Histone deacetylase inhibitors trichostatin A and valproic acid induce cell cycle arrest and p21 expression in immortalized human endometrial stromal cells.
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.
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ABSTRACT: To assess the literature on preclinical and clinical efficacy and safety data of pharmacologic groups proposed in the treatment of endometriosis, we performed a systematic review of publications from March 2002 to January 2012 via PubMed search. Additional relevant articles were identified from citations within these publications. A high number of medications were tested in preclinical models of endometriosis due to their theoretic capacity of disrupting important pathophysiologic pathways of the disease, such as inflammatory response, angiogenesis and cell survival, proliferation, migration, adhesion, and invasion. Tumor necrosis factor α-blockers, nuclear factor κB inhibitors, antiangiogenic agents, statins, antioxidants, immunomodulators, flavonoids, histone deacetylase inhibitors, matrix metalloproteinase inhibitors, metformin, novel modulators of sex steroids expression, and apoptotic agents were all effective in in vitro/animal models. Most of these agents have not been tried in the clinical setting, mainly because of the high risk of adverse effects. However, some of them can be used in humans. Dopamine agonists and valproic acid have already been tested in pilot studies with good results. Etanercept, metformin, and statins are used in humans for other indications, and endostatin is now being tested in phase 2 oncologic trials. These drugs may constitute alternatives to conventional therapy with estrogen inhibitors and anti-inflammatory agents.Fertility and sterility 09/2012; 98(3):529-55. · 3.97 Impact Factor
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ABSTRACT: Epigenetic mechanisms have been ascribed important roles in endometriosis. Covalent histone modifications at lysine residues have been shown to regulate gene expression and thus contribute to pathological states in many diseases. In endometriosis, histone deacetylase inhibition (HDACi) resulted in reactivation of E-cadherin, attenuation of invasion, decreased proliferation of endometriotic cells, and caused lesion regression in an animal model. This study was conducted to assess basal and hormone-regulated gene expression levels of HDAC1 and HDAC2 (HDAC1/2) in cell lines and protein expression levels in tissues. Basal and steroid hormone-regulated HDAC1/2 gene expression levels were determined by quantitative polymerase chain reaction in cell lines and tissues. Protein levels were measured by immunohistochemistry (IHC) in tissues on an endometriosis tissue microarray (TMA). Basal HDAC1/2 gene expression levels were significantly higher in endometriotic versus endometrial stromal cells, which was confirmed by Western blot analysis. Estradiol (E2) and progesterone (P4) significantly downregulated HDAC1 expression in endometrial epithelial cells. Levels of HDAC2 were upregulated by E2 and downregulated by E2 + P4 in endometrial stromal cells. Hormone modulation of HDAC1/2 gene expression was lost in the endometriotic cell line. Immunohistochemistry showed that HDAC1/2 proteins were expressed in a substantial proportion of lesions and endometrium from patients, and their expression levels varied according to lesion localization. The highest proportion of strong HDAC1 immunostaining was seen in ovarian, skin, and gastrointestinal lesions, and of HDAC2 in skin lesions and endometrium from patients with endometriosis. These studies suggest that endometriosis etiology may be partially explained by epigenetic regulation of gene expression due to dysregulations in the expression of HDACs.Reproductive sciences (Thousand Oaks, Calif.) 02/2012; 19(5):483-92. · 2.31 Impact Factor
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ABSTRACT: Combination of low doses of histone deacetylases inhibitors and chemotherapy drugs is considered as one of the most promising strategies to increase the anticancer efficacy. Chidamide is a novel benzamide chemical class of HDAC inhibitor that selectively inhibited HDAC1, 2, 3 and 10. We sought to determine whether chidamide may enhance platinum-induced cytotoxicity in NSCLC cells. In this study, the combination of chidamide with carboplatin showed a good synergism on growth inhibition with the mean combination index value as 0.712 and 0.639 in A549 and NCI-H157 cells, respectively. The used concentration of chidamide was non-toxic on cells by itself as low as 0.3 μM. All of our experiments were comparisons between combination regimen and single carboplatin regimen in A549 and NCI-H157 cell lines. Phosphorylated histone H2A.X (γH2A.X), a hall marker of DNA damage response, was dramatically increased by the combination treatment. Cell cycle analysis by flow cytometry and phosphorylation level analysis of histone H3 (Ser10) by western blotting showed that combination treatment significantly increased the percentage of G2/M phase of cells. Mitochondrial membrane potential and cleaved-PARP1 level analysis indicate that chidamide synergistically enhances carboplatin-induced apoptosis. Additionally, synergistic effects of chidamide were found when it was combined with two other platinum drugs (cisplatin and oxaliplatin). The results suggest that Chidamide in combination with platinum drugs may be a novel therapeutic option for NSCLC.Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 01/2014; · 2.24 Impact Factor
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
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 determinewhether TSA and VPA do so by inducing cell cycle arrest and p21
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 inp21 expression but joint treatment of ATRA and TSA/VPAdid 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
Endometriosis, characterized by the presence of endo-
metrium-like tissue in ectopic sites outside the uterus, is a
common, estrogen-dependent, gynecological disease . It
is a leading cause of disability in women of reproductive
age, resulting in dysmenorrhea, pelvic pain, and subfertility
. The current medical treatment for endometriosis has so
with a major goal to create hypoestrogenic environment .
Yet only 50% of women with endometriosis achieve pain
relief in response to existing hormonal treatments or
conservativesurgery . 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-associated pains is most likely
through suppression of proliferation of the implants 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 potential of a promising compound in endome-
triosis research, either in vitro or in vivo, even though this
outcome measure is quite different from ones used in
clinical trials .
We have previously reported that HOXA10 gene is
aberrantly methylated in eutopic endometrium of women
with endometriosis , which may be responsible for its
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: firstname.lastname@example.org (S.-W. Guo).
0301-2115/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
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-
DNMT1, 3A, and 3B, the three genes coding for DNA
methyltransferases that are responsible for DNA methyla-
tion, are over-expressed in ectopic endometrium ,
suggesting that aberrant methylation may be rampant in
endometriosis. Since demethylation agents and histone
deacetylase inhibitors (HDACIs) can reactivate genes
silenced by promoter hypermethylation , we wondered
whether HDACIs can be used potentially for treating
endometriosis by suppression of proliferation, as reported in
cancer research .
Using a human endometrial stromal cell line established
by Krikun et al.  as a model system, we recently found
that TSA suppresses proliferation of endometrial stromal
cells . In addition, HDACIs such as trichostatin A (TSA)
has been shown recently to suppress IL-1b-induced COX-2
expression in endometrial cells . These observations
have recently been replicated in two endometriotic cell lines
11Z and 22B , 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
, 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
Suppression of proliferation can be achieved through
several ways, including apoptosis, cell cycle arrest, and
senescence. In this study, we attempt to seek possible
mechanisms of proliferation suppression induced by RAs
and two HDACIs, TSA and VPA. TSA, a natural product
isolated from Streptomyces hygroscopicus that was
initially used as an antifungal antibiotic , is a potent,
specific and reversible HDACI yet it appears to have a
rather poor bioavailability . Therefore, it is used
mainly as a benchmark HDACI. VPA also is 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
. VPA has been shown to have potent in vitro and in
vivo antiproliferative effect in various cancers . A
recent pilot study has shown that it is a promising drug in
treating adenomyosis . The effects of TSA and VPA
treatment on cell cycles will be compared with that of
CDB2914, a selective progesterone receptor modulator
 that has been shown to be promising in treating
endometriosis , and N-acetylcysteine, an antioxidant
that has been shown to be anti-proliferative in endometrial
stromal cells .
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 solutions
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. , was kindly
provided by Dr. Asgi Fazleabas of University of Illinois at
Chicago. As in Krikun et al. , 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 (Gibco) at
37 8C with 5% CO2in 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 .
2.3. Cell-cycle analysis
After 16 h incubation with vehicle (ethanol or DMSO),
TSA (10 mM), VPA (3 mM),
(10 mM) + ATRA (1 mM), VPA (3 mM) + ATRA (1 mM),
CDB (10 mM), or NAC (10 mM), cells were harvested by
trypsinization and fixedin ice-cold 70% ethanol overnight at
4 8C. The cells were centrifuged down and resuspended in
0.2 ml propidiumiodide  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,
each sample. Data were analyzed using WINMDI (Version
2.8, The Scripps Research Institute, La Jolla, CA).
ATRA (1 mM),TSA
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 electrophoresis on 4–15% SDS
(sodium dodecylsulphate)–polyacrylamide gels. The protein
was then transferred onto polyvinylidene fluoride (PVDF)
membranes (Millipore, 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 washed 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,
G0/G1, S and G2/M, a linear combination of G0/G1and S
percentages (both arcsin square-root transformed to enhance
normality) with weights 1 and ?1 assigned to the G0/G1and
S percentage, respectively, was used and t-test was
performed. The use of only two percentages (G0/G1and
S)was duetothe fact thatallthreepercentages addup to100
and that the G2/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 G0/G1and S phases represent two contrasting phases in
cell cycle progression, the former being a relatively dormant
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.
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 G0/G1
phases of the cell cycle (all p’s < 0.05), with a concomitant
decrease of percentage of cells in the S and G2/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
alone reduced the accumulation of cells in the S phase from
26% to 18% without appreciably increasing the accumula-
tion in the G0/G1phase. 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 induced by TSA or
VPA through further increasing the percentage of cells in the
G0/G1phase by 2–2.5% while reducing the percentage of
cells in the S phase by 1–1.5%, the change was not
statistically significant (p > 0.05, see also Fig. 1). A linear
regression analysis (R2= 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) 198–203 200
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 (G0/G1, S, and G2/M) are graphically depicted. The numbers after the ‘‘?’’ sign are standard
ATRA and TSA/VPA (p > 0.05) even though ATRA
treatment alone does have an effect on the cell cycle
change (p = 0.0002).
(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 G0/G1
phases of the cell cycle, with a concomitant decrease of
percentage of cells in the S and G2/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
cycle arrest. Since TSA is a benchmark HDACI and usually
it agrees very well with other HDACIs such as VPA , 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).
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 G0/G1phase, 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 G0/G1cannot account for 5–25% (depending on
dosage) more suppression of proliferation as compared with
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 andmay 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 , 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 . 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 . Our finding that TSA and VPA induce cell cycle
arrest and p21 protein expression is consistent with
numerous similar reports in cancer cells .
Retinoic acid modulates the expression of genes
necessary for cellular growth and differentiation . In
human endometrium, RA plays a critical role in the
regulation of matrix metalloproteinases (MMPs)  which
are dysregulated in endometriosis. It also suppresses IL-6
production , which is high in endometriotic cells and
peritoneal fluid . 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 neutrophil differentiation . Since
endometriotic cells appear to be dedifferentiated  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 rationale for retinoid therapy for endometriosis, in
conjunction with HDACIs.
Y. Wu, S.-W. Guo/European Journal of Obstetrics & Gynecology and Reproductive Biology 137 (2008) 198–203201
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 analysiswas performedwith antibodyto 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 enhancedby ATRA. This induction
of cell cyclearrest, alongwith possibleapoptosis or terminal
differentiation, accounts for, at least in part, the observed
suppression of proliferation of endometrial and endome-
The authors would like to thank Dr. Graciela Krikun and
Dr. Asgi Fazleabas for providing the YHES cell line which
made this work possible. Theyalsowould like to thank three
anonymous reviewers for their helpful comments. The
financial support from the Children’s Research Institute of
from The Children’s Research Institute, Milwaukee,
Wisconsin is acknowledged.
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