Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells
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Jian Chen1,2,*, Qitian Mu1,2,3,*, Xia Li1,2,*, Xiufeng Yin1,2, Mengxia Yu1,2, Jing Jin1,2, Chenying Li1,2, Yile Zhou1,2, Jiani Zhou1,2,4, Shanshan Suo1,2, Demin Lu1,2 and Jie Jin1,2
1Department of Hematology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
2Institute of Hematology, Zhejiang University, Hangzhou, China
3Laboratory of Stem Cell Transplantation, Ningbo First Hospital, Zhejiang, China
4Hematology Department of Ningbo Medical Center Lihuili Estern Hospital, Ningbo, China
*These authors contributed equally to this work
Jie Jin, email: [email protected]
Keywords: homoharringtonine, smad3, TGF-β, acute myeloid leukemia
Received: April 04, 2016 Accepted: March 29, 2017 Published: April 08, 2017
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.
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