ISSN 1916-6958

2010; Volume 1 (Number 1):
Pages 27-36

Differential effects of histone
deacetylase inhibitors on cell cycle regulation and apoptosis in human prostate
cancer cells

Hee Jin KIMa, Wahn Soo CHOIb,
Jaewon LEEa, Jung Yeol HANc, Hyung Sik KIMa,*

aCollege
of Pharmacy, Pusan National University, Busan, South Korea; bDepartment of Immunology, College of
Medicine, Konkuk University, Chungju-Si, South Korea; cThe Korean Motherisk Program,
Cheil Hospital & Women's Health-care Center, Kwandong University School of
Medicine, Seoul, Korea

*Corresponding author:
[email-masked]

ABSTRACT

Objectives: Histone deacetylase (HDAC) inhibitors may suppress the
proliferation of cancer cells and induce apoptosis. This study examined the
molecular mechanism of how HDAC inhibitors modulate the cell cycle regulators
and tumor suppressor genes in prostate cancer cells. Methods: HDAC inhibitors were evaluated according to the following
parameters and techniques: (1) cell
viability determined by a microculture tetrazolium assay (MTA); (2) induction
of apoptosis according to 4’-6-diamidine-2’-phenylindole dihydrochloride (DAPI)
staining; (3) the expression of cell cycle related proteins such as CDK2, CDK4,
cyclin D1, and cyclin E by Western blot analysis; (4) p16 mRNA expression by
RT-PCR; (5) the acetylation of the histone protein by nuclear protein
extraction; and (6) cell cycle analysis by flow cytometry. Results: The HDAC inhibitors decreased the level of cell
proliferation and induced apoptosis in human prostate cancer cell lines.
Trichostatin A (TSA) caused a significant decrease in cyclin D1/CDK4 expression
in LNCaP cells. Cell cycle analysis showed that the TSA treatment increased the
proportion of LNCaP cells in the G1 phase and decreased the proportion of cells
in the S phase. The TSA treatment resulted in the induction of apoptotic cell
death which was confirmed by DAPI staining. TSA increased the level of p16 and
WT1 expression in the androgen-dependent LNCaP or androgen-independent PC3 and
DU145 cells. Conclusions: HDAC
inhibitors may prevent the proliferation of prostate cancer cells by altering
the expression of the cell cycle regulators and tumor suppressor genes, which
might be associated with epigenetic regulation.

Key words

Apicidin; Apoptosis; Cell cycle regulator;
Histone deacetylase; Trichostatin A

RÉSUMÉ

Objectif: Les inhibiteurs de l’histone-désacétylase (HDCA)
peuvent réduire la prolifération de cellules cancéreuses et induire une
apoptose. Nous avons examiné dans cette étude les mécanismes moléculaires
impliqués par les inhibiteurs de l’HDAC dans la modulation du cycle cellulaire
et des gènes répresseurs des tumeurs dans les cellules du cancer de la
prostate. Méthodes: Les effets des
inhibiteurs de l’HDAC ont été étudiés par les techniques suivantes: (1) la
viabilité cellulaire par le TTM (test de tétrazolium sur microculture), (2)
l’induction de l’apoptose cellulaire par marquage avec le DAPI
(4’-6-diamidine-2’-phénylindole dihydrochlorure), (3) l’expression des
protéines liées au cycle cellulaire telles que les CDK2, CDK4, cycline D1 et
cycline E par immunotransfert, (4) l’expression de l’ARN messager p16 par
RT-PCR, (5) l’acétylation des histones par extraction des protéines nucléaires,
et (6) l’analyse du cycle cellulaire par cytométrie en flux. Résultats: Les inhibiteurs de l’HDAC
réduisent la prolifération et induisent l’apoptose des cellules du cancer de la
prostate. La trichostatine A (TSA) produit une réduction significative de
l’expression de cycline D1/CDK4 dans les cellules LNCaP. L’analyse du cycle
cellulaire montre une proportion augmentée de cellules LNCaP en phase G1 et une
diminution des cellules en phase S suite an traitement par la TSA. Ce
traitement induit la mort cellulaire par apoptose confirmée par marquage par le
DAPI. La TSA augmente le taux de p16 et l’expression de WT1 tant dans les cellules
androgéno-dépendantes LNCaP que dans les cellules androgéno-indépendantes PC3
et DU145. Conclusions: Les
inhibiteurs de l’HDAC peuvent prévenir la prolifération des cellules du cancer
de la prostate par la modulation du cycle cellulaire et des gènes répresseurs
des tumeurs vraisemblablement par régulation épigénétique.

Mots clés

Apicidine;
Apoptose; Régulateur du cycle cellulaire; Histone-  déacétylase; Trichostatine A

RESUMEN

Objetivos: Los inhibidores de la histona desacetilasa
(HDAC) suprimen la proliferación de las células cancerígenas e inducen la
apoptosis. Este estudio examinó los mecanismos moleculares de los reguladores
del ciclo celular y los genes supresores tumorales por los inhibidores de la
HDAC, en las células prostáticas cancerígenas. Métodos: Para evaluar el efecto de los inhibidores de HDCA,
nosotros estudiamos (1) la viabilidad celular determinada mediante la prueba de
microcultivo con tetrazolium; (2) la inducción de la apoptosis de acuerdo a la
tinción de cloruro de 4’-6-diamino-2’-fenilindol (DAFI); (3) la expresión de
CDK2, CDK4, ciclina D1, y ciclina E, mediante análisis de Western blot; (4) la
expresión de p16 RNA mediante RT-PCR; (5) la acetilación de la histona mediante
extracción proteica nuclear; y (6) el ciclo celular mediante citometría de
flujo. Resultados: Los inhibidores
de la HDAC disminuyeron los niveles de proliferación celular e indujeron la
apoptosis en las células de cáncer de próstata. La tricostatina A (TSA)
disminuyó significativamente la expresión de ciclina D1/CDK4 en las células
LNCaP. El tratamiento con TSA incrementó la proporción de células LNCaP en la
fase G1, disminuyó la proporción de células en la fase S del ciclo celular, y
resultó en la inducción de la muerte celular por apoptosis confirmada mediante
la tinción de DAFI. La TSA incrementó los niveles de p16 y la expresión de WT1
en las células dependientes de andrógenos LNCaP así como en las células
independientes de andrógenos PC3 y DU145. Conclusiones:
Los inhibidores de la HDAC pueden prevenir la proliferación de las células del
cáncer de próstata mediante la alteración de la expresión de los reguladores
del ciclo celular y de los genes supresores tumorales, los cuales podrían estar
asociados con regulaciones epigenéticas.

Palabras clave

Apicidina;
Apoptosis; Reguladores del ciclo celular; Histona deacetilasa; Trichostatina A

INTRODUCTION

Prostate cancer is one of the most common
malignancies and the second leading cause of death among men in the United
States [1] [2]. Although the incidence of prostate cancer in Korea
is still much lower than in Western populations, it has increased rapidly over
the past 10 years [3]. Epidemiological studies have shown that a high
incidence of prostate cancer may be related to dietary factors. Excessive
consumption of polyunsaturated fats has been associated with prostate cancer in
humans [4]. Recent studies suggest that additional factors,
including environmental agents (endocrine disrupting chemicals), aging, and
oxidative stress are causally related to the development of prostate cancer [5]. In the initial stages, prostate cancer cells depend
on androgens for their survival. Therefore, the main form of systemic treatment
is androgen-ablation [6]. However, within a few years, most patients progress
to an androgen-refractory state, which is capable of growing without androgens [7]. Hence, there is a need for alternative therapeutic
strategies for the treatment of hormone-refractory prostate cancer with several
chemotherapy trials being carried out for this purpose [8].

DNA methyltransferases (DNMTs) and HDACs are
potential targets for regulating these epigenetic changes. DNA methylation and
histone modification, which are regulated by several classes of enzymes, play
an important role in the epigenetic mechanisms of gene regulation in various
tumor initiation and progression stages. Epigenetic changes are defined as
heritable changes in gene expression that occur without any changes in DNA
sequence. The importance of an epigenetic alteration to cancer has probably
been understated, since there has been more than ten years of remarkable
advances in the knowledge of the mutational events that lead to the activation
of protooncogenes or the inactivation of tumor suppressor genes [9]. Therefore, DNMT and HDAC inhibitors are believed to be
one of the most promising classes of new anticancer agents in current clinical
trials.

Recently, either naturally derived or
synthetic compounds exhibiting HDAC inhibitory activity were shown to have
anti-proliferative, pro-differentiating, and pro-apoptotic properties in a
variety of cancer cells [10] [11] [12] [13]. TSA, which was originally developed as an antifungal
agent, is a potent and reversible HDAC inhibitor. TSA has HDAC inhibitory
effect at low concentrations; it targets the cell cycle progression of several
cell types, induces cell growth arrest at both the G1 and G2/M phases, and in
some cases, induces apoptosis [14] [15].

Apicidin
[cyclo(N-O-methyl-L-tryptophanyl-L-iso
leucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)] is a fungal metabolite shown
to inhibit both mammalian and protozoan HDACs. It has also been reported to
inhibit the proliferation of variety of tumor cells and selectively up-regulate
the expression of p21WAF/Cip1, which allows tumor cells to undergo cell cycle
arrest at the G0/G1 phase and cause morphological changes [16] [17]. However, the precise mode of actions of these agents
in prostate cancer cells is unclear.

Therefore, this study examined the molecular
mechanism for the epigenetic alterations of the cell cycle regulator proteins
and tumor suppressor genes in prostate cancer cells by DNMT and HDAC
inhibitors.

METHODS

Materials

The TSA and apicidin were obtained from Sigma
Chemical Co. (St. Louis, MO, USA). The medium and its supplements containing
antibiotics, fetal bovine serum was obtained from Gibco Invitrogen Corporation
(CA, USA). Western blot detection reagents were obtained from Amersham
Bioscience Corporation (Bucks, United Kingdom). The polyvinylidene difluoride
(PVDF) membranes were obtained from Bio-Rad (CA, USA). The RT-PCR reagents were
supplied by Invitrogen Corporation. All other chemical reagents were purchased
from Sigma Chemical Co. (St. Louis, MO, USA).

Cell lines and culture media

The LNCaP, PC3 and DU145 cells, which are
human prostate cancer cells, were obtained from ATCC (American Type Culture
Collection, Rockville, MD, USA). The cells were grown in Minimum Essential
Medium (MEM), pH 7.2, containing 2.2 g L-1 NaHCO3, 1 mM
sodium pyruvate, 100 U mL-1 antibiotics, and 10% heat-inactivated
fetal bovine serum (FBS). The cells were maintained at 37oC in a
humidified atmosphere containing 5% CO2. The cells were plated on
plastic dishes. The culture medium was replaced every 2 days. Apicidin and TSA
were dissolved in dimethyl sulfoxide (DMSO) and absolute ethanol, respectively.
These agents were diluted to the appropriate concentrations with the culture
medium containing 5% charcoal-dextran treated FBS (CD-FBS). The final concentration
of DMSO or ethanol was < 0.1% (vol vol-1).

Microculture tetrazolium assay

The cell viability was determined using an
MTA. The cultures were initiated in 96 well plates at a density of 2500 cells
per well. Cells were allowed to reattach for 48 hr and then exposed to the test
compounds. At the end of the treatment period, 15 mL of a 5 mg mL-1 MTT reagent [3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide in PBS] was added to each well.
The plates were incubated again for 4 h at 37oC in the dark. The
supernatant was aspirated, and formazan crystals were dissolved in 100 mL DMSO at 37oC for 10 min with gentle agitation. The
absorbance per well was measured at 540 nm using a VersaMax Microplate Reader
(Molecular Devices Corp, CA, USA).

Nuclear protein preparation

Nuclear protein from the cells was prepared
using the following procedure. All solutions, tubes, and centrifuges were
maintained at 4oC. The cells were harvested by tripsinization,
washed with cold PBS, centrifuged at 3,000 rpm for 5 min, and incubated in 50 mL of lysis buffer I (10 mM HEPES, pH 7.9, 1.5 mM MgCl2,
10 mM KCl, 0.5 mM DTT and 0.5 mM PMSF) for 20 min. After centrifugation at
12,000 rpm for 10 min, supernatant was removed and the pellet was re-suspended
in 30 mL of a lysis
buffer II (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT,
0.5 mM PMSF and 0.05% NP-40) for 20 min. After centrifugation at 12,000 rpm for
10 min, the pellet was re-suspended in 40 mL of lysis buffer III (5 mM HEPES, pH 7.9, 300 mM NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF and 26% glycerol) for 30 min. After the
final centrifugation at 12,000 rpm for 30 min, the protein concentration was
quantified using a Bio-Rad protein assay reagent.

Western blot analysis

The cells were harvested and washed twice
with PBS at 4oC. The total proteins were prepared using a PRO-PREP
protein extract solution (Intron, Seongnam, Korea), and quantified using the
protein assay reagent (Bio-Rad). Fifty microgram of the proteins were denatured
by boiling at 96oC for 5 min in a sample buffer (0.5 M Tris-HCl, pH
6.8, 4% SDS, 20% glycerol, 0.1% bromphenol blue, 10% β-mercaptoethanol) at
1:1 ratio. Each sample was separated using a 10-15% polyacrylamide gel
(SDS-PAGE), and transferred to a PVDF membrane at 100 V for 1 h in a transfer
buffer. The membrane was blocked using a blocking buffer containing 5% non-fat
dry milk in a TNT buffer (10 mM Tris-Cl, pH 7.6, 100 mM NaCl and 0.5% Tween 20)
at room temperature for 1 h. The membrane was then incubated overnight with the
primary antibodies specific to CDK4, cyclin D1, CDK2, cyclin E, Rb, p-Rb, WT1
(Santa Cruz Biotechnology Inc., CA, USA), and Ac-H3 (Upstate Biotechnology) at
4oC.

After washing for 1 hr with TNT buffer, the
membrane was incubated with horseradish peroxidase-conjugated anti-mouse
or anti-rabbit antibodies (Santa Cruz Biotechnology Inc., 1:10,000) at room
temperature for 30 min, and then washed for 60 min with the TNT buffer. Antibody
labeling was detected using ECL plus Western blotting detection reagents
(Amersham Bioscience Corporation).

Assay of RT-PCR

The total RNA was extracted using Trizol
Reagent (Gibco). cDNA was generated using the Superscript II reverse
transcriptase (Invitrogen) according to the manufacturer’s protocol. cDNA was
synthesized using a reverse transcription method. Briefly, 2 mg of the total RNA was reverse transcribed for 50 min at 42oC
in a 20 mL reaction
mixture (containing 1 mL of
oligo primer (0.5 g), 2 mL of 10
mM dNTP mix, 2 mL of 10x RT
buffer, 4 mL of 25 mM MgCl2,
2 mL of 0.1 M DTT, 40 U of RNase Out inhibitor, and 100 U of
Superscript II), followed by denaturation at 70oC for 15 min. RNase
inhibitor (2 U) was then added and incubated at 37oC for 20 min.
cDNA was used for the subsequent PCR reaction, 2 mL of cDNA was amplified in a 25 mL reaction mixture containing 2.5 mL of 10x PCR buffer, 0.75 mL of 50
mM MgCl2, 0.5 mL of 10
mM dNTP mix, 1 U Taq polymerase, and 0.25 mL of sense and anti-sense primer (20 mM).

The primers were p16 (F; 5’-CAACGCACCGAATAGT
TACG-3’, R; 5’-ACCAGCGTGTC CAGGAA-3’) and b-actin (F; 5’-CCTCGCCTTTGCCG
ATCC-3’, R; 5’-GGATCTTCATGA GGTAG TCAGTC-3’). The reaction was initiated at 94oC
for 5 min and PCR was performed using a variable number of cycles of
amplification defined as follows: denaturation at 94oC for 45 sec,
annealing at 55oC (p16, 177 bp) or 60oC (b-actin, 626 bp)
for 45 sec and extension at 72oC for 45 sec. Electrophoresis was
performed in 2% agarose gel containing 1 μg of ethidium bromide (EtBr).
The molecular sizes of the amplified products were determined by comparison
with the molecular weight markers, a 100 bp DNA ladder (iNtRON) run in parallel
with the RT-PCR products.

DAPI staining

Morphological changes in the nuclear chromatin
of cells undergoing apoptosis were detected by staining with the DNA binding
fluorochrome DAPI. The cells were grown in 6 well plate at a density of 4×104
cells per well. After treatment, cells were fixed in absolute methanol, and
stained with 300 mL of DAPI
solution

(1 g mL-1) at 37oC for
15 min. After removing the staining solution, the cells were examined by
fluorescence microscopy (Axiovert 200, ZEISS Inc., Germany).

Flow cytometry analysis

Cells were harvested and washed in 1% BSA before
being fixed in 75% ethanol containing 0.5% Tween 20 for at least 1 h at 4oC.
The cells were washed in 1% BSA and re-suspended in a cold propidium iodine
(PI) staining solution (100 g mL-1 RNase and 10 g mL-1 PI
in PBS) 1 mL for 40 min at 4oC. Data acquisition and analysis was
carried out using a flow cytometry system (Becton Dickinson, CA, USA).

Statistical analysis

The results are reported as the means ± S.E.
of three independent triplicate measurements. Statistically significant
differences between untreated control and treated groups were determined using
one-way analysis of variance (ANOVA).

RESULTS

The antiproliferative effect of TSA and
apicidin on human prostate cancer cells was determined using a MTT assay and
the results are shown in Figure 1. The prostate cancer
cell lines (LNCaP, PC3, and DU145 cells) were treated with either TSA or
apicidin at the consecutive serial concentrations. TSA and apicidin inhibited
the proliferation of LNCaP and DU145 cells in a concentration-dependent manner
and significantly decreased the level of cell proliferation after a 48 h
treatment at the lowest concentration of 50 nM. In contrast, the PC3 cells were
resistant under these conditions (Figure 1, plots A and B).

The effect of apicidin on the level of
histone acetylation was then analyzed by Western blotting using the specific
antibody to acetylated histone H3. Treatment of the prostate cancer cells with
TSA (10 nM) and apicidin (10 nM) for 12 h resulted in a significant increase in
acetylated H3 levels (Figure 2).

In order to determine if the decrease in cell
viability was related to apoptosis, the cells were treated with 50 nM TSA and
50 nM apicidin for different times. The cells were then stained with DAPI,
which enters the cells and binds to the fragmented DNA. The nuclear changes
were observed by fluorescence microscopy. TSA and apicidin markedly increased
the number of apoptotic bodies after a 24 h treatment (Figure
3). Whole cell protein extracts were made from each cell line after
apicidin and TSA treatment for 24 h in order to determine if the cell death
observed in prostate cancer cell lines related to cell cycle regulation. In the
LNCaP cells treated with TSA at 100 nM, there was a significant decrease in
cyclin D1 and CDK4 expression (Figure 4A). When
treated with 10, 50, and 100 nM TSA for 24 h, there was a significant decrease
in cyclin D1, CDK4, cyclin E, and CDK2 expression in the LNCaP cells but not in
the PC3 and DU145 cells (Figure 4B). However, the apicidin
treatment had no effect on cyclin E and CDK2 expression (which act in the G1
late stage) in the LNCaP, PC3 and DU145 cells (Figure 4A).

Figure 1

Growth
inhibition of human prostate cancer cell lines by trichostatin A and
apicidin.

The
cells were treated with different concentrations of trichostatin A (TSA) and
apicidin for 48 h and the cell viability was determined by a MTT assay. The
error bars show a S.D. *P < 0.005, **P <
0.001 as determined by a Student’s t test, difference between the treated and
the control group.

Figure 2

Accumulation
of acetylated histone proteins by treatment with the HDAC inhibitors.

The
cells were treated with 10 nM trichostatin A (TSA) and apicidin for 12 h. The
acetylated histone tails were determined by western blot analysis using
anti-acetylated H3 antibodies.

Figure 3

Apoptosis induction in prostate cancer cell lines
by trichostatin
A and apicidin treatment.

The induction of apoptosis
by trichostatin A (TSA) and apicidin in LNCaP, PC-3, and DU-145 was analyzed
by fluorescence microscopy after nuclear staining with DAPI. The cells were
incubated with or without TSA (50 nM) and apicidin (50 nM) for 24 hr. The
cells undergoing apoptosis and nuclear fragmentation are identified with
arrows.

The
magnification is 400X.

The tumor suppressor, Wilm’s tumor suppressor
gene (WT1), is essential for normal kidney and gonadal development and
regulates cell differentiation and apoptosis in vitro. The expression of WT1 was examined to determine the
effect of TSA on prostate cancer cells. The results showed that the expression
of WT1 was induced dose-dependently in androgen-independent PC3 and DU145
cells. In contrast, the androgen-dependent LNCaP cells did not express a
detectable level of WT1 (Figure 4B). In general, the
p16 gene regulates progression through the G1/S phase of the cell cycle by
binding CDK 4/6, which limits the formation of the CDK 4/6-cyclin D complex.
The mRNA levels of the tumor suppressor gene, p16, were measured by RT-PCR to
determine if the prostate cancer cell death, when treated with TSA, is related
to p16 expression. The levels of p16 mRNA in LNCaP cells increased after TSA
treatment in a dose-dependent manner, whereas there was no effect observed in
the androgen-independent PC3 and DU145 cells (Figure 5,
plots A and B). However, apicidin did not affect the levels of p16
expression in the LNCaP cells (Figure 5C).

Finally, flow cytometry analysis was used to
examine the cell cycle progression of prostate cancer cells treated with TSA or
apicidin for 24 h. TSA induced cell cycle arrest in the G1 phase in the LNCaP
and DU145 cells. However, this was not observed in PC3 cells.In PC3 cells, the
cell cycle arrest in the G1 phase was induced only after 24 h of treatment (Figure 6A). On the other hand, 100 nM of apicidin for 24
h induced cell cycle arrest in the G1 phase in PC3 cells only. In contrast,
apicidin decreased the proportion of LNCaP and DU145 cells in the S phase and
increased the proportion of cells in the G2 phase (Figure
6B). Finally, flow cytometry analysis was used to examine the cell cycle
progression of prostate cancer cells treated with TSA or apicidin for 24 h. TSA
induced cell cycle arrest in the G1 phase in the LNCaP and DU145 cells.
However, this was not observed in PC3 cells. In PC3 cells, the cell cycle arrest
in the G1 phase was induced only after 24 h of treatment (Figure
6A). On the other hand, 100 nM of apicidin for 24 h induced cell cycle
arrest in the G1 phase in PC3 cells only. In contrast, apicidin decreased the
proportion of LNCaP and DU145 cells in the S phase and increased the proportion
of cells in the G2 phase (Figure 6B).

Figure 4

Effect
of the HDAC inhibitors on cell cycle regulators.

The
human prostate cancer cells were treated with 10, 50 and 100 nM trichostatin
A (TSA) and apicidin (100 nM) for 24 hr. The proteins were isolated from
LNCaP, PC-3, and DU145 cells and lysates (50 μg) of the cells were
examined by 10% SDS-PAGE and analyzed by immunoblotting using specific antibodies.

Figure 5

Effect
of HDAC inhibitors on tumor suppressor gene p16 expression in prostate cancer
cells.

The
cells were treated with the indicated concentrations of trichostatin A (TSA) and
apicidin for 48 h. Total RNA was isolated, and RT-PCR was performed using the
specific primers described in the Materials and Methods section. Actin was
used as a housekeeping control gene.

Figure 6

Effect of apicidin on cell cycle distribution.
Human prostate cancer cells were treated with
trichostatin A (TSA) (A) and apicidin (B) at the indicated concentrations for
24 h. The cells were fixed, permeabilized and stained with propidium iodide
to measure DNA content by fluorescence-activated cell sorting (FACS)
analysis. The percentages of cells in each phase of the cell cycle (G1, S and
G2) are indicated.

DISCUSSION

This study examined whether or not HDAC
inhibitors induced apoptotic cell death and cell cycle arrest in various human
prostate cancer cell lines.  It was demonstrated that the inhibition of
histone deacetylation leads to cell death, which is associated with nuclear
chromatin condensation and cell cycle arrest in the G1 or G2 phase [18] [19] [20]. In this study, TSA suppressed the growth of prostate
cancer cells in a dose-dependent manner. These effects were related to
epigenetic regulation of the specific genes associated with cell cycle
regulation. In particular, TSA exhibited strong anticancer efficacy against
cultured human androgen-dependent and -independent prostate cancer cells.
Additionally, TSA inhibited cell growth and induced apoptosis and cell cycle arrest,
induced a decrease in cyclin D1 and CDK4 expression in LNCaP cells, and
restored the levels of p16 expression in prostate cancer cells. The p16 gene is
localized to chromosome 9p21 and encodes the CDK inhibitor. This protein
regulates cell cycle progression through the G1/S by binding CDK 4/6, which
limits the formation of the CDK 4/6-cyclin D complex and the phosphorylation of
Rb [21]. Recently, aberrant DNA methylation of the 5'
promoter region, an epigenetic event, was associated with the transcriptional
silencing of p16 expression in several cancer cell lines including lung,
bladder, colon, and prostate cancers [22] [23] [24]. Based on these results, the level of p16 expression
was measured to determine if its expression is related to the cell cycle regulation
of prostate cancer cells treated with TSA. The results showed that p16
expression was induced in androgen-dependent LNCaP cells in a dose-dependent
manner, whereas there was no expression observed in the androgen-independent PC3
and DU145 cells. However, apicidin did not affect the expression of p16 in
LNCaP cells. Therefore, TSA can restore the expression of p16 by inducing the
relaxed chromatin conformation in androgen-dependent prostate cancer cells.
This restored p16 expression induces prostate cancer cell death by inhibiting
the cell cycle regulators, cyclin D1 and CDK4 in G1 phase of the cell cycle.

Another tumor suppressor gene, WT1 is
essential for normal kidney and gonadal development and regulates cell
differentiation and apoptosis in vitro [25] [26]. WT1 represses the androgen-signaling pathway.
Moreover, WT1 expression in prostate cancer cell lines is limited to those cell
lines lacking functional AR [27] [28]. PC3 and DU145 cells express high levels of WT1 mRNA
and protein, whereas the androgen-dependent LNCaP cell did not express WT1 [29]. Furthermore, WT1 modulates the expression or
activity of apoptotic genes, such as the prostate apoptosis response gene,
par-4 [30] [31] or bcl-2 [32] [33]. In this study, the WT1 protein level was measured to
determine the anticancer effect of TSA on human prostate cancer cells. The
results showed that expression of WT1 was induced in androgen-independent PC3
and DU145 cells in a dose-dependent manner, whereas androgen-dependent LNCaP
cells did not express detectable levels of WT1. Therefore, the HDAC inhibitor
TSA would restore WT1 expression by inducing a relaxed chromatin conformation
in androgen-independent prostate cancer cells. In addition, the intracellular
level of acetylated histone H3 in prostate cancer cell lines induced by HDAC
inhibitors was measured. Low levels of histone acetylation were observed in the
absence of TSA, whereas hyperacetylated H3 was observed in all prostate cancer
cells tested. This suggests that the induction of histone hyperacetylation by
TSA due to the inhibition of HDAC and the activation of the transcriptional
factors for WT1 gene expression.

TSA was found to dramatically induce cell
cycle arrest in the G1 phase and increase the level of apoptosis in LNCaP
cells. The transition from the G1 to S phase is believed to be triggered mainly
by CDK4 after binding to its regulatory subunit, cyclin D1 [27] [28]. In general, G1 arrest depends on the ability of p16
to activate the transcription of specific genes, while G2/M arrest involves the
repression of the transcription of CDK1/cyclin B1 complex [30]. This shows that TSA decreases the expression of
cyclin B1 and CDK1 at both the mRNA and protein levels, and apicidin also increased
the expression p21 and p27, which play a key role in the cell cycle arrest at
the G2/M phase and apoptosis. In conclusion, HDAC inhibitors suppress the
proliferation of prostate cancer cells by altering the expression of cell cycle
regulators and inducing apoptosis. In particular, TSA may have different
mechanism of action in either androgen-dependent or androgen-independent
prostate cancer cells by altering the tumor suppressor genes, which might be
associated with the epigenetic regulations.

AUTHORS’ PARTICIPATION

HS Kim and HJ Kim conceived of and designed
the study. WS Choi and JY Han analyzed and interpreted the data. H.J. Kim
drafted the paper and J Lee and JY Han revised it for important intellectual
content. All authors gave final approval of the version to be published.

ACKNOWLEDGEMENTS

This study is supported by the grants for the
Korea Research Foundation (KRF-2007-313-E00622) funded by Korean Government. HJ
Kim is supported by a grant from the post Brain Korea 21 project.

CONFLICT OF INTERESTS/DISCLAIMERS

HS Kim is member of the Editorial Board of
the journal.

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