title: Nickel pyrithione induces apoptosis in chronic myeloid leukemia cells resistant to imatinib via both Bcr/Abl-dependent and Bcr/Abl-independent mechanisms.
abstract: Acquired imatinib (IM) resistance is frequently characterized by Bcr-Abl mutations that affect IM binding and kinase inhibition in patients with chronic myelogenous leukemia (CML). Bcr-Abl-T315I mutation is the predominant mechanism of the acquired resistance to IM. Therefore, it is urgent to search for additional approaches and targeting strategies to overcome IM resistance. We recently reported that nickel pyrithione (NiPT) potently inhibits the ubiquitin proteasome system via targeting the 19S proteasome-associated deubiquitinases (UCHL5 and USP14), without effecting on the 20S proteasome. In this present study, we investigated the effect of NiPT, a novel proteasomal deubiquitinase inhibitor, on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.
  
  
  
  Cell viability was examined by MTS assay and trypan blue exclusion staining assay in KBM5, KBM5R, K562, BaF3-p210-WT, BaF3-p210-T315I cells, and CML patients' bone marrow samples treated with NiPT. Cell apoptosis in CML cells was detected with Annexin V-FITC/PI and rhodamine-123 staining followed by fluorescence microscopy and flow cytometry and with western blot analyses for apoptosis-associated proteins. Expression levels of Bcr-Abl in CML cells were analyzed by using western blotting and real-time PCR. The 20S proteasome peptidase activity was measured using specific fluorogenic substrate. Active-site-directed labeling of proteasomal DUBs, as well as the phosphorylation of USP14 was used for evaluating the inhibition of the DUBs activity by NiPT. Mouse xenograft models of KBM5 and KBM5R cells were analyzed, and Bcr-Abl-related proteins and protein biomarkers related to proliferation, differentiation, and adhesion in tumor tissues were detected by western blots and/or immunohistological analyses.
  
  
  
  NiPT induced apoptosis in CML cells and inhibited the growth of IM-resistant Bcr-Abl-T315I xenografts in nude mice. Mechanistically, NiPT induced decreases in Bcr-Abl proteins, which were associated with downregulation of Bcr-Abl transcription and with the cleavage of Bcr-Abl protein by activated caspases. NiPT-induced ubiquitin proteasome system inhibition induced caspase activation in both IM-resistant and IM-sensitive CML cells, and the caspase activation was required for NiPT-induced Bcr-Abl downregulation and apoptotic cell death.
  
  
  
  These findings support that NiPT can overcome IM resistance through both Bcr-Abl-dependent and Bcr-Abl-independent mechanisms, providing potentially a new option for CML treatment.
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Lan
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Peiquan
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zang
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Jinjie
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Jinghong
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Huidan
authors.initials: H
authors.lastname: Long
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.
authors.forename: Hongbiao
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Leukemia, Section of Molecular Hematology and Therapy, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA.
authors.forename: Bing Z
authors.initials: BZ
authors.lastname: Carter
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China.|Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Vermillion, SD, 57069, USA.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China. shixp612@163.com.|Department of Pathophysiology, Protein modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 510182, People's Republic of China. shixp612@163.com.
authors.forename: Xianping
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Pathophysiology, State Key Lab of Respiratory Disease, Protein Modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 511436, China. jliu@gzhmu.edu.cn.|Department of Pathophysiology, Protein modification and Degradation Laboratory, Guangzhou Medical University, Guangzhou, Guangdong, 510182, People's Republic of China. jliu@gzhmu.edu.cn.
authors.forename: Jinbao
authors.initials: J
authors.lastname: Liu
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D016044:Fusion Proteins, bcr-abl / Q000494:pharmacology*; D064593:Heterografts; D006801:Humans; D000068877:Imatinib Mesylate / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000473:pathology* / Q000503:physiopathology; D009532:Nickel; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology*; D013871:Thiones / Q000494:pharmacology*
chemical_list: D061988:Proteasome Inhibitors; D011725:Pyridines; D013871:Thiones; C008704:pyrithione; D009532:Nickel; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl; D020169:Caspases
keywords: Apoptosis; Bcr-Abl; Chronic myelogenous leukemia; Imatinib resistance; Nickel pyrithione