Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

Jingjing Zhang,1,* Huayun Geng,2,* Ling Liu,1 Hao Zhang1 1Department of Hematology, Affiliated Hospital of Jining Medical University, Jining 272029, Shandong Province, China; 2Department of Hematology, Dongchangfu People’s Hospital of Liaocheng, Liaocheng 252000, Shandong Province, China *These authors contributed equally to this work Background/Aims: Cytotoxicity induced by reactive oxygen species (ROS) is critical for the effectiveness of chemotherapeutic drugs used in the treatment of acute myeloid leukemia (AML). This study aimed to investigate whether ROS contributes to cytotoxicity in AML cells when treated with homoharringtonine (HHT) and etoposide (ETP) in combination.Methods: AML cell lines THP1 and HL60 and primary AML cells from patients were treated with HHT and ETP alone or in combination, and cell viability was determined by trypan blue exclusion test, and apoptosis was analyzed by annexin-V/propidium iodide double staining as well as Western blot for measuring expression of cleaved caspase-9 and cleaved caspase-3. Intracellular ROS level was detected by DCFH-DA fluorescence assay, and N-Acetyl-L-cysteine (NAC) was used to scavenge intracellular ROS. Retroviral infection was applied to mediate stable overexpression in AML cells.Results: We show that HHT and ETP exhibit synergistic cytotoxicity in AML cell lines and primary AML cells in vitro, and meanwhile, HHT causes elevated ROS generation in ETP-treated AML cells. We next reveal that the elevated ROS is a critical factor for the synergistic cytotoxicity, since ROS scavenge by NAC remarkably diminishes this effect. Mechanistically, we demonstrate that HHT causes elevated ROS generation by disabling thioredoxin-mediated antioxidant defense. Finally, similar to HHT treatment, depletion of thioredoxin sensitizes AML to ETP treatment.Conclusion: These results provide the foundation for augmenting the efficacy of ETP in treating AML with HHT, and also highlight the importance of targeting ROS in improving treatment outcome in AML. Keywords: homoharringtonine, etoposide, synergistic cytotoxicity, acute myeloid leukemia, antioxidant defense, reactive oxygen species, thioredoxin