Gynecologic Oncology 106 (2007) 541 548

www.elsevier.com/locate/ygyno

Curcumin down-regulates Ets-1 and Bcl-2 expression in human

endometrial carcinoma HEC-1-A cells
Ziming Yu , Dinesh M. Shah
Department of Obstetrics and Gynecology, University of Wisconsin-Madison, 7E-Meriter Hospital, 202 South Park Street, Madison, WI 53715, USA
Received 14 January 2007
Available online 27 June 2007

Abstract

Objective. Curcumin has been demonstrated to have an anti-tumor activity but the underlying molecular mechanisms are not fully uncovered.
The present study was undertaken to determine the effect of curcumin on the expression of the proto-oncogene Ets-1 and the anti-apoptotic
molecule Bcl-2 in human endometrial adenocarcinoma HEC-1-A cells.
Methods. Confluent HEC-1-A cells were treated with curcumin at various doses for 16 h or at 60 M for various time points. At the end of the
designated treatments, changes in cell morphology, DNA fragmentation and protein contents of Ets-1 and Bcl-2 were determined, respectively, by
light microscopy, DNA laddering assay and Western blot analysis. As an initial step towards understanding whether Ets-1 was a possible up-
stream regulator of Bcl-2 expression in HEC-1-A cells and if so, whether curcumin could attenuate the Ets-1-induced up-regulation of Bcl-2
expression, cells were transiently transfected with an Ets-1/GFP (Green Fluorescence Protein) fusion construct and the transfectants were treated
with 60 M curcumin for 16 h, followed by whole cell lysate preparation for Western blot analysis of Bcl-2 protein contents.
Results. Curcumin induced apoptosis-like morphological changes and DNA degradation and decreased basal levels of Ets-1 and Bcl-2 protein
contents in HEC-1-A cells in a time- and dose-dependent manner. Overexpression of Ets-1 in the cell resulted in an increase in Bcl-2 protein
contents and that increase was attenuated by curcumin treatment.
Conclusions. Curcumin down-regulates Ets-1 and Bcl-2 expression and induces apoptosis in HEC-1-A cells, suggesting a novel molecular
mechanism for the anti-tumor activity of curcumin.
2007 Elsevier Inc. All rights reserved.

Keywords: Curcumin; Endometrial adenocarcinoma; HEC-1-A; Ets-1; Bcl-2

Introduction apeutic agents [3], capable of inhibiting proliferation of a

Curcumin or diferuloylmethane is an orange-yellow crystal-
line (1,7-bis-[4-hydroxy-3-methoxyphenol]-1,6-heptadiene-
3,5-dione) presented in the rhizome (i.e. root) of the plant
Curcuma longa, commonly known as Turmeric [1]. For
thousands of years, curcumin has been widely used in Asian
countries as a dietary spice as well as a home remedy for
treatment of numerous ailments such as biliary disorders,
anorexia, cough, diabetic wounds, hepatic disorders, rheuma-
tism and sinusitis [1]. Because of its relatively low or non-
cytotoxicity [2], curcumin has recently emerged as one of the
most promising and powerful chemopreventive and chemother-
Corresponding author. Fax: +1 608 267 5773.
E-mail address: zyu1@wisc.edu (Z. Yu).
0090-8258/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygyno.2007.05.024
variety of tumor cells in vitro [4] and suppressing in vivo tumor
formation in the skin [5], the stomach [6], the colon [7], the
breast [8] and the liver [9] in animal models.
Over the last few decades, one of the remarkable advances in
tumor biology is the recognition that tumor cells have acquired
capacities: (1) to be self-sufficient in mitogenic/growth signals;
(2) to be insensitive to anti-growth signals; (3) to perform
limitless replication; (4) to undergo sustained angiogenesis and
(5) to achieve tissue invasion and metastasis [10]. All together
these acquired capacities render tumor cells an ability to evade
apoptosis, a cell suicide program to eliminate damaged and
unwanted cells in order to maintain an issue homeostasis [11],
thus performing limitless proliferation in an uncontrolled
manner. As a result, targeting tumor cell apoptosis while
leaving normal cells unaffected has long been the focus in the
search for effective cancer therapies. While most of the agents
542 Z. Yu, D.M. Shah / Gynecologic Oncology 106 (2007) 541548
commonly used at the present time in the chemotherapy of
cancer patients cannot differentiate the tumor from the normal
cell and thus generate intolerable adverse effects [12], curcumin
has differential effects on apoptosis/survival in tumor versus
normal cells; it selectively induces apoptosis in the fast-
proliferating human mammary epithelial carcinoma cells at the
G2 phase of the cell cycle while arresting the normal human
mammary epithelial cells at the G0 phase, thus allowing the
normal cell to escape from the treatment-induced apoptosis
[13].
There are two distinct apoptotic signaling pathways in
mammalian cells [14]. One is the extrinsic or death receptor
pathway that involves members of the tumor necrosis factor
receptor (TNFR) family such as CD95/Fas and TNF-related
apoptosis-inducing ligand-R1 or R2 (TRAIL-R1 or TRAIL-R2)
and the recruitment of procaspase 8 [15] and the other is the
intrinsic or mitochondrial pathway that involves release of
cytochrome c from the mitochondria and a subsequent
activation of caspase 9 through the interaction of cytochrome
c with the apoptotic protease activating factor-1 (Apaf-1) [16].
Curcumin induces tumor cell apoptosis perhaps mainly through
the mitochondrial pathway [4], though it may also activate Fas
receptor/caspase 8 [17].
Several categories of molecules are involved in the
mitochondrial apoptotic cascade and one of them is the Bcl-2
family of proteins [18] consisting of the anti-apoptotic members
Bcl-2, Bcl-XL and Mcl-1 and the pro-apoptotic members Bax,
Bak, Bim, Bid and Bik. It is known that Bcl-2 and Bcl-XL
inhibit release of cytochrome c from the mitochondria, thus
achieving their anti-apoptotic activities [19]. Curcumin has
been demonstrated to down-regulate the expression of Bcl-2 in
several types of carcinoma cells [2022]. Bcl-2 is expressed in
the human endometrial hyperplasia and adenocarcinoma [23],
but no information is available regarding the effect of curcumin
on the expression of Bcl-2 in the human endometrial carcinoma
cells.
The expression of Bcl-2 is tightly regulated. Among the up-
stream regulators of Bcl-2 expression are transcription factors
NF-B and AP-1. Another family of transcription factors that
also play an important role in oncogenesis are the Ets family
proteins [24]. Ets is the mammalian homologue of the viral v-
ets, originally identified in the avian leukemia retrovirus E26
[25]. In the human there are three Ets genes [26], named Ets-1,
Ets-2 and POINTED, which are located on two different
chromosomes. The human Ets-1 gene encodes two distinct
proteins, p51 and p42, respectively, by the full-length mRNA
and its alternatively spliced form. The wild type Ets-1 protein
functions as a tissue-specific transcription factor, transactivat-
ing its target genes by binding to the consensus recognition
motif, 5-GGA(A/T)-3 [26], in the promoter region of the
targets. It has been reported that some of the Ets family
members may directly regulate expression of Bcl-XL.
However, little is known regarding the direct effect of Ets-1
on Bcl-2 expression in the human endometrial carcinoma cells.
In addition, to date no reports are available in the literature
regarding the effect of curcumin on Ets-1 expression in any
mammalian cells.
The present study was designed: (1) to determine the effect
of curcumin on the basal level expression of Ets-1 and Bcl-2 in
human endometrial carcinomas; (2) to investigate whether Ets-1
was a possible upstream regulator of Bcl-2 expression in these
carcinoma cells and if so whether curcumin could abolish the
Ets-1-induced up-regulation of Bcl-2 expression. The human
endometrial adenocarcinoma cell line, HEC-1-A, was used as a
model. This cell line, originally established from a human
endometrial carcinoma, has been commonly used as an in vitro
model in cellular and biological studies of endometrial
carcinomas [27].
Materials and methods
Cell culture
The HEC-1-A cell line was purchased from the American Type Culture
Collection (ATCC, Manassas, VA). For maintenance, the cells were incubated in
HyQ McCoy's 5A medium (Logan, UT) supplemented with 10% FBS (Sigma,
St. Louis, MO) and 100 U/ml penicillin and 100 g/ml streptomycin
(Invitrogen, Carlsbad, CA) (referred to as basal medium hereafter) at 37 C
under 5% CO2 and humidified atmosphere and sub-cultured every 3 days.
Cell treatment
HEC-1-A cells were plated at 2 106cells/dish in 100-mm dishes in the
basal medium. At confluence the cells were washed briefly with 1 PBS and
then treated with curcumin at 0, 20, 40, 60, 80 or 100 M for 16 h and at
60 M for 0, 1, 3, 8, 16 or 24 h in HyQ McCoy's 5A medium supplemented
with 5% FBS to determine, respectively, the dose- and time-dependent effects
on the cell morphology, DNA degradation and Bcl-2 and Ets-1 expression in
vitro. The 16 h time point and the 60 M concentration chosen, respectively,
for the dose effect and time course experiments were based upon the relevant
reports in the literature [28,29] as well as our morphology and DNA
degradation analyses as discussed in the Results section. Curcumin was
purchased from Sigma (St. Louis, MO) and a 250 mM stock solution was
freshly prepared in DMSO before use. To minimize any potential effect of
DMSO, all the control and treatment cultures contained exactly the same final
concentration of DMSO.
Morphologic evaluation and DNA fragmentation assay
At the end of each of the designated treatment cultures, digital images of the
cells were captured using a Spot2 CCD camera connected to a Zeiss Axioscope
microscope, and then subject to DNA laddering assay as previously described
[30]. Briefly, the cells were harvested and fixed in 70% ethanol at 20 C for
24 h. After centrifugation at 800g for 5 min, the cells were resuspended in 40 l
phosphate-citrate buffer, consisting of 192 parts of 0.2 M Na2HPO4 and 8 parts
of 0.1 M citric acid (pH 7.8) and incubated at room temperature for at least
30 min. The cell suspension was centrifuged at 1000g for 5 min. The
supernatant was transferred to a new tube, followed by addition of 3 l of 0.25%
Nonide NP-40 (in water) and 3 l of RNase (1 mg/ml in water). Upon an
incubation for 30 min at 37 C, 3 l of proteinase K (1 mg/ml) was added and the
sample was incubated for another 30 min at 37 C and then loaded on a 2%
agarose gel with ethidium bromide. After electrophoresis, the DNA bands were
visualized with images captured under UV light using the Bio-Rad VersaDoc
Imaging System.
Transient transfection
HEC-1-A cells, maintained in the above maintenance conditions, were
harvested by trypsinization and centrifugation at 100g for 10 min. The cell
pellet was then resuspended in fresh basal medium and plated in 100-mm dishes
at a density of 1 106cells/dish. At the time of the plating, 10 g Ets-1/GFP
 Z. Yu, D.M. Shah / Gynecologic Oncology 106 (2007) 541548
fusion construct (a generous gift from Dr. Dennis K Watson at the Medical
University of South Carolina, Charleston, SC) or GFP (Clontech, Mountain
Review, CA) control vector that was pre-mixed with the transfection reagent
FuGENE 6 (Roche, Indianapolis, IN) based on the manufacturer's instructions
was added drop by drop to the cells. After incubation at 37 C under 5% CO2
and humidified atmosphere for 24 h, the DNA-containing medium was removed
and the transfectants were washed briefly with 1xPBS before they were treated
with 0 M (control) or 60 M curcumin in HyQ McCoy's 5A medium
supplemented with 5% FBS for 16 h. To monitor the transfection efficiency, the
transfectants were checked under a fluorescence microscope (Zeiss Axioscope)
and digital images, both light and fluorescent, were captured using a Spot2 CCD
camera connected to the microscope. For evaluation of changes in Bcl-2 protein
contents in the transfectants, the whole cell lysates were prepared for Western
blot analysis by lysing the cells in boiling lysis buffer (1% SDS, 1.0 mM sodium
orthovanadate and 10 mM Tris pH 7.4), followed by boiling for additional 5 min
and centrifugation at 12,000g for 5 min.
Preparation of cytosolic and nuclear proteins
This was performed as previously described [31]. Briefly, at the end of each
of the designated treatments, the cells were washed briefly with cold 1 PBS and
lysed by scrapping them in 0.5 ml cold hypotonic buffer (10 mM HEPES,
40 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, 0.2% NP-40, 1 g/ml aprotinin,
2 M leupeptin, 1 mM phenylmethylsulfonyl fluoride, 40 mM p-nitrophenyl
phosphate, 1 mM sodium orthovanadate and 50% glycerol). The lysates were
collected and incubated on ice for 5 min and centrifuged at 15,000g for 20 s.
The supernatant was collected and saved as the cytosolic fraction. The pellet (i.e.
cell nuclei) was resuspended in hypertonic buffer (20 mM HEPES, 4200 mM
KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2% NP-40, 1 g/
ml aprotinin, 2 M leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 40 mM
p-nitrophenyl phosphate, 1 mM sodium orthovanadate and 25% glycerol). After
incubation on ice for 60 min, the samples were centrifuged at 15,000g for
20 min and the supernatants were collected and saved as nuclear extracts. Both
the cytosolic factions and nuclear extracts were stored at 80 C before
analyses.
Western blotting
The total protein concentration in the nuclear extracts, the cytosolic fractions
and the whole cell lysates prepared above was determined by the Bradford assay.
Fifteen micrograms of total proteins from each sample was separated by
electrophoresis on 10% SDS polyacrylamide gels. The separated proteins were
transferred to PVDF membranes (Amersham, Piscataway, NJ), using a Semi-dry
blotting system (Portsmouth, NH). After blocking in 5% non-fat milk powder in
Tris Buffered Saline with Tween 20 (TBST) (Sigma, St. Louis, MO) at room
temperature for 1 h, the blots prepared from the nuclear extracts and from the
cytosolic fractions and the whole cell lsyates were incubated at 4 C overnight,
respectively, with a rabbit IgG against the human Ets-1 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) and with a mouse IgG against the
human Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). These two first
antibodies were respectively diluted 1:750 and 1:500 in TBST containing 1%
bovine serum albumin (BSA). After washing in TBST 3 20 min, the blots were
incubated respectively with a goat anti-rabbit and a goat anti-mouse second
antibody conjugated with the horseradish peroxidase diluted in TBST (1:2,500)
containing 1% BSA at room temperature for 45 min. After washing 3 20 min in
TBST, the blots were incubated with the substrate in the mixture of ECL
reagents (Amersham, Piscataway, NJ) at room temperature for 5 min, followed
by exposure to CL-X Posure films (Pierce, Rockford, IL). The chemilumines-
cent signals for Ets-1 and Bcl-2 were developed and analyzed using the NIH
Image J. As a loading control for the cytosolic and whole cell lysate samples, the
same blots were probed for glyceraldehyde-3-phosphate dehydrogenase
(GADPH) with a rabbit polyclonal antibody against human GAPDH from
Abcam Inc. (Cambridge, MA) after being probed for Bcl-2. For nuclear proteins,
no good loading controls are available. As an alternative, the blots were stained
by Ponceau S (Sigma, St. Louis, MO) after being probed for Ets-1. Since
Ponceau S non-specifically stained all protein bands in the blots, the dominant
band with the strongest stain was used as a reference to normalize the density of
Ets-1 band.
543
Statistical analyses
The experiments were repeated four times. The data on the density of
the Bcl-2 and Ets-1 bands in the Western blot analyses, expressed as the fold
(mean SD) of the treatment over the control (i.e. 0 M curcumin dose and/or 0
time point) in the adjusted density of the Bcl-2 or the Ets-1 band (i.e. the ratio
of Bcl-2 to GAPDH or Ets-1 to the dominant band in the Ponceau S staining),
were analyzed by ANOVA together with Bonferroni's t test for multiple
comparisons. Differences at a level of P 0.05 were considered as significant.
Results
Effect of curcumin on the morphology and DNA degradation in
HEC-1-A cells
Curcumin-induced proliferation inhibition and apoptosis in
HEC-1-A cells have been documented [29]. To validate the
apoptotic activity of curcumin in this endometrial carcinoma
cell line, we determined the dose- and time-dependent effect of
curcumin on the cell morphology and DNA fragmentation in
vitro. When cultured in the control medium (i.e. HyQ
McCoy's 5A medium supplemented with 5% FBS), HEC-1-
A cells kept proliferating with time and became over-crowded
by 24 h (Fig. 1A). Compared with the control, treatment of the
cells with curcumin at 60 M in HyQ McCoy's 5A medium
supplemented with 5% FBS resulted in evident disassociation
of the cells and presence of empty spaces in the monolayer
cultures (Fig. 1) as well as DNA degradation (Fig. 2),
indicating cell proliferation inhibition and/or apoptosis, in a
time-dependent manner; the effect appeared at 3 h and became
increasingly obvious at 8 h through 24 h. Based upon this time
course pattern, the 16 h time point was chosen for the dose-
dependent study. Although the effect could be detectable at 3 h
and became more evident at 8 h, treatment for 16 h, on one
hand, would ensure an adequate exposure of the cells to
curcumin so that the treatment effect, particularly in lower
doses, would be detectable and, on the other hand, would not
result in a severe loss in the cell viability. When treated for
16 h, the cells underwent apoptosis-like changes in morphol-
ogy (Fig. 1B) as well as DNA degradation (Fig. 2) in a dose-
dependent manner; the effect was hardly detectable at 20 M
but emerged at 40 M and became increasingly evident at
60 M through 100 M. Based upon the dose-dependent
effect on the cell morphology and DNA fragmentation, a
concentration of 60 M was used in experiments to determine
the time-dependent effect of curcumin on Bcl-2 and Ets-1
expression in that this concentration would induce evident
apoptosis-like morphologic changes and DNA degradation but
not cause a severe loss in the cell viability as 80 M and
100 M would.
Effect of curcumin on the basal level expression of Bcl-2
As presented above, treatment of HEC-1-A cells with
curcumin resulted in proliferation inhibition and apoptosis as
exemplified by the loss in the cell-to-cell contact and cell
viability as well as DNA degradation. We next sought to address
whether the curcumin-induced apoptosis was associated with
544 Z. Yu, D.M. Shah / Gynecologic Oncology 106 (2007) 541548

Fig. 1. Effect of curcumin on the cell morphology. (A) Time-dependent effect. HEC-1-A cells were treated with curcumin at 0 (control) or 60 M. At each of the
indicated time points, the cell morphology was evaluated under a phase contrast microscope with digital images captured. Note that the images at different time points
within the control or curcumin treatment group were captured from the cells in exactly the same field. (B) Dose-dependent effect. HEC-1-A cells were treated with
curcumin at the indicated doses. At the end of the 16 h treatment, the cells were evaluated under a phase contrast microscope with digital images captured.

changes in the anti-apoptotic molecule Bcl-2. To this end, we
determined the protein contents of Bcl-2 in the cytosolic
fractions prepared from the cells treated with curcumin at 0, 20,
40, 60, 80 or 100 M for 16 h or at 60 M for 0, 1, 3, 8, 16 or
24 h by Western blot analyses. As shown in Fig. 3, a specific
band of Bcl-2 of around 26 kDa was detected in all the cytosolic
preparations. After normalization against the loading control of
GAPDH, the protein contents of Bcl-2 decreased in the
curcumin-treated cells compared with the controls in a manner
dependent on the treatment dose (Fig. 3A) and time (Fig. 3B).
For a 16 h treatment, the decrease was significant (P b 0.05) at
the dose of 40 M and higher. At the dose of 60 M, a
significant (P b 0.05) decrease was detected at the 3 h time point
and afterwards.
Effect of curcumin on the basal level expression of Ets-1
To determine if curcumin had any effect on the expression of
the proto-oncogene Ets-1 in HEC-1-A cells, we performed Ets-1
Western blot analyses on the nuclear extracts prepared at the
same time as the cytosolic fractions were prepared for Bcl-2
analyses from the cells treated with curcumin described above.
As shown in Fig. 4, a dominant band of Ets-1 at 51 kDa was
detected in all the nuclear extract preparations and curcumin
decreased the Ets-1 abundance, expressed as the ratio of the
density of the Ets-1 band over the density of the band that was
stained most strongly by Ponceau S in the blot after probed for
Ets-1, when compared with the controls. The time- and dose-
dependent patterns were very similar to those of changes in Bcl-
 Z. Yu, D.M. Shah / Gynecologic Oncology 106 (2007) 541548
Fig. 2. Effect of curcumin on DNA degradation. HEC-1-A cells were treated
with curcumin at 60 M for the indicated time periods or at the indicated doses
for 16 h and then subject to DNA laddering analyses as described in the
Materials and methods section. M: DNA standard marker.
2; for a 16 h treatment, the decrease was significant (P b 0.05) at
the dose of 40 M and higher and at the dose of 60 M, a
significant (P b 0.05) decrease was detected at the 3 h time point
and afterwards.
Fig. 3. Effect of curcumin on the basal level expression of Bcl-2. HEC-1-A cells
were treated with curcumin at the indicated doses for 16 h (A) or at 60 M for
the indicated time periods (B) and then subject to Western blot analyses using
antibodies respectively against human Bcl-2 and GAPDH as described in the
Materials and methods section. The bar graph (n = 4) shows the treatment-
induced fold changes against the control (with the value set arbitrarily as 1) in
the density of the Bcl-2 bands after normalization against that of the GAPDH
bands.
545
Ets-1-induced up-regulation of Bcl-2 expression and
attenuation of this up-regulation by curcumin
To determine whether the transcription factor Ets-1 might be
one of the upstream regulators of Bcl-2 expression in HEC-1-A
cells and if so whether curcumin is able to attenuate the Ets-1-
induced Bcl-2 up-regulation, we transfected the cells transiently
with an Ets-1/GFP fusion construct, treated the transfectants
with curcumin at 0 M or 60 M for 16 h and then analyzed
Bcl-2 protein contents in the whole cell lysates prepared from
the transfectants by Western blot analysis. To eliminate the
possible effect of the expression vector, the transfection reagent
and the transfection procedure on the expression of Bcl-2,
transfection of the cells with the native GFP construct was
included as a control.
As shown in Fig. 5, the cells were transfected at a reasonably
high efficiency. It is estimated that 45% (160/356) of the cells
Fig. 4. Effect of curcumin on the basal level expression of Ets-1. HEC-1-A
cells were treated with curcumin at the indicated doses for 16 h (A) or at
60 M for the indicated time periods (B) and then subject to Western blot
analyses using an antibody against human Ets-1 as described in the Materials
and methods section. The bar graph (n = 4) shows the treatment-induced fold
changes against the control (with the value set arbitrarily as 1) in the density of
the Ets-1 bands after normalization against that of the bands (top) stained most
strongly by Ponceau S.
546 Z. Yu, D.M. Shah / Gynecologic Oncology 106 (2007) 541548
Fig. 5. Micrographs of the HEC-1-A transfectants, showing the cellular localization of Ets-1 and the transfection efficiency. The GFP is located in the whole cell while
the Ets-1/GFP is dominantly in the cell nucleus. It was estimated that 45% of the cells were transfected with the GFP construct and 30% with the Ets-1/GFP fusion
construct.
were transfected with the GFP construct and 30% (102/348)
with the Ets-1/GFP fusion construct. This estimation was based
on the ratio of total number of cells in five random fields
Fig. 6. Effect of overexpression of Ets-1 on Bcl-2 expression in the presence or
absence of curcumin. HEC-1-A cells overexpressing GFP or Ets-1/GFP were
treated with curcumin at 60 M for 16 h and then subject to Western blot
analyses using antibodies respectively against human Bcl-2 and GAPDH. The
bar graph (n = 4) shows the fold changes against the GFP transfectants in the
absence of curcumin (with the value arbitrarily set as 1) in the density of the Bcl-
2 bands after normalization against that of the corresponding GAPDH bands.
(revealed by DAPI staining, mini-graphs not presented) to the
number of cells carrying Ets-1 in the same five fields. Since the
GFP is a small protein with a molecular weight of approxi-
mately 29 kDa and has no nuclear localization signal, capable of
diffusing back and forth between the cytoplasm and the nucleus,
it was distributed uniformly throughout the whole cells. In
contrast, the Ets-1/GFP fusion was predominantly localized in
the cell nucleus, confirming the nuclear localization of the
transcription factor Ets-1 [32].
Presented in Fig. 6 are the results of the Western blot
analyses of Bcl-2 protein contents in the HEC-1-A cells
overexpressing Ets-1/GFP or GPF in the presence or absence of
curcumin. Compared with the cells transfected with the GFP
control vector, the cells transfected with the Ets-1/GFP fusion
construct had a significantly higher level of Bcl-2 protein
contents (P 0.05) and curcumin at 60 M attenuated the Ets-1
overexpression-induced up-regulation of Bcl-2 and decreased
significantly Bcl-2 protein contents in the cells transfected with
the native GFP as well, when compared with the control (i.e.
0 M curcumin).
Discussion
As stated in the National Cancer Institute's Web site [33],
endometrial cancer is the most common gynecologic malig-
nancy, accounting for 6% of all cancers in women. Patients with
endometrial cancer are usually curable by hysterectomy
provided that the lesion is identified at an early, non-invasive
stage and confined locally to the uterus [34]. However, for
 Z. Yu, D.M. Shah / Gynecologic Oncology 106 (2007) 541548
metastatic or recurrent endometrial cancers, the outcome of the
commonly used treatment strategies at the present time such as
radiotherapy, chemotherapy and immunotherapy is not satis-
factory. This is because these conventional therapies are often
associated with significant intolerable cytotoxicity [12].
Due to their potential chemopreventive and chemotherapeu-
tic potency with low toxicity to normal cells, several natural
botanical products have recently drawn a great attention from
both the scientific community and the pharmaceutical industry
in the course of search for effective anti-tumor agents with less
adverse effects. Among them, curcumin has been subject to
intensive investigations. Accumulating evidence suggests that
this polyphenol derived from the spice, turmeric, possesses an
anti-tumor activity while it is not toxic to normal cells [13].
Various categories of gene products have been documented to
be involved in the suppression of tumor cell proliferation
induced by curcumin. These include transcription factors (e.g.
NF-B, AP-1, -catenin and peroxisome proliferator-activated
receptor-), cell cycle proteins (e.g. cyclin D1 and p21), protein
kinases (e.g. protein kinases A and C, Jun N-terminal kinase and
growth factor receptor protein tyrosine kinases), enzymes (e.g.
cyclooxygenase 2, iNOS and hemeoxygenase-1), cell surface
adhesion molecules (e.g. intercellular adhesion molecule-1 and
vascular cell adhesion molecule-1) and cytokines (e.g. TNF, IL-
1 and IL-6) in various cell systems [1]. However, before
curcumin-based cancer therapies are established, more thorough
studies on the molecular mechanisms responsible for the anti-
tumor activity of curcumin are needed. In the present study, we
sought to investigate the functional significance of changes in
the proto-oncogene Ets-1 expression in association with
changes in the anti-apoptotic molecule Bcl-2 expression in
curcumin-induced apoptosis in human endometrial cancers
using the HEC-1-A as a model.
Curcumin-induced apoptosis has been demonstrated in
HEC-1-A cells by Wei et al. [29]. In the present study, we
observed that curcumin induced evident apoptosis-like changes
in the cell morphology (Fig. 1) and DNA degradation (Fig. 2) in
HEC-1-A cells in a time- and dose-dependent manner,
confirming the apoptotic activity of curcumin in this human
endometrial carcinoma cell line.
Bcl-2 is a key anti-apoptotic molecule and it has been
suggested that curcumin-induced tumor cell apoptosis may be
achieved through a Bcl-2-associated mechanism. For example,
curcumin-induced down-regulation of Bcl-2 expression has
been observed in tumor cells in the head and neck, the lung and
the prostate [2022]; overexpression of Bcl-2 has been
demonstrated to be able to completely block the curcumin-
induced apoptotic pathway involving cytochrome c release and
activation of caspases 8 and 3 in the human myeloid leukemic
cell line, HL-60, that is otherwise susceptible to curcumin-
induced apoptosis [35]. In present study we observed that
curcumin down-regulated the basal level expression of Bcl-2 in
a time- and dose-dependent manner in HEC-1 cells (Fig. 3),
supporting a Bcl-2-associated mechanism for the anti-tumor
activity of curcumin.
The transcription factor Ets-1 is known to regulate a wide
variety of biological processes including cell proliferation,
547
differentiation, transformation and apoptosis under both
physiologic and pathologic conditions. Although Ets-1-
mediated apoptosis through up-regulation of pro-apoptotic
genes and down-regulation of anti-apoptotic genes has been
reported in human vascular endothelial cells [36], it is generally
believed that Ets-1 is an anti-apoptotic molecule, as originally
identified as a proto-oncogene, in many other cell types
including immune T- and B-cells [37] as well as tumor cells
[38]. Despite a large number of reports on apoptosis induced by
curcumin in various tumor cells, no literature is available
regarding the effect of curcumin on the expression and
functional activity of this important transcription factor to
date. Here we demonstrated, for the first time, that curcumin
down-regulated the constitutive expression of Ets-1 (Fig. 4) in a
time- and dose-dependent manner in HEC-1-A cells, suggesting
a novel Ets-1-associated mechanism for the anti-tumor activity
of curcumin.
No direct evidence is available regarding the regulation of
Bcl-2 expression by Ets-1. As an initial step towards deter-
mining whether Ets-1 was a possible up-stream regulator of Bcl-
2 expression, we transiently transfected HEC-1-A cells with
either an Ets-1/GFP fusion vector or the native GFP vector and
observed a significant increase in Bcl-2 protein contents in
white whole lysates of the cells transfected with the Ets-1/GFP
construct when compared with the cells transfected with the
native GFP construct (Fig. 5). As one step further, we
determined whether this Ets-1 overexpression-induced up-
regulation of Bcl-2 expression could be attenuated by curcumin.
We observed that when the cells overexpressing the Ets-1/GFP
fusion protein were treated with curcumin at 60 M for 16 h, the
protein contents of Bcl-2 dropped to the level similar to that of
the non-transfected cells. These together indicate that Ets-1 may
be an upstream positive regulator of Bcl-2 expression in HEC-
1-A cells, but this needs to be further validated in the future.
In summary, we have demonstrated, for the first time, in the
present study that overexpression of Ets-1 up-regulates Bcl-2
expression and curcumin down-regulates the constitutive and
Ets-1-induced expression of Bcl-2 as well as the constitutive
expression of Ets-1 in the human endometrial adenocarcinoma
cell line HEC-1-A, in addition to having confirmed curcumin-
induced apoptosis in this cell line. Our data suggest a novel Ets-
1-associated and Bcl-2-dependent mechanism for the anti-
tumor action of curcumin.
Acknowledgments
The authors would like to express thanks to Dr. Dennis K
Watson at the Medical University of South Carolina, Charles-
ton, SC for providing the Ets-1/GFP expression vector. This
study was supported by the New Faculty Start-up Fund awarded
to ZY from the Department of Obstetrics and Gynecology and
the Medical School at the University of Wisconsin-Madison.
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