氯硝柳胺下调CLOCK引起胃癌细胞节律紊乱诱发支链氨基酸代谢失调的机制研究  

Mechanism Research of Niclosamide Induced Maladjustment of BCAA through Circadian Disorder by Down-Regulating CLOCK

作　　者：陈通钻 戴瑞霞 金高巍 梁琪 朱晓晖 戴纪辰 洪培彬 张加骐 陈通克 方合志 陈祥建[1] 谢彬涛 CHEN Tongzuan;DAI Ruixia;JIN Gaowei;LIANG Qi;ZHU Xiaohui;DAI Jichen;HONG Peibin;ZHANG Jiaqi;CHEN Tongke;FANG Hezhi;CHEN Xiangjian;XIE Bintao(The First Affiliated Hospital,Wenzhou Medical University,Wenzhou 325000,China;Second School of Clinical Medicine,Wenzhou Medical University,Wenzhou 325000,China;Laboratory Animal Centre,Wenzhou Medical University,Wenzhou 325000,China;College of Laboratory Medicine and Life sciences,Wenzhou Medical University,Wenzhou 325000,China;Basic Research Center,Eye Hospital,Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]温州医科大学附属第一医院,温州325000 [2]温州医科大学第二临床医学院,温州325000 [3]温州医科大学实验动物中心,温州325000 [4]温州医科大学检验医学院、生命科学学院,温州325000 [5]温州医科大学附属眼视光医院基础研究中心,温州325000

出　　处：《中国细胞生物学学报》2022年第5期817-825,共9页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:82103367);温州市基础科学研究项目(批准号:Y20190055、Y20190085)资助的课题。

摘　　要：生物节律是周期性的生命活动现象。肿瘤细胞内普遍存在代谢重编程,有研究表明生物节律与代谢调控密切相关。氯硝柳胺(Niclosamide)是一种传统抗蠕虫药,具有氧化磷酸化解偶联的特性,近年来已步入抗癌药物研究领域。为探究氯硝柳胺抑制胃癌发生发展的具体机制,该研究通过CCK-8增殖、ROS和凋亡等实验发现氯硝柳胺可抑制增殖促进凋亡。通过生物信息学筛选出胃癌与癌旁生物节律差异基因Clock、Per、Dbp等,Western blot实验检测发现它们所编码的蛋白在药物作用后表达均有明显变化,其中CLOCK变化最为显著,提示药物作用后胞内生物节律紊乱。通过海马能量代谢和代谢组学分析进一步探讨药物作用后细胞代谢特性及其与生物节律的相关性,结果提示细胞内氧化磷酸化整体趋势呈药物浓度依赖性下降,同时证实了三羧酸循环中间产物延胡索酸在氯硝柳胺抑癌机制中的重要度。通过Western blot实验发现药物作用后支链氨基酸相关蛋白下调,CLOCK敲低与加药联合作用可进一步抑制支链氨基酸转氨酶1(BCAT1)的表达。综上,该研究认为氯硝柳胺可通过紊乱胃癌细胞节律,诱导支链氨基酸代谢失调从而发挥抑癌效应,有望成为抗癌药物的新星。Circadian rhythm is deemed a cyclical phenomenon of lives,while metabolic reprogramming exists in cancer cells at large.Some researches have found relationship between circadian and metabolic regulation.Niclosamide,a traditional anti-worm drugs,armed with properties to uncouple oxidative phosphorylation,is marching to the researching field of anti-tumour drugs.To illuminate mechanisms of its anti-tumour properties,cell counting,reactive oxygen species and apoptosis assay were performed to find that niclosamide could inhibit proliferation and promote apoptosis.Clock,Per,Dbp were selected as circadian differential expression genes through bioinformatics analyses,and expressions of these proteins were in disorder by Western blot,especially CLOCK,after treatment with Niclosamide.The study went deeper into discussing relevance between circadian disorder and metabolism through seahorse analyses and metabonomics analyses.Intracellular oxidative phosphorylation was tested to be suppressed after treatment with niclosamide in a concentration-dependent way.Besides,the importance of fumaric acid in mechanisms of niclosamide’s anti-tumour effect were also found.Western blot was performed to figure out that BCAA (branched amino acid)-related protein were inhibited after treatment with niclosamide,and combination of niclosamide with knockdown of CLOCK could further suppress the expression of BCAT1 (branched chain amino acid transaminase 1).Taken altogether,this research displayed that niclosamide could contribute to maladjustment of BCAA through circadian disorder to suppress tumour,which had a promising future as a new star of the anti-tumour.

关 键 词：氯硝柳胺 生物节律 代谢重编程 支链氨基酸代谢 CLOCK 

分 类 号：R96[医药卫生—药理学]