基于Hedgehog信号通路探析白头翁汤抗结直肠癌HCT116细胞机制

Baitouweng Tang Suppresses Colorectal Cancer HCT116 Cells by Regulating Hedgehog Signaling Pathway

目的:探讨白头翁汤水煎液通过调控经典Hedgehog(Hh)信号通路诱导人结直肠癌细胞HCT116凋亡的作用及其机制。方法:用白头翁汤(25、50、100、200、500、750、1000 mg·L^(-1))处理HCT116细胞24 h,然后用噻唑蓝(MTT)比色法检测白头翁汤对细胞增殖的影响;分别设置空白组、白头翁汤组(125、250、500 mg·L^(-1))和五氟尿嘧啶(5-FU)组(40 mmol·L^(-1)),显微镜观察细胞给药前后形态变化;划痕实验检测白头翁汤对细胞迁移的影响;Hoechest 33324/碘化丙锭(PI)染色法评价白头翁汤对细胞凋亡的影响;流式细胞术检测白头翁汤对HCT116细胞凋亡的影响;蛋白免疫印迹法(Western blot)检测细胞凋亡相关B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达水平及Hh信号通路关键因子音猬因子(SHh)、GLI家族锌指蛋白1(Gli1)、G蛋白偶联受体蛋白Smoothened(Smo)、苏氨酸蛋白激酶Fused抑制因子(SuFu)、c-核蛋白类基因(c-Myc)蛋白表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测凋亡相关基因Bax、Bcl-2和Hh通路相关基因SHh、Gli1、Smo、SuFu、c-Myc mRNA表达水平。结果:与空白组比较,白头翁汤组(125、250、500 mg·L^(-1))HCT116细胞形态发生明显改变,细胞变圆,其增殖被抑制,且呈浓度依赖性,迁移能力下降(P<0.05,P<0.01),细胞核出现致密浓染,凋亡数量增加。与空白组比较,白头翁汤(500 mg·L^(-1))处理HCT116细胞24 h,Bax蛋白和mRNA表达水平升高(P<0.05,P<0.01),Bcl-2蛋白和mRNA表达水平降低(P<0.05,P<0.01)。与空白组比较,白头翁汤组(500 mg·L^(-1))处理24 h后,Hh信号通路关键因子SHh、Gli1、Smo、c-Myc的mRNA和蛋白水平表达降低(P<0.05,P<0.01),Hh信号通路负调控因子SuFu表达水平升高(P<0.05,P<0.01)。结论:白头翁汤通过降低Hh信号通路活性来抑制结直肠癌细胞HCT116增殖和迁移,诱导癌细胞凋亡。

Objective:To explore the effect of Baitouweng Tang(BTWT)on the apoptosis of human colorectal cancer HCT116 cells and decipher the underlying mechanism based on the Hedgehog(Hh)signaling pathway.Method:HCT116 cells were treated with BTWT(25,50,100,200,500,750,and 1000 mg·L^(-1))for 24 h,and then the cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)colorimetry.Five groups were designed for the treatment of HCT116 cells,including a blank control group,BTWT groups(125,250,and 500 mg·L^(-1)),and a positive control(5-fluorouracil,5-FU,40 mmol·L^(-1))group.The cell morphology was observed under an inverted microscope.The migration of the cells was detected by scratch test,and the apoptosis by Hoechest 33324/propidium iodide(PI)staining and flow cytometry.Western blot was employed to determine the protein levels of sonic hedgehog(SHh),GLI family zinc finger protein 1(Gli1),smoothened(Smo),suppressor of fused(SuFu),cellular-myelocytomatosis viral oncogene(c-Myc),and the apoptosisrelated proteins B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax).The quantitative real-time reverse transcription PCR(Real-time PCR)was employed to determine the mRNA levels of Bax,Bcl-2,SHh,Gli1,Smo,SuFu,and c-Myc.Result:Compared with the blank control group,BTWT changed the cell morphology(making the cell become round with dense nucleus),inhibited the proliferation of HCT116 cells in a dose-dependent manner,decreased the ability of migration(P<0.05,P<0.01),and increased apoptotic cells.Compared with the blank control group,BTWT(500 mg·L^(-1))treatment for 24 h up-regulated the protein and mRNA levels of Bax(P<0.05,P<0.01)and down-regulated the protein and mRNA levels of Bcl-2 in HCT116 cells(P<0.05,P<0.01).Moreover,the treatment down-regulated the mRNA and protein levels of SHh,Gli1,Smo,and c-Myc(P<0.05,P<0.01)and up-regulated the mRNA and protein levels of SuFu(P<0.05,P<0.01).Conclusion:BTWT inhibited the proliferation and migration and induced the apoptosis of colorectal cancer HCT116 cells by down-regulating the Hh signaling pathway.