Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line

Blood Cells, Molecules and Diseases 59 (2016) 77–84

Contents lists available at ScienceDirect

Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd

Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line Sadudee Chotirat a, Tawit Suriyo b, Marianne Hokland c, Peter Hokland d, Jutamaad Satayavivad b,e, Chirayu U. Auewarakul f,⁎ a

Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus 8000, Denmark d Department of Haematology, Aarhus University Hospital, Aarhus 8000, Denmark e Center of Excellence on Environmental Health and Toxicology, Office of Higher Education Commission, Ministry of Education, Bangkok 10400, Thailand f Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand b c

a r t i c l e

i n f o

Article history: Submitted 22 December 2015 Revised 16 April 2016 Accepted 17 April 2016 Available online 20 April 2016 Editor: Mohandas Narla Keywords: Non-neuronal cholinergic system Carbachol NB-4 cells CD11b Differentiation

a b s t r a c t The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p b 0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p b 0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72 h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p b 0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The treatment of leukemia in the developing countries and in countries with restrained economics is a serious stumbling block for traditional cytoreductive measures [1]. Consequently, less expensive modalities are highly sought for. The non-neuronal cholinergic system (NNCS) functions through mediator acetylcholine (ACh) and its components including choline transporter (CHT), muscarinic (mAChRs) and nicotinic receptors (nAChRs: ACh receptors), choline acetyltransferase (ChAT: synthesizing ACh enzyme), and acetylcholinesterase (AChE: degrading ACh enzyme) [2,3]. Importantly, the physiological significance of the NNCS has been documented with respect to growth regulation, differentiation, secretion, barrier functions, immunomodulation, ⁎ Corresponding author at: Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkoknoi, Bangkok 10700, Thailand. E-mail addresses: [email protected] (S. Chotirat), [email protected] (T. Suriyo), [email protected] (M. Hokland), [email protected] (P. Hokland), [email protected] (J. Satayavivad), [email protected], [email protected] (C.U. Auewarakul).

http://dx.doi.org/10.1016/j.bcmd.2016.04.009 1079-9796/© 2016 Elsevier Inc. All rights reserved.

and apoptosis in both healthy and malignant cells including neuroblastoma, lung, colon, cervical, breast, prostate, and bile duct cancers [4–13]. Previously, mAChRs stimulation has also been shown to inhibit K562 chronic myelogenous leukemia cell line proliferation [14,15], while α7-nAChR stimulation has been reported to support HL-60 acute promyelocytic leukemia cell line differentiation [16]. Taken together, these data suggest to us that the cholinergic system could potentially be a modulator of leukemogenesis. In search of a model system to test this, acute promyelocytic leukemia (APL) could be of value. It represents a distinct subtype of acute myeloid leukemia (AML) [17], in which the balanced reciprocal translocation of chromosome 15 and 17 results in a fusion transcript between the promyelocytic leukemia gene (PML) and retinoic acid receptor-α (RARα) gene [18]. The fusion protein causes a differentiation arrest at the promyelocytic stage in addition to deregulation of apoptotic signaling cascade [19]. Importantly, this differentiation arrest can be lifted by a number of reagents. Thus, the combination of alltrans retinoic acid (ATRA) and arsenic trioxide (As2O3) targeting aberrant fusion genes has been shown to induce complete remission in up to 95% of APL patients [20,21]. This treatment modality has proven to

78

S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 77–84

be a cornerstone for APL therapy in the western countries, where subsequent standard cytoreduction is subsequently applied [22]. In some developing countries, such therapy constitutes the only modality in situations where other subsets of AML cannot be addressed due to economic constraints [1,23,24]. Little has been reported on the expression pattern of cholinergic components during promyelocytic maturation and their modulating regulatory effect on promyelocytic cell functions and cell growth. Consequently, in the present study, we analyzed the expression of cholinergic components in selected leukemic cell lines and human leucocyte subsets, and in particular, explored the functional role of NNCS in the APL-NB-4 cell line comprising cell viability, terminal differentiation, and apoptosis by using the combination of ATRA and carbachol treatment. The data raised should be extended to primary APL cells in order to further evaluate the clinical value of the cholinergic system in leukemogenesis.

plates (1 × 104 cells/100 μL/well). Cultures were treated with different concentrations of carbachol (0.01–1000 μM) in the medium containing either growth stimulation (supplemented with 10% serum) or deprivation condition (supplemented with 1% serum after cells were deprived of serum for 24 h). To confirm apoptosis of ATRA-induced differentiated NB-4 cells, cultures with ATRA (1 μM) alone were used as controls whereas As2O3 (1 μM) was utilized as a positive control for cell death. Twenty, 44, and 68 h post treatment, the cells were added 50 μL of XTT sodium salt (C22H16N7NaO13S2) diluent mixed with activator phenazinemethosulfate; PMS, C13H11N2·CH3SO4 (Sigma-Aldrich) and subsequently, incubated for another 4 h in 5% CO2 humidified incubator at 37 °C to achieve 24, 48, and 72 h treatment regimen, respectively. Finally, the optical density (OD) was measured at a wavelength between 450 and 500 nm using a SpectraMax M3 Microplate Reader (Molecular Devices, Sunnyvale, CA) to calculate the percentage of cell viability. 2.5. Flow cytometry

2. Material and methods 2.1. Reagents Carbamylcholine chloride (carbachol), atropine, mecamylamine, all-trans retinoic acid (ATRA), arsenic trioxide (As2O3), absolute ethanol (EtOH), and dimetylsulphoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Stock solution of carbachol was prepared in distilled water at 103 mM whereas 102 mM and 101 mM of ATRA and As2O3, respectively were prepared in ethanol and stored protected from light at − 80 °C. Fetal bovine serum (FBS) was purchased from JR Scientific (Woodland, CA). 2.2. Cell lines Leukemic cell lines (acute promyelocytic leukemia NB-4 (CLS300299); chronic myeloid leukemia K-562 (CLS-820700) were purchased from CLS cell line service (Germany); T-lymphocytic leukemia MOLT3 (ATCC® CRL-1552™); B lymphocyte RPMI1788 (ATCC® CCL156™) were obtained from ATCC®. All cell lines were cultured in RPMI-1640 (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% FBS, 2 mM of L-Glutamine, 1 mM sodium pyruvate, 20 mM D-Glucose, and 100 IU/mL of penicillin and 100 μg/mL streptomycin, (Invitrogen, Carlsbad, CA). SH-SY5Y cell line (ATCC®, CRL-2266™) was cultured in a 1:1 mixture of Ham's F12 and MEM (Gibco, Carlsbad, CA) supplemented with 10% FBS, 2 mM L-Glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. The cells used in the experiments were started at passage 5 and did not exceed passage 30. 2.3. NB-4 cell line differentiation Briefly, 10 μL of NB-4 cell suspension were well-mixed with Muse™ Count & Viability Cell Dispersal Reagent in 1:20 dilution, and analyzed for the total number of living cells by using the Muse® cell analyzer (Merck Millipore, Darmstadt, Germany). Stock solutions of ATRA and As2O3 were further diluted in culture medium to the final concentration (1 μM). The NB-4 cells were induced to granulocytic differentiation by ATRA treatment for 5 days. ATRA-induced differentiated NB-4 cells were counted and maintained at a concentration of approximately 2– 5 × 105 cells/mL and half of the medium was replaced and fresh ATRA added every 2 days. 2.4. Cell viability Cell viability was measured by the XTT cell viability assay according to manufacturer's instruction (Sigma-Aldrich). Briefly, non-differentiated or ATRA-induced differentiated NB-4 cells were generated in 96 well

The expression of CD11b, a differentiation marker for granulocytes, was measured by flow cytometry. One million NB-4 cells were harvested, washed twice with cold PBS (pH = 7.6) and incubated with antiCD11b-PE antibody (clone D12, BD Biosciences, San Jose, CA) for 30 min in the dark at room temperature. Thereafter, the cells were washed twice with cold PBS, resuspended in 500 μL of PBS and analyzed on a FACS Canto flow cytometer (Becton Dickinson, USA) acquiring 3 × 104 events per sample. For AChE expression on primary leukocyte subsets, a total of 5 × 105 mononuclear cells (MNCs) previously blocked with human Ig (CSL Behring AG, Bern, Switzerland) for 30 min in the dark at 4 °C or 100 μL of whole blood (WB) were stained with primary polyclonal rabbit-anti-human AChE antibody (Abgent, San Diego, CA) for 15 min and washed twice. The MNC samples were then incubated for another 30 min with goat-anti-rabbit PE secondary antibody together with anti-CD3 V450 (clone UCHT1, BD Biosciences, San Jose, CA), antiCD19 FITC (clone HD37, Dako, Glostrup, Denmark), anti-CD56 (clone CMSSB, eBioscience, San Diego, CA), and Live/Dead dye (Life Technologies, Oregon, CA), washed twice, and fixed with 0.9% formaldehyde. Following anti-AChE, the WB samples were further incubated for another 30 min with anti-CD11b (clone Bear1, Beckman Coulter, Fullerton, CA) and anti-CD14 (clone MϕP9, BD Biosciences) followed by incubation in FACS Lysis (BD Biosciences) for 10 min, washed once and resuspended in PBS. All samples were analyzed on an LSR Fortessa (Becton Dickinson) acquiring at least 5x104 events per sample. Using single stained compensation beads (eBioscience) a compensation matrix was created. The median fluorescence intensity (MFI) was calculated using the FlowJo software v.10.6 (TreeStar Inc., Ashland, OR). 2.6. Western blot analysis Approximately 5 × 106 cells were washed twice with cold PBS; pH 7.4 (2.7 mM potassium chloride, 1.8 mM potassium phosphate, 137 mM sodium chloride, 10.1 mM sodium phosphate). The cells were harvested on ice and incubated in 120 μL lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP40, 10 μL/mL PI cocktail, 0.1 mM PMSF, 1 mM Na3VO4, 20 mM NaF and 1 × protease inhibitor cocktail set I (Calbiochem, Germany). Cell lysates were sonicated and incubated at 4 °C for 30 min prior to centrifugation at 16,000 × g, at 4 °C for 15 min. The supernatant was collected and the protein concentration measured by Bradford reagent (Bio-Rad, Hercules, CA). Aliquot (35 μg) was electrophoresed onto with 7.5% or 12.5% SDS-polyacrylamide gel followed by electrotransfer to nitrocellulose membrane. The nitrocellulose membrane was incubated in blocking buffer for 1 h (5% non-fat dry milk in TBST; 50 mM of Tris, 150 mM of NaCl, 0.1% of Tween-20, pH 7.6). The membrane was incubated with primary antibodies against anti-cleave caspase-3; Clone Asp175 (1:1000), anti-caspase-3, and anti-MMP-9; Clone G657 (1:1000) purchased from Cell Signaling (Beverly, MA). Anti-PARP;

S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 77–84

Clone 7D3–6 (1:2000) was obtained from BD Pharmingen™ (San Diego, CA). Anti-AChE (1:1000), anti-M3-mAChR; Clone H210 (1:1000), antiα7-nAChR; Clone H302 (1:1000), and anti-p53; Clone DO-1, (1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against ChAT (1:1000), anti-CHT; Clone 62-2E8 (1:2000), anti-BAX (1:1000), and anti-Bcl-2; Clone 8C8 (1:1000) were from Merck Millipore. Mouse anti β-actin (1:40,000) was obtained from Sigma-Aldrich and incubated at 4 °C overnight with gently shaking.

79

The membrane was then washed three times for 10 min with TBST (50 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) and incubated for another 2 h with appropriate HRP-conjugated secondary antibodies conjugated with horseradish peroxidase. The protein-antibody conjugation was visualized by performing enhanced chemiluminescence (ECL) system (GE Healthcare, UK). The intensity of individual proteins was quantified by Image Quant TL software (GE Healthcare).

Fig. 1. The expression of different cholinergic components varies between different human leukemic, non-leukemic cell lines and primary leukocyte subsets. (A) A highly variable expression of ChAT, AChE, CHT, M3-mAChR, and α7-nAChR was detected among all leukemic cell lines tested by Western blotting. ChAT; choline acetyltransferase, AChE; acetylcholinesterase, CHT; choline transporter, M3-mAChR; M3 muscarinic acetylcholine receptor, α7-nAChR; α7 nicotinic acetylcholine receptor in NB-4; acute promyelocytic leukemia, K562; chronic myeloid leukemia, MOLT3; T-acute lymphoblastic leukemia, RPMI 1788; B lymphocyte cell. The cholinergic/dopaminergic neuroblastoma SH-SY5Y cells (positive cells) expressed all of the cholinergic components. (B) Heterogeneous expression of AChE on healthy whole blood and mononuclear cell subsets as analyzed by flow cytometry. Light gray histogram represents fluorescence minus one (FMO), which was used as a negative control for AChE. Dark gray histogram represents median fluorescence intensity (MFI) of AChE expression. WB, Whole blood; MNC, Mononuclear cell.

80

S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 77–84

2.7. Statistical analysis The results from at least three independent experiments were calculated and presented as the means ± SEM. Statistical analysis was tested by one-way ANOVA followed by Bonferroni post tests were used to compare between treatment groups test. Differences were considered significant at p-values b0.05. 3. Results 3.1. Expression of different cholinergic components in leukemic cell lines and human leukocytes We initially set out to determine the steady state levels of cholinergic components in leukocyte subsets. Given the diversity of nonmalignant cells, we chose flow cytometry analysis. For the hematopoietic leukemic lines, we chose Western blotting assay because it provides a clear visual comparison among cell lines. The variable expression of the different cholinergic components were identified including ChAT protein with a 68–70 kDa, AChE with a 71 kDa, CHT with a 65 kDa, M3mAChR with a 75 kDa, and α7-nAChR with a 55 kDa relative molecular mass (Fig. 1A). Notably, the expression of AChE and M3-mAChR proteins were observed at a higher level in the myeloid lineage cells, NB4 and K562, as compared to the MOLT3, T-ALL cell line. The expression of M3-mAChR protein was expressed at the lowest level in RPMI1788, which is a B lymphoblast cell line. Moreover, the expression of α7nAChR protein was expressed at a higher level in K562 and RPMI1788 cell line. By contrast, the expression of ChAT and CHT were similar among all cell lines (Supplementary Table 1.). While within the leukocyte subsets of whole blood, the CD11b+ granulocytes detected the highest AChE expression compared to both CD14+ monocytes and lymphocytes. With respect to lymphocyte subsets, the CD19+ B lymphocytes exhibited the highest AChE expression compared to CD56+ CD3− NK cells and CD3+ T lymphocytes, as shown in Fig. 1B. We conclude that AChE are ubiquitously expressed in all hematopoietic lineages. 3.2. Expression of cholinergic components after cholinergic stimulation in differentiated and non-differentiated NB-4 cells We went on to determine the cholinergic components of NB-4 cells before and after ATRA-induced differentiation, as detailed in Material and methods. Analyzing the expression of cholinergic components by Western blotting revealed that cholinergic stimulation by carbachol treatment (100 μM) induced an increase in the expression of ChAT and a relative decrease in AChE on non-differentiated NB-4 cells (Fig. 2). In contrast, ATRA-induced differentiated NB-4 cells showed an increased expression of ChAT but decreased expression of AChE and CHT, as compared with non-differentiated NB-4 either with or without carbachol addition (Table 1). By contrast, α7-nAChR expression remained unchanged after either ATRA or carbachol administration. Finally, the M3-mAChR expression was dramatically increased in ATRA treatment (P b 0.0001). Collectively, these data show that the leukemic differentiation process can modulate the expression of cholinergic components, especially M3-mAChR. 3.3. Effect of carbachol on the late differentiation marker, CD11b of ATRAinduced differentiated NB-4 cells We next sought to determine the mechanism of cholinergic stimulation on ATRA-induced differentiated NB-4 cells. We employed the analysis of CD11b expression by flow cytometry since this allows for the determination of small changes in median fluorescence intensity (MFI) and because of the high level of CD11b expression in neutrophils (Supplemental Fig. 1). To address this, unstimulated and ATRA-treated NB-4 cells were cultured for five days and then treated with carbachol

Fig. 2. Effect of carbachol and ATRA on the expression of cholinergic components in NB-4 cells. Western blotting of ChAT, AChE, CHT, M3-mAChR, α7-nAChR, and β-actin protein of non-differentiated (untreated), and ATRA-induced differentiated NB-4 cells (ATRA) treated with or without carbachol (100 μM) for 5 days. All the experiments were repeated three times. CCh, carbachol; ATRA, all-trans retinoic acid.

(10 to 1000 μM) for a further 24 h. Notably, the expression of CD11b level increased significantly in ATRA-induced differentiated NB-4 compared to non-differentiated NB-4 cells, validating the differentiation effect of ATRA in this cell line (Fig. 3). Furthermore, the addition of carbachol on ATRA-induced differentiated NB-4 cells resulted in a further increase in CD11b expression as compared to ATRA alone, as shown in Fig. 3A-B. These data show that even in optimally ATRA treated NB-4 cells, cholinergic stimulation can lead to further differentiation. 3.4. Effect of carbachol on viability and apoptosis in ATRA-induced differentiated NB-4 cells We next determined the effect of carbachol on ATRA-induced differentiated NB-4 with respect to their viability in an attempt to distinguish between differentiation and cytoreductive effects. This was accomplished by culturing NB-4 cells with ATRA for five days, before stimulated with carbachol for 24, 48, and 72 h. Here, the viability was significantly reduced when cells were exposed to 1000 μM of carbachol at 72 h post treatment, indicating an additional cytoreductive effect for the compound (Fig. 4). These results contrasted to those observed in non-differentiated NB-4 cells, where carbachol did not affect the Table 1 Effect of carbachol and ATRA on the expression of cholinergic components in NB-4 cells. Protein

ChAT AChE CHT M3-mAChR α7-nAChR

Expression levels of cholinergic components (fold of control) Control

CCh

ATRA

ATRA + CCh

1 1 1 1 1

2.268 ± 0.298 0.737 ± 0.061 1.045 ± 0.120 1.104 ± 0.182 1.31 ± 0.154

1.473 ± 0.589 0.571 ± 0.038* 0.4787 ± 0.093* 6.334 ± 0.535a,# 1.302 ± 0.138

1.036 ± 0.122 0.587 ± 0.099* 0.557 ± 0.176 6.187 ± 0.411 a,# 1.535 ± 0.167

Note: NB-4 cells were treated with 100 μM carbachol (CCh), 1 μM all-trans retinoic acid (ATRA), or the combination of 100 μM CCh and 1 μM ATRA for 5 days. Control group was non-differentiated cells (untreated). Protein expressions of cholinergic components were measured by Western immunobloting assay. Each data point represents mean ± SE of three independent experiments and expressed as a fold of the control. *(p b 0.05) and a(p b 0.0001) represent statistically significant difference from the control.#(p b 0.0001) represent statistically significant difference from the CCh treated group. ChAT; choline acetyltransferase, AChE; acetylcholinesterase, CHT; choline transporter, M3mAChR; M3 muscarinic acetylcholine receptor, α7-nAChR; α7 nicotinic acetylcholine receptor.

S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 77–84

81

Fig. 3. Effect of carbachol (CCA) on the expression of CD11b in non-differentiated and ATRA-differentiated NB-4 cells. Cells were treated with 1 μM ATRA for 5 days to induce differentiation. Non-differentiated and ATRA-differentiated NB-4 cells were treated with 10, 100, and 1000 μM CCA for 24 h. Expression level of differentiation marker CD11b was analyzed by flow cytometry. (A) Representative primary flow data of CD11b expression. The y-axis, cell count as percentage of maximum; x-axis, log scale of mean fluorescent intensity (MFI). (B) MFI of CD11b expression. Each data bar represents MFI mean ± SE of four independent experiments and expressed as a fold of the non-differentiated NB-4 untreated control. * (p b 0.05) represents statistically significant difference from the differentiated NB-4 ATRA treated alone group. # (p b 0.05) represent statistically significant difference from the non-differentiated NB-4 untreated control.

Fig. 4. XTT cell viability assay of ATRA-induced differentiated NB-4 cells. NB-4 cells were induced to differentiate beyond promyelocytic stage with ATRA (1 μM) for 5 days. Then ATRAinduced differentiated NB-4 cells were treated with carbachol at 0.01, 0.1, 1, 10, 100, and 1000 μM for 24, 48, and 72 h. The combination of ATRA (1 μM) and As2O3 (1 μM) treatment was used as a positive control of apoptosis event. Data are expressed as the mean ± SEM of 5 experiments. (* p b 0.05 as compared with control). As2O3, arsenic trioxide.

82

S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 77–84

viability (data not shown). Hence, carbachol reduced the viability of ATRA-treated cells, but did not affect the growth and death of nondifferentiated NB-4 cells. Given that carbachol decreased viability, we finally sought to determine to what extent this involved apoptosis. To this end, we determined by Western blotting the activation state of several pro-apoptotic proteins, including caspase-3, PARP, p53 and BAX. We found that cleaved caspase-3 but not cleaved-PARP was increased in ATRA-treated NB-4 cells following exposure to carbachol at a concentration of 100 μM for 24, 48, and 72 h as compared to ATRA alone (Fig. 5A). Moreover, carbachol did not affect the expression of pro-/anti-apoptotic correlated caspase 3, proteolyzed PARP, p53, BAX, and Bcl-2 proteins expression at the 24-h time point (Supplemental Fig. 2). In this setting, cholinergic stimulation significantly increased the expression of cleaved caspase-3 at 72 h when compared with ATRA alone (Fig. 5B-C), suggesting that carbachol-induced decrease in viability was the result of caspase-3 activation. In support of this inhibitory effect of growth, the increased expression of cleaved caspase-3 was abrogated by the pre-treatment with atropine (a muscarinic receptor antagonist), while mecamylamine (a nicotinic receptor antagonist) failed to inhibit this effect of carbachol (Fig. 5B-C). Collectively, these data show that carbachol mediates its function through muscarinic receptors. Given that carbachol treatment did not induce the activation of PARP after 72 h incubation, cholinergic stimulation in ATRA-induced NB-4 differentiated cells with caspase-3 activation was independent of other apoptosis initiation proteins. Moreover, there was no statistically significant difference in the fold change, either an increase or a decrease of control of cleaved caspase-3 activity and PARP proteolyzation at 48 h post-treatment (data not shown). 4. Discussion

Fig. 5. Effect of carbachol treatment involved in caspase-3 activation. ATRA-induced differentiated NB-4 cells were treated with carbachol 100 μM for 24 and 72 h in the absence or presence of atropine (AT, 1 μM) or mecamylamine (MM, 1 μM). (A) The bar graph indicates the expression of cleavedcaspase-3 of the combination ATRA and CCh as compared to the ATRA alone (dash line) in 24, 48, and 72 h treatment. (B) Western blot bands of apoptotic proteins expression including caspase-3 and PARP proteins were shown at post 72 h treatment. (C) The ratio of cleaved caspase-3 to β-actin was calculated, and expressed as fold of control. Data from four independent experiments (n = 4, *p b 0.05, ATRA alone. ATR, Atropine; MM, Mecamylamine; PARP, poly (ADP-ribose) polymerase.

As a preclinical model for evaluating the possible role of NNCS activation as a differentiation inducing strategy in APL, the present data set aims at investigating the expression of NNCS components expression in leukemic cell lines and to explore the effect of carbachol on differentiation and death of ATRA-induced differentiated NB-4 cells. Our data show that leukemic cell lines express a heterogeneous pattern of cholinergic components including ChAT, AChE, CHT, M3-mAChR, and α7-nAChR. Moreover, the expression is comparable to that seen in normal leukocyte subsets included here as controls. These data thus support and extend the data from a previous study, where α7-nAChR was reported to participate in HL-60 APL cell line differentiation beyond promyelocytic state [16]. Collectively, these findings support the notion that NNCS may regulate leukemic developmental state. Given that AChE can also modulate megakaryopoiesis and thrombopoiesis process [25], it can now be concluded that cholinergic stimulation has pleiotropic effects on hematopoiesis. With regards to the immune system, this is the first time that the expression of AChE has been extensively delineated on human leukocyte subsets. Somewhat surprisingly, their levels differ markedly. CD11b+ granulocytes showed the highest expression, being significantly more expressed than in both CD14+ monocytes and unfractionated lymphocytes. In addition, CD19+ B lymphocytes exhibited a higher AChE expression compared to CD56+/CD3− NK cells and CD3+ T lymphocytes. The reason that these results are different from previous study where AChE activity was higher in T lymphocytes than B lymphocytes may be due to the fact that the subsets were purified before analysis [26]. In any case, further studies are needed to address the biological significance of these differences. With respect to the studies in the leukemic cell lines summarized above (Fig. 1), we believe that the observation of M3-mAChR expression being dramatically increased after ATRA induction compared to nondifferentiated NB-4 cells is of great potential interest. This is in light of the fact that carbachol treatment seems to solely induce elevated ChAT expression whereas the combination of ATRA and carbachol decreased it. Taken together, these findings support the hypothesis that NNCS functions in both leukemia and myeloid cells. Supporting this,

S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 77–84

dendritic cell differentiation has previously been found to be required for the activation of muscarinic cholinergic receptors to mediate their function in antigen presenting cells [7]. Finally, it has been shown that nicotine enhanced differentiation on the HL-60 cell [16]. We chose to study the NB-4 cell line because this cell line harbors t(15:17) translocation whereas HL-60 lacks this rearrangement [27]. While the above findings were largely based on Western blotting, we have extended the notion of a functional role of cholinergic stimulation in myeloid differentiation by multiparameter flow cytometry. It is noteworthy that the combination of carbachol and ATRA significantly increased the expression of the late-stage differentiation CD11b marker as compared with ATRA treatment alone. This finding extends to those in HL-60 cells, where nicotine also increased CD11b [16]. The reason why carbachol failed to promote activated matrix metalloproteinase-9 (MMP-9), a molecule employed by effector neutrophils to degrade the extracellular matrix during migration to an inflammatory site, is not known (data not shown). It might be speculated that MMP-9 activation was independent of cholinergic modulation since statistically insignificant change in MMP-9 activation was observed post-carbachol treatment. Induction of apoptosis can be another effect supporting the use of cholinergic stimulation in inducing differentiation of maturationarrested leukemic cells. The data presented here show that in the NB4 cells, this effect seems to be of lesser importance for several reasons. Firstly, the increase of cleaved caspase-3 expression in the co-treated ATRA and carbachol group compared to ATRA treatment was only slight. Secondly, other correlated apoptosis molecules both pro-/antiapoptotic factors including PARP, p53, BAX and Bcl-2 were unaltered after carbachol treatment. Finally, in data not included here, we found that cholinergic activation had no influence on cell death events as evaluated by Annexin analysis (data not shown). Our data thus differ from previous studies which showed that muscarinic activation either promoted SH-SY5Y or inhibited K562 cell proliferation [14,28]. These contrasting findings can be reconciled by molecular differences in the cell lines applied. In particular, the NB-4 cell line contains missense mutations at the R248Q and R273H of p53 tumor suppressor at p53248/273 codon [29] which may lead to deregulation of NB-4 cell apoptosis [30] and make NB-4 less sensitive to induction of apoptosis than nonp53 mutations. Consistent with this, decreased AChE expression or deficiency of it has been reported in apoptosis resistance and tumor aggressiveness in squamous cell carcinoma, colorectal carcinoma, and hepatocellular carcinoma [9,31–33]. Given that decreased AChE expression may inhibit apoptosis, further experimentation should evaluate the role of AChE by using AChE stimulator in order to confirm the role of non-neuronal cholinergic components in induction of apoptosis in APL. In conclusion, our observation highlights the variable expression of cholinergic components on normal leukocyte subsets and shows that cholinergic stimulation enhances the development of ATRAinduced differentiation in NB-4 cells. The latter finding suggests the potential combination regimen of cholinergic stimulation in standard differentiation-inducing regimens in APL and possibly myelodysplastic syndrome (MDS) patients. In some developing countries with economic constraints, such therapy may constitute alternative therapeutic modality. However, further preclinical studies employing freshly isolated leukemic cells from APL patients to validate these findings are necessary before future clinical trials can be pursued. Conflicts of interests All authors declare that they have no conflicts of interest. Author contributions SC conducted experiments and performed data analysis and manuscript drafting. TS supervised study design, conduction of experiments, data analysis, and manuscript drafting and revision. MH and PH supervised flow cytometric studies and critical revision of the manuscript. JS

83

and CA were responsible for the initiation and monitoring of the project and revision of the final manuscript. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bcmd.2016.04.009. Acknowledgements The authors wish to thank the staffs of the Pharmacology Laboratory, Chulabhorn Research Institute, Thailand for their assistance with molecular technical practice on cell culture, flow cytometry techniques, and Western blotting assay and the staffs of FACS Core facility, Institute of Biomedicine, Aarhus University, Denmark for their supervision on flow cytometric analysis. CUA is a current recipient of Siriraj Chalermprakiat Fund and SC is a graduate student whose thesis is partially supported by Thailand Research Fund, Royal Golden Jubilee PhD Program, grant number; PHD/0241/2553. References [1] P. Hokland, F. Cotter, Real world data on acute myeloid leukaemia therapy from the developing world—an eye-opener, Br. J. Haematol. 170 (1) (2015) 1–2. [2] Y. Horiuchi, R. Kimura, N. Kato, T. Fujii, M. Seki, T. Endo, et al., Evolutional study on acetylcholine expression, Life Sci. 72 (15) (2003) 1745–1756. [3] I. Wessler, C.J. Kirkpatrick, Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans, Br. J. Pharmacol. 154 (8) (2008) 1558–1571. [4] S. Trombino, A. Bisio, A. Catassi, A. Cesario, C. Falugi, P. Russo, Role of the nonneuronal human cholinergic system in lung cancer and mesothelioma: possibility of new therapeutic strategies, Curr. Med. Chem. Anticancer Agents. 4 (6) (2004) 535–542. [5] A.J. Espanol, E. de la Torre, G.L. Fiszman, M.E. Sales, Role of non-neuronal cholinergic system in breast cancer progression, Life Sci. 80 (24–25) (2007) 2281–2285. [6] R.R. Resende, A. Adhikari, Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation, Cell Commun. Sig. CCS. 7 (2009) 20. [7] G. Salamone, G. Lombardi, S. Gori, K. Nahmod, C. Jancic, M.M. Amaral, et al., Cholinergic modulation of dendritic cell function, J. Neuroimmunol. 236 (1–2) (2011) 47–56. [8] A. Belo, K. Cheng, A. Chahdi, J. Shant, G. Xie, S. Khurana, et al., Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion, Am. J. Physiol. Gastrointest. Liver Physiol. 300 (5) (2011) G749–G760. [9] Y. Zhao, X. Wang, T. Wang, X. Hu, X. Hui, M. Yan, et al., Acetylcholinesterase, a key prognostic predictor for hepatocellular carcinoma, suppresses cell growth and induces chemosensitization, Hepatology 53 (2) (2011) 493–503. [10] K. Kawashima, T. Fujii, Y. Moriwaki, H. Misawa, Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function, Life Sci. 91 (21−22) (2012) 1027–1032. [11] E.A. Parnell, I.E. Calleja-Macias, M. Kalantari, S.A. Grando, H.U. Bernard, Muscarinic cholinergic signaling in cervical cancer cells affects cell motility via ERK1/2 signaling, Life Sci. 91 (21–22) (2012) 1093–1098. [12] J. Beckmann, K.S. Lips, The non-neuronal cholinergic system in health and disease, Pharmacology 92 (5–6) (2013) 286–302. [13] S. Amonyingcharoen, T. Suriyo, A. Thiantanawat, P. Watcharasit, J. Satayavivad, Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway, Int. J. Oncol. 46 (6) (2015) 2317–2326. [14] B. Aydin, B. Kan, H. Cabadak, The role of intracellular pathways in the proliferation of human K562 cells mediated by muscarinic receptors, Leuk. Res. 37 (9) (2013) 1144–1149. [15] H. Cabadak, B. Aydin, B. Kan, Regulation of M2, M3, and M4 muscarinic receptor expression in K562 chronic myelogenous leukemic cells by carbachol, J. Recept. Signal Transduct. Res. 31 (1) (2011) 26–32. [16] M. Xu, J.E. Scott, K.Z. Liu, H.R. Bishop, D.E. Renaud, R.M. Palmer, et al., The influence of nicotine on granulocytic differentiation — inhibition of the oxidative burst and bacterial killing and increased matrix metalloproteinase-9 release, BMC Cell Biol. 9 (2008) 19. [17] D. Grimwade, A.R. Mistry, E. Solomon, F. Guidez, Acute promyelocytic leukemia: a paradigm for differentiation therapy, Cancer Treat. Res. 145 (2010) 219–235. [18] A. Kakizuka, W.H. Miller Jr., K. Umesono, R.P. Warrell Jr., S.R. Frankel, V.V. Murty, et al., Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML, Cell 66 (4) (1991) 663–674. [19] U. Testa, R. Riccioni, Deregulation of apoptosis in acute myeloid leukemia, Haematologica 92 (1) (2007) 81–94. [20] Z.X. Shen, Z.Z. Shi, J. Fang, B.W. Gu, J.M. Li, Y.M. Zhu, et al., All-trans retinoic acid/ As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia, Proc. Natl. Acad. Sci. U. S. A. 101 (15) (2004) 5328–5335. [21] F. Lo-Coco, G. Avvisati, M. Vignetti, C. Thiede, S.M. Orlando, S. Iacobelli, et al., Retinoic acid and arsenic trioxide for acute promyelocytic leukemia, N. Engl. J. Med. 369 (2) (2013) 111–121. [22] A.R. Derolf, S.Y. Kristinsson, T.M. Andersson, O. Landgren, P.W. Dickman, M. Bjorkholm, Improved patient survival for acute myeloid leukemia: a population-

84

[23]

[24]

[25]

[26] [27]

S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 77–84 based study of 9729 patients diagnosed in Sweden between 1973 and 2005, Blood 113 (16) (2009) 3666–3672. V. Mathews, B. George, K.M. Lakshmi, A. Viswabandya, A. Bajel, P. Balasubramanian, et al., Single-agent arsenic trioxide in the treatment of newly diagnosed acute promyelocytic leukemia: durable remissions with minimal toxicity, Blood 107 (7) (2006) 2627–2632. C. Philip, B. George, A. Ganapule, A. Korula, P. Jain, A.A. Alex, et al., Acute myeloid leukaemia: challenges and real world data from India, Br. J. Haematol. 170 (1) (2015) 110–117. J. Stephenson, B. Czepulkowski, W. Hirst, G.J. Mufti, Deletion of the acetylcholinesterase locus at 7q22 associated with myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), Leuk. Res. 20 (3) (1996) 235–241. J.G. Szelenyi, E. Bartha, S.R. Hollan, Acetylcholinesterase activity of lymphocytes: an enzyme characteristic of T-cells, Br. J. Haematol. 50 (2) (1982) 241–245. K.H. Lee, M.Y. Chang, J.I. Ahn, D.H. Yu, S.S. Jung, J.H. Choi, et al., Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells, Biochem. Biophys. Res. Commun. 296 (5) (2002) 1125–1133.

[28] P. De Sarno, S.A. Shestopal, T.D. King, A. Zmijewska, L. Song, R.S. Jope, Muscarinic receptor activation protects cells from apoptotic effects of DNA damage, oxidative stress, and mitochondrial inhibition, J. Biol. Chem. 278 (13) (2003) 11086–11093. [29] D.S. Fleckenstein, C.C. Uphoff, H.G. Drexler, H. Quentmeier, Detection of p53 gene mutations by single strand conformational polymorphism (SSCP) in human acute myeloid leukemia-derived cell lines, Leuk. Res. 26 (2) (2002) 207–214. [30] S. Haupt, S. di Agostino, I. Mizrahi, O. Alsheich-Bartok, M. Voorhoeve, A. Damalas, et al., Promyelocytic leukemia protein is required for gain of function by mutant p53, Cancer Res. 69 (11) (2009) 4818–4826. [31] M.F. Montenegro, F. Ruiz-Espejo, F.J. Campoy, E. Munoz-Delgado, M.P. de la Cadena, F.J. Rodriguez-Berrocal, et al., Cholinesterases are down-expressed in human colorectal carcinoma, Cell. Mol. Life Sci. 63 (18) (2006) 2175–2182. [32] H. Qavi, A.A. Al-Rajhi, Acetylcholinesterase and HHV-8 in squamous cell carcinoma and retinoblastoma, In Vivo. 23 (5) (2009) 679–683. [33] X.J. Zhang, D.S. Greenberg, Acetylcholinesterase involvement in apoptosis, Front. Mol. Neurosci. 5 (2012) 40.