Blood Cells, Molecules and Diseases 59 (2016) 7784

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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
b
Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok 10210, Thailand
c
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, Ofce of Higher Education Commission, Ministry of Education, Bangkok 10400, Thailand
f
Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differ-
Submitted 22 December 2015 entiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting
Revised 16 April 2016 and in normal leukocyte subsets by ow cytometry and found a heterogeneous expression of choline acetyltrans-
Accepted 17 April 2016
ferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor
Available online 20 April 2016
(M3-mAChR) and 7 nicotinic acetylcholine receptor (7-nAChR). We then evaluated NNCS role in differentia-
Editor: Mohandas Narla tion 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
Keywords: the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with
Non-neuronal cholinergic system ATRA treatment alone (p b 0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 matu-
Carbachol ration. The combination of carbachol and ATRA treatment for 72 h also resulted in decreased viability and
NB-4 cells increased cleaved caspase-3 expression when compared with ATRA treatment alone (p b 0.05). However, this
CD11b combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4
Differentiation
cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced matura-
tion 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 tra-
ditional cytoreductive measures [1]. Consequently, less expensive mo-
dalities are highly sought for. The non-neuronal cholinergic system
(NNCS) functions through mediator acetylcholine (ACh) and its compo-
nents 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 signi-
cance of the NNCS has been documented with respect to growth regula-
tion, 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: sadudee.chotirat@gmail.com (S. Chotirat), tawit@cri.or.th
(T. Suriyo), mhokland@biomed.au.dk (M. Hokland), phokland@clin.au.dk (P. Hokland),
jutamaad@cri.or.th (J. Satayavivad), chirayuaue@yahoo.com, chirayu.aue@mahidol.ac.th
(C.U. Auewarakul).
and apoptosis in both healthy and malignant cells including neuroblas-
toma, lung, colon, cervical, breast, prostate, and bile duct cancers [413].
Previously, mAChRs stimulation has also been shown to inhibit K562
chronic myelogenous leukemia cell line proliferation [14,15], while
7-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- (RAR) gene [18]. The fusion protein causes a differentia-
tion arrest at the promyelocytic stage in addition to deregulation of ap-
optotic signaling cascade [19]. Importantly, this differentiation arrest
can be lifted by a number of reagents. Thus, the combination of all-
trans retinoic acid (ATRA) and arsenic trioxide (As2O3) targeting aber-
rant fusion genes has been shown to induce complete remission in up
to 95% of APL patients [20,21]. This treatment modality has proven to

http://dx.doi.org/10.1016/j.bcmd.2016.04.009
1079-9796/ 2016 Elsevier Inc. All rights reserved.
78 S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 7784
be a cornerstone for APL therapy in the western countries, where subse-
quent 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. Con-
sequently, in the present study, we analyzed the expression of choliner-
gic 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 treat-
ment. The data raised should be extended to primary APL cells in
order to further evaluate the clinical value of the cholinergic system in
leukemogenesis.
2. Material and methods
2.1. Reagents
Carbamylcholine chloride (carbachol), atropine, mecamylamine,
all-trans retinoic acid (ATRA), arsenic trioxide (As2O3), absolute eth-
anol (EtOH), and dimetylsulphoxide (DMSO) were purchased from
Sigma-Aldrich (St. Louis, MO). Stock solution of carbachol was pre-
pared 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 pur-
chased from JR Scientic (Woodland, CA).
2.2. Cell lines
Leukemic cell lines (acute promyelocytic leukemia NB-4 (CLS-
300299); chronic myeloid leukemia K-562 (CLS-820700) were pur-
chased from CLS cell line service (Germany); T-lymphocytic leukemia
MOLT3 (ATCC CRL-1552); B lymphocyte RPMI1788 (ATCC CCL-
156) 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 g/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 g/mL streptomycin. All cell lines were maintained at 37 C in
a humidied 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
Briey, 10 L 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 nal concentration
(1 M). 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). Briey, non-differentiated
or ATRA-induced differentiated NB-4 cells were generated in 96 well
plates (1 104 cells/100 L/well). Cultures were treated with different
concentrations of carbachol (0.011000 M) in the medium containing
either growth stimulation (supplemented with 10% serum) or depriva-
tion condition (supplemented with 1% serum after cells were deprived
of serum for 24 h). To conrm apoptosis of ATRA-induced differentiated
NB-4 cells, cultures with ATRA (1 M) alone were used as controls where-
as As2O3 (1 M) was utilized as a positive control for cell death. Twenty,
44, and 68 h post treatment, the cells were added 50 L of XTT
sodium salt (C22H16N7NaO13S2) diluent mixed with activator
phenazinemethosulfate; PMS, C13H11N2CH3SO4 (Sigma-Aldrich) and
subsequently, incubated for another 4 h in 5% CO2 humidied incubator
at 37 C to achieve 24, 48, and 72 h treatment regimen, respectively. Final-
ly, the optical density (OD) was measured at a wavelength between 450
and 500 nm using a SpectraMax M3 Microplate Reader (Molecular De-
vices, Sunnyvale, CA) to calculate the percentage of cell viability.
2.5. Flow cytometry
The expression of CD11b, a differentiation marker for granulocytes,
was measured by ow cytometry. One million NB-4 cells were harvest-
ed, washed twice with cold PBS (pH = 7.6) and incubated with anti-
CD11b-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 L of PBS and analyzed
on a FACS Canto ow 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 L 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 togeth-
er with anti-CD3 V450 (clone UCHT1, BD Biosciences, San Jose, CA), anti-
CD19 FITC (clone HD37, Dako, Glostrup, Denmark), anti-CD56 (clone
CMSSB, eBioscience, San Diego, CA), and Live/Dead dye (Life Technolo-
gies, Oregon, CA), washed twice, and xed with 0.9% formaldehyde. Fol-
lowing 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 MP9, BD Biosciences) followed by incubation
in FACS Lysis (BD Biosciences) for 10 min, washed once and resuspend-
ed in PBS. All samples were analyzed on an LSR Fortessa (Becton Dickin-
son) acquiring at least 5x104 events per sample. Using single stained
compensation beads (eBioscience) a compensation matrix was created.
The median uorescence 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 L lysis buffer (10 mM
Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP40,
10 L/mL PI cocktail, 0.1 mM PMSF, 1 mM Na3VO4, 20 mM NaF and
1 protease inhibitor cocktail set I (Calbiochem, Germany). Cell ly-
sates were sonicated and incubated at 4 C for 30 min prior to centri-
fugation at 16,000 g, at 4 C for 15 min. The supernatant was
collected and the protein concentration measured by Bradford re-
agent (Bio-Rad, Hercules, CA). Aliquot (35 g) 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 incu-
bated 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) 7784 79

Clone 7D36 (1:2000) was obtained from BD Pharmingen (San Diego,
CA). Anti-AChE (1:1000), anti-M3-mAChR; Clone H210 (1:1000), anti-
7-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 -actin (1:40,000) was obtained from
Sigma-Aldrich and incubated at 4 C overnight with gently shaking.
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 conju-
gation was visualized by performing enhanced chemiluminescence
(ECL) system (GE Healthcare, UK). The intensity of individual proteins
was quantied 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 7-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, 7-nAChR; 7 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 ow cytometry. Light gray
histogram represents uorescence minus one (FMO), which was used as a negative control for AChE. Dark gray histogram represents median uorescence intensity (MFI) of AChE expres-
sion. WB, Whole blood; MNC, Mononuclear cell.
80 S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 7784
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 con-
sidered signicant 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 choliner-
gic components in leukocyte subsets. Given the diversity of non-
malignant cells, we chose ow cytometry analysis. For the hematopoiet-
ic 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 identied including ChAT pro-
tein with a 6870 kDa, AChE with a 71 kDa, CHT with a 65 kDa, M3-
mAChR with a 75 kDa, and 7-nAChR with a 55 kDa relative molecular
mass (Fig. 1A). Notably, the expression of AChE and M3-mAChR pro-
teins were observed at a higher level in the myeloid lineage cells, NB-
4 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 7-
nAChR 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 M) 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, 7-nAChR expression
remained unchanged after either ATRA or carbachol administration. Fi-
nally, 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 com-
ponents, especially M3-mAChR.
3.3. Effect of carbachol on the late differentiation marker, CD11b of ATRA-
induced differentiated NB-4 cells
We next sought to determine the mechanism of cholinergic stimula-
tion on ATRA-induced differentiated NB-4 cells. We employed the anal-
ysis of CD11b expression by ow cytometry since this allows for the
determination of small changes in median uorescence 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 ve 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, 7-nAChR, and -actin protein of
non-differentiated (untreated), and ATRA-induced differentiated NB-4 cells (ATRA)
treated with or without carbachol (100 M) for 5 days. All the experiments were
repeated three times. CCh, carbachol; ATRA, all-trans retinoic acid.
(10 to 1000 M) for a further 24 h. Notably, the expression of CD11b
level increased signicantly in ATRA-induced differentiated NB-4 com-
pared 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 fur-
ther increase in CD11b expression as compared to ATRA alone, as
shown in Fig. 3A-B. These data show that even in optimally ATRA treat-
ed 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 dis-
tinguish between differentiation and cytoreductive effects. This was ac-
complished by culturing NB-4 cells with ATRA for ve days, before
stimulated with carbachol for 24, 48, and 72 h. Here, the viability was
signicantly reduced when cells were exposed to 1000 M 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 Expression levels of cholinergic components (fold of control)
Control CCh ATRA ATRA + CCh
ChAT 1 2.268 0.298 1.473 0.589 1.036 0.122
AChE 1 0.737 0.061 0.571 0.038* 0.587 0.099*
CHT 1 1.045 0.120 0.4787 0.093* 0.557 0.176
M3-mAChR 1 1.104 0.182 6.334 0.535a,# 6.187 0.411 a,#
7-nAChR 1 1.31 0.154 1.302 0.138 1.535 0.167
Note: NB-4 cells were treated with 100 M carbachol (CCh), 1 M all-trans retinoic acid
(ATRA), or the combination of 100 M CCh and 1 M 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 signicant difference from the control.#(p b
0.0001) represent statistically signicant difference from the CCh treated group. ChAT;
choline acetyltransferase, AChE; acetylcholinesterase, CHT; choline transporter, M3-
mAChR; M3 muscarinic acetylcholine receptor, 7-nAChR; 7 nicotinic acetylcholine
receptor.
 S. Chotirat et al. / Blood Cells, Molecules and Diseases 59 (2016) 7784 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 M ATRA for 5 days to induce differentiation.
Non-differentiated and ATRA-differentiated NB-4 cells were treated with 10, 100, and 1000 M CCA for 24 h. Expression level of differentiation marker CD11b was analyzed by ow
cytometry. (A) Representative primary ow data of CD11b expression. The y-axis, cell count as percentage of maximum; x-axis, log scale of mean uorescent 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 signicant difference from the differentiated NB-4 ATRA treated alone group. # (p b 0.05) represent statistically signicant 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 M) for 5 days. Then ATRA-
induced differentiated NB-4 cells were treated with carbachol at 0.01, 0.1, 1, 10, 100, and 1000 M for 24, 48, and 72 h. The combination of ATRA (1 M) and As2O3 (1 M) 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) 7784
Fig. 5. Effect of carbachol treatment involved in caspase-3 activation. ATRA-induced
differentiated NB-4 cells were treated with carbachol 100 M for 24 and 72 h in the
absence or presence of atropine (AT, 1 M) or mecamylamine (MM, 1 M). (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 -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.
viability (data not shown). Hence, carbachol reduced the viability of
ATRA-treated cells, but did not affect the growth and death of non-
differentiated NB-4 cells.
Given that carbachol decreased viability, we nally sought to deter-
mine to what extent this involved apoptosis. To this end, we determined
by Western blotting the activation state of several pro-apoptotic pro-
teins, 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 M for
24, 48, and 72 h as compared to ATRA alone (Fig. 5A). Moreover, carba-
chol did not affect the expression of pro-/anti-apoptotic correlated cas-
pase 3, proteolyzed PARP, p53, BAX, and Bcl-2 proteins expression at the
24-h time point (Supplemental Fig. 2). In this setting, cholinergic stim-
ulation signicantly 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 acti-
vation. In support of this inhibitory effect of growth, the increased ex-
pression 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. More-
over, there was no statistically signicant 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
As a preclinical model for evaluating the possible role of NNCS acti-
vation as a differentiation inducing strategy in APL, the present data
set aims at investigating the expression of NNCS components expres-
sion in leukemic cell lines and to explore the effect of carbachol on dif-
ferentiation 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
7-nAChR. Moreover, the expression is comparable to that seen in nor-
mal leukocyte subsets included here as controls. These data thus sup-
port and extend the data from a previous study, where 7-nAChR was
reported to participate in HL-60 APL cell line differentiation beyond
promyelocytic state [16]. Collectively, these ndings 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 ef-
fects on hematopoiesis. With regards to the immune system, this is the
rst 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
signicantly more expressed than in both CD14+ monocytes and
unfractionated lymphocytes. In addition, CD19+ B lymphocytes exhibit-
ed 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 puried
before analysis [26]. In any case, further studies are needed to address
the biological signicance 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 non-
differentiated 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 de-
creased it. Taken together, these ndings 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) 7784
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) transloca-
tion whereas HL-60 lacks this rearrangement [27].
While the above ndings were largely based on Western blotting,
we have extended the notion of a functional role of cholinergic stimula-
tion in myeloid differentiation by multiparameter ow cytometry. It is
noteworthy that the combination of carbachol and ATRA signicantly
increased the expression of the late-stage differentiation CD11b marker
as compared with ATRA treatment alone. This nding 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 inammatory site, is not
known (data not shown). It might be speculated that MMP-9 activation
was independent of cholinergic modulation since statistically insigni-
cant change in MMP-9 activation was observed post-carbachol treat-
ment. Induction of apoptosis can be another effect supporting the use
of cholinergic stimulation in inducing differentiation of maturation-
arrested leukemic cells. The data presented here show that in the NB-
4 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-/anti-
apoptotic 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 inuence 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 ndings can be reconciled by molecular differences in the
cell lines applied. In particular, the NB-4 cell line contains missense mu-
tations 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 non-
p53 mutations. Consistent with this, decreased AChE expression or
deciency of it has been reported in apoptosis resistance and tumor
aggressiveness in squamous cell carcinoma, colorectal carcinoma, and
hepatocellular carcinoma [9,3133]. Given that decreased AChE expres-
sion may inhibit apoptosis, further experimentation should evaluate the
role of AChE by using AChE stimulator in order to conrm 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 ATRA-
induced differentiation in NB-4 cells. The latter nding suggests the po-
tential 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 modal-
ity. However, further preclinical studies employing freshly isolated
leukemic cells from APL patients to validate these ndings are necessary
before future clinical trials can be pursued.
Conicts of interests
All authors declare that they have no conicts of interest.
Author contributions
SC conducted experiments and performed data analysis and manu-
script drafting. TS supervised study design, conduction of experiments,
data analysis, and manuscript drafting and revision. MH and PH super-
vised ow 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 nal 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 molecu-
lar technical practice on cell culture, ow cytometry techniques, and
Western blotting assay and the staffs of FACS Core facility, Institute of
Biomedicine, Aarhus University, Denmark for their supervision on ow
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.
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