BIMEL is a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine

Abstract

Background: Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death.

Methods: HL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression.

Results: HL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO.

Conclusions: The enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.

Figure 1

BSO augments ATO-induced cell death via intracellular ROS generation. A, HL60 cells were treated with ATO (3 μM) or ATO/BSO (3 μM/50 μM) in the presence or absence of NAC (5 mM) or DTT (0.6 mM) for 12 h. Cell viability was determined using a XTT assay. *p < 0.05, Treatment with ATO in the presence of NAC or DTT treatment vs. treatment with ATO in the absence of NAC and DTT; **p < 0.01, Treatment with ATO/BSO in the presence of NAC or DTT treatment vs. treatment with ATO/BSO in the absence of NAC and DTT. B, Cells were treated with ATO or ATO/BSO for 6 h. The levels of intracellular ROS were monitored. *p < 0.05, Treatment with ATO vs. treatment with none; **p < 0.01, Treatment with ATO/BSO vs. treatment with none.

Figure 2

BSO augments ATO-induced cell death via ROS-mediated mitochondrial injury. A, After treatment as Figure 1A, the release of cytochrome c and the cleavage of caspase 9 were determined by immunoblotting. B, MOMP with NIR dye and Hoechst staining was analyzed by confocal microscopy. Magnification ×40. The results are presented as % untreated cells with SD. A typical result of 3 independent experiments is shown.

Figure 3

BSO induces conformational change in BAX, but not BAK. The conformational change was detected using immunoprecipitation and immunoblotting with an antibody to conformationally changed BAX or BAK. HL60 cells were treated with ATO/BSO or ATO for 12 h in the presence or absence of DTT. Actin and IgG light chain (IgG(L)) were used as the controls. A typical result of 3 independent experiments is shown. IP, immunoprecipitation; Ppt, immunoprecipitate; Sup, supernatant.

Figure 4

BSO induces phosphorylation of BIM_EL and MCL1 in mitochondria. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NAC or DTT for 12 h. A. The expression and phosphorylation of BIM were determined by immunoblotting. The lower panel indicates a longer exposure of the same blot. B, C. The expression and phosphorylation of BAD, BID, tBID, MCL1, BCLxL and BCL2 were determined by immunoblotting.

Figure 5

BSO induces the dissociation of phosphorylated BIM_EL from MCL1 and the interaction with BAX. A and B, The dissociation of phosphorylated BIM_EL and MCL1, and the interaction with BAX were determined by immunoprecipitation and immunoblotting with antibodies to their normal and phosphorylated forms. Values were normalized to actin or IgG(L), respectively and represent relative changes compared with control. A typical result of 3 independent experiments is shown. IP, immunoprecipitation; Ppt, immunoprecipitate; Sup, supernatant.

Figure 6

Silencing of BIM_EL with si RNA abolishes ATO/BSO-induced cell death. The expression of BIM_EL and cleavage of caspase 3 and PARP were determined by immunoblotting. HL60 cells were transfected with two siRNAs designed against BIM_EL (BIM#1 and BIM#2) or control siRNA, incubated for 48 h, and treated with ATO/BSO for 12 h. A typical result of 3 independent experiments is shown. IP, immunoprecipitation; Ppt, immunoprecipitate.

Figure 7

BSO triggers phosphorylation of MCL1and BIM_EL via activation of JNK. A, The phosphorylation of JNK, ERK1/2 and p38 was determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NAC or DTT for 12 h. B, The phosphorylation of BIM_EL and MCL1, and the cleavage of caspase 3 and PARP, were determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence of SP600125 (10 μM) as a JNK inhibitor, U0126 (2 μM) as an ERK1/2 inhibitor, or SB203580 (10 μM) as a p38 inhibitor, PD035901 (100 nM)as a MEK1/2 inhibitor for 12 h. A typical result of 3 independent experiments is shown.

Figure 8

BSO triggers activation of ASK1 and JNK and induces phosphorylation of BIM_EL and MCL1. A, The phosphorylation of ASK1 was determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NAC or DTT for 12 h. B, The phosphorylation of JNK, BIM_EL, and MCL1, and cleavage of caspase 3 and PARP, were determined by immunoblotting. HL60 cells were treated with ATO/BSO or ATO in the presence or absence of NQDI1 (10 μM) for 12 h. A typical result of 3 independent experiments is shown.

Figure 9

The putative molecular events occurring in mitochondria during ATO/BSO-induced apoptosis.