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Valproic acid inhibits adhesion of vincristine- and cisplatin-resistant
neuroblastoma tumour cells to endothelium
RA Blaheta
1
, M Michaelis
2
, I Natsheh
1
, C Hasenberg
1
, E Weich
1
, B Relja
1
, D Jonas
1
, HW Doerr
2
and J Cinatl Jr*
,2
1
Zentrum der Chirurgie, Klinik fu
¨r Urologie und Kinderurologie, Johann Wolfgang Goethe-Universita¨t, Frankfurt am Main, Germany;
2
Zentrum der
Hygiene, Institut fu
¨r Medizinische Virologie, Johann Wolfgang Goethe-Universita¨t, 60590 Frankfurt am Main, Germany
Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). In pursuit of alternative
treatments for chemoresistant tumour cells, we tested the response of multidrug-resistant SKNSH and of vincristine (VCR)-,
doxorubicin (DOX)-, or cisplatin (CDDP)-resistant UKF-NB-2, UKF-NB-3 or UKF-NB-6 NB tumour cell lines to valproic acid (VPA),
a differentiation inducer currently in clinical trials. Drug resistance caused elevated NB adhesion (UKF-NB-2
VCR
, UKF-NB-2
DOX
,
UKF-NB-2
CDDP
, UKF-NB-3
VCR
, UKF-NB-3
CDDP
, UKF-NB-6
VCR
, UKF-NB-6
CDDP
) to an endothelial cell monolayer, accompanied by
downregulation of the adhesion receptor neural cell adhesion molecule (NCAM). Based on the UKF-NB-3 model, N-myc proteins
were enhanced in UKF-NB-3
VCR
and UKF-NB-3
CDDP
, compared to the drug naı
¨ve controls. p73 was diminished, whereas the p73
isoform deltaNp73 was upregulated in UKF-NB-3
VCR
and UKF-NB-3
CDDP
. Valproic acid blocked adhesion of UKF-NB-3
VCR
and UKF-
NB-3
CDDP
, but not of UKF-NB-3
DOX
, and induced the upregulation of NCAM surface expression, NCAM protein content and
NCAM coding mRNA. Valproic acid diminished N-myc and enhanced p73 protein level, coupled with downregulation of deltaNp73
in UKF-NB-3
VCR
and UKF-NB-3
CDDP
. Valproic acid also reverted enhanced adhesion properties of drug-resistant UKF-NB-2, UKF-
NB-6 and SKNSH cells, and therefore may provide an alternative approach to the treatment of drug-resistant NB by blocking invasive
processes.
British Journal of Cancer (2007) 96, 1699 –1706. doi:10.1038/sj.bjc.6603777 www.bjcancer.com
Published online 15 May 2007
&2007 Cancer Research UK
Keywords: neuroblastoma; chemoresistance; valproic acid; adhesion; NCAM
Multiple-agent chemotherapy is the conventional therapy for
patients with advanced stages of neuroblastoma (NB) and
disseminated NB. However, drug resistance arises in the majority
of stage IV and relapsed NB, often leading to treatment failure
(Keshelava et al, 1998). Development of novel antitumoural
strategies is therefore highly desired to overcome resistance
mechanisms and to prevent tumour progression. Molecules
modulating cellular function have been identified in the majority
of tumours and their manipulation might be the key to decreasing
malignancy.
Histone deacetylases (HDAC) represent one of the most
important intracellular targets, as these molecules modulate a
wide variety of cellular functions. Abnormal histone acetylation
status can result in undesirable phenotypic changes, including
developmental disorders and cancer. Indeed, aberrant histone
acetylation may be an aetiological factor in several types of cancer
by derepressing gene transcription. Hence, HDAC inhibitors may
be useful for cancer prevention, due to their ability to ‘reactivate’
the expression of epigenetically silenced genes, including those
involved in differentiation, invasion and metastasis. Most notably,
recent data indicate that HDAC inhibition may be successful in
treating refractory or relapsing tumours after conventional
chemotherapy. Histone deacetylase inhibition has been demon-
strated to block cell growth of drug-resistant small-cell lung cancer
lines (Tsurutani et al, 2003), abrogate resistance in breast cancer
cells (Hirokawa et al, 2005) and induce apoptosis in drug-resistant
ovarian cancer cells (Sonnemann et al, 2006), myeloma cells
(Maiso et al, 2006) and hepatoma cell lines (Pathil et al, 2006).
The branched-chain fatty acid valproic acid (VPA) has been
shown to possess HDAC inhibitory properties and to affect the
growth and survival of tumour cells in vitro and in vivo (Cinatl et al,
1996; Blaheta et al, 2005). This is highly relevant since VPA is an
established drug in the long-term therapy of epilepsy. It can be
applied orally, negative side effects are rare and it demonstrates
expedient pharmacokinetic properties. To clearly assess whether
VPA might be of benefit in treating relapsed NB, we evaluated the
potential of therapeutic VPA concentration to block the interaction
of drug-resistant NB cells with vascular endothelium. The experi-
ments were based on an in vitro model of acquired drug resistance
(Kotchetkov et al, 2003, 2005), closely resembling progressive NB
disease through long-term treatment of NB cell lines with vincristine
(VCR), cisplatin (CDDP) or doxorubicin (DOX) to establish the
resistant tumour cell sublines UKF-NB-3
VCR
, UKF-NB-3
CDDP
and
UKF-NB-3
DOX
. A co-culture binding assay allowed the analysis of
NB cells that adhered to an endothelial cell monolayer. Since surface
receptors are strongly involved in tumour invasion and data have
indicated that changes in neural cell adhesion molecule (NCAM,
CD56) expression play an essential part in the progression of NB, we
investigated NCAM expression (Blaheta et al, 2002), and the NCAM
regulating proteins N-myc, p73 and deltaNp73 (Blaheta et al,2004)
under the influence of VPA.
Received 10 January 2007; revised 4 April 2007; accepted 12 April 2007;
published online 15 May 2007
*Correspondence: Professor Dr J Cinatl Jr;
E-mail: cinatl@em.uni-frankfurt.de
British Journal of Cancer (2007) 96, 1699 – 1706
&
2007 Cancer Research UK All rights reserved 0007 – 0920/07
$
30.00
www.bjcancer.com
Translational Therapeutics
MATERIALS AND METHODS
Cell cultures and induction of drug resistance
The N-myc amplified human NB cell lines UKF-NB-2, UKF-NB-3
and UKF-NB-6 were established in our laboratory from bone
marrow metastases. The CDDP-, VCR- and DOX-resistant UKF-
NB-2, UKF-NB-3 and UKF-NB-6 sublines were established by
exposing the parental cells to increasing concentrations of the
drugs. Solutions of CDDP (Gry-Pharma, Kirchzarten, Germany),
VCR (Sigma-Aldrich, Deisenhofen, Germany) and DOX
(Farmitalia, Milan, Italy) were prepared in accordance to the
manufacturer’s instructions. The initial CDDP, VCR and DOX
concentrations were 50, 0.2 and 2 ng ml
1
medium, respectively.
The resistant sublines were grown for more than 6 months in
Iscove’s modified Dulbecco’s medium (Gibco, Karlsruhe, Germany)
supplemented with 10% fetal calf serum (FCS, Gibco) and either
10 ng ml
1
VCR (UKF-NB-2
VCR
, UKF-NB-3
VCR
, UKF-NB-6
VCR
),
20 ng ml
1
DOX (UKF-NB-2
DOX
, UKF-NB-3
DOX
, UKF-NB-6
DOX
), or
1000 ng ml
1
CDDP (UKF-NB-2
CDDP
, UKF-NB-3
CDDP
,UKF-
NB-6
CDDP
). The clinical relevance of drug-resistant UKF-NB-2 and
UKF-NB-3 sublines has been examined before in a xenograft setting
(Kotchetkov et al, 2003, 2005). Multidrug resistant, not N-myc
amplified SKNSH, were derived from LGC Promochem, Wesel,
Germany. Cells were subcultured at 5-day intervals.
Chemoresistance of the indicated cell lines was verified by the
MTT test (Kotchetkov et al, 2003). The resistant cell lines showed at
least 20-fold increase in resistance to the drugs, expressed as IC
50
.
Human endothelial cells (HUVEC) were isolated from human
umbilical veins and harvested by enzymatic treatment with
chymotrypsin. Human endothelial cells were grown in Medium
199 (M199; Biozol, Munich, Germany), supplemented with 10%
FCS, 10% pooled human serum, 20 mgml
1
endothelial cell growth
factor (Boehringer, Mannheim, Germany), 0.1% heparin,
100 ng ml
1
gentamycin and 20 mMHEPES buffer (pH 7.4).
Subcultures from passages 2 –6 were selected for experimental use.
VPA treatment
Parental NB tumour cells and their drug-resistant sublines were
treated with VPA (gift from GL Pharma GmbH, Lannach, Austria)
at a final concentration of 1 mMfor 3 or 5 days. Tumour cell
adhesion, NCAM, p73, deltaNp73 and N-myc expression were then
measured in VPA-treated cells. Results were compared to
untreated controls. Viability of tumour cells in presence of VPA
was assessed by propidium iodide dsDNA intercalation.
Tumour cell adhesion
Human endothelial cells were transferred to six-well multiplates
(Falcon Primaria; Becton Dickinson, Heidelberg, Germany) in
complete HUVEC medium. When confluency was reached,
0.5 10
6
parental NB tumour cells or their drug-resistant sublines
(VPA treated vs non-treated) were carefully added to the HUVEC
monolayer for 60 min. Subsequently, non-adherent tumour cells
were washed off using warmed (371C) M199. The adherent cells
were fixed with 1% glutaraldehyde and counted in five different
fields (5 0.25 mm
2
) using a phase contrast microscope
(20 objective) to calculate the mean cellular adhesion rate.
Cell proliferation
Proliferative activity of NB tumour cells and HUVEC was
estimated by the PicoGreen assay as described elsewhere (Blaheta
et al, 1998). Briefly, at several time points after plating the cells in
six-well multiplates, culture medium was removed and cells were
digested with papain (0.125 mg protein ml
1
) for 20 h at 601C.
Thereafter, the fluorescent dye PicoGreen (MoBiTec, Goettingen,
Germany), which shows high specificity for dsDNA, was added
(1 : 200 dilution) for 10 min at 201C. Fluorescence intensity was
determined using a computer-controlled fluorescence reader
(Cytofluor 2300 plate scanner; Millipore, Eschborn, Germany) at
lex ¼485 nm and lem ¼530 nm.
Evaluation of NCAM surface expression
Neuroblastoma cells were disaggregated mechanically, washed in
blocking solution (PBS, 0.5% BSA) and then incubated for 60 min
at 41C with an FITC-conjugated monoclonal antibody anti-CD56
which detects the NCAM 120, 140 and 180 kDa isoform (clone
16.2). Neural cell adhesion molecule expression of NB cells was
then measured using a FACscan (Becton Dickinson; FL-1 H (log)
channel histogram analysis; 1 10
4
cells/scan) and expressed as
mean fluorescence units (MFU). A mouse IgG1-FITC was used as
the isotype control.
To explore NCAM localisation, tumour cells were transferred to
round cover slips, which were placed in a 24-well multiplate. Upon
reaching confluency, cell cultures were washed two times with PBS
(with Ca
2þ
and Mg
2þ
) and then fixed in cold (201C) methanol/
acetone (60/40 v/v). Subsequently, cells were washed again with
PBS (without Ca
2þ
and Mg
2þ
), and later once with blocking
buffer (0.5% BSA in PBS without Ca
2þ
and Mg
2þ
). After removing
the washing buffer, cells were incubated for 60 min with FITC-
conjugated anti-NCAM monoclonal antibody. To prevent photo-
bleaching of the fluorescent dye, cover glasses with stained cells
were taken out of the wells and the residual liquid was removed.
The cells were then embedded in an antifade reagent/mounting
medium mixture (ProLongtAntifade Kit, MoBiTec) and mounted
on slides. The slides were viewed using a confocal laser-scanning
microscope (LSM 10; Zeiss, Jena, Germany) with a plan-neofluar
100/1.3 oil immersion objective.
Western blot analysis
Total NCAM content in NB cells was evaluated by Western blot
analysis: tumour cell lysates were applied to a 7% polyacrylamide
gel and electrophoresed for 90 min at 60 V. The protein was then
transferred to nitrocellulose membranes. After blocking, the
membranes were incubated overnight with the anti-NCAM anti-
body (dilution 1 : 1000). HRP-conjugated goat anti-mouse IgG
(Upstate Biotechnology, Lake Placid, NY, USA; dilution 1 : 5000)
served as the secondary antibody. The membrane was briefly
incubated with ECL detection reagent (ECLt, Amersham/GE
Healthcare, Mu
¨nchen, Germany) to visualise the proteins and
exposed to an x-ray film (HyperfilmtECt, Amersham).
Semi-quantitative reverse transcription/polymerase chain
reaction
Total RNA was extracted and purified with Trizol reagent
according to the manufacturer’s instructions and treated with
RNase-free DNase. The NCAM primer sequences were as follows:
for NCAM-180: 50CGAGGCTGCCTCCGTCAGCACC 30and 50CCGG
ATCCATCATGCTTTGCTCTCG 30; for NCAM-140: 50GAACCTG
ATCAAGCAGGATGACGG 30and 50CCGGATCCATCATGCTTTGC
TCTCG 30(Kleinschmidt-DeMasters et al, 1999). Internal controls
for the reverse transcription/polymerase chain reaction (RT-PCR)
reaction was performed by running parallel reaction mixtures with
the housekeeping gene GAPDH: 50ATCTTCCAGGAGCGAGATCC
30and 50ACCACTGACACGTTGGCAGT 30. Ribonucleic acid
(1 –10 mg) was reverse transcribed and the resulting cDNA directly
added to the PCR. Amplification reactions (20 ml) were performed
in the presence of 1/10 (2 ml) of the cDNA reaction, with an initial
incubation step at 941C for 1 min. Cycling conditions consisted of
denaturation at 941C for 1 min, annealing at 551C for 1 min and
extension at 721C for 1 min over a total of 30 cycles. The reaction
Valproic acid blocks neuroblastoma adhesion
RA Blaheta et al
1700
British Journal of Cancer (2007) 96(11), 1699 – 1706 &2007 Cancer Research UK
Translational Therapeutics
was completed by another incubation step at 721C for 10 min. The
PCR products were subjected to electrophoresis in 2% agarose gel
and visualised by ethidium bromide.
Evaluation of p73, deltaNp73 and N-myc
N-myc, p73 and deltaNp73 were evaluated by flow cytometry. To
allow intracellular staining, tumour cells were fixed and permea-
bilised by methanol – acetone (1 : 1, 201C) before the antibodies
were added. Monoclonal anti-N-myc antibody was from Calbio-
chem (clone NCM II 100; mouse IgG1; Calbiochem, Bad Soden,
Germany). p73 was measured using monoclonal anti-p73 (clone
ER-15, Becton Dickinson). To identify deltaNp73, the monoclonal
antibody anti-deltaNp73 (clone 38C674) was purchased from
Active Motif (Rixensart, Belgium). Primary antibodies were
labelled with goat anti-mouse IgG-FITC. To evaluate background
staining, goat anti-mouse IgG-FITC was used.
N-myc was also explored by Western blot analysis using
monoclonal antibodies against N-myc (1 : 250, clone NCM II 100;
mouse IgG1). b-Actin (1 : 1.000, mouse; Sigma, Taufkirchen,
Germany) served as the internal control.
To investigate N-myc coding mRNA, RT/PCR has been carried
out as described above. The N-myc primer sequences were as
follows: forward: 50GACCACAAGGCCCTCAGTAC 30; reverse:
50GTGGATGGGAAGGCATCGTT 30.
Statistics
All experiments were performed 3 –6 times. Statistical significance
was investigated by the Wilcoxon –Mann– Whitney U-test. Differ-
ences were considered statistically significant at Po0.05.
RESULTS
VPA downregulates cell adhesion of CDDP- and VCR-
resistant UKF-NB-3 tumour cells
Adhesion of UKF-NB-3, UKF-NB-3
CDDP
, UKF-NB-3
VCR
or UKF-
NB-3
DOX
was quantified 60 min after plating the cells on to an
endothelial cell monolayer (Figure 1). Nearly 200 parental (drug-
sensitive) UKF-NB-3 cells mm
2
were attached to HUVEC during
this time (SD
interassay
o50%, SD
intraassay
o10%). The amount of
adherent cells increased fourfold when UKF-NB-3 became resistant
to CDDP or VCR. Doxorubicin resistance did not induce any
effects on tumour cell binding to HUVEC. Table 1 provides a
profile of cross-resistance among the sublines examined.
The application of 1 mMVPA to UKF-NB-3
CDDP
or UKF-NB-
3
VCR
significantly blocked the cellular adhesion process (Figure 1).
A 5-day incubation period evoked stronger effects than a 3-day
incubation period. Notably, treatment of UKF-NB-3
CDDP
with VPA
completely reverted the elevated adhesion behaviour induced by
drug resistance. Valproic acid also acted on the parental cell lines,
as evidenced by a significant downregulation of the number of
adherent UKF-NB-3. The 60-min adhesion rate was reduced by
59.4718.6% (n¼6).
The PicoGreen assay did not reveal any proliferative activity
during the experiment, which rules out the possibility that
adhesion differences between drug resistant, VPA-treated and
control NB cells may be caused by different cell growth capacity.
VPA upregulates NCAM surface expression
Figure 2 depicts one representative histogram analysis of NCAM
receptor expression. The histogram presentation concentrates on
isotype controls and NCAM-specific fluorescence of untreated vs
VPA-treated (5-day treatment)-resistant tumour cells. Results of
parental UKF-NB-3 and of resistant tumour cells treated for 3 days
with VPA were not inserted, because the histograms overlapped,
making the figures unclear. However, the complete experimental
data set (MFU7s.d.; n¼6) is given below the histogram.
Tumour cell adhesion
0
200
400
600
800
1000
CDDP-VPA
5
CDDP-VPA
3
CDDP
Control
Adherent cells/mm2
Adherent cells/mm2
Adherent cells/mm2
VCR-VPA5
VCR-VPA3
VCR
Control
DOX-VPA5
DOX-VPA3
DOX
Control
Adhesion characteristics of UKF-NB-3VCR Adhesion characteristics of UKF-NB-3DOX
Adhesion characteristics of UKF-NB-3CDDP
0
200
400
600
800
1000
0
200
400
600
800
1000
Figure 1 Valproic acid treatment causes adhesion blockade in CDDP- and VCR-resistant NB cells. The figure depicts adhesion capacity of parental UKF-
NB-3 (control) vs VCR- (UKF-NB-3
VCR
), CDDP- (UKF-NB-3
CDDP
) or DOX-resistant NB subpopulations (UKF-NB-3
DOX
)vs resistant cell lines treated with
1m
MVPA for 3 (VPA3) or 5 days (VPA5). Neuroblastoma cells were added at a density of 0.5 10
6
cells/well to HUVEC monolayers for 60 min. Non-
adherent tumour cells were washed off in each sample, the remaining cells were fixed and counted in five different fields (5 0.25 mm
2
) using a phase
contrast microscope. Adhesion capacity is depicted as tumour cell adhesion mm
2
(mean7s.d.; n¼6).
Table 1 Level of drug resistance, indicated as IC
50
values (ng ml
1
)
Cell line IC
50
VCR IC
50
DOX IC
50
CDDP
UKF-NB-2 0.7470.15 8.272.5 136741
UKF-NB-2
VCR
47.9711.2 114.6720.7 154732
UKF-NB-2
DOX
23.476.4 40.7712.3 225738
UKF-NB-2
CDDP
1.3570.47 17.477.7 819752
UKF-NB-3 0.3570.09 2.2570.78 188722
UKF-NB-3
VCR
52.779.8 69.1715.8 532768
UKF-NB-3
DOX
205751.2 62.5715.4 231722
UKF-NB-3
CDDP
0.8470.31 17.372.4 12787177
UKF-NB-6 1.9170.18 3.570.9 114722
UKF-NB-6
VCR
57.2711.2 108712 523769
UKF-NB-6
DOX
19.573.4 17.276.5 102718
UKF-NB-6
CDDP
3.7270.76 21.474.5 21027150
SKNSH 7.971.3 25.4710.1 47.1713.5
Values are from six independent experiments7s.d.
Valproic acid blocks neuroblastoma adhesion
RA Blaheta et al
1701
British Journal of Cancer (2007) 96(11), 1699 – 1706&2007 Cancer Research UK
Translational Therapeutics
Reduction of NCAM surface expression was observed on UKF-
NB-3
CDDP
and UKF-NB-3
VCR
, when compared to the parental
control cell line. However, no changes were seen with NCAM
expression on UKF-NB-3
DOX
, compared to the controls. Treatment
of CDDP- or VCR-resistant cell lines with VPA led to a significant
increase in NCAM, which exceeded the control values after a 5-day
incubation period.
Valproic acid also acted on the parental cell lines, as evidenced
by a significant upregulation of NCAM by þ56.9722.8% (n¼5).
VPA enhances NCAM protein content
Similar to the modifications of the NCAM surface expression level,
UKF-NB-3
CDDP
and UKF-NB-3
VCR
were characterised by a strong
reduction of NCAM proteins when compared to the parental UKF-
NB-3 control cells (Figure 3). No differences were seen between
UKF-NB-3 and UKF-NB-3
DOX
.
When UKF-NB-3
CDDP
or UKF-NB-3
VCR
were treated with VPA
for 3 or 5 days, NCAM protein content became upregulated,
partially exceeding the control values. Applying VPA to UKF-NB-
3
DOX
did not induce any alterations in NCAM, independent of the
incubation time.
VPA modifies NCAM mRNA expression
Assessment of NCAM mRNA showed distinct expression of mRNA
encoding the 140 kDa isoform in UKF-NB-3 control cells, which
however became down-modulated in UKF-NB-3
CDDP
or UKF-NB-
3
VCR
(Figure 4). Only slight differences were visualised between
UKF-NB-3 and UKF-NB-3
DOX
. The presence of VPA was
accompanied by elevated 140 kDa mRNA levels in UKF-NB-3
CDDP
or UKF-NB-3
VCR
. This effect was not seen in UKF-NB-3
DOX
,
irrespective if VPA was applied for 3 or 5 days.
Messenger ribonucleic acid encoding the 180kDa isoforms was
not detected in parental UKF-NB-3, nor in UKF-NB-3
CDDP
,orUKF-
NB-3
DOX
, and only very weakly expressed in UKF-NB-3
VCR
.
Surprisingly, VPA evoked NCAM 180 kDa mRNA synthesis already
after 3 days in UKF-NB-3
DOX
. The same phenomenon was observed
after a 5-day VPA treatment in UKF-NB-3
CDDP
or UKF-NB-3
VCR
.
VPA alterates p73, deltaNp73 and N-myc expression
p73, deltaNp73 and N-myc have been identified to trigger NCAM
expression (Blaheta et al, 2004). They were therefore used as
biomarkers to further explore the influence of VPA on NCAM-
triggered NB adhesion. p73 was detected in UKF-NB-3, expression
of which was significantly reduced in UKF-NB-3
CDDP
and UKF-
NB-3
VCR
. Doxorubicin resistance was not accompanied by a
distinct p73 downregulation (Table 2). When VPA was added to
the cell cultures, downregulation of p73 seen in UKF-NB-3
CDDP
and UKF-NB-3
VCR
was reverted and the protein became upregu-
lated after 3 and 5 days. deltaNp73 became enhanced in UKF-NB-
3
CDDP
and UKF-NB-3
VCR
compared to the drug naı
¨ve UKF-NB-3
controls. Application of VPA led to a significant reduction of
deltaNp73 in CDDP- or VCR-resistant cell lines.
N-myc proteins were enhanced in UKF-NB-3
CDDP
and UKF-NB-
3
VCR
,butnotinUKF-NB-3
DOX
, compared to the UKF-NB-3 control
cell line (Figure 5). Moderate N-myc downregulation was induced by
UKF-NB-3VCR
UKF-NB-3CDDP
Surface expression of NCAM
IgG
IgG
CDDP VPA-5
VCR
VPA-5 IgG
DOX VPA-5
IgG isotype control:
Parental control:
Acquired resistance:
Resistance + VPA 3 days:
Resistance + VPA 5 days:
2.46 +/− 3.21
60.58 +/− 34.45
44.93 +/− 26.77
67.85 +/− 51.29
123.63 +/− 66.41
2.46 +/− 3.21
60.58 +/− 34.45
50.23 +/− 48.37
69.38 +/− 61.22
118.49 +/− 124.25
2.46 +/− 3.21
60.58 +/− 34.45
56.14 +/− 17.92
61.90 +/− 26.89
71.24 +/− 44.73
120
100
80
60
Counts
40
20
0
100101102
FL1 height
103104
120
100
80
60
Counts
40
20
0
100101102
FL1 height
103104
120
100
80
60
Counts
40
20
0
100101102
FL1 height
103104
UKF-NB-3DOX
Figure 2 Valproic acid treatment enhances NCAM surface expression in CDDP- and VCR-resistant NB cells. Histograms plots show NCAM surface
expression on untreated vs VPA-treated UKF-NB-3
CDDP
, UKF-NB-3
VCR
and UKF-NB-3
DOX
. IgG isotype controls are also included. The complete
experimental data set (MFU7s.d.; n¼6) is given below the histograms. Tumour cells were disaggregated mechanically in each experiment and washed in
blocking solution. An FITC-conjugated monoclonal antibody anti-CD56, clone 16.2, was used to detect the NCAM 120, 140 and 180 kDa isoform. A mouse
IgG1-FITC served as the isotype control (IgG). Fluorescence was analysed using a FACScan flow cytometer, and a histogram plot (FL1-Height) was
generated to show FITC fluorescence.
NCAM
Control
Resistant
VPA3
VPA5
Control
Resistant
VPA3
VPA5
CDDP
VCR
DOX
-actin
NCAM protein expression
Figure 3 Western blot analysis of NCAM from the proteins of UKF-NB-
3, UKF-NB-3
CDDP
UKF-NB-3
VCR
UKF-NB-3
DOX
and resistant subpopula-
tions treated with VPA for 3 (VPA3) or 5 days (VPA5). Cell lysates were
subjected to SDS –PAGE and blotted on the membrane incubated with
anti-NCAM (clone 16.2) monoclonal antibodies. b-Actin served as the
internal control. The figure shows one representative from three separate
experiments.
Valproic acid blocks neuroblastoma adhesion
RA Blaheta et al
1702
British Journal of Cancer (2007) 96(11), 1699 – 1706 &2007 Cancer Research UK
Translational Therapeutics
VPA in UKF-NB-3
VCR
. N-myc became nearly undetectable in UKF-
NB-3
CDDP
after a 5-day treatment with VPA. The distinct influence of
VPA on UKF-NB-3
CDDP
or UKF-NB-3
VCR
was also proven by flow
cytometry, which revealed left shifting of the N-myc-specific
fluorescence intensity, indicating N-myc loss. N-myc histogram
analysis concentrates on isotype controls and specific fluorescence of
untreated vs VPA-treated (5-day treatment) UKF-NB-3
CDDP
,UKF-
NB-3
VCR
and UKF-NB-3
DOX
. The complete experimental data set
(MFU7s.d.; n¼6) is given below the histogram.
VPA acts on further NB tumour cell lines
To strengthen the relevance of our findings, further NB tumour
cell lines were included in the study. Analysis of UKF-NB-2 and
NCAM140
NCAM180
GAPDH
Control
CDDP
CDDP-VPA3
CDDP-VPA5
Vcr
Vcr-VPA3
Vcr-VPA5
Dox
Dox-VPA3
Dox-VPA5
NCAM mRNA analysis
Figure 4 RT – PCR analysis of NCAM 140 and 180 kDa RNA in UKF-
NB-3, UKF-NB-3
CDDP
UKF-NB-3
VCR
UKF-NB-3
DOX
and resistant sub-
populations treated with VPA for 3 (VPA3) or 5 days (VPA5). Ribonucleic
acid were extracted, reverse-transcribed and submitted to semiquantitative
RT – PCR using gene-specific primers as indicated in Materials and Methods.
The internal control for the RT – PCR reaction was performed by running
parallel reaction mixtures with the housekeeping gene GAPDH. The figure
shows one representative from three separate experiments.
Table 2 Mean fluorescence units showing influence of VPA on p73 and
deltaNp73 expression in drug-resistant neuroblastoma cells
Cell line p73 deltaNp73
UKF-NB-3 18.2976.25 11.5178.34
CDDP 3.3873.64
a
19.7177.68
a
CDDP+VPA3 8.1675.66
b
12.4476.94
b
CDDP+VPA5 12.877.32
b
11.0676.72
b
VCR 5.2374.78
a
26.17713.95
a
VCR+VPA3 13.7876.13
b
17.3378.99
b
VCR+VPA5 17.6278.72
b
11.2376.85
b
DOX 15.4678.29 10.0274.29
DOX+VPA3 17.90711.21 10.6476.78
DOX+VPA5 19.95713.96 11.9573.81
Values are from six independent experiments7s.d. CDDP indicates UKF-NB-3
CDDP
,
VCR indicates UKF-NB-3
VCR
, DOX indicates UKF-NB-3
DOX
. VPA was added for 3
(VPA3) or 5 days (VPA5).
a
Indicates significant difference to UKF-NB-3.
b
Indicates
significant difference to the drug-resistant NB sublines.
N-myc
Control
Resistant
VPA3
VPA5
CDDP
Vcr
Dox
CDDP
Vcr
Dox
-actin
N-myc expression level
Western blot analysis Flow cytometry
IgG
IgG
120
100
80
60
40
Counts
20
0
100101102103
FL1 height
104
120
100
80
60
40
Counts
20
0
100101102103
FL1 height
104
120
100
80
60
40
Counts
20
0
100101102103
FL1 hei
g
ht
104
IgG
VPA-5
VPA-5
VPA-5
DOX
VCR
CDDP IgG control:
Cell control:
Resistance:
Resistance + VPA 3:
Resistance + VPA 5:
IgG control:
Cell control:
Resistance:
Resistance + VPA 3:
Resistance + VPA 5:
IgG control:
Cell control:
Resistance:
Resistance + VPA 3:
Resistance + VPA 5:
3.69 +/− 2.54
82.36 +/− 57.22
369.25 +/− 146.86
250.99 +/− 123.92
161.58 +/− 92.63
3.69 +/− 2.54
82.36 +/− 57.22
109.72 +/− 78.38
99.76 +/− 56.91
71.72 +/− 42.47
3.69 +/− 2.54
82.36 +/− 57.22
84.08 +/− 66.45
86.35 +/− 72.21
81.07 +/− 36.43
Figure 5 Valproic acid alters N-myc expression in CDDP- and VCR-resistant UKF-NB-3 cells. The right part of the figure depicts results from flow
cytometry analysis. To allow intracellular staining, tumour cells were fixed and permeabilised by methanol – acetone before antibodies were added. To
identify N-myc, the monoclonal anti-N-myc antibody clone NCM II 100 was used. Primary antibodies were labelled with goat anti-mouse IgG-FITC.
Background staining was evaluated by goat anti-mouse IgG-FITC. The histograms plots show N-myc expression level in untreated vs VPA-treated UKF-NB-
3
CDDP
, UKF-NB-3
VCR
and UKF-NB-3
DOX
. IgG isotype controls are also included. The complete experimental data set (MFU7s.d.; n¼6) is given alongside.
The left part of the figure shows Western blot analysis of N-myc from the proteins of UKF-NB-3, UKF-NB-3
CDDP
UKF-NB-3
VCR
UKF-NB-3
DOX
and resistant
subpopulations treated with VPA for 3 (VPA3) or 5 days (VPA5). Cell lysates were subjected to SDS – PAGE and blotted on the membrane incubated with
anti-N-myc monoclonal antibodies. b-Actin served as the internal control. The figure shows one representative from three separate experiments.
Valproic acid blocks neuroblastoma adhesion
RA Blaheta et al
1703
British Journal of Cancer (2007) 96(11), 1699 – 1706&2007 Cancer Research UK
Translational Therapeutics
drug-resistant sublines indicated a significant increase in UKF-NB-
2
CDDP
, UKF-NB-2
VCR
and UKF-NB-2
DOX
cell adhesion, compared
to the parental controls. Adhesion correlated inversely with the
NCAM surface level (Table 3). A similar phenomenon was
observed in CDDP- (moderate) and VCR-resistant UKF-NB-6 cell
lines. A 3-day VPA treatment blocked cell binding to HUVEC in
UKF-NB-2 and their drug-resistant sublines, and upregulated
NCAM expression. Valproic acid also acted on UKF-NB-6, UKF-
NB-6
CDDP
, UKF-NB-6
VCR
and SKNSH. However, although VPA
(partially) reverted the increased adhesion phenotype and restored
NCAM levels, it did not re-sensitise the drug-resistant NB cells to
VCR, DOX or CDDP (data not shown).
N-myc was significantly diminished by VPA in UKF-NB-2, UKF-
NB-2
CDDP
, UKF-NB-6 and UKF-NB-6
CDDP
, compared to the
controls. However, no differences were seen between VPA-treated
and non-treated VCR or DOX-resistant tumour cell lines.
To better interprete our data, antiproliferative effects of VPA on
the chemosensitive vs chemoresistant NB cells were analysed in
final experiments. Table 4 document growth inhibition by this
compound, whereas higher VPA concentrations were necessary to
reach 50% reduction in chemotherapy-resistant cell lines com-
pared to the parental controls.
DISCUSSION
Based on this cell culture model, we have demonstrated that drug-
resistant NB cancer cells develop an increased malignant
phenotype as evidenced by enhanced adhesion to vascular
endothelial cells, accompanied by significant downregulation of
the adhesion receptor NCAM. Valproic acid reverted this process
by downregulating cell adhesion and upregulating NCAM expres-
sion. Considering clinical utility, it is encouraging that VPA was
active in NB cell lines resistant to existing chemotherapies, since
overcoming resistance to anticancer agents is a major challenge in
the development of novel antitumour protocols.
In fact, several cases have been documented showing antineo-
plastic effects of VPA in patients with relapsed tumours. When VPA
was given as maintenance therapy for childhood malignant glioma
after postoperative combined chemotherapy and irradiation, about
10% of these patients were maintained in continuous complete
remission and an equal number of patients showed at least partial
responses (Blaheta et al, 2005). An additional pediatric patient with
glioblastoma multiforme responded to VPA after showing progres-
sive disease shortly after having received combined chemotherapy
and irradiation as well as topotecane (Witt et al,2004).Another
pediatric patient suffering from a relapsed supratentorial primitive
neuroectodermal tumour while receiving chemotherapy (CCNU,
VCR and cisplatinum) after total resection and irradiation showed
conspicuous signs of glial differentiation induction and a non-
malignant morphology on histological examination. This patient
had received VPA for epilepsy treatment for a period of several
months before the tumour recurred (Driever et al,2004).
In vitro, VPA has been shown to inhibit proliferation in acute
myeloid leukaemia cells expressing P-glycoprotein (P-gp) and
MDR-associated protein 1 (Tang et al, 2004), and to increase
sensitivity towards apoptosis in hepatoma cells resistant to
epirubicin (Schuchmann et al, 2006). Although the underlying
mode of action has not been explored in these studies, the data
clearly indicate that VPA may alter the malignant behaviour of
tumours that do not respond to chemotherapy. With particular
emphasis on NB, VPA significantly prevented the interaction
between tumour cells and endothelium. This finding is important,
because binding of single cancer cells to the vessel wall represents
the first step in the haematogenous invasion cascade proceeding
transendothelial migration and invasion into surrounding tissue.
We therefore conclude that VPA may have a direct impact on
metastasis formation. In good accordance to this hypothesis, VPA
enhanced the NCAM surface level, expression of which is strongly
involved in tumour cell adhesion and penetration.
In primitive neuroectodermal tumour cells, an increase in
NCAM was paralleled by a significant reduction in cellular motility
and adhesion capacity (Owens et al, 1998; Prag et al, 2002). In a rat
model, NCAM-transfected glioma tumour cells became less
invasive and destructive than control cells with a low NCAM
expression level (Edvardsen et al, 1994). Diminished expression of
NCAM was also associated with clinically aggressive colon cancers
(Sampson-Johannes et al, 1996; Roesler et al, 1997; Huerta et al,
2001), and dissemination of pancreatic b-tumour cells (Perl et al,
1999; Cavallaro et al, 2001). Tezel et al (2001) suggested that
NCAM expression in tubular adenocarcinoma of the pancreas has
a significant impact on overall patient survival. We recently
demonstrated an inverse correlation between NCAM expression
and NB cell adhesion, assessed on 11 NB cell lines. In particular,
transfection with a cDNA encoding the human NCAM-140 kD
Table 3 Comparative analysis of adhesion capacity, NCAM and N-myc
expression level of several neuroblastoma cells and drug-resistant sublines
Cell line
Cell adhesion
to HUVEC
(cells mm
2
)
NCAM
expression
(MFU)
N-myc
expression
(MFU)
UKF-NB-2 75.2 188.2 40.1
UKF-NB-27VPA 60.8
c
212.6
c
28.6
c
UKF-NB-2
CDDP
318.4
a
82.8
a
39.6
UKF-NB-2
CDDP
7VPA 175.2
b
168.9
b
32.2
b
UKF-NB-2
VCR
245.6
a
99.7
a
44.7
UKF-NB-2
VCR
7VPA 121.6
b
154.6
b
42.3
UKF-NB-2
DOX
172.6
a
127.9
a
38.8
UKF-NB-2
DOX
7VPA 124.5 143.5 39.4
UKF-NB-6 72.4 256.8 339.3
UKF-NB-67VPA 41.0
c
316.8
c
238.0
c
UKF-NB-6
CDDP
81.6 248.7 305.8
UKF-NB-6
CDDP
7VPA 56.8
b
283.8
b
224.2
b
UKF-NB-6
VCR
115.6
a
181.0
a
298.9
UKF-NB-6
VCR
7VPA 79.2
b
284.2
b
331.6
UKF-NB-6
DOX
73.8 251.3 344.0
UKF-NB-6
DOX
7VPA 71.9 254.5 321.7
SKNSH 56.8 318.9 17.7
SKNSH7VPA 27.2
c
812.1
c
18.6
MFU ¼mean fluorescence units.
a
Indicates significant difference to the parental
neuroblastoma cell line.
b
Indicates significant difference to the drug-resistant tumour
sublines.
c
Indicates significant difference between VPA-treated parental cells and their
non-treated controls. Mean standard deviations were as follows: cell adhesion
intra-assay
o25%, cell adhesion
inter-assay
o80%. NCAM expression
intra-assay
o5%, NCAM
expression
inter-assay
o30%. N-myc expression
intra-assay
o5%, N-myc expres-
sion
inter-assay
o50%.
Table 4 Antiproliferative effects of VPA, indicated as IC
50
values (mM)
Cell line IC
50
UKF-NB-2 0.4470.31
UKF-NB-2
CDDP
1.8570.58
UKF-NB-2
VCR
1.3670.62
UKF-NB-2
DOX
1.4470.42
UKF-NB-3 1.0370.37
UKF-NB-3
CDDP
1.5170.44
UKF-NB-3
VCR
1.8870.57
UKF-NB-3
DOX
2.1870.76
UKF-NB-6 1.2770.53
UKF-NB-6
CDDP
1.5370.46
UKF-NB-6
VCR
2.3670.71
UKF-NB-6
DOX
0.9370.39
SKNSH 3.1670.78
Values are from six independent experiments7s.d.
Valproic acid blocks neuroblastoma adhesion
RA Blaheta et al
1704
British Journal of Cancer (2007) 96(11), 1699 – 1706 &2007 Cancer Research UK
Translational Therapeutics
isoform enhanced NCAM expression and diminished initial NB
cell adhesion, treatment with NCAM antisense oligonucleotides
reduced NCAM surface level and induced upregulation of NB cell
adhesion to endothelium (Blaheta et al, 2002). It is currently
assumed that NCAM, in its function as a homophilic receptor,
stabilises the primary tumour or tumour cell aggregates, while
circulating in the blood vessels. Reduction of the NCAM
expression level might lead to a reduction in cell – cell binding
forces, and hence to the release of tumours as single cells. The less
NCAM, the more metastatic cells leave the tumour mass, and the
more penetration events can take place (Blaheta et al, 2002).
Consequently, NCAM upregulation observed in CDDP- and VCR-
resistant NB tumour cells under VPA might reduce cell
transmigration and extravasation processes.
There is some evidence from the literature that N-myc down-
regulates NCAM expression, thus increasing the invasiveness of
NB cells. Transfection of the rat NB cell line B104 with an N-myc
expression vector resulted in a dramatic reduction in the levels of
NCAM polypeptides and mRNAs, and increased metastatic ability
(Akeson and Bernards, 1990). Cytomegalovirus-induced accelera-
tion of NB adhesion and transendothelial penetration was evoked
by increasing N-myc protein content, accompanied by a dimin-
ished NCAM surface level (Blaheta et al, 2004). The present data
reveal strong upregulation of N-myc in UKF-NB-3
CDDP
, compared
to the parental cells, process of which was coupled to NCAM loss
and enhanced adhesion capacity. It may therefore be concluded
that N-myc plays an important role in NCAM-driven NB adhesion,
and that VPA has an impact on N-myc protein expression.
Nevertheless, the situation is more complex than initially thought.
Our analysis on further NB cell lines indicated that VPA reverts
cell adhesion, restored NCAM and suppressed N-myc expression
level on UKF-NB-2, UKF-NB-6 and their CDDP-resistant sublines.
However, VPA effects on VCR-resistant sublines were not
accompanied by N-myc alterations. Furthermore, incubation of
multidrug-resistant SKNSH with VPA induced very strong
adhesion blockade and NCAM upregulation, although N-myc
was detected in non-treated controls just very slightly over
threshold values. Based on this, we assume that endogenous N-
myc expression level of NB cells may not correlate with their
responsiveness to VPA-induced NCAM upregulation, and VPA-
induced loss of N-myc may be limited to CDDP-resistant and
drug-sensitive tumour cells.
p73 and deltaNp73, an isoform of p73 lacking the N-terminal
transactivation domain, are both suggested to be associated with
NCAM expression. In vitro experiments demonstrated that
transfected full-length p73 cDNA induces expression of NCAM
and downregulation of N-myc in N1E-115 NB cells. Inversely,
transfection of dominant-negative p73 abrogated the transactiva-
tion of the NCAM promoter (De Laurenzi et al, 2000). Based on
UKF-NB-4 tumour cells, we have recently postulated a direct
association between tumour progression, upregulation of N-myc
and deltaNp73, and downregulation of p73 and NCAM (Blaheta
et al, 2004). According to this statement, VPA evoked UKF-NB-3
adhesion blockade was accompanied by diminished N-myc and
deltaNp73, and an enhanced p73 and NCAM level. With special
respect to the UKF-NB-3 model, VPA may interfere in the N-myc/
deltaNp73 signalling system that causes, as at least one conse-
quence, distinct upregulation of NCAM biosynthesis and receptor
processing. Neural cell adhesion molecule processing finally
attributes to the lowered invasive capacity of the tumour cells.
To summarise, evidence is presented showing that drug-
resistant NB cells are sensitive to VPA treatment. Valproic acid
distinctly reduced the invasive properties of NB and may
therefore be well suited to amend the current treatment protocol
with particular emphasis on those tumours that do not respond
to chemotherapy. Nevertheless, our data are particularly limited
to four NB cell lines. Therefore, the hypothetical possibility that
VPA might overcome drug resistance in general needs further
investigation. Remarkably, VPA blocked cell adhesion of
parental, but not of DOX-resistant UKF-NB-3 or UKF-NB-6
tumour cells in our assay. This implies that DOX may, under
certain circumstances, induce resistance to VPA. A similar
phenomenon was found using NB cells with ‘naturally’ arised
DOX resistance, SMS-KANR and SMS-KCNR (Reynolds et al,
1986). Tabe et al (2006) demonstrated in this context that the
HDAC-inhibitor FK228 induces P-gp expression and prevents
growth inhibition and apoptosis in acute promyelocytic leukae-
mia cells subsequently incubated with DOX. Okada et al (2006)
observed resistance development in DOX-resistant clones of
osteosarcoma and Ewing’s family of tumours after exposure
to FK228. Nevertheless, FK228 is chemically different from VPA
and, therefore, further experiments are necessary to explore
this issue.
Differences between VCR/CDDP- and DOX-resistant cell lines
may also point to different resistance mechanisms that are
operational in these cell lines. Suppression of MAP kinase (MEK-
ERK) signalling has been observed in NB cells with acquired
resistance to DOX (Mattingly et al, 2001; Armstrong et al, 2006).
Down-modulation of ERK1/2 phosphorylation has further been
documented in NB cells with acquired resistance to CDDP
or VCR. However, these cell lines were additionally characterised
by a distinct Akt activation (Kotchetkov et al, 2005; Servidei
et al, 2006). Therefore, although purely speculative, fine-tuned
alterations of the ERK and Akt signalling system may be – at
least partially – responsible for establishing VCR and CDDP,
but not DOX resistance in our NB cell model. However, detailed
knowledge of resistance mechanism in individual cancer
cells is necessary to allow better prediction of the clinical use
of VPA.
Other HDAC inhibitors have also been shown to inhibit tumour
growth and to overcome multidrug resistance. Notably, suberoyla-
nilide hydroxamic acid (SAHA) has been demonstrated to act on
VCR-resistant leukaemia cell lines (Ruefli et al,2002),adriamycin-
resistant breast (Castro-Galache et al, 2003) and paclitaxel-resistant
ovarian cancer cells (Sonnemann et al,2006).SAHAat5mM
significantly diminished UKF-NB-3
CDDP
or UKF-NB-3
VCR
prolifera-
tion and cell adhesion to HUVEC in our own experiments (data not
shown). Histone deacetylase inhibitors different from VPA may
therefore be considered to become additional options for the
treatment of drug-resistant NB. However, detailed studies are
necessary to explore their clinical value.
ACKNOWLEDGEMENTS
We thank Karen Nelson for critically reading the manuscript. This
work was supported by the foundation ‘Hilfe fu
¨r krebskranke
Kinder Frankfurt e. V’, the ‘Heinrich und Erna Schaufler-Stiftung’,
the ‘Horst Mu
¨ggenburg-Stiftung’, the ‘Matthias Lackas-Stiftung’
and the ‘Jung-Stiftung’.
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Translational Therapeutics
