Deoxynucleosides with benzimidazoles as aglycone moiety are potent anticancer agents

Article abstract of DOI:10.1016/j.ejphar.2017.12.018

Abnormally high levels of CK2 and PIM-1 serine/threonine kinases have been documented in many cases of cancer. The elevation of CK2 and PIM-1 in cells entails suppression of apoptosis and implies a protective role for the kinases against cell death. Downregulation of these enzymes by chemical methods promotes apoptosis in cells. The aim of the present study was to explore the anticancer activity of inhibitors of protein kinases CK2 and PIM-1 on neoplastic cell lines in vitro. We studied a series of deoxynucleosides with various tetrahalobenzimidazoles as aglycone moiety. Cytotoxicity, induction of apoptosis by the tested inhibitors, mitochondrial membrane potential, activity of caspases, changes in cell cycle progression, as well as a mechanism of action were determined by flow cytometry and other methods. The results indicate that the studied compounds, e.g., 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole called K164 (also termed TDB), showed diverse cytotoxicity and proapoptotic efficacy in cell lines. Our results showed that the tested compounds are potential anticancer agents for targeted therapy, particularly in the treatment of myeloid leukaemia and androgen-responsive prostate cancer.

Full text of DOI:10.1016/j.ejphar.2017.12.018

Author’s Accepted Manuscript
Deoxynucleosides with benzimidazoles as aglycone
moiety are potent anticancer agents
Mirosława Koronkiewicz, Zdzisław Chilmonczyk,
Zygmunt Kazimerczuk, Andrzej Orzeszko
www.elsevier.com/locate/ejphar
PII:
S0014-2999(17)30809-9
https://doi.org/10.1016/j.ejphar.2017.12.018
EJP71565
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 16 October 2017
Revised date: 1 December 2017
Accepted date: 8 December 2017
Cite this article as: Mirosława Koronkiewicz, Zdzisław Chilmonczyk, Zygmunt
Kazimerczuk and Andrzej Orzeszko, Deoxynucleosides with benzimidazoles as
aglycone moiety are potent anticancer agents, European Journal of
Pharmacology, https://doi.org/10.1016/j.ejphar.2017.12.018
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Deoxynucleosides with benzimidazoles as aglycone moiety are potent anticancer agents
Mirosława Koronkiewicza, Zdzisław Chilmonczyka,
Zygmunt Kazimerczukb, Andrzej Orzeszkob
aDepartment of Cell Biology, National Medicines Institute, Chełmska St. 30/34,
00-725 Warsaw, Poland
bInstitute of Chemistry, Warsaw University of Life Sciences, Nowoursynowska St. 159C,
02-787 Warsaw, Poland
Correspondence to: Mirosława Koronkiewicz, Department of Cell Biology,
National Medicines Institute, Chełmska St. 30/34, 00-725 Warsaw, Poland,
Tel.: + 4822 851 43 79, Fax: + 4822 841 06 52, e-mail: m.koronkiewicz@nil.gov.pl
1

1Abstract. Abnormally high levels of CK2 and PIM-1 serine/threonine kinases have been
documented in many cases of cancer. The elevation of CK2 and PIM-1 in cells entails
suppression of apoptosis and implies a protective role for the kinases against cell death.
Downregulation of these enzymes by chemical methods promotes apoptosis in cells. The aim
of the present study was to explore the anticancer activity of inhibitors of protein kinases CK2
and PIM-1 on neoplastic cell lines in vitro. We studied a series of deoxynucleosides with
various tetrahalobenzimidazoles as aglycone moiety. Cytotoxicity, induction of apoptosis by
the tested inhibitors, mitochondrial membrane potential, activity of caspases, changes in cell
cycle progression, as well as a mechanism of action were determined by flow cytometry and
other methods. The results indicate that the studied compounds, e.g., 1-(β-D-2′-
deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole called K164 (also termed TDB),
showed diverse cytotoxicity and proapoptotic efficacy in cell lines. Our results showed that
the tested compounds are potential anticancer agents for targeted therapy, particularly in the
treatment of myeloid leukaemia and androgen-responsive prostate cancer.
Key Words: Protein kinase inhibitors, tetrahalobenzimidazoles, anticancer effects, flow
cytometry
1 Preliminary results were presented as a poster at the EMBO conference series. Cellular Signaling & Cancer
Therapy. May 27-31, 2016 | Cavtat-Dubrovnik, Croatia. Abstract was published in the Abstracts of poster
presentations.
2

1. Introduction
Constitutively active serine/threonine kinases such as CK2 (casein kinase 2) and the PIMs
family (Proviral Integration site of Moloney Virus) with special reference to PIM-1 show
abnormally high levels in a number of cancers and mediate important hallmarks of cancer,
particularly suppression of apoptosis (Ahmed et al., 2008; Pinna, 2012; Brault et al., 2010;
Nawijn et al., 2011; Amaravadi et al., 2005). Overexpression of kinases CK2 and PIM-1 in
patient tumours frequently correlates with poor prognosis and is considered an unfavourable
prognostic marker in prostate and lung cancers, acute myeloid leukaemia, and pancreatic
ductal adenocarcinoma (O-charoenrat at al., 2004; Kim et al., 2007; Laramas et al., 2007;
Dhanasekaran et al., 2001; Cibull et al., 2006; Reiser-Erkan et.al., 2008). Downregulation of
these kinases by chemical methods was found to promote apoptosis in cells and targeted CK2
and PIM-1 for cancer therapy (Koronkiewicz et al., 2010, 2013; Wang et al., 2008; Kaminska
at al. 2009; Trembley at al. 2010; Duncan at al. 2013).
A number of highly specific heterocyclic compounds have been developed to target CK2
including 4,5,6,7-tetrabromo-1H-benzotriazole (TBB also termed TBBt) (Sarno et al. 2001;
Bretner et al. 2008) 4,5,6,7-tetrabromo-1H-benzimidazole (TBI also termed TBBz), 2-
dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) (Pagano et al. 2004; Zien et
al. 2003), as well as novel tetrahalogenated benzimidazoles (Gianoncelli et al. 2009; Janeczko
et al. 2012; Schneider et al. 2012). The first orally available small molecule inhibitor of CK2
protein in clinical trials for cancer was CX-4945 (Cylene Pharmaceuticals, San Diego, CA,
USA) (Siddiqui-Jain et al. 2010). ATP-competitive small-molecule inhibitors of the PIM1
kinase with growth inhibitory activity against leukaemia and prostate cancer cells have also
been reported (Pogacic et al. 2007; Fedorov et al. 2007). Quercetagetin has been recognized to
be a moderately potent and selective cell permeable inhibitor of PIM-1 kinase that is able to
inhibit PIM-1 activity in prostate cancer cells in a dose-dependent fashion (Holder et al.
3

2007). SGI-1776 (Astex) was found to be a potent adenosine-5’-triphosphate (ATP)
competitive inhibitor of Pim-1, Pim-2, and Pim-3 kinases with half maximal inhibitory
concentrations of 7 nM, 363 nM and 69 nM, respectively (Chen et al. 2009; Cervantes-Gomez
et al. 2013), and a novel series of meridianin C derivatives substituted at C-5 position was
prepared (More et al. 2014).
Previous research has shown that the majority of tetrahalogenated benzimidazoles,
particularly tetrabromo- and tetraiodobenzimidazoles, showed strong activity as inhibitors of
CK2, PIM-1, DYRK1A and other kinases, such as Rio1 (Kubiński et al. 2017), in vitro.
Unfortunately, these compounds are not cell permeable and thus cannot be used for medical
purposes. To avoid this fault, selective powerful cell-permeable dual inhibitors of protein
kinases CK2 and PIM-1 were synthesized (Lopez-Ramos et al. 2010; Pagano et al. 2008;
Cozza et al. 2013, 2014; Girardi et al. 2015). The addition of a sugar moiety (deoxyribose) to
halogenobenzimidazole molecules via C-N bonds improves their solubility and provides
effective intercellular transport (Cozza et al. 2013). Among the compounds synthesized, 1-(β-
D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole, called K164 (also termed
TDB), seems to be the most promising. 1`Cozza G. and co-authors demonstrated that the
cytotoxic efficacy of TDB is almost entirely due to apoptosis, is accompanied by parallel
inhibition of cellular CK2 and PIM-1 and is superior to that observed either by combining
individual inhibitors of CK2 and PIM-1 or by treating cells with the CK2 inhibitor CX4945
(Cozza et al. 2014). Comparing the persistence of CK2 inhibition after their administration to
cells showed that the effect of TDB on CK2 activity is more long-lasting than that of CX-
4945, with important consequences for cellular processes affected by CK2 such as cell
survival, proliferation and migration (Cozza et al. 2015).
In our previous publications we presented that many substituted tetrahalobenzimidazoles
(e.g., 4,5,6,7-tetraiodo-1H-benzimidazole; 4,5,6,7-tetrabromo-1H-benzimidazole; 4,5,6,7-
4

tetrabromo-2-(4-methylpiperazin-1-yl)-1H-benzimidazole; 4,5,6,7-tetrabromo-2-(4-methyl-
piperazin-1-yl)-1H-benzimidazole; 2-aminoethyleneamino-4,5,6,7-tetrabromo-1H-benzimi-
dazole) not only inhibited isolated kinases but also showed, in some cases, activity against
prostate cancer and leukaemia cell lines (HL-60, KG-1, LNCaP) (Koronkiewicz et al. 2010,
2013; Schneider et al. 2012). These interesting findings, particularly for TDB (Cozza et al.
2013, 2014; Girardi et al. 2015), encouraged us to continue and extend our investigations. In
the present study, we reported the anticancer activity of TDB (K164) and its five analogues
against leukaemia and prostate cell lines in vitro. We examined the activity of the compounds
against two leukaemia (KG-1, human acute myelogenous leukaemia and K562, human
chronic erythromyeloblastoid leukaemia) and two prostate (PC-3, androgen-independent
human prostate adenocarcinoma and LNCaP, androgen-responsive human prostate
adenocarcinoma) cell lines. It should be noted that the pro-apoptotic activity of the very same
compounds was recently examined on human T lymphoblastoid (CEM) and its multi-drug
resistance variant (R-CEM), human cervical cancer (HeLa), human embryonic kidney (HEK-
293T) cells and CCD34Lu human neonatal lung fibroblasts (CCd34Lu) (Cozza et al. 2014).
2. Materials and methods
2.1. Chemistry
All nucleosides (1-6) were synthesized using the same procedure as described previously
(Cozza et al. 2013, 2014). Analytical data for these compounds have also been published in
cited publications. In this paper, we recall the general method of synthesis, as well as the data
for 1, as an example. The synthesis scheme and chemical structure of the studied compounds
are shown in Fig. 1.
5

2.1.1. General procedure for preparation of inhibitors 1-6
To a suspension of proper benzimidazole (4 mmol) in dry acetonitrile (70 ml), sodium
hydride (200 mg, 5 mmol, 60% in oil) was added portion-wise. The mixture was stirred and
refluxed for 10 min. After cooling, 2-deoxy-3,5-bis-O-(4-methylbenzoyl)-β-D-erythro-
pentafuranosyl chloride (4 mmol) was added in portions. The mixture was stirred for 20 min
at room temperature. Then, methylene chloride (70 ml) was added and the mixture filtered
through Cellite. The solvent was evaporated and the residue was chromatographed on a silica
gel column with toluene-acetone (95:5, v/v) as an eluent. The fractions containing the main
product were evaporated, and the residue crystallized from methanol to give white needles.
By way of example analytical data for 1-[2-deoxy-3,5-di-O-(4-methylbenzoyl)-β-D-erythro-
pentofuranosyl]-4,5,6,7-tetrachlorobenzimidazole are shown: Yield: (1.09 g, 45%); mp 191-
1
192 C; H NMR (DMSO-d6): δ 2.37 (s, 3H), 2.40 (s, 3H), 2.96 (m, 1H), 3.07 (m, 1H), 4.52
(m, 2H), 4.58 (m, 1H), 5.72 (m, 1H), 7.01 (t, J = 6.4 Hz, 1H), 7.30-8.0 (4d, arom. H, 8H),
8.82 (s, 1H). Anal. Calcd. for C28H22Cl4N2O5 (608.31): C, 55.29; H, 3.65; N, 4.61. Found: C,
55.25; H, 3.60; N, 4.52 (Cozza et al. 2013).
2.1.2. 1-(2-Deoxy-β-D-erythro-pentofuranosyl)-4,5,6,7-tetrachlorobenzimidazole (1).
A
mixture of 1-[2-deoxy-3,5-di-O-(4-methylbenzoyl)-β-D-erythro-pentofuranosyl] (2.63 mmol)
and methanolic sodium methanolate (50 ml, 0.1 M) was stirred and refluxed for 15 min.
Methanol was evaporated, and the residue purified by flash chromatography on silica gel
using chloroform as an eluent. The fractions containing product were evaporated, and the
residue crystallized from methanol water to give colourless needles. Yield: (685 mg, 70%),
mp 141-144 C; 1H NMR (DMSO-d6): δ 2.46 (m, 1H), 2.58 (m, 1H), 3.65 (2m, 2H), 3.89 (q, J
= 3.9 Hz, 1H), 4.47 (m, 1H), 5.04 (t, J = 5.2 Hz, 1H), 5.36 (d, J = 4.5 Hz, 1H), 6.85 (t, J = 5.9
Hz, 1H), 8.88 (s, 1H). uv (MeOH): λ (ε) 225 (22 400). 270 (9 200), 298 (3 100). Anal. Calcd.
6

for C12H10Cl4N2O3 (372.04): C, 38.74; H, 2.71; N, 7.53. Found: C, 38.66; H, 2.77; N, 7.47
(Cozza et al. 2013).
2.2. Cell culture and treatment with agents
The following cell lines were used in this study: KG-1 (human acute myelogenous
leukaemia), K562 (human chronic erythromyeloblastoid leukaemia), PC-3 (androgen-
independent human prostate adenocarcinoma) and LNCaP (androgen-responsive human
prostate adenocarcinoma). Cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA).
KG-1 cells were grown in IMDM medium (CytoGen, Sinn, Germany) supplemented with
20% (v/v) heat-inactivated foetal bovine serum (CytoGen), 1% (v/v) antibiotic–antimycotic
solution (CytoGen) and 1% (v/v) stable glutamine (L-alanyl-L-glutamine) solution
(CytoGen). K562 and PC-3 cell lines were cultured in RPMI 1640 (CytoGen) supplemented
with 10% (v/v) heat-inactivated foetal bovine serum (CytoGen), 1% (v/v) antibiotic–
antimycotic solution (CytoGen) and 1% (v/v) stable glutamine (L-alanyl-L-glutamine)
solution (CytoGen). LNCaP cell line was cultured in RPMI 1640 (CytoGen) supplemented
with 10% (v/v) heat-inactivated foetal bovine serum (CytoGen) and 1% (v/v) antibiotic–
antimycotic solution (CytoGen), 1% (v/v) stable glutamine (L-alanyl-L-glutamine) solution
(CytoGen), 1% (v/v) MEM non-essential amino acid solution (Sigma-Aldrich, St. Louis, MO,
USA), and 1 mM sodium pyruvate (Sigma-Aldrich).
Cell lines were cultured at 37°C in a humidified atmosphere of 5% CO2 in air. All
experiments were performed in exponentially growing cultures. The compounds studied were
added to the cultures as solutions in dimethyl sulfoxide (DMSO) (Sigma-Aldrich); control
cultures were treated with the same volume of the solvent. The final concentration of DMSO
7

was maintained ca. 0.1%. After culturing the cells with the studied compounds for 24 or 48 h,
the cells were collected and used for labelling.
2.3. Cell viability (MTT - colorimetric assay)
Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (Sigma-Aldrich). All cells were cultured in 96-well plates with the addition
of compounds and incubated for 48 h. MTT stock solution was added to each well to a final
concentration of 0.5 mg/ml. After 4 h of incubation at 37°C, formazan crystals were dissolved
by the addition of SDS-HCl solution (10% SDS in 0.001 M HCl, final concentration). MTT
and SDS were added directly to the cell culture. The solubilized formazan product was
spectrophotometrically quantified in a Power Wave XS (Bio Tek, Winooski, VT, USA)
microplate reader at a wavelength of 570 nm. The MTT data were analysed to determine the
IC50 values (concentration required to reduce the viability of cells by 50% compared with the
control cells) of each compound. Regression analysis was performed with SigmaPlot software
(San Jose, CA, USA).
2.4. Apoptosis assay by annexin V/propidium iodide (PI) labelling
Apoptosis was measured using the FITC Annexin V Apoptosis Detection Kit I (BD
Pharmingen). After a 24- or 48-hour incubation with the tested agents, the cells were collected
by centrifugation, rinsed twice with cold phosphate-buffered saline (PBS) and suspended in
binding buffer at 1 × 106 cells/ml. Then, 100-µl aliquots of the cell suspension were labelled
according to the kit manufacturer’s instructions. Briefly, 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 μl of cold binding buffer was added, and the cells were
8

vortexed again and kept on ice. Flow cytometry measurements were performed within 1 h
after labelling.
2.5. Mitochondrial membrane potential (ΔΨm) assay
Mitochondrial membrane potential was assessed by flow cytometry using JC-1 (5,5’,6,6’-
tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide; Sigma). JC-1 undergoes
potential-dependent accumulation in mitochondria. In healthy cells, the dye accumulates in
mitochondria, forming aggregates with red fluorescence (FL-2 channel), whereas in dead and
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 × 106 cells/ml and
incubated with 2.5 µl of JC-1 solution in DMSO (1 mg/ml) for 15 min at 37°C in the dark.
The stained cells were then washed with cold PBS, suspended in 400 µl of PBS and then
examined by flow cytometry.
2.6. Caspase activity
Active caspases in apoptotic cells were detected using Fluorescent Labelled Inhibitors of
CAspases (FLICA) that covalently bind with active caspase enzymes. FLICA measures the
intracellular process of apoptosis. If an active caspase enzyme is inside the cell, it will
covalently bind with FLICA and retain the green fluorescent signal within the cell. Since non-
apoptotic cells lack active caspases, these cells quickly return to their non-fluorescent status
after the wash step. The activity of caspases was measured using the FAM-FLICA™ in vitro
Caspase Detection Kit (ImmunoChemistry Technologies, LLC. MN, USA) applicable to flow
cytometry. To detect specific caspases, cells were labelled by adding FLICA caspase-8
inhibitor reagent FAM-LETD-FMK (ImmunoChemistry Technologies, LLC.) or FLICA
9

caspase-9 inhibitor reagent FAM-LEHD-FMK (ImmunoChemistry Technologies, LLC.)
according to the kit manufacturer’s instructions. The cells were then washed twice and
analysed directly on a flow cytometer.
2.7. Morphological evaluation (fluorescence and inverted microscopy)
After exposure to inhibitors, the cells were collected, washed with cold PBS and fixed with
70% ethanol at -20°C for at least 24 h. Next, ethanol was washed out, and the cells were
stained with 1.0 μg/ml DAPI and 20 μg/ml sulforhodamine 101. Then, cell morphology was
evaluated using a BX60 fluorescence microscope equipped with a DP50 digital camera
(Olympus, Japan). Cell morphology was evaluated also using an ITM-2 inverted microscope
equipped with a DP10 digital camera (Olympus, Japan).
2.8. Western blot analyses
The cells were washed with cold PBS buffer, and then, whole cell extractions were prepared
using M-PER reagent (Pierce, Rockford, IL, USA). Protein concentration in the samples was
measured using a BCA protein assay kit (Pierce). Equal amounts of proteins were loaded on
10% or 12% SDS-PAGE. After electrophoresis the proteins were transferred to a
nitrocellulose membrane and probed with primary anti-human antibodies specific to: BAD,
BCL-2, XIAP (Apoptosis I Sampler Kit) (BD Biosciences), PARP (113 kDa) (Apoptosis II
Sampler Kit) (BD Biosciences), PARP (89 kDa) (BD Biosciences), phospho-Akt1 (Ser129)
(Cell Signaling Technology), phospho-Bad (Ser112) (Cell Signaling Technology). Secondary
antibodies conjugated with HRP were used. Protein bands were visualized using a CN/DAB
substrate kit (Pierce) or PierceTM 1-Step Ultra TMB-Blotting Solution (Pierce) or were
detected by chemiluminescence on a MicroChemi (Bio-Imaging Systems).
10

2.9. Cell cycle analysis
After exposure to the tested compounds, the cells were washed with cold PBS and fixed
at - 20 ºC in 70% ethanol for at least 24 h. Next, the cells were washed in PBS and stained
with 50 µg/ml PI and 100 µg/ml RNase solution in PBST (PBS supplemented with 0.1% v/v
Triton X-100) by 30 min incubation in the dark at room temperature. Cell DNA content and
the distribution of the cells in different phases of the cell cycle were determined by flow
cytometry employing CellQuest, BD FACSDiva software packages and MacCycle (Phoenix
Flow Systems, San Diego, CA, USA).
2.10. Flow cytometry
Flow cytometry analyses were run on a FACSCalibur or FACSCanto II flow cytometer (BD
Biosciences, San Jose CA, USA) and analysed using CellQuest, BD FACSDiva and WinMDI
2.9 software. The DNA histograms obtained were analysed using MacCycle software.
3. Results
3.1. Cytotoxic activity
Cytotoxic activity was determined for a series of deoxynucleosides carrying various
tetrahalobenzimidazoles as aglycone moiety (see Fig. 1) by assessing the number of viable
cells (KG-1, K562, LNCaP, PC-3) with the MTT assay. The MTT data were analysed to
calculate the IC50 value of each compound. The results are given in Table 1. The studied
inhibitors [K156, K157, K163, K164 (TDB) and K165] expressed IC50 values in the
concentration range from 4.01 μM to 60.09 μM. Tetrabromo- (K164) and tetraiododerivatives
(K165) appeared to be most cytotoxic, whereas a tetrachloroderivative (K156) exhibited
rather low cytotoxicity. Compound K170 (dichloroderivative) appeared to be quite nontoxic
(IC50 value >200 μM). The IC50 values of the most cytotoxic compound (K164) in cell lines
11

KG-1 and K562 were 4.01 μM and 8.51 μM, respectively (as compared to K165 where the
respective values were 20.12 μM and 10.69 μM). K562 cells exhibited greater sensitivity to
all tested compounds (except K164) compared to the KG-1 cell line. Among prostate cancer
cell lines, LNCaP showed high sensitivity to the tested compounds, whereas PC-3 cells are
resistant (Table 1).
3.2. Induction of apoptosis
Induction of apoptosis was assessed using Annexin V (FITC)/PI labelling after 24 and 48 h
treatment of cells with the tested compounds. The studied inhibitors, used in the concentration
range from 10 to 50 μM, evoked various proapoptotic effects in the leukaemia and prostate
cell lines (Fig. 2). The observed apoptotic effects were time-dependent (K562, KG-1). Among
the tested compounds, the most effective apoptosis inducers were again tetrabromo- (K164)
and tetraiododerivatives (K165). On the other hand, compound K170 (dichloroderivative)
exhibits no proapoptotic activity on any of the tested cell lines. Induction of apoptosis was
highest in the LNCaP cell line. The percentage of apoptotic cells was 91.80 after 48 h
exposure with 10 μM K164. Surprisingly, there is a lack of response from the PC-3 cells to
any of the studied compounds, especially compared to another prostate cell line (LNCaP cell
line) (Fig. 2). However, it should be noted that LNCaP cells responded to the compounds that
were most active in cytotoxicity tests, tetrabromo- (K164) and tetraiododerivatives (K165),
regardless of the dose but in a time-dependent manner (Fig. 2).
3.3. Mitochondrial membrane potential (ΔΨm)
Incubation with compound K164 and all remaining inhibitors (data not shown) caused
mitochondrial membrane depolarization (as evidenced by the shift from red-to-green
fluorescence ratio) in the KG-1, K562, LNCaP and PC-3 cell lines. This effect was dose-
12

dependent and may be linked to the intrinsic apoptotic pathway. Representative cytograms for
the tetrabromoderivative (K164) and leukaemia as well as prostate cell lines are shown in
Fig. 3.
3.4. Activity of caspases
The FLICA method was used to assess and identify apoptosis based on the detection of
caspase activation in cell lines treated with compound K164 for 48 h. We found that in the
process of apoptosis both caspase-8 and -9 are involved (Fig. 4).
3.5. Morphological changes in cells
The influence of compounds on the morphology of cells was observed by fluorescence
microscope and inverted light microscope. No changes were observed for control cells.
Characteristic apoptotic changes in the morphology of treated cells (changes in chromatin
concentration and apoptotic body formation) were observed by fluorescence microscope, and
changes in confluence of adherent cells were also observed. The morphology of cell lines
treated with K164 is shown in Fig. 5.
3.6. Effect of compounds on the levels of proteins
To determine the levels of proteins important for the apoptosis process and two proteins, Akt1
and Bad, that are directly phosphorylated by protein kinase CK2 (Akt1) and PIM1 (Bad), we
performed Western blot analysis of whole-cell extracts obtained from cells cultured in the
presence of the most promising inhibitor K164 (concentration 10 μM, 20 μM, 50 μM) after 48
h of incubation. We found that levels of pro-apoptotic (Bad) and anti-apoptotic (BCL-2,
XIAP) proteins decreased after treatment with K164 in KG-1, K562 and LNCaP cells with the
exception of prostate cancer line PC-3. Interestingly, protein BCL-2 was not detected in
13

extracts obtained from K562 cells. Western blots tests showed that compound K164 at 20 μM
and 50 μM induces PARP cleavage in the KG-1, K562 and LNCaP cell lines but no changes
in the level of this protein in PC-3 cells. PARP cleavage (89 kDa) was dose-dependent. We
can see that with the increase in the level of PARP (89 kDa), we observe a decrease in the
level of PARP (113 kDa) in the KG-1 and K562 cell lines. The difference is in the case of
LNCaP cells where the levels of both PARPs (89 kDA and 113 kDa) increased. Interestingly,
there is a very high level of PARP (113 kDa) after treatment of LNCaP cells with K164 at a
concentration of 20 μM and 50 μM, in line with its proapoptotic activity decrease. There was
also a decrease in phosphorylated Akt1 (Ser129) and Bad (Ser112) protein levels in cell
lysates obtained after treatment of cells (KG-1, K562, LNCaP) with the test compound.
However, in KG-1 cells, the result is ambiguous in the case of phospho-Akt1 (Ser129) protein
levels. An exemplary plot of Western blot analysis for K164 is presented in Fig. 6.
3.7. Effect of compounds on cell-cycle progression
Exemplary DNA histograms shown in Figure 7 demonstrate changes in cell-cycle progression
of cells after 48 h of incubation with the most cytotoxic compound K164. This compound at
concentrations of 20 μM and 50 μM induced large changes and perturbations in the cell cycle
distribution; precise statistical analysis of the DNA histograms by the MacCycle computer
programme was often impossible. Accumulation of cells in sub-G1 indicates of DNA
degradation often seen in apoptotic cells. The compound K164 causes a concentration-depend
accumulation of PC-3 cells in the G2M phase and the border of S and G2M phases, reducing
the percentage of cells in G1 phase of the cell cycle.
14

4. Discussion
In this paper, we have shown potential anticancer activity of selective inhibitors of protein
kinases CK2 and PIM-1 on neoplastic cell lines. Compounds K156, K157, K163, K164 and
K165 exhibited moderate to high cytotoxic and pro-apoptotic activity in KG-1 and K562
myeloid and LNCaP (but not PC-3) prostate cell lines (compound K170 was ineffective in all
cell lines) (Table 1, Fig. 2). Among the compounds studied, K164 (1-(β-D-2′-
deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole) appears to be the most promising.
According to the previous publication, K164 inhibits CK2 by two mechanisms: an ATP site-
directed mechanism and an allosteric one. K164 is purely competitive with respect to ATP (Ki
= 0.015 µM) and to PIM-1 (Ki = 0.040 µM). To assess the selectivity of K164, this compound
has been profiled at 1 μM concentration on a panel of 124 kinases. Only CLK2 and DYRK1A
are inhibited by K164 as drastically as CK2 and PIM-1. K164 reduces towards the two protein
kinases (IC50 = 0.032 μM with CK2 and 0.086 μM with PIM-1) (Cozza et al. 2014). Previous
studies have also shown the cytotoxic efficiency of K164 with DC50 (concentrations inducing
50% cell death) values from 2.45±0.84 µM to 28.91±3.74 µM on different cell lines (Cozza et
al. 2014).
These data encouraged us to pursue broader research on the series of inhibitors on neoplastic
myeloid and prostate cell lines. We found that the most active compound K164 evoked high
cytotoxic and pro-apoptotic effects in K562, a Philadelphia (Ph) chromosome-positive (BCR-
ABL-positive) leukaemia cell line derived from chronic myeloid leukaemia (CML) in blast
crisis (Lozzio et al. 1975) as well as in the BCR-ABL negative acute myelogenous leukaemia
cell line KG-1. Compounds K156, K157, K163 and K165 exhibited higher cytotoxic and pro-
apoptotic activity in the K562 cell line (as compared to KG-1 cell line). The results of
investigations by Meeker et al. showed that among 37 human cell lines tested, the myeloid
cell lines with highest expression of the PIM-1 gene are K562 and KG-1 (Meeker et al. 1987,
15

1990). Moreover, kinase CK2 is highly expressed in proliferating myeloblastic cells from
patients with acute myelogenous leukaemia (AML) or chronic myelogenous leukaemia
(CML) in blastic crisis (Phan-Dinh-Tuy et al. 1985). The better efficiency of compounds in
the K562 cell line could be explained in terms of a key role of PIM1 and CK2 kinases in the
BCR/ABL-mediated cell protection from apoptosis. It should be noted that PIM1 can promote
proliferation of BCR/ABL-transformed cells, inhibit activation of caspase-3 and stimulate cell
cycle progression (Nieborowska-Skorska et al. 2002). CK2 is an essential mediator of BCR-
ABL oncogenic signals. The BCR-ABL/CK2 complex is indeed responsible for mediating
BCR-ABL induced cellular proliferation and survival. It was demonstrated that BCR-ABL is
able to physically interact with CK2 in the K562 cell line via the ABL portion of the chimeric
protein (Morotti et al. 2015).
In our study, we have shown that most of the tested inhibitors evoke cytotoxic and pro-
apoptotic effects in two myeloid cell lines and one prostate cell line (Table 1, Fig. 2). An
increase of mitochondrial permeability, cleavage of caspases-8 and -9 and characteristic
changes in the morphology of cells accompanied their apoptosis process (Fig. 3-5).
These results may suggest intrinsic and extrinsic pathways of apoptosis induced by the dual
inhibitors of CK2 and PIM-1. Unfortunately, compound K170 [1-(β-D-2′-
deoxyribofuranosyl)-5,6-dichloro-1H-benzimidazole] does not exhibit cytotoxicity or
proapoptotic activity on any of the cell lines tested (Table 1, Fig. 2), which may be due to its
high IC50 value (>40 μM) for both CK2 and PIM-1 kinases (Cozza et al. 2014).
Additionally, the lack of response of PC-3 cells to all studied compounds in comparison to
prostate cell line LNCaP should be noted (Fig. 2). This result may be related to the
characteristics of the cell lines, for example PC-3 is an androgen-independent cell line that
was derived from an advanced prostate carcinoma and expresses null p53, while LNCaP is an
androgen-responsive human prostate adenocarcinoma derived from an early stage of prostate
16

carcinoma and expresses wild-type p53. It was found that the status of p53 is associated with
the differential sensitivity observed in prostate cancer cells in response to treatment (Liu et al.
2013). On the other hand, other studies have shown that p53 is not required for the induction
of apoptosis (Schneider et al. 2010).
Kinase CK2 plays a specific role in the proliferation regulation mechanism of prostate cancer.
It has been shown that alterations in CK2 activity are dependent on the androgenic status were
due to a rapid shutting of CK2 from chromatin structures into the nucleus and to the nuclear
matrix. Moreover, it has been shown that androgen withdrawal in rats was followed by a rapid
loss of CK2 from nuclear compartments and induced apoptosis. The increased responsiveness
of androgen-sensitive cells compared with androgen-insensitive cells may relate to the
observation that androgen receptor-mediated transcriptional activity is modulated by CK2.
These data indicated tissue-specific signalling by CK2 (Wang et al. 2008; Ahmed et al. 2015).
In our study, we observed a decrease in pro-apoptotic activity of compound K164 in LNCaP
cells at 20 µM concentration compared to 10 µM (Fig. 2). Based on our results it could be
suggested that the above fact has an association with strong expression of the Poly(ADP-
ribose) polymerase (PARP), which participates in DNA repair (Fig. 6).
We can see not only high levels of PARP protein (113 kDa) but also increased levels of
PARP cleavage (89 kDa), proteins characteristic of apoptosis (Fig. 6). Western blot analysis
of whole-cell extracts obtained from KG-1 and K562 cells cultured in the presence of K164
showed increased levels of PARP (89 kDa) and decreased levels of PARP (113 kDa). We
discovered decreased levels of pro-apoptotic protein Bad (total) and phospho-Bad (Ser112)
(Fig. 6). PIM-1 kinase promotes inactivation of the Bad protein by phosphorylating it on the
Ser112 gatekeeper site and is one of several mechanisms via which the PIM-1 kinase can
enhance BCL-2 activity and promote cell survival (Aho et al. 2005). Moreover, the level of
phospho-AKT1 (Ser129) protein is decreased by the K164 inhibitor (Fig. 6). We have also
17

shown high levels of the anti-apoptotic proteins BCL-2 and X-linked inhibitor of apoptosis
protein (XIAP) in PC-3 cell extracts (Fig. 6), which may affect resistance to apoptosis.
Whereas our results also showed decreases in the levels of BCL-2 and XIAP in extracts
obtained from cell lines (KG-1, LNCaP cells) after the treatment of cells with K164. The
change in XIAP protein level in LNCaP cells seems to be very interesting. There is observed
the very low level of this protein at 10 μM K164 compared to concentration of 20 μM and 50
μM. Protein BCL-2 was not detected in K562 cells in contrast to other cell lines. As shown in
previous studies, K562 cells have high levels of BCL-xL without expression of BCL-2
(Benito et al. 1996; Yin et al. 2011). XIAP is a member of the inhibitors of apoptosis family
of proteins (IAPs), and it is regulated by kinase CK2. Downregulation of CK2 results in loss
of cellular IAPs in a distinct manner, and overexpression of CK2 protects IAPs from such
downregulation induced by apoptotic signals (Wang et al. 2008).
In conclusion, our results showed that the majority of the tested inhibitors exhibited cytotoxic
properties and induced apoptotic death in myeloid leukaemia and androgen-responsive
prostate cell lines. The pro-apoptotic effects of the tested inhibitors may be related to the
intrinsic and extrinsic pathway of apoptosis. Among all examined compounds, 1-(β-D-2′-
deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (K164) appears most promising
for anticancer treatment, whereas its 5,6-dichloroanalogue is completely inactive.
Acknowledgements
This research was partly supported by the Foundation of Development of Diagnosis and
Therapy (Warsaw, Poland).
18

References
Ahmed, K.A., Wang, G., Unger, G., Slaton, J., Ahmed, K., 2008. Protein kinase CK2 - A key
suppressor of apoptosis. Advavces in Enzyme Regulation. (48), 179-87.
https://dx.doi.org/10.1016%2Fj.advenzreg.2008.04.002.
Ahmed, K., Issinger, O.G., Szyszka, R., 2015. Protein Kinase CK2 Cellular Function in
Normal and Disease States. (eds.). Springer. 111.
Aho, T.L., Sandholm, J., Peltola, K.J., Mankonen, H.P., Lilly, M., Koskinen, P.J., 2004. Pim-
1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it
on the Ser112
https://10.1016/j.febslet.2004.06.050.
Amaravadi, R., Thompson, C.B., 2005. The survival kinases Akt and Pim as potential
pharmacological targets. J. Clin. Invest. 115 (10), 2618–2624.
https://dx.doi.org/10.1172%2FJCI26273
gatekeeper
site.
FEBS
Lett.
571
(1-3),
43-49.
Benito, A., Silva, M., Grillot, D., Nuñez, G., Fernández-Luna, J.L., 1996. Apoptosis induced
by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL.
Blood. (87), 3837-3843.
Brault, L., Gasser, C., Bracher, F., Huber, K., Knapp, S., Schwaller, J., 2010. PIM
serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies
and
solid
cancers.
Haematologica
95
(6),
1004–1015.
https://dx.doi.org/10.3324%2Fhaematol.2009.017079.
Bretner, M., Najda-Bernatowicz, A., Łebska, M., Muszyńska, G., Kilanowicz, A., Sapota, A.,
2008. New inhibitors of protein kinase CK2, analogues of benzimidazole and
benzotriazole. Mol
Cell
Biochem.
316
(1),
87-89.
http://link.springer.com/article/10.1007%2Fs11010-008-9827-0.
19

Cervantes-Gomez, .F, Chen, L.S., Orlowski, R.Z., Gandhi, V., 2013. Biological Effects of the
Pim Kinase Inhibitor, SGI-1776, in Multiple Myeloma, Clinical Lymphoma,
Myeloma
&
Leukemia
13
(2),
317-329.
http://dx.doi.org/10.1016%2Fj.clml.2013.05.019.
Chen, L.S., Redkar, S., Bearss, D., Wierda, W.G., Gandhi, V., 2009. Pim kinase inhibitor,
SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells. Blood. 114 (19)
4150-4157. https://dx.doi.org/10.1182%2Fblood-2009-03-212852.
Cibull, T.L., Jones, T.D., Li, L., Eble, J.N., Ann Baldridge L., Malot,t S.R., Luo, Y., Cheng,
L. 2006. Overexpression of Pim-1 during progression of prostatic adenocarcinoma. J
Clin Pathol. 59 (3), 285-288. https://dx.doi.org/10.1136%2Fjcp.2005.027672.
Cozza, G., Sarno, S., Ruzzene, M., Girardi, C., Orzeszko, A., Kazimierczuk, Z., Zagotto, G.,
Bonaiuto, E., Di Paolo, M.L., Pinna, L.A., 2013. Exploiting the repertoire of CK2
inhibitors to target DYRK and PIM kinases. Biochim Biophys Acta 1834 (7), 1402–
1409. https://dx.doi.org/10.1016/j.bbapap.2013.01.018.
Cozza, G., Girardi, C., Ranchio, A., Lolli, G., Sarno, S., Orzeszko, A., Kazimierczuk, Z.,
Battistutta, R., Ruzzene, M., Pinna, L.A., 2014. Cell-permeable dual inhibitors of
protein kinases CK2 and PIM-1: structural features and pharmacological potential.
Cell Mol Life Sci. 71 (16), 3173-3185. https://dx.doi.org/10.1007/s00018-013-1552-5
Dhanasekaran, S.M., Barrette, T.R., Ghosh, D., Shah, R., Varambally, S., Kurachi, K., Pient,
K.J., Rubin, M.A., Chinnaiyan, A.M., 2001. Delineation of prognostic biomarkers in
prostat cancer. Nature. 412, 822-826. https://dx.doi.org/10.1038/35090585.
Duncan, J.S., Litchfield, D.W., 2008. Too much of a good thing: the role of protein kinase
CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2. Biochim.
Biophys. Acta 1784 (1), 33–47. https://dx.doi.org/10.1016/j.bbapap.2007.08.017.
20

Fedorov, O., Marsden, B., Pogacic, V., Rellos, P., Müller, S., Bullock, A.N,, Schwaller, J.,
Sundström, M., Knapp, S., 2007. A systematic interaction map of validated kinase
inhibitors with Ser/Thr kinases. Proc Natl Acad Sci U S A 104 (51), 20523–20528.
https://dx.doi.org/10.1073/pnas.0708800104.
Gianoncelli, A., Cozza, G., Orzeszko, A., Meggio, F., Kazimierczuk, Z., Pinna, L.A., 2009.
Tetraiodobenzimidazoles are potent inhibitor of protein kinase CK2. Bioorg Med
Chem 17 (20), 7281-7289. https://dx.doi.org/10.1016/j.bmc.2009.08.047.
Girardi, C., Ottaviani, D., Pinna, L.A., Ruzzene, M., 2015. Different Persistence of the
Cellular Effects Promoted by Protein Kinase CK2 Inhibitors CX-4945 and TDB.
Biomed Res Int. Article ID 185736, 9 pages. https://dx.doi.org/10.1155/2015/185736.
Holder, S., Zemskova, M., Zhang C, Tabrizizad, M., Bremer, R., Neidigh, J.W., Lilly, M.B.,
2007. Characterization of a potent and selective small-molecule inhibitor of the PIM
kinase. Mol Cancer Ther 6 (1), 163–172. https://dx.doi.org/10.1158/1535-7163.MCT-
06-0397.
Janeczko, M., Orzeszko, A., Kazimierczuk, Z., Szyszka, R., Baier, A., 2012. CK2α and
CK2α' subunits differ in their sensitivity to 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-
1H-benzimidazole derivatives. European Jurnal of Medicinal Chemistry. 47 (1), 345-
350. https://dx.doi.org/10.1016/j.ejmech.2011.11.002.
Kaminska, B., Ellert-Miklaszewska, A., Oberbek, A., Wisniewski, P., Kaza, B., Makowska,
M., Bretner, M., Kazimierczuk, Z., 2009. Efficacy and mechanism of anti-tumor
action of new potential CK2 inhibitors toward glioblastoma cells. Int J Oncol. 35 (5),
1091-100. DOI: 10.3892/ijo_00000424.
Kim, J.S., Eom, J.I., Cheong, J.W., Cho,i A.J,, Lee, J.K., Yang, W.I., Min, Y.H., 2007.
Protein Kinase CK2α as an Unfavorable Prognostic Marker and Novel Therapeutic
21

Target in Acute Myeloid Leukemia. Clin. Cancer Res. 13 (3), 1019-1028.
https://dx.doi.org/10.1158/1078-0432.CCR-06-1602.
Koronkiewicz, M., Żukowska, M., Chilmonczyk, Z., Orzeszko, A., Kazimierczuk, Z., 2010.
Synthesis and proapoptotic properties if new casein kinase II inhibitors. Acta Pol
Pharm 67 (6), 635-641. http://www.ptfarm.pl/pub/File/Acta_Poloniae/2010/6/635.pdf.
Koronkiewicz, M., Chilmonczyk, Z. Kazimierczuk, Z., 2013. Synergistic Anti-leukemic
Effects of CK2 Inhibitors and Pentabromobenzylisothioureas In Vitro. Anticancer
Res. 33 (11), 4891-4899. http://ar.iiarjournals.org/content/33/11/4891.full.pdf+html.
Kubiński, K., Masłyk, M., Orzeszko, A., 2017. Benzimidazole inhibitors of protein kinase
CK2 potently inhibit the activity of atypical protein kinase Rio1. Mol Cell Biochem
426 (1-2), 195–203. https://dx.doi.org 10.1007/s11010-016-2892-x.
Laramas, M., Pasquie,r D., Filhol, O., Ringeisen, F., Descotes, J.L., Cochet, C., 2007. Nuclear
localization of protein kinase CK2 catalytic subunit (CK2a) is associated with poor
prognostic factors in human prostate cancer. Eur. J. Cancer 43 (5), 928-934.
https://dx.doi.org/10.1016/j.ejca.2006.11.021.
Liu, C., Zhu, Y., Lou, W., Nadiminty, N., Chen, X., Zhou, Q., Shi, X.B., deVere White, R.W.,
Gao, A.C., 2013. Functional p53 determines docetaxel sensitivity in prostate cancer
cells. Prostate. 73 (4), 418-427. https://dx.doi.org/10.1002/pros.22583.
Lopez-Ramos, M., Prudent, R., Moucadel, V., Sautel, C.F., Barette, C., Lafanechere, L.,
Mouawad, L., Grierson, D., Schmidt, F., Florent, J.C., Filippakopoulos, P., Bullock,
A.N., Knapp, S., Reiser, J.B., Cochet, C., 2010. New potent dual inhibitors of CK2
and Pim kinases: discovery and structural insights. The FEBS Journal 24 (9), 3171-
3185. https://dx.doi.org/10.1096/fj.09-143743.
22

Lozzio, C.B., Lozzio, B.B., 1975. Human chronic myelogenous leukemia cell-line with
positive Philadelphia-chromosome. Blood 45 (3), 321–334.
http://www.bloodjournal.org/content/bloodjournal/45/3/321.full.pdf.
Meeker, T.C., Nagarajan, L., ar-Rushdi, A., Rovera, G., Huebner, K., Croce, C.M., 1987.
Characterization of the human PIM-1 gene: a putative proto-oncogene coding for a
tissue specific member of the protein kinase family. Oncogene Res. 1 (1), 87-101.
Meeker, T.C., Loeb, J., Ayres, M., Sellers, W., 1990. The human Pim-1 gene is selectively
transcribed in different hemato-lymphoid cell lines in spite of a G + C-rich
housekeeping
promoter.
Mol
Cell
Biol.
10
(4),
1680-1688.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC362273/pdf/molcellb00040-0390.pdf.
More, K.N., Jang, H.W., Hong, V.S., Lee, J., 2014. Pim kinase inhibitory and antiproliferative
activity of a novel series of meridianin C derivatives. Bioorganic & Medicinal
Chemistry Letters 24 (11), 2424-2428. https://dx.doi.org/10.1016/j.bmcl.2014.04.035.
Morotti, A., Carrà, G., Panuzzo, C., Crivellaro, S., Taulli, R., Guerrasio, A., Saglio, G., 2015.
Protein Kinase CK2: A Targetable BCR-ABL Partner in Philadelphia Positive
Leukemias. Adv Hematol. 2015, 612567. https://dx.doi.org/10.1155/2015/612567.
Nawijn, M.C., Alendar, A., Berns, A., 2011. For better or for worse: the role of Pim
oncogenes in tumorigenesis.
Nat.
Rev.
Cancer
11
(1),
23–34.
https://dx.doi.org/10.1038/nrc2986.
Nieborowska-Skorska, M., Hoser, G., Kossev, P., Wasik, M.A., Skorski, T., 2002.
Complementary functions of the antiapoptotic protein A1 and serine/threonine kinase
pim-1 in the BCR/ABL-mediated leukemogenesis. Blood 99 (12), 4531-4539.
http://www.bloodjournal.org/content/bloodjournal/99/12/4531.full.pdf.
O-charoenrat, P., Rusch, V., Talbot, S.G., Sarkaria, I., Viale, A., Socci, N., Nga, I,, Rao, P., d
Singh, B., 2004. Casein Kinase II Alpha Subunit and C1-Inhibitor Are Independent
23

Predictors of Outcome in Patients with Squamous Cell Carcinoma of the Lung. Clin
Cancer Res. 10 (17), 5792-803. https://dx.doi.org/10.1158/1078-0432.CCR-03-0317.
Pagano, M.A,, Meggio, F., Ruzzene,, M., Andrzejewska, M., Kazimierczuk, Z., Pinna, L.A.,
2004. 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and
selective inhibitor of protein kinase CK2. Biochem. Biophys. Res. Commun. 321 (4),
1040–1044. https://dx.doi.org/10.1016/j.bbrc.2004.07.067.
Pagano, M.A., Bain, J., Kazimierczuk, Z., Sarno, S., Ruzzene, M., Di Maira, G., Elliott, M.,
Orzeszko, A., Cozza, G., Meggio, F., Pinna, L.A., 2008. The selectivity of inhibitors
of protein kinase CK2: an update. Biochem J. 415 (3), 353-365.
https://dx.doi.org/10.1042/BJ20080309.
Phan-Dinh-Tuy, F., Henry, J., Boucheix, C., Perrot, J. Y., Rosenfeld, C., Kahn, A., 1985.
Protein kinases in human leukemic cells. American Journal of Hematology 19 (3),
209–218. https://dx.doi.org/10.1002/ajh.2830190302.
Pinna, L.,A., 2012. Protein kinase CK2. Wiley, New York.
Pogacic, V., Bullock, A.N., Fedorov, O., Filippakopoulos, P., Gasser, C., Biondi, A., Meyer-
Monard, S., Knapp, S., Schwaller, J., 2007. Structural analysis identifies imidazo[1,2-
b]pyridazines as PIM kinase inhibitors with in vitro antileukemic activity. Cancer Res
67 (14), 6916–69124. https://dx.doi.org/10.1158/0008-5472.CAN-07-0320.
Reiser-Erkan, C.1., Erkan, M., Pan, Z., Bekasi, S., Giese, N.A., Streit, S., Michalski, C.W.,
Friess, H., Kleeff, J., 2008. Hypoxia-inducible proto-oncogene Pim-1 is a prognostic
marker in pancreatic ductal adenocarcinoma. Cancer Biol Ther. 7 (9), 1352-1359.
Sarno, S., Reddy, H., Maggio, F., Ruzzene, M., Davies, S.P., Donella-dean, A., Shuger, D.,
Pinna, A., 2001. Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed
inhihitor of protein kinase CK2 (“casein kinase-2”). FEBS Lett 496: 44-48.
24

Schneider, C.C., Hessenauer, A., Montenarh, M., Götz, C., 2010. p53 is dispensable for the
induction of apoptosis after inhibition of protein kinase CK2. The Prostate 70 (2), 126-
134. https://dx.doi.org/10.1002/pros.21044.
Schneider, C.C., Kartarius, S., Montenarh, M., Orzeszko, A., Kazimierczuk, Z., 2012.
Modified tetrahalogenated benzimidazoles with CK2 inhibitory activity are active
against human prostate cancer cells LNCaP in vitro. Bioorganic & Medicinal
Chemistry. 20 (14), 4390-4396. https://dx.doi.org/10.1016/j.bmc.2012.05.038.
Siddiqui-Jain, A., Drygin, D., Streiner, N., Chua, P., Pierre, F., O’Brien, S.E., Bliesath, J.,
Omori, M., Huser, N., Ho, C., Proffitt, Ch., Schwaebe, M.K., Ryckman, D.M.,
Rice, W.G., Anderes, K., 2010. CX-4945, an Orally Bioavailable Selective Inhibitor of
Protein Kinase CK2, Inhibits Prosurvival and Angiogenic Signaling and Exhibits
Antitumor
Efficacy.
Cancer
Res
70
(24),
10288-10298.
https://dx.doi.org/10.1158/0008-5472.CAN-10-1893.
Trembley, J.H., Chen, Z., Unger, G., Slaton, J., Kren, B.T., Van Waes, C., Ahmed, K., 2010.
Emergence of Protein Kinase CK2 as a Key Target in Cancer Therapy. Biofactors.
36 (3), 187-195. https://dx.doi.org/10.1002/biof.96.
Wang, G., Ahmad, K.A., Harris, N.H., Ahmed, K., 2008. Impact of protein kinase CK2 on
inhibitor of apoptosis proteins in prostate cancer cells. Mol Cell Biochem 316 (1-2),
91–97. https://dx.doi.org/10.1007/s11010-008-9810-9.
Yin, S., Wang, R., Zhou, F., Zhang, H., Jing, Y., 2011. Bcl-xL is a dominant antiapoptotic
protein that inhibits homoharringtonine-induced apoptosis in leukemia cells. Mol
Pharmacol. 79 (6), 1072-1083. https://doi.org/10.1124/mol.110.068528.
Zien, P., Bretner, M., Zastapilo, K., Szyszka, R., Shugar, D., 2003. Selectivity of 4,5,6,7-
tetrabromobenzimidazole as an ATP-competitive potent inhibitor of protein kinase
25

CK2 from various sources. Biochem. Biophys. Res. Commun. 306 (1), 129–133.
http://www.sciencedirect.com/science/article/pii/S0006291X03009288.
Figures and legend:
Fig. 1. Synthesis of 2’-deoxyribo-nucleosides of 4,5,6,7-tetrahalogeno- and 5,6-dichloro-
benzimidazoles 1-6.
Fig. 2. Induction of apoptosis (sum of early and late apoptosis) by agents in KG-1, K562,
LNCaP, and PC-3 cells. The data were determined by FACS cytometer after 24 and 48 h of
treatment. Cells were stained with annexin V-FITC and PI. Each bar represents the mean ±
S.D. (n ≥ 4).
Fig. 3. Representative flow cytograms demonstrating changes in mitochondrial membrane
potential (ΔΨm) of all cells induced by 48 h of culturing with K164 compound. The cells
were stained with JC-1 dye. The cells in the lower right region (R2) showed increased green
fluorescence (apoptotic cells).
Fig. 4. Representative flow histograms demonstrating active caspases in KG-1 cells (panel A),
K562 cells (panel B) , LNCaP cells (panel C) and PC-3 cells (panel D) treated with K164 (50
µM) compound for 48 h. Marker M1 designates the negative cell population whereas M2
designates the positive cell population indicating caspase activity. Cells were labelled by
adding caspase-8 inhibitor reagent (FAM-LETD-FMK) or FLICA caspase-9 inhibitor reagent
(FAM-LEHD-FMK).
26

Fig. 5. Morphology of cells cultured for 48 h. Panel A. Inverted microscopy (light
observation, 100x magnification). Panel B. Fluorescence microscopy (400x magnification).
Morphological examination of cells stained with DAPI/sulforhodamine 101. Cells were
harvested, fixed and stained with DAPI for DNA (blue fluorescence) and sulforhodamine 101
for protein (red fluorescence). Arrows indicate apoptotic bodies (Panel B).
Fig. 6. Western blot analysis of B-cell lymphoma 2 (BCL-2) family proteins (pro- and anti-
apoptotic), PARP proteins (panel A) and phosphorylated proteins (panel B) in whole cell
extracts obtained from cell lines cultured in the presence of K164 (concentration 10 µM, 20
µM, 50 µM) after 48 h of incubation. Preparation of cell extracts and protein detection are
described in Materials and Methods. All bands were quantified by densitometric analysis and
their intensity normalized with respect to β-actin. The ratio of the studied proteins to β-actin
for cell lines incubated in the absence of K164 was assumed to be 1.
Fig. 7. Exemplary DNA histograms of cell lines treated for 48 h with K164 compound
(stained with propidium iodide). The data obtained from the flow cytometer were analysed
using MacCycle software to determine the percentage of cells in each phase of the cell cycle.
Table 1. IC50 values of compounds studied (MTT - assay; n ≥ 3).
IC50 [µM]; 48h
Compound
K156
KG-1
K562
LNCaP
22.50 ± 0.13
29.27 ± 5.96
30.08 ± 5.36
ND*
PC-3
> 50
> 50
> 50
> 50
ND
60.09 ± 12.16
34.65 ± 4.98
32.91 ± 5.73
4.01 ± 0.70
20.12 ± 7.65
> 100
45.06 ± 6.86
12.51 ± 4.33
23.65 ± 1.57
8.51 ± 0.62
10.69 ± 3.63
> 100
K157
K163
K164
K165
8.23 ± 0.75
> 200
K170
> 200
*Not Designated (cells respond regardless of the dose)
27

Fig. 1.
28

Fig. 2.
29