Histone deacetylase inhibitors valproate and trichostatin A are toxic to neuroblastoma cells and modulate cytochrome P450 1A1, 1B1 and 3A4 expression in these cells.

Abstract

Histone deacetylase inhibitors such as valproic acid (VPA) and trichostatin A (TSA) were shown to exert antitumor activity. Here, the toxicity of both drugs to human neuroblastoma cell lines was investigated using MTT test, and IC50 values for both compounds were determined. Another target of this work was to evaluate the effects of both drugs on expression of cytochrome P450 (CYP) 1A1, 1B1 and 3A4 enzymes, which are known to be expressed in neuroblastoma cells. A malignant subset of neuroblastoma cells, so-called N-type cells (UKF-NB-3 cells) and the more benign S-type neuroblastoma cells (UKF-NB-4 and SK-N-AS cell lines) were studied from both two points of view. VPA and TSA inhibited the growth of neuroblastoma cells in a dose-dependent manner. The IC(50) values ranging from 1.0 to 2.8 mM and from 69.8 to 129.4 nM were found for VPA and TSA, respectively. Of the neuroblastoma tested here, the N-type UKF-NB-3 cell line was the most sensitive to both drugs. The different effects of VPA and TSA were found on expression of CYP1A1, 1B1 and 3A4 enzymes in individual neuroblastoma cells tested in the study. Protein expression of all these CYP enzymes in the S-type SK-N-AS cell line was not influenced by either of studied drugs. On the contrary, in another S-type cell line, UKF-NB-4, VPA and TSA induced expression of CYP1A1, depressed levels of CYP1B1 and had no effect on expression levels of CYP3A4 enzyme. In the N-type UKF-NB-3 cell line, the expression of CYP1A1 was strongly induced, while that of CYP1B1 depressed by VPA and TSA. VPA also induced the expression of CYP3A4 in this neuroblastoma cell line.

Full text

Page 1

interdisciplinary
Histone deacetylase inhibitors valproate and
trichostatin A are toxic to neuroblastoma
cells and modulate cytochrome P450 1A1,
1B1 and 3A4 expression in these cells
Jana HŘEBAČKOVÁ 1, Jitka POLJAKOVÁ 1,2, Tomáš ECKSCHLAGER 1, Jan HRABĚTA 1,
Pavel PROCHÁZKA 1, Svatopluk SMUTNÝ 3, Marie STIBOROVÁ 2
1 Department of Pediatric Hematology and Oncology, 2nd Medical School, Charles University and University Hospital Motol, Prague 5, Czech Republic
2 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic
3 1st Department of Surgery, 2nd Medical School, Charles University and University Hospital Motol, V Úvalu 84, 150 06 Prague 5, Czech Republic
ITX020309A06 • Received: 04 August 2009 • Revised: 26 August 2009 • Accepted: 27 August 2009
ABSTRACT
Histone deacetylase inhibitors such as valproic acid (VPA) and trichostatin A (TSA) were shown to exert antitumor activity. Here, the
toxicity of both drugs to human neuroblastoma cell lines was investigated using MTT test, and IC50 values for both compounds were
determined. Another target of this work was to evaluate the effects of both drugs on expression of cytochrome P450 (CYP) 1A1, 1B1
and 3A4 enzymes, which are known to be expressed in neuroblastoma cells. A malignant subset of neuroblastoma cells, so-called
N-type cells (UKF-NB-3 cells) and the more benign S-type neuroblastoma cells (UKF-NB-4 and SK-N-AS cell lines) were studied from
both two points of view. VPA and TSA inhibited the growth of neuroblastoma cells in a dose-dependent manner. The IC50 values
ranging from 1.0 to 2.8 mM and from 69.8 to 129.4 nM were found for VPA and TSA, respectively. Of the neuroblastoma tested here,
the N-type UKF-NB-3 cell line was the most sensitive to both drugs. The different effects of VPA and TSA were found on expression
of CYP1A1, 1B1 and 3A4 enzymes in individual neuroblastoma cells tested in the study. Protein expression of all these CYP enzymes in
the S-type SK-N-AS cell line was not influenced by either of studied drugs. On the contrary, in another S-type cell line, UKF-NB-4, VPA
and TSA induced expression of CYP1A1, depressed levels of CYP1B1 and had no effect on expression levels of CYP3A4 enzyme. In the
N-type UKF-NB-3 cell line, the expression of CYP1A1 was strongly induced, while that of CYP1B1 depressed by VPA and TSA. VPA also
induced the expression of CYP3A4 in this neuroblastoma cell line.
KEY WORDS: histone deacetylase inhibitors; valproate; trichostatin A; neuroblastoma; cytotoxicity
neuroblastoma grows relentlessly and may be rapidly fatal.
Prognosis of high-risk form of cancer is poor, because drug
resistance arises in the majority of those patients, initially
responding to chemotherapy (Brodeur, 2003).
Neuroblastoma consists of two principal neoplastic cells
(Voigt et al., 2000; Hopkins-Donaldson et al., 2004): i) neu-
roblastic or N-type: undifferentiated, round and small scant
cytoplasm cells; and ii) stromal or S-type: large hyaline, flat-
tened and adherent differentiated cells. As neuroblastoma
cells seem to have the capacity to differentiate spontane-
ously in vivo and in vitro (Morgenstern et al., 2004), their
heterogeneity could affect treatment outcome, in particular
the response to apoptosis induced by chemotherapy.
To achieve the most suitable concept of treatment, drugs
are usually used in various combinations. Agents commonly
used in neuroblastoma treatment are platinum compounds
(cisplatin, carboplatin), alkylating agents (cyclophospha-
mide, ifosfamide, melphalan), topoisomerase II inhibitors
(etoposide), anthracycline antibiotics (doxorubicin) and
Introduction
Neuroblastoma is the major cause of death from neoplasia in
infancy (Maris and Mathay, 1999). These solid extracranial
tumors are biologically heterogeneous, with cell popula-
tions differing in their genetic programs, maturation stage
and malignant potential (Brodeur, 2003). Low-risk neuro-
blastoma is one of the rare human malignancies that are
known to demonstrate spontaneous regression in infants
from an undifferentiated state to a completely benign
cellular formation (ganglioneuroma), whereas high-risk
Interdisc Toxicol. 2009; Vol. 2(3): 205–210.
doi: 10.2478/v10102-009-0019-x
Published online in:
www.setox.eu/intertox & www.versita.com/science/medicine/it/
Copyright©2009 Slovak Toxicology Society SETOX
Correspondence address:
Prof. Marie Stiborová, DSc.
Department of Biochemistry, Faculty of Science, Charles University,
Albertov 2030, 128 40 Prague 2, Czech Republic
TEL: +420-221951285 • E-MAIL: stiborov@natur.cuni.cz
TEL: +420-221951241 • E-MAIL: jitka.poljakova@seznam.cz
ORIGINAL ARTICLE

Page 2

206
J. Hřebačková, J. Poljaková, T. Eckschlager, J. Hraběta, P. Procházka, S. Smutný, M. Stiborová
Histone deacetylase inhibitors valproate and trichostatin A are toxic to neuroblastoma cells
ISSN: 1337-6853 (print version) | 1337-9569 (electronic version)
vinca alkaloids (vincristine). Some novel regimen include
also topoisomerase I inhibitors (topotecan and irinotecan)
that are effective against recurrent disease (Brodeur, 2003).
Because the epigenetic structure of DNA and its lesions
play a role in the origin of human neuroblastomas, phar-
maceutical manipulation of the epigenome may offer other
treatment options also for neuroblastomas (Furchert et al.,
2007). Histone deacetylases (HDAC) and histone acetyl
transferases modify histone proteins and contribute to an
epigenetic code recognized by proteins involved in regula-
tion of gene expression (Marks et al., 2003; 2004; Hooven
et al., 2005). Indeed, former studies demonstrated the
cytotoxicity of HDAC inhibitors to several neuroblastoma
cells, resulting in growth inhibition of these tumor cells
(Cinatl et al., 1996; Michaelis et al, 2004; 2007; Furchert et
al., 2007). In neoplastic cells, where overexpression of dif-
ferent HDACs was frequently detected (for summary see,
Bolden et al, 2006), the abundance of deacetylated histones
is usually associated with DNA hypermethylation and gene
silencing (Santini et al., 2007). Treatment with HDAC
inhibitors induced the reactivation of growth regulatory
genes and consequently apoptosis in these cells. One of the
HDAC inhibitors, valproic acid (VPA), inhibits growth and
induces differentiation of human neuroblastoma UKF-NB-2
and UKF-NB-3 cells in vitro at concentrations ranging from
0.5 to 2 mM that have been achieved in human with no
significant adverse effects (Cinatl et al., 1996). However,
information on effects of VPA and other HDAC inhibitors
on additional neuroblastoma cells are scarce. Therefore,
here we extended this study by investigating the effect of
VPA and another HDAC inhibitor, trichostatin A (TSA),
on other neuroblastoma cell lines. Because heterogeneity of
neuroblastoma cells could affect their treatment, two types
of neuroblastoma cell lines were tested for their response
to VPA and TSA treatment. Besides the effect of VPA and
TSA on UKF-NB-3 cells (the invasive N-type), that on the
UKF-NB-4 and SK-N-AS cell lines (the non-invasive and
less-aggressive S-type) was investigated in this work.
In addition, VPA and TSA are known to be metabolized
by cytochrome P450 (CYP) biotransformation enzymes
and can increase and/or decrease their activities and/or
expression, thereby affecting mechanisms that control
drug disposition (Fisher et al., 1991; Rogiers et al., 1992;
1995; Isojärvi et al., 2001; Wen et al., 2001; Bort et al.,
2004; Cerveny et al., 2007; Nelson-DeGrave et al., 2004;
Hooven et al., 2005; Snykers et al., 2007; Kiang et al., 2006).
Because several CYP enzymes metabolizing a variety of
drugs (CYP1A1, 1B1 and 3A4) were found to be expressed
in neuroblastoma cells (Poljaková et al., 2009), here we also
investigated whether their expression is influenced by VPA
and TSA in these cells.
Material and methods
Chemicals
Valproate and trichostatin A were obtained from Sigma (St.
Louis, MO, USA). All other chemicals used in the experi-
ments were of analytical purity or better.
Cell cultures
The UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines,
established from bone marrow metastases of high-risk
neuroblastoma, were a gift of prof. J. Cinatl, Jr. (J. W. Goethe
University, Frankfurt, Germany). Cell line UKF-NB-4 was
established from infiltrated bone marrow of chemoresistant
high-risk neuroblastoma recurrence and have high expres-
sion of P-glycoprotein. SK-N-AS, derived from bone marrow
metastases of neuroblastoma, was of the commercial source
(ECACC, Salisbury, UK). Cells were grown at 37 °C and 5% CO2
in Iscove’s modified Dulbecco’s medium (IMDM) (KlinLab Ltd,
Prague, Czech Republic), supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, 100 units/ml of penicilline and 100
μg/ml streptomycine (PAA Laboratories, Pasching, Austria).
MTT assay
The cytotoxicity of valproate and trichostatin A was deter-
mined by MTT test. For a dose-response curve, culture
medium stock solutions of valproate (200 mM) and DMSO
solutions of trichostatin A (1 mM) were dissolved in culture
medium to final concentrations of 0 – 50 mM and 0 – 1 μM
for valproate and trichostatin A, respectively. Cells in expo-
nential growth were seeded at 1 × 104 per well in a 96-well
microplate. After incubation (72 hours) at 37 °C in 5% CO2
saturated atmosphere the MTT solution (2 mg/ml PBS) was
added, the microplates were incubated for 4 hours and cells
lysed in 50% N,N-dimethylformamide containing 20% of
SDS, pH 4.5. The absorbance at 570 nm was measured for
each well by multiwell ELISA reader Versamax (Molecular
devices, CA, USA). The mean absorbance of medium controls
was subtracted as a background. The viability of control cells
was taken as 100% and the values of treated cells were calcu-
lated as a percentage of control. The IC50 values were calcu-
lated from at least 3 independent experiments using linear
regression of the dose-log response curves by SOFTmaxPro.
Estimation of contents of cytochromes P450 1A1, 1B1
and 3A4 in neuroblastoma cells by Western blot
To determine the expression of CYP1A1, 1B1 and 3A4
proteins, cells were homogenized in RIPA buffer. Protein
concentrations were assessed using the DC protein assay
(Bio-Rad, Hercules, CA, USA) with serum albumin as a
standard. 10–45 μg of extracted proteins were subjected to
SDS-PAGE electrophoresis on a 10% gel. After migration,
proteins were transferred to a nitrocellulose membrane
and incubated with 5% non-fat milk to block non-specific
binding. The membranes were then exposed to specific
anti-CYP1A1 (1:1000, Millipore, MA, USA) anti-CYP1B1
(1:500, AbCam, MA, USA) and anti-CYP3A4 (1:5000, AbD
Serotec, Oxford, UK) rabbit polyclonal antibodies overnight
at 4 °C. Membranes were washed and exposed to peroxidase-
conjugated anti-IgG secondary antibody (1:3000, Bio-Rad,
Hercules, CA, USA), and the antigen-antibody complex was
visualized by enhanced chemiluminiscence’s detection sys-
tem according to the manufacturer’s instructions (Immun-
Star HRP Substrate, Bio-Rad, Hercules, CA, USA). Films
(MEDIX XBU, Foma, Hradec Králové, Czech Republic)
were scanned with a computerized image-analyzing system
(ElfoMan 2.0, Ing. Semecký, Prague, Czech Republic).

Page 3

207
Also available online on setox.eu/intertox & versita.com/science/medicine/it
Interdisciplinary Toxicology. 2009; Vol. 2(3): 205–210
Copyright © 2009 Slovak Toxicology Society SETOX
Results
Cytotoxicity of valproate and trichostatin A
to human neuroblastoma cells
To evaluate the cytotoxicity of VPA and TSA to human
neuroblastoma cells (UKF-NB-3, UKF-NB-4 and
SK-N-AS), these cells were treated with increasing con-
centrations of both drugs (0–50 mM for VPA and 0–1 μM
for TSA). We first determined the effect of VPA and TSA
on growth of human neuroblastoma cell lines cultured
for 72 hours in the presence of both drugs, using MTT
assay. As shown in Figures 1 and 2, all three neuroblas-
toma cell lines were sensitive to VPA and TSA. Both drugs
inhibited the growth of neuroblastoma cell lines in a
dose-dependent manner. The IC50 values for VPA and TSA
0
0
10
20
30
40
50
60
70
80
90
100
2468 10 1214
viability [%]
VPA concentration [mM]
UKF-NB-3
UKF-NB-4
SK-N-AS
0
0.15
1
1.5
2
2.5
3
3.5
VPA [mM]
UKF-NB-3
1.02
2.77
2.62
UKF-NB-4 SK-N-AS
Figure 1. Cytotoxicity (viable cells as percentage of control) of val-
proate to UKF-NB-3, UKF-NB-4 and SK-N-AS after 72 h exposure to the
compound, determined by the MTT assay (A) and the values of IC50
(B). Values are means and standard deviations of 8 determinations.
Figure 2. Cytotoxicity (viable cells as percentage of control) of trichos-
tatin A to UKF-NB-3, UKF-NB-4 and SK-N-AS after 72 h exposure to the
compound, determined by the MTT assay. (A) and the values of IC50
(B). Values are means and standard deviations of 8 determinations.
AA
BB
0
0
10
20
30
40
50
60
70
80
90
100
50 100150200250 300
viability [%]
TSA concentration [nM]
UKF-NB-3
UKF-NB-4
SK-N-AS
0
20
60
40
80
100
120
140
160
180
TSA [nM]
UKF-NB-3
69.8
129.4
118.0
UKF-NB-4SK-N-AS
calculated from the dose-log response curves are shown in
Figures 1B and 2B.
Among the neuroblastoma cell lines tested in this study,
the UKF-NB-3 cell line was the most sensitive to both drugs,
with IC50 values of 1.02 mM and 69.8 nM for VPA and TSA,
respectively. The IC50 values indicating the toxicity of VPA
and TSA to UKF-NB-4 cells were similar to those found for
the SK-N-AS cell line, being up to a 2.7-fold lower than for
the UKF-NB-3 cell line (Figures 1 and 2). Nevertheless, the
curves showing the viability of SK-N-AS cells under treat-
ment with increasing concentrations of VPA and TSA sig-
nificantly differed from those of UKF-NB-3 and UKF-NB-4
cell lines. At higher VPA and TSA concentrations, the
sensitivity of SK-N-AS cells was much lower than that of
other two neuroblastoma cell lines analyzed in this work.

Page 4

208
J. Hřebačková, J. Poljaková, T. Eckschlager, J. Hraběta, P. Procházka, S. Smutný, M. Stiborová
Histone deacetylase inhibitors valproate and trichostatin A are toxic to neuroblastoma cells
ISSN: 1337-6853 (print version) | 1337-9569 (electronic version)
Eff ect of VPA and TSA on expression of cytochrome
P450 1A1, 1B1 and 3A4 proteins
Using Western blot analysis with antibodies raised against
CYP1A1, 1B1 and 3A4, the effects of VPA and TSA on
protein expression levels of these enzymes were analyzed in
the tested neuroblastoma cell lines.
Expression of CYP1A1 protein in neuroblastoma
UKF-NB-3 and UKF-NB-4 cells was elevated by increasing
concentrations of VPA and/or TSA in a dose-dependent
manner (Figure 3). A 1.7-, 4.0- and 8.1-fold increase in
CYP1A1 expression was caused by treating the UKF-NB-3
cells for 48 hour with 0.5, 1.0 and 2.0 mM VPA, respec-
tively, while lower, only up to a 1.7-fold increase in levels
of this CYP was produced by VPA in UKF-NB-4 cells. In
the SK-N-AS cells, even no effect of VPA on the CYP1A1
expression was detectable.
Similar effects on CYP1A1 expression in neuroblastoma
UKF-NB-3 and UKF-NB-4 cell lines were detected when
cells were treated for 48 hours with TSA. Up to a 4.4-fold
increase in expression levels of CYP1A1 was produced by
50–200 nM TSA in these cells (Figure 3). No effects of TSA on
the expression of CYP1A1 protein in SK-N-AS were found.
Figure 3. Expression of CYP1A1, 1B1 and 3A4 in human neuroblas-
toma cell lines UKF-NB-3, UKF-NB-4 and SK-N-AS by Western blot.
Expression of CYP1B1 protein was decreased in
UKF-NB-3 and UKF-NB-4 cells after their 48-hour treat-
ment with increasing concentrations of VPA and/or TSA,
being decreased in a dose-dependent manner. Similar to
CYP1A1, no effect of both HDAC inhibitors on expres-
sion of CYP1B1 was produced in SK-N-AS cells (Figure 3).
Interestingly, two protein bands detectable by antibody
against CYP1B1 were found in SK-N-AS cells.
In the case of the effects of VPA and TSA on expres-
sion of CYP3A4 protein in neuroblastoma cells, both
these drugs essentially did not influence its expression in
S-type UKF-NB-4 and SK-N-AS cell lines. The N-type of
neuroblastoma cell line, UKF-NB-3, was only the excep-
tion; whereas increased concentrations of VPA increased
CYP3A4 expression in this cell line, TSA had no effect
(Figure 3).
Discussion
The results of this work show that human neuroblastoma
UKF-NB-3, UKF-NB-4 and SK-N-AS cell lines are sensitive
to two tested HDAC inhibitors, VPA and TSA. In the case
of VPA, its concentrations that were toxic to neuroblastoma
cells are clinically applicable, since concentrations between
0.35–0.7 mM in patients serum are commonly therapeuti-
cally used (Duenas-Gonzalez et al., 2007). The IC50 values
for VPA and TSA indicate that a UKF-NB-3 cell line was
the most sensitive to both HDAC inhibitors, while their
toxicity to the UKF-NB-4 and SK-N-AS cell lines was up
to a 2.7-fold lower. Thus, the sensitivity to the two drugs
seems to be related to the phenotype, with the S-type cells
(UKF-NB-4 and SK-N-AS) being less sensitive than the
N-type (UKF-NB-3), probably because of their partly lower
capability of undergoing apoptosis (Servidei et al., 2004).
However, the results shown here indicate that it seems to
be questionable to evaluate the toxic effects of chemicals
to cells in culture using only the IC50 values. The question
arises, whether the IC50 value is a real appropriate sensi-
tivity marker. Namely, of the S-type neuroblastoma cells
utilized in this study, the SK-N-AS cell line seems to be
even less sensitive to VPA and TSA than the second S-type
cell line, UKF-NB-4, even though the IC50 values for VPA
and TSA were similar for both these cell lines. At higher
VPA and TSA concentrations, the sensitivity of SK-N-AS
cells was much lower than that of UKF-NB-4. This less
sensitive SK-N-AS line seems to be, at least in part, capable
of overcoming treatment with VPA and TSA at concentra-
tions that cause almost complete eradication of UKF-NB-4
cells. These results suggest that caution should be exerted
to sort neuroblastoma cells into their types. Even in one
type of neuroblastoma cells (S-type in this case), biological
heterogeneity should be taken into account. This suggestion
is also supported by further features found in this cell line.
Namely, the SK-N-AS cell line behaves differently from the
other S-type cell line, UKF-NB-4, from the point of view of
the effects of VPA and TSA on CYP expression; no effects of
both drugs was found on levels of individual CYP enzymes.
Moreover, in this cell line, the two CYP1B1 protein bands

Page 5

209
Also available online on setox.eu/intertox & versita.com/science/medicine/it
Interdisciplinary Toxicology. 2009; Vol. 2(3): 205–210
Copyright © 2009 Slovak Toxicology Society SETOX
were detectable by antibody against this CYP. Now, we can
only speculate on the origin of the second protein band in
SK-N-AS cells. The questions, whether it might follow from
a degradation of the CYP1B1 protein in this cell line or it
is the artifact caused by the method used (Western blot),
remain to be answered.
The expression of all CYP enzymes analyzed in this
work was modulated by VPA and TSA only in the N-type
UKF-NB-3 cell line. Whereas the CYP1A1 enzyme was
induced by both drugs, expression of CYP1B1 was depressed
by both drugs. The CYP3A4 enzyme was increased by VPA,
but TSA had no influence on the expression of this enzyme.
The expression of CYP1A1 and 1B1 was also similarly
affected by VPA and TSA in the UKF-NB-4 cell line, but no
effect on expression levels of CYP3A4 was produced in this
line. Similarity in response of UKF-NB-3 and UKF-NB-4
cells to the effects of VPA and TSA on CYP1A1 and 1B1
expression might probably be caused by their similar effects
on state (degree) of acetylation of histones and, therefore,
transcription activity. But differences between these two
cell lines and particularly SK-N-AS cells in response to
CYP enzyme expression and its affecting by VPA and TSA
are still valuable. The question whether such differences
are caused by the fact that cells vary in the broad spectrum
of metabolic and signalling pathways that might also be
affected by VPA and TSA in a different way, independently
of a cell type (N- or S-type), remains to be answered.
Further studies with these and other neuroblastoma cell
lines and various HDAC inhibitors and broader spectrum
of CYP enzymes have to be performed in order to shed
more light on this field.
Since CYP enzymes are involved in biosynthesis and
metabolism of many endogenous physiologically active
substances and in biotransformation of xenobiotics with
pharmacological and/or toxic effects (Myasodeova, 2008),
a change in their expression might affect the cells signifi-
cantly. In the case of oncology, the participation of CYPs
in drug metabolism seems to be their most important role.
A variety of CYP enzymes is involved in metabolism of a
broad spectrum of drugs that can, moreover, either increase
or decrease their expression levels. The finding that VPA
and TSA are capable of inducing and depressing CYP
enzyme expression in neuroblastoma cells (CYP1A1, 1B1
and 3A4 tested in our work) might have great importance.
This feature might be utilized mainly in the combination
therapy with other drugs whose pharmacological effects
are dependent on their CYP-mediated metabolism. Such a
study with one of these drugs, ellipticine, is under way in
our laboratory.
Acknowledgements
We thank Prof. J. Cinatl, Jr. (J. W. Goethe University,
Frankfurt, Germany) for providing cell lines. This work
was supported by the Internal Grant Agency of Ministry
of Health of the Czech Republic (grant NR9522-3/2007)
and Ministry of Education of the Czech Republic (grant
MSM0021620813 and RP MSMT 14/63).
REFERENCES
Batty N, Malouf GG, Issa JP. (2009). Histone deacetylase inhibitors as anti-neo-
plastic agents. Cancer Lett 280: 192–200.
Blaheta RA, Cinatl J Jr. (2002). Anti-tumor mechanisms of valproate: a novel role
for an old drug. Med Res Rev 22: 492–511.
Bolden JE, Peart MJ, Johnstone RW. (2006). Anticancer activities of histone
deacetylase inhibitors. Nat Rev Drug Discov 5: 769–784.
Bort R, Gomez-Lechon MJ, Castell JV, Jover R. (2004). Role of hepatocyte nu-
clear factor 3 gamma in the expression of human CYP2C genes. Arch Biochem
Biophys 426: 63–72.
Brodeur GM. (2003). Neuroblastoma: biological insights into a clinical enigma.
Nat Rev Cancer 3: 203–216.
Cerveny L, Svecova L, Anzenbacherova E, Vrzal R, Staud F, Dvorak Z, Ulrichova
J, Anzenbacher P, Pavek P. (2007). Valproic acid induces CYP3A4 and MDR1
gene expression by activation of constitutive androstane receptor and preg-
nane X receptor pathways. Drug Metab Dispos 35: 1032–1041.
Cinatl J Jr, Cinatl J, Scholz M, Driever PH, Henrich D, Kabickova H, Vogel JU, Do-
err HW, Kornhuber B. (1996). Antitumor activity of sodium valproate in cul-
tures of human neuroblastoma cells. Anticancer Drugs 7: 766–773.
Condorelli F, Gnemmi I, Vallario A, Genazzani AA, Canonico PL. (2008). Inhibitors
of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma
cells. Br J Pharmacol 153: 657–668.
Duenas-Gonzalez A, Candelaria M, Perez-Plascencia C, Perez-Cardenas E, de
la Cruz-Hernandez E, Herrera LA. (2008). Valproic acid as epigenetic cancer
drug: preclinical, clinical and transcriptional eff ects on solid tumors. Cancer
Treat Rev 34: 206–222.
Fisher JE, Nau H, Löscher W. (1991). Alterations in the renal excretion of valpro-
ate and its metabolites after chronic treatment. Epilepsia 32: 146–150.
Furchert SE, Lanvers-Kaminsky C, Juürgens H, Jung M, Loidl A, Frühwald MC.
(2007). Inhibitors of histone deacetylases as potential therapeutic tools for
high-risk embryonal tumors of the nervous system of childhood. Int J Cancer
120: 787–1794.
Hooven LA, Mahadevan B, Keshava C, Johns C, Pereira C, Desai D, Amin S,
Weston A, Baird WM. (2005). Eff ects of suberoylanilide hydroxamic acid and
trichostatin A on induction of cytochrome P450 enzymes and benzo[a]py-
rene DNA adduct formation in human cells. Bioorg Med Chem Lett 15: 1283–
1287.
Hopkins-Donaldson S, Yan P, Bourloud KB, Muhlethaler A, Bodmer JL and Gross
N. (2002). Doxorubicin-induced death in neuroblastoma does not involve
death receptors in S-type cells and is caspase-independent in N-type cells.
Oncogene 21: 6132–6137.
Isojärvi JI, Turkka J, Pakarinen AJ, Kotila M, Rättyä J, Myllylä VV. (2001). Thyroid
function in men taking carbamazepine, oxcarbazepine, or valproate for epi-
lepsy. Epilepsia 42: 930–934.
Kiang TK, Ho PC, Anari MR, Tong V, Abbott FS, Chang TK. (2006). Contribution
of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic mi-
crosomes from individuals with the CYP2C9*1/*1 genotype. Toxicol Sci 94:
261–271.
Marchion DC, Bicaku E, Daud AI, Sullivan DM, Munster PN. (2005). Valproic acid
alters chromatin structure by regulation of chromatin modulation proteins.
Cancer Res 65: 3815–3822.
Maris JM, Mathay KK. (1999). Molecular biology of neuroblastomas. J Clin Oncol
17: 2264–2279.
Marks PA, Miller T, Richon VM. (2003). Histone deacetylases. Curr Opin Pharma-
col 3: 344–351.
Marks PA, Richon VM, Miller T, Kelly WK. (2004). Histone deacetylase inhibitors.
Adv Cancer Res 91: 137–168.
Michaelis M, Doerr HW, Cinatl J Jr. (2007). Valproic acid as anti-cancer drug. Curr
Pharm Des 13: 3378–3393.
Michaelis M, Suhan T, Cinatl J, Driever PH, Cinatl J Jr. (2004). Valproic acid and in-
terferon-alpha synergistically inhibit neuroblastoma cell growth in vitro and
in vivo. Int J Oncol 25: 1795–1799.
Morgenstern BZ, Krivoshik AP, Rodriguez V and Anderson PM. (2004). Wilms’ tu-
mor and neuroblastoma. Acta Paediatr Suppl 93: 78–85.
Myasoedova KN. (2008). New fi ndings in studies of cytochromes P450. Bio-
chemistry (Mosc) 73: 965–969.
Nelson-DeGrave VL, Wickenheisser JK, Cockrell JE, Wood JR, Legro RS, Strauss
JF 3rd, McAllister JM. (2004). Valproate potentiates androgen biosynthesis in
human ovarian theca cells. Endocrinology 145: 799–808.

Page 6

210
J. Hřebačková, J. Poljaková, T. Eckschlager, J. Hraběta, P. Procházka, S. Smutný, M. Stiborová
Histone deacetylase inhibitors valproate and trichostatin A are toxic to neuroblastoma cells
ISSN: 1337-6853 (print version) | 1337-9569 (electronic version)
Poljakova J, Eckschlager T, Hrabeta J, Hrebackova J, Smutny S, Frei E, Martinek V,
Kizek R, Stiborova M. (2009). The mechanism of cytotoxicity and DNA adduct
formation by the anticancer drug ellipticine in human neuroblastoma cells.
Biochem Pharmacol 77: 1466–1479.
Rogiers V, Akrawi M, Vercruysse A, Phillips IR, Shephard EA. (1995). Eff ects of
the anticonvulsant, valproate, on the expression of components of the cyto-
chrome-P-450-mediated monooxygenase system and glutathione S-trans-
ferases. Eur J Biochem 231: 337–343.
Rogiers V, Callaerts A, Vercruysse A, Akrawi M, Shephard E, Phillips I. (1992). Ef-
fects of valproate on xenobiotic biotransformation in rat liver. In vivo and in
vitro experiments. Pharm Weekbl Sci 14: 127–131.
Santini V, Gozzini A, Ferrari G. (2007). Histone deacetylase inhibitors: molecular
and biological activity as a premise to clinical application. Curr Drug Metab 8:
383–393.
Servidei T, Morosetti R, Ferlini C, Cusano G, Scambia G, Mastrangelo R, Koeffl er
HP. (2004). The cellular response to PPARgamma ligands is related to the phe-
notype of neuroblastoma cell lines. Oncol Res 14: 345–354.
Silva MF, Aires CC, Luis PB, Ruiter JP, Ijlst L, Duran M, Wanders RJ, Tavares de
Almeida I. (2008). Valproic acid metabolism and its eff ects on mitochondrial
fatty acid oxidation: A review. J Inherit Metab Dis 31: 205–216.
Snykers S, Vanhaecke T, De Becker A, Papeleu P, Vinken M, Van Riet I, Rogiers
V. (2007). Chromatin remodeling agent trichostatin A: a key-factor in the he-
patic diff erentiation of human mesenchymal stem cells derived of adult bone
marrow. BMC Dev Biol 7: 24–39.
Van Lint C, Emiliani S, Verdin E. (1996). The expression of a small fraction of cel-
lular genes is changed in response to histone hyperacetylation. Gene Expr 5:
245–253.
Voigt A, Hartmann P and Zintl F. (2000). Diff erentiation, proliferation and adhe-
sion of human neuroblastoma cells after treatment with retinoic acid. Cell Ad-
hes Commun 7: 423–440.
Wen X, Wang JS, Kivistö KT, Neuvonen PJ, Backman JT. (2001). In vitro evalu-
ation of valproic acid as an inhibitor of human cytochrome P450 isoforms:
preferential inhibition of cytochrome P450 2C9 (CYP2C9). Br J Clin Pharmacol
52: 547–553.