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 ´ ´ ˜ ´ Roma A Giron1, Luis F Montano2, Marıa L Escobar3 and Rebeca Lopez-Marure1 ´ ´ ´ ´ ´ Departamento de Biologıa Celular, Instituto Nacional de Cardiologıa ‘Ignacio Chavez’, Mexico D.F., Mexico ´ ´ Laboratorio de Inmunobiologıa, Departamento de Biologıa Celular y Tisular, Facultad de Medicina, Universidad Nacional Autonoma de ´ ´ Mexico (UNAM), Mexico ´ ´ ´ Departamento de Biologıa Celular, Facultad de Ciencias, Universidad Nacional Autonoma de Mexico (UNAM), Mexico 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: rlmarure@yahoo.com.mx (Received 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 5598 FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R A Giron et al Results 160 % of proliferation 140 CASKI HeLa C33A 120 100 80 * * 60 * * 40 * *** 20 *** 0 6.25 12.5 25 50 70 100 200 µ DHEA (µM) Fig 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 6.25 12.5 25 50 DHEA (µM) µ 70 Fig 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 FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS 5599 ´ R A Giron et al 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 and Table 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 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 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 10 100 1+D Concentration (nM) 5600 10 + D Fig 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 h before DHEA D, DHEA *P < 0.01 compared with the control FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R A Giron et al 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 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 13 15 15 14 9 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 10 Counts 20 G2/M 10 C33A S Counts 20 30 G1 CD 200 400 600 FL2-A 800 1000 200 400 600 FL2-A 800 1000 400 600 FL2-A 800 1000 200 400 600 FL2-A 800 1000 200 400 600 FL2-A 800 1000 Counts 20 10 0 40 40 30 30 Counts 20 Counts 20 10 10 HeLa Fig 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 40 40 0 200 400 600 FL2-A FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS 800 1000 5601 ´ R A Giron et al 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 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 FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ R A Giron et al 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 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 FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS 5603 ´ R A Giron et al DHEA and cervical cancer Table 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 63.06 46.78 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, 5604 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 FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS ´ 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 FEBS Journal 276 (2009) 5598–5609 ª 2009 The Authors Journal compilation ª 2009 FEBS 5605 ´ 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 at °C Plates were washed three times by immersion in de-ionized water, air-dried and stained for 20 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 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 on ice Cells were washed twice with NaCl ⁄ Pi and incubated with RNAse (50 lgỈmL)1 in NaCl ⁄ Pi) for h at 37 °C Cells were then stained with propidium iodide (200 mgỈL)1) for 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 · 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 h at 37 °C in a 5% CO2 humid atmosphere At the end of the incubation, mL of wash buffer were added to each tube Cells were mixed and centrifuged at 180 g for at room temperature The cell pellet was resuspended in mL of wash buffer, centrifuged at 180 g at room temperature for and resuspended again in 400 lL of wash buffer Cells were then stained with 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 at °C, washed three times, and labeled with biotin-dUTP by incubation with reaction buffer containing terminal deoxinucleotidyl transferase enzyme for 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) References Hayashi T, Esaki T, Muto E, Kano H, Asai Y, Thakur NK, Sumi D, Jayachandran 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Dehydroepiandrosterone inhibits the proliferation of HUVEC by enhancing the expression of p53 and p21, restricting the phosphorylation of RB, and is androgen- and estrogen-receptor independent FEBS... 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... independent Cancer J 12, 160–165 Liu D, Iruthayanathan M, Homan LL, Wang Y, Yang L, Wang Y & Dillon JS (2008) Dehydroepiandrosterone stimulates endothelial proliferation and angiogenesis through