Disulfiram targeting lymphoid malignant cell lines via ROS-JNK activation as well as Nrf2 and NF-κB pathway inhibition

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. Method: 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 M) of Cu2+, DS induced cytotoxicity in Raji cells with an IC50 of 0.085 +/- 0.015 mu M and in Molt4 cells with an IC50 of 0.435 +/- 0.109 mu M. 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 M)/Cu (1 mu M) 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 mu M 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. Conclusion: 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-kappa B and Nrf2 may also contribute to the DS/Cu induced apoptosis.