5-氟尿嘧啶对结肠癌干细胞干性和增殖的影响及相关机制  

Effects of 5-fluorouracil on colon cancer stem cells stemness and proliferation and the related mechanism

作　　者：蔡尚党[1] 蔡满地 娄宁[3] 郭艳萍[4] 殷涛[5] CAI Shang-Dang;CAI Man-Di;LOU Ning(Department of Anus-intestines,Henan Province Traditional Chinese Medicine Hospital,Zhengzhou 450002,Henan,China)

机构地区：[1]河南省中医院肛肠科,河南郑州450002 [2]河南大学医学院临床医学系 [3]中山大学附属肿瘤医院ICU [4]河南省人民医院病理科 [5]华中科技大学同济医学院附属协和医院胰腺病研究所

出　　处：《中国老年学杂志》2019年第24期6055-6059,共5页Chinese Journal of Gerontology

基　　金：国家自然科学基金(No.81372665)

摘　　要：目的探讨5-氟尿嘧啶(5-FU)抑制结肠癌干细胞(CCSCs)干性对结肠癌细胞生长的影响及相关分子机制。方法将慢病毒介导的p53特异性短发来RNA(shRNA)转导入CCSCs,构建敲低p53表达的shRNA-p53-CCSCs细胞系。然后将正常CCSCs和shRNA-p53-CCSCs分别分成以下处理组:阴性对照组、DMSO组、舒林酸组和5-FU组,舒林酸组细胞经10μg/ml舒林酸处理,5-FU组细胞经2.5μg/ml 5-FU处理,然后通过BrdU检测试剂盒检测正常CCSCs和shRNA-p53-CCSCs活性,末端转移酶dUTP切口末端标记检测细胞凋亡情况,球体形成实验检测细胞干性变化,Western印迹检测处理后细胞中线粒体凋亡途径相关蛋白Bax/Bcl-2和细胞色素c,β-连环蛋白及其下游靶蛋白c-Myc和cyclinD1表达情况。结果与阴性对照组相比,5-FU组正常CCSCs增殖速率下降了52%,凋亡率为31%(P<0.05),而shRNA-p53-CCSCs增殖速率下降了73%,凋亡率为42%(P<0.05),舒林酸组正常CCSCs增殖速率下降了47%(P<0.05)。球体形成实验检测结果表明,5-FU和舒林酸均显著抑制CCSCs球体形成,且与阴性对照组相比,5-FU和舒林酸组球体体积显著下降(P<0.05),5-FU处理后正常CCSCs中Bax/Bcl-2比例升高约4倍,正常CCSCs与shRNA-p53-CCSCs中细胞色素c表达分别升高约2.8倍,2.5倍,β-连环蛋白在正常CCSCs的胞质和胞核中分别下降75%,73%,在shRNA-p53-CCSCs中分别下降81%,80%,而c-Myc和cyclinD1在上述两种细胞中的表达分别下降83%,52%和47%,53%。结论 5-FU能通过抑制Wnt/β-连环蛋白信号通路并上调线粒体介导的细胞凋亡抑制CCSCs增殖并促进细胞凋亡,具有潜在的抗肿瘤性。Objective To explore the effects of 5-fluorouracil on colon cancer stem cells(CCSCs) stemness and proliferation and the related mechanism. Methods Lentiviral-mediated p53-specific short hairpin RNA(shRNA) was transduced into CCSCs to construct a shRNA-p53-CCSCs line that knocked down p53 expression. The normal CCSCs and shRNA-p53-CCSCs were then divided into the following treatment groups: negative control group, DMSO treatment group, sulindac treatment group and 5-FU treatment group, and sulindac treatment group was treated with 10 μg/ml sulindac. 5-FU treatment group was treated with 2.5 μg/ml 5-FU. The activity of normal CCSCs and shRNA-p53-CCSCs was detected by BrdU detection kit, and the terminal transferase dUTP nick end label was used to detect apoptosis. The spheroid formation assay was used to detect the change of cell dryness. The expressions of mitochondrial apoptosis pathway-related protein Bax/Bcl-2 and cytochrome c, β-catenin and its downstream target proteins c-Myc and cyclinD1 were detected by Western blot.Results Compared with the negative control group, the proliferation rate of 5-FU-treated normal CCSCs decreased by 52%, the apoptotic rate was 31%(P<0.05), and the proliferation rate of shRNA-p53 CCSCs decreased by 73%, the apoptotic rate of shRNA-p53 CCSCs decreased by 42%(P<0.05), the proliferation rate of normal CCSCs in sulindac treatment group decreased by 47%(P<0.05). The results of spheroid formation assay showed that both 5-FU and sulindac significantly inhibited the formation of CCSCs, and the volume of spheres in 5-FU and sulindac treated groups decreased significantly compared with the negative control group(P<0.05). The ratio of Bax/Bcl-2 in normal CCSCs increased by about 4 times after 5-FU treatment. The expression of cytochrome c in normal CCSCs and shRNA-p53-CCSCs increased by about 2.8-fold and 2.5-fold, respectively, and β-catenin decreased by 75% and 73% in the cytoplasm and nucleus of normal CCSCs, respectively, in shRNA-p53-CCSCs. The decrease was 81% and 80%, respective

关 键 词：结肠癌干细胞 5-氟尿嘧啶 P53 Β-连环蛋白 

分 类 号：R285[医药卫生—中药学]