title: Disulfiram targeting lymphoid malignant cell lines via ROS-JNK activation as well as Nrf2 and NF-kB pathway inhibition.
abstract: BACKGROUND
  Disulfiram (DS), an anti-alcoholism drug, demonstrates strong antitumor activity in a copper (Cu)-dependent manner. This study investigates the cytotoxicity of DS/Cu complex in lymphoid malignant cell lines in vitro and in vivo.
  
  
  METHODS
  Raji cells were subjected to different treatments and thereafter MTT assay, flow cytometry were used to determine IC50 and apoptotic status. We also tested the cytotoxicity of DS/Cu in acute lymphoblastic leukemia cell line Molt4 in vitro. In vivo experiments were also performed to demonstrate the anticancer efficacy of DS/Cu in Raji cells xenografted nude mice.
  
  
  RESULTS
  In combination with a low concentration (1 μM) of Cu2+, DS induced cytotoxicity in Raji cells with an IC50 of 0.085 ± 0.015 μM and in Molt4 cells with an IC50 of 0.435 ± 0.109 μM. The results of our animal experiments also showed that the mean tumor volume in DS/Cu-treated mice was significantly smaller than that in DS or control group, indicating that DS/Cu inhibits the proliferation of Raji cells in vivo. DS/Cu also induced apoptosis in 2 lymphoid malignant cell lines. After exposure to DS (3.3 μM)/Cu (1 μM) for 24 hours, apoptosis was detected in 81.03 ± 7.91% of Raji cells. DS/Cu induced significant apoptosis in a concentration-dependent manner with the highest apoptotic proportion (DS/Cu: 89.867 ± 4.69%) at a concentration of 2 μM in Molt4 cells. After 24 h exposure, DS/Cu inhibits Nrf2 expression. Flow cytometric analysis shows that DS/Cu induced ROS generation. DS/Cu induced phosphorylation of JNK and inhibits p65 expression as well as Nrf2 expression both in vitro and in vivo. N-acetyl-L-cysteine (NAC), an antioxidant, can partially attenuate DS/Cu complex-induced apoptosis and block JNK activation in vitro. In addition, NAC is able to restore Nrf2 nuclear translocation and p65 expression.
  
  
  CONCLUSIONS
  Our study manifests that DS/Cu complex targets lymphoid malignant cells in vitro and in vivo. Generation of ROS might be one of core steps in DS/Cu induced apoptosis. Moreover, ROS-related activation of JNK pathway and inhibition of NF-κB and Nrf2 may also contribute to the DS/Cu induced apoptosis.
authors.forename: Jie
authors.initials: J
authors.lastname: Zha
authors.forename: Feili
authors.initials: F
authors.lastname: Chen
authors.forename: Huijuan
authors.initials: H
authors.lastname: Dong
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Shi
authors.forename: Yao
authors.initials: Y
authors.lastname: Yao
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Rongwei
authors.initials: R
authors.lastname: Li
authors.forename: Shiyun
authors.initials: S
authors.lastname: Wang
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Weiguang
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. xubingzhangjian@126.com.
authors.forename: Bing
authors.initials: B
authors.lastname: Xu
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D004221:Disulfiram / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; C495635:NFE2L2 protein, human; D017382:Reactive Oxygen Species; D048670:MAP Kinase Kinase 4; D004221:Disulfiram