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 papillom
Trang 1induces the death of HPV-positive and HPV-negative
cervical cancer cells through an androgen- and
estrogen-receptor independent mechanism
Roma A Giro´n1, Luis F Montan˜o2, Marı´a L Escobar3and Rebeca Lo´pez-Marure1
1 Departamento de Biologı´a Celular, Instituto Nacional de Cardiologı´a ‘Ignacio Cha´vez’, Me´xico D.F., Me´xico
2 Laboratorio de Inmunobiologı´a, Departamento de Biologı´a Celular y Tisular, Facultad de Medicina, Universidad Nacional Autonoma de Me´xico (UNAM), Me´xico
3 Departamento de Biologı´a Celular, Facultad de Ciencias, Universidad Nacional Autonoma de Me´xico (UNAM), Me´xico
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 Lo´pez-Marure, Departamento de Biologı´a
Celular, Instituto Nacional de Cardiologı´a
‘Ignacio Cha´vez’, Juan Badiano No 1,
Colonia Seccio´n 16, Tlalpan, C.P 14080,
Me´xico D.F., Mexico
Fax: +52 55 73 09 26
Tel: +52 55 73 29 11 ext 1337
E-mail: rlmarure@yahoo.com.mx
(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 IC50doses 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.
Trang 2DHEA 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 necroapopto-tic 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
*
*
* *
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
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.
Trang 3DHEA 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
* *
*
ICI Flutamide Letrozol
CASKI
80
100
120
0
20
40
60
* * * * * * * * * * * *
ICI Flutamide Letrozol
HeLa
80
100
120
* * * * * * * * * *
0
20
40
* * * * * *
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.
Trang 4decrease 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
0 200 400 600
FL2-A
FL2-A
800 1000
0 200 400 600
FL2-A
FL2-A
800 1000
0 200 400 600
FL2-A
FL2-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 cell cycle
Trang 5A
B
C
C33A
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 (IC 50 ) 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.
Trang 6as 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 estroandro-gen 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
C33A
CASKI
HeLa
FL1-H
10 4
10 2 10 3
10 1
10 0
4
10 2 10 3
10 1
10 0
4
10 2 10 3
10 1
10 0
4
102 103
101
100
4
10 2 10 3
10 1
10 0
FL1-H
10 4
10 2 10 3
10 1
10 0
4
10 2 10 3
10 1
10 0
FL1-H
10 4
10 2 10 3
10 1
10 0
FL1-H
10 4
10 2 10 3
10 1
10 0
Fig 6 DHEA also induces necrotic death Cells were cultured with and without DHEA (IC 50 ) 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.
Trang 7crystal 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 estroestro-gen 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 receprecep-tor 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
Trang 8unknown 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
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 (Me´xico City, Me´xico) The Apoptag Red in situ apoptosis detection kit was obtained
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
Trang 9various 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
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 lL
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
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
for 48 h Then, cells were trypsinized and fixed with 50%
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
IC50previously determined for each cell line in the
prolifera-tion assays for 48 h Then cells were recovered from the
cellsÆmL)1in NaCl⁄ Pibefore 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
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
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
and labeled with biotin-dUTP by incubation with reaction
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 Auto´noma de Me´xico, and is supported by a postgraduate scholarship from the Consejo Nacional
de Ciencia y Tecnologı´a (CONACyT)
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