Thymoquinone exerts potent growth-suppressive activity on leukemia through DNA hypermethylation reversal in leukemia cells

// Jiuxia Pang 1 , Na Shen 1 , Fei Yan 1 , Na Zhao 1 , Liping Dou 1, 2 , Lai-Chu Wu 3 , Christopher L. Seiler 4 , Li Yu 2 , Ke Yang 5 , Veronika Bachanova 6 , Eric Weaver 7 , Natalia Y. Tretyakova 4 , Shujun Liu 1 1 The Hormel Institute, University of Minnesota, Austin, MN 55912, USA 2 Department of Hematology, Chinese PLA General Hospital, Medical School of Chinese PLA, Beijing 100853, China 3 Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH 43021, USA 4 Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA 5 Chongqing Engineering Research Center of Stem Cell Therapy, The Children’s Hospital of Chongqing Medical University, Chongqing 400014, China 6 Division of Hematology, Oncology and Transplantation, Minneapolis, MN 55455, USA 7 Prairie Pharms LLC, Nora Springs, IA 50458, USA Correspondence to: Shujun Liu, email: sliu@umn.edu Keywords: DNA methylation, leukemia, thymoquinone, DNA methyltransferase, bioactive compounds Received: February 10, 2017      Accepted: March 14, 2017      Published: March 21, 2017 ABSTRACT Thymoquinone (TQ), a bioactive constituent of the volatile oil of Monarda fistulosa and Nigella sativa , possesses cancer-specific growth inhibitory effects, but the underlying molecular mechanisms remain largely elusive. We propose that TQ curbs cancer cell growth through dysfunction of DNA methyltransferase 1 (DNMT1). Molecular docking analysis revealed that TQ might interact with the catalytic pocket of DNMT1 and compete with co-factor SAM/SAH for DNMT1 inhibition. In vitro inhibitory assays showed that TQ decreases DNMT1 methylation activity in a dose-dependent manner with an apparent IC50 of 30 nM. Further, exposure of leukemia cell lines and patient primary cells to TQ resulted in DNMT1 downregulation, mechanistically, through dissociation of Sp1/NFkB complex from DNMT1 promoter. This led to a reduction of DNA methylation, a decrease of colony formation and an increase of cell apoptosis via the activation of caspases. In addition, we developed and validated a sensitive and specific LC-MS/MS method and successfully detected a dynamic change of TQ in mouse plasma after administration of TQ through the tail vein, and determined a tolerable dose of TQ to be 15 mg/kg in mouse. TQ administration into leukemia-bearing mice induced leukemia regression, as indicated by the reversed splenomegaly and the inhibited leukemia cell growth in lungs and livers. Our study for the first time demonstrates that DNMT1-dependent DNA methylation mediates the anticancer actions of TQ, opening a window to develop TQ as a novel DNA hypomethylating agent for leukemia therapy.