title: BIMEL is a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine.
abstract: BACKGROUND
  Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death.
  
  
  METHODS
  HL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression.
  
  
  RESULTS
  HL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO.
  
  
  CONCLUSIONS
  The enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, Aichi Medical University, 1-1 Yazako-Karimata, Nagakute, Aichi, Japan. koma@aichi-med-u.ac.jp.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Komatsu
authors.forename: Hiroko
authors.initials: H
authors.lastname: Shigemi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamauchi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Fujii
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000008:administration & dosage*; D000072224:Bcl-2-Like Protein 11; D019328:Buthionine Sulfoximine / Q000008:administration & dosage*; D004338:Drug Combinations; D055785:Gene Knockdown Techniques / Q000379:methods; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010087:Oxides / Q000008:administration & dosage*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D001152:Arsenicals; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D004338:Drug Combinations; D008565:Membrane Proteins; D010087:Oxides; D011518:Proto-Oncogene Proteins; D019328:Buthionine Sulfoximine; D000077237:Arsenic Trioxide