连翘苷通过Wnt/β-catenin通路抑制体内外结肠癌增殖和干细胞特性

Phillyrin Inhibits Colon Cancer Proliferation and Stem Cell Properties by Inhibiting Wnt/β-catenin Pathway in vitro and in vivo

[目的]探究连翘苷对结肠癌的抑制作用及其可能的机制。[方法]将结肠癌SW480细胞分为对照组,连翘苷低、中、高浓度组和5-氟尿嘧啶(5-fluorouracil,5-Fu)组。以噻唑蓝(methy thiazolyl tetrazolium,MTT)法检测细胞活性,克隆形成试验检测连翘苷对SW480细胞增殖的作用,微球体形成试验检测干细胞成球直径和成球数量,定量反转录聚合酶链式反应(quantitative reverse transcription-polymerase chain reaction,qRT-PCR)检测Nanog、性别决定区Y盒子2(sex-determining region Y box 2,SOX2)、醛脱氢酶1(aldehyde dehydrogenase 1,ALDH1)、八聚体结合转录因子4(octamer binding transcription factor 4,OCT4)、Wnt、β-catenin和糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)mRNA的相对表达水平,免疫印迹检测Nanog、SOX2、ALDH1、OCT4、Wnt、β-catenin、磷酸化GSK-3β(phospho-GSK-3β,p-GSK-3β)和GSK-3β蛋白的相对表达水平。建立结肠癌异种移植瘤模型,观察连翘苷对体内结肠癌生长的效应,免疫组化检测移植瘤组织中Ki-67、Nanog和SOX2的阳性表达量,免疫印迹检测结肠癌异种移植瘤组织中Wnt和β-catenin蛋白的表达量。[结果]与对照组比较,连翘苷各浓度组SW480细胞活性和克隆形成数降低(P<0.05,P<0.05),干细胞成球直径和成球数量降低(P<0.05,P<0.05),Nanog、SOX2、ALDH1、OCT4、Wnt、β-catenin mRNA和蛋白相对表达水平降低(P<0.05,P<0.05),p-GSK-3β蛋白表达水平降低(P<0.05),而GSK-3β蛋白相对表达水平升高(P<0.05)。在体内实验中,与控制组比较,实验组的结肠癌异种移植瘤质量减轻(P<0.05),异种移植瘤组织中Ki-67、Nanog和SOX2的阳性表达下调(P<0.05,P<0.05,P<0.05),Wnt和β-catenin蛋白表达下调(P<0.05,P<0.05)。[结论]连翘苷能抑制体内外结肠癌细胞增殖及干细胞特性,其机制可能与抑制Wnt/β-catenin通路有关。

[Objective]To explore the anti-tumor effect of phillyrin on colon cancer and its possible mechanism.[Methods]The colon cancer SW480 cells were divided into control group,low,medium and high dose phillyrin groups as well as 5-fluorouracil(5-Fu)group.The viability of SW480 cells was detected by methy thiazolyl tetrazolium(MTT)method.The proliferation of SW480 cells was detected by colony formation assay.Cell stemness of SW480 cells was analyzed by sphere formation assay.The mRNA levels of Nanog,sex-determining region Y box 2(SOX2),aldehyde dehydrogenase 1(ALDH1),octamer binding transcription factor 4(OCT4),Wnt,β-catenin and glycogen synthase kinase-3β(GSK-3β)were detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).The protein levels of Nanog,SOX2,ALDH1,OCT4,Wnt,β-catenin,phospho-GSK-3β(p-GSK-3β)and GSK-3βwere analyzed via Western blot.Colon cancer xenograft tumor model was established to explore the anti-tumor effect of phillyrin in vivo.The positive expression of Ki-67,Nanog and SOX2 were detected by immunohistochemistry,and the levels of Wnt andβ-catenin protein were detected by Western blot.[Results]Compared with control group,the cell viability and clonal formation rate of SW480 cell were decreased after phillyrin treatment(P<0.05,P<0.05).Phillyrin effectively inhibited the spheres number and the size of spheres of SW480 cells(P<0.05,P<0.05).The mRNA and protein expression of Nanog,SOX2,ALDH1,OCT4,Wnt andβ-catenin were decreased after phillyrin treatment(P<0.05,P<0.05).Phillyrin decreased the protein relative expression of p-GSK-3β(P<0.05),while the level of GSK-3βwas increased(P<0.05).The tumor weight was reduced in treatment group in comparison with dominant group(P<0.05).The positive expression levels of Ki-67,Nanog and SOX2 in tumor tissues were down-regulated by phillyrin treatment(P<0.05,P<0.05,P<0.05),and phillyrin decreased the levels of Wnt andβ-catenin in xenograft tumors(P<0.05,P<0.05).[Conclusion]Phillyrin can inhibit the proliferation and stem cell properties of colon cancer cells in vitro and in vivo,the mechanism may be related to the inhibition of Wnt/β-catenin pathway.