Proapoptotic effects of novel pentabromobenzylisothioureas in human leukemia cell lines

Article abstract of DOI:10.1007/s00044-011-9841-8

A series of new pentabromobenzylisothioureas [ZKK-1-ZKK-5; (ZKKs)] carrying additional substituents on nitrogen atoms has been synthesized. The ZKKs were found to induce apoptosis in HL-60 (human promyleocytic leukemia) and K-562 (human chronic erythromyeloblastoid leukemia) cell lines in a concentration-dependent manner at low micromolar concentrations. ZKK-3 [(N,N'- dimethyl-S-2,3,4,5,6-pentabromobenzyl)isothiouronium bromide] showed the highest proapoptotic activity in HL-60 cells, whereas ZKK-2 [N-methyl-S-(2,3,4,5,6- pentabromobenzyl)isothiouronium bromide] was most effective in this respect in K-562 cells. During the ZKKsinduced apoptosis, an 85 kDa fragment of cleaved PARP (caspase-3 and caspase-7 substrate) was detected in both cell lines tested. The studied compounds also decreased mitochondrial transmembrane potential in both these cell lines and caused the cells to accumulate in G₁ and at the G₁/S border of the cell cycle in a concentration-dependent manner. These results show promise for their study as new compounds in the treatment of leukemia, after an appropriate preclinical toxicity profile.

Full text of DOI:10.1007/s00044-011-9841-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3111–3118
DOI 10.1007/s00044-011-9841-8
ORIGINAL RESEARCH
Proapoptotic effects of novel pentabromobenzylisothioureas
in human leukemia cell lines
•
Mirosława Koronkiewicz Zdzisław Chilmonczyk
•
Zygmunt Kazimierczuk
Received: 3 January 2011 / Accepted: 25 October 2011 / Published online: 11 November 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract A series of new pentabromobenzylisothioureas
[ZKK-1–ZKK-5; (ZKKs)] carrying additional substitu-
ents on nitrogen atoms has been synthesized. The ZKKs
were found to induce apoptosis in HL-60 (human promy-
leocytic leukemia) and K-562 (human chronic erythromy-
eloblastoid leukemia) cell lines in a concentration-dependent
manner at low micromolar concentrations. ZKK-3 [(N,N0-
dimethyl-S-2,3,4,5,6-pentabromobenzyl)isothiouronium
bromide] showed the highest proapoptotic activity in
HL-60 cells, whereas ZKK-2 [N-methyl-S-(2,3,4,5,6-
pentabromobenzyl)isothiouronium bromide] was most
effective in this respect in K-562 cells. During the ZKKs-
induced apoptosis, an 85 kDa fragment of cleaved PARP
(caspase-3 and caspase-7 substrate) was detected in both
cell lines tested. The studied compounds also decreased
mitochondrial transmembrane potential in both these cell
lines and caused the cells to accumulate in G1 and at the
G1/S border of the cell cycle in a concentration-dependent
manner. These results show promise for their study as new
compounds in the treatment of leukemia, after an appro-
priate preclinical toxicity profile.
Introduction
Isothioureas are a class of amphiphilic compounds carrying
a highly basic isothiourea function of pKa & 10. There-
fore, at physiological pH these compounds exist in a pro-
tonated (cationic) form that may be important for their
specific biological effects. In solid state they form salts of
usually better water solubility then that of the substrates
used for their synthesis.
Reports on anticancer activity of isothioureas are very
scarce. S-(10-undecen-1-yl)isothiouronium iodide was
found to be effective against Walker carcinoma cells in rats
(Carmona and Gonzalez-Cadavid, 1978; Gonzalez-Cada-
vid and Herrera Quijada, 1974), and bisisothiouronium
derivatives of thiophene were reported to show activity
against Yoshida sarcoma (Gogte et al., 1967). Recently, a
report showing proapoptotic activity of a number of pen-
tabromobenzylisothiourea derivatives with substantial
cytotoxicity toward human glioblastoma cells has been
published. The efficacy of the latter compounds was higher
than that of the well-known casein kinase 2 (CK2) inhibitor
4,5,6,7-tetrabromo-1H-benzotriazole (TBB), and was sim-
ilar to that of 4,5,6,7-tetrabromo-1H-benzimidazole (TBI).
Cell death induced in rat and human malignant glioma cells
by the pentabromobenzylisothiourea derivatives was asso-
ciated with a decrease in mitochondrial membrane poten-
tial and with activation of caspase-3 and caspase-7
followed by PARP cleavage (Kaminska et al., 2009).
More attention was given to isothioureas as inhibitors of
nitric oxide synthase (NOS) isoforms. These enzymes play
a significant and multifaceted role in both physiology and
pathology; therefore, there is an ongoing search for their
effective inhibitors (Garvey et al., 1994; Jin et al., 2009;
Rairigh et al., 1998; Kalish et al., 2002). For instance,
some isothioureas show selectivity for neuronal NOS and a
Keywords Pentabromobenzylisothioureas ꢀ
Pentabromobenzylisothiouronium ꢀ Apoptosis ꢀ
Human leukemia cell lines ꢀ Flow cytometry
Z. Kazimierczuk (&)
Mossakowski Medical Research Centre,
5 Pawinskiego St., 02-106 Warsaw, Poland
e-mail: ZKazimierczuk@gmail.com
M. Koronkiewicz ꢀ Z. Chilmonczyk
Department of Cell Biology, National Medicines Institute,
30/34 Chełmska St., 00-725 Warsaw, Poland
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Med Chem Res (2012) 21:3111–3118
promise for utility in the treatment of neurodegenerative
diseases (Schulz et al., 1997; Castano et al., 2008),
whereas some selective inhibitors of inducible NOS,
including few S-substituted isothioureas, were found
effective as chemopreventive agents in rat tracheal epi-
thelial cells treated with the carcinogen benzo[a]pyrene
(Sharma et al., 2002). We have recently reported that some
substituted benzylisothioureas, including the proto-
type pentabromobenzylisothiourea, are potent inhibitors of
Ca2?/calmodulin-dependent NOSs (endothelial NOS
and neuronal NOS) in normal rat brain homogenates
(Kazimierczuk et al., 2010). Moreover, another group of
S-benzylisothioureas has been recently shown to inhibit
indoleamine-2,3-dioxygenase, which is overexpressed and
may play an important role in a variety of illnesses,
including cancer and some neurodegenerative diseases
(Matsuno et al., 2010).
used for NMR spectra was deuteriodimethylsulfoxide.
Elemental analyses were performed at the Faculty of
Chemistry, Warsaw Technical University, using a Heraeus
CHN-Rapid analyzer.
General procedure for the preparation of N-substituted
S-(2,3,4,5,6-pentabromobenzyl)isothiouronium
bromides (ZKK 1-5)
To a hot solution of thiourea derivative (5.1 mmol) in
anhydrous ethanol (20 mL) 2,3,4,5,6-pentabromobenzyl
bromide (5 mmol) was added. The mixture was refluxed
for 20 min and then the solvent was partially evaporated to
a final volume of about 10 mL. This was left refrigerated
overnight. The chromatographically pure crystals that
formed were filtered off and washed with a small volume
of cold ethanol/ethyl ether mixture (1:1, v/v). For elemental
analysis, a small amount of the product was recrystallized
from ethanol. Synthesis scheme and chemical structure of
ZKKs are shown in Fig. 1.
In this study we examined proapoptotic and cytostatic
effects of the previously described S-(2,3,4,5,6-pentabro-
mobenzyl)isothiourea (ZKK-1) and its four newly syn-
thesized congeners ZKK-2, ZKK-3, ZKK-4, and ZKK-5
(ZKKs) in HL-60 (human promyleocytic leukemia) and
K-562 (human chronic erythromyeloblastoid leukemia) cell
lines.
S-(2,3,4,5,6-pentabromobenzyl)isothiouronium bromide
(ZKK-1)
Synthesis of this compound has been described previously
(Kazimierczuk et al., 2010).
Materials and methods
N-Methyl-S-(2,3,4,5,6-pentabromobenzyl)isothiouronium
bromide (ZKK-2)
Chemistry
Melting points were determined in open capillary tubes on
a model MFB-595-030G Gallenkamp melting point appa-
Yield: 88%; mp 266–268°C. 1H-NMR (DMSO-D6):
d = 2.96 (s, 3H, N–CH3), 4.92 (s, 2H, –CH2–), 9.25, 9.64
and 9.96 (3 bs, 3H, NH and NH2). Anal. for C9H8N2SBr6
(575.81): Calc. C: 16.49, H, 1.23, N, 4.27. Found C: 16.35,
H, 1.28, N, 4.16.
1
ratus. H-NMR spectra were recorded on a Bruker AMX-
400 instrument at 25°C. Chemical shifts are reported in
ppm from internal tetramethylsilane standard. The solvent
Fig. 1 Synthesis and structure
of N-substituted
pentabromobenzylisothioureas
(ZKKs)
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Med Chem Res (2012) 21:3111–3118
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N,N0-Dimethyl-S-(2,3,4,5,6-
pentabromobenzyl)isothiouronium bromide (ZKK-3)
Apoptosis assay by annexin V/propidium iodide (PI)
labeling
Yield 85%, mp 242–244°C. 1H-NMR (DMSO-D6):
d = 3.00 (s, 6H, 2 9 CH3), 4.91 (s, 2H, –CH2–), 9.42 and
9.62 (2 bs, 2H, 2 9 NH). Anal. for C10H10N2SBr6
(588.79): Calc. C: 17.94, H, 1.51, N, 4.18. Found C: 17.90,
H, 1.55, N, 4.09.
Apoptosis was measured using the Annexin-V FITC
Apoptosis Kit (Invitrogen). Twenty-four or 48 h post-
treatment the cells were collected by centrifugation, rinsed
twice with cold PBS and suspended in binding buffer at
2 9 106 cells/ml. One-hundred-ll aliquots of the cell sus-
pension were labeled according to the kit manufacturer’s
instructions. In brief, annexin V-FITC and PI were added
to the cell suspension and the mixture was vortexed and
incubated for 15 min at room temperature in the dark.
Then, 400 ll of cold binding buffer was added and the cells
were vortexed again and kept on ice. Flow cytometry
measurements were performed within 1 h after labeling.
N-Ethyl-S-(2,3,4,5,6-pentabromobenzyl)isothiouronium
bromide (ZKK-4)
Yield 77%, mp 229–231°C. 1H-NMR (DMSO-D6):
d = 1.19 (t, 3H, J = 7.2 Hz, –CH3), 3.35 (q, 2H, overlap.
HOD, N–CH2–), 4.91 (s, 2H, –CH2–), 9.28, 9.60 and 9.40
(3bs, 3H, NH and NH2). Anal. for C10H10N2SBr6 (588.79):
Calc. C: 17.94, H, 1.51, N, 4.18. Found C: 17.88, H, 1.57,
N, 4.08.
Morphological evaluation
After exposure to drugs, the cells were collected, washed
with cold PBS and fixed at -20°C in 70% ethanol for at
least 24 h. Next, ethanol was washed out and the cells were
stained with 1.0 lg/ml DAPI and 20 lg/ml sulforhodamine
101. Cell morphology was evaluated using a BX60 fluo-
rescence microscope equipped with a DP50 digital camera
(Olympus, Japan).
N-Allyl-S-(2,3,4,5,6-pentabromobenzyl)isothiouronium
bromide (ZKK-5)
Yield 75%, mp 250–252°C. 1H-NMR (DMSO-D6):
d = 4.02 (d, 2H, J = 4.7 Hz, –N–CH2), 4.94 (s, 2H,
–CH2–), 5.26 (s, 1H, =CH), 5.29 (d, 1H, J = 6.1 Hz, =CH),
5.86 (m, 1H, –CH=), 9.34, 9.69 and 10.15 (3bs, 3H, NH and
NH2). Anal. for C11H10 N2SBr6 (600.80): C, 19.38, H, 1.48,
N, 4.11. Found: C, 19.29, H, 1.55, N, 4.03.
Mitochondrial membrane potential (DWm) assay
Mitochondrial membrane potential was assessed by flow
cytometry using JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetra-
ethylbenzimidazolocarbocyanine iodide; Sigma). JC-1
undergoes potential-dependent accumulation in mitochon-
dria. In healthy cells, the dye accumulates in mitochondria,
forming aggregates with red fluorescence (FL-2 channel),
whereas in apoptotic cells the dye remains in the cytoplasm
in a monomeric form and emits green fluorescence (FL-1
channel). Cells were harvested by centrifugation 48 h post-
treatment, suspended in 1 ml of complete culture medium
at approximately 1 9 106 cells/ml and incubated with
2.5 ll JC-1 solution in DMSO (1 mg/ml) for 15 min at
37°C in the dark. Stained cells were washed with cold PBS,
suspended in 400 ll of PBS and then examined with a
FACSCalibur flow cytometer equipped with CellQuest
software (BD Biosciences, San Jose, CA, USA).
Antileukemic activity studies
Cell lines and treatments
HL-60 (human promyelocytic leukemia) cell line was
obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA), and K-562 (human chronic
erythromyeloblastoid leukemia) cell line was obtained
from the German Collection of Microorganisms and Cell
Cultures (DSMZ). The cells were grown in RPMI-1640
medium (Gibco, Grand Island, NY, USA) supplemented
with 10% (v/v) of heat-inactivated fetal bovine serum
(Gibco, Grand Island, NY, USA) and 1% (v/v) of antibi-
otic–antimycotic solution (Gibco), at 37°C in a humidified
atmosphere of 5% CO2 in air. For experiments, 3 ml ali-
quots per well of cell suspension in the same medium
(2.5 9 105 cells/ml), were seeded onto 6-well plates
(Nunc, Denmark). All experiments were performed in
exponentially growing cultures. The compounds studied
were added to the cultures as solutions in dimethyl sulf-
oxide (DMSO; Sigma), and control cultures were treated
with the same volume of the solvent. After culturing the
cells with the studied compounds for 24 or 48 h, the cells
were collected and used for labeling.
PARP cleavage assay
Caspase-3 and caspase-7 cleave poly(ADP-ribose) poly-
merase (PARP). PARP cleavage was detected by flow
cytometry using Anti-PARP CSSA FITC Apoptosis
Detection Kit (Invitrogen) according to manufacturer’s
protocol. The FITC-conjugated anti-PARP antibody
employed in the kit specifically recognizes the 85 kDa
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Med Chem Res (2012) 21:3111–3118
was expressed as fluorescence intensity that was assessed
using CellQuest and the free WinMDI software package
written by Joseph Trotter of the Scripps Institute (La Jolla,
CA, USA).
Cell cycle analysis
After exposure to the tested compounds, the cells were
washed with cold PBS and fixed at -20°C in 70% ethanol
for at least 24 h. Next, the cells were washed free of eth-
anol and stained with 50 lg/ml PI and 100 lg/ml RNase
solution in PBST (PBS supplemented with 0.1% v/v Triton
X-100) by 30 min incubation in the dark at room temper-
ature. Cell DNA content and the distribution of the cells in
different phases of the cell cycle were determined by flow
cytometry employing MacCycle (Phoenix Flow Systems,
San Diego, CA, USA) and CellQuest software packages.
Flow cytometry
Flow cytometry analyses were run on a FACSCalibur flow
cytometer (BD Biosciences, San Jose CA, USA), and ana-
lyzed by CellQuest software (BD Biosciences, San Jose,
CA, USA) and WinMDI 2.9 software. The DNA histograms
obtained were analyzed using the MacCycle software.
Results
Chemistry
Fig. 2 Induction of apoptosis by ZKKs in HL-60 cells (a) and K-562
cells (b). The data were determined by FACS cytometer after 24 and
48 h treatment. Cells were stained with annexin V-FITC and PI. Each
bar represents the mean SD (n C 4)
The N-substituted pentabromobenzylisothioureas were
obtained following the direct strategy shown in Fig. 1. The
reaction was performed using pentabromobenzyl bromide
and the respective thiourea. The products—isothiouronium
bromides—crystallized from the reaction mixture after
concentrating. The compounds were characterized using
1H-NMR and elemental analyses.
fragment of cleaved PARP. The cells meant for the assay
were harvested 48 h post-treatment and washed twice with
PBS just before use. The level of cleaved PARP protein
Fig. 3 Morphology
(fluorescence microscopy
employing DAPI/
sulforhodamine 101 labeling) of
HL-60 cells cultured for 48 h in
the absence (control, a) and
presence of ZKK-3 (8 lM, b).
Arrows indicate apoptotic
nuclei
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Med Chem Res (2012) 21:3111–3118
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Fig. 4 Representative flow cytograms demonstrating changes in
mitochondrial membrane potential (DWm) of HL-60 cells (upper
panels) and K-562 cells (lower panels) induced by 48 h culturing with
various ZKKs compounds. The cells were stained with JC-1 dye. The
cells in the lower right (R3) quadrant showed increased red-to-green
fluorescence ratio (apoptotic cells)
Fig. 5 Effect of ZKKs on proteolytic cleavage of PARP protein in
cells were exposed for 48 h to ZKKs. Representative histograms
showing increased level of 85 kDa fragment of PARP protein
indicating induction of apoptosis after ZKKs treatment. a: Histogram
of HL-60 control cells and overlay histogram of treated cells at 8 lM
ZKK-3. b: Histogram of K-562 control cells and overlay histogram
of treated cells at 10 lM ZKK-2. Marker M1 designates negative cell
populations whereas M2 designates positive cell populations (indicate
apoptosis). Thin line control cells, thick line ZKK-treated cells
Induction of apoptosis by ZKKs in HL-60 and K-562
leukemia cells
in K-562 cells. Fluorescence microscopy showed typical
concentrating chromatin and apoptotic bodies’ formation
(Fig. 3).
ZKKs compounds induced apoptosis in both cell lines
utilized, but the effect was more prominent in HL-
60 cells (Fig. 2). The observed apoptotic effect was dose-
and time-dependent. ZKK-3 [(N,N0-dimethyl-S-2,3,4,5,
6-pentabromobenzyl)isothiouronium bromide] was the
most effective in HL-60 cell line, whereas ZKK-2
[N-methyl-S-(2,3,4,5,6-pentabromobenzyl)isothiouronium
bromide] showed the most potent cytotoxic apoptotic effect
Changes in mitochondrial membrane potential (DWm)
Analysis of the respective cytograms (for a representative
cytogram see Fig. 4) showed that the tested compounds
caused mitochondrial membrane depolarization (as evi-
denced by increased green-to-red fluorescence intensity
ratio) in both cell lines studied.
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Fig. 6 Changes in cell cycle progression in HL-60 (a) and K-562
(b) cells after 48 h treatment with ZKKs. Each bar represents the
mean SD (n C 4). The data obtained from FACSCalibur flow
cytometer were analyzed using MacCycle software to determine the
percentage of cells in each phase of the cell cycle
ZKKs-induced cleavage of PARP protein
Discussion
The enhancement of apoptosis was confirmed by detecting
PARP cleavage after 48 h incubation with the tested
compounds. During ZKKs-induced apoptosis, the presence
of 85 kDa PARP fragments was revealed in both cell lines
with the use of specific antibody (Fig. 5).
We decided to synthesize modified pentabromobenzyli-
sothioureas in a search for new inhibitors of the antiapo-
ptotic enzyme casein kinase 2 (CK2), structurally similar to
such known polyhalogenobenzimidazole CK2 inhibitors as
4,5,6,7-tetrabromobenzimidazole (TBI) or 4,5,6,7-tetra-
bromo-2-dimethylaminobenzimidazole (DMAT) (Szyszka
et al., 1995; Pagano et al., 2004; Gianoncelli et al., 2009).
We expected that the new compounds would show the
advantage of increased water solubility while retaining
high CK2 inhibitory activity. However, the novel com-
pounds showed only moderate CK2 inhibitory activity
(Ki & 4 lM, Dr. F. Meggio, personal communication),
whereas, surprisingly, they revealed a considerable anti-
leukemic action in vitro. It should be noted that other
known benzylisothioureas with substituents in the benzene
Effect of ZKKs on cell cycle progression
Figures 6a, b, and 7 demonstrate changes in the cell cycle
progression of HL-60 and K-562 cells after 48 h incubation
with the tested compounds. The compounds exerting
cytostatic effect caused a concentration-dependent accu-
mulation of cells in G1 and at the border of G1 and S
phases, decreasing the number of cells in the G2M phase of
the cell cycle.
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Med Chem Res (2012) 21:3111–3118
3117
G1
A
B
C
G1
S
37,6 3,2
43,3 3,4
G1
S
55,5 2,0
35,5 5,5
G1
S
55,0 2,0
42,5 0,5
G2M 19,1 3,9
G2M 9,0 4,5
G2M 2,5 2,5
G2M
Sub
-G1
S
DNA
Fig. 7 Exemplary DNA histograms of K-562 cells treated for 48 h
with ZKK-3. The data obtained from FACSCalibur flow cytometer
and analyzed using MacCycle software to determine the percentage of
cells in each phase of the cell cycle. a: Control (no ZKK-3 added);
b: 10 lM ZKK-3; c: 20 lM ZKK-3
part of the molecule (for example, 2,3,4,5,6-pentafluoro-
and 3,4- and 2,4-dichlorobenzylisothioureas) showed only
weak cytotoxic activity. Apparently, the introduction of a
bulky substituent (e.g., phenyl or benzyl group) at one of
the nitrogen atoms considerably reduces cytotoxicity of
pentabromobenzylisothioureas (data not shown).
seems to be related to overexpression of the antiapoptotic
protein Bcl-xL (Horita et al., 2000). In general, K562 cells
are highly resistant to multiple anticancer agents and easily
transform to drug-resistant lines during treatment by novel
drugs (McGahon et al., 1994; Bedi et al., 1995; Amarante-
Mendes et al., 1998).
As we previously reported, modified benzylisothioureas
are also inhibitors of the Ca2?/calmodulin-dependent NO
synthase (Kazimierczuk et al., 2010). The role of NO in
cancer initation and progression is still debated and it is not
yet decided whether NO should be considered as a poten-
tial anticancer agent or instead a carcinogen (Mocellin,
2009). When comparing the NOS inhibitory activity and
anticancer activity of other tested benzylisothioureas, we
did not find a straightforward correlation between these
attributes (data not shown).
Concluding remarks
Our results suggest that N-substituted pentabromobenzyli-
sothioureas might be promising anticancer agents. The
study on anticancer activity of this compound class in solid
tumors is in progress, and further investigations are needed
to evaluate their clinical potential.
Acknowledgment This study was supported by the Ministry of
Science and Higher Education (Poland) grants: PBZ-MIN 014/P05/
2004 and N N209 371439.
ZKKs showed considerable cytotoxic and cytostatic
effects in both HL-60 (human promyleocytic leukemia)
and K-562 (human chronic erythromyeloblastoid leukemia)
cells. Proapoptotic effects were higher in HL-60 than in
K-562 cells. Apoptotic death was associated with increased
depolarization of the mitochondrial membrane and with
increase in the level of 85 kDa fragments of PARP protein.
The latter effect is an indirect measure of activation of
the effector caspase-3 and caspase-7 that proteolytically
cleave native 116 kDa PARP protein into 85 and 25 kDa
fragments. Decreasing mitochondrial transmembrane
potential suggests an activation of ‘‘the intrinsic’’ pathway
of apoptosis by the tested compounds.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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