Effect of combined treatment with progesterone and tamoxifen on the growth and apoptosis of human ovarian cancer cells

Department of Obstetrics and Gynecology, Sisters of Charity Hospital, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Oncology Reports (Impact Factor: 2.3). 09/2011; 27(1):87-93. DOI: 10.3892/or.2011.1460
Source: PubMed
ABSTRACT
Progesterone has a potential protective effect against ovarian carcinoma induced by estrogen. Progesterone is also known to cause apoptosis while tamoxifen induces growth arrest. Therefore, we attempted to determine whether combined treatment with progesterone and tamoxifen has a synergistic effect on anti-cancer activity. Although progesterone is known to cause apoptosis while tamoxifen induces growth arrest in many cancer cells, the detailed action of progesterone and tamoxifen and the anticancer effect of combined treatment have not been tested in ovarian cancer cells. Therefore, we tested the growth and apoptosis activity of progesterone and tamoxifen and the anticancer effect of combined treatment of progesterone and tamoxifen in ovarian cancer cells. Ovarian cancer cells, PA-1, were treated with progesterone, tamoxifen, or a combination of progesterone and tamoxifen. The anti-cancer effects were investigated by use of flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, DNA fragmentation analysis, and Western blot analysis. We found that 100 µM progesterone induced typical apoptosis in PA-1 cells. Treatment of PA-1 cells with 10 µM tamoxifen resulted in an increase in the levels of p21, p27, p16 and phospho-pRb, indicating typical G1 arrest. Co-treatment of PA-1 cells with 100 µM progesterone and 10 µM tamoxifen resulted in typical apoptosis, similar to that induced by treatment with 100 µM progesterone alone. These results indicate that progesterone caused apoptosis and tamoxifen induced G1 arrest. Combined treatment with tamoxifen and progesterone caused apoptosis similar to that induced by treatment with progesterone alone and had no additional anti-cancer effect in ovarian cancer cells.

Full-text

Available from: Seong-Hoon Park, Mar 18, 2016
ONCOLOGY REPORTS 27: 87-93, 2012
Abstract. Progesterone has a potential protective effect
against ovarian carcinoma induced by estrogen. Progesterone
is also known to cause apoptosis while tamoxifen induces
growth arrest. Therefore, we attempted to determine whether
combined treatment with progesterone and tamoxifen
has a synergistic effect on anti-cancer activity. Although
progesterone is known to cause apoptosis while tamoxifen
induces growth arrest in many cancer cells, the detailed action
of progesterone and tamoxifen and the anticancer effect of
combined treatment have not been tested in ovarian cancer
cells. Therefore, we tested the growth and apoptosis activity
of progesterone and tamoxifen and the anticancer effect of
combined treatment of progesterone and tamoxifen in ovarian
cancer cells. Ovarian cancer cells, PA-1, were treated with
progesterone, tamoxifen, or a combination of progesterone
and tamoxifen. The anti-cancer effects were investigated by
use of ow cytometry, terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL) assay, DNA fragmentation
analysis, and Western blot analysis. We found that 100 µM
progesterone induced typical apoptosis in PA-1 cells.
Treatment of PA-1 cells with 10 µM tamoxifen resulted in
an increase in the levels of p21, p27, p16 and phospho-pRb,
indicating typical G
1
arrest. Co-treatment of PA-1 cells with
100 µM progesterone and 10 µM tamoxifen resulted in typical
apoptosis, similar to that induced by treatment with 100 µM
progesterone alone. These results indicate that progesterone
caused apoptosis and tamoxifen induced G
1
arrest. Combined
treatment with tamoxifen and progesterone caused apoptosis
similar to that induced by treatment with progesterone alone
and had no additional anti-cancer effect in ovarian cancer cells.
Introduction
Ovarian carcinoma is one of the most common fatal gyneco-
logic malignancies and is characterized by an insidious onset
and a lack of early specic symptoms. About two-thirds of
patients with ovarian carcinoma usually present with advanced
ovarian carcinoma and have widespread tumor dissemination.
Unfortunately, the most effective strategy for the management
of ovarian carcinoma is yet to be determined. Although Taxol
and platinum-based combination chemotherapy is a standard
treatment for ovarian carcinoma and has achieved a high
response rate, its success is limited by the development of drug
resistance (1). Therefore, it is important to explore alternative
treatment modalities that have favorable cost benet ratios in
terms of toxicity and do not lead to the development of drug
resistance or disease relapse.
It has been reported that estrogen and progesterone are
involved in the etiology and long-term survival of patients with
ovarian carcinoma. In 1963, Long and Evans (2) suggested for
the rst time that ovarian carcinoma might be sensitive to
hormones. According to their report, the use of diethylstilbes-
trol in 14 patients with advanced ovarian carcinoma led to a
partial response in 4 (28%) patients. The incidence of ovarian
carcinoma is increased among women after menopause
because of lower levels of sex steroids. Estrogen replacement
therapy in postmenopausal women does not reduce the risk
of ovarian carcinoma. However, the risk may be reduced by
use of combination-type oral contraceptives, which contain
estrogen and a high dose of progesterone. Recent studies
have reported the presence of estrogen, progesterone, and
androgen receptors in varying concentrations and combina-
tions in ovarian carcinoma (3). According to the results of
many studies, although estrogen is proposed to facilitate the
induction of ovarian carcinoma, progesterone has a potential
protective effect (4,5). Progesterone has strong effects on
Effect of combined treatment with progesterone and tamoxifen
on the growth and apoptosis of human ovarian cancer cells
JI-YOUNG LEE
3
, JONG-YEON SHIN
4
, HYUN-SEOK KIM
5
, JEE-IN HEO
1,2
, YOON-JUNG KHO
2
,
HONG-JUN KANG
6
, SEONG-HOON PARK
1,5
and JAE-YONG LEE
1,2
1
Department of Biochemistry and
2
Institute of Natural Medicine, College of Medicine, Hallym University, Chuncheon,
Gangwon-do 200-702, Republic of Korea;
3
Department of Obstetrics and Gynecology, Sisters of Charity Hospital,
State University of New York at Buffalo, Buffalo, NY 14214, USA;
4
Genomic Medicine Institute, Medical Research
Center, Seoul National University, Seoul, Republic of Korea;
5
Molecular Radiation Oncology, Radiation Oncology Branch,
Center for Cancer Research, NCI, NIH, Bethesda, MD 20892;
6
Genetic Disease Research Section,
NIDDK, National Institutes of Health, Building 10, Room 9D11, Bethesda, MD 20892, USA
Received June 9, 2011; Accepted July 28, 2011
DOI: 10.3892/or.2011.1460
Correspondence to: Dr Jae-Yong Lee, Department of Biochem-
istry, College of Medicine, Hallym University, 1 Okcheon-dong,
Chuncheon, Gangwon-do 200-702, Republic of Korea
E-mail: jyolee@hallym.ac.kr
Key words: ovarian cancer cell line, progesterone, tamoxifen,
apoptosis, growth arrest
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LEE et al: COMBINED TREATMENT OF PROGESTERONE AND TAMOXIFEN IN OVARIAN CANCER CELLS
88
human hormone-responsive endometrial and breast cancers. It
has also been reported that progesterone signicantly inhibits
cell proliferation and reduces the risk of ovarian carcinoma (6).
In particular, because progesterone has been reported to have
antiproliferative and apoptotic effects on ovarian cancer cell
lines, progesterone has been widely used in the clinical treat-
ment of ovarian carcinoma. Van der Vange et al (7) reported
that the response rate was approximately 7% in a series with
an adequate number of patients and response criteria. In
addition to progesterone, tamoxifen, which is a competitive
estrogen antagonist, has been successfully used in the manage-
ment of early and advanced breast cancer. The clinical activity
of anti-estrogens in ovarian carcinoma was rst reported by
Myers et al (8). The reported response rates to tamoxifen
were between 0 and 28%, and the overall response rate was
approximately 8%. Although Marth et al suggested a better
response for endometrioid adenocarcinomas of the ovary, a
literature review revealed no apparent differences in histo-
logical subtype, grade of tumor, or hormone receptor values
between responders and non-responders and no correlation
between response rate and the presence of estrogen or proges-
terone receptors in the tumors were evident (9-11). However,
the estrogen receptor-negative/progesterone receptor-positive
phenotype predicts favorable tumor biology and long term
survival, probably reecting the functional effects on tumor
proliferation, differentiation, and apoptosis (12). Tamoxifen
causes cell cycle arrest (G
0
/G
1
arrest) with up-regulation of
both p21 and p27 levels in susceptible estrogen-receptor posi-
tive breast cancer cells (13). The exact molecular mechanisms
of apoptosis or growth arrest of ovarian cancer cells induced
by progesterone and tamoxifen are not clear. In addition, the
effect of combined treatment of progesterone and tamoxifen
has not been tested.
In this study, the molecular mechanisms of apoptosis and
growth arrest of ovarian cancer cells induced by progesterone
and tamoxifen were investigated. In addition, the effect of
combined treatment of progesterone and tamoxifen was tested
since combined treatment seems to have synergistic effects.
Materials and methods
Cell lines and cell cultures. PA-1, a human ovarian cancer cell
line with wild-type p53, was obtained from ATCC (American
Type Culture Collection, Manassas, VA, USA). Cells were
cultured in Dulbecco's modied essential medium (DMEM,
Gibco-BRL, NY) supplemented with 10% fetal bovine serum
(FBS; Life Technologies, Inc.), streptomycin (100 µg/ml), and
penicillin G (100 IU/ml), and maintained at 37˚C in a humidi-
ed incubator with 5% CO
2
.
RT-PCR. Total-RNA was isolated from 2x10
6
cells using
RNA Plus™ (Quantum Biotechnologies, Inc., CA). cDNA
was synthesized by incubation at 52˚C for 20 min in a 20-µl
reaction volume containing RNA (100 ng), 10 mM deoxynu-
cleotide triphosphates (Boehringer Mannhein), 10X reaction
buffer (provided by Takara Shuzo Co., Japan) and Avian
Myeloblastosis Virus (AMV) reverse transcriptase (Takara).
Primers for hormone receptor gene were designed that amplify
mRNAs between exons 7 and 8 of the estrogen receptor
gene (5'-GCACCCTGAAGTCTCTGGAA-3', 5'-TGGCTA
AAGTGGTGCATGAT-3'), exons 3 and 4 of progesterone
receptor mRNA (5'-TGTCAGGCTGGCATGGTCCTTGG-3',
5'-GACGGGTGACTGCAGAAACATCC-3'). The PCR reac-
tion was performed as follows; incubation at 95˚C for 8 min
followed by 40 cycles of reaction (denaturation at 95˚C for
1 min, annealing at different temperature for 1 min, extension
at 72˚C for 1 min), and a nal extension at 72˚C for 10 min.
Annealing temperatures for the estrogen receptor and proges-
terone receptor were 55 and 65˚C, respectively. The amplied
PCR products were resolved by electrophoresis on 1% agarose
gel and visualized by ethidium bromide staining.
Treatment of PA-1 cells with tamoxifen, progesterone, or both.
Progesterone (Sigma, St. Louis, MO) and tamoxifen (Sigma)
were dissolved in ethanol and kept at C until use. Cells were
plated and medium was changed to DMEM with 10% charcoal-
stripped FBS and antibiotics (streptomycin and penicillin G) to
deplete medium steroid hormones and estrogenic compounds
for the remainder of the assay period. After 48 h, cultures were
replenished with fresh charcoal-stripped DMEM, and then
progesterone, tamoxifen, or both drugs were added to each dish.
Methyl thiazole tetrazolium (MTT) assay. Cells were seeded in
96-well plates at a density of 2x10
6
cells/well and incubated for
24 h. After treatment of drugs, the cell growth was assessed by
staining with MTT dye at 37˚C for 2 h. The staining medium
was then replaced by phosphate-buffered saline (PBS) with
2.5% of protamin sulfate stock solution (Sigma), and the plates
were incubated at C overnight. The formazan was extracted
with 250 µl of dimethyl sulfoxide (DMSO, 10%, Sigma) at
room temperature for 4 h. Then 150 µl of the extract was
transferred to a at-bottomed 96-well plate for reading the
absorbance at 570 nm in a microplate reader (Dynatech, UK).
Cell cycle analysis. Cells were harvested by trypsinization at
0, 12, 24, and 48 h after drug treatment, collected gently, and
resuspended in 2 ml of PBS. Cells were then xed by adding
gradually 5 ml of 95% ethanol with vortexing. After incuba-
tion at room temperature for 30 min, cells were stored at 4˚C.
Cells were then collected by centrifugation and stained by
adding 1 ml of 50 µg/ml propidium iodide solution (Sigma).
RNase A (Sigma) was then added at a nal concentration of
100 µg/ml and samples were incubated at room temperature
for 30 min. The DNA contents of the cells were analyzed by a
Becton-Dickinson FACScan ow cytometer.
TUNEL assay: terminal deoxynucleotidyl transferase-
mediated dUTP nick end labeling. PA-1 cells were attached
to Poly-Prep slides (Sigma) and treated with progesterone
100 µM for 48 h. The cells were xed with 10% buffered
formalin and permeabilized by immersing the slides in 0.2%
Triton X-100 solution. After the cells were washed in PBS, the
cells were incubated in equilibrium buffer (200 mM potassium
cacodylated, pH 6.6, 25 mM Tris-HCl, pH 6.6, 0.2 mM DTT,
0.25 mg/ml BSA and 2.5 mM cobalt chloride) containing
biotinylated nucleotide mix and 25 units of terminal deoxy-
nucleotidyl transferase (TdT) at 37˚C for 1 h. The reaction
was stopped by immersing the slides in 2X SSC (0.3 M NaCl
and 30 mM sodium citrate, pH 7.0). The cells on slides were
incubated in 0.3% hydrogen peroxide and then with strepta-
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ONCOLOGY REPORTS 27: 87-93, 2012
89
vidin horseradish-peroxidase (HRP) diluted in PBS. The cells
were incubated with diaminobenzidine (DAB) components,
rinsed several times with deionized water, and mounted in
an aqueous or permanent mounting medium. Each slide was
observed under a uorescence microscope and photographs
were taken at appropriate magnication. All batches of experi-
ments were repeated at least three times.
Analysis of DNA fragmentation. Drug treated-cells were washed
twice with PBS and resuspended in 25 µl PBS. Cells were lysed
by the addition of 25 µl lysis buffer (60 mM Tris, pH 7.4, 50 mM
ethylene diamine tetraacetic acid, and 1.6% sodium lauryl
sarcosine) containing proteinase K, and incubation was carried
out at 50˚C for 3 h, and digestion with 200 µg/ml DNase-free
RNase A for an additional 20 min. DNA from the cell lysates
was then analyzed on a 2% agarose gel containing ethidium
bromide, and visualized and photographed under ultraviolet
light. All batches of experiments were repeated at least three
times.
Detection of apoptosis-related proteins and cyclin-dependent
kinase inhibitors. Cells were lysed in lysis buffer (10 mM
Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 10% glycerol,
and 1% NP-40, 0.1 mM PMSF, 10 µg/ml each leupeptin,
aprotinin, and pepstatin A). Equal amounts of proteins were
subjected to sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and transferred to PVDF membranes
(Millipore, USA). PVDF membranes were incubated with
primary antibodies and then with horseradish peroxidase-
conjugated secondary antibodies. The resulting bands were
visualized by ECL system (Amersham Biosciences).
Primary antibodies used in this study were anti-caspase-3,
anti-poly (ADP-ribose) polymerase (PARP), anti-cyto-
chrome c, anti-BAX, anti-BCL-2, anti-p53, anti-p21, anti-p27,
anti-p16 and anti-Rb. All batches of experiments were repeated
at least three times.
Subcellular fractionation. Cell pellets were resuspended in
sucrose-supplemented cell extract buffer (300 mM sucrose,
10 mM HEPES, pH 7.4, 50 mM KCl, 5 mM EGTA, 5 mM
MgCl
2
, 1 mM DTT, and protease inhibitor mixture). The
cells were homogenized on ice with a dounce homogenizer.
Unbroken cells and nuclei were removed by centrifugation at
2,000 x g at 4˚C for 10 min. The post-nuclear supernatant was
further collected at 10,000 x g for at 4˚C 10 min. The purity
of the mitochondria fraction was determined by the absence of
cytosolic β-actin using Western blot analyses. The supernatant
was further ultracentrifuged at 14,000 x g for 10 min and then
filtered by passing through a 0.22-µm filter (Millipore) to
generate puried cytosolic fraction.
Results
mRNA expression of estrogen and progesterone receptors in
PA-1 cells. Before the effects of progesterone and tamoxifen
were tested, the expression of estrogen and progesterone recep-
tors in PA-1 cells, which have wild type p53, was rst assessed
by RT-PCR. The expression of progesterone and estrogen
receptor mRNAs in PA-1 cells was evident when compared
with the control samples without reverse transcriptase (Fig. 1).
The estrogen receptor mRNA amplicon was 470 base pairs
(bp), and the progesterone receptor mRNA amplicon was
460 bp.
Effects of progesterone and tamoxifen on cell proliferation
and viability. After treatment with progesterone, tamoxifen,
or both hormones, the viability of ovarian cancer cells was
evaluated by using the MTT assay. Progesterone was added at
nal concentrations of 10, 30 or 100 µM to PA-1 cells growing
in DMEM with charcoal-stripped FBS. The PA-1 cells were
Figure 2. Viability of ovarian cancer PA-1 cells when treated with tamoxifen,
progesterone or both. (A) Tamoxifen (0, 1, 3 and 10 µM), (B) progesterone
(0, 10, 30 and 100 µM) or (C) both drugs (10 µM tamoxifen and 100 µM
progesterone) were added to PA-1 cells incubated in charcoal-stripped
DMEM. After incubation for indicated periods (0, 12, 24, 36, 48 and 72 h),
cell growth was assessed by staining with methyl thiazole tetrazolium (MTT)
dye as described in ‘Materials and methods. Viability (%) was dened as the
percent of the absorbance of the drug-treated cells over that of control cells.
All batches of experiments were repeated at least three times and average
values with standard deviations were plotted.
Figure 1. Expression of estrogen and progesterone receptors in ovarian
cancer PA-1 cells. The expression of estrogen and progesterone receptors in
ovarian cancer PA-1 cells was examined through RT-PCR. RT (-) indicates the
absence of reverse transcriptase. The sizes of RT-PCR products for estrogen
and progesterone receptors were 470 and 460 bp. ER, estrogen receptor; PR,
progesterone receptor.
Page 3
LEE et al: COMBINED TREATMENT OF PROGESTERONE AND TAMOXIFEN IN OVARIAN CANCER CELLS
90
incubated for 0, 12, 24, 48 or 72 h. The control cells were not
treated with progesterone. Treatment with 10 µM progesterone
slightly inhibited cell growth as compared to the control cells.
Signicant cell growth inhibition was observed at 100 µM of
progesterone at 48 h of incubation (Fig. 2B).
Similarly, PA-1 cells were treated with 1, 3 or 10 µM
tamoxifen for 0, 12, 24, 48 or 72 h, and the number of the
viable cells was counted. As was observed with progesterone,
the effects of tamoxifen were dependent on concentration.
Treatment with 1 or 3 µM tamoxifen had little effect, but
10 µM of tamoxifen signicantly decreased cell viability at
48 h (Fig. 2A). The effective concentrations of progesterone
and tamoxifen for inhibition of cell growth were 100 and
10 µM, respectively, and these concentrations were used for
subsequent experiments. Co-treatment with 10 µM tamoxifen
and 100 µM progesterone resulted in less pronounced inhi-
bition of growth than observed with either progesterone or
tamoxifen treatment alone (Fig. 2C).
Cell cycle analysis. As 100 µM progesterone and 10 µM
tamoxifen were most effective in inhibiting cell proliferation,
these concentrations were used for cell cycle analysis. The
nuclear DNA content of individual cells was analyzed by
ow cytometry after treatment with 100 µM progesterone,
10 µM tamoxifen, or both to evaluate cell viability and cell
cycle distribution. The peak representing cells in sub-G
0
phase was evaluated after 0, 12, 24 and 48 h of incubation
(Fig. 3). The number of cells in sub-G
0
(M1 phase) increased
markedly after treatment with 100 µM progesterone as incuba-
tion time increased, whereas the fraction of cells in S-phase
(M3 phase) decreased. When PA-1 cells were treated with
10 µM tamoxifen, most cells were in the G
1
stage of the cell
cycle (M2 phase). After co-treatment with tamoxifen and
progesterone, the number of cells in the sub-G
0
stage increased
markedly as incubation time increased, but the fraction of
cells in S-phase decreased. These results suggest that treat-
ment with progesterone and co-treatment with progesterone
Figure 3. Cell cycle analysis of ovarian cancer PA-1 cells treated with tamoxifen, progesterone and both. PA-1 cells were treated with 10 µM tamoxifen, 100 µM
progesterone, or both drugs (tamoxifen 10 µM and progesterone 100 µM), respectively, for 0, 12, 24 and 48 h. Cells were harvested and stained with propidium
iodide as described in ‘Materials and methods. The DNA content of the cells was analyzed and plotted using a Becton-Dickinson FACScan (Caliber) ow
cytometer. Percent populations of M1, M2 and M3 were also calculated and summarized. M1 represents the population of G
0
apoptotic cells. M2 represents
the population of G
1
phase cells. M3 represents the population of S, G
2
and M phase cells.
Page 4
ONCOLOGY REPORTS 27: 87-93, 2012
91
and tamoxifen caused apoptosis because of the increased cell
population in the sub-G
0
phase. Treatment with tamoxifen
resulted in G
1
arrest because of the increased number of cells
in the G
1
phase.
Analysis of progesterone-induced apoptosis. To confi r m
progesterone-induced apoptosis in PA-1 cells, terminal deoxy-
nucleotidyl transferase dUTP nick end labeling (TUNEL)
assay, DNA fragmentation analysis, and Western blot analysis
of apoptotic markers were performed. After treatment with
100 µM progesterone, 10 µM tamoxifen, or both for 48 h,
the number of apoptotic cells with fragmented DNA were
quantitatively determined by TUNEL assay. Apoptotic cells
(uorescent cells) were detected by use of a uorescence micro-
scope (Fig. 4A). Although apoptotic cells were not detected in
the control cells and the cells treated with 10 µM tamoxifen,
apoptotic cells were detected among the cells treated with
progesterone and the combination of progesterone and tamox-
ifen. The results showed that progesterone induced apoptosis
and DNA fragmentation.
Apoptosis ultimately induces the activation of DNA endo-
nuclease, which cleaves DNA into fragments of approximately
180-200 bp. Agarose gel electrophoresis of DNA isolated from
progesterone-treated cells showed the characteristic DNA
fragmentation ladder, indicating that progesterone-treated
cells had the typical characteristics of apoptosis (Fig. 4B).
Apoptosis mediated by p53 is known to involve transcrip-
tional repression of BCL-2 and activation of BAX. To test
whether progesterone-induced apoptosis of PA-1 cells is facili-
tated by this mechanism, PA-1 cells treated with progesterone
were subjected to Western blot analysis with anti-BCL-2 and
anti-BAX antibodies. Changes in the levels of BCL-2 or BAX
were not observed in cells treated with 100 µM of proges-
terone. Western blot analysis of p53 and p21 proteins showed
a time-dependent up-regulation in p53 and p21 proteins. After
incubation for 24 h, the intensities of the p53 and p21 bands
reached their peaks (Fig. 5A).
To further explore the detailed molecular processes of
progesterone-induced apoptosis, the expression of caspase-3,
PARP, and cytochrome c was evaluated in progesterone-
treated cells. Caspase-3 is an effector caspase that is critical
in many apoptotic pathways. When caspase-3 is activated, it is
cleaved into fragments of 11 and 20 kDa. Activated caspase-3
cleaves several key enzymes that are required for normal cell
maintenance, including a DNA repair enzyme PARP (14).
Western blot analysis of PARP showed that PARP is cleaved
from its intact form (116 kDa) into fragments of 85 and 25 kDa.
Western blot analysis also showed decreased intensity of the
caspase-3 band after progesterone treatment (Fig. 5A). This
result suggests that progesterone-induced apoptosis of PA-1
cells involves caspase-3-mediated apoptosis.
To examine the involvement of BAX and cytochrome C,
the cell cytosolic and mitochondrial fractions were separated,
and the content of BAX protein in each fraction was analyzed
by Western blot analysis. The results showed that cytosolic
BAX had mostly disappeared after 24 h and had completely
disappeared after 48 h of progesterone treatment, while the
mitochondrial content of BAX increased after 48 h (Fig. 5B).
These results indicate that translocation of BAX from the
cytosol to mitochondria mediates progesterone-induced
Figure 4. Apoptosis analysis of PA-1 cells treated with tamoxifen and progesterone. (A) The TUNEL assay was performed in PA-1 cells treated with tamoxifen
and progesterone as described in ‘Materials and methods. PA-1 cells were attached to Poly-Prep slides and treated with tamoxifen (10 µM), progesterone
(100 µM), or both (10 µM tamoxifen and 100 µM progesterone) for 0, 12, 24 and 48 h. The cells were then incubated in equilibrium buffer containing biotinylated
nucleotide mix and terminal deoxynucleotidyl transferase (TdT). The cells on slides were incubated with streptavidin horseradish-peroxidase (HRP) and
diaminobenzidine (DAB) and observed under a uorescence microscope. Images were captured at appropriate magnication. (B) DNA fragmentation analysis
was performed to conrm progesterone-induced apoptosis of PA-1 cells. Progesterone or tamoxifen/progesterone-treated cells were lysed using lysis buffer
containing proteinase K and digested with DNase-free RNase A for 20 min. DNA from the cell lysates was then analyzed on a 2% agarose gel containing
ethidium bromide, and visualized and photographed under ultraviolet light.
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LEE et al: COMBINED TREATMENT OF PROGESTERONE AND TAMOXIFEN IN OVARIAN CANCER CELLS
92
apoptosis, presumably through cytochrome C release from the
mitochondria.
Apoptotic mechanisms were also observed after simulta-
neous treatment with tamoxifen and progesterone. Up-regulation
of p53, p21 and p27 proteins was observed, but the levels of BAX
and BCL-2 were unchanged. Cleavage of caspase-3 and PARP
were conrmed by Western blot analysis (Fig. 5C).
Analysis of tamoxifen-induced cell growth arrest. Molecular
changes induced by tamoxifen were analyzed by Western
blot analysis. The p53, p21 and p27 protein levels increased
within 24 h after tamoxifen treatment (Fig. 6). However, the
levels of these proteins decreased after 24 h. Increased p16
and dephosphorylated Rb protein levels were observed 24 h
after tamoxifen treatment. These results suggest that typical G
1
arrest is involved in tamoxifen-induced cell cycle arrest.
Discussion
To investigate the effects of combined progesterone and
tamoxifen treatment on ovarian carcinoma, the growth and
death of epithelial ovarian cancer cells with wild-type p53
were tested. Progesterone has been reported to promote
apoptosis in ovarian carcinoma cells (14) in a p53-dependent
manner (15). Tamoxifen causes cell cycle arrest (G
0
/G
1
arrest)
with up-regulation of both p21 and p27 in susceptible estrogen
receptor-positive breast cancer cells (13). However, whether
combined treatment with progesterone and tamoxifen has
synergistic anti-cancer effects has also not been investigated.
In this study, we demonstrated that progesterone treatment
of ovarian carcinoma cells triggers apoptosis and tamoxifen
treatment induces growth arrest at G
1
. Combined treatment
induced apoptosis similar to that induced by progesterone
alone and had no synergistic effect on progesterone treatment
alone. Flow cytometric analysis showed that progesterone
treatment and combined treatment with both hormones
induced apoptosis after 48 h while tamoxifen treatment alone
induced mostly G
1
arrest (Fig. 3). Furthermore, the TUNEL
assay and DNA fragmentation analysis conrmed that proges-
terone treatment and combined treatment induced apoptosis
(Fig. 4). Western blot analysis revealed the digestion of PARP
and caspase-3 in both progesterone treatment and combined
treatment (Fig. 5). Tamoxifen treatment resulted in G
1
arrest-
related induction of p21 and p16 and dephosphorylation of pRB
(Fig. 6). Combined treatment showed no synergistic effect as
compared to treatment with progesterone alone. We expected
that combined treatment would induce more apoptosis or cell
cycle arrest or cause these events to occur at a faster rate than
either treatment alone. However, the results showed that this
Figure 5. Time course changes in levels of apoptosis-related proteins during
progesterone or progesterone/tamoxifen-treated PA-1 cells. (A) PA-1 cells
were treated with progesterone (100 µM) for 0, 6, 12, 24, 36 and 48 h and
harvested by trypsinization. Cell lysates were subjected to Western blot
analysis using anti-p53, anti-p21, anti-BCL-2, anti-BAX, anti-caspase 3,
anti-PARP and anti-actin antibodies. Actin was used as an internal control.
(B) Cellular localization of BAX was analyzed by subcellular fractionation
and Western blot analysis. Cell pellets were fractionated into mitochondria
and cytosol by centrifugation as described in ‘Materials and methods.
Mitochondrial and cytosolic fractions were analyzed by Western blot analysis
using anti-BAX antibody. (C) PA-1 cells were treated with tamoxifen (10 µM)
and progesterone (100 µM) for 0, 6, 12, 24, 36 and 48 h and harvested by
trypsinization. Cell lysates were subjected for Western blot analysis using
anti-p53, anti-p21, anti-bcl2, anti-bax, anti-caspase 3, anti-PARP and anti-
actin antibodies. Actin was used as an internal control. The experiments were
repeated 3 times.
Figure 6. Time course changes in levels of growth arrest-related proteins
during tamoxifen-induced growth arrest of PA-1 cells. Cells were treated
with tamoxifen (10 µM) for 0, 6, 12, 24, 36 and 48 h and harvested by
trypsinization. Cell lysates were prepared as described in ‘Materials and
methods’. Cell lysates were analyzed for Western blot analysis using anti-p53,
anti-p21, anti-p27, anti-p16, anti-Rb and anti-actin antibodies. Actin was used
as an internal control. Anti-Rb antibody is able to detect both phosphorylated
and dephosphorylated Rb. The experiments were repeated 3 times.
Page 6
ONCOLOGY REPORTS 27: 87-93, 2012
93
did not happen. Currently, we do not have an answer as to why
this occurred.
One plausible explanation is the following. It has been
proposed that the binding of p53 to specific p53 response
elements differs greatly. Low-affinity sites appear to be
associated with growth arrest-related genes, while high-
afnity sites are more related to proapoptotic genes (16,17).
Ubiquitination of p53 in response to mild damage is known to
be associated with growth arrest-related genes, and acetylated
and phosphorylated p53 proteins activate proapoptotic genes
in response to severe damage. In addition, p53-interacting
proteins like HZF interact directly with the p53 DNA-binding
domain to activate p21 while proapoptotic genes are attenu-
ated. In contrast, CAS is associated with p53 on the promoters
of several proapoptotic genes. This relieves the inhibitory
H3K27 methylation within the transcribed region of those
genes, thereby increasing their transcription and facilitating
apoptosis (18,19). Therefore, tamoxifen treatment appears
to cause mild damage in which p53 induces growth arrest
whereas progesterone treatment causes severe damage, which
results in apoptosis. Combined treatment seems to induce a
severe form of damage, resulting in apoptosis similar to the
case of progesterone treatment.
We still do not know the detailed mechanism of how p53
decides between growth arrest and apoptosis in PA-1 cells
subjected to these treatments. Combined treatment is used in
many therapeutic cancer regimes since it sometimes results in
synergistic effects on cancer. Some cell death-inducing agents
that induce the death of cancer cells via different pathways
will exert a synergistic effect. Even combined treatment with
apoptosis-inducing agents will be advantageous if the two
agents involve different apoptosis pathways. However, the
combination of an anti-cancer medicine that induces growth
arrest and another that induces apoptosis may not be recom-
mended as the combination will not have an advantageous
effect. Further detailed characterization of the mechanisms
of action of these medicines in p53-deleted or p53-mutated
ovarian cancer cells may provide a better understanding of
these treatment methods.
Acknowledgements
This study was supported by Priority Research Centers Program
through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2010-0029642).
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