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A novel Bcl-2 small molecule inhibitor 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB)-induced apoptosis in leukemia cells

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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 IC50 = 0.70 μM, 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 μM 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 (ΔΨmt), 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.

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Acknowledgements

This work was supported in part by Department of Defense. The authors thank Dr. Karen Cresewell of the Lombardi Cancer Center, Georgetown University for operating FACScan and Ms. Karen Kreutzer of the University of Michigan Medical School for proofreading the original manuscript.

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Authors and Affiliations

  1. Department of Biochemistry, West Virginia University, Morgantown, WV, 26506, USA

    Manchao Zhang

  2. Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC, 20007, USA

    Yan Ling & Feng Zhu

  3. University of Michigan Medical School, Ann Arbor, MI, 48109, USA

    Chao-Yie Yang, Hongpeng Liu, Renxiao Wang, Xihan Wu, Ke Ding, Shaomeng Wang & Dajun Yang

  4. West Virginia School of Osteopathic Medicine, Lewisburg, WV, 24901, USA

    Brian N. Griffith

  5. Karmanos Cancer Institute, Wayne State University, Detroit, MI, 48202, USA

    Ramzi M. Mohammad

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  1. Manchao Zhang
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Correspondence to Manchao Zhang.

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Zhang, M., Ling, Y., Yang, CY. et al. A novel Bcl-2 small molecule inhibitor 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB)-induced apoptosis in leukemia cells. Ann Hematol 86, 471–481 (2007). https://doi.org/10.1007/s00277-007-0288-4

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  • DOI: https://doi.org/10.1007/s00277-007-0288-4

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