摘要
目的:研究熊果酸对结直肠癌细胞自噬的影响及其基于Wnt信号通路的调节自噬机制.方法:MTT法检测熊果酸对SW480细胞增殖的影响;细胞划痕试验检测熊果酸5.47、10.94、21.88 μmol/L对SW480细胞迁移能力的影响;MDC/PI法检测其对SW480细胞自噬体形成的影响;qRT-PCR法检测对细胞自噬相关基因微观相关蛋白轻链3(Lc3)、苄氯素1(Beclin1)、自噬相关基因5(Atg5)和自噬蛋白(P62)以及Wnt信号通路相关基因β连环蛋白(Bcatenin)、T-细胞因子4(Tcf4)、糖原合成酶激酶3β(Gsk3β)、Wnt家族成员5A(Wnt5a)和Myc原癌基因(Cmyc)mRNA表达的影响;Western Blot法检测熊果酸对细胞自噬相关蛋白LC3-Ⅱ、BEC-LIN-1、ATG5 和 P62 以及 Wnt 信号通路相关蛋白 β-catenin、NUCLEAR β-catenin、TCF4、GSK-3β、p-GSK-3β、WNT5A 和 C-MYC 表达的影响.结果:熊果酸能显著抑制SW480细胞增殖和迁移(P＜0.01);增强SW480细胞自噬体荧光强度(P＜0.01);熊果酸能显著上调SW480 细胞Lc3、Beclin1、Atg5 和 Gsk3b mRNA 表达,下调 P62、Bcatenin、Tcf4、Wnt5a 和 Cmyc mRNA 表达(P＜0.05),显著上调 SW480细胞 LC3-Ⅱ、BECLIN-1、ATG5 和 GSK-3β 蛋白表达,下调 P62、β-catenin、NUCLEAR β-catenin、TCF4、WNT5A、p-GSK-3β(Ser389)和 C-MYC蛋白表达(P＜0.05);熊果酸抗SW480细胞增殖作用可被Wnt信号通路抑制剂IWR-1协同增强(P＜0.01),而被Wnt信号通路激动剂SKL2001拮抗减弱(P＜0.01).结论:熊果酸能够抑制SW480细胞增殖、迁移,增强SW480细胞自噬,其机制与降低结直肠癌细胞Wnt信号通路活性相关.
Abstract
Objective:To investigate the effect of ursolic acid(UA)on autophagy in colorectal cancer cells and its mechanism of regulation of auto-phagy based on Wnt signaling pathway.Methods:The effect of UA on the proliferation of SW480 cells was detected by MTT assay.In vitro scratch assay was used to measure the effect of UA(5.47,10.94 and 21.88 μmol/L)on the migration of SW480 cells.The influence of UA on autophagosome formation in SW480 cells was observed with MDC/PI assay.The effects of UA on the mRNA levels of autophagy-related genes including microtubule-associated protein light chain3(Lc3),Beclin-1,autophagy-related gene5(Atg5)and P62,and Wnt signaling pathway-related genes involving β-catenin,Tcf4,Gsk-3β,Wnt5a and C-myc were detected by qRT-PCR.Western blot was employed to analyze the influence of UA on the expression of autophagy-related proteins including LC3-Ⅱ,Beclin-1,ATG5 and P62,and Wnt signaling pathway-re-lated proteins involving β-catenin,nuclear β-catenin,TCF4,GSK-3β,p-GSK-3β,Wnt5 A and C-Myc.Results:U A significantly inhibited the proliferation and migration of SW480 cells(P＜0.01),and enhanced the fluorescence intensity of autophagosomes in SW480 cells(P＜0.01).UA significantly up-regulated the mRNA expression levels of Lc3,Beclin-1,Atg5 and Gsk-3β,while down-regulated the mRNA levels of P62,β-catenin,Tcf4,Wnt5a and C-myc in SW480 cells(P＜0.05).Also,UA significantly up-regulated the protein expressions of LC3-Ⅱ,Bec-lin-1,ATG5 and GSK-3β,while down-regulated the protein expressions of P62,β-catenin,nuclear β-catenin,TCF4,Wnt5A,p-GSK-3β(Ser389)and C-Myc in SW480 cells(P＜0.05).The inhibition of UA on the proliferation of SW480 cells was synergized by Wnt signaling in-hibitor IWR-1(P＜0.01),but antagonized by Wnt signaling activator SKL2001(P＜0.01).Conclusion:UA may inhibit the proliferation and migration,and enhance the autophagy of SW480 cells.Its mechanism may be related to decreasing Wnt signaling pathway activity in colorectal cancer cells.
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