Dehydroepiandrosterone inhibits the proliferation and induces the death of HPV-positive and...

Dehydroepiandrosterone inhibits the proliferation andinduces the death of HPV-positive and HPV-negativecervical cancer cells through an androgen- andestrogen-receptor independent mechanismRoma A. Giron1, Luis F. Montano2, Marıa L. Escobar3 and Rebeca Lopez-Marure1

1 Departamento de Biologıa Celular, Instituto Nacional de Cardiologıa ‘Ignacio Chavez’, Mexico D.F., Mexico

2 Laboratorio de Inmunobiologıa, Departamento de Biologıa Celular y Tisular, Facultad de Medicina, Universidad Nacional Autonoma de

Mexico (UNAM), Mexico

3 Departamento de Biologıa Celular, Facultad de Ciencias, Universidad Nacional Autonoma de Mexico (UNAM), Mexico

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.

Keywords

androgen receptor; cell proliferation; DHEA;

estrogen-receptor; HPV

Correspondence

R. Lopez-Marure, Departamento de Biologıa

Celular, Instituto Nacional de Cardiologıa

‘Ignacio Chavez’, Juan Badiano No. 1,

Colonia Seccion 16, Tlalpan, C.P. 14080,

Mexico D.F., Mexico

Fax: +52 55 73 09 26

Tel: +52 55 73 29 11 ext. 1337

E-mail: [email protected]

(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 epithelial-

derived carcinomas; however, the mechanisms remain unknown. We deter-

mined 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-(4,5-

dimethylthiazol-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 apop-

tosis 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.

Abbreviations

DHEA, dehydroepiandrosterone; FLICA, fluorochrome-labeled inhibitors of caspases; HPV, human papilloma virus; MTT, 3-(4,5-dimethyl-

thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

5598 FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS

DHEA is considered to exert its action through con-

version 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 antiprolifera-

tive, 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 sponta-

neous 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 can-

cer 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 apopto-

tic and necrotic cell death. Taken together, these

results suggest that DHEA could be used in the treat-

ment of cervical cancer.

Results

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 HPV-

positive cell lines to achieve a similar inhibitory

decrease (Fig. 1). The effect of DHEA was dose-depen-

dent, 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 inhibi-

tory effect commenced in the 25 lm range in all three

cell lines, indicating a decrease in cell viability. This

120

140

160

60

80

100

CASKI HeLa C33A

* *

* *

*

*

*

0

20

40

0 6.25 12.5 25 50 70 100 200

% o

f p

rolif

erat

ion

* *

* *

DHEA (µµM)

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.

120

140

60

80

100

CASKI HeLa C33A

* *

* *

* *

* * * *

0

20

40 * *

* *

0 6.25 12.5 25 50 70 100 200 DHEA (µµM)

MT

T r

edu

ctio

n (

%)

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.

R. A. Giron et al. DHEA and cervical cancer

FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS 5599

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 conver-

sion to testosterone or estradiol. In order to assess

this, antagonists to androgen and estrogen receptors

(flutamide and ICI 182,780, respectively), and an inhib-

itor of the aromatase responsible for conversion of

androgen to estrogen (letrozol), were used alone or in

combination with DHEA before evaluation of cell

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 conver-

sion to other metabolites (Fig. 3).

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

C33A

80

100

120

* *

* * * * * * * * * *

0

20

40

60

ICI Flutamide Letrozol

* *

*

* *

0 1 10 100 1 + D 10 + D 100 + D DHEA Concentration (nM)

% o

f p

rolif

erat

ion



CASKI

80

100

120

0

20

40

60

* * * * * * *

* * * * *

% o

f p

rolif

erat

ion



HeLa

80

100

120

* * * * * * * * * *

0

20

40

60 * * * * * * * * *

% o

f p

rolif

erat

ion

A

B

C

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.

DHEA and cervical cancer R. A. Giron et al.


decrease was associated with an increase in the per-

centage 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 (HPV-

negative) and HeLa cells (HPV-positive) showed a

lower percentage of cell death in comparison with con-

trol cells (Table 1). These results suggest that the effect

of DHEA upon CASKI cells is more cytotoxic than

cytostatic.

DHEA induces apoptotic and necrotic death

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 con-

trol to induce apoptotic cell death. Cisplatin and

DHEA treatments resulted in apoptosis of both

HPV-positive cells and C33A cells, in comparison with

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

Control DHEA

G1

C33A

S G2/M CD

CASKI

HeLa

4030

20C

ount

s10

0

4030

20C

ount

s10

0

4030

20C

ount

s10

0

4030

20C

ount

s10

0

0 200 400 600FL2-A

800 1000 0 200 400 600FL2-A

800 1000

0 200 400 600FL2-A

800 1000 0 200 400 600FL2-A

800 1000

4030

20C

ount

s10

0

4030

20C

ount

s10

0

0 200 400 600FL2-A

800 1000 0 200 400 600FL2-A

800 1000

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 cytom-

etry (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.

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 S G2 ⁄ M Cell death

C33A Control 40 21 15 24

DHEA 36 18 14 32

CASKI Control 46 14 9 31

DHEA 22 9 4 65

HeLa Control 47 13 9 31

DHEA 41 15 8 36



Control

A

B

C

TUNEL DAPIC33A

Phase contrast

Cisplatin

DHEA

CASKI

Control

Cisplatin

DHEA

Control

HeLa

Cisplatin

DHEA

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 proce-

dures. Cells were counterstained with

4¢,6-diamidino-2-phenylindole. The images

were obtained using a phase contrast

microscope, and correspond to an experi-

ment 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 andro-

gen 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 dramati-

cally 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

Control Cisplatin DHEA

NC LAC

C33A

LC EAC

CASKI

HeLa

104

102

FL2

-H10

310

110

010

410

2F

L2-H

103

101

100

104

102

FL2

-H

FL1-H

103

101

100

104

102

FL1

-H10

310

110

010

410

2F

L1-H

103

101

100

104

102

FL1

-H10

310

110

0

104

102

FL1

-H10

310

110

010

410

2F

L1-H

103

101

100

104

102

FL1

-H10

310

110

0

104102 103101100

FL1-H104102 103101100

FL1-H104102 103101100

FL1-H104102 103101100

FL1-H104102 103101100

FL1-H104102 103101100

FL1-H104102 103101100

FL1-H104102 103101100

FL1-H104102 103101100

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.



crystal staining and MTT reduction, independently of

the HPV type. Several studies have found an antipro-

liferative effect induced by DHEA in normal cells such

as T lymphocytes, isolated neurons and endothelial

cells, or malignant cell lines such as human hepatoblas-

toma cells (HepG2), colon adenocarcinoma cells

(HT-29) and breast cancer cells (MCF-7) [3,23–26].

Our results are the first evidence for an antiprolifera-

tive 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 muta-

tions in p53 [28]. C33A cells are known to have a non-

functional p53 protein due to mutations [29], whereas

CASKI and HeLa cells possess a non-mutant p53

protein. Given that p53 is associated with an antipro-

liferative 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 mam-

mary 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,

immortalization is dramatically increased in HPV16-

infected 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 differ-

ent 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 can-

cers 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 estro-

gen receptor b to the same degree as estrogen at its

circulating concentration [42]. We showed that the

inhibition of proliferation induced by DHEA is inde-

pendent 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; how-

ever, our results cannot discount the possible conver-

sion 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 recep-

tor 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 oppo-

site 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]; how-

ever, the mechanism of this differential effect is

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

cytometry.

Percentage of cells

Alive Early apoptosis Late apoptosis Necrosis

C334 Control 93.1 0 0.12 6.78

Cisplatin 63.06 0 0.44 36.50

DHEA 46.78 0 0.38 52.84

CASKI Control 91.54 4.88 2.24 1.34

Cisplatin 65.46 19.9 8.84 5.8

DHEA 78.52 8.08 7.04 6.36

HeLa Control 93 1.8 4.08 1.12

Cisplatin 40.28 47.1 10.16 2.46

DHEA 34.98 21.46 31.68 11.88



unknown. This differences have also been observed in

neuronal cell cultures, in which DHEA has a protec-

tive role at concentrations ranging from 0.1–1 lm, but

a pro-oxidant ⁄ cytotoxic effect is seen at higher concen-

trations [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 hor-

mones. 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 intra-

cellular 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 ste-

roid structures such as sulfated DHEA, androstenedi-

one, 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 sug-

gests 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 recep-

tors or is converted to other metabolites.

The antiproliferative effect of DHEA has been asso-

ciated 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 protec-

tive role against apoptosis has been shown at physio-

logical concentrations of DHEA in neurons [56];

similar DHEA concentrations act as a survival factor

in endothelial cells by triggering the G-alpha-1 G-pro-

tein-phosphoinositide 3-kinase/AKT protein family-

Bcl-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

kind of resistance to the necrotic process, although one

would imagine that HPV-infected cells possess mecha-

nisms that immortalize them more easily than non-

HPV-infected cells. It has recently been demonstrated

that HPV protein E7 induce S-phase entry in keratino-

cytes [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 there-

fore believe that alternative therapeutic approaches

should be considered in the treatment of cervical can-

cer. 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 tryp-

sin were purchased from Gibco ⁄BRL (Grand Island, NY,

USA). Fetal bovine serum was purchased from HyClone

(Loga, UT, USA). The carboxyfluorescein FLICA apopto-

sis detection kit was purchased from Immunology Techno-

logies (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 stain-

ing. Cells were plated in 96-well plates and cultured with



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 ice-

cold 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-dimethylthiaz-

oil-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 lLper well of a MTT solution (5 mgÆmL)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 inhibi-

tory 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 lgÆmL)1

in NaCl ⁄Pi) for 1 h at 37 �C. Cells were then stained with

propidium iodide (200 mgÆL)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 prolifera-

tion assays for 48 h. Then cells were recovered from the cul-

ture plate, and adjusted to a final concentration of 3 · 106

cellsÆmL)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 tempera-

ture. 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 lgÆmL)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% paraformal-

dehyde 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-phenylin-

dole 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 signifi-

cance, 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 Tecnologıa (CONACyT).

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