)、积雪草苷低、中、高浓度组(ATS-L组、ATS-M组、ATS-H组)、积雪草苷高浓度+Wnt激活剂组(ATS-H+LiCl组);Edu检测细胞增殖;流式细胞仪检测细胞凋亡;划痕实验检测细胞迁移;Transwell实验检测细胞侵袭;Western blot检测细胞核增殖抗原标记物(Ki67)、细胞周期调控因子(P21)、细胞周期蛋白D1(CyclinD1)、B细胞淋巴瘤-2相关X蛋白(Bax)、c-Myc癌基因(c-Myc)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、Wnt3a、β-catenin蛋白表达;裸鼠移植瘤实验检验ATS对结肠癌移植瘤生长的影响.探究积雪草苷(ATS)通过调节Wnt/β-catenin信号通路对结肠癌细胞恶性生物学行为的影响.通过筛选选择ATS浓度为25、50、100μmol/L进行后续实验.结果显示与Control组比较,ATS-L、ATSM、ATS-H组Edu阳性率、细胞划痕愈合率、细胞侵袭数量及Ki67、CyclinD1、c-Myc、MMP-2、MMP-9、Wnt3a、β-catenin表达水平依次降低,细胞凋亡率及P21、Caspase-3、Bax表达水平显著依次升高(P<0.05);与ATS-H组相比,ATS-H+LiCl组Edu阳性率、划痕愈合率、细胞侵袭数量及Ki67、CyclinD1、c-Myc、MMP-2、MMP-9、Wnt3a、β-catenin表达水平升高,细胞凋亡率及Caspase-3、P21、Bax表达水平显著降低(P<0.05).裸鼠移植瘤实验结果显示,ATS可显著抑制结肠癌移植瘤的生长(P<0.05).表明ATS可以通过抑制Wnt/β-catenin信号通路抑制结肠癌细胞增殖、迁移与侵袭等恶性生物学行为.

Human colon cancer cells(HCT-116)were treated with asiaticoside(ATS)at a concentration of 2.5-100.0μmol/L,and CCK-8 method was applied to detect cell activity to screen the optimal drug concentration;HCT-116 cells were separated into Control group,low,medium,and high concentration asiaticoside groups(ATS-L group,ATS-M group,ATS-H group),and high concentration asiaticoside+Wnt activator group(ATS-H+LiCl group);Edu was applied to detect cell proliferation;flow cytometry was applied to detect cell apoptosis;scratch experiments were applied to detect cell migration;Transwell experiment was applied to detect cell invasion;Western blot was applied to detect the expression of nuclear proliferating antigen markers(Ki67),cell cycle regulatory factors(P21),cyclinD1(CyclinD1),B-cell lymphoma 2 associated X protein(Bax),c-Myc oncogene(c-Myc),matrix metalloproteinase 2(MMP-2),matrix metalloproteinase 9(MMP-9),Wnt3a,andβ-catenin proteins;nude mouse transplantation tumor experiment was applied to test the effect of ATS on the growth of colon cancer transplantation tumors.The effect of ATS on the malignant biological behavior of colon cancer cells was investigated by regulating the Wnt/β-catenin signaling pathway.By filtering,ATS concentrations of 25.0μmol/L,50.0μmol/L,and 100.0μmol/L were selected for subsequent experiments.The results showed that compared with the control group,the positive rate of Edu,cell scratch healing rate,the number of cell invasions,and the expression levels of Ki67,CyclinD1,c-Myc,MMP-2,MMP-9,Wnt3a,andβ-catenin in the ATS-L,ATS-M,and ATS-H groups decreased sequentially,the apoptosis rate and the expression levels of P21,Caspase-3,and Bax increased significantly in sequence(P<0.05);compared with the ATS-H group,the positive rate of Edu,cell scratch healing rate,the number of cell invasions,and the expression levels of Ki67,CyclinD1,c-Myc,MMP-2,MMP-9,Wnt3a,andβ-catenin in the ATS-H+LiCl group increased,the apoptosis rate and the expression levels of Caspase-3,P21,and Bax reduced significantly(P<0.05).The results of nude mouse tumor transplantation experiment showed that ATS was able to significantly inhibit the growth of colon cancer transplanted tumors(P<0.05).In conclusion,the results demostrated ATS can inhibit the proliferation,migration and invasion of colon cancer cells by inhibiting the Wnt/β-catenin signaling pathway.