Role of polyamines in determining the cellular response to chemotherapeutic agents: modulation of protein kinase CK2 expression and activity.

Abstract

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

Full text

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Role of polyamines in determining the cellular response
to chemotherapeutic agents: modulation of protein kinase
CK2 expression and activity
Jan N. Kreutzer•Birgitte B. Olsen•
Karolina Lech•Olaf-Georg Issinger•
Barbara Guerra
Received: 13 June 2011/Accepted: 24 June 2011
? Springer Science+Business Media, LLC. 2011
Abstract
compounds stimulate the expression of the polyamine
catabolic enzyme spermidine/spermine N1-acetyltransfer-
ase resulting in anti-proliferative activity and apoptosis. As
many cancer cell types including pancreatic cancer cells
express high levels of polyamines, the possibility to
develop anti-tumor strategies to deplete polyamine pools
has drawn considerable attention in recent years. This has
been effectively accomplished by treating cells with plat-
inum drugs in combination with polyamine analogs such as
N1,N11-diethylnorspermine (DENSPM). The present study,
examined the cytotoxic effects of oxaliplatin in combina-
tion with stimulators of polyamine catabolism in human
pancreatic cancer cells, that are notoriously resistant to
chemotherapeutic treatment, and colorectal cancer cells.
Additionally, as protein kinase CK2 has been shown to be
an anti-apoptotic and pro-survival enzyme regulated by the
intracellular polyamine pools, we aimed to investigate the
effect of combined DENSPM and oxaliplatin treatment on
CK2-mRNA and -protein levels. Results reported here
show that treatment with oxaliplatin and DENSPM in
combination impairs cell viability particularly in the case
of colorectal cancer cells. The analysis of CK2 expression
and activity indicates that the response to a specific treat-
ment may depend on the impact that individual compounds
exert on pro-survival and pro-death proteins at the
Numerous studies have shown that platinum
transcription and translation levels that should be carefully
evaluated in view of subsequent clinical studies.
Keywords
analogs ? Spermidine/spermine N1-acetyltransferase
Protein kinase CK2 ? Oxaliplatin ? Polyamine
Introduction
Polyamines are amino acid-derived organic molecules that
are synthesized in all organisms [1]. Proteins involved in
the synthesis of polyamines, e.g., ornithine decarboxylase
(ODC) which is the rate-limiting enzyme, are expressed in
all tissues [1]. Polyamines are essential molecules for many
physiological processes such as growth, development and
tissue repair. In the late 1960s the association of increased
polyamines synthesis and high levels of ODC activity with
cell growth and cancer arising from epithelial tissues such
as skin, prostate and colon was reported [1]. Despite the
fact that a link between cancer and polyamines deregula-
tion has been reported more than 30 years ago, it is only
recently that evidence emerged about a causative rather
than an associative effect of polyamines in tumor devel-
opment. Studies conducted on colon cancer have revealed
that ODC is negatively modulated by the adenomatous
polyposis coli tumor suppressor gene (APC). Loss of APC
function leads to increased expression of Myc transcription
factor which, in turn, stimulates the synthesis of ODC
[2, 3]. Oncogenic mutations in KRAS, result in aberrant
activation of the enzyme, suppresses the polyamine
catabolism by inhibiting the expression of the peroxisome-
proliferator-activated receptor-c (PPARc) which stimulates
the transcription of the spermidine/spermine N1-acetyl-
transferase (SSAT) gene by binding to its promoter [4, 5].
Polyamines contribute to the regulation of numerous
Jan N. Kreutzer and Birgitte B. Olsen contributed equally to the study.
J. N. Kreutzer ? B. B. Olsen ? K. Lech ? O.-G. Issinger ?
B. Guerra (&)
Department of Biochemistry and Molecular Biology, University
of Southern Denmark, Campusvej 55, 5230 Odense, Denmark
e-mail: bag@bmb.sdu.dk
123
Mol Cell Biochem
DOI 10.1007/s11010-011-0949-4

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cancer-related functions such as cellular proliferation and
blood-vessel development in growing tumor tissues [6].
Increasing evidence indicates that polyamine metabolic
pathways are frequently deregulated in cancer. Hence, it is
not surprising that the development of polyamine meta-
bolic inhibitors has gained interest in recent years leading
to the synthesis of selective inhibitors of ODC such as
2-difluoromethylornithine (DFMO, reviewed in [7]). The
cytotoxicity of DFMO observed with various cancer cell
types encouraged clinical investigations in which the
activity of DFMO as single agent or in combination with
other chemotherapeutic agents, was tested against several
tumor models ([8] and for a review see [9]). As the target
inhibition of enzymes involved in the active synthesis of
polyamines did not seem to confer a significant clinical
success in the treatment of malignancies, subsequent
strategies have focused on the suppression of polyamines
by polyamine analogs such as N1,N11-diethylnorspermine
(DENSPM) and N1,N14-diethylhomospermine (DEHSPM)
[10]. Numerous studies have shown that cells respond
to exposure to polyamine analogs with a large increase
in SSAT expression in multiple tumor cell types such
as melanoma, pancreatic, bladder and breast cancers
(reviewed in [9]). Interestingly, treatment of colorectal
cancer cells with DENSPM in combination with oxaliplatin
used as standard chemotherapeutic agent, led to a signifi-
cant increase in SSAT mRNA levels as compared to oxa-
liplatin and DENSPM single treatments, respectively,
indicating a synergistic effect with respect to the expres-
sion of SSAT [11, 12].
Protein kinase CK2 is a pleiotropic and constitutive
active serine/threonine enzyme expressed in all eukaryotic
organisms. It is composed of two catalytic a- and/or a0-
subunits and two regulatory b-subunits. The regulatory
subunits have been shown to confer stability, modulate
substrate specificity as well as the activity of the holoen-
zyme [13]. CK2 appears distributed in tetrameric com-
plexes inside the cells but significant evidence indicates
that the individual subunits also exist as free proteins. CK2
phosphorylates and/or interacts with many intracellular
proteins involved in a variety of biological processes
including gene expression, cell cycle regulation, transfor-
mation, apoptosis, DNA sensing and repair (for reviews see
[14–16]). Although CK2 is considered a constitutively
active enzyme, evidence indicates that certain structural
conditions, intracellular distribution or effector molecules
such as polyamines can modulate the activity of CK2 (for a
recent review see [16]). CK2 has been shown to be acti-
vated in vitro by polyamines by directly binding to the
b-subunits while in vivo studies have revealed that
increased polyamine levels result in enhanced CK2 activity
through mechanisms distinct from the direct interaction
with the regulatory subunits observed earlier [17–19].
In this study, as pancreatic cancer cells are notoriously
resistant to treatment with chemotherapeutic agents such as
gemcitabine and platinum analogs used in various combi-
nations (reviewed in [20]), we aimed to determine whether
the incubation of cells with oxaliplatin and other com-
pounds that modulate polyamine metabolism may have
synergistic effects in inducing significant cytotoxicity as
reported in the case of colorectal cancer cells [11, 21]. In
addition, as CK2 expression and activity are elevated in
highly proliferating normal and cancer cells and stimulated
by increased cellular polyamine levels, we evaluated the
effects on CK2 at the mRNA and protein levels following
treatment with the aforementioned compounds used alone
or in combination. The data presented here, indicate that
cell treatment with compounds regulating polyamine
metabolism does not affect the viability and proliferation of
pancreatic cancer cells to the same extent as reported in
colorectal cancer cells. Moreover, the analysis of CK2
mRNA levels, protein expression and activity suggests that
the outcome of a specific combination treatment largely
depends on the effect exerted by the individual compounds
on the transcriptional and translational regulation of the
expression of key survival proteins.
Materials and methods
Cell culture and treatments
The human pancreatic cancer PANC-1 and colorectal
cancer HCT116 cell lines were obtained from the Ameri-
can Type Culture Collection (Rockville, MD, USA). The
cells were cultured in Dulbecco’s modified Eagle’s med-
ium (Invitrogen, Taastrup, Denmark) supplemented with
10% fetal bovine serum and maintained in a humidified
atmosphere containing 5% CO2at 37?C. Cells were treated
with DENSPM (Tocris Bioscience, Bristol, United King-
dom) and oxaliplatin (Sigma, MO, USA) as indicated in the
figure legends.
Viability and proliferation assays
Cell viability was determined by the WST-1 assay (Roche,
Penzberg, Germany) in 96-well plates. Twenty-four hours
after seeding, cells were treated with various concentra-
tions of oxaliplatin and DENSPM for 72 h as indicated in
the figure legends. The viability assay was performed
according to the manufacturer’s recommendations. In brief,
cells were incubated with the WST-1 reagent for 2 h at
37?C before absorbance was measured with a microtiter
plate reader (VERSAmax, Molecular Devices, Sunnyvale,
CA, USA) at wavelengths indicated in the figure legends.
Similarly to the WST-1 test, the cell proliferation assay
Mol Cell Biochem
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was performed in 96-well plates. Twenty-four hours after
seeding, cells were treated with various compounds as
described in the figure legends. Afterward, cells were
labeled with 5-bromo-20deoxyuridine (BrdU) for 3 h
(Calbiochem, Nottingham, United Kingdom). Cells were
then fixed, the DNA was denatured and cells were incu-
bated with a peroxidase-conjugated anti-BrdU antibody
according to the manufacturer’s instructions. The immune
complexes were revealed in a microtiter plate reader as
indicated in the figure legends.
Cell cycle analysis
Thedistributionofcellsinthevariousphasesofthecellcycle
was estimated by flow cytometry. Cells were fixed in 70%
ethanolat-20?Covernightandsubsequentlyincubatedwith
20 lg/ml propidium iodide (Sigma) and 40 lg/ml RNase A
(Roche) in PBS for 30 min in the dark. Cells were then
analyzed on a FACS-Calibur flow cytometer (Becton–
Dickinson, CA, USA). The acquired data were analyzed by
CellQuest Pro Analysis software (Becton–Dickinson). For
each measurement, 10,000 cells were analyzed.
Western blot analysis and densitometric analysis
Whole cell extracts were prepared as described in [22]. For
protein detection on western blot membranes, the following
antibodies have been used: mouse monoclonal anti-CK2a
and -b were from Calbiochem, mouse monoclonal anti-b-
actin was from Sigma, rabbit polyclonal anti-CK2a0was
obtained by immunizing rabbits with a specific peptide
(SQPCADNAVLSSGTAAR), rabbit polyclonal anti-SSAT
(H-77) was from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). Densitometric analysis of protein bands on
western blot was performed with the UVIsoft analysis
software (UVItec, Cambridge, UK).
In vitro protein kinase assay
The activity of native protein kinase CK2 present in whole
extracts from HCT116 and PANC-1 cells was determined
as described in [23].
Quantitative real-time PCR
Total RNA from cells treated as indicated in the figure
legends was isolated using TRIzol reagent (Invitrogen).
300 ng of total RNA was used for reverse transcription
using the GeneAmp?RNA PCR Core Kit (Applied Bio-
systems by Life Technology, Carlsbad, CA, USA). The
obtained cDNAs were then used as a template for the
subsequent PCR. PCR reactions were performed in a 20 ll
volume consisting of 30 ng template, 1x SYBR?Green
JumpStart
primers relative to the cDNA of the analyzed proteins each
at 200 nM final concentration. All samples were prepared
in triplicates. The reactions consisted of a 10 min initial
denaturation (95?C) followed by 40 cycles of denaturation
(95?C, 15 s) and annealing/extension (60?C, 1 min).
Measurement of SSAT, b-actin, CK2a, -a0and -b gene
expression levels was carried out according to the quanti-
fication method of the StepOnePlus real-time PCR System
(Applied Biosytems). All mRNA expression values are
ratios relative to b-actin. Fold changes in samples derived
fromdrug-treatedcellsweredeterminedrelativetountreated
(control) samples. Primer pairs against the human SSAT1,
b-actin, CK2a, -a0and -b mRNAs were as follows: for
SSAT-1, forward primer 50-TCTAAGCCAGGTTGCAAT
GAGGTGT-30and reverse primer 50-ACAGTCTCCAACC
CTCTTCACTGGAC-30; b-actin, forward primer 50-GACA
GGATGCAGAAGGAGATTACT-30and reverse primer
50-TGATCCACATCTGCTGGAAGGT-30; CK2a, forward
primer 50-ACGAGTCACATGTGGTGGAA-30and reverse
primer50-TTTACCTCGGCCTAATTTTCG-30;
forward primer 50-TCAAGGTACTTCAAGGGACCAG-30
and reverse primer 50-TCCACATGTCCAAGCTATAAT
CA-30; CK2b, forward primer 50-ACCTGCCAACCAGT
TTGTG-30and reverse primer 50-GACTGGGCTCTTGA
AGTTGC-30.
TMTaq ReadyMix
TM(Sigma), forward and reverse
CK2a0,
Statistical analysis
The statistical significance of differences between the mean
of two sets of data was evaluated by the two-tailed-t-test
(Student’s t-test) and the level of significance is indicated
in the figure legends.
Results
The combination of oxaliplatin and the spermine analog
DENSPM results in synergistic effects on pancreatic
and colorectal cancer cell lines
Changes in SSAT-mRNA and -protein expression levels
were investigated in the human pancreatic adenocarcinoma
PANC-1 cell line following treatment with oxalipatin and
DENSPM as single agents or in combination. By com-
parison, the evaluation included also the analysis of the
human colorectal cancer HCT116 cell line previously
reported to respond to the aforementioned treatment by a
marked increase in SSAT mRNA levels [21]. As shown in
Fig. 1a, treatment of both cell lines with combined oxa-
liplatin and DENSPM markedly increased the SSAT
mRNA level up to 48 h upon drug treatment. The increase
was more pronounced in the case of HCT116 cells, where a
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*15-fold increase in the expression with respect to control
experiment was observed. In PANC-1 cells, the combina-
tion treatment led to a *8-fold increase in SSAT mRNA
level in comparison to control cells. The level of SSAT
mRNA returned almost to control values by 72 h after drug
treatment in both cell lines (Fig. 1b). Oxaliplatin slightly
increased the amount of SSAT mRNA transcripts as
compared to control experiments (*3- and *4-fold in
HCT116 and PANC-1 cells, respectively, Fig. 1a). Next,
the expression of SSAT protein was analyzed in both cell
lines 72 h after adding the drugs to the culture. The com-
bination of oxaliplatin and DENSPM significantly induced
SSAT protein expression (Fig. 1c). DENSPM alone
slightly induced SSAT expression in PANC-1 while the
protein expression level was higher in HCT116 cells.
Although oxaliplatin treatment resulted in enhanced SSAT
mRNA levels in both cell lines, a concomitant increase in
SSAT protein expression under the indicated experimental
conditions was not observed. As induction of SSAT has
been linked to the anti-proliferative effects of DENSPM
[24, 25], dose–response experiments were performed by
measuring the metabolic activity of cells treated with ox-
aliplatin and DENSPM as single agents or in combination
for 72 h (Fig. 2). Oxaliplatin at 0.25 lM concentration in
combination with 2.5 lM DENSPM led to severe cyto-
toxicity of HCT116 cells resulting in *34% viable cells
with respect to untreated cells. Incubation at higher con-
centration of oxaliplatin (i.e. 0.5 lM) dropped the survival
to almost 0%. PANC-1 were more resistant as *61% cells
were still viable in comparison to HCT116 cells. Although
the combination treatment showed a synergistic effect on
cell viability, a higher concentration of oxaliplatin (i.e.
0.5 lM) did not further increase the cytotoxicity of PANC-
1 cells as observed in the case of HCT116 cells. The anti-
proliferative effects of oxaliplatin and DENSPM were also
investigated. 0.25 lM oxaliplatin in combination with
2.5 lM DENSPM for 72 h exerted a major effect on
HCT116 cells as their proliferation decreased by *75%
with respect to control experiments while the proliferation
of PANC-1 cells decreased about 38% under the same
SSAT
Actin
0
4
8
12
16
mRNA (fold change)
HCT116
HCT116
0
4
8
12
16
mRNA (fold change)
A
C
SSAT
PANC-1
Actin
PANC-1
0
4
8
12
16
mRNA (fold change)
B
mRNA (fold change)
PANC-1
0
4
8
12
16
HCT116
Fig. 1 Effect on SSAT mRNA
and protein levels following
cellular treatment with
oxalipatin and DENSPM.
a Human HCT116 and PANC-1
cells were incubated singly with
oxaliplatin (Oxa) and DENSPM
or in combination for 24 h
followed by 24 h incubation in
drug-free medium. SSAT
mRNA was quantified
(triplicate measurements)
relative to b-actin by
quantitative RT-PCR. Fold
changes are relative to control
experiments (CT).
b Experiments were performed
as in (a) except that cells were
harvested after 48 h incubation
in drug-free medium. c Whole
protein extracts from cells
treated as in (b) were analyzed
by SDS-PAGE followed by
Western blot analysis. The
expression of SSAT was
verified by probing the
membrane with a rabbit
polyclonal anti-SSAT antibody.
Detection of b-actin protein was
used as a loading control. Three
separate experiments were
performed obtaining similar
results. Data from one
representative assay are shown.
Bars indicate the average of
three independent
measurements ± standard
deviation (SD)
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treatment
above, revealed dose–response effects of oxaliplatin and
DENSPM alone, respectively, but also synergistic effects
when compounds were used in combination.
Subsequent experiments were conducted with PANC-1
cells by applying conditions described in the legend to
Fig. 1 as they led to the observations reported in the sub-
sequent figures. In parallel, HCT116 cells were also
employed confirming data from previous studies on the
induction of SSAT-gene and -protein expression levels in
these cells [21]. The analysis of cell death by flow
cytometry induced by 10 lM oxaliplatin incubation of
conditions(Fig. 3).Experimentsdescribed
HCT116 cells for 24 h followed by 48 h in drug-free
medium, led to *15% cell death. This percentage further
increased to *40% in the oxaliplatin/DENSPM combina-
tion treatment (Fig. 4). In the case of PANC-1 cells, oxa-
liplatin alone induced a 15% increase in cell death with
respect to control experiment while the addition of
DENSPM did not lead to a further change in the percentage
of cells with reduced DNA content observed with the sole
oxaliplatin treatment.
0
1
2
3
4
HCT116
PANC-1
0
1
2
3
4
Viability (arbitrary units)
Viability (arbitrary units)
3.09 2.902.872.672.372.712.142.601.871.98
2.80 2.73 2.47 1.86 1.30 2.99 2.43 2.06 0.99 0.14
Fig. 2 Effect of oxaliplatin and DENSPM on the viability of human
colorectal HCT116 and pancreatic adenocarcinoma PANC-1 cell
lines. Cells were treated with the indicated concentrations of
oxaliplatin and DENSPM for 72 h. Proportions of viable cells
determined by the WST-1 assay are shown in arbitrary units as the
difference in absorbance measured at 450 and 690 (reference) nm
wavelengths, respectively. Each bar indicates the average of six
independent measurements ± SD. Experiments were repeated three
times obtaining similar results and data from one representative
experiment are shown
HCT116
0
0.5
1
1.5
2
PANC-1
0
0.5
1
1.5
2
Proliferaion (arbitrary units)
Proliferation (arbitrary units)
1.66 1.68 1.71 1.72 0.99 1.27 1.40 1.31 0.36 0.04
1.26 1.24 1.27 1.19 0.92 1.14 1.07 1.07 0.88 0.73
Fig. 3 Effect of oxaliplatin and DENSPM on cell proliferation. Cell
proliferation for the indicated cell lines treated as described in Fig. 2,
was determined by BrdU incorporation into genomic DNA. Results
are expressed in arbitrary units as the difference in absorbance
measured at 450 and 540 (reference) nm wavelengths, respectively.
Experiments were repeated three times obtaining similar results and
data from one representative experiment (bars indicate means ± SD
of three measurements) are shown
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Cellular treatment with oxaliplatin and DENSPM
affects the CK2-mRNA and -protein expression levels
Previously, it has been reported that the activity of protein
kinase CK2 is enhanced as a result of polyamines binding
to the regulatory b-subunit in vitro [17, 18]. However, later
studies showed that overexpression of ODC resulted in a
3-fold increase in CK2 mRNA molecules suggesting that
the regulation of endogenous CK2 may occur through
mechanisms distinct from the direct stimulation by poly-
amines as reported earlier [19]. Because of the role of CK2
as anti-apoptotic and pro-survival protein in cancer cells
and the effects mediated by polyamine levels, we first
investigated whether the simultaneous treatment of cells
with oxaliplatin and DENSPM affected the kinase activity
of CK2. In Fig. 5, treatment of HCT116 cells with oxa-
liplatin and DENSPM resulted in a slightly higher activity
of CK2 with respect to control experiment. This effect was
observed also in PANC-1 cells at longer incubation time
(i.e. up to 144 h, results not shown).
To test whether oxaliplatin and DENSPM treatments
used as individual agents or in combination had an effect
on CK2-gene transcription, real-time PCR analysis was
performed to quantify the transcript levels in both cell
lines. As shown in Fig. 6a, oxaliplatin alone produced a
slight increase of the individual CK2 subunit mRNA
levels in HCT116 cells while the induction was much
more significant in PANC-1 cells compared to control
experiment. DENSPM itself did not cause any effect
while the combination treatment did not result in a higher
increase in mRNA, with respect to oxaliplatin alone, in
both cell lines indicating that oxaliplatin is responsible for
the marked induction of CK2-gene transcription. Unex-
pectedly, increased expression of the individual CK2
subunits transcripts was not accompanied by higher pro-
tein expression levels (Fig. 6b) as anticipated from the
results shown in Fig. 6a. The indicated treatments caused
decreased expression of the individual CK2 subunits, with
respect to control cells, in both cell lines which was
significant in the case of CK2a with respect to the
0
20
40
60
80
100
0
20
40
60
80
100
HCT116 PANC-1
Sub-G1 (%)
Sub-G1 (%)
Fig. 4 Effect on cell death in cells treated with oxaliplatin and
DENSPM. Cells treated singly with oxaliplatin and DENSPM or in
combination for 24 h were subsequently incubated in drug-free
medium for additional 48 h prior to harvesting. Fixed cells were
subjected to flow cytometry analysis following propidium iodide
staining. The fraction of cells in sub-G1 (i.e. with reduced level of
DNA indicative of late apoptosis and/or necrosis) is reported in
percentage and calculated with respect to the total number of cells.
Experiments were performed three times obtaining similar results.
Data from one representative experiment is shown
0
0.5
1
CK2 activity
(pmol/min/µ µg)
0
0.5
1
CT 10 M 10 M 10 M
CK2 activity
(pmol/min/µ µg)
HCT116 PANC-1
0.59 0.79 0.71 0.67
0.55 0.58 0.51 0.55
Fig. 5 Determination of protein
kinase CK2 activity following
incubation of cells with
oxalipatin and DENSPM. Cells
were treated as indicated in
Fig. 4. Total lysates (20 lg)
were subjected to a radioactive
CK2 kinase assay with a
specific substrate peptide as
indicated in the materials and
methods section. The average ±
SD of three independent
experiments is shown
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combination treatment as also indicated by the densito-
metric analysis of protein bands.
Discussion
Previous studies reported that incubation of colorectal
cancer cells with platinum drugs such as oxaliplatin results
in a slight induction of SSAT mRNA which significantly
increases when cells are treated with the drug in combi-
nation with the polyamine analog DENSPM. In the present
work, we observed a similar effect in the pancreatic cancer
cell line PANC-1 although not as pronounced as in the case
of HCT116 cells. The analysis of SSAT protein expression
levels in both type of cells revealed that induction of SSAT
mRNA by oxaliplatin apparently did not result in a con-
comitant expression of the protein unless DENSPM was
present. Moreover, the combination of DENSPM with
oxaliplatin led to a significant increase in SSAT protein
level compared to the treatment with the single agents, only
in PANC-1 cells. In HCT116 cells there was no difference
in the expression of SSAT induced by DENSPM alone or
in combination with oxaliplatin. While it is unclear what
the underlying mechanisms are, it is likely that differences
observed with the two cell lines might be related to their
biology and that DENSPM appears to facilitate the trans-
lation of SSAT mRNA. The cytotoxic and cell proliferation
effects of oxaliplatin and DENSPM used as single agents
or in combination were extensively studied in both cell
lines at various concentrations. Our results indicate that the
effects were synergistic at the concentrations employed in
the study although marked cytotoxicity and reduced cell
proliferation were observed only in HCT116 cells. Results
obtained with PANC-1 cells suggest that these cells might
0
1
2
3
A
0
1
2
3
4
5
mRNA (fold change)
CK2
CK2
CK2
B
HCT116
CK2
CK2
Actin
CK2
PANC-1
PANC-1
CK2
Actin
CK2
CK2
CK2
CK2
CK2
mRNA (fold change)
HCT116
1.0 0.6 0.7 0.2
1.0 0.8 0.8 0.5
1.0 0.8 0.7 0.6
1.0 1.0 0.7 0.5
1.0 1.1 0.6 0.6
1.0 1.1 0.7 0.7
*
*
*
*
*
*
Fig. 6 Effect of oxaliplatin and DENSPM alone and in combination
on CK2 mRNA (a) and protein levels (b) in HCT116 and PANC-1
cells. Cells were exposed to 10 lM oxaliplatin, 10 lM DENSPM
singly or in combination for 24 h followed by 48 h incubation in
drug-free medium. Cells were harvested and subsequently processed
for either quantitative RT-PCR or Western blot analysis. In (a), CK2
mRNA was quantified (triplicate measurements) relative to b-actin.
Fold-changes are relative to untreated controls. In the two graphs, the
vertical axis have different scales to better highlight differences in
gene expression levels between control and treated cells in the two
cell lines. Statistical significance of the differences between control
and oxaliplatin-treated groups, respectively, was determined by the
Student’s t-test which resulted in *P\0.005 (statistically significant)
for all the determinations. In (b), protein expression levels were
quantified by densitometric analysis from a representative Western
blot deriving from one out of three separate experiments. Fold
changes were calculated with respect to protein levels in untreated
cells. b-actin was used as loading control
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be not highly dependent on the intracellular level of
polyamines although numerous studies have shown that
pancreatic cancer cell lines have significantly higher levels
of polyamines than normal pancreatic tissue. In this
respect, Black and colleague demonstrated a positive cor-
relation between the levels of ODC, protein content and
chemoresistance of the various pancreatic cancer cell lines
analyzed in their studies [26, 27]. However, despite the
enhanced expression of SSAT protein in PANC-1 cells,
one cannot exclude the possibility that the polyamine levels
might still be elevated, thus, counteracting the effects
induced by the oxaliplatin/DENSPM combination treat-
ment. Moreover, despite the higher expression, SSAT
enzyme activity might be comparable to basal values. In
this respect, studies on human lung carcinoma cell lines
sensitive and resistant to the effects of DENSPM, respec-
tively, have demonstrated a correlation between SSAT
activity and cytotoxicity induced by the polyamine
analog [28].
Numerous studies have shown that protein kinase CK2
is an important pro-survival enzyme since its overexpres-
sion protects against drug-induced apoptosis and chemical
inhibition or siRNA-mediated downregulation can sensitize
cancer cells toward cell death (reviewed in [29]). In our
study, treatment with oxaliplatin alone stimulates tran-
scription of the CK2-gene and the transcripts are not
translated into proteins in both cell lines (Fig. 6). Instead,
DENSPM seems to negatively contribute to the regulation
of CK2 protein expression and the lowest expression of the
individual CK2 subunits is observed following incubation
of cells with DENSPM in combination with oxaliplatin. In
this respect, it would be interesting to investigate the role
of DENSPM with respect to the modulation of CK2 protein
synthesis and degradation. In HCT116 cells, a significant
percentage of cell death is observed with oxaliplatin and
DENSPM co-treatment (Fig. 4). This effect is accompa-
nied by marked down-regulation of CK2 subunits expres-
sion supporting the notion that the kinase plays an
important pro-survival role in colorectal cancer cells.
Decreased CK2 expression was also observed in PANC-1
cells but this was not accompanied by a concomitant
increase in cell death under the same experimental condi-
tions (Fig. 6). The present data suggest that apparently
CK2 does not seem to have any influence on the survival of
pancreatic cancer cells as reported earlier (Fig. 4, [30] and
reviewed in [31]). However, data obtained from experi-
ments where PANC-1 cells were exposed to lower con-
centrations of the aforementioned compounds for an
Oxa/DENSPM
Impaired synthesis/
Enhanced degradation
Inactive pool Active protomers
Fig. 7 Model of the proposed
effects on the CK2 holoenzyme
molecules following the
simultaneous exposure to
oxalipatin and DENSPM.
Equilibrium exists inside cells
between CK2 molecules present
in the form of catalytically
active protomers (i.e. the
holoenzyme) and inactive
oligomeric aggregates.
Simultaneous exposure to
oxaliplatin and DENSPM
negatively affects the
expression of the free tetrameric
forms causing the release of
active protomers. Only this
latter fraction accounts for the
kinase activity measured in vitro
while both pools (i.e. oligomeric
aggregates and protomers)
contribute to the expression
level of CK2 detected by
Western blot
Mol Cell Biochem
123

Page 9

extensive amount of time (i.e. up to 144 h) led to signifi-
cant cell death also in this cell line (data not shown).
As mentioned above, HCT116 cells showed enhanced
cell death in the combination treatment and this was
accompanied by down-regulation of protein kinase CK2
expression levels. However, slight increased CK2 activity
was observed under the same experimental conditions
(Fig. 5). It is conceivable that the expression of the indi-
vidual CK2 subunits might contribute to the regulation
of cell survival through the interaction with specific intra-
cellular proteins. However, in cell-fate decision one cannot
exclude that there might be a threshold beyond which the
activity of CK2 does not play a decisive role with respect to
survival. The mechanism by which drug combination leads
to slightly higher kinase activity remains to be elucidated.
However, as previously suggested by biochemical and
structural studies [32–35] it is conceivable that the cellular
regulation of CK2 catalytic activity might be based on the
presence of an inactive pool of high molecular mass
aggregates of CK2 molecules in equilibrium with the active
tetrameric form. Treatment of cells with oxaliplatin and
DENSPM in combination would negatively affect the
expression of the individual CK2 holoenzyme molecules
causing the release of tetramers from the supra-molecular
aggregates (Fig. 7).
In summary, our study shows that the combination of
oxaliplatin and DENSPM provides an effective treatment
by inducing high cytotoxicity primarily in colon cancer
cells through modulation of the polyamine pathway.
Moreover, the analysis of protein kinase CK2 at the mRNA
and protein expression levels indicates that the outcome of
a specific treatment strategy is the result of molecular
changes at both transcriptional and translational levels that
should be carefully analyzed and interpreted.
Acknowledgments
Danish Cancer Society (DP08152) and the Danish Natural Science
Research Council (272-07-0258) to BG.
This work was supported by grants from the
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