BACKGROUND Notch activation requires proteolytic cleavage of the receptor by γ-secretase protein complex. Inhibition of Notch receptor activation (e.g. Notch3) with γ-secretase inhibitor is a potential new therapeutic approach for the targeted therapy of non-small cell lung cancer (NSCLC). However, only a few safe and effective γ-secretase inhibitors have been discovered. Evodiamine (EVO), a compound derived from Euodiae Fructus (Chinese name, Wu-Zhu-Yu), exhibits remarkable anti-NSCLC activities. However, the underlying mechanisms of action have yet to be fully elucidated. PURPOSE We sought to determine the involvement of Notch3 signaling in the anti-NSCLC effects of EVO, and to explore whether EVO suppressed Notch3 signaling by inhibiting γ-secretase in cultured A549 and H1299 NSCLC cells and in urethane-induced lung cancer FVB mouse model. METHODS Cell viability, migration, stemness and cell cycle distribution of EVO were examined by the MTT assay, wound healing assay, soft agar colony assay and flow cytometry analysis, respectively. The binding affinity of EVO and γ-secretase complex was analyzed by molecular docking. Cellular thermal shift assay (CETSA) was performed to study the drug-target interactions in NSCLC cells. Protein levels were determined by Western blotting. RESULTS EVO dramatically inhibited cell viability, induced G2/M cell cycle arrest, suppressed cell migration, and reduced stemness in NSCLC cells. Mechanistic studies indicated that EVO prevented the γ-secretase cleavage of Notch3 at the cell surface and hence inhibited Notch3 activation. Moreover, EVO notably reduced tumor growth in the mouse model and inhibited Notch3 activity in the tumors. CONCLUSION This study provides new insights into the anti-NSCLC action of EVO, and suggests that suppressing Notch3 signaling by inhibiting γ-secretase is a mechanism of action underlying the anti-NSCLC effect of EVO.

中文翻译：

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Evodiamine通过直接与γ-分泌酶结合来抑制肺肿瘤发生过程中的Notch3信号传导。

背景技术缺口激活需要γ-分泌酶蛋白复合物对受体进行蛋白水解切割。γ-分泌酶抑制剂抑制Notch受体激活（例如Notch3）是针对非小细胞肺癌（NSCLC）靶向治疗的潜在新治疗方法。但是，仅发现了几种安全有效的γ-分泌酶抑制剂。乙二胺（Evodiamine（EVO））是一种从大叶草（Eudiaae Fructus）（中文名称为吴竹鱼）衍生的化合物，具有非NSCLC的显着活性。但是，根本的作用机理尚未完全阐明。目的我们试图确定Notch3信号传导参与EVO的抗NSCLC作用，并探讨EVO是否通过抑制γ-分泌酶在培养的A549和H1299NSCLC细胞以及聚氨酯诱导的肺癌FVB小鼠模型中抑制Notch3信号传导。方法分别用MTT法，伤口愈合法，软琼脂菌落法和流式细胞仪分析EVO的细胞活力，迁移，干性和细胞周期分布。通过分子对接分析EVO和γ-分泌酶复合物的结合亲和力。进行细胞热位移分析（CETSA）以研究NSCLC细胞中的药物-靶标相互作用。通过蛋白质印迹测定蛋白质水平。结果EVO可以显着抑制细胞活力，诱导G2 / M细胞周期停滞，抑制细胞迁移，并降低NSCLC细胞的干性。机理研究表明，EVO阻止了Notch3在细胞表面的γ-分泌酶裂解，因此抑制了Notch3的活化。此外，EVO明显降低了小鼠模型中的肿瘤生长，并抑制了肿瘤中的Notch3活性。