A novel Bcl-2 small molecule inhibitor 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB)-induced apoptosis in leukemia cells.
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.
Zhang
Manchao
M
Department of Biochemistry, West Virginia University, Morgantown, WV 26506, USA. mzhang2@hsc.wvu.edu
Ling
Yan
Y
Yang
Chao-Yie
CY
Liu
Hongpeng
H
Wang
Renxiao
R
Wu
Xihan
X
Ding
Ke
K
Zhu
Feng
F
Griffith
Brian N
BN
Mohammad
Ramzi M
RM
Wang
Shaomeng
S
Yang
Dajun
D
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*
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