High concentrations (greater than 5 μ M) of arsenic trioxide (As2O3) have been reported to be able to induce apoptosis in several malignant cells. We explored cell lines in which apoptosis was induced with a therapeutic concentration (1–2 μ M) of As2O3, and found that 1 μ M of As2O3 induced apoptosis in the NKM-1 cell line, which was established from a patient with acute myeloid leukemia (M2). Apoptosis induced by 1 μ M of As2O3 in NKM-1 cells was accompanied by an increased cellular content of H2O2, a decreased mitochondrial membrane potential (Δψm), and activation of caspase-3. C-Jun-terminal kinase (JNK) was activated only in NKM-1 cells and arsenic-sensitive NB4 cells, but not in arsenic-insensitive HL-60 cells. Activation of JNK in NKM-1 was sustained from 6 to 24 h after As2O3 treatment, and preceded changes in cellular H2O2, Δψm, and caspase-3 activation. Moreover, addition of a JNK inhibitor reduced the percentage of apoptotic cells after the As2O3 treatment. Taken together, in the M2 cell line NKM-1, 1 μ M of As2O3 induced sustained activation of JNK and apoptosis. This finding may provide a basis to select a subgroup other than acute promyelocytic leukemia, which can benefit from As2O3 treatment.
Arsenic trioxide (As2O3) has been shown to be effective for relapsed acute promyelocytic leukemia (APL) patients after all-trans retinoic acid (ATRA) therapy and conventional chemotherapy have failed.1,2,3,4 In all, 85% of relapsed APL patients achieved a complete remission with As2O3 alone safely; also, the 18-month overall and relapse-free survivals were 66 and 56%, respectively.5 The mechanisms of action of As2O3 differ from those of ATRA. ATRA induces differentiation in APL cells, whereas As2O3 mainly induces apoptosis.2 Up to now, it has been reported that As2O3 can induce apoptosis in several malignant cells including APL cell lines. As2O3-induced apoptosis has been executed mainly by three mechanisms: caspases, reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPKs).6,7,8,9,10,11,12,13,14
Caspases, a family of cysteine aspartic acid proteases, are central regulators of apoptosis. Caspases are activated by cleavage; in turn, activated caspases cleave downstream substrates including other caspases, cytoskeletal and nuclear proteins, and thereby induce apoptosis. ROS, including the superoxide anion, hydrogen peroxide (H2O2), hydroxyl radical, nitric oxide, and peroxynitrite have been implicated in the etiology of a large number of diseases, such as cancer, inflammation, aging, and diabetic complications.15,16 Recently, generation of ROS has been reported to regulate As2O3-induced apoptosis.17,18,19 The ability of As2O3 to induce apoptosis in leukemic cells is dependent on the activity of enzymes that regulate the cellular H2O2 content.20,21 The MAPK family is involved in signal transduction of apoptosis as well as cell growth and differentiation.22,23 Two different MAPK cascades that converge on c-Jun N-terminal kinase (JNK; also known as SAPK, stress-activated protein kinase) and p38MAP kinase are activated preferentially by cytotoxic stressors and by proinflammatory cytokines.24,25,26 However, these studies were performed using concentrations greater than 5 μ M, often 50 μ M of As2O3, depending on the cell lines used; therefore, the relevance to therapeutic levels (1–2 μ M) remains to be determined.
NKM-1 was established from a patient with acute myeloid leukemia (AML, FAB classification M2). The cells were positive for myeloperoxidase staining, and proliferated in response to exogenous granulocyte-colony-stimulating factor or macrophage-colony-stimulating factor in a dose-dependent manner, while interleukin-3 or granulocyte–macrophage-colony-stimulating factor had no effect.27 We found that 1 μ M of As2O3 induced apoptosis in NKM-1 cells. Sustained activation of JNK and inactivation of ERK were involved in As2O3-induced apoptosis in NKM-1 cells, while no activation of JNK was observed in leukemia cell lines which were less sensitive to As2O3. These findings indicated that sustained JNK activation is closely related to arsenic-induced apoptosis.
Materials and methods
Cells and viability
NKM-1 (AML (M2)), NB4 (APL), HL-60 (AML), KASUMI-1 (AML (M2)), and K562 (chronic myeloid leukemia (CML)) were maintained in RPMI 1640 (GIBCO BRL, Life Technologies, NY, USA) with 10% heat-inactivated fetal calf serum (FCS) at 37°C in a humidified atmosphere containing 5% CO2. Cell viability and proliferation were determined by trypan blue dye exclusion assay. The cells were mixed with an equal volume of phosphate-buffered saline (PBS) containing 0.05% trypan blue dye, and manually counted in triplicate.
2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was from Acros Organics (Piscataway, NJ, USA). 3,39-Dihexy-loxacarbocyanine (DiOC6) was obtained from Molecular Probes (Interchim, France). Carbonyl cyanide m-chlorophenylhydrazone (mCl-CCP) was purchased from Sigma (St Louis, MO, USA). (L)-JNKI1 (JNK inhibitor-1) was purchased from Calbiochem (Bad Soden, Germany).
Apoptosis assessment by annexin-V staining
After drug treatment, 1 × 106 cells were washed in PBS, and resuspended in 125 μl staining solution containing annexin-V-fluorescein and PI in a HEPES buffer (annexin-V-FLUOS Staining Kit; Roche, Japan), following the manufacturer's instructions. After a 15-min incubation at room temperature, the cells were analyzed by FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
Western blotting analysis
The cells were lysed by adding an equal volume of a two-fold concentrated sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol); then, protein samples were subjected to 8–12% sodium dodecyl sulfate-polyacrylamide gel electorophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membrane was incubated with primary antibodies as follows: antiprocyclic acid repetitive protein (PARP), antiphosphospecific p38 (Thr180/Tyr182), anti-p38, antiphosphospecific SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, antiphospho-specific ERK1/2 (Ser217/221), anti-ERK1/2, antiphospho-specific AKT, and anti-AKT, which were all purchased from Cell Signaling (Beverly, MA, USA). Primary antibodies were detected by horseradish peroxidase-conjugated secondary antibody (1:2000), and were visualized by chemiluminescence (ECL; Amersham, Buckinghamshire, UK). For reprobing, the membranes were stripped (2% SDS, 62.5 mM Tris, pH 6.8, 100 mM 2-ME, 50°C, 30 min) and reprobed with the corresponding antibodies.
Measurement of H2O2 production
Production of H2O2 was detected using DCFH-DA, an uncharged cell-permeable fluorescent probe. Inside the cells, DCFH-DA is cleaved by nonspecific esterases forming DCFH, which is the nonfluorescent form, and is oxidized to the fluorescent compound 28,78-dichlorofluorescein (DCF) in the presence of H2O2.28 Exponentially growing cells (1 × 105 cells/ml) were labeled with 0.5 μmol/l DCFH-DA for 1 h, and then incubated in the absence or presence of As2O3 at 37°C for various periods of time. After washing with PBS, 10 000 cells were analyzed in every sample by FACScan (Becton Dickinson), with excitation and emission settings of 495 and 525 nm, respectively.29
Flow-cytometric analysis of mitochondrial membrane potential (Δψm)
To evaluate the mitochondrial transmembrane potential (Δψm), the cells were incubated for 15 min at 37°C in PBS containing 40 nM DiOC6.30 After washing with PBS, 10 000 cells were analyzed in every sample by flow cytometry. In control experiments, the cells were labeled after preincubation with mCl-CCP (50 μ M, 37°C, 30 min). Analysis was performed by FACScan (Becton Dickinson), with excitation and emission settings of 495 and 525 nm, respectively.
Measurement of intracellular glutathione (GSH) concentration
The intracellular GSH content was measured using a Glutathione Assay Kit (Wako, Japan).31 Briefly, 5 × 106 cells were homogenized in 5% metaphosphoric acid using a Teflon pestle; cell debris and membranes were separated from the cytosol by centrifugation at 4000 g. The supernatant was used for GSH measurement according to the manufacturer's instructions. The pellet was dissolved in 1 mol/l NaOH, and analyzed for protein by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The GSH content was expressed as nanomoles per milligram of protein.
As2O3 induces apoptosis in the human AML cell line NKM-1
It was reported that 1 μ M of As2O3 induced apoptosis in NB4 cells, but not in other leukemia cell lines. On the other hand, 5 μ M of As2O3 induced apoptosis in almost every leukemia cell line. Treatment of NKM-1 cells with As2O3 led to dose- and time-dependent cell death, as determined using trypan blue exclusion (Figure 1a). At a concentration of 5 μ M, As2O3 reduced the viability of NKM-1 cells rapidly; approximately 10% of control cells were viable after 2 days. At a concentration of 1 μ M, the percentage viability of NKM-1 cells was 43.4% after 3 days and 7.9% after 5 days. Flow-cytometry analysis for annexin-V staining showed that cell death was mainly due to apoptosis (Figure 1b).
Caspase-3 activation was examined by immunoblotting analysis with an antibody specific to PARP as an intracellular substrate of caspase-3. After 12-h treatment, only full-length PARP with a molecular mass of 116 kDa was detected; thereafter, a cleaved fragment form with a molecular mass of 85 kDa appeared as time progressed (Figure 1c). These results suggested that As2O3-induced apoptosis in NKM-1 was mediated through caspase-3 activation.
Effect of As2O3 on the cellular H2O2 content and the mitochondrial membrane potential (Δψm) of NKM-1 cells
The ability of As2O3 to induce apoptosis in leukemic cells was reported to be dependent on the activity of enzymes that regulate cellular H2O2 content.20,21 In order to measure the level of cellular H2O2, the cells labeled with DCFH-DA were analyzed by flow cytometry. Without As2O3 treatment, the cellular content of H2O2 of NKM-1 cells was lower than that of NB4, KASUMI-1, and K562, but higher than that of HL-60. Although the cellular H2O2 content of NKM-1 cells increased slightly 6 h after the treatment, it increased up to two times after 24 h and four times after 48 h. In NB4 cells, the cellular content of H2O2 increased slightly after 24 h, but it increased to about three times after 48 h. Compared with these remarkable increases, no change in the cellular H2O2 content of the other three cell lines was observed (Figure 2a). The Δψm was measured by FACS analysis with cells labeled with DiOC6,13,30,32,33,34,35 showing that the decrease in Δψm proceeded almost concomitant with the increase in the cellular H2O2 content (Figure 2b).
Reduced GSH has an antioxidant function, and plays an important role in the growth-inhibitory effects of As2O3. It was reported that the human APL cell line NB4, in which 1 μ M As2O3 can induce apoptosis, has a lower level of cellular GSH content than other leukemia cell lines.18,36,37,38 As shown in Figure 2c, the level of cellular GSH of NKM-1 cells was lower than that of NB4 cells and K562 cells. On the other hand, the level of cellular GSH of NKM-1 was higher than HL-60 and KASUMI-1. These findings suggest that GSH contents are not correlated with the sensitivity to As2O3.
As2O3-induced activation of MAP kinases
To determine the involvement of MAPKs in As2O3-induced apoptosis, we next examined whether As2O3 could promote the phosphorylation of three classes of MAPK family, JNK, p38MAP kinase, and ERK in NKM-1 cells, arsenic-sensitive NB4 cells, and arsenic-insensitive HL-60 cells by Western blot analysis. In the treatment of NKM-1 cells with 1 μ M As2O3, activation of JNK was detected 6 h after the start of treatment and sustained until 24 h, after which it decreased (Figure 3a). In NB4 cells, JNK was also activated from 24 h after the start of treatment; also, the activation was sustained to 48 h (Figure 3b). On the other hand, no remarkable activation of JNK was detected in HL-60 cells treated with 1 μ M As2O3 (Figure 3c). In contrast to JNK, the second stress-induced kinase, p38MAP kinase, was markedly activated in 0.3 h, and this activation was sustained to 24 h in these three cell lines. In NKM-1 only, the activation of p38MAP kinase decreased after 48 h. Rapid decreases in the levels of activated forms of ERK were observed in NKM-1, but not in NB4 and HL-60 in the treatment of 1 μ M As2O3. Inactivation of ERK in NKM-1 was seen from 0.3 to 24 h. These findings suggested that sustained JNK activation is involved in As2O3-induced apoptosis.
Involvement of JNK in As2O3-induced apoptosis in NKM-1 cells
To determine the effect of JNK on As2O3-induced apoptosis in NKM-1 further, JNK inhibitor-1, a cell-permeable peptide inhibitor of JNK, was utilized. Apoptosis and the decrease in Δψm induced by As2O3 were inhibited in the cells treated with 1 μ M of JNK inhibitor, compared to the cells treated without the inhibitor (Figure 4a–c). JNK inhibitor-1 suppressed both rapid and sustained activation of JNK induced by As2O3 (Figure 4d). Thus, JNK activation is involved in As2O3-induced apoptosis.
Involvement of ROS in As2O3-induced apoptosis in NKM-1 cells
To evaluate the relationship between ROS and As2O3-induced apoptosis, N-acetylcysteine (NAC), which is a potent antioxidant, was utilized. Treatment with NAC restored As2O3-induced cell growth inhibition in NKM-1 cells remarkably (Figure 5a). Moreover, the NAC treatment suppressed the JNK activation (Figure 5b) and increase in cellular H2O2 content (Figure 5c) induced by As2O3. These findings suggest that the generation of ROS is involved in As2O3-induced apoptosis in NKM-1 cells. In addition, like NAC, JNK inhibitor-1 suppressed As2O3-induced cellular H2O2 generation in NKM-1. This result and the time course of cellular H2O2 generation in As2O3-treated NKM-1 suggest that cellular H2O2 generation exists downstream of JNK activation.
Since the discovery of As2O3-induced apoptosis, its molecular mechanisms have been studied in various malignant tumors.6,7,8,9,10,11,12,13 The concentration used in those studies, however, was higher than that used for APL cells. At therapeutic levels (1–2 μ M), there has been no report that As2O3 induced apoptosis in myeloid leukemia cell lines except for APL cell lines. To induce apoptosis in such a line, 5 and often 50 μ M of As2O3 is needed. This study revealed that 1 μ M As2O3 induced apoptosis in NKM-1 cells derived from a patient with AML (FAB classification M2).27
It is reported that the ability of As2O3 to induce apoptosis in leukemic cells is dependent on the activity of enzymes that regulate the cellular H2O2 and GSH contents.39,40 Without As2O3 treatment, the level of cellular H2O2 content of NKM-1 cells was not higher than that of the other leukemia cells. The cellular level of H2O2 in NKM-1 cells increased much more than that of the others after treatment with 1 μ M As2O3. In addition, the decreased Δψm in NKM-1 cells paralleled the increase in cellular H2O2 content. These changes in both H2O2 content and Δψm were inhibited by agents that reduce the generation of ROS, such as dithiothreitol, and NAC. GSH is the major antioxidant of the cells, and functions to scavenge free radicals and to detoxify toxins and chemotherapeutic agents.41,42 In NKM-1 cells, the cellular GSH level was lower than that of NB4 but higher than that of HL-60 and KASUMI-1, in which apoptosis was not induced by 1 μ M As2O3. These findings suggest that the generation of ROS, specifically H2O2, but not GSH content, might play an important role in As2O3-induced apoptosis.
Activation of MAPKs is also involved in the signaling of many types of apoptosis; JNK and p38MAP kinase are activated during apoptosis induced by cytotoxic stressors, and by proinflammatory cytokine stimulation.43,44,45 JNK and p38MAP kinase were shown to be activated by treatment with 5–50 μ M of As2O3.9,40,46 We found that 1 μ M As2O3 activated JNK only in As2O3-sensitive cell lines, NKM-1 and NB4. In addition, this activation of JNK preceded the change in the cellular H2O2 content and Δψm. Addition of a cell-permeable peptide inhibitor of JNK inhibited As2O3-induced apoptosis in NKM-1 cells, and this inhibition continued for 48 h. On the other hand, p38MAP kinase was phosphorylated not only in NKM-1 and NB4, but also in a less-sensitive cell line, HL-60. Regarding the classical MAP kinase ERK, the activation of which prevents cell apoptosis, it has been argued whether inactivation of ERK is required for As2O3-induced apoptosis.24,40,46 In our study, ERK was inactivated in NKM-1 cells with 1 μ M of As2O3. In contrast to the inactivation of ERK in NKM-1, no change was detected in the status of ERK activation in the other two cell lines, suggesting that ERK does not seem to be involved in As2O3-induced apoptosis. These results suggest that only sustained activation of JNK is important for As2O3-induced apoptosis in leukemia cells.
The sustained activation of JNK for apoptosis has been shown in other settings. For example, apoptosis signal-regulating kinase (ASK1), a mitogen-activated kinase kinase kinase (MAPKKK), is required for sustained activation of JNK/p38MAP kinase and apoptosis by oxidative stress.47 H2O2-induced activation of JNK and p38MAP kinase, especially sustained activation, is dramatically suppressed in embryonic fibroblasts of ASK1-deleted mice (ASK1−/−), and ASK1−/− cells are less sensitive than ASK1+/+ cells to death-inducing activities resulting from oxidative stress. Taken together, these results indicated that, by whatever stressors, the sustained activation of JNK may be required for apoptosis induction.
Recently, JNK phosphatase was reported to be important in several types of stress stimuli-induced JNK activation.48 In that report, inactivation of JNK phosphatase, M3/6, was involved in JNK activation. At high concentration (500 μ M) of sodium, arsenite inactivates this phosphatase and activates the JNK pathway. The therapeutic concentration of As2O3 remains to be explored.
As to the relationship between ROS and JNK activation, NAC, a scavenger of ROS, suppressed As2O3-induced JNK activation in NKM-1 cells. As NAC inhibited H2O2-induced activation of ASK1,49 it was thought that the inhibition of ASK1 activation by NAC suppressed As2O3-induced JNK activation. While NAC completely prevented As2O3-induced apoptosis in NKM-1 cells, JNK inhibitor partly suppressed As2O3-induced apoptosis in NKM-1, suggesting that a second pathway of As2O3-induced apoptosis in NKM-1 that is dependent on ROS but not JNK may exist. Further investigation of the relationship between ROS and As2O3-induced apoptosis will be needed.
In the M2 cell line NKM-1, 1 μ M of As2O3 induced sustained activation of JNK and apoptosis. This finding may provide a basis to select a subgroup other than APL, which can benefit from As2O3 treatment.
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We thank Satoshi Suzuki and Chika Wakamatsu for their excellent technical assistance.
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