Chen Jian, Mu Qitian, Li Xia, Yin Xiufeng, Yu Mengxia, Jin Jing, Li Chenying, Zhou Yile, Zhou Jiani, Suo Shanshan, Lu Demin, Jin Jie
Department of Hematology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Institute of Hematology, Zhejiang University, Hangzhou, China.
Oncotarget. 2017 Jun 20;8(25):40318-40326. doi: 10.18632/oncotarget.16956.
Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT.
高三尖杉酯碱（HHT）在中国长期广泛用于治疗急性髓系白血病（AML），临床治疗效果显著，但作用机制尚不清楚。本研究旨在通过虚拟筛选寻找HHT可能的靶点，并通过细胞实验验证结果。使用了包括Autodock、Python和MGL工具在内的软件，以HHT为配体，PI3K-Akt通路、Jak-stat通路、TGF-β通路和NK-κB通路的蛋白为受体。培养人AML细胞系U937、KG-1、THP-1作为实验细胞系。采用MTT法检测细胞增殖，流式细胞术检测HHT作用后的细胞凋亡和细胞周期阻滞，蛋白质印迹法检测蛋白质水平变化，病毒shRNA转染抑制候选靶蛋白的表达水平，病毒mRNA转染用于过表达。虚拟筛选显示，TGF-β通路的smad3可能是HHT结合的候选靶点。在AML细胞系U937和KG-1中，HHT可诱导smad3的Ser423/425磷酸化，这种磷酸化随后可激活TGF-β通路，导致U937细胞的细胞周期阻滞于G1期，KG-1细胞凋亡，敲低smad3可削弱U937细胞对HHT的敏感性，过表达smad3可在两种细胞系中重新建立敏感性。我们得出结论，smad3是HHT可能的靶蛋白，在HHT的作用机制中起重要作用。
