新型多胺代谢酶抑制剂SI-4650对结肠癌CT-26细胞增殖的影响及其机制  

Effects of a novel polyamine metabolic enzyme inhibitor SI-4650 on proliferation of colon cancer CT-26 cells and its mechanism

作　　者：黄娇娇 王艳林[1] 孙丽丹[1] 曹春雨[1] 秦烨[1] 杨建林[1] HUANG Jiao-jiao;WANG Yan-lin;SUN Li-dan;CAO Chun-yu;QIN Ye;YANG Jian-lin(Medical College of China Three Gorges University,Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,Yichang 443002,China)

机构地区：[1]三峡大学医学院肿瘤微环境与免疫治疗湖北省重点实验室,湖北宜昌443002

出　　处：《中国应用生理学杂志》2020年第4期330-335,共6页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金资助项目(81372265,30772590);湖北省卫生健康科研基金(WJ2019H532);肿瘤微环境与免疫治疗湖北省重点实验室开放基金项目(2019KZL01,2016KZL04)。

摘　　要：目的:探讨本实验室新发现的新型多胺代谢酶小分子抑制剂SI-4650对结肠癌CT-26细胞增殖、自噬和凋亡的影响。方法:体外培养CT-26细胞,以0μmol·L-1 SI-4650处理48 h细胞为正常对照组,单独2.5 mmol·L-13-MA处理细胞为自噬抑制对照组,40、80μmol·L-1 SI-4650处理48 h细胞以及3-MA联合40、80μmol·L-1 SI-4650处理48 h细胞为4个实验组,化学发光法检测CT-26细胞中多胺代谢酶SMO和APAO酶活性的变化,HPLC法检测细胞中多胺含量的变化,CCK8法检测CT-26细胞增殖能力变化;PI单染结合流式细胞术分析细胞周期;Western blot法分析细胞自噬;PI/FITC-Annexin V双染、JC-1荧光探针和Fluo-3 AM钙离子荧光探针分别结合流式细胞术以及Western blot法分析细胞凋亡。结果:与正常对照组比较,40、80μmol·L-1 SI-4650实验组细胞生长抑制率分别为36.98%、46.91%,有效抑制肿瘤细胞增殖(P<0.01);同时细胞中SMO和APAO酶活性下降(P<0.01);细胞中多胺总含量减少(P<0.01);CT-26细胞被阻滞在G0/G1期(P<0.01);凋亡细胞数分别为7.69%和16.87%,细胞中钙浓度增加(P<0.01)、线粒体膜电位下降(P<0.01),c-PARP、Bax表达增加(P<0.01)、Bcl-2含量减少(P<0.01);以及CT-26细胞中自噬相关蛋离子白Beclin-1、LC3-Ⅱ以及P62含量显著上升(P<0.01)。与单独40、80μmol·L-1 SI-4650处理组相比,2.5 mmol·L-13-MA联合40、80μmol·L-1 SI-4650实验组细胞自噬水平下降(P<0.01),凋亡相关蛋白、线粒体膜电位和钙离子浓度变化均减弱(P<0.01),凋亡细胞数减少(P<0.01)。结论:SI-4650有效抑制结肠癌CT-26细胞增殖,机制可能与抑制多胺代谢酶活性,干扰多胺代谢,减少多胺总含量以及诱导细胞周期阻滞、细胞自噬和凋亡相关。Objective:To investigate the effects of a novel polyamine metabolism enzyme inhibitor SI-4650 on autophagy and apoptosis of colon cancer CT-26 cells as well as their correlation.Methods:CT-26 cells treated with 40,80μmol·L-1 SI-4650 alone or in combination with 3-MA were used as experimental group.CT-26 cells treated with 0μmol·L-1 SI-4650 alone or in combination with 3-MA were used as control group.Chemiluminescence was used to analyze the effect of SI-4650 on spermine oxidase(SMO)and acetylpolyamine oxidase(APAO)activity.High performance liquid chromatography(HPLC)was performed to detect cellular polyamine content.The CCK8 method was used to detect the inhibitory effect of SI-4650 on proliferation of CT-26 cells.PI single-staining/flow cytometry(FCM)were used to analyze cell cycle.Western blot were used to analyze autophagy.Apoptosis was analyzed by PI/FITC-Annexin V double staining,JC-1 fluorescent probe and Fluo-3 AM calcium ion fluorescent probe combined with flow cytometry and Western blot.Results:CCK8 assay showed that 24-,48-,72-hours treated with SI-4650 all could inhibit the proliferative activity of CT-26 cells in a dose-and time-dependent manner(P<0.01).The inhibition rate was 36.98%and 46.91%in 40μmol·L-1 SI-4650 group and 80μmol·L-1 SI-4650 group respectively.SI-4650 could significantly inhibit the activities of SMO and APAO interfere with polyamine metabolism and reduce the content of total polyamine in CT-26 cells(P<0.01).SI-4650 could block CT-26 cells in G0/G1 phase,significantly reduce the number of cells in S phase(P<0.01),and lead to a significant increase in the contents of autophagy-related Beclin-1,LC3-II in CT-26 cells(P<0.01);At the same time,the concentration of calcium in CT-26 cells was increased,the mitochondrial membrane potential was decreased,the expressions of c-PARP and Bax were increased,the content of Bcl-2 was decreased,and the number of apoptotic cells was increased.After SI-4650 combined with autophagy inhibitor 3-MA treatment of CT-26 cells,the level of autophagy,t

关 键 词：多胺代谢酶抑制剂SI-4650 结肠癌 CT-26细胞 凋亡 自噬 

分 类 号：R966[医药卫生—药理学]