title: A novel Bcl-2 small molecule inhibitor 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB)-induced apoptosis in leukemia cells.
abstract: A novel small molecule inhibitor, 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB), competes with the Bak BH3 peptide to bind Bcl-2 protein with a binding affinity of IC(50) = 0.70 microM, as assessed by a fluorescence polarization based binding assay. HL-60 cells express the highest levels of Bcl-2 among the cell lines examined. Treated with 5 microM of MNB only for 6 h, 85% of HL-60 cells were detected to undergo apoptosis. Pan-caspase inhibitor, Z-VAD-FMK, blocks MNB-induced apoptosis in HL-60 cells. Caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, and PARP activation were observed at as early as 4 to 6 h of MNB treatment. In addition, it has been confirmed that the caspase-3 specific inhibitor, Z-DEVD-FMK, blocks the activation of caspase-8 in MNB-treated HL-60 cells. MNB treatment does not change Bcl-2 or Bax expression level in HL-60 cells, but causes Bid cleavage. Further experiments have illustrated that MNB inhibits the heterodimerization of Bcl-2 with Bax or Bid, reduces the mitochondrial membrane potential (DeltaPsimt), and induces cytochrome c release from mitochondria in HL-60 cells. These results suggest that MNB induces apoptosis in HL-60 by inhibiting the heterodimerization of Bcl-2 with pro-apoptosis Bcl-2 members, resulting in a decrease in the mitochondrial membrane potential and cytochrome c release, activation of caspases and PARP; it is a caspase-dependent process in which the activation of caspase-8 is dependent on the mitochondrial apoptosis signal transduction pathway. MNB prolongs the life spans of HL-60 bearing mice, potently kills fresh AML and ALL cells, indicating that it has the potential to be developed to treat leukemia.
authors.affiliation: Department of Biochemistry, West Virginia University, Morgantown, WV 26506, USA. mzhang2@hsc.wvu.edu
authors.forename: Manchao
authors.initials: M
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Ling
authors.forename: Chao-Yie
authors.initials: CY
authors.lastname: Yang
authors.forename: Hongpeng
authors.initials: H
authors.lastname: Liu
authors.forename: Renxiao
authors.initials: R
authors.lastname: Wang
authors.forename: Xihan
authors.initials: X
authors.lastname: Wu
authors.forename: Ke
authors.initials: K
authors.lastname: Ding
authors.forename: Feng
authors.initials: F
authors.lastname: Zhu
authors.forename: Brian N
authors.initials: BN
authors.lastname: Griffith
authors.forename: Ramzi M
authors.initials: RM
authors.lastname: Mohammad
authors.forename: Shaomeng
authors.initials: S
authors.lastname: Wang
authors.forename: Dajun
authors.initials: D
authors.lastname: Yang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000187:drug effects*; D053143:Caspase 2 / Q000187:drug effects*; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D004195:Disease Models, Animal; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000188:drug therapy; D051379:Mice; D008819:Mice, Nude; D010069:Oxadiazoles / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects*
chemical_list: C522711:4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D010069:Oxadiazoles; D019253:Proto-Oncogene Proteins c-bcl-2; C490360:CASP2 protein, human; D053143:Caspase 2; D003546:Cysteine Endopeptidases