| Background : Acute myeloid leukemia(AML)is an aggressive hematologic malignancy with an incidence of 19.4/100,000 in the elderly population,and the incidence of AML will further increase with the aging of China’s population.Traditional AML treatment is based on chemotherapy regimens,and the development of targeted drugs has brought AML to a new era of targeted therapy.Venetoclax,a Bcl-2 inhibitor,has received great attention because it is not limited to specific mutations and targets the mitochondrial apoptosis pathway.However,Venetoclax has limited efficacy alone and is effective in combination with other chemotherapeutic agents,so combinations are generally recommended to allow patients to achieve deeper remission.Disulfiram has been in clinical use for 70 years,and several studies in recent years have demonstrated antitumor activity in a variety of cancers,including leukemia.Given that both Disulfiram and Venetoclax have apoptosis-inducing effects in leukemia cells,it is necessary to clarify whether these two drugs have synergistic anti-AML effects and provide new therapeutic options for targeted AML therapy.Objective: The aim of this study was to investigate the effects of Disulfiram and Venetoclax alone and in combination on the viability and apoptosis levels of AML cells,to clarify whether the combination of the two drugs has a synergistic effect,to further explore the synergistic mechanism of the two drugs,and to provide new therapeutic strategies for the treatment of AML.Methods: Disulfiram and Venetoclax were used alone or in combination in AML cell lines(OCI-AML3,OCI-AML2,HL-60,MOLM-13,MV4-11)and AML primary cells,and cell viability was measured by Cell Titer-Lumi,cell apoptosis,mitochondrial membrane potential,intracellular reactive oxygen species(ROS)production by flow cytometry.The expression levels of apoptotic proteins PARP and c-PARP and anti-apoptotic proteins Bcl-2,Mcl-1 and Bcl-x L were measured by Western blot.Results:(1)Both Disulfiram and Venetoclax were cytotoxic to the five AML cell lines in a dosedependent manner.(2)The combination of Disulfiram and Venetoclax could inhibit cell proliferation,and Compu Syn software calculated the combination index(CI)of the two drugs,and the results showed that the CI of the combination in two drugs was <1,and the two drugs had a synergistic effect.(3)After selecting appropriate concentrations,the apoptosis rate was detected by flow cytometry after 48 h of single drug and combination,and the experimental results showed that both single drugs could lead to apoptosis,and the apoptosis rate was further enhanced after the combination.(4)Primary cells from patients with diagnosed AML were collected,and appropriate concentrations of single drug and combination were selected to act on primary cells,and the results showed that cell proliferation was further inhibited by the combination of Disulfiram in Venetoclax-sensitive and Venetoclax-resistant primary cells.(5)The combination of Disulfiram and Venetoclax leads to increased intracellular ROS production and decreased cellular mitochondrial membrane potential,resulting in cell apoptosis.(6)Western blot assay showed that Disulfiram combined with Venetoclax resulted in increased expression of apoptotic proteins and decreased expression of anti-apoptotic proteins Bcl-2,Mcl-1,and Bcl-x L.Conclusion:In this study,we firstly verified that Disulfiram and Venetoclax alone have dose-dependent toxicity to AML cell lines,and Disulfiram combined with Venetoclax has synergistic effects in AML cell lines and primary cells,and the apoptosis rate is further enhanced by the combination of the two drugs compared with that of the single drug,and the combination of the two drugs induces ROS generation,decreases mitochondrial membrane potential,and increases apoptotic protein expression and decreases anti-apoptotic protein expression. |
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