Abstract 1030: Arsenic trioxide plus ethacrynic acid-butyl ester is synergistic to induce apoptosis in myeloid leukemia cells through upregulation of NOXA

Abstract

Abstract Arsenic trioxide (As2O3) induces complete remission in acute promyelocytic leukemia (APL) patients. However, other types of myeloid leukemia patients are less responsive to As2O3 treatment. APL t(15;17) NB4 cells are more sensitive to As2O3 apoptosis induction than other types of myeloid leukemia cells such as HL-60 and K562 cells. Previously we reported that As2O3 induces apoptosis in NB4 cells through a reactive oxygen species (ROS)-mediated pathway which is attenuated by the expression of glutathione-s-transferase π (GSTπ). NB4 cells contain lower levels of GSTπ than other types of myeloid leukemia cells. Therefore, inhibition of GSTπ should enhance As2O3 induction of apoptosis in other types of myeloid leukemia cells. We investigated the combined effect of As2O3 with ethacrynic acid (EA) and a more potent non-diuretic EA analogue ethacrynic acid-butyl ester (EABE), known GSTπ inhibitors, to induce apoptosis in HL-60 and K562 cells. As2O3 at clinical achievable concentrations (1-2 µM) did not induce apoptosis in HL-60 cells nor in K562 cells. EA at a concentration lower than 60 μM did not induce apoptosis in both cell lines either. As2O3 at 2 μM plus EA at 60 μM are synergistic to induce apoptosis in both HL-60 and K562 cells as measured by Annexin V staining, PARP cleavage and caspase activation. More intriguing, EABE at 1 μM (60 fold lower than EA), which inhibits GSTπ activity, exhibited synergistic induction with As2O3 in K562 cells. Combined index of As2O3 with EABE to induce apoptosis in K562 cells calculated by CompuSyn software was less than 1.0 which indicated synergism. Apoptosis induction by the combination treatment of As2O3 with EABE correlated with the upregulation of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knock-down of NOXA using siRNA blocked apoptosis induction of As2O3 plus EABE, while, knock-down of Mcl-1 enhanced the apoptosis induction of As2O3 plus EABE. Immunoprecipiation with Mcl-1 antibody revealed that NOXA bond to Mcl-1 in the K562 cells treated with As2O3 plus EABE. Inhibition of ROS production did not inhibit the upregulation of NOXA nor the apoptosis induction of As2O3 plus EABE treatment. Our data suggest that As2O3 plus EABE induce apoptosis through a novel mechanism which induces NOXA and sequesters Mcl-1 leading to the activation the intrinsic apoptotic pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1030.