东北刺人参falcarindiol制备及其抗结肠癌作用机制研究

为建立一种快速高效的falcarindiol(FAD)制备程序并探讨其抑制结肠癌细胞HCT-116增殖作用与调控细胞周期阻滞相关基因之间的关系。研究使用硅胶柱色谱富集及制备HPLC分离纯化得到了FAD单体,依据波谱数据鉴定其结构;采用MTS法检测FAD对结肠癌细胞HCT-116的细胞毒活性,运用流式细胞术、RT-qPCR以及Westernblotting法分别检测FAD对结肠癌细胞HCT-116周期影响、周期阻滞基因β-catenin、cyclinD1和c-myc的mRNA水平及蛋白表达的作用。结果显示建立制备HPLC方法可以较快速稳定地得到较高纯度的FAD。FAD对结肠癌细胞HCT-116具有明显的细胞毒活性,与HCT-116细胞作用24、48、72h的IC50值分别为8.1±1.4、4.6±0.5、3.2±0.4μmoL/L。此外,FAD将HCT-116细胞周期阻滞在G2/M期,且能够显著下调Wnt/β-catenin通路中的β-catenin、cyclinD1和c-myc基因的转录及表达。据此推断FAD可能是通过调节Wnt/β-catenin信号通路阻滞HCT-116细胞生长周期进而抑制其细胞的增殖来产生抗结直肠癌的作用。

Preparation of falcarindiol from Oplopanax elatus and its mechanism on anti-colorectal cancer

This study aimed to develop a rapid and efficient preparative method for falcarindiol (FAD) and investigate the relationship of the anti-proliferative effect of FAD on colorectal cancer HCT-116 cells and regulating cell cycle arrest related genes.The separation and purification were performed on silica gel chromatography and preparative HPLC in order to get FAD with high purity,which was identified by spectral data.MTS method was employed to test the cytotoxity of FAD on colorectal cancer HCT-116 cells.Subsequently,the cell cycle arrest,the mRNA level,protein expression of β-catenin,cyclin D1 and c-myc of HCT-116 cells were analyzed by flow cytometry,RT-qPCR and Western blotting,respectively.As the results,a rapid and efficient HPLC method has been validated for the preparation and purification of FAD from O.elatus .FAD showed cytotoxity on HCT-116 cells with the IC 50 values of 8.1±1.4,4.6±0.5 and 3.2±0.4 μmoL/L at 24,48 and 72 h,respectively,which could arrest the cell cycle of HCT-116 cell line at G2/M phase and significantly down-regulate the mRNA level and protein expressions of β-catenin,cyclin D1 and c-myc.These results suggested that FAD possessed a strong cytotoxic activity against HCT-116 cells,and arrested its cell cycle at G2/M phase,while its mechanism referred to Wnt/β-catenin signaling pathway.