Role of Mitochondrial Apoptotic Pathway in Disulfiram/Copper Mixture-Induced Cell Apoptosis in Human B-Lineage Acute Lymphoblastic Leukemia in Vitro

Backgrounds: The long term prognosis of adult B-lineage acute lymphoblastic leukemia (B-ALL) is poor when compared with pediatric B-ALL. The current therapeutic regimen for adult B-ALL often results in refractory and relapsing diseases. Therefore, it is urgently needed to explore novel approaches to treat adult B-ALL. Disulfiram (DS) has been used clinically as a safe anti-alcoholism drug for over 6 decades. Recent studies demonstrated that disulfiram/cooper mixture (DS/Cu) was cytotoxic to multiple solid cancers, but its effects on B-ALL cells are still unclear. Moreover, the molecular mechanism of the cytotoxicity of DS/Cu to tumor cells was poorly defined. In this study, we investigated the effects of DS/Cu on B-ALL cells in vitro and its related cytotoxic mechanism.

Results: Firstly, CCK8 assay indicated that DS/Cu markedly inhibited Nalm6 cell proliferation in a dose-dependent manner. Secondly, colony-forming assay showed that DS/Cu also abolished the clonogenicity of Nalm6 cells (P<0.001). Thirdly, FACS analyses revealed that DS/Cu mixture could induce apoptosis of Nalm6 cells, as well as primary B-ALL cells (n=16) in a dose-dependent manner. We additionally analyzed the relationship between clinical characteristics of B-ALL patients, including age, WBC counts, immunophenotype, cytogenetics, risk stratification and Ph chromosome, with the efficacy of DS/Cu on B-ALL cells. The apoptosis isolated from pro-B and cytogenetic abnormality B-ALL pastients was higher. Therefore, our results demonstrated that DS/Cu mixture could induce significant cytotoxicity against B-ALL cells in vitro.

To decipher the cytotoxic mechanism of DS/Cu mixture, JC-1 staining was done and the results showed that DS/Cu mixture could significantly reduce the mitochondrial membrane potential in Nalm6 cells (P<0.01) and 7 cases of primary B-ALL cells (P<0.05). Consistently, Western Blot analysis showed that DS/Cu induced B-ALL cell apoptosis by down-regulating the expression of anti-apoptotic protein Bcl2 and Bcl-XL, as well as activating caspase-3 and its substrate PARP. Hence, our results indicated that DS/Cu induced apoptosis of B-ALL cells at least partly through the intrinsic mitochondrial apoptotic pathway.

Conclusion: Our results demonstrated that DS/Cu not only significantly inhibit proliferation and clonogenicity, but also induce apoptosis of B-ALL cells in vitro.The mitochondrial apoptotic pathway might be the molecular mechanism of DS/Cu-induced apoptosis of B-ALL cells.