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Departments of Pharmaco-Biology and Cell Biology, University of Calabria, 87036 Arcavacata di Rende (CS), Italy
1 Department of Histology, Microbiology and Medical Biotechnologies and
2 Department of Medical and Surgical Sciences, Clinica Medica 3, University of Padova, 35128 Padova, Italy
(Requests for offprints should be addressed to V Pezzi, Department of Pharmaco-Biology, University of Calabria, Arcavacata di Rende (CS) 87036, Italy; Email: v.pezzi{at}unical.it)
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Abstract |
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and ERß in the growth regulation of adrenocortical tumors. We demonstrated that H295R cells are able to convert androgens to estrogens by a constitutive expression of active cytochrome P450 aromatase protein and express ERß to a greater extent than ER. Moreover, physiological concentrations of 17ß-estradiol (E2) determined an increase of thymidine incorporation, suggesting the presence of an autocrine mechanism in maintaining H295R cell proliferation. Evaluating the response to ER antagonists like 4-hydroxytamoxifen (OHT) and ICI 182 780 (ICI), we observed an up-regulation of ERß and a dose-dependent inhibition of H295R cell proliferation. Whereas ICI determined the growth arrest of H295R cells, OHT induced morphological changes that were characteristic of apoptosis. According to the above-mentioned observations, OHT but not ICI clearly induced a marked expression of FasL and the cleavage of both caspase-8 and caspase-3. Interestingly, the apoptotic effects of OHT in H295R cells may be consequent to the enhanced levels of ERß which stimulate the expression of FasL interacting with activating protein (AP)-1 sites located within its promoter sequence. In conclusion, we have demonstrated that H295R cells are able to transform androgens to estrogens that activate an autocrine mechanism, mediated by their own receptors, and contribute to regulate the proliferation of these cells. Moreover, this study points towards a role for ERß as an important mediator of the repressive effects exerted by antiestrogens on H295R cells; however, further studies are needed to clarify its role in the control of adrenocortical cell proliferation and on the potential benefits of antiestrogens for treatment of adrenocortical cancer.
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Introduction |
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The H295R adrenocortical carcinoma cell line (Rainey et al. 1994) was derived from H295 cells, which were established from a primary hormonally active adrenocortical carcinoma (Gazdar et al. 1990), and has been used as a model system to investigate the role of different signaling pathways in the growth regulation of adrenocortical tumors (Rossi et al. 1998, Logié et al. 1999, Bourcigaux et al. 2000). The IGF system has been well characterized (Weber et al. 2000): the IGF-II but not the IGF-I gene is strongly expressed in H295R cells as well as in human adrenocortical tumors and exhibits paternal isodisomy (loss of the maternal-derived allele and duplication of the IGF-II active paternal allele) or less frequently the loss of imprinting (Gicquel et al. 1997). IGF-II regulates the growth of adrenal cells by binding to the IGF receptor and inducing the activation of kinase systems as well as mitogen-activated protein kinases or protein kinase B/Akt (Kirschner 2002). Recently, in several tumoral cells the presence of a cross-talk has been reported between the IGF system and estrogens, which is able to activate the same pathway through the action of estrogen receptors (ERs; Hamelers & Steenbergh 2003).
It has been largely demonstrated that the effects of estrogens on target tissues are mediated by the ER and ERß, which act as transcription factors (Nilsson et al. 2001). In the human fetal adrenal gland the mRNA of ERß was much more expressed than that of ER and the ERß protein was detected in the definitive zone of the adrenal cortex (Takeyama et al. 2001). The highly estrogenic environment during pregnancy has been reported to influence steroidogenesis of the primate fetal gland (Hirst et al. 1992, Albrecht et al. 1999) and it has been suggested that the effects of estrogens via ERß may play an important role in modulating the development of both human and primate fetal adrenal glands (Albrecht et al. 1999, Takeyama et al. 2001). However, it remains to be elucidated whether estrogens can influence adrenocortical growth and function in the adult, and even tumorigenesis like that observed in other hormone-dependent tissues (Gao et al. 2002). A possible involvement of estrogens in adrenocortical tumor development is suggested by epidemiological and experimental studies. Adrenal tumors, and especially functioning tumors, are more frequently found in women than in men (Barzon et al. 2003). Moreover, adrenocortical cancers show a different distribution among genders, with functioning tumors, which represent about half of adrenocortical carcinomas, significantly more frequent in females, and non-functioning carcinomas more frequent in males. Interestingly, a case-control study demonstrated that use of estro-progestins was a risk-factor for the development of adrenocortical carcinomas (Hsing et al. 1996). Moreover, a recent study on H295R cells (Somjen et al. 2003) has shown that these cells are sensitive to low doses of estrogens in terms of proliferation and that they express mRNA of both ER isoforms.
In the present study, using the H295R cell as a model system, we investigated the involvement of estrogen and ERs in the growth regulation of adrenocortical carcinoma. We have demonstrated that H295R cells are able to transform androgens to estrogens that activate an autocrine mechanism, mediated by their own receptors, and contribute to the regulation of proliferation of these cells. Moreover, for the first time we have revealed that in H295R cells antiestrogens such as 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI) exert relevant growth-inhibitory effects through different pathways. For instance, OHT induces apoptosis in H295R cells, up-regulating the expression of FasL and determining the autocrine activation of caspases.
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Materials and methods |
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Forskolin (FSK), 17ß-estradiol (E2) and OHT were purchased from Sigma (St Louis, MO, USA), ICI was a gift from Astra-Zeneca (Italy) and Letrozole was a gift from Novartis Pharma AG (Basel, Switzerland). All these reagents were dissolved in dimethylsulfoxide (DMSO).
Cell cultures
H295R cells, a cell line established from a human adreno-cortical carcinoma, was obtained from Dr W E Rainey (University of Texas Southwestern Medical Center, Dallas, TX, USA) and cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12; 1:1; Eurobio, Les Ulis, France) supplemented with 1% ITS Liquid Media Supplement (100x; Sigma), 10% calf serum and antibiotics (Eurobio), at 37 °C in an atmosphere of humidified air containing 5% CO2. MCF-7 breast cancer cells were incubated in the same conditions but maintained in DMEM/F12 supplemented with 10% calf serum.
Plasmids and in vitro transcription/translation
The coding regions of ER and ERß were excised from their vector and subcloned into the pcDNA3.1 zeo(+) expression plasmid (Invitrogen, Carlsbad, CA, USA). 1 µg each plasmid was used for in vitro transcription/translation using T7 RNA polymerase in the rabbit reticulocyte system following manufacturer’s protocol for the TNT kit (Promega, Madison, WI, USA) in a final volume of 50 µl.
Aromatase activity assay
The aromatase activity in subconfluent H295R cell culture medium was measured by tritiated water-release assay using 0.5 µM [1ß-3H(N)]androst-4-ene-3,17-dione (25.3 Ci/mmol; DuPont NEN, Boston, MA, USA) as a substrate (Lephart & Simpson 1991). Incubations were performed at 37 °C for 2 h under a 95%:5% air/CO2 atmosphere. The results obtained were expressed as pmol/h and normalized to milligram of protein (pmol/h per mg protein).
Estradiol measurement
Subconfluent H295R cells, seeded in 12-well/plates at the concentration of 5 x 105 cells/well, were washed twice with PBS and grown in medium without serum. After 24 h, cells were washed again with PBS and grown in 0.5 ml/well fresh medium without serum for 48 and 72 h. The experiment was performed three times in sextuplicate. Total E2 was measured in the super-natant of H295R cells by competitive immunoassays on ADVIA Centaur (Bayer Diagnostics, Tarrytown, NY, USA).
RNA isolation and reverse transcriptase (RT)-PCR
Total cellular RNA was extracted from H295R cells with Trizol reagent (Invitrogen, Life Technologies, San Giuliano Milanese, Italy) according to the protocol provided by the manufacturer. All the RNA was DNase-treated using the DNA-freeTM kit (Ambion, Austin, TX, USA), and purity and integrity of the RNA was checked spectroscopically and by gel electrophoresis before carrying out the analytical procedures. The evaluation of gene expression was performed by semiquantitative RT-PCR (Maggiolini et al. 1999). For cytochrome P450 aromatase (henceforth called P450 aromatase), ERß and internal control gene 36B4 the primers were 5'-CTGGAAGAATGTATGGACTT-3' (P450 aromatase forward), 5'-GATCATTTCCAGCAT GTTTT-3' (P450 aromatase reverse), 5'-CAGCATTC CCAGCAATGTCAC-3' (ERß forward), 5'-GCAGAA GTGAGCATCCCTCTTTG-3' (ERß reverse), 5'-CTC AACATCTCCCCCTTCTC-3' (36B4 forward) and 5'-CAAATCCCATATCCTCGTCC-3' (36B4 reverse), to yield products of 660, 281 and 408 bp, respectively, with 30, 20 and 15 PCR cycles of 1 min at 95 °C for all genes, 1 min at 64 °C for P450 aromatase, 1 min at 58 °C for ERß and 1 min at 57 °C for 36B4, followed by 1 min at 72 °C for all genes.
Western-blot analysis
Total cell protein extracts from H295R and MCF-7 cells were lysed in ice-cold Ripa buffer containing protease inhibitors (20 mM Tris, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS, 1 mM PMSF, 0.15 units/ml aprotinin and 10 µM leupeptin). Nuclear extracts were prepared from H295R and MCF-7 as previously described (Andrews & Faller 1991). Briefly, cells plated onto 60 mm2 dishes were scraped into 1.5 ml cold PBS. Cells were pelleted for 10 s and resuspended in 400 µl cold buffer A, containing protease inhibitors, 10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF, 10 µM leupeptin and 0.15 units/ml aprotinin, by flicking the tube. The cells were allowed to swell on ice for 10 min and then vortexed for 10 s. Samples were centrifuged for 10 s and the supernatant fraction discarded. The pellet was resuspended in 50 µl cold buffer C (20 mM Hepes, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, 10 µM leupeptin and 0.15 units/ml aprotinin) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation for 2 min at 4 °C and the supernatant fraction (containing DNA-binding proteins) was stored at –70 °C. The yield was determined by Bradford method (Bradford 1976). The proteins were separated on 11% SDS/polyacrylamide gel and then electroblotted onto a nitrocellulose membrane. The blots were incubated overnight at 4 °C with (1) mouse anti-(human P450 aromatase) antibody, raised against a conserved epitope within P450 aromatase (1:1000; Serotec, Oxford, UK), (2) anti-ERß antibody against the C-terminal region of the ERß (1:500; Serotec), (3) anti-ER (F-10) antibody against the N-terminal region of the ER (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), (4) anti-caspase-8 1C12 monoclonal antibody (1:1000; Cell Signaling Technology, Celbio, Milan, Italy), (5) anti-caspase-3 antibody (1:1000; Cell Signaling Technology), (6) anti-Fas (FL-335) antibody (1:1000; Santa Cruz Biotechnology) and (7) anti-FasL antibody (1:1000; DB Transduction Laboratories, Lexington, KY, USA). The antigen–antibody complexes were detected by incubation of the membranes at room temperature with goat anti-mouse IgG coupled to peroxidase, developed using the ECL Plus Western-blotting detection system (Amersham Biosciences, Cologno Monzese, Italy). As a loading control, membranes were stripped and reprobed with ß-actin antiserum.
Cell-proliferation assay
A total of 1 x 106 cells were seeded onto six-well plates in complete medium, for proliferative analysis. After 3 days the medium was replaced with DMEM lacking Phenol Red as well as serum and ITS Liquid Media Supplement and treated with different concentrations of E2, ICI 182 780 and OHT alone or in combination for 96 h. Control cells were treated with the same amount of vehicle alone (DMSO) that never exceeded the concentration of 0.01% (v/v).
[3H]Thymidine incorporation was evaluated after a 24-h incubation period with 1 µCi [3H]thymidine (PerkinElmer Life Sciences, Boston, MA, USA) per well. Cells were washed once with 10% trichloroacetic acid, twice with 5% trichloroacetic acid and lysed in 1 ml 0.1 M NaOH at 37 °C for 30 min. The total suspension was added to 10 ml optifluor fluid and was counted in a scintillation counter.
Staining for apoptosis detection
The apoptotic cells were stained using Hoechst 33342 staining (Sigma). H295R cells, plated on chamber slides, were fixed with 4% paraformaldehyde and stained with Hoechst 33342 dye to a final concentration of 5 µg/ml (150 µM) for 20 min at room temperature. Following washing with PBS, the cells were observed under an Olympus BX51 fluorescence microscope.
Chromatin immunoprecipitation (ChIP)
This assay was performed using the ChIP assay kit from Upstate Biotechnology (Lake Placid, NY, USA; http://www.nature.com/cgi-taf/DynaPage.taf?file=/onc/journal/v23/n45/full/1208014a.html-bib23#bib23) with minor modifications in the protocol. H295R cells were grown in 100 mm plates. Confluent cultures (90%) were treated for 24 h with 10 nM E2, 10 µM OHT or 10 µM ICI. Control cells were treated with the same amount of vehicle alone (DMSO) that never exceeded 0.01% (v/v). Following treatment DNA–protein complexes were cross-linked with 1% formaldehyde at 37 °C for 10 min. Next, cells were collected and resuspended in 400 µl SDS lysis buffer (Upstate Biotechnology) and left on ice for 10 min. Cells were sonicated four times for 10 s at 30% maximal power (Vibra Cell VCX 500; Sonics & Material, Inc. Newtown, CT, USA) and collected by centrifugation at 4 °C for 10 min at 11 000 g. Of the supernatants 20 µl were kept as input (starting material, to normalize results) and 100 µl were diluted 1:10 in 900 µl ChIP dilution buffer (Upstate Biotechnology) and immunocleared with 80 µl sonicated salmon sperm DNA–Protein A agarose (Upstate Biotechnology) for 6 h at 4 °C. The precleared chromatin was immunoprecipitated overnight with 2 µg specific anti-ERß antibody. The following day 60 µl salmon sperm DNA–Protein A agarose was added and precipitation was continued at 4 °C until the day after. After pelleting, precipitates were washed sequentially for 5 min with the following buffers: High Salt Immune Complex Wash Buffer, Low Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer and then twice with TE buffer (all buffers are contained in the kit). The immune complexes were eluted with elution buffer (1% SDS and 0.1 M NaHCO3). The eluates and the 20 µl input were reverse cross-linked by heating at 65 °C overnight and digested with proteinase K (0.5 mg/ml) at 45 °C for 1 h. DNA was recovered by phenol/chloroform extractions. A 2 µl aliquot of 10 mg/ml yeast tRNA was added to each sample and DNA was precipitated with ethanol for 1 h at –20 °C and then resuspended in 50 µl TE buffer. A 5 µl volume of each sample and 2 µl of input were used for PCR using the following FasL promoter primers (GenBank accession no. AF035584 [GenBank] ): sense, 5'-AAACTG AGGCAGGAGGATGT-3', and antisense, 5'-TCG TAGTTAACTGCAGCCTC-3'. The PCR conditions were 1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C for 30 cycles. The amplification products of 200 bp were analyzed on a 1% agarose gel and visualized by ethidium bromide staining. In control samples, nonimmune rabbit IgG was used instead of the specific antibody.
Statistical analysis
Statistical analysis was performed using one-way ANOVA. Data were analyzed using software from STATPAC (Minneapolis, MN, USA).
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Results |
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We initially examined whether H295R cells, used as a model system, were able to synthesize estrogens. H295R cells were cultured for 48 and 72 h in serum-free medium; E2 content was measured with a competitive immuno assay and revealed a time-dependent increase. E2 medium content was 512 ± 36 pg/ml at 48 and 834 ± 67 pg/ml at 72 h.
We next investigated the expression and the activity of P450 aromatase in the absence or presence of the adenylate cyclase activator FSK. A 24-h treatment with FSK significantly increased aromatase mRNA expression (Fig. 1A and B
) and protein levels (Fig. 1C and D). This behavior was reproduced with aromatase activity, which was 7.43 ± 0.4 pmol/h per mg protein in basal conditions and increased to 23.28 ± 2.5 pmol/h per mg protein after incubating cells for 24 h with FSK. The presence of the aromatase inhibitor Letrozole, used 1 h before and during the incubation with the tritiated aromatase substrate, inhibited basal and, in particular, FSK-induced aromatase activity to 1.087 ± 0.1 pmol/h per mg protein (Fig. 1E).
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and ERß in H295R cells
Since the effects of estrogens on target tissues are mediated by the estrogen receptors (ER and ERß), we next investigated the expression of these factors in H295R cells. We subcloned the ER and ERß genes into the expression vectors pcDNA3.1 zeo(+) and used these vectors for in vitro transcription, and the synthetized proteins were utilized as a standard curve for ER and ERß in Western-blot analysis. Standard points were obtained using increasing amounts of the synthetized proteins (0.5, 1, 3 and 6 µl). Densitometric analysis, reference band intesities to the curve where the standard point with the lowest concentration was taken as 100%, indicated that in H295R cells ERß is significantly higher than ER. We also used MCF-7 human breast cancer cells, a known ER-positive cell type, as a control, and found as expected a high expression of the ER isoform, whereas ERß was barely detectable (Fig. 2). Based on these results we focused our attention on the role of ERß in mediating estrogen signaling in this adrenal cancer cell line.
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We then aimed to investigate the effects of E2 on the proliferation of H295R cells assayed using thymidine incorporation. Treatment with different concentrations of E2 (0.1–1000 nM) for 96 h exhibited a slight but significative increase of thymidine incorporation (Fig. 3A), while shorter exposures (24, 48 and 72 h) did not determine significant effects on DNA synthesis (data not shown). To demonstrate the involvement of ERs, we also investigated the effects of antiestrogens on H295R proliferation. Treatment with increasing amounts of ICI (0.1–10 µM) or OHT (0.1–10 µM) determined a dose-dependent inhibition of thymidine incorporation both under the basal conditions and in the presence of E2 (10 nM) (Fig. 3B). To further confirm that H295R growth depends on estrogens we investigated the effect of aromatase inhibitor Letrozole on cell proliferation. As shown in Fig. 3C treatment with increasing amounts of Letrozole (1–10 µM) determined a dose-dependent inhibition of thymidine incorporation.
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The above results prompted us to evaluate whether ERß expression can be modulated by E2 and/or antiestrogens such as ICI and OHT. Performing a semiquantitative RT-PCR we observed that a 24-h exposure to ICI or OHT was clearly able to up-regulate the mRNA levels of ERß, which were not modified by E2 (10 nM) treatment (Fig. 4A and B). In fact, E2 dose-response (0.1–1000 nM) or time-course (12, 24 and 48 h) experiments did not reveal any significant differences with respect to controls (data not shown). Besides, Western-blot analysis revealed an increase in ERß protein levels after a 96-h exposure to antiestrogens (Fig. 4C and D), which paralleled the evaluation of cell proliferation.
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Microscopic observation of H295R cells treated with 10 µM OHT and stained with Hoechst 33342 showed morphological changes characteristic of apoptosis after only 24 h (Fig. 5). These changes were not observed in cells treated with estradiol or ICI (data not shown).
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), however the expression of FasL was up-regulated only by OHT (Fig. 6B and E). The autocrine mechanism of apoptosis in H295R cells by the Fas/FasL system was further supported by the expression of the active forms of caspase-8 (p43/45 and p28) and caspase-3 (p20 and p17) induced only by OHT treatment (Fig. 7).
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Since for several ER-dependent genes transcription has been shown to be regulated through ER binding to the AP-1 complex, we used chromatin immunoprecipitation analysis to show ERß binding to the AP-1 site on the FasL promoter in H295R cells. Samples were immunoprecipitated with the ERß antibody and the FasL promoter region containing the AP-1 site was amplified with specific primers. The results obtained show that ERß is able to interact with the protein complex bound to the AP-1 site in basal conditions and that this binding is maintained in treated cells (Fig. 8). Bands are specific as shown by the lack of amplification in samples immunoprecipitated with nonimmune rabbit IgG.
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Discussion |
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Androgens and estrogens determine various biological activities on mammalian tissues controlling cellular growth and differentiation through different signal transduction pathways. Previous studies have demonstrated that androgens inhibit H295R cell proliferation through the androgen receptor (Rossi et al. 1998), whereas the role of estrogens and ERs in both normal adult and malignant adrenocortical cells remains to be elucidated. Our results give a functional emphasis to recent studies which revealed that the H295R cell line is able to transform androgens into estrogens (Watanabe & Nakaijn 2004) and express ER and ERß mRNA (Somjen et al. 2003). We demonstrated that high levels of basal aromatase activity are present in H295R cells, allowing these cells to produce E2. Moreover, H295R cells also express both ER protein isoforms, with a prevalence of ERß, thus reproducing the same ER ratio present in the fetal adrenal gland (Takeyama et al. 2001). The exposure to a physiological concentration of E2 determined a slight but significant increase of thymidine incorporation, confirming the results of a previous study (Somjen et al. 2003) and revealing the presence of an autocrine mechanism which could contribute to H295R cell proliferation. The observation that both antiestrogens and the aromatase inhibitor Letrozole determined a dose-dependent arrest of these proliferative effects supports this hypothesis. However, only OHT was responsible for the morphological changes associated with apoptosis as observed in previous studies performed on MCF-7 breast cancer cells using tamoxifen and its analogs (Bardon et al. 1987, Warri et al. 1993, Wilson et al. 1995). On the contrary, apoptotic events were shown to be induced also by ICI in ER-positive primary breast cancer cells (Ellis et al. 1997). Moreover a study performed on six malignant rhabdoid tumor cell lines revealed that OHT but not ICI induced apoptosis (Koshida et al. 2002). To explain these apparently controversial data we have to take into account that the pharmacological potency elicited by antiestrogens is dependent on the cellular context as well as on the interaction of these molecules with the two different ER isoforms. This hypothesis is supported by studies which have shown that ER agonists and antagonists are able to induce distinct conformations and biological activity of both ER isoforms (Van Den Bemd et al. 1999). A study using a fingerprint assay clearly indicates that OHT determines, for both ER and ERß, the exposure of unique peptide-binding surfaces that are not exposed in the presence of ICI (Paige et al. 1999). So, different ER–ligand complexes may be able to recruit different coactivator and corepressor proteins within the cell, and the overall biological response is determined by unique combinations of protein–protein interactions that occur in a given cell and promoter context (Jones et al. 1999). The present study contributes to the understanding of the biological activity of OHT and ICI, extending to adrenocortical cancer cells the different response to antiestrogens.
We ascertained that the antiestrogens OHT and ICI were able to induce the up-regulation of ERß, whereas E2 did not show any change in our experimental conditions. What is the molecular mechanism by which antiestrogens can enhance ERß expression and what is the biological counterpart of this response? It is worth noting that Paech et al.(1997) reported an antiestrogen-dependent transcriptional activity of ERß at AP-1 sites which were demonstrated to be located within the promoter region of ERß gene (Li et al. 2000). In line with these findings, our data may provide new insight on ERß autologous regulation by antiestrogens as displayed even in H295R cells. Moreover, could this up-regulation of ERß mediate the above reported down-regulatory effects of ICI and OHT on H295R cell proliferation? This question opens an additional intriguing area of research that we are investigating currently.
In H295R cells we also observed that ICI determined a dose-dependent inhibition of proliferation. This cytostatic effect could be explained by the inhibitory effects exerted by ICI on the IGF signaling pathway, which is activated strongly in H295R cells by the autocrine action of IGF-II through the type 1 IGF receptor (IGF-1R; Logié et al. 1999). In fact, in mammary tissue it has been demonstrated that the effect of estrogen on cell growth is mediated by the up-regulation of IGF-1R (Stewart et al. 1990), insulin receptor substrate (IRS)-1 and IRS-2 (Lee et al. 1999) expression and/or by the down-regulation of the inhibitory IGF-binding protein 3 (IGFBP-3; Huynh et al. 1996), while ICI reduced basal phosphorylation of IGF-1R, IRS-1, IRS-2, Akt-1 and the p85 subunit of phosphoinositide 3-kinase (Chan et al. 2001). These observations indicate that inhibition of cell growth by ICI may not only be attributable to competition between estrogens and ICI for ER but also to the interruption of the IGF signaling pathway. By doing so, ICI may also block a possible cross-talk between the ER and IGF-1R signaling pathways (Dupont et al. 2000). The molecular mechanism determining the cytostatic effect of ICI on H295R cells is currently under investigation, but preliminary results seem to confirm this hypothesis.
Interestingly, we demonstrated the involvement of the Fas/FasL system as a signal transduction pathway mediating the OHT-induced apoptosis. FasL is a membrane protein belonging to the tumor necrosis factor family (Suda et al. 1993), which is able to induce apoptosis by cross-linking with the Fas receptor (Takahashi et al. 1994). FasL is normally expressed on activated cells of the immune system and is used for killing cells infected with viruses or cancer cells expressing Fas (Suda et al. 1993). It has also been demonstrated that tumor cells can express FasL by which they kill Fas-positive immune cells evading the immune system (Walker et al. 1997, Nagarkatti 2000). Recently, it has been described that, in certain Fas-positive tumor cells, OHT induces apoptotic effects by up-regulating FasL (Nagarkatti & Davis 2003). Our study recalls these observations since we have shown that H295R cells are Fas-positive and that OHT is able to increase the expression of FasL which may activate pro-apoptotic events. This autocrine mechanism is further substantiated by the cleavage of caspase-8 and caspase-3 observed upon OHT treatment. Notably, these phenomena could be consequent to the interaction of the ERß–OHT complex with the promoter of FasL, which contains a complete AP-1 sequence (Mor et al. 2000), while in our cellular context the ERß–ICI complex seems to be unable to activate FasL transcription.
A previous study (Mor et al. 2000) reported that OHT inhibits the expression of FasL in MCF-7 and in T47D breast tumor cells through ER. In contrast, other authors showed that OHT is able to up-regulate FasL in T47D cells (Nagarkatti & Davis 2003), depending on the concentrations of treatments and culture conditions. A role for ERß in the regulation of FasL has been hypothesized by a study on neuronal cells demonstrating that ERß mediates apoptosis induction in cells expressing Fas/FasL proteins, while ERß has antiapoptotic and neuroprotective effects (Nilsen et al. 2000). Mechanisms controlling the activation of ER-dependent genes through AP-1 sites are not completely clear. ERß shows a unique capacity to enhance AP-1 activity in response to selective antiestrogens (Paech et al. 1997, Weatherman & Scanlan 2001); this is due to ERß interactions with corepressors, as such interactions are inhibited by antiestrogens and increased by agonists (Webb et al. 2003, Uht et al. 2004). Our data show that ERß is able to interact with the proteins bound to the AP-1 complex on human FasL promoter and confirm that transcriptional activity does not depend only on ERß binding to the promoter but instead depends on ERß’s ability to recruit specific cofactors. In our cellular context, the interaction between ERß and OHT may be able to recruit corepressors bound to the AP-1 complex determining the activation of FasL expression that in turn promotes apoptosis. Nevertheless we cannot exclude that high doses of OHT are also able to induce apoptosis involving other factors such as p53 or c-Myc in a direct manner (Mandlekar & Kong 2001). Whatever the mechanism involved, what clearly emerges from this study is that the H295R adrenocortical cancer cell line exhibits estrogen-sensitive proliferation which can be inhibited by exposure to antiestrogens ICI and OHT or the aromatase inhibitor Letrozole. The present findings and our preliminary observations (Barzon et al., unpublished observations), which clearly reveal (by real-time PCR and western-blot analysis) the expression of ERß in human adrenocortical carcinoma tissues, open new perspectives on the potential therapeutic benefits of antiestrogens as pharmacological agents in antagonizing adrenocortical carcinoma cell growth and progression. However, further studies are needed to clarify the role for ERß as a possible mediator of adrenocortical cell proliferation.
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Acknowledgements |
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Received 25 May 2005
Accepted 14 June 2005
Made available online as an Accepted Preprint 23 June 2005
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