0013-7227/06/$15.

00/0 Endocrinology 147(9):4179 – 4191

Printed in U.S.A. Copyright © 2006 by The Endocrine Society
doi: 10.1210/en.2006-0168

Pigment Epithelium-Derived Factor Is Estrogen

Sensitive and Inhibits the Growth of Human Ovarian
Cancer and Ovarian Surface Epithelial Cells
Lydia W. T. Cheung, Simon C. L. Au, Annie N. Y. Cheung, Hextan Y. S. Ngan, Joyce Tombran-Tink,
Nelly Auersperg, and Alice S. T. Wong
Departments of Zoology (L.W.T.C., A.S.T.W.), Pathology (A.N.Y.C.), and Obstetrics and Gynecology (H.Y.S.N.), University of
Hong Kong, Hong Kong; Department of Physiology (S.C.L.A.), Chinese University of Hong Kong, Hong Kong; Division of
Pharmaceutical Sciences (J.T.-T.), University of Missouri, Kansas City, Missouri 64110; Department of Ophthalmology
(J.T.-T.), Yale University School of Medicine, New Haven, Connecticut 06520; and Department of Obstetrics and Gynecology
(N.A.), University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Epithelial ovarian carcinoma is the most lethal gynecological tor of PEDF in human OSE. Treatment of the cultured cells
cancer. However, little is known about the molecular mech- with 17␤-estradiol (E2) inhibited the expression of PEDF pro-
anisms underlying the disease development and progression. tein and mRNA in a dose- and time-dependent manner, which
In this study, we found that the expression of pigment epi- could be reversed by the specific estrogen receptor antago-
thelium-derived factor (PEDF) was greatly reduced in ovar- nist, ICI 182,780, indicating that the regulation was estrogen
ian tumors and in ovarian cancer cell lines when compared receptor-mediated. We further showed that this down-regu-
with their normal precursor, ovarian surface epithelium lation of PEDF gene transcription was a direct, primary effect
(OSE). In addition, we showed that exogenous PEDF inhibited of E2. E2 promoted OSE and ovarian cancer cell growth,
the growth of cultured human OSE as well as ovarian cancer whereas simultaneous treatment with E2 and PEDF abro-
cell lines, whereas targeted inhibition of endogenous PEDF gated the estrogenic growth stimulation of these cells. This
using small interfering RNA or neutralizing PEDF antibody study is the first to demonstrate a role of PEDF in OSE biology
promoted the growth of these cells, confirming that the and ovarian cancer and suggests that the loss of PEDF may e of
growth-inhibitory effect was PEDF specific. We also report for relevance in carcinogenesis. (Endocrinology 147: 4179 – 4191,
the first time that estrogen is an important upstream regula- 2006)

O VARIAN CANCER IS the most lethal gynecological

cancer among women in Western countries, and the
vast majority of human ovarian carcinomas are derived from
nal). PEDF action appears to be cell specific. To neurons,
PEDF is neurotrophic, promoting cell survival and differen-
tiation (6 – 8). However, when endothelial cells are exposed
the ovarian surface epithelium (OSE) (1). The OSE is a simple to PEDF, their cell migration and proliferation are inhibited
squamous to cuboidal mesothelial cells that overlies the (9), and they undergo apoptosis (10, 11). Although the spe-
ovary. During each reproductive cycle, OSE on the preovu- cific functions of PEDF in cancer are not known, there is some
latory follicle undergoes apoptosis at the time of ovulation evidence that PEDF could have inhibitory effects on tumor
and then proliferates rapidly to repair the ruptured follicle and growth. First, the levels of PEDF are highest in normal tissue
reconstitutes an intact mesothelium (2). Repeated ovulation is (prostate, liver, and melanocyte), and they decrease during
believed to contribute to OSE neoplastic transformation (3), carcinogenesis (12–15). Second, PEDF has been reported to
indicating that the process of healing damaged OSE may con- have a direct antitumor effect on melanoma (13), osteosar-
tribute to the disease. Therefore, there is great interest in un- coma (16), endometrial (17), and prostate (12, 14) cancer cells
derstanding the cell survival and death signals in these cells. by inhibiting angiogenesis, cell growth, and migration. Fi-
Pigment epithelium-derived factor (PEDF) is a secreted nally, in PEDF-knockout mice, vascular density is markedly
50-kDa glycoprotein first described as an ocular neurotro- increased, and epithelial cell hyperplasia is evident in the
phic protein synthesized by fetal retinal pigment epithelial prostate and exocrine pancreas (12).
cells (4, 5). Subsequent studies demonstrate that it is widely PEDF appears to play an important role in determining the
expressed in most normal tissues (neuronal and nonneuro- tissue growth of many organs, and this may include the
ovary, where a high basal level of PEDF mRNA expression
First Published Online June 15, 2006
Abbreviations: ActD, Actinomycin D; CHX, cycloheximide; E2, 17␤- is found (18). Interestingly, allelic loss in the 17p13.3 region,
estradiol; ER, estrogen receptor; FBS, fetal bovine serum; ICI, ICI 182,780; where the PEDF gene is located, is frequently encountered in
MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide; early-stage ovarian carcinoma (19), implicating PEDF in this
OSE, ovarian surface epithelium; PEDF, pigment epithelium-derived fac- region as a potential tumor suppressor gene regulating the
tor; rPEDF, recombinant PEDF; siRNA, small interfering RNA; TUNEL,
terminal deoxynucleotidyl transferase-mediated nick-end labeling. behavior of ovarian cancers. As yet, however, no study has
Endocrinology is published monthly by The Endocrine Society (http://
examined the regulation of PEDF expression in normal ova-
www.endo-society.org), the foremost professional society serving the ries or ovarian tumors.
endocrine community. The mechanism underlying the regulation of PEDF is

4179

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4180 Endocrinology, September 2006, 147(9):4179 – 4191
largely unknown. Several lines of evidence prompted us to
investigate that one of the mechanisms regulating PEDF
expression in OSE or ovarian cancer cells might involve
estrogen. 1) Estrogen is the major female hormone with mi-
togenic activities on a variety of tissues including the ovary.
Normal OSE and most ovarian carcinomas have specific
receptors for estrogen (20 –22). A role of estrogen in ovarian
carcinogenesis has been suggested; however, the epidemio-
logical data are inconsistent. Some past studies have linked
the use of postmenopausal estrogen replacement therapy to
an increased risk, whereas other reports detected an un-
changed or reduced risk of developing the cancer. Estrogen
taken as oral contraceptives during premenopausal years
seems to offer protection (23–25). Although further investi-
gations are required to resolve how exogenous estrogens
affect cancer risk, numerous studies have demonstrated the
mitogenic action of estrogen in ovarian cancer. For instance,
experimental ovarian tumor could be induced by estrogens
(26, 27), and estrogen stimulated the growth of several es-
trogen receptor (ER)-positive ovarian carcinoma cell lines in
vitro (21, 28, 29). However, little is known about the estrogen-
regulated genes in ovarian tissue. 2) It is of interest to note that
a putative estrogen-response element has been identified in the
5⬘-flanking region of the PEDF gene, further implying that
PEDF may be linked to the regulation of hormone-dependent
growth in the ovary and that in the ovarian cancer (18).
In this study, we aimed to elucidate the role of PEDF in the
tumorigenesis of ovarian cancer and in particular to inves-
tigate the possible regulation of PEDF by 17␤-estradiol (E2)
in ovarian epithelial cells. Our findings indicate that PEDF
plays an important role in regulating the normal OSE cell
function, and its decrease or loss in ovarian tumors could
contribute to tumor development and progression. In addi-
tion, we demonstrate that estrogen-mediated down-regula-
tion of PEDF is a novel way of reducing PEDF levels in OSE
and mediates the effect of E2 on proliferation of these cells.
Materials and Methods
Cell culture and tissue samples
Two immortalized nontumorigenic human OSE cell lines, IOSE-29
and IOSE-397 (30), and the human ovarian epithelial carcinoma cell lines
Caov-3 and SKOV-3 were cultured at 37 C in Medium 199:105 (Sigma,
St. Louis, MO) supplemented with 5% fetal bovine serum (FBS) (Hyclone
Laboratories Ltd., Logan, UT), 100 U/ml penicillin, and 100 ␮g/ml
streptomycin (Invitrogen, San Diego, CA) in a humidified atmosphere
of 5% CO2 in air. All cells were passaged using 0.06% trypsin/0.01%
EDTA (Invitrogen). All experiments were performed using both OSE
lines and repeated two to three times with each experiment yielding
essentially similar results. The results from a representative cell line
(IOSE-397) are shown in Figs. 3 and 5–7.
Samples of primary ovarian carcinomas obtained after surgery were
collected from patients at Queen Mary Hospital, Hong Kong. The his-
tology of all samples was verified by surgical pathologists and found to
contain more than 70% tumor cells. There were 11 serous carcinomas,
two endometrioid carcinomas, and one mixed endometrioid and serous
carcinoma (ages, 31– 62 yr; mean age, 44.9 yr). Normal human OSE cells
(ages, 34 – 47 yr; mean age, 40.5 yr) were derived from surface scrapings
of normal ovaries from women with nonmalignant gynecological dis-
eases. OSE-2 and OSE-3 were cultured in Medium 199:105 with genta-
micin (50 ␮g/ml) and 15% FBS (1). RNA was extracted from cells har-
vested in passage 2. The use of these tissues was approved by the
Institutional Ethical Review Board for Research on the use of human
subjects. Informed consent was obtained from all patients.
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Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF
Treatments
To study the regulation of PEDF expression by E2, 5 ⫻ 105 cells were
plated onto six-well dishes. After 48 h preincubation in phenol red-free
medium supplemented with 2.5% charcoal/dextran-treated FBS (for
removal of endogenous steroids in the serum), the cells were treated
with 0.01–100 nm E2 (Sigma) in fresh medium for 24 h. To test the
specificity of E2 effect, some cells were exposed to a blocker of classical
ER, ICI 182,780 (ICI; Tocris Cookson Ltd., Bristol, UK), 30 min before and
during treatment with E2. To study the stability of PEDF mRNA, cells
were treated with 100 nm E2 for 3 h, and cells without pretreatment
served as controls. Actinomycin D (ActD; 4 ␮g/ml) (Calbiochem, San
Diego, CA) was then added to the cultures, and total RNA was prepared
at the times indicated for up to 8 h. To inhibit protein synthesis, cells were
incubated with 4 ␮g/ml cycloheximide (CHX) (Sigma) for 1 h followed
by the addition of 100 nm E2 for 24 h. Control cultures were treated with
the vehicle (i.e. ethanol) alone. To examine the effect of E2 and recom-
binant PEDF (rPEDF) (Upstate Biotechnology Inc., Lake Placid, NY) on
cell growth, cells were plated for 3-(4,5-dimethyl-thiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) and apoptosis assays. Twenty-four
hours later, the cultures were treated with E2 in the presence or absence
of rPEDF for 5 d, with fresh hormone added every other day.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues that include three normal
ovarian tissues (ages, 23– 47 yr; mean age, 31.3 yr), 13 benign tumors
(ages, 20 – 67 yr; mean age, 36.8 yr), 12 borderline tumors (ages, 24 –57
yr; mean age, 35.9 yr), and 24 ovarian carcinomas (ages, 30 – 69 yr; mean
age, 49.6 yr) (10 serous, six endometrioid, four mucinous, and four clear
cell) were obtained with Internal Review Board approval from the ar-
chives of the Department of Pathology of Queen Mary Hospital, the
University of Hong Kong. Patients were identified anonymous reference
numbers. Five-micrometer sections were deparaffinized and rehydrated
in a graded series of ethanol following standard protocol. Sections were
treated with 3% H2O2 for 10 min to eliminate endogenous peroxidase
activity and then incubated with an anti-PEDF antibody (1:200) (Bio-
products, Middletown, MD) for 30 min at room temperature before
exposure to biotinylated secondary antibodies for 10 min. Antigen-
antibody complexes were visualized using the substrate-chromogen
mixture (Zymed Laboratories Inc., San Francisco, CA) and counter-
stained with hematoxylin. Omission or substitution of the primary
antibody with preimmune serum was used as a negative control. Im-
munoreactivity was assessed by the intensity and percentage of positive
staining. Staining intensity was scored as 0 (negative), 1 (faint), 2 (mod-
erate), and 3 (strong). A case was considered to be negative if there was
no staining or weak staining involving less than 10% cells.
Silencing of PEDF by small interfering RNA (siRNA)
The Smart-pool siRNA for silencing PEDF (catalog no. M-010153-00)
was purchased from Dharmacon (Lafayette, CO). Transfection of the
PEDF-specific siRNA (20 nm) was performed using SilentFect (Bio-Rad,
Carlsbad, CA) per the manufacturer’s instructions. As a nonspecific
siRNA control, scrambled siRNA (Dharmacon) was used. To determine
the efficiency of siRNA transfection, RT-PCR and Western blot analyses
were performed to detect PEDF expression level at 5 d posttransfection.
To examine the role of PEDF in cell proliferation and viability, trans-
fected cells were plated for MTT and apoptosis assays 24 h after siRNA
transfection.
Semiquantitative RT-PCR
Total RNA was isolated from cultured cells using the TRIzol reagent
(Invitrogen) according to the manufacturer’s procedure. First strand
cDNA synthesis was performed from 2.5 ␮g total RNA using Super-
Script Reverse Transcriptase (Invitrogen). cDNA was amplified in a
15-␮l PCR mixture containing 1 mm dNTPs, 1⫻ PCR buffer, 2.5 mm
MgCl2, and 1 U DNA Taq polymerase (Promega, Madison, WI) with 5
pmol each primer for human PEDF (sense, 5⬘-CATTCACCGGGCTCTC-
TAC-3⬘; antisense: 5⬘-GGCAGCTGGGCAATCTTGCA-3⬘). The condi-
tion in the logarithmic phase of PCR amplification was as follows: 5 min
initial denaturation at 94 C, 1 min denaturation at 94 C, 35 sec annealing
Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF
at 67 C, and 1.5 min extension at 72 C for 30 cycles. The number of
amplification cycles during which PCR product formation was limited
by template concentration was determined in pilot experiments. PCR
products were analyzed by agarose gel electrophoresis. ␤-Actin was
used as the internal control (sense, 5⬘-TCACCGAGGCCCCTCTGAAC-
CCTA-3⬘; antisense, 5⬘-GGCAGTAATCTCCTTCTGCATCCT-3⬘). The
reproducibility of the quantitative measurements was evaluated by
three independent cDNA syntheses and PCR amplification from each
preparation of RNA. Densitometric analysis was performed using Scion
Image software (Scion Corp., Frederick, MD), and the relative PEDF
mRNA expression levels were determined as the ratio of the signal
intensity of PEDF to that of ␤-actin.
Real-time PCR
Real-time PCR was performed using the iCycler iQ Real-Time de-
tection system and the IQ SYBR Green Supermix (Bio-Rad). PCR primers
for PEDF were as described above. ␤-Actin was analyzed in the same run
as internal control. Fluorescent measurements were recorded during
each annealing step. The PCR quality and specificity were verified by
melting curve analysis and gel electrophoresis. For quantitative com-
parison, the relative abundance of the PEDF mRNA was determined by
dividing the threshold of each sample by the threshold of the internal
control ␤-actin. These experiments were carried out in duplicate and
independently repeated three times.
Western blot analysis
Cellular extracts were prepared using RIPA [1% Triton X-100, 50 mm
Tris-HCl (pH 7.4), 0.1% SDS, 150 mm NaCl, and 5 mm EDTA, supple-
mented with protease inhibitors containing 1 mm phenylmethylsulfonyl
fluoride, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 1 ␮g/ml pepstatin
A]. Conditioned media were collected and clarified by centrifugation.
Total protein content in each sample was determined using the Bradford
assay (Bio-Rad). Forty micrograms extracted proteins from each cell
culture or 100 ␮g total proteins from the conditioned media was sep-
arated by 7.5% SDS-PAGE and then transferred to nitrocellulose mem-
brane. Membranes were blocked using 5% nonfat dry milk in PBS
containing 0.05% Tween 20 and incubated overnight at 4 C with rabbit
polyclonal human PEDF antibody (1:1000) (Bioproducts) or rabbit poly-
clonal ␤-actin antibody (1:1000) (Sigma) as a loading control. For con-
ditioned media, parallel gels were stained with Coomassie blue to con-
firm equal loading. The immunocomplex was detected with horseradish
peroxidase-conjugated antirabbit IgG (Bio-Rad) and visualized using an
enhanced chemiluminescence detection system (Amersham Biosciences,
Little Chalfont, UK).
MTT assay
Cell viability was assessed colorimetrically by the mitochondria-de-
pendent reduction of MTT (Sigma) to formazan (31). Cells were seeded
at 2000 per well in 96-well plates. After a 5-d incubation period, 10 ␮l
MTT was added to each well, and plates were incubated at 37 C for 4 h.
Finally, culture medium was aspirated, and 100 ␮l DMSO was added to
extract the dye. Conversion of MTT to formazan by metabolically viable
cells was monitored by the absorbance of each well at 570 nm with 630
nm as the reference wavelength. Relative cell viability was expressed as
the fold change over control cultures. Assays were performed in trip-
licates, and data points represent the mean values ⫾ sd from three
independent experiments.
Terminal deoxynucleotidyl transferase-mediated nick-end
labeling (TUNEL) staining
TUNEL assay was performed using an in situ cell death detection kit
(Roche Biochemical, Indianapolis, IN) per the manufacturer’s instruc-
tions. This system end-labels the fragmented DNA of apoptotic cells.
Omission of the enzyme in the TUNEL reaction was used as a negative
control, and cells treated with DNAse I were used as a positive control.
The number of TUNEL-positive cells was counted in five different fields
under a light microscope at ⫻40 magnification, and representative fields
were photographed.
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Endocrinology, September 2006, 147(9):4179 – 4191 4181
Hoechst staining
To confirm the findings of TUNEL assay, apoptotic cells were also
detected by Hoechst staining. Briefly, cells were trypsinized and fixed
with Carnoy’s fixative (methanol/acetic acid, 1:3) for 10 min at room
temperature. After two PBS washes, DNA-specific fluorochrome
Hoechst 33342 (Sigma) was added at a final concentration of 5 ␮g/ml,
and the suspension was incubated at room temperature for 30 min in the
dark. Cell suspensions were mounted on slide glasses and subjected to
immunofluorescence microscopic examination. Apoptotic cells were iden-
tified by chromatin condensation and nuclei fragmentation. The percent-
ages of apoptotic cells were calculated from the ratio of apoptotic cells to
total cells counted. At minimum, 500 cells were counted in five different
fields, and assays were performed in duplicate at least three times.
Promoter constructs and luciferase assay
The PEDF promoter region between positions ⫺864 and ⫹63 was
cloned into promoterless luciferase reporter vector pGL3-Basic as pre-
viously described (32). Cells were transiently transfected with 1 ␮g of the
construct using Lipofectamine 2000 (Invitrogen) as directed by the man-
ufacturer. After 6 h, the cells were incubated with or without E2 (100 nm)
for an additional 24 h before being harvested for luciferase activity
measurement. The pSV-␤-galactosidase plasmid was cotransfected as
internal control. Luciferase units were calculated as luciferase activity/
␤-galactosidase activity and are presented as the mean ⫾ sd of three
individual experiments with triplication. The fold change was calculated
by comparison with the promoterless luciferase vector (pGL3-Basic).
Statistical analysis
Every experiment was repeated at least twice in either duplicate or
triplicate with different cell preparations to ensure consistency of the find-
ings. Statistical analysis was carried out using Fisher’s exact test and
ANOVA followed by Tukey’s post hoc test (GraphPad Software, San Diego,
CA) where applicable. P ⬍ 0.05 was considered statistically significant.
Results
Loss of PEDF in human ovarian cancer
To understand the significance of PEDF in ovarian carci-
nogenesis, we first evaluated the expression of the PEDF
protein in normal (n ⫽ 3), benign (n ⫽ 13), borderline (n ⫽
12), and malignant (n ⫽ 24) ovarian tissues by immunohis-
tochemistry. Our analyses revealed strong PEDF immuno-
staining in normal OSE, cortical stroma, and endothelium
(Fig. 1A, a; and Table 1). In benign serous and mucinous
cystadenomas, a comparable strong epithelial expression
was detected (Fig. 1A, b; and Table 1). Of these, six showed
strong expression of PEDF (46.1%), and five showed mod-
erate expression (38.5%). In contrast, tumors of borderline
malignancy exhibited no or only weak expression level of
PEDF (Fig. 1A, c), and loss of PEDF was confirmed in 22 of
24 tumors (91.7%) (Fig. 1A, d–f; and Table 1). There was no
significant correlation between the PEDF expression and the
clinical data of the tumor histological type (P ⫽ 0.74). Omis-
sion or substitution of the primary antibody with preimmune
serum did not produce any staining (data not shown). We
also performed real-time PCR to verify our immunohisto-
chemistry results with ovarian cancer tissue specimens (OC-
1–14) and normal ovarian epithelium scrapings (OSE-1) or
culture of OSE scrapings (OSE-2 and -3). As shown in Fig. 1B,
PEDF mRNA expression was significantly higher in normal
OSE (n ⫽ 3) compared with ovarian cancer (n ⫽ 14). No sig-
nificant difference was seen in uncultured and cultured OSE
brushings, in which all cases had relatively high levels of PEDF
4182 Endocrinology, September 2006, 147(9):4179 – 4191 Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF

FIG. 1. Immunohistochemical staining

of PEDF in human ovarian tissues. Rep-
resentative tissue sections are shown.
A, Paraffin-embedded tissue sections
of: a, human normal ovary (surface ep-
ithelium indicated by arrows); b, benign
cystadenoma; c, borderline tumor; and
d to f, ovarian adenocarcinomas were
immunostained for PEDF. Sections
were counterstained with hematoxylin
to reveal the nuclei. All sections are
shown at ⫻400 magnification. B, PEDF
mRNA expression was assessed in
three normal OSE (OSE-1–3) and 14
human ovarian cancer cells (OC-1–14)
by real-time PCR with specific primers,
as described in Materials and Methods.
␤-Actin was used as internal control. In
all cases, fold change in PEDF expres-
sion was compared with the ovarian
cancer cell line Caov-3, which was given
an arbitrary value of 1.

expression. Together, these results suggest that PEDF protein with Coomassie blue (Fig. 2C). Consistently, OSE cells se-
diminishes with ovarian neoplastic progression. creted the most PEDF, and ovarian cancer cells secreted
Corroborating the observations in vivo, PEDF expression much less to no detectable PEDF. The presence of a specific
was further confirmed in established ovarian cell lines; the 50-kDa band indicated that PEDF was appropriately syn-
immortalized normal OSE cell lines were found to express thesized and processed.
PEDF at high levels by real-time PCR (Fig. 2A) and Western
blot analyses (Fig. 2B). In contrast, the levels were minimal
Suppression of PEDF leads to increased cell proliferation
to absent in ovarian cancer cell lines Caov-3 and SKOV-3,
and viability
suggesting that the decreased PEDF protein expression may
result from decreased mRNA synthesis. Media conditioned To study the physiological functions of PEDF, we per-
by these cells were tested for PEDF expression by Western formed the MTT cell viability and proliferation assay. As
blot. Equal loading was confirmed by parallel gels stained shown, rPEDF induced a significant decrease in cell growth

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Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF Endocrinology, September 2006, 147(9):4179 – 4191 4183

TABLE 1. Summary of immunoreactivity of PEDF in the ovary

sections

Staining intensity scorea

n
0 (%) 1 (%) 2 (%) 3 (%)
Normal ovary 3 3 (100)
Benign tumors 13 2 (15.4) 5 (38.5) 6 (46.1)
Borderline tumors 12 6 (50) 5 (41.7) 1 (8.3)
Ovarian carcinoma
Serous 10 9 (90) 1 (10)
Endometrioid 6 5 (83.3) 1 (16.7)
Mucinous 4 4 (100)
Clear cell 4 4 (100)
Total 24 22 (91.7) 2 (8.3)
a
0, No staining; 1, weak staining; 2, moderate staining; 3, strong
staining.

when compared with untreated controls (Fig. 3A) (P ⬍ 0.05).

The growth-inhibitory effect of rPEDF was dose dependent
because the effect (decreased by 42.2, 61.2, and 78.9% re-
spectively) increased with concentration of treatment (1, 10,
and 100 nm) (Fig. 3A). The antiproliferative effect of rPEDF
was blocked by the addition of antibodies specific to PEDF
(P ⬍ 0.05), confirming the effect of PEDF was specific (Fig.
3A). The antibody by itself had no effect on cell number (Fig.
3A). The ability of rPEDF to decrease cell number could result
from either decreased cell cycle progression or increased
apoptosis. Our analyses did not detect a significant change
in cell cycle progression as assessed by propidium iodide
staining (data not shown); however, exposure to rPEDF re-
sulted in a concomitant increase of apoptosis with 55.7% of
the cells being TUNEL positive (at 100 nm rPEDF) when
compared with 16.8% in untreated controls (Fig. 3B). The
apoptotic activity was greatly reduced by the addition of
PEDF antibodies. The ability of rPEDF to induce apoptosis
was examined also by counting Hoeschst-stained cells with
fragmented DNA. Similar to the TUNEL staining experi-
ments, a significant increase in rPEDF-induced apoptosis
was observed (Fig. 3B).
RNA interference assay was conducted to further verify
the role of endogenous PEDF in the regulation of cell pro-
liferation and viability. siRNA oligos that specifically target
human PEDF were transiently transfected into OSE cells. As
revealed by real-time PCR and Western blot analyses (Fig.
3C), transfection of PEDF siRNA efficiently repressed PEDF
expression by over 80% at both mRNA and protein levels,
whereas nonspecific siRNA had no effect. Importantly, in-
hibition of PEDF expression by RNA interference resulted in
a significant increase (98.6% increase) in cell proliferation
(Fig. 3D). In addition, PEDF siRNA suppressed apoptosis FIG. 2. PEDF expression in ovarian cells. Expression of PEDF in
(Fig. 3E). No inhibition was observed for nonspecific siRNA immortalized normal OSE and ovarian cancer cell lines was detected
(Fig. 3E). by: A, real-time PCR using primers specific for PEDF and ␤-actin; B,
To examine whether PEDF might also target tumor epi- cell lysates (40 ␮g); and C, conditioned media (100 ␮g) were analyzed
thelial cells, we treated Caov-3 and SKOV-3 ovarian cancer by Western blot for the presence of PEDF proteins. Immunoblotting
for ␤-actin and staining of proteins by Coomassie blue were included
cells with rPEDF proteins in vitro. As shown in Fig. 4A, as loading controls for B and C, respectively. The signal intensity was
rPEDF at 100 nm substantially decreased cell proliferation in determined by densitometry and expressed as the ratio of PEDF
both cancer cell lines (P ⬍ 0.05) when compared with results relative to loading control for each sample (lower panels of B and C).
obtained with untreated controls, and this response was Experiments were repeated three times, and data are shown as
mean ⫾ SD. **, Statistically significant difference between mean
blocked by treatment with an antibody against PEDF (Fig. PEDF expression levels in OSE cells and those observed in ovarian
4A). We also observed a 3.5-fold increase in the rate of ap- cancer cells with P ⬍ 0.005. The fold change in PEDF expression was
optosis in rPEDF-treated cells, with two apoptosis assays compared with the ovarian cancer cell line Caov-3, which was given
confirming the same finding (P ⬍ 0.005) (Fig. 4B). Consistent an arbitrary value of 1.

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4184 Endocrinology, September 2006, 147(9):4179 – 4191 Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF

FIG. 3. Effect of PEDF on cell proliferation and apoptosis of OSE cells. A, OSE cells were treated with increasing concentrations of rPEDF,
rPEDF ⫹ anti-PEDF antibody (anti-PEDF), or anti-PEDF alone for 5 d. Cell growth was assessed by MTT assay as described in Materials and
Methods. The absorbance of wells not exposed to treatments was arbitrarily set as 1, and cell growth after treatment was expressed as the fold
changes compared with the control. B, Samples were stained for TUNEL using immunofluorescent labels according to the manufacturer’s
protocol and Hoechst stains, then photographed (left) and counted to determine the percentage of apoptotic cells (right). C, OSE cells were
transfected with PEDF siRNA or a nonspecific (NS) siRNA for 5 d. siRNA-mediated depletion of PEDF was analyzed by: a, real-time PCR; and
b, Western blot analysis. D, In parallel experiments, cells were collected for MTT assay. E, Apoptosis was detected by TUNEL and Hoechst
staining. The bar graphs summarize the percentage of apoptotic cells counted in five fields from three experiments. Experiments were repeated
three times, and data are shown as mean ⫾ SD. *, P ⬍ 0.05; or **, P ⬍ 0.005 compared with untreated controls or as indicated.

with these results, siRNA-mediated down-regulation of served for nonspecific siRNA (Figs. 4, C–E). These findings
PEDF increased proliferation and decreased apoptosis of indicate a functional role for PEDF in OSE and cancer cell
Caov-3 and SKOV-3 cells (Fig. 4, C–E). No effects were ob- growth and survival.

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Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF Endocrinology, September 2006, 147(9):4179 – 4191 4185

FIG. 4. rPEDF induces apoptotic cell death in ovarian cancer cells. Caov-3 and SKOV-3 cells were cultured and treated with 100 nM rPEDF
and/or antihuman PEDF (anti-PEDF) for 5 d. A, Cell proliferation was assessed by MTT assay as described and expressed as relative fold changes
compared with control. B, In parallel experiments, samples were harvested for TUNEL assay and Hoechst staining, and the bar graphs
summarize the percentage of apoptotic cells counted in five fields from three experiments. C, Cells were transfected with PEDF siRNA or
nonspecific (NS) siRNA. Real-time PCR analysis and Western blotting were performed 5 d after transient gene transfer. In parallel experiments,
cells were collected for MTT assay (D) and TUNEL assay and Hoechst staining (E). Experiments were repeated three times, and data are shown
as mean ⫾ SD. *, P ⬍ 0.05; or **, P ⬍ 0.005 compared with untreated controls or as indicated.

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4186 Endocrinology, September 2006, 147(9):4179 – 4191 Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF

PEDF expression is down-regulated by E2 after the addition of E2, which could be reversed by the
To evaluate the possible effect of E2 on PEDF expression, addition of ICI (Fig. 6D) (P ⬍ 0.05 compared with untreated
cells were preincubated for 48 h in phenol red-free DMEM control). These results demonstrate a direct effect of E2 on the
medium with 2.5% charcoal-stripped FBS to eliminate any human PEDF gene promoter.
endogenous estrogenic effect. Cells were then treated with
different concentrations (0.01–100 nm) of E2 for 24 h. RT-PCR PEDF abrogates the proliferative effect of E2 on OSE cells
analysis was performed using PEDF sequence-specific prim- To evaluate the functional role of E2 in OSE cells, the cells
ers as well as immunoblot analysis for PEDF. Results showed were treated with E2 at a dose range between 0.01 and 100
that E2 markedly reduced PEDF mRNA expression (Fig. 5A, nm for 5 d and assessed by MTT dye conversion. As shown
a). The inhibition was dose-dependent, with more than 90% in Fig. 7A, a dose-dependent increase in cell growth was
decrease at 100 nm. This significant down-regulation of gene clearly observed in E2-treated OSE cultures, with an approx-
expression at the mRNA level is consistent with the changes imately 3-fold increase in cell growth at 100 nm E2. Exposure
in protein levels (Fig. 5A, b and c). To examine the specificity of cells to ICI abolished the E2-induced cell growth enhance-
of the effects imposed by E2, the studies were repeated in the ment (Fig. 7B). ICI by itself had no effect on cell growth (Fig.
presence of a pure ER antagonist, ICI. Treatment with in- 7B). TUNEL staining showed significant decreases of apo-
creasing concentrations (10 nm, 100 nm, or 1 ␮m) of ICI ptosis in E2-treated cells (5.3%) compared with untreated
attenuated E2-induced down-regulation of PEDF mRNA ex- controls (21.8%) (at 100 nm E2) (P ⬍ 0.005) (Fig. 7C). The
pression by 20, 60, and 100%, respectively, reflecting the E2-induced apoptosis was found to be dose-dependent and
mediation of E2 effect through classical ER (Fig. 5B, a). The reversible by cotreatment of cells with ICI (Fig. 7C). The
compound alone did not alter PEDF mRNA expression, even percentage of apoptotic cells determined by Hoechst nuclear
at a concentration (1 ␮m) that caused complete abolition of staining is approximately the same as determined by TUNEL
the E2 effect (Fig. 5B). Similar results were obtained by West- assay (Fig. 7C).
ern blotting with antibodies to PEDF (Fig. 5B, b and c). To demonstrate that the regulation of PEDF expression
could explain some of the effects of E2 on OSE cell growth,
E2 directly regulates PEDF transcription we tested the ability of E2 to trigger proliferation of OSE cells
The down-regulation of PEDF was time-dependent; in the absence or presence of rPEDF proteins. Cotreatment of
mRNA was dramatically reduced by 50% at 3 h, tapering cells with E2 and rPEDF blocked the estrogenic action on
gradually afterward, and reaching a maximum at 24 h after growth stimulation, suggesting a negative role of PEDF in
E2 treatment (Fig. 6A). Decreased PEDF mRNA levels lasted estrogen-induced OSE cell proliferation (Fig. 7, B and C).
at least 48 h after E2 addition (Fig. 6A). This rapid effect of
E2 on PEDF mRNA levels suggests that E2 could directly Effect of PEDF on estrogen-induced cell proliferation in
regulate the expression of PEDF. ovarian cancer cells
To evaluate whether the observed decrease in PEDF Next, we asked whether the effect of estrogen on the reg-
mRNA expression was a direct effect of E2, cells were stim- ulation of PEDF seen in OSE could also be found in ovarian
ulated with E2 in the absence or presence of the protein cancer cells. As shown in Fig. 8A, E2 treatment decreased the
synthesis inhibitor CHX. Treatment of OSE cells with E2 expression of PEDF mRNA in the ER-positive Caov-3 cells
alone or in combination with CHX resulted in a strong re- (2.25-fold). This effect was blocked by ICI (Fig. 8A). In con-
duction of PEDF mRNA expression, suggesting that the in- trast, E2 did not affect PEDF mRNA expression in the estro-
hibition of PEDF gene expression by E2 was not dependent gen-insensitive SKOV-3 cell line (20, 33) (data not shown). To
upon de novo synthesis of an intermediate protein (Fig. 6B). determine the effects of PEDF on estrogen-induced cell pro-
Next, to test whether the effect of E2 on PEDF mRNA ex- liferation, we performed MTT assays. The results indicated
pression was the result of decreased mRNA stability upon E2 that treatment of Caov-3 cells with E2 caused an approxi-
treatment, we performed ActD chase experiments to deter- mately 4-fold increase in cell proliferation, which could be
mine the half-life and stability of PEDF mRNA. OSE cells reversed by the addition of ICI (P ⬍ 0.05) (Fig. 8B). The
were preincubated with 100 nm E2 for 3 h and then treated presence of rPEDF proteins completely attenuated the pro-
with ActD. Total RNA was isolated at 0, 1, 2, 4, 6, and 8 h after liferative effect of estrogen, suggesting the growth-inhibitory
the addition of ActD and analyzed by semiquantitative RT- role of PEDF in modulating the mitogenic action of estrogen
PCR. The apparent half-life of PEDF mRNA was approxi- in ovarian cancer cells. Parallel TUNEL and Hoechst staining
mately 2–3 h in the absence of E2 and was not changed upon demonstrated a decrease in number of apoptotic cells (from
E2 treatment (Fig. 6C). Thus, the decreased PEDF mRNA 13.7 to 3.8%) upon estrogen treatment (Fig. 8C). This effect
level in response to E2 was not due to an effect on PEDF was blocked by ICI and rPEDF.
mRNA stability.
To assess whether E2 causes transcriptional repression at
Discussion
the PEDF promoter, we transfected OSE cells with a lucif-
erase reporter construct of the PEDF promoter region ⫹63 to This study demonstrates that PEDF is an estrogen-respon-
⫺864 and measured luciferase activity after treatment with sive gene and represents the first direct implication of this
100 nm E2. The luciferase reading of the promoterless pGL3- protein in ovarian cancer development and progression.
Basic vector was arbitrary shown as 1. The results showed PEDF is able to induce growth inhibition and apoptosis of
that the activity of PEDF promoter strongly reduced (10-fold) OSE and ovarian cancer cells, and a marked decrease or loss

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Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF Endocrinology, September 2006, 147(9):4179 – 4191 4187

FIG. 5. E2 regulation of PEDF expression in OSE cells. A, OSE cells were incubated in culture medium supplemented with 2.5% charcoal-
stripped FBS for 48 h and, subsequently, were treated with vehicle only or increasing concentrations of E2 for 24 h. B, OSE cells were pretreated
with 10 nM, 100 nM, or 1 ␮M ICI for 30 min before the addition of 100 nM E2 for 24 h. a, Changes in PEDF mRNA levels were determined by
semiquantitative RT-PCR. b, PEDF protein levels were detected by Western blot using specific antibodies to PEDF. c, PEDF in conditioned
media collected from OSE cells treated with increasing concentrations of E2 or E2 and ICI were also analyzed by immunoblot. Immunoblotting
for ␤-actin and staining of proteins by Coomassie blue were included as loading controls for b and c, respectively. The signal intensity was determined
by densitometry and expressed as the ratio of PEDF relative to ␤-actin for each sample. Experiments were repeated three times, and data are shown
as mean ⫾ SD. *, Statistically significant difference when treated samples were compared with untreated controls (P ⬍ 0.05).
of PEDF is noted in most primary tumors and ovarian cancer The cause for the loss of PEDF expression in cancer cells
cell lines when compared with their normal precursor, OSE. is unknown, but it appears that estrogen may be one of the
These results extend the study of Phillips et al. (19), impli- mechanisms in this down-regulation. We showed here that
cating PEDF as a potential tumor suppressor gene regulating E2 treatment resulted in a decrease of PEDF in both OSE and
the behavior of ovarian cancers. ovarian cancer cell lines and that this repression was re-

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4188 Endocrinology, September 2006, 147(9):4179 – 4191 Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF

FIG. 6. E2 reduces PEDF mRNA synthesis through modulation of transcription but not PEDF mRNA stability. A, OSE cells were incubated
in culture medium supplemented with 2.5% charcoal-stripped FBS for 48 h and then cultured in the presence of 100 nM E2 for time points
indicated. B, OSE cells were treated with or without 4 ␮g/ml CHX for 1 h before adding 100 nM E2 for 24 h. Total RNA was extracted and reversed
transcribed followed by PCR with PEDF and ␤-actin sequence-specific primers. C, OSE cells were pretreated with 100 nM E2 for 3 h followed
by the posttreatment with 4 ␮g/ml ActD over a time course of 0, 1, 2, 4, 6, and 8 h. The PEDF mRNA levels before the addition of ActD were
set to be 100%. The signal intensity was determined by densitometry, and the level of PEDF mRNA was normalized against that of ␤-actin.
Experiments were repeated three times, and data are shown as mean ⫾ SD. D, OSE cells were transfected with 1 ␮g reporter construct of PEDF
promoter region from nucleotide ⫹63 to ⫺864. All cells were cotransfected with 0.5 ␮g pSV-␤Gal as an internal control for transcription efficiency.
The results shown are statistics of three repeated experiments. Cells were treated with 100 nM E2 for 24 h. The relative luciferase activities
were calculated relative to the pGL3-Basic vector, which was arbitrarily assigned a value of 1, and data are shown as mean ⫾ SD. *, Statistically
significant difference when treated samples were compared with untreated controls (P ⬍ 0.05).

versed by the addition of a specific ER antagonist ICI, sug- However, in the study of ovarian cancer cell lines, at least one
gesting that ER is involved. It is not known whether this is (SKOV-3) that is ER positive but estrogen insensitive (20, 33)
a mechanism common to all ER-expressing ovarian tumors. showed greatly reduced constitutive expression of PEDF,

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Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF Endocrinology, September 2006, 147(9):4179 – 4191 4189

FIG. 7. Effect of PEDF on E2-mediated cell proliferation and apoptosis in OSE. A, OSE cells were cultured at a density of 2000 cells/well in
a 96-well plate in culture medium supplemented with 2.5% charcoal-stripped FBS. After preincubation for 48 h, the cells were treated with
increasing concentrations of E2 for 24 h. B, OSE cells were treated with E2, ICI, E2 ⫹ ICI, or E2 ⫹ rPEDF for 5 d. Control cells were treated
with vehicle alone. The cell growth was assessed by MTT assay as described in Materials and Methods. The absorbance of wells not exposed
to treatments was arbitrarily set as 1, and cell growth after treatment was expressed as the fold changes compared with the control. C, In parallel
experiments, samples were stained for apoptotic cells with TUNEL and Hoechst stains. *, P ⬍ 0.05; or **, P ⬍ 0.005 compared with untreated
controls or as indicated.

suggesting that other mechanisms might also contribute to the
loss of PEDF expression. Notably, the PEDF gene was mapped
at the 17p13.3, a chromosomal region suspected to harbor tu-
mor suppressor genes and recurrently deleted in ovarian car-
cinomas (19, 34, 35), but whether the ovarian carcinoma cells
and cancer lines that we studied exhibit allelic loss or somatic
mutation of the PEDF gene has yet to be defined. Epigenetic
changes such as promoter hypermethylation may be an alter-
native mechanism (our preliminary observation). It is also pos-
sible that other factors (cytokines and growth factors) may
modulate PEDF expression. Irrespective of the underlying
mechanisms, the findings of frequent loss of PEDF expression
in epithelial ovarian carcinomas suggest that this change in
PEDF may confer a selective advantage to the cells and could
be an important event in the pathogenesis of ovarian cancer.
The OSE that surrounds the ovary is the source of epithe-
lial ovarian carcinomas. Our results indicate that OSE pro-
duces a large amount of PEDF. Moreover, we show that
PEDF is a potent growth inhibitor and/or inducer of apo-
ptosis in OSE cells. Although the regulation of in vivo PEDF
activity in the ovary has not been established, it is tempting
to speculate that the OSE’s response to PEDF is intimately
linked to the apoptosis and mitotic activity of OSE before and
after ovulation. In this regard, our data would suggest that
lower levels of PEDF, which are found in response to high
levels of estrogens released with the follicular fluid at the
time of ovulation, may play a role in the survival and/or
proliferation of OSE cells next to the rupture site (1). Con-
versely, high levels of PEDF, which are present before ovu-
lation, may induce apoptosis in OSE cells surrounding the
apex of the follicle to facilitate the release of oocyte (2). The
process of apoptosis is also critical in eliminating OSE that
are sequestered in inclusion cysts, thereby removing poten-
tial sites of ovarian tumors (36). Therefore, one could spec-
ulate that the loss of PEDF in these cells, and the subsequent
survival of damaged cells, could increase the risk of devel-
oping cancer. Intriguingly, PEDF is also found to be a potent
inducer of apoptosis in ovarian cancer cells. Although the
number of cell lines being examined is too small for definitive
conclusion to be drawn, the fact that PEDF is equally potent

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4190 Endocrinology, September 2006, 147(9):4179 – 4191 Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF

FIG. 8. Effect of PEDF on E2-mediated cell proliferation and apoptosis in ovarian cancer cells. A, Caov-3 cells were pretreated with or without
1 ␮M of ICI for 30 min before the addition of 100 nM E2 for 24 h. Changes in PEDF mRNA levels were determined by semiquantitative RT-PCR.
The signal intensity was determined by densitometry, and the level of PEDF mRNA was normalized against that of ␤-actin (right). B, Effects
of E2 and rPEDF on Caov-3 cell proliferation were assessed by MTT assays. Caov-3 cells were treated with E2, ICI, E2 ⫹ ICI, or E2 ⫹ rPEDF
for 5 d. Control cells were treated with vehicle alone. C, In parallel experiments, samples were harvested for TUNEL assay and Hoechst staining.
Experiments were repeated three times, and data are shown as mean ⫾ SD. *, Statistically significant difference when treated samples were
compared with untreated controls (P ⬍ 0.05) or as indicated.

to induce apoptosis in both estrogen dependent (Caov-3) and
independent (SKOV-3) ovarian cancer cell lines would sug-
gest this protein has potential as new therapeutic strategies
for treatment of estrogen-resistant ovarian tumors. A 32-bp
deletion in exon 1 of ER transcript has been found in the
SKOV-3 cell line, which is ER positive but insensitive to E2
with respect to cell proliferation and induction of gene ex-
pression (20, 33). This may explain the lack of responsiveness
and resistance to E2 in some ovarian cancer. Of particular
interest, our findings may also be pertinent to human breast
cancer. In ER-negative breast cancer cells, exogenous PEDF
also reduces cell proliferation (our unpublished data), sup-
porting a general growth suppressor role for this protein, in
addition to its well-known effect on the tumor vasculature.
Estrogen is a major regulator of growth and differentiation in
normal ovaries. Despite intensive studies on estrogen-induced
growth, very little is known about potential estrogen-regulated
genes in ovarian tissue (21, 26 –29, 37). In the present study, we
demonstrate that E2 lowers the expression of PEDF in OSE and
ovarian cancer cells in vitro. Furthermore, this newly discovered
effect of E2 appears to confer these cells with a growth advan-
tage because in the presence of exogenous PEDF, their mito-
genic response to E2 is attenuated. Thus, the present findings
not only confirm others, which indicate that E2 stimulation of
OSE and tumor cell proliferation (21, 26 –29, 37), but also reveal
its possible interaction with PEDF. Interestingly, we observed
that growth factors such as hepatocyte growth factor and epi-
dermal growth factor could also regulate the expression of
mRNA for PEDF and at least in part counteract the growth-
inhibitory effects of PEDF on OSE (data not shown), suggesting
that other mitogenic signaling pathways may converge with
PEDF to modulate its effects. These factors may also be present
and act to inhibit PEDF signaling in vivo, contributing to the
mitogenic activity of OSE after ovulation. Indeed, the level of
hepatocyte growth factor is highest in the late follicular phase
and during the luteal phase (38). Although the combined effects
of growth factors on OSE cell growth are starting to be inves-
tigated, it has been known for a long time that expression and
action of one factor may affect the expression of other factors
and the cellular response of OSE at any one time depends on
the combined influences of numerous growth factors (39).
In other cell types, E2 has been shown to regulate transcrip-
tional activation of downstream genes as well as transcript
stability (40). Our current findings indicate that E2 decreases
PEDF mRNA in human OSE cells through a predominantly
transcriptional mechanism. 1) There is a rapid decrease of
mRNA level within 3 h after E2 treatment. 2) The use of ActD
suggests that the observed changes in mRNA are due to E2-
regulated mRNA synthesis rather than mRNA stability. Pre-
treatment of cells with CHX has no effect on the E2-mediated
decrease of PEDF mRNA expression, suggesting that E2 effect
does not require de novo protein synthesis. 3) Furthermore, E2
causes transcriptional repression at the PEDF promoter, which
could be reversed by ICI, indicating the regulation is ER me-
diated. This is the first demonstration that PEDF is a target gene
of ER activation. Investigations are in progress to identify and

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Cheung et al. • Down-Regulation and Antiproliferative Role of PEDF
characterize the mechanisms responsible for the E2-mediated
transcriptional repression of PEDF.
In summary, we have shown that PEDF is consistently down-
regulated in ovarian cancer. The identification of PEDF as a new
ER-regulated target gene implicates a novel mechanism to reg-
ulate PEDF expression. Furthermore, our studies are the first to
establish a role of PEDF in normal OSE and tumor growth by
influencing cell proliferation and apoptosis. This highlights the
potential of PEDF as a tumor suppressor and may provide a
target for therapeutic intervention in ovarian cancer.
Acknowledgments
We thank Ms. Mary Chiu and Ms. Stephanie Liu for their help in
tissue sample preparation and Dr. Liao (Pathology, University of Hong
Kong) for assessing immunohistochemical staining.
Received February 9, 2006. Accepted June 7, 2006.
Address all correspondence and requests for reprints to: Alice S. T.
Wong, University of Hong Kong, Department of Zoology, 4S-14 Ka-
doorie Biological Sciences Building, Pokfulam Road, Hong Kong. E-
mail: awong1@hku.hk.
This work was supported by the Hong Kong Research Grants Council
(grants to A.S.T.W.).
The authors have nothing to disclose.
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