A novel Bcl-2 small molecule inhibitor 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB)-induced apoptosis in leukemia cells
- 131 Downloads
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.
KeywordsBcl-2 Small molecule Apoptosis Leukemia
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.
- 5.Salomons GS, Smets LA, Verwijs-Janssen M, Hart AA, Haarman EG, Kaspers GJ, Wering EV, Der Does-Van Den Berg AV, Kamps WA (1999) Bcl-2 family members in childhood acute lymphoblastic leukemia: relationships with features at presentation, in vitro and in vivo drug response and long-term clinical outcome. Leukemia 13:1574–1580PubMedCrossRefGoogle Scholar
- 11.Campos L, Sabido O, Liang H, Vasselon C, Guyotat D (1996) Expression of human Bcl-xL, an inhibitor of programmed cell death. Nature 33:335–341Google Scholar
- 19.Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ, Joseph MK, Kitada S, Korsmeyer SJ, Kunzer AR, Letai A, Li C, Mitten MJ, Nettesheim DG, Ng S, Nimmer PM, O’Connor JM, Oleksijew A, Petros AM, Reed JC, Shen W, Tahir SK, Thompson CB, Tomaselli KJ, Wang B, Wendt MD, Zhang H, Fesik SW, Rosenberg SH (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435:677–681PubMedCrossRefGoogle Scholar
- 30.Wieder T, Essmann F, Prokop A, Schmelz K, Schulze-Osthoff K, Beyaert R, Dorken B, Daniel PT (2001) Activation of caspase-8 in drug-induced apoptosis of B-lymphoid cells is independent of CD95/Fas receptor-ligand interaction and occurs downstream of caspase-3. Blood 97:1378–1387PubMedCrossRefGoogle Scholar
- 43.Becattini B, Culmsee C, Leone M, Zhai D, Zhang X, Crowell KJ, Rega MF, Landshamer S, Reed JC, Plesnila N, Pellecchia M (2006) Structure–activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid. Proc Natl Acad Sci U S A 103:12602–12606PubMedCrossRefGoogle Scholar
- 47.Marsden VS, O’Connor L, O’Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K, Tomaselli KJ, Roy S, Nicholson DW, Vaux DL, Bouillet P, Adams JM, Strasser A (2002) Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature 419:634–637PubMedCrossRefGoogle Scholar