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Cell growth inhibition by 3-deoxysappanchalcone is mediated by directly T targeting the TOPK signaling pathway in colon cancer
Ran Zhaoa,b,1, Hai Huanga,1, Bu Young Choic,1, Xuejiao Liua, Man Zhanga, Silei Zhoua, Mengqiu Songa,b, Fanxiang Yina,b, Hanyong Chend, Jung-Hyun Shime, Ann M. Boded, Zigang Donga,b,d,f, , Mee-Hyun Leea,b,f,
a Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan 450001, P.R. China
b China-US (Henan) Hormel Cancer Institute, No.127, Dongming Road, Jinshui District, Zhengzhou, Henan, 450008, China
c Department of Pharmaceutical Science and Engineering, School of Convergence Bioscience and Technology, Seowon University, Chungbuk, South Korea
d The Hormel Institute, University of Minnesota, Austin MN55912, USA
e Department of Pharmacy, College of Pharmacy, Mokpo National University, Muan, Jeonnam 58554, South Korea
f The Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, Zhengzhou, China
G2/M cell cycle
Background: Colorectal cancer is one of the most common causes of cancer death worldwide. Unfortunately, chemotherapies are limited due to many complications and development of resistance and recurrence. The T-lymphokine-activated killer cell-originated protein kinase (TOPK) is highly expressed and activated in colon cancer, and plays an important role in inflammation, proliferation, and survival of cancer cells. Therefore, suppressing TOPK activity and its downstream signaling cascades is considered to be a rational therapeutic/ preventive strategy against colon cancers.
Purpose: 3-Deoxysappanchalcone (3-DSC), a component of Caesalpinia sappan L., is a natural oriental medicine. In this Phosal50PG study, we investigated the eﬀects of 3-DSC on colon cancer cell growth and elucidated its underlying molecular mechanism of targeting TOPK.
Study design and methods: To evaluate the eﬀects of 3-DSC against colon cancer, we performed cell proliferation assays, propidium iodide- and annexin V-staining analyses and Western blotting. Targeting TOPK by 3-DSC was identified by a kinase-binding assay and computational docking models.
Results: 3-DSC inhibited the kinase activity of TOPK, but not mitogen-activated protein kinase (MEK). The direct binding of 3-DSC with TOPK was explored using a computational docking model and binding assay in vitro and ex vivo. 3-DSC inhibited colon cancer cell proliferation and anchorage-independent cell growth, and induced G2/ M cell cycle arrest and apoptosis. Treatment of colon cancer cells with 3-DSC induced expression of protein that are involved in cell cycle (cyclin B1) and apoptosis (cleaved-PARP, cleaved-caspase-3, and cleaved-caspase-7), and suppressed protein expressions of extracellular signal-regulated kinase (ERK)-1/2, ribosomal S6 kinase (RSK), and c-Jun, which are regulated by the upstream kinase, TOPK.
Conclusion: 3-DSC suppresses colon cancer cell growth by directly targeting the TOPK- mediated signaling pathway.
Introduction kinase kinase (MAPKK) family (Abe et al., 2000). It plays important
roles in many cellular processes, such as growth, development, apop- The lymphokine-activated killer T (T-LAK)-cell-originated protein tosis and inflammation (Li et al., 2011; Nandi et al., 2004; Simons-
lated to the serine-threonine kinases of the mitogen-activated protein ERKs, histone H3 (Ser10), histone H2AX (Ser139), peroxiredoxin (PRX,
Abbreviations: ERK, Extracellular signal-regulated kinase; MBP, Myelin binding protein; MEK, Mitogen-activated protein kinase; TOPK, T–lymphokine-activated killer cell–originated protein kinase; 3-DSC, 3-Deoxysappanchalcone
E-mail addresses: [email protected] (Z. Dong), [email protected], [email protected] (M.-H. Lee). 1 These authors contributed equally.
Fig. 1. 3-DSC specifically targets TOPK. (A, B) Eﬀects of 3-DSC on TOPK or MEK kinase activity. (C) The binding of 3-DSC to TOPK or MEK in HCT-15 cell lysates was determined using Sepharose 4B or 3-DSC-conjugated Sepharose 4B beads. (D, E) The specific binding of 3-DSC to active TOPK and its ATP competitive binding. (F) Modeling of 3-DSC binding with TOPK. Left: 3-DSC binding at the ATP binding pocket of TOPK. TOPK structure is shown as ribbon representation and 3-DSC is shown as a stick model.
Ser32) 1, c-Jun-NH2-kinase (JNK, Thr183/Tyr185) 1 and p53-related protein kinase (PRPK, Ser250). The binding between TOPK and these substrates was verified by in vitro and ex vivo binding assays or
prediction by computational docking models (Oh et al., 2007; Zhu et al., 2007; Zykova et al., 2006; Zykova et al., 2010). The phosphor-ylation of downstream eﬀectors by TOPK activates various signaling