European Journal of Pharmacology 747 (2015) 36–44

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Rosmarinic acid potentiates ATRA-induced macrophage differentiation in acute promyelocytic leukemia NB4 cells Sook-Kyoung Heo a,1, Eui-Kyu Noh b,1, Dong-Joon Yoon a, Jae-Cheol Jo b, SuJin Koh b, Jin Ho Baek b, Jae-Hoo Park c, Young Joo Min a,b, Hawk Kim a,b,n a

Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan 682-060, Republic of Korea Department of Hematology and Oncology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan 682-714, Republic of Korea c Department of Hematology and Oncology, Myongji Hospital, Gyeonggi-do 412-270, Republic of Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 26 May 2014 Received in revised form 17 October 2014 Accepted 22 October 2014 Available online 3 December 2014

Rosmarinic acid (RA, an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid) has a number of biological activities, but little is known about anti-leukemic activities of RA combined with all-trans retinoic acid (ATRA) against acute promyelocytic leukemia (APL) cells. We examined the differentiation marker, CD11b, in bone marrow cells (BMC) of an APL patient, in NB4 cells (APL cell line), and in normal BMC and peripheral blood mononuclear cells (PBMC) of healthy subjects by flow cytometric analysis. ATRA/RA induced expression of CD11b in the BMC of the APL patient and in NB4 cells, but not in normal BMC or PBMC. Therefore, we realized that RA potentiated ATRA-induced macrophage differentiation in APL cells. Further characterization of the induced macrophages showed that they exhibited morphological changes and were able to phagocytose and generate reactive oxygen species. Th also had typical expression of C–C chemokine receptor type 1 (CCR1), CCR2, and intercellular adhesion molecule-1 (ICAM-1). Moreover, the expression of CD11b þ and CD14 þ cells depended on ERK-NF-κB axis activation. Together, these results indicate that RA potentiates ATRA-induced macrophage differentiation in APL cells. Thus, RA may play an important role as an appurtenant differentiation agent for functional macrophage differentiation in APL. Additionally, the differentiated macrophages might have a normal life span and, they could die. These data indicate that co-treatment with RA and ATRA has potential as an anti-leukemic therapy in APL. & 2014 Elsevier B.V. All rights reserved.

Chemical compounds studied in this article: Rosmarinic acid (Pubchem CID 5281792). Keywords: Rosmarinic acid All-trans retinoic acid Macrophage differentiation Acute promyelocytic leukemia Anti-leukemic activity Differentiation therapy

1. Introduction Acute promyelocytic leukemia (APL, also called M3 subtype) is a type of acute myeloid leukemia (AML) (Mi et al., 2012) and is a hematological malignancy driven by a chimeric oncoprotein containing the C-terminus of the retinoic acid receptor-α (RARα) fused to an N-terminal partner, generally promyelocytic leukemia protein (PML). This PML–RARα fusion acts as a transcriptional repressor and regulates various signaling pathways (Dos Santos et al., 2013). APL is also characterized by an accumulation of abnormal promyelocytes and by a t (15;17) translocation that is observed in more than 90% of APL cases; this translocation leads to the PML–RARα fusion gene, which can be a molecular target in the treatment of APL (Shima et al., 2013).

n Correspondence to: Division of Hematology and Hematological Malignancies, Department of Hematology and Oncology, Ulsan University Hospital, University of Ulsan College of Medicine, 877 Bangeojinsunhwan-doro, Ulsan 682-714, Republic of Korea. Tel.: þ82 52 250 8892; fax: þ82 52 251 8235. E-mail address: [email protected] (H. Kim). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ejphar.2014.10.064 0014-2999/& 2014 Elsevier B.V. All rights reserved.

All-trans retinoic acid (ATRA) was identified as a differentiation agent of APL cells in 1981 (Petrie et al., 2009). ATRA is used to treat APL but is not normally effective in non-APL patients because the drug does not cause proper transcriptional activation of retinoic acid receptor target genes (Harrison, 2012). ATRA has been used effectively to treat APL for many years, but some patients can develop resistance to the treatment (Congleton et al., 2012). Therefore, alternative or combination therapies are necessary to improve prognosis and survival. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, has been isolated as a pure compound from the plant Rosmarinus officinalis and was named accordingly (Petersen and Simmonds, 2003). Biological activities of RA have been extensively examined and include anti-oxidative, anti-inflammatory, anti-mutagen, anti-bacterial, and anti-viral activities (Moon et al., 2010). Further, RA inhibited Ca2 þ -dependent pathways of T-cell antigen receptormediated signaling (Kang et al., 2003) and induced apoptosis of activated T-cells from patients with rheumatoid arthritis (Hur et al., 2007). Recent reports indicate that RA has neuroprotective (Ghaffari et al., 2014), chemopreventive (Furtado et al., 2014), radiosensitizing

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(Alcaraz et al., 2014), and nephroprotective effects (Domitrovic et al., 2014). However, little is known about the activities of RA in combination with ATRA and the potential anti-leukemic activities on APL cells. We suggest here that RA promotes ATRA-induced differentiation of NB4 cells and stimulates macrophage functions including reactive oxygen species production, phagocytic activity, and expression of the chemokine receptors, C-C chemokine receptor type-1 (CCR-1) and CCR-2, and of the adhesion molecule, intercellular adhesion molecule1 (ICAM-1). Moreover, the expression of CD11b þ and CD14 þ cells depends on ERK-NF-κB axis activation.

2. Materials and methods 2.1. Materials RA, ATRA, zymosan A, 2-7-dichlorodihydrofluorescein diacetate (DCF-DA), polymyxin B, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich Co. (St. Louis, MO). The inhibiters PD98059, SB203580, and PP2 were from Merck Millipore (Billerica, MA). Fetal bovine serum (FBS), RPMI 1640 medium, and penicillin–streptomycin were obtained from GibcoBRL (Grand Island, NY). The phycoerythrin (PE)-conjugated anti-human CD11b and CD14 monoclonal antibodies (mAb) were purchased from BD Bioscience (San Diego, CA). Alexa Fluors 488 mouse anti-NF-κB p65 (phospho-S529) was purchased from BD Bioscience. Antibodies against phospho-ERK, ERK, IκBα, and β-actin were obtained from Cell Signaling Technology (Beverly, MA). DRAQ5TM was purchased from Abcam (Cambridge, MA).

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plates in the same medium with 10% FBS and 1% penicillin– streptomycin in a 5% CO2 humidified atmosphere at 37 1C. 2.6. Detection of CD11b and CD14 expression Cells (5  105 cells/ml) were cultured with 10 nM ATRA and/or 40 mM RA for 72 h. Then, cells were washed twice with flow cytometry buffer (PBS containing 0.1 NaN3 and 0.2% bovine serum albumin) and were incubated with the appropriate fluorochromelabeled mAb, including anti-human PE-conjugated CD11b, CD14, and isotype control, on ice for 30 min. After incubation, cells were washed three times with cold flow cytometry buffer. The cells were suspended in flow cytometry buffer and analyzed using the FACSCalibur flow cytometer and CellQuestPro software (BD Biosciences, San Jose, CA). In some experiments, several inhibitors were used to prevent mitogen-activated protein kinase (MAPK) or SRC activation; cells were preincubated with 10 μM MAPK inhibitor, PD98059 and SB203580, or SRC inhibitor, PP2, for 1 h at 37 1C prior to the addition of ATRA and RA. 2.7. Measurement of reactive oxygen species Cells were cultured with 10 nM ATRA and/or 40 mM RA for 72 h. Then, cells were washed twice with PBS. Reactive oxygen species production was evaluated by flow cytometry using the reactive oxygen species-sensitive fluorescent dye, DCF-DA. Cells were stained with 5 mM DCF-DA and 200 nM of PMA at 37 1C for 30 min. Cells were then washed twice with PBS. After washing, florescence intensity was analyzed with the FACSCalibur flow cytometer and CellQuestPro software using FL-1 detector (BD Biosciences, San Jose, CA).

2.2. Cell culture 2.8. Phagocytosis assay Human acute promyelocytic leukemia NB4 cells were grown as suspension cultures in 100-mm culture dishes or 24-well plates in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin (100 mg/ml streptomycin, 100 U/ml penicillin) under a 5% CO2 humidified atmosphere at 37 1C. 2.3. Human samples Blood and bone marrow samples were obtained once from healthy volunteers participating in this study at Ulsan University Hospital, Ulsan, South Korea; bone marrow cells (BMCs) were obtained from a 44-year-old male healthy control (HC)-1 and peripheral blood mononuclear cells (PBMCs) from a 42-year-old female HC-2. One patient recently diagnosed with APL (other diseases not specified) (APL-1, a 47-year-old female) at Ulsan University Hospital, Ulsan, South Korea participated in this study. Bone marrow sample with APL was collected prior to her first round of chemotherapy. 2.4. Ethics statement The two healthy volunteers and the patient with APL provided informed written consent before the study's commencement. The study protocol and patient consent form and information were approved by the Ulsan University Hospital Ethics Committee and Institutional Review Board (UUH-IRB-11-18). 2.5. Isolation of primary cells and culture The peripheral blood and bone marrow obtained from the subjects were drawn into heparinized tubes and were separated via density gradient centrifugation at 400g using LymphoprepTM (Axis-Shield, Oslo, Norway). PBMC and BMC were isolated, washed with RPMI 1640 medium, and then cultured in 24-well culture

Cells were cultured with 10 nM ATRA and/or 40 mM RA for 72 h. Then, cells were washed twice with PBS. Opsonized FITC-labeled zymosan particles were used to measure complement-mediated phagocytosis. Cells were incubated in PBS containing 2% FBS and 1 mg/ml FITC-zymosan at 37 1C for 40 min. After incubation, cells were washed twice with PBS and were analyzed by FlowSight (Millipore, Billerica, MA). In some experiments, to exclude the effect of endotoxin, the cells were pretreated with 25 μg/ml lipopolysaccharide (LPS) receptor antagonist polymyxin B at 37 1C for 1 h before measuring phagocytosis. 2.9. Quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) Total RNA was isolated from NB4 cells using the TRIzol Reagent (Invitrogen, Grand Island, NY), and the cDNA was synthesized using Superscript III Reverse Transcriptase according to the manufacturer's instructions (Invitrogen). cDNA was analyzed with the CFX96 real-time PCR system using iQ SYBR Green supermix (BioRad, Hercules, CA). The following primer pairs were used for the PCR: Homo sapiens intercellular adhesion molecule 1 (ICAM1): forward, 50 -ACCGCCAGCGGAAGATCAAGAAAT-30 and reverse, 50 CGTGGCTTGTGTGTTCGGTTTCAT-30 . Homo sapiens chemokine (C-C motif) receptor 1 (CCR1): forward, 50 -TCTTGGCTTCCATGCCAGGCTTAT-30 and reverse, 50 -AGGCAATACCAGCCCAAAGAGGTT-30 . Homo sapiens chemokine (C-C motif) receptor-like 2 (CCR2): forward, 50 TGCTCTGCTGTGTTTGTGATCGGT-30 and reverse, 50 -AGGGCAGGGTAAGCAAGAAACACA-30 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward, 50 -CATGTTCGTCATGGGTGTGAACCA-30 and reverse, 50 -AGTGATGGCATGGACTGTGGTCAT-30 . PCR conditions were an initial heat-denaturing step at 95 1C for 5 min, and 40 cycles at 95 1C for 20 s, 62 1C for 30 s, and 72 1C for 30 s. SYBR Green fluorescence data were acquired by optical detection during

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the 72 1C extension step. Expression of the target genes relative to the endogenous control gene, GAPDH, was calculated using the difference in threshold cycle (ΔCt ¼ Ct' target Ct' control) method as described in our previous report (Heo et al., 2008), in which the ΔΔ relative expression equaled 2  Ct (ΔΔCt ¼ ΔCt’ target ΔCt’ untreated). Each assay was performed in triplicate.

2.10. Immunoblotting Cell extracts were prepared in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO) for 20 min on ice and were centrifuged at 18,000g for 10 min to isolate total protein. Protein concentrations were determined using the BCA protein assay reagent (Thermo Scientific, Rockford, IL). Equal amounts of cell extracts (40–80 μg) were analyzed by SDS-PAGE and were electrotransferred to nitrocellulose membranes for 1.5 h. The membranes were blocked with 4% milk/0.05% PBST (PBS with 0.05% Tween 20) at room temperature for 1 h and blotted with their respective primary antibodies, antiphospho-ERK, ERK, IκBα, and β-actin, for 2 h. The membranes were washed three times with PBST, followed by incubation with HRP-conjugated anti-rabbit IgG secondary antibody for 1 h. Proteins were detected using the Immune-star WesternC kit (Bio-Rad, Hercules, CA).

2.11. Intracellular staining of NF-κB Cells were cultured with 10 nM ATRA and 40 mM RA for several time periods. Cells were harvested and were washed twice with flow cytometry buffer. Next, cells were fixed with 4% paraformaldehyde in PBS, after which they were added to a solution of 0.1% Triton X-100 in PBS for permeabilization. Cells were stained with anti-NF-κB p65 (phospho S529)-Alexa Fluors 488 mAb or isotype control mAb at 4 1C for 30 min. Samples were then analyzed using the FACSCalibur flow cytometer and Cell-Quest PRO software. After flow cytometry, the nuclei of cells were stained by DRAQ5TM (5 μM) to indicate the nucleus location. Stained cells were analyzed using FlowSight and IDEAS software (Millipore, MA). To prevent ERK activation, cells were preincubated with the MEK/ERK inhibitor, PD98059, for 1 h at 37 1C prior to the addition of ATRA and RA.

2.12. Detection of CD11b þ Annexin V þ cells NB4 cells were cultured with 10 nM ATRA, 40 mM RA, and/or 1 or 5 μM dasatinib for 72 h at 37 1C. Cells then were washed twice with flow cytometry buffer (PBS containing 0.3% BSA and 0.1% NaN3). First, cells were stained anti-hCD11b-PE on ice for 30 min. After incubation, they were washed twice with flow cytometry buffer. Second, cells were incubated with Annexin V-FITC from the Apoptosis Detection Kit I (BD Bioscience, San Diego, CA) on ice for 30 min. Cells then were washed twice with FACS buffer and, finally, were analyzed using the FACSCalibur flow cytometer and CellQuestPro software.

2.13. Statistical analysis Statistical analysis was performed using GraphPad Prism ver. 6.0 (La Jolla, CA). Results were expressed as mean 7standard error of mean (S.E.M) from at least three independent experiments. Data values were evaluated by one-way ANOVA, followed by Turkey range tests. The differences between treatment groups were considered as significant at P o0.05.

3. Results 3.1. Phenotype of ATRA and RA-treated NB4 cells To determine the effect of RA treatment on myeloid differentiation, NB4 cells were incubated with RA in the presence or in the absence of ATRA and, then, were monitored by flow cytometric analysis of CD11b and CD14 expression as previously described (Noh et al., 2010). Cotreatment with ATRA and RA markedly increased expression of CD11b and CD14 (19-fold and 16-fold higher than DMSO-treated control, respectively), while treatment with RA alone had no effect on differentiation marker expression in NB4 cells (Table 1). We also confirmed the expression of CD16, CD56, CD83, CD14/ CD16, and HLA-DR in the ATRA/RA-activated NB4 cells. As shown in Table 1, combination treatment significantly increased the expression of CD16 (7-fold higher than control). There are three types of CD16 þ cells in the immune cell subsets including macrophages, dendritic cells (DC), and natural killer (NK) cells. Thus, we examined the expression of the DC marker, CD83, and the NK cells marker, CD56, in the ATRA/RA-activated NB4 cells. Their expression level did not change. By contrast, the population of CD14 þ /CD16 þ cells increased significantly by RA or ATRA/RA treatment. Finally, combination treatment significantly increased the expression of HLA-DR (6.3-fold higher than control). These results indicate that RA enhances ATRA-induced APL cell differentiation and, at the same time, induces activated macrophage differentiation. Moreover, the ATRA/RA-induced HLA-DR þ activated macrophages can act as professional antigen-presenting cells and, thus, as fully functional macrophages. Therefore, RA plays a crucial role in differentiation of NB4 cells. Next, we wanted to confirm that these effects were APL specific. We examined the combination effects of ATRA and RA on CD11b expression in BMC from a patient with APL and in BMC and PBMC from healthy controls. As shown in Fig. 1, ATRA and RA stimulation induced expression of CD11b in the BMC of APL, but not in the BMC or PBMC of healthy controls. Thus, ATRA/RA-induced macrophage differentiation was found only in APL cells. These results indicate that the synergistic effects of the ATRA/RA combination appear to be APL specific. 3.2. RA enables morphological change, reactive oxygen species production, and phagocytosis in ATRA-activated NB4 cells We confirmed the synergistic effects of RA and ATRA on morphological changes, reactive oxygen species production, and phagocytosis. Cells were stimulated with 10 nM ATRA and/or 40 mM RA for 72 h and were analyzed using an inverted microscope for morphologic changes and flow cytometry for reactive oxygen species production against PMA. As seen in Fig. 2A, expression of jagged-edged cells, such as DC, Table 1 Phenotypes of ATRA and RA -treated NB4 cells by using flow cytometer. Cell surface antigen

Control

ATRA

CD11b þ CD14 þ CD16 þ CD56 þ CD83 þ CD14 þ CD16 þ HLA-DR þ

2.47 0.3 2.37 0.2 3.07 0.6 5.77 1.1 6.27 1.7 0.87 0.4 1.17 0.5

18.07 4.8a, 26.37 3.4b 1.97 0.8d 5.27 1.2 8.57 1.7 1.17 0.6d 1.77 0.8c

d

RA

ATRA þ RA

4.57 0.9d 7.97 1.7d 15.27 1.1b 6.07 0.1 7.27 2.2 5.87 1.9b 3.97 1.8

46.0 73.1b 37.1 73.6b 20 72.7b 5.1 71.4 5.5 71.6 4.8 72.2b 7.1 73.6b

These data represent the means 7S.E.M. ATRA: all-trans-retinoic acid, RA: Rosmarinic acid. a

Po 0.01 vs compared with DMSO-treated control. P o 0.001 vs compared with DMSO-treated control. c P o0.01 vs compared with combination of ATRA and RA. d P o0.001 vs compared with combination of ATRA and RA. b

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phagocytic activity against FITC-labeled zymosan was determined. We found that combination of ATRA and RA significantly stimulated phagocytosis by the differentiated macrophages (48% enhancement compared with control, Fig. 2C). These results indicate that ATRA and RA contribute to macrophage differentiation of NB4 cells (Table 1 and Fig. 1). As shown in Fig. 2C, pretreatment with polymyxin B or with PBS had no effect on the phagocytosis activity that was measured against opsonized FITC-labeled zymosan, demonstrating that there was no endotoxin contamination in our system. 3.3. ATRA and RA increases CCR-1, CCR-2, and ICAM-1 expression in NB4 cells Chemokine receptors play important roles in the defense against exogenous antigens (Baggiolini et al., 1997). We examined the effect of ATRA and RA on CCR-1, CCR-2 and ICAM-1 mRNA expression. Cells were cultured with ATRA and/or RA for several time periods and were analyzed by QRT-PCR. As shown in Fig. 3, CCR-1, CCR-2, and ICAM-1 mRNA expression increased following ATRA and RA activation, and these changes occurred in a time-dependent fashion. The maximal signals for CCR-1, CCR-2, and ICAM-1 expression were at 6 h of ATRA and RA treatment in NB4 cells. 3.4. Induction of CD11b and CD14 by ATRA/RA is via ERK pathway In Table 1 and Fig. 1A, we show that myeloid differentiation markers such as CD11b and CD14 were induced significantly by ATRA and RA stimulation in APL cells. Mitogen-activated protein kinases (MAPKs) are associated with a number of cellular signaling pathways (Boutros et al., 2008). We examined these signaling pathways using several inhibitors including MEK/ERK inhibitor (10 μM PD98059), p38 MAPK inhibitor (10 μM SB203580), and SRC family inhibitor (10 μM PP2). The NB4 cells were preincubated with PD98059, SB203580, and PP2 for 1 h and, then, were activated with ATRA and RA. We found that the populations of ATRA/RA-induced CD11b þ and CD14 þ cells markedly decreased by treatment with PD98059, but not with SB203580 or PP2 (Fig. 4A and B). On the contrary, SB203580 and PP2 significantly increased the populations of ATRA/RA-induced CD11b þ and CD14 þ cells. All inhibitors alone had no effect on CD11b and CD14 expression in NB4 cells. Based on the results shown in Fig. 4A and B, we measured ERK phosphorylation following ATRA and RA stimulation. The combination of ATRA and RA increased ERK phosphorylation, and the maximum signal was reached at 4 h (Fig. 4C). Therefore, these results suggested that expression of CD11b and CD14 in ATRA/RAtreated APL cells depended on ERK pathway (Fig. 4). 3.5. ERK is essential for ATRA/RA-induced NF-κB activation Fig. 1. Effects of RA and ATRA on CD11b expression in APL patient and healthy control samples. Cells were incubated with 10 nM ATRA and/or 40 μM of RA for 72 h. Cells then were harvested and were immunostained with anti-human CD11b mAb. The expression of CD11b then was measured by flow cytometry. (A) BMC of APL patient. (B) BMC of HC-1. (C) PBMC of HC-2. Data represent the mean 7 S.E.M. Statistically significant difference from the DMSO-treated control (n) or combination of ATRA and RA (#); ##: Po 0.01; nnn, ###: P o0.001. BMC, bone marrow cells; PBMC, peripheral blood mononuclear cells; HC, healthy control.

increased after exposure to ATRA and RA. Additionally, reactive oxygen species production in ATRA and RA-treated cells was 4.7-fold higher than in the control group (Fig. 2B). Based on results shown in Fig. 1A and B, we tested whether phagocytic activity of the differentiated macrophage increased in cells stimulated by ATRA and/or RA treatment. Cells were collected and treated under the same conditions described above, and

NF-κB is a ubiquitously expressed transcription factor and is crucial for elementary cellular responses. Optimal activation of NFκB requires phosphorylation of p65 (Rel A) (Perkins, 2007). After cells were stimulated with ATRA and RA for 72 h, we measured the levels of phospho-p65 (S529) in NB4 cells over a time course. As shown in Fig. 5A, the maximum level of expression was at 16 h, and this level was 4.5-fold higher than at 0 h. We also compared the levels of p65phosphorylation following stimulation with ATRA/RA or LPS, a wellknown, classical stimulus. The largest signal phospho-p65 (S529) in ATRA/RA-treated cells was much higher than that of LPS (fold of control, ATRA/RA at 16 h: 4.5-fold vs LPS at 8 h: 3.4-fold). These results indicate that ATRA and RA combination is very powerful to stimulate NF-кB and is a stronger stimulus than LPS. Simultaneously, the location of phospho-p65 (S529) by ATRA and RA activation was determined by FlowSight imaging flow cytometry. The phospho-p65 (S529) was located in the nucleus at 16 h (Fig. 5B).

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Fig. 2. Synergistic effects of ATRA and RA on morphological changes, reactive oxygen species production, and phagocytosis. (A) Morphological changes of NB4 cells by ATRA and RA. Cells were stimulated with 10 nM ATRA and/or 40 mM RA for 72 h and were analyzed for morphological changes using an inverted microscope. (B) Reactive oxygen species production. Cells were incubated with ATRA and RA for 72 h. To evaluate reactive oxygen species production, cells were stained with the reactive oxygen speciessensitive fluorescent dye, DCF-DA, for 30 min. After incubation, cells were harvested and washed before the percentages of reactive oxygen species-positive cells were assessed by flow cytometry. (C) Phagocytosis. Opsonized FITC-labeled zymosan particles were used to measure complement-mediated phagocytosis. Cells were incubated in PBS containing 2% FBS and 1 mg/ml FITC-zymosan at 37 1C for 40 min. After incubation, cells were washed twice with PBS and analyzed by FlowSight. Data represent the mean 7S.E.M. Statistically significant difference from control (n) or combination of ATRA and RA (#); n: Po 0.05; nnn, ###: P o 0.001. polyB, Incubation with polymyxin B.

To determine the role that ATRA/RA-induced ERK played in NF-

κB activation (IκBα degradation), we pretreated NB4 cells with the MEK/ERK inhibitor, PD98059, prior to ATRA and RA stimulation. As shown in Fig. 5C, western blot analysis revealed that the ATRA/RAinduced IκBα degradation was reduced by pretreatment with PD98059. Once again, cells were collected and treated under the same conditions described above, and intracellular staining of phospho-p65 (S529) was determined by flow cytometry. We found a similar result to that shown in Fig. 5C; blocking ERK signaling inhibits ATRA/RA-induced NF-κB activation (Fig. 5D). Therefore, these results indicate that ERK is essential for ATRA/RA-induced

NF-кB activation, and activation of differentiated macrophages by ATRA/RA treatment depends on ERK-NF-кB cascade. 3.6. Synergistic effects of ATRA, RA, and dasatinib on the differentiation and cell death of APL cells Recently, it has been suggested that dasatinib could be used for the differentiation of AML cells (Fang et al., 2013) and that dasatinib enhances ATRA-induced differentiation of AML cells (Kropf et al., 2010). Thus, we also evaluated the effects of dasatinib on ATRA/RA-treated NB4 cells. Dasatinib significantly increased

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Fig. 4. ERK signaling is critical for CD11b and CD14 induction by ATRA/RA. Cells were preincubated with 10 μM of the MAPK inhibitors, PD98059 or SB203580, or the SRC inhibitor, PP2, for 1 h at 37 1C prior to the addition of ATRA and RA. Cells were cultured with 10 nM ATRA and/or 40 mM RA for 72 h. Then, cells were washed twice with flow cytometry buffer and were stained with the appropriate mAb. (A) Expression of CD11b þ cells. (B) Expression of CD14 þ cells. (C) Western blot analysis of phospho-ERK1/2 and ERK1/2 expression following ATRA and RA treatment. The results are representative of three independent experiment. Data represent the mean7 S.E.M. Statistically significant difference from control (n) or combination of ATRA and RA (#); ##: P o0.01; nnn, ###: Po 0.001.

4. Discussion Fig. 3. ATRA/RA increases mRNA expression of CCR1, CCR2, and ICAM-1. The mRNA expression of CCR1 (A), CCR2 (B), and ICAM-1 (C) was determined by quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR). Data were normalized to GAPDH mRNA and represent the fold change relative to the control. Data represent the mean 7 S.E.M. Statistically significant difference from control (n); n: Po 0.05; nn: Po 0.01; nnn: Po 0.001. CCR1, C-C chemokine receptor type 1; CCR2, C-C chemokine receptor type 2; ICAM-1, intercellular adhesion molecule-1.

the population of CD11b þ cells in ATRA/RA-treated NB4 cells (Fig. 6A). RA also markedly increased the population of CD11b þ cells in ATRA/dasatinib-treated NB4 cells in a dose-dependent manner (Fig. 6B). Moreover, combination of ATRA, RA, and dasatinib induced apoptosis of differentiated CD11b þ NB4 cells, as shown in Fig. 6C and D. Therefore, using ATRA, RA, and dasatinib in combination therapy could be more effective than dual-combination therapy in the treatment of APL. Together, all three materials have a synergistic effect on CD11b þ cell differentiation and cell death.

Since the 1970s, the concept of differentiation therapy has been regarded as a promising and new approach for the treatment of acute myeloid leukemia (AML) and other cancers. However, the successful clinical application of differentiation therapy has been recognized only since the late 1980s and only in one subtype of AML, acute promyelocytic leukemia (APL) (Petrie et al., 2009). ATRA induces differentiation in an AML cell line (Breitman et al., 1980) and in APL (but not other AML subtypes) patient samples (Breitman et al., 1981). Furthermore, the use of ATRA, which induces degradation of the PML–RARα fusion oncoprotein, in combination with chemotherapy is currently the accepted treatment of APL, presenting a potential paradigm for differentiation therapy in clinical oncology (Petrie et al., 2009). Therefore, we sought to determine if RA could have anti-leukemic activities against APL cells in combination with ATRA treatment. As shown in Table 1 and Fig. 1, induction of CD11b expression by ATRA and RA stimulation was observed in the NB4 APL cell line and in the BMC of the APL patient, but not in the BMC or PBMC of the

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Fig. 5. NF-кB is downstream of ERK activation in ATRA/RA-treated NB4 cells. Cells were intracellularly stained with anti-NF-κB (phospho-p65 S529)-Alexa Fluors 488 or isotype control mAb, followed by flow cytometery analysis. (A) The expression of intracellular phospho-p65 (S529) by ATRA/RA- and LPS-stimulation over the time course. (B) After 16 h of stimulation, cells were monitored by Flowsight analysis. (C) The expression of IκBα was measured by western blot analysis, and β-actin was used to confirm equal loading. (D) Mean fluorescence intensity (MFI) of phospho-p65 (S529). The results are representative of three independent experiment. Data represent the mean 7 S.E.M. Statistically significant difference from control (n) or combination of ATRA and RA (#); nnn, ###: P o 0.001.

healthy controls. Thus, ATRA/RA-induced macrophage differentiation was found only in APL cells. These results indicate that the synergistic effects of the ATRA/RA combination are APL specific. Thus, RA plays an important role as an adjuvant for potentiating ATRA-induced differentiation. Combination treatment significantly increased the expression of CD14 and CD16, whereas CD56 and CD83 expression was unchanged. Conversely, RA or ATRA/RA treatment increased significantly the population of CD14 þ /CD16 þ cells. This subpopulation represents approximately 975% of total monocytes, and these cells have very strong effects on the anti-tumor response (Szaflarska et al., 2004). Moreover, combination treatment significantly increased the expression of HLA-DR (Table 1). These results indicate that RA enhances ATRA-induced APL cell differentiation and, simultaneously, induces macrophage differentiation. Furthermore, ATRA/RA-induced HLA-DR þ activated macrophages can be professional antigen-presenting cells. Usually, ATRA treatment alone is not used for patients with APL because ATRA alone has a lower complete remission rate and a higher incidence of ‘ATRA syndrome’ than when used in combination with cytotoxic chemotherapeutic agents or arsenic trioxide. Risk of opportunistic infections in the presence of leukopenia during therapy is a very important clinical consideration, especially when ATRA is combined with cytotoxic agents. Thus, RA-potentiated macrophage differentiation might strengthen the host defense system. It is well-known that most cancer cells have immune system evasion mechanisms and can escape cytotoxic T lymphocytes (CTL)mediated immune surveillance (Vonderheide and Bayne, 2013). As

such, because ATRA and RA treatment can induce functional, antigen-presenting macrophages, ATRA/RA therapy might increase the immune surveillance capacity in patients with APL. Next, we confirmed the synergistic effects of ATRA and RA on morphological changes, reactive oxygen species production, and phagocytosis. As shown in Fig. 1A, jagged-edged cells significantly increased after activation by ATRA and RA. Because the cells looked like DC, we evaluated the cell surface antigen, CD83, a well-known DC marker. However, it was not changed by RA and ATRA stimulation as shown in Table 1. According to ZieglerHeitbrock, monocytes can give rise to macrophages or DC; when cultured for 2 days in the presence of TLR2 ligands, CD16 þ monocytes, especially, developed preferentially into CD11b þ DC with high antigen-presenting capacity (Ziegler-Heitbrock, 2007). In our study, the ATRA/RA-induced CD16 þ monocytes/macrophages could have the potential to differentiate to DC if TLR2 signals also were provided in the presence of ATRA and RA. As shown in Fig. 3, combination of ATRA and RA increased the mRNA expression of CCR1, CCR2, and ICAM-1, which would affect macrophage migration. Collectively, as we summarize in Fig. 7, activated macrophages were induced by ATRA and RA and were characterized by several activities including morphological changes (Fig. 2A), reactive oxygen species generation (Fig. 2B), phagocytosis (Fig. 2C), and the expression of CCR1, CCR2, and ICAM-1 (Fig. 3), which might contribute to differentiate the functional macrophages. Moreover, the expression of CD11b þ and CD14 þ cells depended on ERK-NF-κB axis activation. Together these results indicate that RA

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Fig. 6. RA and ATRA sensitize dasatinib-induced CD11b þ expression and cell death. Cells were cultured with 10 nM ATRA, 40 mM RA, and/or 1 or 5 μM of dasatinib for 72 h. Then, cells were washed twice with flow cytometry buffer and were stained with anti-human CD11b mAb. (A) ATRA and RA effects on dasatinib-induced CD11b expression are more potent. (B) RA potentiates CD11b induction by ATRA/dasatinib. (C) Cells were stained with CD11b and Annexin V, and were analyzed by flow cytometry. (D) Mean of the values depicted in (C). Data are represented as the mean 7 S.E.M. Statistically significant difference from control (n) or combination of ATRA and RA (#); n :Po 0.05; nn : Po 0.01; nnn, ###: P o 0.001.

potentiates ATRA-induced macrophage differentiation in APL cells including NB4 cells and BMC from a patient with APL (Figs. 4 and 5). It has been reported that dasatinib enhanced ATRA-induced differentiation of AML cells (Kropf et al., 2010). As shown in Fig. 6A, dasatinib significantly increased the percentage of CD11b þ cells in ATRA/RA-treated NB4 cells. RA also prominently increased the percentage of CD11b þ cells in ATRA/dasatinib-treated NB4 cells in a dose-dependent manner (Fig. 6B). Interestingly, combination of ATRA, RA, and dasatinib induced apoptosis in differentiated CD11b þ NB4 cells (Fig. 6C and D). Therefore, using of ATRA, RA, and dasatinib in combination therapy could be more effective than dual-combination therapy in treating for APL patients. Recently, research has focused on the ability of natural compounds to sensitize cancer cells, such as the ability of curcumin to chemosensitize 5-fluorouracil-resistant human colon cancer cells (Shakibaei et al., 2014) and modified arabinoxylan from rice bran,

MGN-3/Biobran, to sensitize metastatic breast cancer cells to paclitaxel therapy (Ghoneum et al., 2014). According to Fig. 6, RA might be a useful chemosensitizer to some chemotherapy drugs such as ATRA or dasatinib. In conclusion, the present results demonstrated for the first time that RA and ATRA treatment induced functional macrophage differentiation in APL cells including NB4 cells and BMC of a patient with APL, but not in BMC and PBMC of healthy controls. Fig. 7 summarizes our results and shows the ATRA/RA-based differentiation therapy as an APL therapeutic strategy. APL-blasts are immature cells that have blocked differentiation capacity and accumulate in bone marrow. ATRA/RA-based differentiation therapy might help APLblasts to become matured cells (CD11 þ macrophages), which could have a normal life span or become chemosensitive. Consequently, they are susceptible to death. These differentiation therapies have a low toxicity and, thus, are very useful ways to treat APL patients.

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Fig. 7. Differentiated macrophages by ATRA and RA played several roles including morphological change, reactive oxygen species generation, phagocytosis, and the expression of CCR1, CCR2 and ICAM-1 that might be functional macrophages. Moreover, the expression of CD11b þ and CD14 þ cells depended on ERK-NF-κB axis activation. These results indicate that RA potentiates ATRA-induced macrophage differentiation in APL cells including NB4 cells and BMC from a patient with APL.

These results indicate that RA potentiates ATRA-induced macrophage differentiation in APL cells. At the same time, RA can play an important role as appurtenant differentiation agent in APL. Finally, co-treatment with RA and ATRA together has potential as an antileukemic therapy in APL.

Acknowledgment This work was supported by Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20090094050).

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