摘要
在经典的Wnt/β-catenin信号通路中,β-catenin/TCF4(T-cell factor 4)相互作用在非小细胞肺癌(Non-small cell lung cancer,NSCLC)的生长分化、化疗耐药、转移复发等过程中发挥着重要的促进作用,已成为新型靶向性抗NSCLC转移药物开发的理想靶标之一。为了高效地发现抑制β-catenin/TCF4相互作用的苗头化合物,本研究在应用酶联免疫吸附实验(Enzyme-linkedimmunosorbentassay,ELISA)原理的基础上,通过优化GST-TCF4βBD包被浓度和β-catenin反应浓度,建立ELISA高通量筛选模型并成功应用于苗头化合物的筛选。ELISA筛选模型优化实验结果表明,选用2μg/mL GST-TCF4βBD和0.5μg/mLβ-catenin建立ELISA高通量筛选模型,其Z′因子值为0.83,并成功筛选到具有良好抑制活性的白花丹素(Plumbagin)。肿瘤细胞增殖实验结果表明,白花丹素对A549、H1299、MCF7和SW480细胞具有明显的细胞毒性。TOPFlash实验结果证实,白花丹素对转染的HEK293细胞内β-catenin介导的转录活性具有显著的抑制作用,β-catenin/TCF4相互作用可能是白花丹素抗癌活性的潜在分子靶标之一。文中以β-catenin/TCF4相互作用为靶标,通过系统的实验优化方案,成功地建立了适用于药物高通量筛选的ELISA筛选模型,为高效筛选靶向β-catenin/TCF4相互作用的小分子抑制剂奠定了实验基础。
In canonical Wnt/β-catenin signaling pathway,β-catenin/TCF4(T-cell factor 4)interaction plays an important role in the pathogenesis and development of non-small cell lung cancer(NSCLC),and it is tightly associated with the proliferation,chemoresistance,recurrence and metastasis of NSCLC.Therefore,suppressingβ-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis.In this study,considering the principle of enzyme-linked immunosorbent assay(ELISA),an optimized high-throughput screening(HTS)assay was developed for the discovery ofβ-catenin/TCF4 interaction antagonists.Subsequently,this ELISA-like screening assay was performed using 2μg/mL GST-TCF4βBD and 0.5μg/mLβ-catenin,then a high Z′factor of 0.83 was achieved.A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potentialβ-catenin/TCF4 interaction antagonist.Plumbagin remarkably inhibited the proliferation of A549,H1299,MCF7 and SW480 cell lines.More importantly,plumbagin significantly suppressed theβ-catenin-responsive transcription in TOPFlash assay.In short,this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targetingβ-catenin/TCF4 interaction,and this interaction is a potential anticancer target of plumbagin in vitro.
作者
付正豪
闫干干
朱小红
刘晓平
陈云雨
Zhenghao Fu;Gangan Yan;Xiaohong Zhu;Xiaoping Liu;Yunyu Chen(Institute for Drug Screening and Evaluation,Wannan Medical College,Wuhu 241002,Anhui,China;Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2021年第8期2878-2889,共12页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.81703546)
安徽省自然科学基金(No.1808085QH265)
皖南医学院大学生科研资助金(No.WK2021XS54)
吉林省科技发展计划项目(No.20160520045JH)
安徽省高校自然科学研究重大项目(No.KJ2019ZD30)
安徽省重点研究与开发计划项目(No.202004a07020041)资助。
