Báo cáo khoa học: Dehydroepiandrosterone inhibits the proliferation and induces the death of HPV-positive and HPV-negative cervical cancer cells through an androgen- and estrogen-receptor independent mechanism pptx

Dehydroepiandrosterone inhibits the proliferation and
induces the death of HPV-positive and HPV-negative
cervical cancer cells through an androgen- and
estrogen-receptor independent mechanism
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Roma A. Giron1, Luis F. Montano2, Marıa L. Escobar3 and Rebeca Lopez-Marure1
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1 Departamento de Biologıa Celular, Instituto Nacional de Cardiologıa ‘Ignacio Chavez’, Mexico D.F., Mexico
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2 Laboratorio de Inmunobiologıa, Departamento de Biologıa Celular y Tisular, Facultad de Medicina, Universidad Nacional Autonoma de
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Mexico (UNAM), Mexico
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3 Departamento de Biologıa Celular, Facultad de Ciencias, Universidad Nacional Autonoma de Mexico (UNAM), Mexico

Keywords
androgen receptor; cell proliferation; DHEA;
estrogen-receptor; HPV
Correspondence
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R. Lopez-Marure, Departamento de Biologıa
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Celular, Instituto Nacional de Cardiologıa
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‘Ignacio Chavez’, Juan Badiano No. 1,
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Colonia Seccion 16, Tlalpan, C.P. 14080,
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Mexico D.F., Mexico
Fax: +52 55 73 09 26
Tel: +52 55 73 29 11 ext. 1337
E-mail:
(Received 3 June 2009, revised 21 July
2009, accepted 30 July 2009)
doi:10.1111/j.1742-4658.2009.07253.x

Dehydroepiandrosterone (DHEA) has a protective role against epithelialderived carcinomas; however, the mechanisms remain unknown. We determined the effect of DHEA on cell proliferation, the cell cycle and cell
death in three cell lines derived from human uterine cervical cancers
infected or not with human papilloma virus (HPV). We also determined
whether DHEA effects are mediated by estrogen and androgen receptors.
Proliferation of C33A (HPV-negative), CASKI (HPV16-positive) and HeLa
(HPV18-positive) cells was evaluated by violet crystal staining and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction.
Flow cytometry was used to evaluate the phases of the cell cycle, and cell
death was detected using a commercially available carboxyfluorescein apoptosis detection kit that determines caspase activation. DNA fragmentation
was determined using the terminal deoxynucleotidyl transferase dUTP
nick-end labeling (TUNEL) assay. Flutamide and ICI 182,780 were used to
inhibit androgen and estrogen receptors, respectively, and letrozol was used
to inhibit the conversion of DHEA to estradiol. Our results show that
DHEA inhibited cell proliferation in a dose-dependent manner in the three

cell lines; the DHEA IC50 doses were 50, 60 and 70 lm for C33A, CASKI
and HeLa cells, respectively. The antiproliferative effect was not abrogated
by inhibitors of androgen and estrogen receptors or by an inhibitor of the
conversion of testosterone to estradiol, and this effect was associated with
an increase in necrotic cell death in HPV-negative cells and apoptosis in
HPV-positive cells. These results suggest that DHEA strongly inhibits the
proliferation of cervical cancer cells, but its effect is not mediated by
androgen or estrogen receptor pathways. DHEA could therefore be used as
an alternative in the treatment of cervical cancer.

Introduction
Dehydroepiandrosterone (DHEA) is an adrenal steroid
hormone, a precursor of sex steroids [1], with a wide

variety of biological effects both in vivo and in vitro
however, its physiological role remains unknown.

Abbreviations
DHEA, dehydroepiandrosterone; FLICA, fluorochrome-labeled inhibitors of caspases; HPV, human papilloma virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

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R. A. Giron et al.

Results

160

% of proliferation

140

CASKI
HeLa
C33A

120
100
80

*

*

60

*
*

40

*

***

20

***

0
0

6.25

12.5

25

50

70

100

200

µ
DHEA (µM)
Fig. 1. DHEA inhibits cell proliferation. Cervical cancer cell lines
were treated with 6.25, 12.5, 25, 50, 70, 100 or 200 lM of DHEA
for 48 h. Cell proliferation was evaluated by crystal violet staining
as described in Experimental procedures. The results are expressed
as percentages with respect to untreated cells (0). The results
shown are for an experiment representative of three independent
assays. Asterisks indicate P values < 0.01 compared with control
cells.

140
120

MTT reduction (%)

DHEA is considered to exert its action through conversion to other steroids [1], but there is evidence
showing that DHEA activity is estrogen-independent
[2–4]. In animal models, DHEA has been shown to
have chemoprotective properties against a variety of
diseases: obesity, diabetes, immune disorders, cancer
and atherosclerosis [5,6], as a result of its antiproliferative, anti-inflammatory and anti-oxidant effects [7–9].
DHEA is a powerful inhibitor of carcinogenesis, in
the early- and late-progression stages, of liver, colon,
lung, skin, thyroid, mammary and prostate cancers
[10–16]. DHEA also decreases the incidence of spontaneous breast cancer development in C3H female mice
[17] and the spontaneous emergence of lymphomas in
p53-negative mice [18], and inhibits partially cervical
carcinogenesis induced by methylcholanthrene in mice
[19]. Long-term use of intravaginal DHEA (150 mg
per day) promoted regression of low-grade cervical
dysplasia in 83% of the patients; its local application
was shown to be safe and well tolerated [20].
Cervical cancer is the most common gynecological
cancer in women between 25 and 55 years old, and it
is the second most common cause of death from cancer among Mexican women [21]. Therefore, the aim of
this work was to evaluate the effect of pharmacological
doses of DHEA on the proliferation and death of
three cell lines derived from human cervical cancers
associated with human papilloma virus (HPV) and

positive for the estrogen receptor, and to determine
whether the effect of DHEA was dependent on its
conversion into testosterone or estradiol.
We found that DHEA inhibits the proliferation of
HPV-positive and HPV-negative cervical cancer cell
lines independently of its conversion to testosterone or
estradiol, and also found that DHEA induces apoptotic and necrotic cell death. Taken together, these
results suggest that DHEA could be used in the treatment of cervical cancer.

DHEA and cervical cancer

CASKI
HeLa
C33A

100
80

*

60

*

*
*

*

*

*

*

40

**
*

*

*

20

*

100

200

0
0

6.25

12.5

25

50

DHEA (µM)
µ

70

Fig. 2. DHEA decreases cell viability. Cells were cultured without
and with DHEA at concentrations of 6.25, 12.5, 25, 50, 70, 100 and
200 lM. The percentage MTT reduction was evaluated 48 h later,
as described in Experimental procedures. The results are expressed
as percentages with respect to untreated cells (0). The results
shown are for an experiment representative of three independent
assays. Asterisks indicate P values < 0.01 compared with control
cells.

DHEA inhibited cell proliferation and decreased
cell viability
Three cell lines were evaluated: non-HPV-infected cells
(C33A) and cells infected with human papilloma virus
type 16 (CASKI) or type 18 (HeLa). DHEA inhibited
the proliferation of all the cell lines. It induced a 40%
decrease at 25 lm concentration in C33A cells; higher
concentrations of DHEA were required in the HPVpositive cell lines to achieve a similar inhibitory
decrease (Fig. 1). The effect of DHEA was dose-dependent, with half maximal inhibitory concentrations

(IC50) of 50, 60 and 70 lm for C33A, CASKI and
HeLa cells, respectively. The sulfate ester form of
DHEA had no effect on proliferation (data not

shown).
As shown in Fig. 2, treatment of cells with DHEA
inhibited the reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). The inhibitory effect commenced in the 25 lm range in all three
cell lines, indicating a decrease in cell viability. This

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DHEA and cervical cancer

DHEA concentration induced a 50% inhibitory effect,
but a three-fold increase in DHEA concentration was
needed to obtain 75% inhibition.
The antiproliferative effect induced by DHEA is
independent of androgen and estrogen receptors
DHEA is converted to sex steroids, and cervical cancer
cell lines have estrogen and progesterone receptors
[1,22]; therefore, we evaluated whether the DHEA
antiproliferative effect was related to possible conversion to testosterone or estradiol. In order to assess
this, antagonists to androgen and estrogen receptors
(flutamide and ICI 182,780, respectively), and an inhibitor of the aromatase responsible for conversion of
androgen to estrogen (letrozol), were used alone or in
combination with DHEA before evaluation of cell
A

C33A

% of proliferation

120

**

60

DHEA did not induce cell-cycle arrest
Figure 4 and Table 1 show that DHEA decreased the
percentage of cells in the G1 phase of the cell cycle
compared with non-DHEA-treated cell lines. This
ICI
Flutamide
Letrozol

100
80

proliferation. Our results showed that, at the highest
concentration, letrozol modified the cell proliferation
in the three cell lines, but not significantly (Fig. 3).
Androgen and estrogen receptor inhibitors did affect
proliferation but not significantly, and there was no
difference between the response of each cell line. When
the inhibitors were used in combination with DHEA,
none of them was able to abrogate the inhibition
induced by DHEA, indicating that DHEA has a direct

effect on the proliferation independent of its conversion to other metabolites (Fig. 3).

*

** *

*
** * * *

40
20

*

0
0

1

10

100

1+D

10 + D

100 + D

DHEA

Concentration (nM)

% of proliferation

B 120

CASKI

100

ICI
Flutamide
Letrozol

80
60

** * * *
** * * *
*
*

40
20
0
0

1

10

100

1+D

10 + D

100 + D

DHEA

Concentration (nM)

HeLa

120

% of proliferation

C

ICI
Flutamide
Letrozol

100
80

* **

60
40

* *
*

* **

*
* *

100 + D

DHEA

20
0
0

1

10

100

1+D

Concentration (nM)

5600

10 + D

Fig. 3. The antiproliferative effect induced
by DHEA is independent of androgen and
estrogen receptors. C33A (A), CASKI (B)
and HeLa (C) cells were cultured with half
the maximal inhibitory concentration of
DHEA (IC50) alone or in combination with
flutamide, ICI 182,780 or letrozol at 1, 10
and 100 nM. Cell proliferation was measured
by crystal violet staining 48 h later, and the
results of the experiments are expressed as
percentages with respect to untreated cells
(0). All inhibitors were added 2 h before
DHEA. D, DHEA. *P < 0.01 compared with
the control.

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DHEA and cervical cancer

decrease was associated with an increase in the percentage of cells with a smaller amount of DNA in the
so-called sub-G1 phase, thus indicating cell death.
CASKI cells were the most responsive to the toxic

effect induced by DHEA, with an increase of cells in
the sub-G1 phase of 34%; interestingly, C33A (HPVnegative) and HeLa cells (HPV-positive) showed a
lower percentage of cell death in comparison with control cells (Table 1). These results suggest that the effect
of DHEA upon CASKI cells is more cytotoxic than
cytostatic.

Table 1. Percentage of cells in each phase of the cell cycle as
evaluated by flow cytometry.
Percentage of cells in the phases of the
cellcycle
G1
C33A
CASKI
HeLa

DHEA induces apoptotic and necrotic death

G2 ⁄ M

Cell death

40
36
46
22
47
41

Control
DHEA

Control
DHEA
Control
DHEA

S
21
18
14
9
13
15

15
14
9
4
9
8

24
32
31
65
31
36

untreated cells (Fig. 5). The morphology of CASKI
and C33A cells changed strongly after treatment with
cisplatin or DHEA, and the cell number was reduced

dramatically (Fig. 5A,B), whereas HeLa cells showed
fewer morphological modifications and were more
resistant to treatment with cisplatin and DHEA
(Fig. 5C). Because the TUNEL assay detects DNA
fragmentation, which can occur as a result of necrotic

To determine the type of death induced by DHEA,
cells were analyzed for apoptosis using the terminal
deoxynucleotidyl transferase dUTP nick-end labeling
(TUNEL) assay. Cisplatin was used as a positive control to induce apoptotic cell death. Cisplatin and
DHEA treatments resulted in apoptosis of both
HPV-positive cells and C33A cells, in comparison with

40

DHEA

40

Control

30
0

0

10

Counts
20

G2/M

10

C33A

S

Counts
20

30

G1
CD

200

400
600
FL2-A

800

1000

0

200

400
600
FL2-A

800

1000

400
600
FL2-A

800

1000

0

200

400
600
FL2-A

800

1000

0

200

400
600
FL2-A

800

1000

Counts
20
10
0

0

40

40

30

30

Counts
20

Counts

20

10

10

0

HeLa

0

Fig. 4. DHEA does not induce cell-cycle
arrest. Cells were cultured with and without
(control) DHEA (IC50) for 48 h. Histograms
show the percentage of cells in each phase
of the cell cycle as evaluated by flow cytometry (see Experimental procedures). The
percentage of cells in each phase of the cell
cycle was analyzed using Modift software
(Becton Dickinson). The results shown are
for an experiment representative of three
independent assays. CD, cell death.

200

30

30
Counts
20

10

CASKI

0
40

40

0

0

200

400
600
FL2-A

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800

1000

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DHEA and cervical cancer

A

TUNEL

DAPI
C33A

Phase contrast

Control

Cisplatin

DHEA

B

CASKI

Control

Cisplatin

DHEA

C

HeLa

Control

Cisplatin

DHEA

5602

Fig. 5. DHEA induces apoptotic death.
C33A (A), CASKI (B) and HeLa (C) cells
were cultured with and without DHEA (IC50)
for 48 h. Cisplatin (40 nM) was used as a
positive control to induce death. DNA
fragmentation was detected by TUNEL
assay as described in Experimental procedures. Cells were counterstained with
4¢,6-diamidino-2-phenylindole. The images
were obtained using a phase contrast
microscope, and correspond to an experiment representative of three independent
assays.

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DHEA and cervical cancer

104

101

102
FL1-H

103

104

100

104
FL1-H
102 103
101
104

101

102
FL1-H

103

104

101

102
FL1-H

103

104

100

101

102
FL1-H

103

104

101

102
FL1-H

103

104

101

102

FL1-H

103

104

FL1-H
102 103
FL1-H
102 103

104

100

101

101

FL1-H
102 103

104

100

100

101

FL1-H
102 103
100

101

FL2-H
102 103
100
FL2-H
102 103
100

101

HeLa

103

104

103

102
FL1-H

100

102
FL1-H

104

100

101

100

101

101

CASKI

100

100

101
104

104

103

100

EAC

102
FL1-H

100

100

LC
101

104

100

DHEA

FL1-H
102 103

LAC

FL2-H
102 103

NC

101

C33A

Cisplatin
104

104

Control

100

Fig. 6. DHEA also induces necrotic death. Cells were cultured with and without DHEA (IC50) for 48 h. Cisplatin (40 nM) was used as a
positive control to induce cell death. Cells were labeled with FLICA (FL1-H) and propidium iodide (PI) (FL2-H). Left lower panels, living cells
(LC); right lower panels, early apoptotic cells (EAC); left upper panels, necrotic cells (NC); right upper panels, late apoptotic cells (LAC).
Non-stained cells served as negative control. Results correspond to an experiment representative of three independent assays.

as well as apoptotic degradation, the type of cell death
was determined using fluorochrome-labeled inhibitors
of caspases (FLICA) and propidium iodide, which can
distinguish between apoptotic and necrotic cells,
respectively. In C33A cells, DHEA was a more potent
inducer of cell death by necrosis than cisplatin was
(Fig. 6). On the other hand, CASKI and HeLa cells
showed higher early and late apoptosis than C33A
cells (Table 2). These results indicate that DHEA can
induce early and late apoptosis and also necrosis.

Discussion
DHEA is an intermediate in the biosynthesis of androgen and estrogen hormones. It was originally isolated
from the adrenal gland, but it is also synthesized in
extra-adrenal tissues such as the ovary and testis; due
to its solubility, it diffuses into the bloodstream where

it is found in equilibrium with its sulfated form [1]. The

levels of DHEA and sulfated DHEA decline dramatically with age in humans of both sexes, as the incidence
of most cancers rises. Low levels of these adrenal
steroids have been associated with the presence and risk
of development of cancer. Oral administration of
DHEA to mice inhibits spontaneous breast cancer and
chemically induced tumors of the lung and colon [7];
however, its effect in cervical cancer remains unknown.
Therefore, we evaluated the effect of DHEA on three
cell lines of cervical cancer that are positive to estrogen
receptor [22]: (a) an invasive carcinoma of the cervix,
with poorly differentiated cells but negative for HPV
(C33A), (b) a small bowel metastasis of an epidermoid
carcinoma of the cervix, which was HPV16-positive
(CASKI), and (c) an epithelial-like cell line derived
from an cervical adenocarcinoma at IV-B metastatic
stage and positive for HPV type 18 (HeLa).
The results show that DHEA strongly inhibits the
proliferation of all cell lines, as determined by violet

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DHEA and cervical cancer

Table 2. Percentage of cells alive and dead as determined by flow
cytometry.
Percentage of cells
Alive
C334

Control
Cisplatin
DHEA
CASKI Control
Cisplatin
DHEA
HeLa Control
Cisplatin
DHEA

Early apoptosis Late apoptosis Necrosis

93.1
0
63.06 0
46.78 0
91.54 4.88
65.46 19.9
78.52 8.08
93
1.8
40.28 47.1
34.98 21.46

0.12
0.44
0.38
2.24
8.84
7.04
4.08
10.16
31.68

6.78
36.50
52.84
1.34
5.8
6.36
1.12
2.46
11.88

crystal staining and MTT reduction, independently of
the HPV type. Several studies have found an antiproliferative effect induced by DHEA in normal cells such
as T lymphocytes, isolated neurons and endothelial
cells, or malignant cell lines such as human hepatoblastoma cells (HepG2), colon adenocarcinoma cells
(HT-29) and breast cancer cells (MCF-7) [3,23–26].
Our results are the first evidence for an antiproliferative effect of DHEA on cervical tumor cells. There was
a non-statistically significant difference in the response
of the cell lines to treatment with DHEA. C33A and
CASKI cells were more responsive to DHEA, and

HeLa cells were the most resistant. This might be
related to the malignant state of the cells. HeLa cells
are an advanced-stage cervical cell carcinoma [27], in
comparison with the other cell lines used for which no
stage is specified; therefore, HeLa cells could be more
resistant to antiproliferative factors. The E6 protein
from HPV18 is related to the regulation of G0 ⁄ G1
phases in the cell cycle; this effect is altered by mutations in p53 [28]. C33A cells are known to have a nonfunctional p53 protein due to mutations [29], whereas
CASKI and HeLa cells possess a non-mutant p53
protein. Given that p53 is associated with an antiproliferative effect, the high resistance of both cell lines to
DHEA might be associated with a non-p53-related
mechanism. It has been shown that p53 protein levels
are quite low in cell lines derived from cervical tumors
[30]. DHEA-induced cellular effects in hyperplastic
and premalignant (carcinoma in situ) lesions in mammary gland of rats are associated with increased
expression of p16 and p21, but not p53, implying a
p53-independent mechanism of action [31]. It will be
interesting to determine whether other proteins that
control the cell cycle are involved in the effects induced
by DHEA in cervical cancer.
It has been suggested that HPV18 increases the
susceptibility of cells to inhibitory factors. Similarly,
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immortalization is dramatically increased in HPV16infected human keratinocytes [32]. It is probable that
our HPV-infected cell lines could not respond to low
DHEA concentrations because of the presence of a
multidrug resistance gene that is expressed in a different way [33]. Nevertheless, it is interesting to observe
that HeLa cells, which are HPV18-positive are also
resistant to the antiproliferative effect of ceramide [34].

Resistance to apoptosis and radiation in cervical cancers are also determined by transcription factors such
as hypoxia inducible factor-1 alpha [35], and DHEA is
known to alter this transcription factor, decreasing its
accumulation in human pulmonary artery cells [36].
DHEA can be converted to testosterone and then to
estradiol by the P450 aromatase. It has been shown
that approximately 35% of cervical carcinomas express
aromatase [37] and that DHEA binds to the androgen
receptor and estrogen receptors a or b [38–41]. DHEA
at 30 nm is sufficient to activate transcription of estrogen receptor b to the same degree as estrogen at its
circulating concentration [42]. We showed that the
inhibition of proliferation induced by DHEA is independent of its conversion to estrogen and androgen,
because use of antagonists to androgen and estrogen
receptors (flutamide and ICI 182,780, respectively),
and letrozol, an inhibitor of the aromatase responsible
for converting androgen to estrogen, did not abrogate
the antiproliferative effect induced by DHEA; however, our results cannot discount the possible conversion of DHEA to 5-androstenediol, a steroid that has
been demonstrated to be a biologically active estrogen
[43,44]. Despite the fact that the cervical cancer cell
lines used in this investigation express estrogen receptor and progesterone receptor genes [45,46], our results
showed that DHEA does have a direct inhibitory effect
in these cells. A direct effect of DHEA is supported by
the fact that progesterone and estradiol have an opposite effect on the growth of cervical cancer, i.e. they
induce their proliferation [47].
DHEA can exert various effects depending on its
concentration. In this work, the effects induced by
DHEA were seen at concentrations between 50 and
70 lm. We also observed that low concentrations of
DHEA (physiological concentrations) increased the
proliferation of CASKI cells. We previously showed

that DHEA plays differential roles depending on its
concentration. In MCF-7 cells, DHEA at 100 lm
inhibits cell proliferation, but has a proliferative effect
at physiological concentrations. Other studies have
also shown that DHEA at concentrations of 25–50 lm
inhibits the proliferation of MCF-7 cells [48], and that
lower concentrations induce stimulation [49,50]; however, the mechanism of this differential effect is

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R. A. Giron et al.

unknown. This differences have also been observed in
neuronal cell cultures, in which DHEA has a protective role at concentrations ranging from 0.1–1 lm, but
a pro-oxidant ⁄ cytotoxic effect is seen at higher concentrations [25]. It has been shown that the HPV status in
cervical cancer cell lines is related to a differential
expression of IGF/insulin receptors [51]. We previously
showed that the antiproliferative effect induced by
DHEA in MCF-7 cells is also androgen and estrogen
receptor-independent [3]. These results indicate that
DHEA acts through activation of a putative receptor
rather than through conversion to other steroid hormones. Recently, Liu et al. [4] showed a cytoprotective
role of DHEA on endothelial cells which is estrogen
receptor-independent. They also showed that DHEA
binds to specific receptors on plasma membranes of
endothelial cells, and that this receptor activates intracellular G proteins (specifically Gai2 and Gai3) and
endothelial nitric oxide synthase [52]. There is evidence
showing that the binding of [3H]-DHEA to plasma

membranes is highly specific [53]. Closely related steroid structures such as sulfated DHEA, androstenedione, 17a-hydroxypregnenalone, testosterone and
17b-estradiol did not compete with [3H]-DHEA for
binding at various concentrations. The absence of
competition between DHEA and sulfated DHEA suggests that the 3-position of the A ring may be an
important component of the functional group for this
receptor [39]. More recently, it has been shown that
the anti-atherogenesis effect of dehydroepiandrosterone
does not occur via its conversion to estrogen [53].
These results support the conclusion that DHEA is the
active form and can act in a direct way, independent
of whether it is bound to androgen or estrogen receptors or is converted to other metabolites.
The antiproliferative effect of DHEA has been associated with an arrest of the cell cycle and cell death in
BV-2 cells, a murine microglial cell line, in hepatoma
cell lines and in HepG2 cells [25,54,55]. Our results
showed that pharmacological concentrations of DHEA
interfere with cell proliferation by inducing cell death
without inducing cell-cycle arrest. In contrast, a protective role against apoptosis has been shown at physiological concentrations of DHEA in neurons [56];
similar DHEA concentrations act as a survival factor
in endothelial cells by triggering the G-alpha-1 G-protein-phosphoinositide 3-kinase/AKT protein familyBcl-2 protein (Gai-PI3K ⁄ Akt-Bcl-2) pathway to protect
cells against apoptosis [4]. An interesting observation
was that, in the HPV-negative cell line, cell death was
primarily due to necrosis, whereas the death was
secondary to apoptosis in both HPVpositive cell lines.
It is not known whether HPV infection confers some

DHEA and cervical cancer

kind of resistance to the necrotic process, although one
would imagine that HPV-infected cells possess mechanisms that immortalize them more easily than nonHPV-infected cells. It has recently been demonstrated
that HPV protein E7 induce S-phase entry in keratinocytes [57], thus favoring activated proliferation of the

cells, and thus major resistance to the cytotoxic effects
of DHEA.
Our results suggest that the cell-death mechanism in
cervical cancer is dependent on the presence or not of
HPV, and also demonstrate that DHEA is highly
effective in non-HPV-infected cancer cells. We therefore believe that alternative therapeutic approaches
should be considered in the treatment of cervical cancer. DHEA could be useful in the treatment of cervical
cancer, either alone or in synergy with other drugs,
depending on the HPV status.

Experimental procedures
Materials
RPMI-1640, Dulbecco’s modified Eagle’s medium and trypsin were purchased from Gibco ⁄ BRL (Grand Island, NY,
USA). Fetal bovine serum was purchased from HyClone
(Loga, UT, USA). The carboxyfluorescein FLICA apoptosis detection kit was purchased from Immunology Technologies (Bloomington, MN, USA). Sterile plastic material for
tissue culture was purchased from NUNC (Rochester, NY,
USA) and COSTAR (Lowell, MA, USA). Flow cytometry
reagents were purchased from Becton Dickinson Immuno´
cytometry Systems (San Jose, CA, USA). ICI 182,780 was
purchased from Tocris Cookson Inc. (Ellisville, MO, USA)
´
´
and letrozol from Novartis (Mexico City, Mexico). The
Apoptag Red in situ apoptosis detection kit was obtained
from Chemicon International (Temecula, CA, USA).
DHEA and all other chemicals were purchased from Sigma
Aldrich (St Louis, MO, USA).

Cell culture
CASKI, HeLa and C33A cells were purchased from the

American Type Culture Collection (Manassas, VA, USA).
CASKI and HeLa cell lines were maintained in RPMI-1640
medium and C33A cells in Dulbecco’s modified Eagle’s
medium, both supplemented with 5% fetal bovine serum
and l-glutamine (2 mm). Cells used for the experiments
were cultured in their respective medium supplemented with
5% charcoal-stripped serum and without red phenol.

Cell proliferation
The number of cells was evaluated by crystal violet staining. Cells were plated in 96-well plates and cultured with

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R. A. Giron et al.

DHEA and cervical cancer

various concentrations of DHEA alone or in combination
with either the androgen or the estrogen receptor inhibitor.
After 48-h incubation, cells were fixed with 100 lL of icecold glutaraldehyde (1.1% in NaCl ⁄ Pi) for 15 min at 4 °C.
Plates were washed three times by immersion in de-ionized
water, air-dried and stained for 20 min with 100 lL of a
0.1% crystal violet solution (in 200 mm phosphoric acid
buffer, pH 6). After careful aspiration of the crystal violet
solution, the plates were extensively washed with de-ionized
water, and air-dried prior to solubilization of the bound

dye with 100 lL of a 10% acetic acid solution for 30 min.
The absorbance was measured at 595 nm using a multiplate
spectrophotometer (EL311; Bio-Tek Instruments, Winooski,
VT, USA).

Cell viability assay
Cell viability was determined using the 3-(4,5-dimethylthiazoil-2-yl)-2,5-diphenyltentrazolium bromide (MTT) reduction
assay. MTT is reduced in metabolically active cells to yield
an insoluble purple formazan product. Cells were cultured
in 96-well culture dishes with DHEA for 48 h. Then 20 lL
per well of a MTT solution (5 mgỈmL)1) was added. Four
hours later, the supernatants were discarded and 100 lL of
acidic isopropyl alcohol (HCl 0.04 N) per well were added
to dissolve the formazan. The absorbance was measured
using a multiplate spectrophotometer (Bio-Tek Instruments)
at 570 nm against a reference wavelength (630 nm). The
background absorbance (630 nm) was subtracted before
calculating MTT reduction (MTTR) according to the
following formula: MTTR = (1 ) mean absorbance of
tested cells ⁄ mean absorbance of control cells) · 100.

Determination of the phases of the cell cycle
DNA content was analyzed by propidium iodide staining
followed by cytometric analysis using the DNA reagent kit
from Becton Dickinson. Cells were treated with the inhibitory concentration for DHEA-induced proliferation (IC50)
for 48 h. Then, cells were trypsinized and fixed with 50%
methanol in NaCl ⁄ Pi for 10 min on ice. Cells were washed
twice with NaCl ⁄ Pi and incubated with RNAse (50 lgỈmL)1
in NaCl ⁄ Pi) for 1 h at 37 °C. Cells were then stained with
propidium iodide (200 mgỈL)1) for 2 min, washed twice with

NaCl ⁄ Pi, and immediately subjected to cytometric analysis
using a Becton Dickinson Facscalibur instrument.

Cell death assay
Cell death was evaluated using the carboxyfluorescein
FLICA apoptosis detection kit. Cells were treated with the
IC50 previously determined for each cell line in the proliferation assays for 48 h. Then cells were recovered from the culture plate, and adjusted to a final concentration of 3 · 106

5606

cellsỈmL)1 in NaCl ⁄ Pi before transferring 300 lL of each cell
suspension to sterile tubes, to which 10 lL of a 30· FLICA
solution were added. The tubes were covered with alum
paper, manually agitated and incubated for 1 h at 37 °C in a
5% CO2 humid atmosphere. At the end of the incubation,
2 mL of wash buffer were added to each tube. Cells were
mixed and centrifuged at 180 g for 5 min at room temperature. The cell pellet was resuspended in 1 mL of wash buffer,
centrifuged at 180 g at room temperature for 5 min and
resuspended again in 400 lL of wash buffer. Cells were
then stained with 2 lL propidium iodide (250 lgỈmL)1) and
analyzed by flow cytometer using the cell quest software
program (Becton Dickinson, Franklin Lakes, NJ, USA).
Detection of DNA fragmentation was performed by
TUNEL assay using the Apoptag Red in situ apoptosis
detection kit. Cells were cultured on cover slips and treated
with cisplatin (40 nm) as a positive control and DHEA for
48 h. Afterwards, the cells were fixed with 2% paraformaldehyde for 20 min, washed three times, permeabilized with
0.05% Triton X-100 for 5 min at 4 °C, washed three times,
and labeled with biotin-dUTP by incubation with reaction
buffer containing terminal deoxinucleotidyl transferase

enzyme for 1 h at 37 °C. Biotinylated nucleotides were
detected using streptavidin conjugated with rhodamine.
Cells were counterstained using 4¢,6-diamidino-2-phenylindole to determine DNA distribution. Cell fluorescence was
determined using an E600 Nikon Eclipse microscope
(Melville, NY, USA) with red and blue filters.

Statistical analysis
All experiments were performed in triplicate in at least
three independent trials. The results are expressed as the
mean ± standard deviation of the mean. Student’s t, ANOVA
and Bonferroni tests were used to determine statistical significance, with a P value < 0.01. spss software (release 12;
SPSS Inc., Chicago, IL, USA) was used.

Acknowledgements
R.A.G. is a postgraduate student at the Universidad
´
´
Nacional Autonoma de Mexico, and is supported by a
postgraduate scholarship from the Consejo Nacional
de Ciencia y Tecnologı´ a (CONACyT).

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