BIMELis a key effector molecule in oxidative stress-mediated apoptosis in acute myeloid leukemia cells when combined with arsenic trioxide and buthionine sulfoximine
© Tanaka et al.; licensee BioMed Central Ltd. 2014
Received: 17 September 2013
Accepted: 10 January 2014
Published: 15 January 2014
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.
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.
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.
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.
KeywordsArsenic trioxide Buthionine sulfoximine Mitochondrial apoptosis BIMEL MCL1 BAX
Arsenic trioxide (ATO) has been reported to be an effective therapeutic agent in both newly diagnosed and relapsed patients with acute promyelocytic leukemia (APL) [1, 2]. This success has prompted an interest in understanding the molecular mechanisms of action underlying the clinical effectiveness of ATO. ATO is reported to induce apoptosis in leukemic promyelocytes [3, 4]. ATO-induced apoptosis appears to be dependent on the intracellular redox homeostasis. In particular, the effectiveness of ATO in inducing to apoptosis is associated with an increased generation of intracellular reactive oxygen species (ROS) [5, 6]. However, the antitumor effect of ATO is limited in other types of leukemia and solid tumor cells, since these cancer cell types have low susceptibility to ATO [7, 8]. Previous studies suggest that the ineffectiveness of ATO in ATO-resistant tumors may be due to low ROS levels, preventing the triggering of effective apoptosis [5, 9, 10]. These early studies thus provide a rationale for utilizing ATO in combination with oxidative pathway modulators to extend the use of ATO for treating non-APL malignacies. Buthionine sulfoximine (BSO), which is known to effectively deplete cellular glutathione , is used to augment ATO-induced apoptosis [12–14]. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis remains unclear. In particular, the molecular events in mitochondria involved in increased apoptotic susceptibility are unknown. In this study we investigated the detailed molecular mechanism of mitochondrial injury-mediated cell death by treating HL60 with ATO/BSO, compared with that with ATO alone. We report that the dissociation of BIMEL and MCL1 and the subsequent interaction of BIMEL and BAX play a critical role in BSO-mediated augmentation of ATO-induced apoptosis.
ATO, BSO, n-acetylcysteine (NAC), dithiothreitol (DTT), SP600125, U0126, PD035901 and SB203580 were purchased from Sigma Chemical (St. Louis, MO, USA). The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): antibodies to the cleaved form of caspase 3 (C-cas3), caspase 9 (C-cas9), poly (ADP-ribose) polymerase (C-PARP); antibodies to normal and phosphorylated forms of MCL1 (MCL1, P-MCL1 at Ser159/Thr163), BCL2 (BCL2, P-BCL2 at Ser70), BIM (BIM, P-BIM at Ser69), JNK (JNK, P-JNK at Thr183/Tyr185), c-JUN (c-JUN, P-c-JUN at Ser63), p38 (p38, P-p38 at Thr180/Tyr182) and ERK1/2 (ERK1/2, P-ERK1/2 at Thr202/Tyr204); antibodies to actin, BAD, BID and BOK. Antibodies to BAK, ASK, and normal and phosphorylated forms of BCLxL (BCLxL, P-BCLxL at Ser62) were obtained from Abcam (Cambridge, MA, USA). Antibodies to mouse and human phosphorylated forms of ASK1 (P-ASK1 at Thr845 or at Thr838)  was provided by Dr. H. Ichijo, the University of Tokyo.
The HL60 cell line, which was derived from peripheral blood cells of a 36-year old Caucasian female with APL, was obtained from ATCC (Manassas, VA, USA). Cells were maintained in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum.
Cell viability was determined using a cell proliferation kit (XTT) (Roche Applied Sciences, Rotkreuz, Switzerland) as described elsewhere . The half-maximal inhibitory concentration (IC50) was calculated using Graphpad PRISM software (GraphPad, San Diego, CA, USA). The nontoxic concentrations of various reagents were confirmed by the XTT test.
Identification of apoptotic cell death
Apoptotic cells were identified using a cell death detection kit (Roche Applied Sciences) using mouse monoclonal antibodies against fragmented DNA and histones according to the manufacturer’s instructions.
Determination of intracellular ROS level
The generation of intracellular ROS was determined using a redox-sensitive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate probe (CM-H2DCFDA) (Molecular probes, Eugene, Oregon, USA) according to the manufacturer’s instructions.
Determination of mitochondrial outer membrane permeability (MOMP)
Cells were incubated with NIR dye supplied in a NIR mitochondrial membrane potential assay kit (Abcam) and 1 μM Hoechst 33342 dye for 30 min at 37˚C. Stained cells were subjected to confocal microscopy (Leica TCS SP II, Wetzlar, Germany). The fluorescence ratio of the two dyes was determined for quantitative analysis of MOMP. The Leica confocal software, a MetaMorph ver.7.8 (Molecular Devices) was used for the analysis.
Determination of cytochrome crelease
The release of cytochrome c was determined using an ApoAlert cell fractionation kit (Clontech, Mountain View, USA). The cells were processed according to the manufacturer’s instructions and the concentration of released cytochrome c in the cytosolic fractions was determined by immunoblotting with anti-cytochrome c antibody.
Immunoprecipitation and immunoblotting analysis
Cells were lysed in CHAPS buffer (10 mM Hepes, pH7.5, 150 mM NaCl, 2% CHAPS) or RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1.0% NP-40) containing protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor cocktail I (Wako Pure Chemical, Osaka, Japan). Protein lysates (500 μg of protein) in CHAPS buffer were subjected to immunoprecipitation using an antibody to BAX 6A7 (BD Biosciences, San Jose, CA, USA), BAK Ab1 (Abcam), BIM (Cell Signaling Technology) or MCL1 (BD Pharmingen). The immunoprecipitates or whole cell lysates (10 μg of protein) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 7.5-15% gels and electroblotted onto nitrocellulose membranes (BIO-RAD, Hercules, CA, USA). Immunoblots were treated with primary and secondary antibodies and then analyzed using an ECL-Advance Western blotting kit (GE Healthcare, Little Chalfont, England). In several experiments, band intensities were quantified using a LAS4000 imaging system (GE Healthcare).
Transfection with small interfering (si) RNA
HL60 cells (1 × 106 cells) were transfected with two distinct siRNA (BIM#1 and BIM#2) specifically designed against human BIMEL, or with a control non-silencing siRNA (Silencer Select; Life Technologies, Carlsbad, CA, USA) using hemagglutinating virus supplied in a Japan-envelope vector kit (GenomONE-Si; Ishihara Sangyo, Osaka, Japan), according to the manufacturer’s instructions. The cells were used for ATO/BSO treatment 48 h after transfection with siRNA.
Experimental values are represented as the mean ± standard deviation in triplicate. The experiments were carried out at least 3 times. The significance of difference between experimental and control groups was determined by the Student’s t-test. A value of p < 0.05 was considered statistically significant.
BSO augments ATO-induced cell death via intracellular ROS generation
BSO augments ATO-induced cell death via ROS-mediated mitochondrial injury
BSO induces conformational change in BAX, but not in BAK
Immunoprecipitation analysis using an antibody to whole BAK or conformationally changed BAK demonstrated no presence of conformationally changed BAK in either ATO/BSO or ATO alone treatment (Figure 3, lower panel).
BSO induces phosphorylation of BIMELand MCL1 in mitochondria
Second, the effect of BSO addition on the expression and activation of MCL1, an anti-apoptotic protein of the BCL2 family, was examined. BSO addition augmented the expression and phosphorylation of MCL1 at Ser159 and/or Thr163, whereas ATO alone did it only minimally (Figure 4C). The BSO-mediated augmentation of MCL1 expression and phosphorylation was abolished by antioxidants. Similar augmentation was seen in phosphorylation of BCLXL (Figure 4C). In addition, there was no significant difference in the BCL2 expression in ATO/BSO treatment in the presence or absence of antioxidants (Figure 4C).
BSO induces the dissociation of phosphorylated BIMELfrom MCL1
BSO induces the interaction of phosphorylated BIMELwith BAX
Since BIM promotes apoptosis through binding directly to BAX and inducing conformational changes [24, 25], the interaction between BIMEL dissociated from MCL1 and BAX in ATO/BSO treatment was examined using immunoprecipitation. As shown in Figure 5A, BSO reduced the amount of non-phosphorylated (basal) BIMEL and increased the amount of BIMEL slower migrating forms (phosphorylated BIMEL) in cell lysate (Figure 5B, left panel). The BIMEL slower migrating form was detected in immunoprecipitates of BAX in ATO/BSO-treated cells but few in ATO alone-treated cells (Figure 5B, right panel). To confirm the interaction between BAX and phosphorylated BIMEL, BAX immunoprecipitates were analyzed by immunoblotting with an anti-phosphorylated BIMEL antibody (Figure 5B, right panel). Phosphorylated BIMEL was detected in BAX immunoprecipitates but not in ATO-treated cells. BSO was suggested to augment the interaction between phosphorylated BIMEL and BAX.
Silencing of BIMELwith si RNA abolishes ATO/BSO-induced cell death
BSO triggers phosphorylation of MCL1 and BIMELvia activation of JNK
BSO triggers activation of ASK1 and JNK and induces phosphorylation of BIMELand MCL1
In the present study, we have demonstrated that BSO augments ATO-induced cell death in HL60 cells and that the augmentation is responsible for ROS-mediated mitochondrial apoptosis. The detailed molecular mechanism of BSO-mediated mitochondrial injury was studied by comparing ATO cell death in the presence or absence of BSO. We here report that BSO augments intracellular ROS production in mitochondria and induces a series of molecular events, such as conformational change of BAX, phosphorylation and dissociation of BIMEL and MCL1, and interaction of BIMEL and BAX.
Previously several groups showed that BSO decreased the levels of glutathione and enhanced ATO-induced apoptosis [29, 30]. Chen et al. reported that ATO/BSO induced apoptosis in ATO-sensitive and insensitive leukemia cells through activation of JNK, which up-regulated death receptor (DR) 5 and the caspase 8 pathway . However, they did not report the accumulation of ROS in ATO/BSO-induced apoptosis, nor the associated molecular events occurring in mitochondria. We have demonstrated that ATO/BSO induces the dissociation of BIMEL from MCL1, and that its interaction with BAX plays a critical role in ATO/BSO-induced apoptosis via conformational changes in BAX. Our results demonstrate that BSO causes ROS-mediated mitochondrial injury, accompanied by cytochrome c release and reduced MOMP. This is the first report showing the involvement of ROS-mediated mitochondrial injury in BSO-mediated augmentation of ATO-induced apoptosis. Moreover, we show the pivotal role played by the pro-apoptotic molecule, BIMEL, in ATO/BSO-induced apoptosis, and confirm it by the finding that knockdown of BIMEL abolishes ATO/BSO-induced apoptosis. The remarkable behavior of pro-apoptotic BIMEL in mitochondria provides new insights into the molecular mechanism of ATO/BSO-induced apoptosis. Pro-apototic effects are reported to be associated with BIML activation . However, we could not confirm the activation of BIML in this study. Rather, the role of BIMEL might be more important than that of BIML, although we do not exclude the involvement of BIML in ATO/BSO-induced apoptosis.
The critical role of ASK1 in apoptosis induction has been reported [36–38]. We have found that BSO triggers activation of ASK1 in ATO-treated cells. The involvement of ASK1 activation in ATO/BSO-induced apoptosis is confirmed by using pharmacological inhibitors, although it must be confirmed by more specific technique. Yan et al.  reported that ASK1 is activated by ATO through ROS accumulation, and that it negatively regulates apoptosis in leukemia cells without activating JNK and p38. In contrast, our results clearly show that ASK1 activated by BSO causes the activation of JNK and p38. The difference between the two studies might be due to excessive ROS generation in response to ATO/BSO. ASK1 is a member of the MAPK kinase kinase family and activates JNK and p38 MAPKs in response to an array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx . It is reasonable that BSO activates ASK1 via oxidative stress and then activates JNK and p38. Inhibition of p38 with a pharmacological inhibitor induces the activation of caspase 3 and PARP in ATO/BSO-induced apoptosis, suggesting negative feedback of p38 against ATO/BSO-induced apoptosis. The precise role of ASK1 and MAPKs in ATO/BSO-mediated apoptosis must await further characterization.
ATO/BSO combined treatment induces ROS-mediated mitochondrial apoptosis in HL60 cells. ATO/BSO-induced mitochondrial apoptosis is caused by successive BIMEL alterations consisting of phosphorylation, dissociation from MCL1, and interaction with BAX. The enhancing effect of BSO on ATO-induced apoptosis was characterized at the molecular level for clinical use.
A combined treatment of ATO and BSO
Reactive oxygen species
Acute promyelocytic leukemia
Acute myeloid leukemia
BCL2-interacting mediator of cell death-extra long protein
Myeloid cell leukemia-1 protein
BCL2-assocated X protein
BCL2-associated death promoter protein
BH3-interacting domain death agonist
B cell lymphoma 2 protein
BCL2-like X protein
c-JUN N-terminal kinase
Extracellular signal-regulated kinase 1/2
Apoptosis signal-regulating kinase 1.
We thank Dr. H. Ichijo for kindly providing anti-phopho-ASK1 antibody. We thank Dr. M. Urasaki for excellent technical advice. Ms. T. Sugino provided outstanding technical assistance.
Role of the funding source
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (23590147, 2011).
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