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Clinical Cancer Research 13, 5436, September 15, 2007. doi: 10.1158/1078-0432.CCR-07-0481
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Myeloperoxidase Is a Key Regulator of Oxidative Stress–Mediated Apoptosis in Myeloid Leukemic Cells

Tomonori Nakazato1, Morihiko Sagawa1, Kenji Yamato3, Mingji Xian1, Takehiro Yamamoto2, Makoto Suematsu2, Yasuo Ikeda1 and Masahiro Kizaki1

Authors' Affiliations: 1 Division of Hematology, Department of Internal Medicine and 2 Department of Biochemistry, Keio University School of Medicine; and 3 Molecular Cellular Oncology and Microbiology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Requests for reprints: Masahiro Kizaki, Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3785; Fax: 81-3-3353-3515; E-mail: makizaki{at}sc.itc.keio.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We reported previously that reactive oxygen species (ROS) are key mediators of apoptosis induced by a polyphenol, (–)-epigallocatechin-3-gallate (EGCG), in myeloid leukemic cells. This study aimed to further examine the mechanism of ROS-mediated apoptosis induced by EGCG and its relationship to the heme enzyme myeloperoxidase (MPO).

Experimental Design: We established stably transfected K562 cells expressing wild-type and mutant MPO. Then, sensitivity against EGCG and other ROS-inducing agent was examined and further investigated the detailed molecular mechanism of ROS-inducing apoptosis in MPO-positive leukemic cells.

Results: EGCG rapidly induced apoptosis in MPO-positive leukemia cells. Preincubation of myeloid leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide, and the heme biosynthesis inhibitor, succinylacetone, resulted in inhibition of the intracellular MPO activity, ROS production, and induction of apoptosis following addition of EGCG. Overexpression of MPO sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. In contrast, an enzymatically inactive MPO mutant–expressing K562 cell could not respond to EGCG, suggesting that MPO is important for determining the sensitivity to EGCG-induced oxidative stress. Hypochlorous acid scavengers and the hydroxyl radical (·OH) scavenger inhibited EGCG-induced apoptosis in myeloid leukemic cells. The fluorescence intensity of both aminophenyl fluorescein– and hydroxyphenyl fluorescein–loaded myeloid leukemic cells significantly increased on stimulation with EGCG, indicating that EGCG generated highly toxic ROS in myeloid leukemic cells.

Conclusions: These results indicated that highly toxic ROS such as ·OH generated via the hydrogen peroxide/MPO/halide system induce apoptosis and that ROS may be the direct mediators of EGCG-induced apoptosis in MPO-positive leukemic cells.

Acute myelogenous leukemia (AML) is a heterogeneous group of malignant disorders of hematopoietic progenitor cells by an accumulation of granulocyte and monocyte precursors in the bone marrow and peripheral blood. Despite scientific advances in our understanding of the epidemiologic, genetic, and biological features of AML, the disease remains fatal in a majority of patients and especially in older individuals. The therapeutic approach to AML is usually chemotherapy, but severe side effects and complications, including infection and bleeding induced by the anticancer drugs, are major problems in the clinical setting. Recently, more specifically targeted agents have been developed for the treatment of AML, including anti-CD33 antibodies and immunoconjugate drugs, inhibitors of multidrug resistance proteins, farnesyl transferase inhibitors, tyrosine kinase inhibitors, histone deacetylase, and proteosome inhibitors (1). However, these candidate agents for targeted therapy have yet to be translated into clinical application. In the future, in addition to doing therapeutic trials of these agents, it will be important to identify other highly specific therapeutic agents based on our evolving understanding of the biology of AML.

Myeloperoxidase (MPO), a microbicidal protein in the primary granules of neutrophils, is the hallmark enzyme of the myeloid lineage. Expression of the MPO gene is specific for myeloid precursors and their leukemic counterparts. The percentage of MPO-positive blast cells has been generally considered to be important for the diagnosis of AML. MPO catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant derived from chloride ions and hydrogen peroxide (H2O2). HOCl then may interact with other small molecules including NH3 to form monochloramines (NH2Cl) or with other ROS to yield peroxynitrite (ONOO–), hydroxyl radical (·OH), singlet oxygen (1O2), and ozone (O3; ref. 2). There are many species of ROS, but they tend to be considered collectively as "oxidative stress" when their effects in living cells are discussed. However, each species of ROS is likely to have a specific role in living cells. Further, it has been reported that the percentage of MPO-positive blast cells has an effect on the prognostic significance for clinical outcomes of patients with AML (3–6). The Japan Adult Leukemia Study Group study showed that patients with >50% MPO-positive blast cells have a significantly better outcome (7). However, the biological significance of the relationship between MPO and prognosis of AML is totally unknown.

We have reported previously that the green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG) rapidly induces apoptosis of AML cells via modulation of reactive oxygen species (ROS) production in vitro and in vivo (8). In this study, we further investigated the molecular mechanisms of oxidative stress–mediated apoptosis by EGCG and other ROS-producing agents. We found that highly toxic ROS (hROS), such as hydroxyl radical generated via the H2O2/MPO/halide system induce apoptosis and that such ROS may directly mediate EGCG-induced apoptosis in myeloid leukemic cells.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture. The human myeloid leukemic cell lines HL-60, U937, Kasumi-1, KG-1, K562, and THP-1 were obtained from the Japan Cancer Research Resources Bank (Tokyo, Japan). The retinoic acid–resistant acute promyelocytic leukemia cell line UF-1 was established in our laboratory from a relapsed patient with acute promyelocytic leukemia who received treatment with all-trans-retinoic acid (9). The retinoic acid–sensitive acute promyelocytic leukemia cell line NB4 was a gift of Dr. M. Lanotte (Hôpital St. Louis, Paris, France; ref. 10). Bone marrow or peripheral blood samples from patients with AML were obtained after appropriate Human Protection Committee validation at the Keio University School of Medicine (Tokyo, Japan) and with written informed consent. Mononuclear cells were separated by Lymphoprep (Nycomed Pharma AS). Cells were maintained in RPMI 1640 (Life Technologies) with 10% FCS (Hyclone Laboratories), 100 units/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere with 5% CO2. The morphology was evaluated by cytospin slide preparations with Giemsa staining and the viability was assessed by trypan blue dye exclusion.

Reagents. Catechin derivative EGCG (Fig. 1A ), taurine, methionine, and thiourea were purchased from WAKO Chemical Co. Catalase, manganese superoxide dismutase (MnSOD), 4-aminobenzoic acid hydrazide (ABAH), ebselen, N-nitro-L-arginine methyl ester hydrochloride, desferrioxamine, apocynin, succinyl acetone (ScAc), H2O2, and arsenic trioxide (As2O3) were obtained from Sigma Chemical Co.


Figure 1
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  		Fig. 1. A, structures of EGCG. B, EGCG inhibited the proliferation of myeloid leukemic cells. Various myeloid leukemic cells (NB4, HL-60, U937, and K562) were treated with EGCG for various concentrations (0-100 µmol/L) for 24 h. Clonogenic assay was done as described in Materials and Methods. Columns, mean of three different experiments; bars, SD (within 5% of the mean).

 
Clonogenic assay. Colony growth inhibition of EGCG-treated leukemic cells was analyzed as reported previously (11). Briefly, MPO-sensitive and MPO-resistant leukemic cells were exposed to various concentrations (0-100 µmol/L) of EGCG for 24 h and washed with RPMI 1640. The cells (1,400 per plate) were then plated in triplicated wells in a 12-well plate containing 0.6 mL of Methocult medium (Veritas). The plates were incubated at 37°C with 5% CO2 for 7 days. Colonies (greater double equals30 cells) were counted with an inverted microscope and the percentage colony growth inhibition compared with the untreated control cells was calculated.

Assays for apoptosis. Apoptosis was determined by morphologic changes as well as by staining with an Annexin V-FITC and propidium iodide double-labeling kit purchased from PharMingen.

Measurement of intracellular generation of ROS. To assess the production of ROS, control and EGCG-treated cells were incubated with 10 µmol/L dichlorodihydrofluorescein diacetate (DCFH-DA; Molecular Probes), which was oxidized to the fluorescent compound, dihydrofluorescein, by cellular ROS. Cells (1 x 105) were stained with 10 µmol/L DCFH-DA for 30 min at 37°C and then washed and resuspended in PBS. The oxidative conversion of DCFH-DA to dihydrofluorescein was measured by flow cytometry (Becton Dickinson).

Cell lysate preparation and Western blotting. Cells were collected by centrifugation at 700 x g for 10 min and then the pellets were resuspended in lysis buffer [1% NP40, 1 mmol/L phenylmethylsulfonyl fluoride, 40 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl] at 4°C for 15 min. Protein concentrations were determined using a protein assay DC system (Bio-Rad). Cell lysates (15 µg protein per lane) were fractionated in 12.5% SDS-polyacrylamide gels before transfer to the membranes (Immobilon-P membranes, Millipore) using a standard protocol. Antibody binding was detected by using an enhanced chemiluminescence kit for Western blotting detection with hyper-ECL film (Amersham). Blots were stained with Coomassie brilliant blue to confirm that there were equal amounts of protein extract on each lane. The following antibodies were used in this study: anti-MPO (Upstate Biotechnology) and anti-ß-actin (Santa Cruz Biotechnology).

MPO staining and measurement of MPO activity. A 3,3'-diaminobenzidine staining kit (Muto Chemical Co.) was used for cytochemical staining of MPO according to the manufacturer's instructions. Briefly, the peroxidase reaction was developed with 0.05% 3,3'-diaminobenzidine in 50 mmol/L Tris-HCl (pH 7.6) with 0.03% H2O2 for 1 to 2.5 min. The sections were counterstained with hematoxylin, dehydrated, and mounted. Cytospin slides were prepared and stained with Giemsa. Oxidized 3,3'-diaminobenzidine (a brown, highly insoluble indamine polymer) is visible under light microscopy. Cells were harvested by centrifugation, washed with PBS, and resuspended in 50 µL PBS containing Triton X-100 (0.2%) and phenylmethylsulfonyl fluoride (1 mmol/L). Protein concentrations were determined using a Bio-Rad protein assay. MPO activity was measured by using a MPO ELISA kit (Calbiochem) according to the manufacturer's instructions.

Measurement of MPO expression by flow cytometry. Control and EGCG-treated cells were prepared for flow cytometry. Cytoplasmic staining was done to detect the expression of MPO. For fixation and permeabilization of the cells, an Intraprep cell permeabilization kit (Immunotech) was used. Cells (1 x 106) were incubated with FITC-conjugated anti-MPO antibody (Beckman Coulter) for 30 min at 4°C, and isotype-matched mouse IgG served as a control. Flow cytometric analysis was done by using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson).

Transfection of K562 cells with normal and mutant MPO cDNAs. Plasmids with normal and mutated cDNA for MPO were transfected into log-phase K562 cells by Amaxa Nucleofector apparatus (Amaxa) according to the manufacturer's procedure. Cells were washed, resuspended at 1 x 106 in 100 µL Cell Line Nucleofector V buffer (Amaxa) with 20 µg pREP10 plasmid only as a control (K562/Cont) and containing normal or mutated cDNA, and then electroporated. After electroporation, cells were immediately cultured with complete medium in 12-well plates at 37°C. By site-directed mutagenesis, we introduced changes affecting His-502 (CAC) to Ala (GCC) in pro-MPO (K562/H502A). His-502 in pro-MPO is located at calcium-binding domain of the enzyme, and it has been reported that this site is important for the enzymatic activity (12). Stable transfectants (K562/Cont, K562/MPO, or K562/H502A) were selected using 0.7 mg/mL neomycin for 48 h after transfection and stable lines expressing the desired protein for up to 6 weeks were obtained 72 h after selection.

Detection of hROS. To selectively detect the hROS, such as ·OH and ONOO–, cells were loaded with aminophenyl fluorescein (APF) or hydroxyphenyl fluorescein (HPF; 10 µmol/L) by incubation for 30 min at room temperature according to the previously published method (13). APF and HPF themselves are not highly fluorescent, but when reacted with hROS, APF and HPF exhibit strong dose-dependent fluorescence. Furthermore, using these probes together, hypochlorite (OCl–) can be selectively detected from ·OH and ONOO–. HPF/APF can be used to differentiate hROS from H2O2, nitric oxide (NO), and O2–. Dye-loaded cells were treated with EGCG, and fluorescence images were acquired twice in each experiment (before and 30 min after the treatment with EGCG) using an LSM510 confocal laser scanning unit. The excitation wavelength was 488 nm, and the emission was filtered using a 505 to 550 nm barrier filter.

Statistical analysis. Results are expressed as mean ± SD. The Student's t test was used to compare quantitative data population with normal distribution and equal variance. P < 0.05 was considered statistically significant otherwise specified.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
EGCG significantly inhibited cellular proliferation of MPO-positive myeloid leukemic cells. We first investigated the effects of EGCG on the cellular proliferation of various leukemic cell lines (HL-60, NB4, UF-1, Kasumi-1, K562, and U937) by a clonogenic assay. EGCG inhibited the cellular proliferation of all leukemic cells in a dose-dependent manner (Fig. 1B; data not shown). Interestingly, EGCG was particularly sensitive to NB4 and HL-60 leukemic cells (Fig. 1B), all of which expressed MPO protein (Fig. 2A ). These MPO-positive cell lines also showed strong MPO activity as detected by MPO ELISA assay and cytochemical staining (Fig. 2B, inset). Annexinn V–positive apoptotic fractions were detected 24 h after exposure to EGCG in all MPO-positive leukemic cell lines (Fig. 2C). These results showed that EGCG-induced growth inhibition of MPO-positive leukemic cells was mediated by apoptosis. In contrast, EGCG failed to induce apoptosis in MPO-negative leukemic cell lines (Fig. 2C).


Figure 2
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  		Fig. 2. Expression of MPO protein in various myeloid leukemic cells. A, MPO protein expression was measured using Western blotting. Rabbit polyclonal anti-MPO antibody detected precursor MPO at 85 to 90 kDa (immature MPO) and glycosylated heavy subunit at ~59 kDa (mature MPO) in EGCG-sensitive cell lines (top). Detection of intracellular MPO protein by fluorescence-activated cell sorting. For fixation and permeabilization of the cells, an Intraprep cell permeabilization kit was used. Intracellular MPO protein was detected by using FITC-conjugated anti-MPO antibody. Percentage of MPO-positive cells was expressed in the top right corner (bottom). B, MPO activity of various myeloid leukemic cells. MPO activity of the cells was measured by using a MPO ELISA kit according to the manufacturer's instructions. MPO activity was detected by cytochemical staining in HL-60 and K562 cells. Cytospin slides were prepared and cytoplasmic MPO was stained by using a 3,3'-diaminobenzidine staining kit. Then, each slide was stained with Giemsa. Original magnification, x1,000 (inset). C, detection of apoptotic cells by Annexin V staining. Cells were cultured with 50 µmol/L EGCG for 24 h, stained with Annexin V-FITC, and analyzed by flow cytometry. Three independent experiments were done and all gave similar results.

 
Effects of EGCG on primary cells from patients with various types of AML. Among the myeloid leukemic cell lines, MPO-positive leukemic cells were more sensitive to EGCG compared with MPO-negative leukemic cells. We therefore analyzed the association between the sensitivity to EGCG and the expression of MPO in freshly isolated cells from patients with AML and found that the expression of active MPO also contributed to the sensitivity to EGCG-induced apoptosis in the primary myeloid leukemic cells (Supplementary Fig. S1).

ROS production triggers EGCG-induced apoptosis in MPO-positive leukemic cells. We have reported previously that EGCG induces apoptosis of myeloid leukemic cells via modulation of ROS production in vitro and in vivo (8). To investigate the role of ROS in EGCG-induced apoptosis in myeloid leukemic cells, we analyzed the production of intracellular ROS in both MPO-positive and MPO-negative cells. Treatment with EGCG caused a significant ROS production in the MPO-positive HL-60 cell line. In contrast, intracellular ROS production was less modulated by EGCG in MPO-negative U937 cells (Fig. 3A, left ). Enhancement of cellular antioxidant status by preincubation of MPO-positive leukemic cells with a superoxide (O2–) and a H2O2 scavenger, catalase and MnSOD, respectively, largely suppressed EGCG-induced ROS production and cell growth (Fig. 3A, middle). In addition, H2O2 itself induced growth inhibition of MPO-positive leukemic cells (Fig. 3A, right). These results indicate that EGCG-induced apoptosis is mediated by the production of H2O2 and O2–.


Figure 3
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  		Fig. 3. EGCG induced apoptosis via the production of ROS in myeloid leukemic cells. A, left, detection of apoptotic cells by Annexin V staining. Cells were cultured with 50 µmol/L EGCG for 24 h, stained with Annexin V-FITC, and analyzed by flow cytometry. Three independent experiments were done and all gave similar results. To determine the intracellular concentration of ROS, leukemic cells were cultured with DCFH-DA and the fluorescence was measured by flow cytometry. Leukemic cells were treated for 1 h with 50 µmol/L EGCG. Middle, the antioxidants, catalase and MnSOD, blocked EGCG-induced cell growth in MPO-positive leukemic cells. Cells were treated with 50 µmol/L EGCG alone or together with 500 units/mL catalase or 500 units/mL MnSOD for 24 h. Cell viability was measured by trypan blue dye exclusion. Columns, mean of at least three different experiments; bars, SD. *, P < 0.01. Right, direct effect of H2O2 on the cellular growth of leukemic cells. Cells were treated with H2O2 (0-300 µmol/L) for 24 h. Cell viability was assessed by trypan blue dye exclusion test. Points, mean of three different experiments; bars, SD (within 5% of the mean). B, MPO activity was detected by cytochemical staining. Pretreatment of HL-60 cells with 100 µmol/L ABAH and 0.5 mmol/L ScAc for 24 and 3 h, respectively, significantly inhibited MPO enzymatical activity. C, ABAH blocked EGCG-induced cell growth in MPO-positive leukemic cells. Cells were treated with 50 µmol/L EGCG alone or together with 100 µmol/L ABAH for 24 h. Cell viability was measured by trypan blue dye exclusion. Columns, mean of at least three different experiments; bars, SD. *, P < 0.01 (top).

 
To investigate the role of MPO in ROS-mediated apoptosis induced by EGCG, we further investigated the effects of the MPO-specific inhibitor ABAH on proliferation of various leukemic cells. ABAH represents a highly selective, mechanism-based inhibitor for MPO, which is first oxidized by the enzyme and then causes its irreversible inactivation (14). Preincubation of MPO-positive myeloid leukemic cells (HL-60, NB4, and UF-1) with ABAH resulted in significant inhibition of intracellular MPO activity, ROS production, and apoptosis following addition of EGCG (Fig. 3B; Fig. 3C, top).

A heme biosynthesis inhibitor significantly inhibited MPO activity and blocked EGCG-induced apoptosis. ScAc, a potent inhibitor of 5-aminolevulinic acid dehydratase, has been reported to be an inhibitor of heme biosynthesis in MPO processing (15, 16). It has also been reported that treatment of HL-60 cells with ScAc resulted in a loss of MPO enzymatic activity and disruption of the posttranslational processing of the enzyme (17). Untreated myeloid leukemic cells expressed both precursor (85-90 kDa) and heavy subunit (mature) MPO (60 kDa). In contrast, ScAc-treated cells expressed large amounts of precursor MPO but only very small amounts of mature MPO (Supplementary Fig. S2, top). Furthermore, ScAc significantly inhibited intracellular MPO activity, ROS production, and induction of apoptosis following addition of EGCG to MPO-positive myeloid leukemic cells (Fig. 3C, bottom; Supplementary Fig. S2, bottom). However, both ABAH and ScAc did not affect cellular growth of EGCG-treated MPO-negative parent K562 cells (data not shown). These results strongly suggest that MPO plays a central role in EGCG-induced apoptosis.

Overexpression of normal MPO in K562 cells enhanced MPO activity and ROS production and sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. To investigate the role of MPO in EGCG-induced apoptosis, we transfected the full-length MPO cDNA and empty vector into MPO-negative K562 cells and designated these cells as K562/MPO and K562/Cont, respectively. MPO protein was expressed in the three independent K562/MPO clones but not in the K562/Cont clones or parent K562 cells based on Western blotting and flow cytometry analysis (Fig. 4A, top ). In addition, K562/MPO cells exhibited significantly more MPO activity, but K562/Cont and parent K562 cells showed very little MPO activity (Fig. 4B). K562/MPO cells were positive for cytochemical staining (Fig. 4B, top), suggesting that K562 cells transfected with normal MPO cDNA synthesized the enzymatically active MPO protein. In contrast to K562/Cont cells, K562/MPO cells enhanced MPO activity and ROS production and sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG (Fig. 4A, bottom; Supplementary Fig. S3). In addition, catalase, SOD, and MPO inhibitor ABAH inhibited the EGCG-induced suppression of cell growth in K562/MPO cells (Supplementary Fig. S3).


Figure 4
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  		Fig. 4. Overexpression of MPO in K562 cells enhanced MPO activity and ROS production and sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. A, top, MPO protein expression in MPO-transfected K562 cells (K562/MPO cells). Western blotting and fluorescence-activated cell sorting analysis showed MPO protein expression in K562/MPO cells, but MPO protein was not detected in K562/Cont cells or parent K562 cells. Bottom, overexpression of MPO in K562 cells sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. Cells were treated with 50 µmol/L EGCG for 24 h. Cell viability was measured by trypan blue dye exclusion. Points, mean of at least three different experiments; bars, SD. B, measurement of MPO activity by cytochemical staining (top) and ELISA (bottom). K562/MPO cells synthesized enzymatically active MPO protein. In contrast, K562/Cont cells expressed only a low level of MPO activity, which was similar to the level in parent K562 cells. Enzymatically active MPO protein in K562/MPO cells was detected by cytochemical staining. C, EGCG-induced apoptosis in K562/H502A cells. The indicated cells were cultured with EGCG (50 µmol/L) for 24 h and then stained with Annexin V-FITC and analyzed by flow cytometry.

 
To confirm the role of MPO on EGCG-induced apoptosis in K562 cells, we made stably transfected K562 cells expressing mutant MPO (K562/H502A). It has been reported that no MPO activity was detected in the cells infected with the virus containing MPO/H502A cDNA (12). EGCG induced apoptosis of K562/MPO and MPO-positive HL-60 cells; however, EGCG did not induce apoptosis of MPO-negative parent K562 cells as well as K562/H502A cells expressing enzymatically defective MPO activity (Fig. 4C). These results suggested that MPO expression and its activity determined the sensitivity to EGCG in myeloid leukemic cells.

hROS are key regulators of EGCG-induced apoptosis. MPO catalyzes the formation of HOCl, a powerful oxidant formed from chloride ions and H2O2. We next examined the relationship between EGCG-induced apoptosis and the H2O2/MPO/halide system in MPO-positive myeloid leukemic cells. H2O2 scavenger (catalase), O2– scavenger (MnSOD), HOCl scavenger (taurine and methionine), and ABAH significantly blocked EGCG-induced apoptosis in HL-60 cells (Fig. 5A ), indicating that H2O2, O2–, HOCl, and MPO all participate in EGCG-induced apoptosis. Interestingly, the ·OH scavengers, thiourea and dimethyl thiourea, also inhibited EGCG-induced apoptosis in HL-60 cells. Thiourea and its derivative, dimethyl thiourea, have been widely introduced as a specific scavenger of ·OH (18). These results suggest that hydroxyl radical also may play a key role in EGCG-induced apoptosis. Therefore, we further used the novel fluorescence probes APF and HPF, which can selectively detect hROS, to determine the more detailed mechanism of MPO-mediated apoptosis in myeloid leukemic cells (13). It is noteworthy that the fluorescence intensity of both APF- and HPF-loaded HL-60 cells greatly increased on stimulation with EGCG (Fig. 5B). Our results suggested that EGCG generated hROS (·OH and ONOO–) intracellularly in MPO-positive HL-60 cells. Both NO synthesis inhibitor (NG-monomethyl-L-arginine) and peroxynitrite scavenger (ebselen) failed to block this apoptosis (Fig. 5A), suggesting that ONOO– is not a mediator of MPO-mediated apoptosis. Taken together, these observations indicated that hROS such as the hydroxyl radical generated via the H2O2/MPO/halide system induce apoptosis and that such ROS may be the direct mediators of EGCG-induced apoptosis in MPO-positive myeloid leukemic cells.


Figure 5
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  		Fig. 5. Various antioxidants blocked EGCG-induced apoptosis. A, the addition of HOCl scavengers (methionine and taurine) inhibited EGCG-induced apoptosis in MPO-positive (EGCG sensitive) myeloid leukemic cells. HL-60 cells were treated with 50 µmol/L EGCG alone or together with various antioxidants for 24 h. The antioxidants were as follows: 500 units/mL catalase (H2O2 scavenger), 500 units/mL MnSOD (O2– scavenger), 25 mmol/L taurine (HOCl scavenger), 25 mmol/L methionine (HOCl scavenger), 25 mmol/L thiourea (TU; ·OH scavenger), 100 µmol/L ABAH (MPO inhibitor), 4 µmol/L ebselen (ONOO– scavenger), 1 mmol/L NG-monomethyl-L-arginine (L-NMMA; inducible nitric oxide synthase inhibitor), 100 µmol/L desferrioxamine (DFO; iron chelator), and 200 µmol/L apocynin (NADPH inhibitor). Apoptotic cells were detected by double staining with Annexin V and propidium iodide (PI). Three independent experiments were done and all gave similar results. B, hROS were selectively detected by staining with the novel fluorescence probes APF and HPF. Cells were seeded onto a glass-bottomed dish and then loaded with APF or HPF (10 µmol/L) by incubation for 30 min at room temperature. Dye-loaded cells were treated with EGCG. The fluorescence intensity images were acquired twice in each experiment (before and 30 min after the treatment with EGCG) using an LSM510 confocal laser scanning unit and flow cytometry.

 
EGCG markedly enhances As2O3-mediated apoptosis in myeloid leukemic cells. As2O3 has been reported to inhibit the proliferation of human acute promyelocytic leukemia cells by inducing apoptosis via intracellular ROS production (19–21). We thus tested the possibility of using a ROS-generating agent, EGCG, to enhance the activity of As2O3. The combination of low-dose As2O3 (1 µmol/L) and EGCG (15 µmol/L) resulted in a significant increase in apoptosis compared with low-dose As2O3 or EGCG treatment alone in HL-60 cells (Fig. 6A ). We also found that the combination of low-dose As2O3 and EGCG resulted in a higher level of hROS production than did either As2O3 or EGCG alone (Fig. 6B). Treatment of HL-60 cells with antioxidants blocked the combination of low-dose As2O3 and EGCG-induced hROS production and apoptosis (Fig. 6B). These results suggested that EGCG increased the production of hROS and enhanced As2O3-induced cytotoxicity.


Figure 6
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  		Fig. 6. A, EGCG potentiated As2O3-mediated apoptosis in myeloid leukemic cells. Cells were cultured in the absence or the presence of As2O3 (1 µmol/L), EGCG (15 µmol/L), or (As2O3 + EGCG) for 48 h. Apoptotic cells were detected by Annexin V and propidium iodide double staining and analyzed by fluorescence-activated cell sorting. Three independent experiments were done and all gave similar results. B, the combination of As2O3 and EGCG resulted in a higher level of hROS production than did either As2O3 or EGCG alone. To detect hROS, the novel fluorescence probes APF and HPF were used.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Redox reactions regulate a broad array of signal transduction pathways. Various ROS, including O2–, H2O2, ·OH, and ONOO–, are now thought of as signaling molecules that are mobilized in response to various stimuli (22–24). ROS modulate Ca2+ signaling and protein phosphorylation events and thereby function as regulators for various biological processes, such as gene expression, cell growth, differentiation, and chemotaxis (25). ROS are known to mediate apoptosis in many different cell types. Indeed, most anticancer drugs kill their target cells at least in part through the generation of elevated amounts of intracellular ROS (26–28). ROS can stimulate proapoptotic signal molecules, such as apoptosis signal regulating kinase 1, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase, activate the p53 protein pathway, or initiate the mitochondrial apoptotic cascade (29, 30). The various ROS can exert different effects according to their nature and to their intracellular levels, which are determined by both their production rate and the activity of antioxidant enzymes.

We have reported previously that green tea polyphenol, EGCG, rapidly induces apoptosis of AML cells via modulation of ROS production in vitro and in vivo (8). EGCG has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells (31–35). Although EGCG is generally well known as an antioxidant, it can also behave as a pro-oxidant under certain conditions (36). Recently, it has also been reported that EGCG may induce the production of H2O2 in the culture medium (37). In this study, we found that EGCG rapidly induced apoptosis of MPO-positive myeloid leukemic cell lines (HL-60, UF-1, NB4, and Kasumi-1), whereas EGCG failed to induce apoptosis of MPO-negative leukemia cells (U937, THP-1, KG-1, and K562). The MPO-specific inhibitor ABAH and the heme biosynthesis inhibitor ScAc resulted in significant inhibition of intracellular MPO activity and ROS production and apoptosis following addition of EGCG. In addition, overexpression of normal MPO enhanced MPO activity and ROS production and sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. These results suggest that MPO plays a key role in the oxidative stress–mediated apoptosis induced by EGCG.

MPO is a heme protein that is abundant in the granules of hematopoietic cells, including neutrophils, neutrophil precursors, and macrophages, where its activity in the presence of H2O2 is important in the killing of ingested organisms (2). It is contained in the neutrophil primary azurophilic granules, which appear at the promyelocyte stage of myeloid maturation, and this fact explains its abundance in the HL-60, NB4, and UF-1 cell lines. It was recently reported that a drug-induced cytotoxicity was correlated with the amount of MPO in HL-60 cells (38, 39). However, the role of MPO in oxidative stress–induced apoptosis of myeloid leukemic cells is still unclear.

To determine the species responsible for the MPO-mediated apoptosis induced by EGCG, we examined the detailed effects of various antioxidants on the apoptosis in this investigation. As judged from results indicating radical scavengers, O2– (MnSOD), H2O2 (catalase), HOCl or monochloramines (methionine and taurine), and ·OH scavengers (thiourea and dimethyl thiourea) are involved in the mechanisms for EGCG-induced apoptosis. Considering that O2– or H2O2 are primary species of ROS generated through oxidase and oxygenase systems, the MPO-derived chlorinated oxidants and ·OH resultantly generated from them seemed to account for ROS responsible for triggering the apoptosis. Among ·OH-generating systems in biological systems, the reaction of NO with O2– is unlikely to be involved in the mechanisms because neither an inhibitor of NO synthesis, NG-monomethyl-L-arginine, nor a peroxynitrite scavenger, ebselen, blocked the apoptosis. Furthermore, Fenton reaction involving free ion is also unlikely to be involved in the mechanisms, thus far as judged from the absence of the effects of deferoxamine, an iron chelator (data not shown). Excluding these two possibilities of ·OH-generating systems, we hypothesized that EGCG induces the production of H2O2, and then H2O2 is converted to HOCl by MPO (H2O2 + Cl– + H+ -> H2O + HOCl). HOCl per se is unlikely to account for the ultimate species responsible for apoptosis inducer because MPO-induced apoptosis was blocked by an O2– scavenger (SOD) and an ·OH scavenger (thiourea) in MPO-positive leukemic cells. If HOCl acts directly to induce the apoptosis of leukemic cells, SOD should have an accelerating rather than an inhibitory effect due to the induction of H2O2 by the treatment with SOD and the ·OH scavenger. Subsequent interaction of HOCl with O2– causes generation of hydroxyl radical (HOCl + O2– -> ·OH + Cl– + O2) that may directly induce apoptosis of MPO-positive leukemic cells. It has reported that ·OH participates in various biological processes (40). ·OH can damage DNA bases and mediates redox alteration of cell membrane Ca2+ channels, resulting in the induction of apoptosis in HL-60 cells (41). However, because of the lack of an effective method for directly detecting ·OH, its participation in these events has been established only indirectly by several scavengers. Several fluorescence probes to detect ROS, such as DCFH and dihydrorhodamine 123, have been developed, but both can react with oxidizing species (O2–, H2O2, NO, ferrous ion, and others). In addition, DCFH is easily auto-oxidized, resulting in a spontaneous increase in fluorescence on exposure to light (42). Therefore, it is not to be appropriate probes for detecting a specific oxidizing species in cells, such as H2O2 or NO, but rather they should be considered as detecting a broad range of oxidizing reactions that may be increased during intracellular oxidative stress. To investigate the molecular mechanisms of oxidative stress–mediated apoptosis by EGCG and its relationship to MPO, we used novel fluorescence probes (AFP and HPF) in this study. The fluorescence intensity of both APF- and HPF-loaded HL-60 cells greatly increased on stimulation with EGCG. These results suggested that EGCG generated hROS in MPO-positive HL-60 cells. Taken together, these observations indicated that hROS such as hydroxyl radical generated via the H2O2/MPO/halide system induce apoptosis and that such ROS may be direct mediators of EGCG-induced apoptosis in MPO-positive myeloid leukemic cells.

Oxidative damage has been suggested to be a key mechanism by which As2O3 causes cell death (19–21). As2O3-induced apoptosis is associated with the generation of ROS in several experimental models. Antioxidants and free radical scavengers are able to inhibit apoptosis induced by As2O3. These observations suggest the possibility of developing new therapeutic strategies using the free radical-mediated mechanism of As2O3 to selectively kill cancer cells. Based on the ability of both EGCG and As2O3 to cause free radical generation in cells, we hypothesized that the combination of low-dose As2O3 (1 µmol/L) and EGCG (15 µmol/L) should enhance the cytotoxic activity. The combination of low-dose As2O3 and EGCG resulted in a significant increase in apoptosis compared with As2O3 or EGCG treatment alone in MPO-positive myeloid leukemic cell lines. We also found that the combination of As2O3 and EGCG resulted in higher levels of hROS than did As2O3 or EGCG alone. Furthermore, treatment of myeloid leukemic cells with various antioxidants completely blocked the combination of As2O3- and EGCG-induced apoptosis. These findings suggest that low-dose As2O3-EGCG combination treatment enhances apoptosis through an increase in the production of hROS in myeloid leukemic cells. In the clinical setting, low-dose EGCG will enhance positive responses and will limit the toxicity of As2O3.

Many cytotoxic drugs function selectively to kill cancer cells by the abrogation of proliferative signals, leading to cell death, and numerous reports have shown that ROS are generated following treatment with these drugs (43, 44). For example, anticancer drugs such as the anthracyclines daunorubicin or doxorubicin have been shown to induce apoptosis in various tumor cells via ROS generation (45). It is indeed possible to combine EGCG with ROS-generating agents, such as As2O3, anthracycline, bortezomib, or 2-methoxyestradiol (known as a SOD inhibitor; ref. 46) to enhance therapeutic activity and overcome drug resistance.

Because MPO is a major component of myeloid precursor cells, the enzymatic activity of MPO is used to classify acute leukemias (47). Several studies have shown that the percentage of MPO-positive blast cells is a strong independent factor in AML (3–6). The Japan Adult Leukemia Study Group reported that overall survival and disease-free survival were significantly better in a high-MPO group than a low-MPO group (7). Our experimental results strongly support the clinical significance of MPO. MPO may be important to determine the sensitivity to oxidative stress–mediated apoptosis of myeloid leukemic cells by several anticancer drugs. According to our findings, it is expected that ROS-generating agents should be selected to overcome drug resistance in high-MPO AML cells. Furthermore, differentiation-inducing agents such as all-trans-retinoic acid or several growth factors that trigger the induction of MPO may enhance the production of hROS and the cytotoxicity of ROS-generating agents in low-MPO or even in MPO-negative AML cells.

We have herein proposed a novel strategy by which EGCG and other ROS-generating agents may serve as an enhancer of chemotherapy via MPO-mediated production of hROS in myeloid leukemic cells. Furthermore, the combination of differentiation-inducing agents and ROS-generating agents may enhance the MPO-mediated production of hROS and their cytotoxicity. "MPO-targeted therapy" will lead to new insights that should help in overcoming drug resistance in refractory AML.


   Acknowledgments
 
We thank Chika Nakabayashi for her helpful technical assistance.


   Footnotes
 
Grant support: Ministry of Education, Culture, Sports, Science and Technology of Japan, Takeda Science Foundation, and Keio University Medical Science Fund of Keio University (M. Kizaki).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Received 2/26/07; revised 5/21/07; accepted 5/30/07.


   References
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 Abstract
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 References
 

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