JAK1/STAT1/p21通路在姜黄素诱导结肠癌细胞HCT116周期阻滞中的作用  

Curcumin Induces Cycle Arrest of Colon Cancer HCT116 Cells via JAK1/STAT1/p21 Pathway

作　　者：李天硕 席作武[2] 董文洁 史登辉 刘云蓉 林子栋 LI Tianshuo;XI Zuowu;DONG Wenjie;SHI Denghui;LIU Yunrong;LIN Zidong(Second Clinical Medical College,Henan University of Chinese Medicine,Zhengzhou 450002,China;Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南中医药大学第二临床医学院,郑州450002 [2]河南省中医院,郑州450002

出　　处：《中国实验方剂学杂志》2024年第9期74-82,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：张仲景经方合方的临床应用及其作用机制研究项目(GZY-KJS-2022-041);河南中医药学科领军人才项目(豫卫中医函[2021]8号)。

摘　　要：目的:探讨姜黄素对人结肠癌HCT116细胞周期阻滞的影响及其可能的分子作用机制。方法:噻唑蓝(MTT)比色法检测姜黄素(0、12.5、25、50、75、100μmol·L^(-1))和5-氟尿嘧啶(5-FU)(600μmol·L^(-1))处理不同时间(24、48、72 h)对HCT116细胞增殖的影响;流式细胞术检测姜黄素(0、25、50、75μmol·L^(-1))和5-FU对HCT116细胞周期的影响;蛋白免疫印迹法(Western blot)检测HCT116细胞中Janus酪氨酸蛋白激酶1(JAK1)/信号传导及转录激活因子1(STAT1)/细胞周期依赖性激酶抑制因子1A(p21)通路相关蛋白表达的影响;染色质免疫沉淀法(ChIP)检测STAT1与p21启动子区的结合情况;采用小干扰核糖核酸(siRNA),分别通过Western blot和细胞免疫荧光检测STAT1在调控p21表达中的作用及JAK1蛋白在调控STAT1活化中的作用。结果:与空白组比较,姜黄素各组和5-FU组HCT-116细胞活性明显降低(P<0.05)。与空白组比较,姜黄素组和5-FU组DNA合成前期(G0/G1)细胞比例明显升高(P<0.05),DNA复制期(S)和DNA合成后期(G2/M)细胞比例及磷酸化p21(p-p21)蛋白水平明显降低(P<0.05),p21蛋白表达明显升高(P<0.05)。与姜黄素组比较,p21 siRNA+姜黄素组G0/G1期细胞明显降低(P<0.05)。与空白组比较,姜黄素处理后细胞中磷酸化STAT1(p-STAT1)水平明显升高(P<0.05)。与姜黄素组比较,姜黄素+STAT1 siRNA组HCT116细胞中p21蛋白表达水平升高(P<0.05)。机制研究表明,与空白组比较,姜黄素处理明显增强了STAT1蛋白在p21启动子区的富集(P<0.05)。与空白组比较,姜黄素处理后细胞中磷酸化JAK1(p-JAK1)水平明显升高(P<0.05)。与姜黄素组比较,姜黄素+STAT1 siRNA组HCT116细胞中的p-STAT1和p21蛋白水平明显升高(P<0.05)。结论:姜黄素诱导的人结肠癌HCT116细胞周期阻滞,其机制可能与激活JAK1/STAT1/p21信号通路有关。Objective:To investigate the effect of curcumin on the cycle arrest of human colon cancer HCT116 cells and decipher the possible molecular mechanism.M ethod:The methyl thiazolyl tetrazolium(MTT)method was employed to examine the effects of curcumin(0,12.5,25,50,75,100μmol·L^(-1))and 5-fluorouracil(5-FU,600μmol·L^(-1))on the proliferation of HCT116 cells at different time points(24,48,72 h).Flow cytometry was employed to examine the cycle of HCT116 cells treated with curcumin(0,25,50,75μmol·L^(-1))and 5-FU.Western blot was employed to determine the expression of proteins in the Janus kinase 1(JAK1)/signal transducer and activator of transcription 1(STAT1)/cyclin-dependent kinase inhibitor 1A(p21)pathway in HCT116 cells.The binding of STAT1 to p21 promoter region was detected by chromatin immunoprecipitation(ChIP).Small interfering RNA(siRNA)was employed to measure the role of STAT1 in regulating the expression of p21 and that of JAK1 in regulating the activation of STAT1 by Western blot and cellular immunofluorescence,respectively.Result:Compared with the blank group,the HCT-116 cells treated with curcumin and 5-FU showed decreased viability(P<0.05),increased proportions of cells in the G0/G1 phase(P<0.05),decreased proportions of cells in the S phase and G2/M phase(P<0.05),down-regulated protein level of phosphorylated p21(P<0.05),and up-regulated protein level of p21(P<0.05).Compared with the curcumin group,the p21 siRNA+curcumin group presented decreased proportion of cells in G0/G1 phase(P<0.05).Compared with the blank group,curcumin elevated the level of phosphorylated STAT1(p-STAT1)(P<0.05).Compared with the curcumin group,thecurcumin+STAT1 siRNA group showcased up-regulated protein level of p21 in HCT116 cells(P<0.05).The mechanism study showed that curcumin treatment enhanced the enrichment of STAT1 in the p21 promoter region(P<0.05)compared with the blank group.Compared with the blank group,curcumin up-regulated the level of phosphorylated JAK1(p-JAK1)(P<0.05).Compared with the curcumin group,the cu

关 键 词：姜黄素 人结肠癌细胞 细胞周期阻滞 Janus酪氨酸蛋白激酶1/信号传导及转录激活因子1(JAK1/STAT1)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22R242R285.5