三叶青总黄酮通过Wnt/β-catenin通路抑制结肠癌细胞周期及增殖  

Total flavonoids of tetrastigma hemsleyanum inhibits the cycle and proliferation of colon cancer cells through the Wnt /β-catenin pathway

作　　者：余纳[1] 张钰坪 叶院宁 孙文荣[1] 刘畅[1] 汪芳裕[1] 

机构地区：[1]南京大学医学院附属金陵医院(南京军区南京总医院)消化内科,南京医学硕士210002

出　　处：《医学研究生学报》2016年第6期582-586,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81270453)

摘　　要：目的三叶青总黄酮(Total Flavonoids of Tetrastigma Hemsleyanum,TFTH)对多种肿瘤具有较强的抑制作用。分析TFTH对结肠癌细胞周期及细胞增殖能力的影响,并进一步探索其分子机制。方法采取CCK-8试验及克隆形成试验检测不同浓度TFTH(0、1.6、3.2及6.4 mg/m L)对人结肠癌细胞HT29及SW620细胞增殖能力的影响,并运用流式细胞术检测不同浓度TFTH对肿瘤细胞周期的调节作用;通过免疫印迹及实时荧光定量聚合酶链反应等方法证实TFTH对Wnt/β-catenin通路有效调控及细胞周期相关蛋白Cylin D1和c-myc的表达。结果在0、1.6、3.2和6.4 mg/m L TFTH孵育后,SW620细胞在6孔板中形成的克隆数量分别为(196±25.06)、(75.33±7.64)、(19±6.08)、0个/孔,HT29细胞为(206.67±30.55)、(106±12.17)、(9.67±4.04)、0个/孔,各浓度间两两比较,差异均有统计学意义(P<0.05)。结肠癌细胞S+G2/M期细胞的比例降低(P<0.05),且呈浓度依赖性。同时,TFTH干预的HT29细胞及SW620细胞内活化的β-catenin蛋白的表达随药物浓度增高而逐渐降低,将0、1.6、3.2和6.4 mg/m L TFTH作用于SW620细胞后,a-β-catenin蛋白的IOD分别为231.46±20.66、109.42±15.58、63.14±2.33、6.38±11.05;作用于HT29细胞时,其IOD分别为817.28±76.55、566.10±96.80、324.23±53.51、141.67±34.35,与0 mg/m L相比,差异均有统计学意义(P<0.01),进一步研究发现,与0 mg/m L相比,1.6、3.2、6.4 mg/m L的TFTH干预后细胞内Cyclin D1和c-myc在mRNA及蛋白质水平的表达也显著下调(P<0.05)。结论 TFTH抑制结肠癌细胞增殖能力,其作用机制与下调肿瘤细胞Wnt/β-catenin信号通路活性,抑制细胞周期有关。Objective The total flavonoids of tetrastigma hemsleyanum （TFTH）, a traditional herbal medicine, has a strong inhibitory effect on various tumors. This study was to explore the effects of TFTH on the cycle and proliferation of colon cancer cell lines and its underlying mechanisms. Methods SW620 and HT29 human colon cancer cell lines were treated with TFTH at the concentrations of 0, 1.6, 3.2, and 6.4 mg/mL. The proliferation of colon cancer cells was evaluated by plate colony formation assay and the cell cycle distribution determined by flow cytometry. The Wnt/β- catenin pathway activation and the expressions of the cell cycle-related proteins cyclin D1 and c-myc were measured by Western blot and quantitative real-time PCR （qRT-PCR）. Results After treatment with TFTH at 0, 1.6, 3.2, and 6. d mg/mL, the numbers of cloned SW620 cells were 196 ±25.06, 75.33 ±7.64, 19 ±6.08, and 0, and those of the HT29 cells were 206.67 ± 30.55, 106 ± 12.17, 9.67 ± 4.04, and 0, respectively, with statistically significant difference between every two concentrations （P 〈 0.05 ） The colon cancer cells were markedly reduced in G0/G1 phase in a dose-dependent manner （P 〈 0.05）. Compared with the control group, less activation of the Wnt/β-catenin pathway was detected in the HT29 cells treated with different concentrations of TFTH （817.28 ± 76.55 vs 566.10 -± 96.80,324.23 ± 53.51, and 141.67 ± 34.35, P 〈 0.01 ） as well as in the SW620 cells （231.46 ± 20.66 vs 109.42 ± 15.58, 63.14 ± 2.33, and 6.38 ± 11.05, P 〈 0.01 ）. Western blot and qRT-PCR revealed lower levels of cyclin D1 and c-myc after TFTH treatment （ P 〈 0.05 ）. Conclusion TFTH suppresses the proliferation of colon cancer cells by down-regulating the activation of the Wnt/β-catenin pathway and inhibiting their cell cycle.

关 键 词：三叶青总黄酮 结肠癌 细胞周期 细胞增殖 

分 类 号：R285[医药卫生—中药学]