厚朴酚衍生物CT2-3对结肠癌细胞的抑制作用及其机制  

Inhibitory Effect of Magnolol Analogue CT2-3 on Colon Cancer Cells and Its Mechanism

作　　者：黄小飞 陈子德 陶成 陈健[1,2] HUANG Xiao-fei;CHEN Zi-de;TAO Cheng;CHEN Jian(School of Basic Medical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Integrated Chinese and Western Medicine Postdoctoral Research Station,Jinan University,Guangzhou 510632,China;Shenzhen Institute of Geriatrics,Shenzhen 518020,China)

机构地区：[1]广州中医药大学基础医学院,广州510006 [2]暨南大学中西医结合博士后流动站,广州510632 [3]深圳市老年医学研究所,广东深圳518020

出　　处：《中国实验方剂学杂志》2020年第23期113-119,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：广东省基础与应用基础研究基金区域联合基金项目(2019A1515110874);中国博士后科学基金项目(2019M663396)。

摘　　要：目的:研究厚朴酚衍生物CT2-3对结肠癌细胞的抑制作用及其机制,为CT2-3在治疗结肠癌中的应用奠定基础。方法:体外培养SW480和LoVo细胞,不同浓度(10,20,40,80μmol·L-1)CT2-3和厚朴酚分别干预24,48 h,采用细胞增殖-毒性检测(CCK-8)法检测CT2-3和厚朴酚对结肠癌细胞增殖的影响;采用平板克隆形成实验检测CT2-3对结肠癌细胞克隆形成能力的影响;进一步采用流式细胞术和蛋白免疫印迹法(Western blot)检测CT2-3对结肠癌细胞凋亡和DNA损伤标志物磷酸化组蛋白H2AX(γH2AX)蛋白表达的影响;采用活性氧(ROS)试剂盒检测CT2-3对结肠癌细胞内ROS产生的影响;最后用实时荧光定量聚合酶链式反应(Real-time PCR)检测CT2-3对结肠癌细胞内线粒体凋亡相关基因B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X基因(Bax)表达的影响。结果:给药24,48 h,厚朴酚对两株结肠癌细胞SW 480和LoVo的半数抑制浓度(IC50)均>80μmol·L-1,CT2-3对SW480细胞的IC50分别为(54.59±1.73)μmol·L-1和(29.82±1.13)μmol·L-1,对LoVo细胞的IC50分别为(66.68±2.11)μmol·L-1和(46.70±1.81)μmol·L-1;与空白组比较,CT2-3(20,40μmol·L-1)组结肠癌细胞克隆形成能力显著下降(P<0.01),且呈浓度依赖性。CT2-3组凋亡细胞显著增加(P<0.01),γH2AX蛋白相对表达显著增加(P<0.01);CT2-3组细胞内ROS水平显著增加(P<0.01);CT2-3组细胞Bcl-2 mRNA相对表达显著下调(P<0.01),Bax mRNA相对表达显著上调(P<0.01)。结论:CT2-3对结肠癌细胞具有显著抑制作用,其机制可能是通过激活Bcl-2/Bax信号通路使线粒体功能受损,促进ROS的产生,进一步诱导DNA损伤,从而导致结肠癌细胞凋亡。Objective: To study the anti-colon cancer effect and mechanism of magnolol analogue CT2-3,in order to lay a foundation for the application of CT2-3 in anti-colon cancer area. Method: Colon cancer cells SW480 and LoVo were cultured in vitro. Different concentrations(10,20,40,80 μmol·L-1)of CT2-3 and magnolol were used to stimulate colon cancer cells for 24,48 h. The effect of CT2-3 and magnolol on the cell viability of colon cancer cells was detected by cell counting kit(CCK-8). Colony formation assay was used to detect the colony formation capacity of CT2-3 on colon cancer cells. Flow cytometry and Western blot were used to determine the effect of CT2-3 on the apoptosis of colon cancer cells and the expression of DNA damage marker phosphorylated histone H2 AX(γH2 AX). Reactive oxygen species(ROS)generation was measured by ROS assay kit. Real time quantitative polymerase chain reaction(Real-time PCR)was used to detect the effect of CT2-3 on expressions of mitochondrial apoptosis-related genes B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X(Bax)in colon cancer cells. Result:The half maximal inhibitory concentration(IC50)of magnolol in two kinds of colon cancer cells after treatment for 24,48 h were both higher than 80 μmol·L-1.While the IC50 of CT2-3 in SW480 cells after treatment for 24,48 h were(54.59±1.73)μmol·L-1 and(29.82±1.13)μmol·L-1,respectively. The IC50 of CT2-3 in LoVo cells after treatment for 24,48 h were(66.68±2.11)μmol·L-1 and(46.70±1.81)μmol·L-1,respectively. Compared with the blank group,the colony formation capacity of colon cancer cells in CT2-3 groups(20,40 μmol·L-1)was significantly decreased in a dosedependent manner(P<0.01),apoptotic colon cancer cells were significantly increased(P<0.01),relative expression of DNA damage marker γH2 AX was significantly increased(P<0.01),ROS was significantly increased(P<0.01). In addition,relative mRNA expression of Bcl-2 was significantly decreased(P<0.01),while relative mRNA expression of Bax was significantly increased(P<0.01). Conclusi

关 键 词：厚朴酚 厚朴酚衍生物CT2-3 结肠癌 细胞凋亡 活性氧 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]