Histone Deacetylase Inhibitor, Valproic Acid has an Anti

Histone Deacetylase Inhibitor, Valproic Acid has an Anti-Proliferative Effect in Synovial Sarcoma
+1Yoshida, A; 1Sato, C; 1Morimoto, Y; 1Kunisada, T; 1Ozaki, T
+1Okayama University Graduate School, Okayama, Japan
Senior author [email protected]
on SYO-1 cell (data not shown). VPA treatment induced acetyl-histone
H3 and apoptosis in SYO-1 cells (Fig.1).
INTRODUCTION:
Histone Deacetylases (HDACs) are a critical family of proteins
In vivo: Tumor growth suppression of VPA in SYO-1 cells
involved in the transcriptional regulation of a large number of genes, and
Mean serum concentration of VPA of high dose and low dose
in the functional regulation of multiple proteins1) . HDAC inhibitors
group were 43.4±16.3µg/ml and 19.1±4.7µg/ml, respectively.
(HDACI) play multifunctional roles in a carcinogenesis, such as
The increase in the size of tumor xenograft of high dose group
upregulating tumor suppressor gene expression, reducing tumor growth,
was significantly inhibited compared with that of low dose group on Day
inhibiting angiogenesis and inducing apoptosis and cellular
25 (p<0.01) (Fig.2). There was not statically difference of the change of
differentiation.2) Our previous study demonstrated that HDACI
weight among all groups.
significantly suppressed the growth of synovial sarcoma cells. 3)
The histological analysis showed positive TUNEL staining and
Valproic acid (VPA) is one of drugs for epileptic seizures and
acetyl-histone H3 staining after the administration of VPA, indicating
bipolar disorder, and recently has been reported to inhibit HDAC.
that VPA can induce apoptosis, and activation of histone acetylation.
According to clinical trials of epigenetic therapy, VPA has a potential
The areas of tumor necrosis rate of the control group, VPA of low dose
antitumor effect in human cancer 4), but the effect of VPA on
and high dose groups were 45.7%, 47.6%, and 53.6% respectively (n.s).
musculoskeletal sarcoma remains unclear.
(Fig.3).
The aim of this study was to assess in vitro and in vivo effect of
VPA on the musculoskeletal sarcoma cells.
Fig.1
Fig.2
MATERIALS AND METHODS:
In vitro
Cell culture and reagents: We used four cell lines, such as synovial
sarcoma (SYO-1), chondrosarcoma (OUMS-27), malignant peripheral
nerve sheath tumor (FUSFT9817 and FUSFT8611). These four cell lines
were maintained in DMEM supplemented with 10% fetal bovine
serum.VPA was kindly supplied by Kyowa Hakko Kogyo Co., LTD.
Detection of proliferation: Four cell lines were seeded in 96-well
plates ,these cell lines were cultured with the indicated doses of VPA
(0.001, 0.1, 1 and 10 mM). After cultured with VPA for 72 hours, a
Fig.3 Histological analysis of SYO-1 xenograft model
growth inhibition was evaluated using WST-1 assay (Roche), and a
cytotoxicity was detected using LDH assay (Roche).
Immunocytochemistry: SYO-1 cell was cultured in 8-well culture slide
Acetyl-histone H3
in the medium with 2mM VPA for 24 hours and fixed in 1% PFA.
positive staining
Apoptosis was assessed using TUNEL staining, and histone acetylation
was measured as
was demonstrated to acetyl-histone H3 (Cell signaling) staining.
follows, +++ diffuse
In vivo
positive signals in
Tumor growth and treatment in nude mice: 1x107 of SYO-1 cells were
<80% of the tumor
injected into the left flank of 5-week-old BALB/c nu/nu mice. After the
cells; ++ positive
tumor was more than 5mm in diameter, twelve mice were divided to
signals in <50%
three groups: oral administration of vehicle only (control group),
tumor cells; combination of VPA gavage (200mg/kg) by daily with VPA in drinking
negative.
water (0.1%) (low dose group), and combination of VPA gavage by
daily (400mg/kg) with VPA in drinking water (0.4%) (high dose group)
(Table.1). Tumor volumes were calculated, and the body weight of mice
was measured every three days. These mice were scarified on Day 25 of
the treatment by CO2 overdose.
The plasma level of VPA in nude mice: When these mice were sacrificed,
blood was collected from these treatment mice by cardiac puncture, and
the plasma level of VPA was examined.
Histologic analysis: Tumor xenografts were resected, fixed in 4% PFA
and embedded in paraffin. Serial sections were stained with hematoxylin
and eosin, TUNEL staining and acetyl-histone H3 (Cell signaling)
staining. Tumor area and necrosis rate was evaluated using Image J
software.
Statistical analysis: To test for significant differences in animal models,
histological analysis and to determine the differences between all pairs
of groups in vitro, Students t-test was applied.
Table.1 Treatment group (n=4 each)
Group
VPA gavage (mg/kg) VPA in Drinking water( %)
Control
0
0
Low
200
0.1
High
400
0.4
DISCUSSION:
The present study demonstrated that VPA had a growth inhibition
on synovial sarcoma cell. The function of HDAC inhibitors remain
unclear, however our previous study reported that HDAC had an
important role of the cell growth induction in synovial sarcoma3).
The inhibition of HDAC by VPA increased the levels of acetylated
H3 and enhanced apoptosis. VPA can activate histone acetylation, and
can induce apoptotic cell death in vitro and in vivo. The effective serum
concentration of VPA in human epilepsy patients was 50-100µg/ml. Our
results showed that mean concentration of VPA of high dose group was
43.4±16.3µg/ml, indication that high dose treatment could inhibit the
tumor growth compared with low dose group that demonstrated mean
concentration of 19.1±4.7µg/ml. These findings suggested that apoptosis
might be influenced by the amount of acetylated histone H3.
As clinical trials for leukemia or MDS reported the safety of oral VPA
treatment,5)6) oral VPA may be a potential chemotherapeutic agent
through targeting of HDAC in Synovial Sarcoma.
RESULTS:
In vitro: Anti-proliferative effect of VPA in human musculoskeletal
tumor cell lines
IC50 of VPA treated cell lines was SYO-1, OUMS-27,
FUSFT9817, and FUSFT8611 were 2.8, >10, 4.2, and >10mM,
respectively. SYO-1cell had the highest suppression effect among them.
VPA showed dose-dependent anti-proliferative effect and cytotoxicity
REFERENCES:
1) Mariadason J. (2008) Epigenetics. 3: 28-37
2) Liu T, et al. (2006 ) Cancer Treat Rev. 32:157-165
3) Ito T, et al. (2005) Cancer Lett. 224, 311- 319.
4) Kuendgen A, et al. (2005) Ann Hematol. 84, S61-6
5) Bug G, et al. (2005) Cancer. 104, 2717-25
6) Duenas-Gonzalez A, et al. (2008) Cancer Treat Rev. 34:206-22.
Poster No. 1584 • 55th Annual Meeting of the Orthopaedic Research Society

Download Report