Histone deacetylase inhibitors trichostatin A and valproic acid induce cell cycle arrest and p21 expression in immortalized human endometrial stromal cells

Article (PDF Available)inEuropean Journal of Obstetrics & Gynecology and Reproductive Biology 137(2):198-203 · May 2008with47 Reads
DOI: 10.1016/j.ejogrb.2007.02.014 · Source: PubMed
Abstract
Following our observation that histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and valproic acid (VPA) can suppress proliferation of endometrial stromal cells, we sought to determine whether TSA and VPA do so by inducing cell cycle arrest and p21 expression. A recently established immortalized endometrial stromal cell line was treated with TSA, VPA, and/or all-trans retinoic acid (ATRA) and the consequent cell cycle progression was measured by flow cytometry and p21 protein expression by Western blot analysis. Both TSA and VPA induced cell cycle arrest and p21 expression in a concentration-dependent manner. Treatment with ATRA alone also induced cell cycle arrest and moderate increase in p21 expression but joint treatment of ATRA and TSA/VPA did not further enhance cell cycle arrest as compared with TSA/VPA treatment alone. HDACIs suppress proliferation of endometrial stromal cells through induction of cell cycle arrest and possibly also through apoptosis as well. RA also induces cell cycle arrest but it does not synergize with HDACIs in inducing cell cycle arrest. HDACIs may be promising compounds for treating endometriosis.
Histone deacetylase inhibitors trichostatin A and valproic acid
induce cell cycle arrest and p21 expression in immortalized
human endometrial stromal cells
Yan Wu, Sun-Wei Guo
*
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, United States
Received 3 October 2006; received in revised form 25 January 2007; accepted 18 February 2007
Abstract
Objective: Following our observation that histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and valproic acid (VPA) can
suppress proliferation of endometrial stromal cells, we sought to determine whether TSA and VPA do so by inducing cell cycle arrest and p21
expression.
Study design: A recently established immortalized endometrial stromal cell line was treated with TSA, VPA, and/or all-trans retinoic acid
(ATRA) and the consequent cell cycle progression was measured by flow cytometry and p21 protein expression by Western blot analysis.
Results: Both TSA and VPA induced cell cycle arrest and p21 expression in a concentration-dependent manner. Treatment with ATRA alone
also induced cell cycle arrest and moderate increase in p21 expression but joint treatment of ATRA and TSA/VPA did not further enhance cell
cycle arrest as compared with TSA/VPA treatment alone.
Conclusions: HDACIs suppress proliferation of endometrial stromal cells through induction of cell cycle arrest and possibly also through
apoptosis as well. RA also induces cell cycle arrest but it does not synergize with HDACIs in inducing cell cycle arrest. HDACIs may be
promising compounds for treating endometriosis.
# 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cell cycle arrest; Differentiation; Endometriosis; Histone deacetylase inhibitor; p21; Proliferation; Retinoic acid
1. Introduction
Endometriosis, characterized by the presence of endo-
metrium-like tissue in ectopic sites outside the uterus, is a
common, estrogen-dependent, gynecological disease [1].It
is a leading cause of disability in women of reproductive
age, resulting in dysmenorrhea, pelvic pain, and subfertility
[2]. The current medical treatment for endometriosis has so
far focused on the hormonal alteration of the menstrual cycle
with a major goal to create hypoestrogenic environment [3].
Yet only 50% of women with endometriosis achieve pain
relief in response to existing hormonal treatments or
conservative surgery [4]. In addition, all current medications
for treating endometriosis have many undesirable, and
sometimes severe, side effects. Hence, novel and effective
therapies for endometriosis are needed.
The major mode of action for all current major therapies
in treating endometriosis-associ ated pains is most likely
through suppression of proliferation of the impl ants and
reduction of adhesion formation [5,6]. Therefore, it is not
surprising that suppression of proliferation is the most
frequently used outcome measurement in evaluating the
therapeutic pot ential of a promising compound in endome-
triosis res ear ch, ei ther in vitro or in vivo, even though this
outcome measure is quite different from ones used in
clinical trials [7].
We have previously reported that HOXA10 gene is
aberrantly methylated in eutopic endometrium of women
with endometriosis [8], which may be responsible for its
www.elsevier.com/locate/ejogrb
European Journal of Obstetrics & Gynecology and
Reproductive Biology 137 (2008) 198–203
* Corresponding author at: Deptartment of Pediatrics, Medical College of
Wisconsin, 8701 Watertown Plank Road, MS 756, Milwaukee, WI 53226-
0509, United States. Tel.: +1 414 456 4992; fax: +1 414 456 6663.
E-mail address: swguo@mcw.edu (S.-W. Guo).
0301-2115/$ see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ejogrb.2007.02.014
aberrant expression [9,10]. Recently, we also found that the
promoter region of the gene coding for progesterone
receptor isoform B (PR-B), but not PR-A, is hypermethy-
lated in ectopic endometrium [11]. In addition, we found that
DNMT1, 3A, and 3B, the three genes codi ng for DNA
methyltransferases that are responsible for DNA methyla-
tion, are over-expressed in ectopic endometrium [12],
suggesting that aberrant methylation may be rampant in
endometriosis. Since demethylation agents and histone
deacetylase inhibitors (HDACIs) can reactivate genes
silenced by promoter hypermethylation [13], we wondered
whether HDACIs can be used potential ly for treating
endometriosis by suppression of proliferation, as reported in
cancer research [14].
Using a human endometrial stromal cell line established
by Krikun et al. [15] as a model system, we recently found
that TSA suppresses proliferatio n of endometrial stromal
cells [16]. In addition, HDACIs such as trichostatin A (TSA)
has been shown recently to suppress IL-1b-induced COX-2
expression in endometrial cells [17]. These observations
have recently been replicated in two endometriotic cell lines
11Z and 22B [18], and in fact endometriotic cells are over
10-fold more sensitive to TSA treatment than endometrial
cells (Wu and Guo, manuscript to be submitted). Since it has
been proposed that endometriotic cells are dedifferentiated
[19], we recently have investigated the effect of retinoic
acids on endometrial stromal cell proliferation and found
that TSA and RA have a synergistic effect in suppression of
proliferation (Wu and Guo, manuscript to be submitted). All
these results raise the question as whether HDACIs such as
TSA and VPA can cause cell cycle arrest in endometrial
cells.
Suppression of prol iferation can be achieved through
several ways, including apoptosis, cell cycl e arrest, a nd
senescence. In this study, we attempt to seek possible
mechanisms of proliferation suppression induced by RAs
and two HDACIs, T SA and VPA. TSA, a natural product
isolated from Streptomyces hygroscopicus that was
initially used as an antifungal antibioti c [20], is a potent,
specific and reversible HDAC I ye t it appears to have a
rather poo r b ioavailability [21]. Therefore, it is used
mainly as a benchmark HDACI. V PA also i s a potent
HDACI and is a well-tolerated anti-epileptic drug with an
extensively characterized toxicity profile. It inhibits both
class I and II HDACs (excluding HDAC6 and HDAC10)
with resultant hyperacetylation of histones H3 and H4
[22]. VPA has been shown to have potent in vitro and in
vivo antiproliferative effect in various canc ers [23].A
recent pilot study has shown that it is a promising drug in
treating adenomyosis [24]. The effects of TSA and VPA
treatment on cell cycles will b e compared with that of
CDB2914, a selective progesterone receptor modulator
[25] that has been shown to be promising in treating
endometriosis [26],andN-acetylcysteine, an antioxidant
that has been shown to be anti-proliferat ive in e ndometrial
stromal cells [27].
2. Materials and methods
2.1. Chemicals and reagents
Stock solutions of TSA (10 mM) (Sigma) and CDB2914
(10 mM, provided as gift from HRA Pharma, Paris, France)
were dissolved in absolute ethanol; stock solution of VPA
(0.2 M), N-acetylcysteine (NAC) (600 mM) and ATRA
(1 mM) (Sigma) were dissolved in distilled water, PBS and
dimethysulfoxide (DMSO), respectively. All stock solu tions
were stored in 20 8C before use.
2.2. Cell line and its maintenance
An immortalized human endometrial stromal cell line,
recently established by Krikun et al. [15], was kindly
provided by Dr. Asgi Fazleabas of University of Illinois at
Chicago. As in Krikun et al. [15], cells were maintained in a
phenol red-free DMEM/F12 (Gibco, Carlsbad, CA) medium
supplemented with 10% charcoal-dextran stripped fetal
bovine serum (Biomeda, Foster City, CA), 1 ml/ml sodium
pyruvate (Gibco) and 1 Pen/Strep/Fungizone (Gibc o) at
37 8C with 5% CO
2
in air in a humidified incubator. The cell
line, referred to as the Yale Human Endometrial Stromal or
YHES cell line hereafter, has been shown to be karyoty-
pically, morphologically, and phenotypically similar to the
primary parent cells [15].
2.3. Cell-cycle analysis
After 16 h incubation with vehicle (ethanol or DMSO),
TSA (10 mM), VPA (3 mM), ATRA (1 mM), TSA
(10 mM) + ATRA (1 mM), VPA (3 mM) + ATRA (1 mM),
CDB (10 mM), or NAC (10 mM), cells were harvested by
trypsinization and fixed in ice-cold 70% ethanol overnight at
4 8C. The cells were centrifuged down and resuspended in
0.2 ml propidium iodide [28] staining solution (50 mg/ml PI,
25 mg/ml RNase A in PBS). After 45 min of incubation at
room temperature, the DNA content of cells was measured
using a BD FACS LSR II flow cytometry (BD Biosciences,
San Jose, CA). A minimum of 10,000 events was counted for
each sample. Data were analyzed using WINMDI (Version
2.8, The Scripps Research Institute, La Jolla, CA).
2.4. Western blot analysis
Western blot analysis was performed to measure the
protein expression of p21. Cells were treated with vehicle,
TSA (1 and 10 mM), ATRA (1 mM) or TSA (10 mM) +
ATRA (1 mm) for 16 h. Whole cell lysates were prepared
using lysis buffer containing 50 mM Tris, pH 7.4, 150 mM
NaCl, 12 mM EDTA, 1% Triton X-100, 0.5% sodium
deoxycholate, and 0.1% SDS. To avoid degradation, a
protease inhibitor cocktail (Sigma, St. Louis, MO) was
added at the final concentration of 2%. Total protein was
quantified using a bicinchoninic acid (BCA) protein assay
Y. Wu, S.-W. Guo / European Journal of Obstetrics & Gynecology and Reproductive Biology 137 (2008) 198–203 199
kit (Pierce, Rockford, IL). Twenty micrograms of total
protein were separated by electrophor esis on 4–15% SDS
(sodium dodecylsulphate)–polyacrylamide gels. The protein
was then transferred onto polyvinylidene fluoride (PVDF)
membranes (Milli pore, Bedford, MA). To inhibit nonspe-
cific binding, the membrane was initially blocked with 5%
nonfat dry milk in PBS (blocking solution) for 1 h at room
temperature. The blot was then subsequently incubated with
primary antibody mouse anti-human p21/Cip1 monoclonal
antibody (BD Biosciences, Franklin Lakes, NJ) and a rabbit
anti-b-actin (Cell Signaling Technology, Beverly, MA),
respectively, in blocking solution overnight at 4 8C. The
membranes were was hed with PBST (PBS plus 0.5%
Tween-20), and were then incubated with the secondary
antibody rabbit anti-mouse IgG HRP or goat anti-rabbit IgG
HRP (Invitrogen, Carlsbad, CA) in blocking solution for 2 h
at room temperature. After incubation, the membrane blot
was then washed with PBST and was developed using the
enhanced chemiluminescence (ECL) protocol (Amersham
Pharmacia Biotech, Piscataway, NJ).
2.5. Statistical analysis
To evaluate the statistical significance of the difference
between two groups in percentages of three different phases,
G
0
/G
1
, S and G
2
/M, a linear combination of G
0
/G
1
and S
percentages (both arcsin square-root transformed to enhance
normality) with weights 1 and 1 assigned to the G
0
/G
1
and
S percentage, respectively, was used and t-test was
performed. The use of only two percentages (G
0
/G
1
and
S) was due to the fact that all three percentages add up to 100
and that the G
2
/M percentage is completely determined once
the other two percentages are known. The contrasting
weights, 1 and 1, were chosen simply due to the fact that
the G
0
/G
1
and S phases represent two contrasting phases in
cell cycle progression, the former being a relatively dormant
state while the latter being a relatively active state. To further
evaluate possible synergistic effect of ATRA and HDACIs
on cell cycle progression, a linear regression analysis of
transformed data was performed through the introduction of
indicator variables for TSA treatment (1 if yes or 0 if not),
VPA treatment (1 if yes or 0 if not), and ATRA treatment (1
if yes or 0 if not). Results were considered to be statistically
significant if p < 0.05.
3. Results
Compared with untreated cells, the YHES cells treated
with ATRA, NAC, CDB, TSA, VPA, TSA + ATRA, or
VPA + ATRA for 16 h all showed statistically significant
substantial increase in percentage of cells in the G
0
/G
1
phases of the cell cycle (all p’s < 0.05), with a concomitant
decrease of percentage of cells in the S and G
2
/M phases,
especially in the S phase (Fig. 1). The cell cycle arrest
induced by TSA and VPA was very similar, and both
HDACIs caused treated cells to accumulate predominantly
in the G
0
/G
1
phase of the cell cycle. ATRA treatment (1 mM)
alone reduced the accumulation of cells in the S phase from
26% to 18% without appreciably increasing the accumula-
tion in the G
0
/G
1
phase. Overall, the ATRA treatment alone
caused significant changes in percentages of cells in
different phases ( p = 0.03). Although it appeared to have
further enhanced the growth arrest effect induc ed by TSA or
VPA through further increasing the percentage of cells in the
G
0
/G
1
phase by 2–2.5% while reducing the percentage of
cells in the S phase by 1–1.5%, the change was not
statistically sign ificant ( p > 0.05, see also Fig. 1). A linear
regression analysis (R
2
= 0.989, p = 2.6 10
11
) further
confirmed that there is no interaction (synergy) between
Y. Wu, S.-W. Guo / European Journal of Obstetrics & Gynecology and Reproductive Biology 137 (2008) 198–203200
Fig. 1. Effects of treatment with TSA, VPA, ATRA, NAC, CDB, TSA + ATRA, and VPA + ATRA on cell cycle progression. Cells were treated for 16 h with
(A) vehicle (as control); (B) TSA (10 mM); (C) TSA (10 mM) + ATRA (1 mM); (D) VPA (3 mM); (E) VPA (3 mM) + ATRA (1 mM); (F) ATRA (1 mM); (G)
CDB (10 mM); (H) NAC (10 mM). The treated cells were then fixed in 70% ethanol, and DNA content was analyzed by flow cytometry after propidium iodide
staining. The percentages of cells in each phase of the cell cycle (G
0
/G
1
, S, and G
2
/M) are graphically depicted. The numbers after the sign are standard
deviations.
ATRA and TSA/VPA ( p > 0.05) even though ATRA
treatment alone does have an effect on the cell cycle
change ( p = 0.0002).
As a comparison, the effect of CDB 2914 or N-acetylsteine
(NAC) on cell cycle progression was also evaluated (Fig. 1).
As with TSA and VPA, both CDB 2914 and NAC treatment
resulted in increased accumulation of cells in the G
0
/G
1
phases of the cell cycle, with a concomitant decrease of
percentage of cells in the S and G
2
/M phases. However,
induction of cell cycle arrest by either CDB 2914 or NAC
appeared to be less than that of TSA or VPA.
We further examined protein level of cyclin-dependent
kinase inhibitor p21 which may play an important role in cell
cycle arrest. Since TSA is a benchmark HDACI and usually
it agrees very well with other HDACIs such as VPA [29],we
only investigated the effect of TSA and ATRA on the p21
protein expression. We found that the protein level of p21
increased in a concentration-dependent manner after
treatment of YHES cells with TSA (Fig. 2). Consistent
with the cell cycle analysis, ATRA treatment increased the
protein expression of p21 but the increase was not as much
as that induced by TSA. The induction of p21 by TSA is
higher than by CDB 2914 (data not shown).
4. Discussion
This study shows that HDACIs and RA suppress
endometrial stromal cell proliferation at least through
induction of cell cycle arrest, accumulating 75% to nearly
80% of cells in the G
0
/G
1
phase, higher than that induced by
either CDB 2914 or N-acetylcysteine (Fig. 1). While RA
treatment alone did cause moderate but statistically
significant cell cycle arrest as compared with untreated
cells, it did not further enhance the effect of cell cycle arrest
induced by HDACIs significantly. Given the strong
synergistic antiproliferative effects induced by TSA and
RA (Wu and Guo, unpublished observation), the slight and
statistically insignificant increase (2.5%) in percentage of
cells in the G
0
/G
1
cannot account for 5–25% (depending on
dosage) more suppression of proliferation as compared with
TSA treatment alone (data not shown). This suggests that the
enhanced proliferation-inhibitory effect conferred by RA in
addition to TSA may not be principally through the
induction of growth arrest, but could be through other
means, such as induction of apoptosis, terminal differentia-
tion, or senescence.
There is mounting evidence suggesting that endome-
triosis also is an epigenetic disease and may be thus treatable
by targeting HDACs. The methylation-silenced PR-B alone
would disrupt apoptotic signals induced by progesterone .
Aberrant methylation may also mediate the down-regulation
of genes involved in apoptosis [30], rendering endometriotic
cells resistant to apoptosis. Therefore, endometriosis
appears to be a good candidate for epigenetic reprogram-
ming through HDACIs.
HDACs play an important role in the regulation of gene
transcription and oncogenesis through remodeling of
chromatin structure and dynamic changes in nucleosomal
packaging of DNA [31]. Inhibition of HDAC increases
histone acetylation and maintains chromatin structure in a
more open conformation, resulting in reactivation of
transcriptionally silenced pathways or suppression of
aberrantly expressed genes through recruitment of repres-
sors [32]. Our finding that TSA and VPA induce cell cycle
arrest and p21 protein expression is consistent with
numerous similar reports in cancer cells [33].
Retinoic acid modulates the expression of genes
necessary for cellular growth and differentiation [34].In
human endometrium, RA plays a critical role in the
regulation of matrix metalloproteinases (MMPs) [35] which
are dysregulated in endometriosis. It also suppresses IL-6
production [36], which is high in endometriotic cells and
peritoneal fluid [37] . All-trans RA has been shown recently
to inhibit vascular endothelial growth factor (VEGF)
expression in a cell model of neutrophil activation,
suggesting that the up-regulated VEGF and angiogenesis
seen in tissue from women with endometriosis may be a
result of failure of neut rophil differentiation [38]. Since
endometriotic cells appear to be dedifferentiated [19] and
may involve a pool of rather undifferentiated cells that are
capable of self-renewal, RA may cause terminal differentia-
tion in endometriotic cells. All these observations provide a
strong ration ale for retinoid therapy for endometriosis, in
conjunction with HDACIs.
Y. Wu, S.-W. Guo / European Journal of Obstetrics & Gynecology and Reproductive Biology 137 (2008) 198–203 201
Fig. 2. p21 protein expression in YHES cells as measured by Western blot
analysis. YHES cells were treated with indicated concentration of TSA and/
or ATRA, and cell lysates were prepared after 16 h of treatment. Western
blot analysis was performed with antibody to p21. Cells treated with vehicle
alone served as control. The amount pf protein was normalized by compar-
ison with levels of b-actin.
In summary, HDACIs such as TSA and VPA induce cell
cycle arrest and p21 expression, but the induction is not
statistically significantly enhanc ed by ATRA. This induction
of cell cycle arrest, along with possible apoptosis or terminal
differentiation, accounts for, at least in part, the observed
suppression of proliferation of endometrial and endome-
triotic cells.
Acknowledgements
The authors would like to thank Dr. Graciela Krikun and
Dr. Asgi Fazleabas for providing the YHES cell line which
made this work possible. They also would like to thank three
anonymous reviewer s for their helpful comments. The
financial support from the Children’s Research Institute of
Wisconsin is gratefully acknowledged. The financial support
from The Children’s Research Institute, Milwaukee,
Wisconsin is acknowledged.
References
[1] Giudice LC, Kao LC. Endometriosis. Lancet 2004;364(9447):1789–
99.
[2] Farquhar CM. Extracts from the ‘clinical evidence’’. Endometriosis
BMJ 2000;320(7247):1449–52.
[3] Olive DL, Pritts EA. Treatment of endometriosis. N Engl J Med
2001;345(4):266–75.
[4] Vercellini P, Cortesi I, Crosignani PG. Progestins for symptomatic
endometriosis: a critical analysis of the evidence. Fertil Steril
1997;68(3):393–401.
[5] Wright JA, Sharpe-Timms KL. Gonadotropin-releasing hormone ago-
nist therapy reduces postoperative adhesion formation and reformation
after adhesiolysis in rat models for adhesion formation and endome-
triosis. Fertil Steril 1995;63(5):1094–100.
[6] Friedlander RL. The treatment of endometriosis with Danazol. J
Reprod Med 1973;10(4):197–9.
[7] Guo SW, Olive DL. Two unsuccessful c linical trials on endome-
triosis and a few lessons learned. Gynecol Obstet Invest 2007;64(1):
24–35.
[8] Wu Y, Halverson G, Basir Z, Strawn Y, Yan P, Guo SW. Aberrant
methylation at HOXA10 may be responsible for its aberrant expres-
sion in the endometrium of patients with endometriosis. Am J Obstet
Gynecol 2005;192.
[9] Taylor HS, Bagot C, Kardana A, Olive D, Arici A. HOX gene
expression is altered in the endometrium of women with endome-
triosis. Hum Reprod 1999;14(5):1328–31.
[10] Gui Y, Zhang J, Yuan L, Lessey BA. Regulation of HOXA-10 and its
expression in normal and abnormal endometrium. Mol Hum Reprod
1999;5(9):866–73.
[11] Wu Y, Strawn E, Basir Z, Halverson G, Guo SW. Promoter hyper-
methylation of progesterone receptor isoform B (PR-B) in endome-
triosis. Epigenetics 2006;1(2):106–11.
[12] Wu Y, Strawn E, Basir Z, Halverson G, Guo SW. Aberrant expression
of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A and
DNMT3B in women with endometriosis. Fertil Steril 2007;87(1):24–
32.
[13] Cameron EE, Bachm an KE, Myohanen S, Herman JG, Baylin SB.
Synergy of demethylation and histone deacetylase inhibition in the
re-expression of genes silenced i n cancer. N at Genet 1999;21(1):
103–7.
[14] Marks PA, Rifkind RA, Richon VM, Breslow R. Inhibitors of histone
deacetylase are potentially effective anticancer agents. Clin Cancer
Res 2001;7(4):759–60.
[15] Krikun G, Mor G, Alvero A, et al. A novel immortalized human
endometrial stromal cell line with normal progestational response.
Endocrinology 2004;145(5):2291–6.
[16] Wu Y, Guo SW. Inhibition of proliferation of endometrial stromal cells
by trichostatin A, RU486, CDB-2914, N-acetylcysteine, and ICI
182780. Gynecol Obstet Invest 2006;62(4):193–205.
[17] Wu Y, Guo SW. Suppression of IL-1ß-induced COX-2 expression by
trichostatin A (TSA) in human endometrial stromal cells. Eur J Obstet
Gynecol Reprod Biol, 10 Feb, 2007 [Epub ahead of print].
[18] Zeitvogel A, Baumann R, Starzinski-Powitz A. Identification of
an invasive N-cadherin-expressing epithelial cell type in endome-
triosis using a new cell culture model. Am J Pathol 2001;159(5):
1839–52.
[19] Starzinski-Powitz A, Zeitvogel A, Schreiner A, Baumann R. In search
of pathogenic mechanisms in endometriosis: the challenge for mole-
cular cell biology. Curr Mol Med 2001;1(6):655–64.
[20] Tsuji N, Kobayashi M, Nagashima K, Wakisaka Y, Koizumi K. A
new antifungal antibiotic, trichostatin. J Antibiot (Tokyo) 1976;29(1):
1–6.
[21] Elaut G, Torok G, Vinken M, et al. Major phase I biotrans-
formation pathways of Trichostatin A in r at hepatocytes and in
rat and human liver microsomes . Drug Metab Dispos 2002;30(12):
1320–8.
[22] Gottlicher 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(24):6969–78.
[23] Bacon CL, Gallagher HC, Haughey JC, Regan CM. Antiproliferative
action of valproate is associated with aberrant expression and nuclear
translocation of cyclin D3 during the C6 glioma G1 phase. J Neu-
rochem 2002;83(1):12–9.
[24] Liu X, Guo SW. A pilot study on the off-label use of valproic acid to
treat adenomyosis. Fertil Steril; 2007.
[25] Gainer EE, Ulmann A. Pharmacologic properties of CDB(VA)-2914.
Steroids 2003;68(10–13):1005–11.
[26] Chwalisz K, Perez MC, Demanno D, Winkel C, Schubert G, Elger W.
Selective progesterone receptor modulator development and use in the
treatment of leiomyomata and endometriosis. Endocr Rev 2005;26(3):
423–38.
[27] Foyouzi N, Berkkanoglu M, Arici A, Kwintkiewicz J, Izquierdo D,
Duleba AJ. Effects of oxidants and antioxidants on proliferation
of endometrial stromal cells. Fertil Steril 2004;82(Suppl 3):
1019–22.
[28] Pi X, Yan C, Berk BC. Big mitogen-activated protein kinase (BMK1)/
ERK5 protects endothelial cells from apoptosis. Circ Res
2004;94(3):362–9.
[29] Marks PA, Richon VM, Miller T, Kelly WK. Histone deacetylase
inhibitors. Adv Cancer Res 2004;91:137–68.
[30] Gopisetty G, Ramachandran K, Singal R. DNA methylation and
apoptosis. Mol Immunol; 2006.
[31] Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK.
Histone deacetylases and cancer: causes and therapies. Nat Rev
Cancer 2001;1(3):194–202.
[32] Richon VM, O’Brien JP. Histone deacetylase inhibitors: a new class of
potential therapeutic agents for cancer treatment. Clin Cancer Res
2002;8(3):662–4.
[33] Richon VM, Sandhoff TW, Rifkind RA, Marks PA. Histone deace-
tylase inhibitor selectively induces p21WAF1 expression and gene-
associated histone acetylation. Proc Natl Acad Sci USA 2000;97(18):
10014–9.
[34] Chambon P. A decade of molecular biology of retinoic acid receptors.
FASEB J 1996;10(9):940–54.
[35] Osteen KG, Keller NR, Feltus FA, Melner MH. Paracrine regulation of
matrix metalloproteinase expression in the normal human endome-
trium. Gynecol Obstet Invest 1999;48(Suppl 1):2–13.
Y. Wu, S.-W. Guo / European Journal of Obstetrics & Gynecology and Reproductive Biology 137 (2008) 198–203202
[36] Sawatsri S, Desai N, Rock JA, Sidell N. Retinoic acid suppresses
interleukin-6 production in human endometrial cells. Fertil Steril
2000;73(5):1012–9.
[37] Wu MY, Ho HN, Chen SU, Chao KH, Chen CD, Yang YS. Increase in
the production of interleukin-6, interleukin-10, and interleukin-12 by
lipopolysaccharide-stimulated peritoneal macrophages from women
with endometriosis. Am J Reprod Immunol 1999;41(1):106–11.
[38] Tee MK, Vigne JL, Taylor RN. All-trans retinoic acid inhibits vascular
endothelial growth factor expression in a cell model of neutrophil
activation. Endocrinology 2006;147(3):1264–70.
Y. Wu, S.-W. Guo / European Journal of Obstetrics & Gynecology and Reproductive Biology 137 (2008) 198–203 203
    • "DNMT1, DNMT3a and DNMT3b mRNAs are overexpressed in endometriosis (Wu et al., 2007). HOXA10 and PR-B genes are hypermethylated in endometriosis (Wu et al., 2005, 2006, 2008). Trichostatin A (HDAC pan inhibitor) inhibits NFκB signaling, COX-2 expression, and cell proliferation ; by contrast, increases expression of PR-B and E-cadherin in endometriotic cells in vitro (Wu and Guo, 2007, 2008; Wu et al., 2007, 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: Endometriosis is an inflammatory gynecological disease of reproductive-age women. The prevalence of endometriosis is 5~10% in reproductive-age women. Modern medical treatments are directed to inhibit the action of estrogen in endometriotic cells. However, hormonal therapies targeting estrogen can be prescribed only for a short time because of their undesirable side effects. Recent studies from our laboratory, using endometriotic epithelial cell line 12Z and stromal cell line 22B derived from red lesion, discovered that selective inhibition of prostaglandin E2 (PGE2) receptors EP2 and EP4 inhibits adhesion, invasion, growth, and survival of 12Z and 22B cells by modulating integrins, MMPs and TIMPs, cell cycle, survival, and intrinsic apoptotic pathways, suggesting multiple epigenetic mechanisms. The novel findings of the present study indicate that selective pharmacological inhibition of EP2 and EP4: (i) decreases expression of DNMT3a, DNMT3b, H3K9me3, H3K27me3, SUV39H1, HP1a, H3K27, EZH2, JMJD2a, HDAC1, HDAC3, MeCP2, CoREST and Sin3A; (ii) increases expression of H3K4me3, H3H9ac, H3K27ac; and (iii) does not modulate the expression of DNMT1, hSET1, LSD1, MBD1, p300, HDAC2, and JMJD3 epigenetic machinery proteins in an epithelial and stromal cell specific manner. In this study, we report for the first time that inhibition of PGE2-EP2/EP4 signaling modulates DNA methylation, H3 Histone methylation and acetylation, and epigenetic memory machinery proteins in human endometriotic epithelial cells and stromal cells. Thus, targeting EP2 and EP4 receptors may emerge as long-term nonsteroidal therapy for treatment of active endometriotic lesions in women. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Apr 2015
    • "At therapeutic levels, valproic acid directly inhibits class I and II HDACs (except HDAC6 and HDAC10), with resultant hyperacetylation of histones H3 and H4. After treatment with valproic acid, there is altered expression of multiple genes, including the cyclin-dependent kinase inhibitor p21Cip1, glycogen synthase kinase-3ß, and peroxisome proliferatoractivated receptors, and down-regulation of the expression of the antiapoptotic protein kinase C α and ε isoforms33343536373839. Valproic acid has displayed potent in vitro and in vivo antitumor activities against neuroblastoma, glioma, leukemia, breast cancer, multiple myeloma, and prostate cancer lines [9,4041424344454647. "
    [Show abstract] [Hide abstract] ABSTRACT: Smokers experience aberrant gene promoter methylation in their bronchial cells, which may predispose to the development of neoplasia. Hydralazine is a DNA demethylating agent, and valproic acid is a histone deacetylase inhibitor, and both have modest but synergistic anticancer activity in vitro. We conducted a phase I trial combining valproic acid and hydralazine to determine the maximally tolerated dose (MTD) of hydralazine in combination with a therapeutic dose of valproic acid in patients with advanced, unresectable, and previously treated solid cancers. Twenty females and nine males were enrolled, with a median age of 57 years and a median ECOG performance status of 0. Grade 1 lymphopenia and fatigue were the most common adverse effects. Three subjects withdrew for treatment-related toxicities occurring after the DLT observation period, including testicular edema, rash, and an increase in serum lipase accompanied by hyponatremia in one subject each. A true MTD of hydralazine in combination with therapeutic doses of valproic acid was not reached in this trial, and the planned upper limit of hydralazine investigated in this combination was 400 mg/day without grade 3 or 4 toxicities. A median number of two treatment cycles were delivered. One partial response by Response Evaluation Criteria In Solid Tumors criteria was observed, and five subjects experienced stable disease for 3 to 6 months. The combination of hydralazine and valproic acid is simple, nontoxic, and might be appropriate for chemoprevention or combination with other cancer treatments. This trial supports further investigation of epigenetic modification as a new therapeutic strategy.
    Full-text · Article · Apr 2014
    • "Moreover, due to the suppression of miR-106b-93- 25 cluster in TSA treated EMC cells, p21 and BIM dramatically increased and eventually led to cell cycle arrest and apoptosis. p21 was reported to be up-regulated after TSA treatment in different cells by several independent groups [30,45,46]. The main explanation of this phenomenon lies in the up-regulation of p53. "
    [Show abstract] [Hide abstract] ABSTRACT: Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the miR-106b-93-25 cluster, together with its host gene MCM7, were obviously down-regulated in EMC cells. p21 and BIM, which were identified as target genes of miR-106b-93-25 cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified MYC as a regulator of miR-106b-93-25 cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of miR-106b-93-25 cluster and up-regulation of p21 and BIM. More important, we found miR-106b-93-25 cluster was up-regulated in clinical EMC samples in association with the overexpression of MCM7 and MYC and the down-regulation of p21 and BIM. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the miR-106b-93-25 cluster and up-regulation of it's target genes p21 and BIM via MYC.
    Full-text · Article · Sep 2012
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