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

backgroundDisulfiram (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.

methodRaji 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.

resultsIn 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.

conclusionOur 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.