参白解毒方基于Wnt/β-catenin信号通路对结直肠癌细胞增殖和迁移的抑制作用

Inhibition effects of Shenbai Jiedu Formula on proliferation and migration of colorectal cancer cells based on Wnt/β-catenin signaling pathway

目的:研究参白解毒方对结直肠癌细胞增殖和迁移的药效及其作用机制。方法:用参白解毒方处理人结直肠癌细胞HT29、SW480和SW620,MTT和平板克隆形成实验检测细胞的增殖和自我更新能力;细胞划痕实验检测HT29细胞的迁移能力;实时定量PCR或Western Blot检测上皮-间充质转化(EMT)相关标记物(E-Cadherin、N-Cadherin和Vimentin)、Wnt5a和β-catenin的表达水平;实时定量PCR检测Wnt/β-catenin信号通路下游的结直肠癌肿瘤干细胞标志物CD44和CD133的表达水平。结果:参白解毒方可抑制结直肠癌细胞HT29、SW480和SW620的增殖(P<0.01,P<0.05),减少HT29细胞形成克隆的数目(P<0.05,P<0.01);细胞划痕实验结果表明参白解毒方可以显著抑制HT29细胞的迁移(P<0.05,P<0.01),且具有剂量依赖性。实时定量PCR和Western Blot结果显示,参白解毒方可以剂量和时间依赖性的上调上皮细胞标志物E-Cadherin mRNA和蛋白的表达,下调间质细胞标志物N-Cadherin、Vimentin mRNA和蛋白的表达;参白解毒方同时可以下调Wnt5a、β-catenin mRNA和蛋白的表达,抑制Wnt/β-catenin信号通路活化,显著抑制结直肠癌肿瘤干细胞标志物CD44、CD133的mRNA表达。结论:参白解毒方可以抑制结直肠癌细胞的增殖和迁移,其作用机制可能与调控Wnt/β-catenin信号通路从而逆转结直肠癌细胞EMT的发生,降低结直肠癌细胞干性相关。

Objective: To explore the efficacy and mechanism of Shenbai Jiedu Formula(SBJDF) in inhibiting the proliferation and migration of colorectal cancer(CRC) cells. Methods: After treating human CRC cells HT29,SW480,and SW620with SBJDF,MTT and clone formation assay were used to detect the proliferation and self-renewal ability of the cells;the wound healing assay was used to detect the migration ability of HT29 cells;Real-time quantitative PCR and Western Blot were used to detect the expression levels of Wnt5a,β-catenin and epithelial-mesenchymal transition(EMT)-related proteins(E-cadherin,N-cadherin and Vimentin);Real-time quantitative PCR was used to detect the expression levels of CRC stem cells markers CD44and CD133 regulated by of Wnt/β-catenin signaling pathway. Results: SBJDF can effectively inhibited the proliferation of CRC cells HT29,SW480 and SW620(P<0.01,P<0.05),and reduced the number of clones formed by HT29 cells(P<0.05,P<0.01).The results of wound healing assay showed that SBJDF can significantly inhibit the migration of HT29 cells(P<0.05,P<0.01) in a dose-dependent manner. The results of real-time quantitative PCR and Western Blot showed that SBJDF can up-regulated the mRNA and protein expression of the epithelial cell marker E-cadherin,and down-regulated the mRNA and protein expression of the mesenchymal cell markers N-cadherin and Vimentin. SBJDF can down-regulated the mRNA and protein expressions of Wnt5a and β-catenin,inhibit the activation of Wnt/β-catenin signaling pathway,and significantly inhibited the mRNA expression of CRC stem cells markers CD44 and CD133. Conclusion: SBJDF can inhibited the proliferation and migration of CRC cells,and its mechanism of action may be through regulating the Wnt/β-catenin signaling pathway to reverse the occurrence of EMT of CRC cells and reduce the stemness of CRC cells.