Mechanism Of Arenobufagin(ARE)-Induced Apoptosis In Acute Leukemia Cells Through Impairing Mitochondrial Function

BackgroundLeukemia is a disease characterized by the malignant clonal proliferation of bone marrow hematopoietic stem cells,and is known for its high lethality and relapse rate,which is higher in children and in elderly patients over 60 years of age.Leukemia is classified as myeloid leukemia and lymphoblastic leukemia.Leukemia cells in the bone marrow proliferate with impaired normal haematopoiesis,extensively infiltrating the liver,spleen,lymph nodes and other organs,mainly manifesting as anaemia,bleeding,infection and other signs.The treatment of leukemia is mainly based on chemotherapy and bone marrow transplantation,and in recent years the development and use of CAR-T technology has provided a new strategy for leukemia treatment.At present,bone marrow transplantation is difficult to match,the source is limited,the treating period is long,and the side effects caused by suppression of immunity to prevent rejection after transplantation seriously affect the quality of survival of patients;CAR-T therapy is technically difficult and expensive,the long-term side effects and adverse reactions are unknown,and the change in overall survival of patients is unknown,making its clinical application very limited.Therefore,the development and application of inexpensive and high-quality new drugs for leukemia remains particularly important,which makes our study full of clinical practicality and application value.Arenobufagin(ARE),one of the active ingredients isolated from the venom secreted from the postauricular and skin glands of the toad Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider,not only acts as a potent Na~+-K~+-ATPase inhibitor that plays a digitalis-like role in the regulation of myocardial cell inotropy,but also inhibits the proliferation and metastasis of a variety of cancer cells,and yet the mechanism of its anti-leukemia effect has not been elucidated.In this study we will investigate the molecular mechanism of ARE-induced apoptosis in acute leukemia cells,and provide a scientific basis for the clinical development of acute leukemia treatment with ARE.ObjectivesTo elucidate the molecular mechanism of ARE-induced apoptosis in acute leukemia Jurkat and MV-4-11 cells through impairing mitochondrial function,providing theoretical and experimental basis for the development of ARE as a clinical therapeutic agent for acute leukemia.MethodsHuman acute leukemia Jurkat and MV-4-11 cells were selected for in vitro experiments.Cell survival was measured by MTT,apoptosis by flow cytometry,apoptosis and mitochondrial damage related protein expression by Western blot,co-localization of BAX/Drp1 with mitochondria by Co IP,mitochondrial morphology by transmission electron microscopy and activity of mitochondrial respiratory chain complex by chemical methods.Results1.ARE inhibits proliferation and induces apoptosis in leukemia cellsMTT assay revealed that ARE inhibited the proliferation of different types of leukemia cells,with significant effects on Jurkat and MV-4-11(P<0.01).Flow cytometry showed that ARE induced apoptosis in Jurkat and MV-4-11(P<0.01),Western blot assay confirmed that ARE caused degradation of apoptosis-related protein PARP1,activation of Caspase 3 and Caspase 7,and downregulation of anti-apoptotic protein XIAP.2.ARE triggers mitochondrial damageTransmission electron microscopy results indicated that mitochondria of Jurkat and MV-4-11 cells showed swelling,irregular fracture and cristae distortion after ARE treatment.Flow cytometry showed that treatment with ARE resulted in decreases in the mitochondrial membrane potential in Jurkat and MV-4-11 cells(P<0.01).Western blot assay clearly showed that ARE treatment triggered mitochondrial damage,caused BAX translocation from cytoplasm to mitochondria and increased AIF and Cyto C release from mitochondria to cytoplasm.3.ARE activates the Rho A/ROCK1 pathway and promotes the translocation of BAX to mitochondria,triggering mitochondrial damage and leading to apoptosisWestern blot showed that ARE treatment activated the Rho A/ROCK1 signaling pathway in Jurkat and MV-4-11 cells,promoted BAX translocation to mitochondria,Cyto C and AIF release into the cytoplasm,increased Caspase 3,Caspase 7 and PARP1 cleavage,and decreased XIAP expression in dose-and time-dependent manners(P<0.01).Pretreatment with the ROCK1 inhibitor Y-27632 significantly attenuated ARE-induced mitochondrial translocation of BAX,release of Cyto C and AIF into the cytoplasm,cleavage of Caspase 3,Caspase 7 and PARP1,downregulation of XIAP,as well as induction of apoptosis(P<0.01).4.ARE activates the AMPK/Drp1 pathway and inhibits mitochondrial respiratory chain complex enzyme activity,triggering impaired mitochondrial energy metabolism and leading to apoptosisWestern blot assay showed that ARE treatment increased AMPK(T172)phosphorylation in Jurkat and MV-4-11 cells,decreased phosphorylation of Drp1(S637),and induced Drp1 translocation to mitochondria in dose-and time-dependent manners(P<0.01).Pretreatment with the AMPK agonist AICAR enhanced ARE-mediated dephosphorylation of Drp1(S637)and mitochondrial translocation of Drp1,while pretreatment with the AMPK inhibitor DM attenuated ARE-mediated dephosphorylation of Drp1(S637)and mitochondrial translocation of Drp1;however,pretreatment with the Drp1inhibitor Mdivi-1 had no effect on AMPK phosphorylation induced by ARE.Additional experimental results suggested that ARE treatment reduced mitochondrial respiratory chain complex enzyme activity and decreased mitochondrial ATP production in Jurkat and MV-4-11 cells,triggering impaired mitochondrial energy metabolism and leading to apoptosis(P<0.01).Pretreatment with the AMPK agonist AICAR enhanced ARE-mediated inhibition of mitochondrial respiratory chain complex enzyme activity and ATP production and induction of apoptosis(P<0.01).Pretreatment with both the AMPK inhibitor DM and the Drp1 inhibitor Mdivi-1 attenuated ARE-mediated inhibition of mitochondrial respiratory chain complex enzyme activity and ATP production and induction of apoptosis(P<0.01).ConclusionARE induces apoptosis in acute leukemia Jurkat and MV-4-11 cells through impairing mitochondrial function.ARE activates the Rho A/ROCK1 signaling pathway,resulting in mitochondrial translocation of BAX,leading to mitochondrial damage and apoptosis;ARE also activates the AMPK/Drp1 pathway,which inhibits mitochondrial respiratory chain complex enzyme activity and reduces mitochondrial ATP production,resulting in impaired mitochondrial energy metabolism,and leading to mitochondrial damage and apoptosis.