基于Akt/FoxO信号通路探讨熊果酸对结直肠癌细胞增殖和凋亡的影响  

Effect of Ursolic Acid on Proliferation and Apoptosis of Colorectal Cancer Cells Based on Akt/FoxO Signaling Pathway

作　　者：郑巧 陈念芝 章刚 赵梓亦[1] 唐健元 ZHENG Qiao;CHEN Nianzhi;ZHANG Gang;ZHAO Ziyi;TANG Jianyuan(Traditional Chinese Medicine(TCM)Regulating Metabolic Diseases Key Laboratory of Sichuan Province,Hospital of Chengdu University of TCM,Chengdu 610032,China;State Key Laboratory of Ultrasound in Medical and Engineering,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]成都中医药大学附属医院代谢疾病中医药调控四川省重点实验室,成都610032 [2]重庆医科大学超声医学工程国家重点实验室,重庆400016

出　　处：《中国实验方剂学杂志》2023年第17期109-115,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(8207152674)。

摘　　要：目的:观察熊果酸对结直肠癌细胞增殖和凋亡的影响及具体的分子机制。方法:选用人源结直肠癌细胞RKO为研究对象,用细胞增殖与活性检测(CCK-8)法检测不同浓度的熊果酸(0、5、10、15、20、25、30μmol·L^(-1))处理RKO细胞的增殖抑制率,计算出24、48 h的半抑制浓度(IC50)。后续实验根据熊果酸处理RKO细胞24 h的IC50,选用2个浓度开展。集落形成实验检测细胞增殖能力,流式细胞术检测熊果酸处理RKO细胞后的凋亡率及细胞周期阻滞情况,蛋白免疫印迹法(Western blot)检测熊果酸作用RKO细胞24 h,RKO细胞中B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)、Raji细胞中Bcl-2、人T淋巴细胞白血病PMA反应基因(Noxa)凋亡蛋白表达情况,细胞周期依赖性蛋白激酶抑制因子1A(p21)、周期蛋白依赖激酶抑制因子1B(p27)、细胞周期蛋白依赖性激酶4(CDK4)周期相关的蛋白表达情况,以及蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、叉头框蛋白O3a(FoxO3a)、磷酸化FoxO3a(p-FoxO3a)通路蛋白表达情况。结果:与空白组比较,熊果酸组能够抑制RKO细胞的活力(P<0.05,P<0.01),熊果酸组RKO细胞的克隆形成率明显降低(P<0.05,P<0.01),呈现浓度依赖性;与空白组比较,熊果酸组熊果酸组(20μmol·L^(-1))细胞发生了周期阻滞,在合成前期即G0/G1期明显升高(P<0.05);与空白组比较,熊果酸组(15、20μmol·L^(-1))p21、p27蛋白表达增加,CDK4蛋白表达降低(P<0.05,P<0.01),熊果酸组细胞凋亡率升高,熊果酸组(20μmol·L^(-1))凋亡相关蛋白Bax、Noxa表达升高,Bcl-2表达降低(P<0.05,P<0.01)。与空白组比较,熊果酸组(20μmol·L^(-1))下调p-Akt蛋白的表达,且上调p-FoxO3a的表达(P<0.05,P<0.01),Akt和FoxO3a总蛋白无明显变化。结论:熊果酸能够有效抑制结直肠癌细胞RKO的增殖,促进细胞凋亡,其机制可能与Akt/FoxO途径有关。Objective:To investigate the effects and molecular mechanism of ursolic acid on the proliferation and apoptosis of colorectal cancer cells.Method:The proliferation inhibition rate of human colorectal cancer RKO cells treated with different concentrations of ursolic acid(0,5,10,15,20,25,30μmol·L^(-1))was detected by cell counting kit-8(CCK-8),and the half maximal inhibitory concentration(IC50)at 24 h and 48 h was calculated.According to the IC50 of RKO cells treated with ursolic acid for 24 h,two concentrations were selected for subsequent experiments.The colony formation assay was used to detect the proliferation ability of the cells and flow cytometry was used to detect the apoptosis rate and cell cycle arrest after treatment of RKO cells with ursolic acid.After treatment of RKO cells with ursolic acid for 24 hours,the expression of B-cell lymphoma 2(Bcl-2)-associated X protein(Bax)in RKO cells,Bcl-2 in Raji cells,PMA responsive gene in T lymphocyte(Noxa),cyclin-dependent kinase inhibitor 1A(p21),cyclin-dependent kinase inhibitor 1B(p27),cyclin-dependent kinase 4(CDK4),protein kinase B(Akt),phosphorylated Akt(p-Akt),forkhead transcription factor O3a(FoxO3a),and phosphorylated FoxO3a(p-FoxO3a)was determined by Western blot.Result:Compared with the blank group,the ursolic acid groups could inhibit the viability of RKO cells(P<0.05,P<0.01),and the colony formation rates of RKO cells in the ursolic acid groups were reduced(P<0.05,P<0.01)in a concentration-dependent manner.The cells in the ursolic acid group(20μmol·L^(-1))experienced cell cycle arrest,which increased in the early stage of synthesis,ie,the G0/G1 phase(P<0.05)as compared with the results in the blank group.Compared with the blank group,the ursolic acid groups(15 and 20μmol·L^(-1))showed increased protein expression of p21 and p27,decreased expression of CDK4 protein(P<0.05,P<0.01),and increased apoptosis rate,and the ursolic acid group(20μmol·L^(-1))showed increased protein expression of Bax and Noxa and decreased expression of Bcl-2(P<0.05,P<0

关 键 词：熊果酸 结直肠癌 增殖 凋亡 蛋白激酶B(Akt)/叉头框蛋白O(FoxO)信号通路 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5