Effects of valproic acid on the cell cycle and apoptosis through acetylation of histone and tubulin in a scirrhous gastric cancer cell line.

Abstract

Management of peritoneal dissemination is the most critical problem in gastric cancer. This study was performed to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA.
The effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition, paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The acetylation status of histone H3 and α-tubulin after exposure to VPA were analyzed by western blotting. The activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western blotting. The effects of VPA in vivo were evaluated in a xenograft model, and apoptotic activity was assessed by TUNEL assay.
OCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and MKN-28. The concentration of VPA required for significant inhibition of cell viability (P < 0.05) was 5 mM at 24 h and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects. VPA increased acetyl-histone H3, acetyl-α-tubulin, and p21WAF1 levels accompanied by upregulation of p27, caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment, the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the control group at 4 weeks after treatment (P < 0.01). The apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (P < 0.001).
VPA induced dynamic modulation of histone H3 and α-tubulin acetylation in relation with the anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer.

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RESEARCHOpen Access
Effects of valproic acid on the cell cycle and
apoptosis through acetylation of histone and
tubulin in a scirrhous gastric cancer cell line
Yasumichi Yagi1*, Sachio Fushida1, Shinichi Harada2, Jun Kinoshita1, Isamu Makino1, Katsunobu Oyama1,
Hidehiro Tajima1, Hideto Fujita1, Hiroyuki Takamura1, Itasu Ninomiya1, Takashi Fujimura1, Tetsuo Ohta1,
Masakazu Yashiro3, Kosei Hirakawa3
Abstract
Background: Management of peritoneal dissemination is the most critical problem in gastric cancer. This study
was performed to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of
human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA.
Methods: The effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition,
paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were
evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The
acetylation status of histone H3 and a-tubulin after exposure to VPA were analyzed by western blotting. The
activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western
blotting. The effects of VPA in vivo were evaluated in a xenograft model, and apoptotic activity was assessed by
TUNEL assay.
Results: OCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and
MKN-28. The concentration of VPA required for significant inhibition of cell viability (P < 0.05) was 5 mM at 24 h
and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects.
VPA increased acetyl-histone H3, acetyl-a-tubulin, and p21WAF1 levels accompanied by upregulation of p27,
caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment,
the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the
control group at 4 weeks after treatment (P < 0.01). The apoptotic index was significantly higher in the VPA-treated
group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (P < 0.001).
Conclusions: VPA induced dynamic modulation of histone H3 and a-tubulin acetylation in relation with the
anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with
PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer.
Background
Gastric cancer remains one of the leading causes of can-
cer death in the world [1]. Particularly, the prognosis of
scirrhous gastric cancer is poorer than those of other
types of gastric cancer [2,3]. In gastric cancer, the most
critical factor responsible for poor prognosis is
peritoneal dissemination. Consequently, the manage-
ment of peritoneal dissemination is an urgent problem
in gastric cancer patients.
The recent development of anticancer drugs and
intraperitoneal chemotherapy improved the clinical out-
comes in gastric cancer patients with peritoneal dissemi-
nation [4,5]. Moreover, molecular targeted therapy has
attracted a great deal of attention as a new class of
anticancer agents. Clinical studies indicated that com-
bining molecular targeted agent with conventional
* Correspondence: y-yagi@live.jp
1Department of Gastroenterologic Surgery, Division of Cancer Medicine,
Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8641,
Japan
Full list of author information is available at the end of the article
Yagi et al. Journal of Experimental & Clinical Cancer Research 2010, 29:149
http://www.jeccr.com/content/29/1/149
© 2010 Yagi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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chemotherapy enhances the inhibition of tumor growth
and metastasis in gastric cancer patients [6,7]. Chemo-
sensitivity is influenced by changes in expression of var-
ious genes, including those known to be associated with
the cell cycle and apoptosis [8]. There is increasing evi-
dence that epigenetic alterations, such as histone acetyla-
tion and promoter methylation, play important roles in
regulation of gene expression associated with the cell
cycle and apoptosis [9]. Chromatin remodeling is physio-
logically regulated by two enzymes, histone acetyltrans-
ferase (HAT) and histone deacetylase (HDAC). The ratio
of these two enzymes regulates the amount of histone
acetylation and controls posttranslational modification of
histones and gene transcription. Acetylation of lysine
residues of the histones weakens their binding to DNA
and induces a change in DNA conformation essential for
binding of transcription factors to the promoter regions
of target genes [10,11]. HDACs are subdivided into three
classes [12,13]. Class I HDACs are composed of HDAC 1
- 3 and 8. Class II HDACs are composed of HDAC 4 - 7
and 9 - 11. Aberrant levels of HDAC activity have been
found in a variety of human malignancies and result in
repression of tumor-suppressor genes and promotion of
tumorigenesis [14]. HDAC inhibitors represent a structu-
rally diverse group of compounds that inhibit the deace-
tylation of histones, permitting the chromatin scaffolding
to assume a more relaxed, open conformation, which
generally promotes gene transcription. Recently, HDAC
inhibitors have been shown to have antiproliferative
activity through cell cycle arrest, differentiation, and
apoptosis in various cancer cell types [15], including gas-
tric cancer cell lines [16,17]. Especially, the combination
of HDAC inhibitor with conventional chemotherapy is
expected to have a synergistic effect, because the
mechanism of action is different from those of conven-
tional chemotherapeutic regimens.
Valproic acid (VPA), which has long been used clini-
cally for treatment of epilepsy and bipolar disorder
without significant toxicity, causes hyperacetylation of
the N-terminal tails of histones H3 and H4 in vitro and
in vivo and inhibits HDAC activity, probably by binding
to the catalytic center and thereby blocking substrate
access [18,19]. VPA inhibits both class I and II HDACs,
with high potency for class I HDACs [20]. Earlier stu-
dies indicated that p21WAF1, one of the target genes
induced by VPA, affects differentiation and decreases
tumor cell growth [21,22]. Another report focused on
the apoptotic activity of VPA [23]. However, the detailed
mechanism of apoptosis by VPA has not been eluci-
dated. On the other hand, recent evidence suggests that
HDAC inhibitors also enhance the acetylation of non-
histone proteins, such as p53, c-Jun, and a-tubulin
[24-26]. It is possible that VPA increases acetylation of
non-histone proteins in relation with apoptosis.
However, no reports have focused on the therapeutic
potential of VPA in gastric cancer. The present study
was performed to investigate the anticancer mechanism
of action of VPA by analyzing the expression of cell
cycle regulatory proteins and apoptosis-modulating pro-
teins in a scirrhous gastric cancer cell line. In addition
to acetylation of histones, the possibility that acetylation
of the non-histone protein a-tubulin contributes to inhi-
bition of tumor growth was also examined.
Paclitaxel (PTX) is an anticancer agent, which stabi-
lizes polymerized microtubules and enhances microtu-
bule assembly, and thus arrests the cell cycle in G0/G1
and G2/M phases, leading to cell death [27], and has
been used for peritoneal dissemination of ovarian and
gastric cancer [4,28]. As tubulin is a target molecule of
PTX, combination of VPA with PTX has the potential
to show synergistic effects. In the present study, we also
evaluated the synergistic effects of PTX with VPA on a
scirrhous gastric cancer cell line. The mechanisms of
these anticancer effects of VPA, which are different
from conventional chemotherapy, may provide a new
strategy to improve the clinical outcome of gastric can-
cer patients.
Methods
Materials
VPA was purchased from Sigma-Aldrich Co. (Japan).
PTX was kindly provided by Bristol-Myers Squibb Com-
pany (Japan).
Cell lines and cell culture
OCUM-2MD3, a highly peritoneal-seeding cell line
derived from human scirrhous gastric cancer, was kindly
provided by the Department of Surgical Oncology of
Osaka City University of Medicine. In this study, we
used mainly OCUM-2MD3 to investigate the efficacy of
VPA on this peritoneal-seeding cell line. Other human
gastric cancer cell lines (MKN28, moderately differen-
tiated adenocarcinoma; TMK-1, poorly differentiated
adenocarcinoma) were obtained from the American
Type Culture Collection (Rockville, MD). These were
seeded in 75-cm2dishes (Becton Dickinson, Japan) and
cultured in 10 mL of medium at 37°C in a humidified
atmosphere of 5% CO2in air. OCUM-2MD3 cells were
grown in DMEM (Invitrogen, Japan) supplemented with
10% heat-inactivated fetal bovine serum (Nichirei
Bioscience Inc., Japan), 100 IU/mL penicillin, 100 mg/
mL streptomycin (Invitrogen), 2 mM glutamine (Nissui
Pharmaceutical Co., Ltd., Japan), and 0.5 mM sodium
pyruvate. The culture medium for MKN28 and TMK-1
cells was RPMI (Nissui) with the same additives as
above. Cells were grown to confluence and harvested by
trypsinization with 0.25 mg/mL trypsin/EDTA (Invitro-
gen) and suspended in culture medium before use.
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Cell growth assay
The viability of OCUM-2MD3 cells treated with VPA
was determined by standard 3-(4, 5-Dimethylthiazol-2-
yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.
OCUM-2MD3 cells were seeded at 5 × 103per well in
96-well plates and incubated overnight at 37°C. After
incubation, the supernatant was discarded and replaced
with fresh serum-free culture medium. VPA was dis-
solved in phosphate buffered saline (PBS) and added to
the cell culture medium at various concentrations (0 -
10 mM). At 24, 48, and 72 h after exposure to VPA, the
supernatant was discarded and MTT solution was added
to each well (500 μg/mL final concentration) and incu-
bated at 37°C for 3 h. Then, the supernatant was
removed, and 150 μL of DMSO was added. The absor-
bance of the solution was read at a wavelength of 540
nm using a microplate reader (BIO-RAD550; BIO-RAD,
Japan). The percentage inhibition was determined by
comparing the cell density of the drug-treated cells with
that of untreated controls. All experiments were
repeated at least three times. In addition, the effects of
VPA combined with PTX were evaluated at various
concentrations.
Animals and xenograft model treated with VPA
BALB/c nu/nu mice (female, 4 - 6 weeks old; Charles
River Laboratories, Japan, Inc) were used for xenograft
models. They were housed under specific pathogen-free
conditions and fed standard chow pellets and water ad
libitum. Experiments were performed according to the
Standard Guidelines for Animal Experiments of Kana-
zawa University. The effects of VPA on the xenograft
model were examined as follows: OCUM-2MD3 cells (2
× 106cells) were inoculated s.c. into the dorsal side of
mice. The mice were divided into two groups: a control
group (PBS i.p., n = 6) and a VPA-treated group (10
mg/mouse i.p. for 5 days per week, n = 6). The treat-
ment was started on day 7 after xenografting and dis-
continued after 5 weeks. Tumors were measured weekly
with Vernier calipers. Tumor volume was calculated
using the following formula: volume = length × width ×
height × 0.5236. At the end of the experiment, tumor
specimens were collected for immunohistochemical
examination and TUNEL assay.
Western blotting
The effects of VPA on acetylation of histone H3 and a-
tubulin, cell cycle regulatory and apoptosis-related pro-
teins, were analyzed in cell lysates by western blotting.
OCUM-2MD3 cells were seeded at a density of 1 × 106
cells per 75-cm2dish and cultured in 10 mL of medium
overnight. Lysates were obtained from the cells har-
vested at 0, 0.5, 1, 3, 6, 12, 24, and 48 h after incubation
with 1 mM VPA, which corresponded approximately to
the level obtained by administrating a clinical dose of
VPA. Whole-cell lysates were prepared in denaturing
SDS sample buffer and subjected to SDS-PAGE (ATTO
Co. Ltd., Japan). As primary antibodies, a rabbit polyclo-
nal HDAC1 antibody (1:5000) (Santa Cruz Biotechnol-
ogy Inc., Santa Cruz, CA), rabbit polyclonal HDAC2
antibody (1:5000) (Santa Cruz), rabbit polyclonal acetyl-
histone H3 (Lys 9) antibody (1:5000) (Cell Signaling,
Beverly, MA), mouse monoclonal acetyl a-tubulin anti-
body (1:5000) (Sigma), and mouse monoclonal b-actin
antibody (1:5000) (Sigma) were used. As antibodies
against apoptosis-related proteins, a rabbit polyclonal
cleaved caspase 3 (Asp175) antibody (1:5000) (Cell Sig-
naling), mouse monoclonal caspase 9 antibody (1:5000)
(Santa Cruz), mouse monoclonal bcl-2 antibody (1:5000)
(Santa Cruz), mouse monoclonal survivin 6E4 antibody
(1:5000) (Cell Signaling), and mouse monoclonal p53
antibody (1:5000) (Sigma) were used. As antibodies
against cell cycle regulatory proteins, a mouse monoclo-
nal p21WAF1 (1:5000) (Pharmingen, San Diego, CA),
mouse monoclonal p27 antibody (Santa Cruz), and
mouse monoclonal cyclin D1 (1:5000) (Sigma) were
used. The immunoblots were visualized using an ECL
Plus kit (GE Healthcare UK Ltd., Japan). The antibody-
antigen complex was detected using an ECL Western-
Blotting detection kit (GE Healthcare) and the Light-
Capture system (ATTO) and then quantified using the
CS analyzer program (ATTO).
Immunohistochemical examination and TUNEL assay
Tumor specimens obtained from xenograft models were
fixed in 10% neutral buffered formalin and embedded in
paraffin. The sections were stained with H&E and
immunostained with a mouse monoclonal p21WAF1
(1:200) (Pharmingen) and a rabbit polyclonal cleaved
caspase 3 antibody (1:200) (Cell Signaling) at 4°C over-
night. The sections were reacted with EnVision reagent
(Dako Co., Japan) for visualization. The degree of apop-
tosis was evaluated using the TdT-mediated dUTP
nick-end labeling (TUNEL) method (Apoptosis in situ
Detection Kit; Wako, Osaka, Japan). For quantitative
analysis, the cells that were TUNEL-positive and also
fulfilled the morphological criteria of apoptosis were
counted under ×400 magnification in 10 randomly cho-
sen fields representing at least 1000 nuclei. The results
were expressed as the mean percentage of apoptosis
cells. These results were used as the apoptotic index (n
= 6 in each group).
Statistical analysis
Values are expressed as means ± standard deviation
(SD). Comparisons among the data sets were made by
Student’s t test using the computer software package
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SPSS10.0 (SPSS, Japan, Inc). In all analyses, P < 0.05 was
taken to indicate statistical significance.
Results
Expression of HDAC1 and HDAC2 in OCUM-2MD3 cells
On western blotting analysis, OCUM-2MD3 cells showed
high levels of HDAC1 and HDAC2 compared with the
other human gastric cancer cell lines (Figure 1). Immu-
nohistologically, both HDAC1 and HDAC2 were
expressed mainly in nuclei. The HDAC2 expression was
characteristically observed in the cells in mitotic phase
(Figure 2).
Effects of VPA on the growth of OCUM-2MD3 cells in vitro
As shown in Figure 3, the inhibition of VPA in OCUM-
2MD3 cells was dependent on the dose and incubation
time. The concentration of VPA required for significant
inhibition of cell viability (P < 0.05) was 5 mM at 24 h,
and 0.5 mM at 48 h and 72 h. Degenerated cancer cells
were observed at high concentrations (> 5 mM at 48
and 72 h) of VPA (data not shown). According to these
results, we examined the western blotting by 1 mM
VPA, which showed the evident decrease of OCUM-
2MD3 cells. VPA in combination with PTX showed
dose-dependent combinatorial effects (Figure 4).
Effects of VPA on acetyl-histone H3 level, cell cycle
regulatory protein
The acetylation status of histone H3 in OCUM-2MD3
cells was determined during 48 h of incubation with 1
mM VPA, using an antibody that specifically recognizes
hyperacetylated forms of histone H3. As shown in Fig-
ure 5, VPA markedly increased acetyl-histone H3
expression with maximal induction at 12 h of incubation
with VPA. In addition, the maximal increase of
p21WAF1 was detected concomitant with activation of
acetyl-histone H3. The level of p27 showed a gradual
increase for up to 48 h. In contrast, VPA showed a gra-
dual decrease in cyclin D1 level.
Effects of VPA on the induction of apoptosis
We analyzed the effects of VPA on apoptotic regulatory
proteins by western blotting (Figure 6). The levels of
cleaved caspase 3 and caspase 9 showed mild increases
up to 24 h, suggesting that the apoptosome pathway
was activated by this VPA treatment. Conversely, the
levels of bcl-2 and survivin gradually decreased. VPA
reduced bcl-2 level by 30% and survivin level by 70%,
suggesting that the antiapoptotic activity was suppressed
by this HDAC inhibitor.
Acetylation of tubulin after exposure to VPA
Figure 7 shows the status of tubulin acetylation deter-
mined by western blotting. Increased acetyl-a-tubulin was
detected by 6 h and the maximal induction was evident by
12 h. Such rapid tubulin acetylation occurred in parallel
with increases in acetyl-histone H3 and p21WAF1.
Effects of VPA on xenograft model in vivo
The time courses of changes in xenografted tumor
volume are shown in Figure 8. The mean tumor volume
of the VPA-treated group (246.3 ± 56.0 mm3) was sig-
nificantly reduced by 36.4%, compared with that of the
control group (387.5 ± 99.6 mm3) at 4 weeks after treat-
ment (P < 0.01). As shown in Figure 9, immunohisto-
chemical examination of the xenografted tumor revealed
upregulation of p21WAF1 in the VPA-treated group.
Moreover, degenerated cells with VPA treatment
showed reactivity for cleaved caspase 3, indicating cas-
pase 3 activation. TUNEL assay showed that the apopto-
tic index was significantly higher in the VPA-treated
group (42.3% ± 3.5%) than in the control group (7.7% ±
2.5%) as shown in Figure 10 (P < 0.001).
Discussion
The results of the present study showed that VPA alone
has an antiproliferative effect on a scirrhous gastric can-
cer cell line (OCUM-2MD3) in vitro and in vivo. VPA
increased the acetylation of histone H3, resulting in a
significant reduction of tumor growth through induction
Figure 1 Expression of HDAC1 and HDAC2 in gastric cancer
cell lines examined by western blotting. OCUM-2MD3 showed
high levels of HDAC1and 2 compared with other cell lines.
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of both p21WAF1 and apoptosis. Furthermore, we also
demonstrated that VPA induces a-tubulin acetylation,
thus stabilizing tubulin, suggesting that VPA in combi-
nation with PTX will have a synergistic effect.
Previous studies showed that the HDAC inhibitor tri-
chostatin A has an antiproliferative effect through cell
cycle regulation and apoptosis [16], and increases che-
mosensitivity of gastric cancer cell lines to anticancer
drugs, including 5-fluorouracil, PTX, and irinotecan
[17]. In the present study, the acetylation of histone H3
was observed with upregulation of p21WAF1 expres-
sion, supporting the suggestion that VPA induces differ-
entiation of cancer cells as reported previously [29]. In
addition, VPA induced alterations in the expression of
other cell cycle-related proteins, such as p27 and cyclin
D1. As p21WAF1 and p27 are cyclin-dependent kinase
inhibitors that bind to cyclin-dependent kinase com-
plexes and decrease kinase activity, they may act as key
regulators of G0/G1accumulation [30]. Most previous
studies indicated that HDAC inhibitors upregulate the
transcription of p53 [31,32]. However, Sami et al.
reported the efficacy of VPA with no effect on p53
expression [18]. In the present study, we also demon-
strated that VPA has an anticancer effect through a
p53-independent pathway. With regard to apoptosis, the
activation of caspase-3 and caspase-9 and the downregu-
lation of bcl-2 and survivin were observed with the
apoptotic activity induced by VPA in the present study.
Taken together, the total effects on the cell cycle and
apoptosis were considered to result in the anticancer
activity of VPA.
Figure 2 Immunostaining of HDAC1 and HDAC2 in OCUM-2MD3 cells. Both HDAC1 and HDAC2 were expressed mainly in nuclei of tumor
cells. Expression of HDAC2 was observed characteristically in tumor cells in the mitotic period (arrows). (a) HDAC1, (b) HDAC2. Original
magnification ×400.
Figure 3 Effects of VPA on the growth of OCUM-2MD3 cells in vitro. Cell viability was assessed by MTT assay. OCUM-2MD3 cells were
treated with the indicated doses of VPA (0 - 10 mM) in serum-free medium.
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Peritoneal dissemination of scirrhous gastric cancer is
characterized by rapid infiltration and proliferation of
cancer cells with abundant fibrosis in the stroma [33].
From the viewpoint of molecular biology, transforming
growth factor-b (TGF-b) is considered a key factor,
which contributes to the invasiveness and morphological
changes in peritoneal dissemination of diffuse-type gas-
tric cancer [34]. Clinically, the expression of TGF-b is
correlated to the malignant potential of scirrhous gastric
cancer [35]. It has also been reported that TGF-b pro-
duced by stromal fibroblasts or gastric cancer cells sti-
mulates both the invasion and adhesion of scirrhous
gastric cancer cells to the peritoneum, resulting in an
increase in the potential for peritoneal dissemination
[36,37]. On the other hand, TGF-b is considered a
major factor that triggers epithelial-mesenchymal transi-
tion (EMT), which promotes invasion and metastasis
with acquiring fibroblastoid features and morphological
Figure 4 Combinatorial effects of VPA with PTX in vitro. The results are means ± SD of three different experiments.
Figure 5 Time courses of changes in protein levels, including acetyl-histone H3, cell cycle regulatory proteins (p21WAF1, p27 and
cyclin D1). OCUM-2MD3 cells were treated with 1 mM VPA, and cell lysates were harvested up to 48 h. Western blotting was performed using
a series of primary antibodies.
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changes [38-40]. Accordingly, TGF-b-induced EMT
could be a target for regulation of aggressiveness in gas-
tric cancer. These changes induced by TGF-b may work
better for formation of peritoneal dissemination. On the
other hand, HDAC4 plays an essential role in epigenetic
regulation of myofibroblastic differentiation by TGF-b
induction [41]. HDAC4 could be a target for interstitial
fibrosis involved in peritoneal dissemination. In addition,
VPA can also inhibit an activity of HDAC4 which is one
of class II HDACs [29]. Therefore, VPA has the poten-
tial to reduce fibrosis by inhibition of HDAC4. However,
further investigations are needed to confirm the effec-
tiveness of VPA on fibrosis.
We found that VPA increases acetylation of a-tubulin
as well as histone H3. Interestingly, tubulin acetylation
has a direct relation with HDAC6 inhibition induced by
the action of VPA [42,43]. HDAC inhibitors also play a
role as microtubule-associated deacetylases and cause
acetylation of lysine40 of a-tubulin [44,45]. Acetylation
of tubulin may contribute to the inhibition of tumor cell
growth in addition to the known effects caused by his-
tone acetylation. On the other hand, the mechanism of
tubulin acetylation by HDAC inhibitors could have a
favorable effect in combination with PTX [26,46], which
is a key drug in the treatment of gastric cancer. As PTX
is a taxane-based drug that interferes with mitosis and
Figure 6 Time courses of changes in apoptosis-related proteins. Cleaved caspase 3, caspase 9, survivin, bcl-2 and p53 were examined by
western blotting with a series of primary antibodies. Lysates were obtained from OCUM-2MD3 cells with exposure to 1 mM VPA up within 48 h
incubation.
Figure 7 Acetylation status of a-tubulin assessed by western blotting. Acetyl-a-tubulin level was increased after exposure to 1 mM VPA.
50 kDa: monomer; 100 kDa: dimer.
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cell replication by binding to a subunit of tubulins, PTX
has the potential to reduce fibrosis by inhibition of
TGF-b/Smad signaling [47-50]. It is noteworthy that the
inhibition of tumor cell proliferation can be achieved by
much higher dosages of PTX. In contrast, the inhibition
of TGF-b/Smad signaling can be attained with very low
doses of PTX [47]. Therefore, we suggest that VPA
enhances the anticancer action in combination with
PTX. However, further clinical studies are required to
determine the clinical applicability of the combination
treatment.
VPA is a safe drug with excellent bioavailability based on
long-term clinical experience in the treatment of epilepsy.
Recent clinical trials for various malignancies have shown
that the serum concentration of VPA, achieved during
therapy of epilepsy with a daily dose, acts as a potent inhi-
bitor of HDACs required for histone acetylation [51,52].
Biomonitoringof peripheralbloodlymphocytes
Figure 8 In vivo effects of VPA on the growth of tumor xenografts. The results are means ± SD of three different experiments.
Figure 9 Effects of VPA on the expression of p21WAF1 and cleaved caspase 3 in xenograft model. Immunohistochemical examination
showed that p21WAF1-positive cells (nuclear staining) were increased compared with the control group. Cleaved-caspase 3-positive cells were
observed as apoptotic cells characterized by cell shrinkage and nuclear fragmentation in the VPA-treated group. Original magnification ×400.
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demonstrated the induction of histone hyperacetylation in
the majority of patients and downregulation of HDAC2
[51]. In addition to the antitumor effect, VPA plays a vari-
ety roles as a mood-stabilizer and analgesic adjuvant for
patients in advanced stages of malignancies [53,54]. How-
ever, continuous oral treatment with VPA at high doses is
not feasible for patients with advanced stages of cancer
due to gastrointestinal disturbance [55,56]. Further devel-
opment of VPA as an HDAC inhibitor in patients with
gastric cancer requires careful consideration of the treat-
ment schedule and synergism with conventional che-
motherapy. Class I HDAC is overexpressed in gastric
cancer patients [57,58]. Both HDAC1 and HDAC2 play
important roles in the aggressiveness and carcinogenesis
of gastric cancer [59,60]. High levels of expression of class
I HDACs, especially HDAC2, are clinically associated with
nodal spread and prognosis of gastric cancer patients [61].
These relationships suggest that the level of class I HDAC
is a reliable maker of prognosis and a specific target for
VPA treatment. Moreover, the effect of VPA, which is a
class I- and class II- specific HDAC inhibitor, may depend
on the expression patterns of HDACs in tumor cells. The
availability of VPA in patients with gastric cancer may
depend on patient selection based on biological para-
meters, such as HDAC2 overexpression. Under pathologi-
cal conditions of peritoneal dissemination characterized by
fibrosis, HDAC4 also may be a target of VPA.
Conclusion
Our data suggested that VPA induces dynamic modula-
tion of histone and tubulin acetylation, in relation to the
anticancer effect and the enhancement of PTX. The
multifunctional effect of VPA provides insight into the
design of suitable drug combination therapies, including
microtubule targeting drugs. Therefore, the combination
of VPA and PTX is expected to be a promising regimen
in cases of peritoneal dissemination of gastric cancer.
Author details
1Department of Gastroenterologic Surgery, Division of Cancer Medicine,
Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8641,
Japan.2Center for Biomedical Research and Education, School of Medicine;
Kanazawa University, Ishikawa 920-8641, Japan.3Department of Surgical
Oncology, Graduate School of Medicine, Osaka City University, Osaka 545-
8585, Japan.
Authors’ contributions
YY carried out most of experiments, participated in the design of the study,
performed the statistical analysis and drafted the manuscript. SF, SH and JK
participated in the design of the study and helped to draft the manuscript.
IM, KO, HT and HF assisted the experiments. HT, IN, TF, TO, MY and KH
participated in its design and coordination. All authors read and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 29 September 2010 Accepted: 17 November 2010
Published: 17 November 2010
References
1.Souza RF, Spechler SJ: Concepts in the prevention of adenocarcinoma of
the distal esophagus and proximal stomach. CA cancer J Clin 2005,
55:334-51.
2. Ikeguchi M, Miyake T, Matsunaga T, et al: Recent results of therapy for
scirrhous gastric cancer. Surg Today 2009, 39:290-4.
3.Chen CY, Wu CW, Lo SS, Hsieh MC, Lui WY, Shen KH: Peritoneal
carcinomatosis and lymph node metastasis are prognostic indicators in
Figure 10 Effects of VPA on apoptosis in the xenograft model. Shrunken tumor cells showed positive reactivity in TUNEL assay. Apoptotic
index of the VPA-treated group was significantly higher than that of the control group. Original magnification ×400.
Yagi et al. Journal of Experimental & Clinical Cancer Research 2010, 29:149
http://www.jeccr.com/content/29/1/149
Page 9 of 11

Page 10

patients with Borrmann type IV gastric carcinoma. Hepatogastroenterology
2002, 49:874-7.
Ishigami H, Kitayama J, Kaisaki S, et al: Phase II study of weekly
intravenous and intraperitoneal paclitaxel combined with S-1 for
advanced gastric cancer with peritoneal metastasis. Ann Oncol 2010,
21:67-70.
Fushida S, Kinoshita J, Yagi Y, et al: Dual anti-cancer effects of weekly
intraperitoneal docetaxel in treatment of advanced gastric cancer
patients with peritoneal carcinomatosis: a feasibility and
pharmacokinetic study. Oncol Rep 2008, 19:1305-10.
Shah MA, Ramanathan RK, Ilson DH, et al: Multicenter phase II study of
irinotecan, cisplatin, and bevacizumab in patients with metastatic gastric
or gastroesophageal junction adenocarcinoma. J Clin Oncol 2006,
24:5201-6.
Pinto C, Di Fabio F, Siena S, et al: Phase II study of cetuximab in
combination with FOLFIRI in patients with untreated advanced gastric
or gastroesophageal junction adenocarcinoma (FOLCETUX study). Ann
Oncol 2007, 18:510-7.
Schniewind B, Christgen M, Kurdow R, et al: Resistance of pancreatic
cancer to gemcitabine treatment is dependent on mitochondria-
mediated apoptosis. Int J Cancer 2004, 109:182-8.
Fang JY, Lu YY: Effects of histone acetylation and DNA methylation on
p21 (WAF1) regulation. World J Gastroenterol 2002, 8:400-5.
Jenuwein T, Alli’s CD: Translating the histone code. Science 2001,
293:1074-80.
Davie JR, Moniwa M: Control of chromatin remodeling. Crit Rev Eukaryotic
Gene Expression 2000, 10:303-25.
Grozinger CM, Schreiber SL: Deacetylase enzymes: biological functions
and the use of small-molecule inhibitors. Chem Biol 2002, 9:3-16.
Gray SG, Ekström TJ: The human histone deacetylase family. Exp Cell Res
2001, 262:75-83.
Monneret C: Histone deacetylase inhibitors. Eur J Med Chem 2005, 40:1-13.
Carey N, La Thangue NB: Histone deacetylase inhibitors:gathering pace.
Curr Opin Pharmacol 2006, 6:369-75.
Suzuki T, Yokozaki H, Kuniyasu H, et al: Effect of Trichostatin A on cell
growth and expression of cell cycle-and apoptosis-related molecules in
human gastric and oral carcinoma cell lines. Int J Cancer 2000, 88:992-7.
Zhang X, Yashiro M, Ren J, et al: Histone deacetylase inhibitor, trichostatin
A, increases the chemosensitivity of anticancer drugs in gastric cancer
cell lines. Oncol Rep 2006, 16:563-8.
Sami S, Höti N, Xu HM, Shen Z, Huang X: Valproic acid inhibits the growth
of cervical cancer both in vitro and in vivo. J Biochem 2008, 144:357-62.
Kramer OH, Zhu P, Ostendorff HP, et al: The histone deacetylase inhibitor
valproic acid selectively induces proteasomal degradation of HDAC2.
EMBO J 2003, 22:3411-20.
Göttlicher M, Minucci S, Zhu P, et al: Valproic acid defines a novel class of
HDAC inhibitors inducing differentiation of transformed cells. EMBO J
2001, 20:6969-78.
Hrzenjak A, Moinfar F, Kremser ML, et al: Valproate inhibition of histone
deacetylase 2 affects differentiation and decreases proliferation of
endometrial stromal sarcoma cells. Mol Cancer Ther 2006, 5:2203-10.
Rocchi P, Tonelli R, Camerin C, et al: p21Waf1/Cip1 is a common target
induced by short-chain fatty acid HDAC inhibitors (valproic acid,
tributyrin and sodium butyrate) in neuroblastoma cells. Oncol Rep 2005,
13:1139-44,.
Takai N, Narahara H: Human endometrial and ovarian cancer cells:
histone deacetylase inhibitors exhibit antiproliferative activity, potently
induce cell cycle arrest, and stimulate apoptosis. Curr Med Chem 2007,
14:2548-53.
Yu X, Guo ZS, Marcu MG, et al: Modulation of p53, ErbB1, ErbB2, and Raf-
1 expression in lung cancer cells by depsipeptide FR901228. J Natl
Cancer Inst 2002, 94:504-13.
Blagosklonny MV, Robey R, Sackett DL, et al: Histone deacetylase inhibitors
all induce p21 but differentially cause tubulin acetylation, mitotic arrest,
and cytotoxicity. Mol Cancer Ther 2002, 1:37-41.
Catalano MG, Poli R, Pugliese M, Fortunati N, Boccuzzi G: Valproic acid
enhances tubulin acetylation and apoptotic activity of paclitaxel on
anaplastic thyroid cancer cell lines. Endocr Relat Cancer 2007, 14:839-45.
Gelmon K: The taxoids: paclitaxel and docetaxel. Lancet 344:1267-72.
Markman M, Bundy BN, Alberts DS, et al: Phase III trial of standard-dose
intravenous cisplatin plus paclitaxel versus moderately high-dose
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
carboplatin followed by intravenous paclitaxel and intraperitoneal
cisplatin in small-volume stage III ovarian carcinoma: an intergroup
study of the Gynecologic Oncology Group, Southwestern Oncology
Group, and Eastern Cooperative Oncology Group. J Clin Oncol 2001,
19:1001-7.
Gurvich N, Tsygankova OM, Meinkoth JL, Klein PS: Histone deacetylase is a
target of valproic acid-mediated cellular differentiation. Cancer Res 2004,
64:1079-86.
Johnson DG, Walker CL: Cyclins and cell cycle checkpoints. Annu Rev
Pharmacol Toxicol 1999, 39:295-312.
Joseph J, Wajapeyee N, Somasundaram K: Role of p53 status in
chemosensitivity determination of cancer cells against histone
deacetylase inhibitor sodium butyrate. Int J Cancer 2005, 115:11-8.
Kitazono M, Bates S, Fok P, Fojo T, Blagosklonny MV: The histone
deacetylase inhibitor FR901228 (desipeptide) restores expression and
function of pseudo-null p53. Cancer Biol Ther 2002, 1:665-8.
Yashiro M, Chung YS, Nishimura S, Inoue T, Sowa M: Fibrosis in the
peritoneum induced by scirrhous gastric cancer cells may act as ‘soil’ for
peritoneal dissemination. Cancer 1996, 77:1668-75.
Shinto O, Yashiro M, Kawajiri H, et al: Inhibitory effect of a TGFbeta
receptor type-I inhibitor, Ki26894, on invasiveness of scirrhous gastric
cancer cells. Br J Cancer 2010, 102:844-51.
Kinugasa S, Abe S, Tachibana M, et al: Over expression of transforming
growth factor-beta1 in scirrhous carcinoma of the stomach correlates
with decreased survival. Oncology 1998, 55:582-7.
Inoue T, Chung YS, Yashiro M, et al: Transforming growth factor-beta and
hepatocyte growth factor produced by gastric fibroblasts stimulate the
invasiveness of scirrhous gastric cancer cells. Jpn J Cancer Res 1997,
88:152-9.
Koyama T, Yashiro M, Inoue T, et al: TGF-beta1 secreted by gastric
fibroblasts up-regulates CD44 H expression and stimulates the
peritoneal metastatic ability of scirrhous gastric cancer cells. Int J Oncol
2000, 16:355-62.
Taylor MA, Parvani JG, Schiemann WP: The pathophysiology of epithelial-
mesenchymal transition induced by transforming growth factor-beta in
normal and malignant mammary epithelial cells. J Mammary Gland Biol
Neoplasia 2010, 15:169-90.
Miyazono K: Transforming growth factor-beta signaling in epithelial-
mesenchymal transition and progression of cancer. Proc Jpn Acad Ser B
Phys Biol Sci 2009, 85:314-23.
Gos M, Miłoszewska J, Przybyszewska M: Epithelial-mesenchymal transition
in cancer progression. Postepy Biochem 2009, 55:121-8.
Glenisson W, Castronovo V, Waltregny D: Histone deacetylase 4 is
required for TGFbeta1-induced myofibroblastic differentiation. Biochim
Biophys Acta 2007, 1773:1572-82.
Khan N, Jeffers M, Kumar S, et al: Determination of the class and isoform
selectivity of small-molecule histone deacetylase inhibitors. Biochem J
2008, 409:581-9.
Zhang Y, Li N, Caron C, et al: HDAC-6 interacts with and deacetylates
tubulin and microtubules in vivo. EMBO J 2003, 22:1168-79.
Blagosklonny MV, Robey R, Sackett DL, et al: Histone deacetylase inhibitors
all induce p21 but differentially cause tubulin acetylation, mitotic arrest,
and cytotoxicity. Mol Cancer Ther 2002, 1:937-41.
Hubbert C, Guardiola A, Shao R, et al: HDAC6 is a microtubule-associated
deacetylase. Nature 2002, 417:455-8.
Dowdy SC, Jiang S, Zhou XC, et al: Histone deacetylase inhibitors and
paclitaxel cause synergistic effects on apoptosis and microtubule
stabilization in papillary serous endometrial cancer cells. Mol Cancer Ther
2006, 5:2767-76.
Zhang D, Sun L, Xian W, et al: Low-dose paclitaxel ameliorates renal
fibrosis in rat UUO model by inhibition of TGF-beta/Smad activity. Lab
Invest 2010, 90:436-47.
Choi HS, Savard CE, Choi JW, Kuver R, Lee SP: Paclitaxel interrupts TGF-
beta1 signaling between gallbladder epithelial cells and myofibroblasts.
J Surg Res 2007, 141:183-91.
Liu X, Zhu S, Wang T, et al: Paclitaxel modulates TGFbeta signaling in
scleroderma skin grafts in immunodeficient mice. PLoS Med 2005, 2:
e354.
Guo W, Shan B, Klingsberg RC, Qin X, Lasky JA: Abrogation of TGF-beta1-
induced fibroblast-myofibroblast differentiation by histone deacetylase
inhibition. Am J Physiol Lung Cell Mol Physiol 2009, 297:L864-70.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
Yagi et al. Journal of Experimental & Clinical Cancer Research 2010, 29:149
http://www.jeccr.com/content/29/1/149
Page 10 of 11

Page 11

51.Atmaca A, Al-Batran SE, Maurer A, et al: Valproic acid (VPA) in patients
with refractory advanced cancer: a dose escalating phase I clinical trial.
Br J Cancer 2007, 97:177-82.
Rocca A, Minucci S, Tosti G, et al: A phase I-II study of the histone
deacetylase inhibitor valproic acid plus chemoimmunotherapy in
patients with advanced melanoma. Br J Cancer 2009, 100:28-36.
Hardy JR, Rees EA, Gwilliam B, Ling J, Broadley K, A’Hern R: A phase II study
to establish the efficacy and toxicity of sodium valproate in patients
with cancer-related neuropathic pain. J Pain Symptom Manage 2001,
21:204-9.
Rodriguez-Menendez V, Tremolizzo L, Cavaletti G: Targeting cancer and
neuropathy with histone deacetylase inhibitors: two birds with one
stone? Curr Cancer Drug Targets 2008, 8:266-74.
Braiteh F, Soriano AO, Garcia-Manero G, et al: Phase I study of epigenetic
modulation with 5-azacytidine and valproic acid in patients with
advanced cancers. Clin Cancer Res 2008, 14:6296-301.
Munster P, Marchion D, Bicaku E, et al: Clinical and biological effects of
valproic acid as a histone deacetylase inhibitor on tumor and surrogate
tissues: phase I/II trial of valproic acid and epirubicin/FEC. Clin Cancer Res
2009, 15:2488-96.
Nakagawa M, Oda Y, Eguchi T, et al: Expression profile of class I histone
deacetylases in human cancer tissues. Oncol Rep 2007, 18:769-74.
Weichert W, Röske A, Gekeler V, et al: Association of patterns of class I
histone deacetylase expression with patient prognosis in gastric cancer:
a retrospective analysis. Lancet Oncol 2008, 9:139-48.
Choi JH, Kwon HJ, Yoon BI, et al: Expression profile of histone deacetylase
1 in gastric cancer tissues. Jpn J Cancer Res 2001, 92:1300-4.
Song J, Noh JH, Lee JH, et al: Increased expression of histone deacetylase
2 is found in human gastric cancer. APMIS 2005, 113:264-8.
Weichert W, Röske A, Gekeler V, et al: Association of patterns of class I
histone deacetylase expression with patient prognosis in gastric cancer:
a retrospective analysis. Lancet Oncol 2008, 9:139-48.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
doi:10.1186/1756-9966-29-149
Cite this article as: Yagi et al.: Effects of valproic acid on the cell cycle
and apoptosis through acetylation of histone and tubulin in a scirrhous
gastric cancer cell line. Journal of Experimental & Clinical Cancer Research
2010 29:149.
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