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Journal of Steroid Biochemistry and Molecular Biology
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The Journal of Steroid Biochemistry & Molecular Biology
1a,25-Dihydroxyvitamin D3 inhibits the human H295R cell proliferation by cell cycle arrest: A model for a protective role of vitamin D receptor against adrenocortical cancer
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Catia Pilonª, Riccardo Urbaneta, Tracy A. Williams b, Takashi Maekawac, Silvia Vettore ª, Rosa Sirianni d, Vincenzo Pezzid, Paolo Mulaterob, Ambrogio Fassinaª, Hironobu Sasanoc, Francesco Falloa,*
a Department of Medicine-DIMED, Clinica Medica 3 and Cythopathology Unit, University of Padova, Padova, Italy
b Department of Medical Sciences, University of Torino, Torino, Italy
” Department of Pathology, Tohoku University School of Medicine, Sendai, Japan
d Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, Arcavacata di Rende (CS), Italy
ARTICLE INFO
Article history: Received 22 July 2013 Received in revised form 28 October 2013 Accepted 7 November 2013
Keywords: Vitamin D H295R cells Adrenocortical cancer
ABSTRACT
Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1,25- dihydroxyvitamin D3 [1a,25(OH)2D3)]-vitamin D receptor (VDR) axis in the growth of adrenocortical cancer (ACC). The presence of VDR in various adrenocortical tissues, including ACC, was also investigated. DNA synthesis was evaluated by [3H]thymidine cell incorporation after treatment with 1x,25(OH)2D3 at increasing doses. The effect of 1a,25(OH)2D3 on cell cycle and apoptosis was analyzed with a flow cytometer. Cyclin-dependent kinase 4 (CDK4) expression, a molecular marker of G1-S cell cycle tran- sition phase, was evaluated in cells treated with 10,25(OH)2D3 before and after VDR gene silencing. 1a,25(OH)2D3 treatment inhibited cell proliferation by 20% at a dose of 1 nM, in parallel with steroid secretion decrease. A cell cycle arrest in G1, with no change in apoptotic cell proportion, was observed after 10 nM 1x,25(OH)2D3 cell exposure. CDK4 activation was reduced by 10 nM 1x,25(OH)2D3 but was not affected by 1a,25(OH)2D3 after VDR gene silencing. Expression of VDR mRNA was lower in ACC than in benign adrenocortical tumors. VDR immunostaining was evident in benign tumors but it was weak in ACC tissues.
Conclusions: Slightly supra-physiological concentrations of 10,25(OH)2D3 have a moderate anti- proliferative effect on H295R cells. Anti-proliferative effect was due to cell cycle arrest in G1 phase, without inducing apoptosis. The low mRNA expression levels at qRT-PCR as well as the weak immunohis- tochemical expression of VDR in ACC, suggests a protective role of VDR against malignant adrenocortical growth.
@ 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Bio-activation of vitamin D3, the inert secosteroid precur- sor (cholecalciferol) obtained from dietary sources or de novo synthesis in the skin as a result of ultraviolet light-induced photolysis, involves two sequential hydroxylations in humans catalyzed by cytochrome P450 (CYP)4-containing enzymes. The first of these conversions is catalyzed in the liver by CYP2R1,
a vitamin D 25-hydroxylase which metabolizes cholecalciferol to 25-hydroxycholecalciferol [25(OH)D3]. The second step is the conversion in the kidney of 25(OH)D3 to active metabolite 1a,25-dihydroxycholecalciferol D3 [1a,25(OH)2D3] by CYP27B1 (25(OH)D3, 1a-hydroxylase) [1]. In addition to the classical role in calcium and bone homeostasis, 1x,25(OH)2D3 (also known as cal- citriol) has been recognized to have several “non-calcemic” effects ina variety of cells after binding to vitamin D receptor (VDR; NR1I1), a member the nuclear receptor superfamily which includes recep- tors for steroids, thyroid hormones and retinoic acid. Depending on the cell type, the VDR forms homodimers or heterodimers with the retinoid X receptor (RXR; NR2B) to allow specific DNA binding. The binding of 1a,25(OH)2D3 with VDR-RXR complex is followed by the attachment of this complex to the vitamin D responsive ele- ments, which then initiate transcription in the promoter of target
* Corresponding author at: Department of Medicine-DIMED, Clinica Medica 3, University of Padova, Via Giustiniani 2, 35128 Padova, Italy. Tel .: +39 049 8212654; fax: +39 049 8218744.
E-mail address: francesco.fallo@unipd.it (F. Fallo).
genes [2,3]. The precise functions of 1x,25(OH)2D3 in non-skeletal tissues remain unclear [4]. Epidemiological surveys have shown that sunlight exposure and consequent increased circulating lev- els of 25(OH)D3, which is used to determine a patient’s vitamin D status, are associated with reduced occurrence and mortality in different types of cancer [5-8]. These studies also provide justifica- tion for the use of 1,25(OH)2 D3 or its analogs in the prevention and treatment of malignancy. In fact, there is extensive evidence that 1a,25(OH)2 D3 can inhibit the growth of normal and malignant cells, and affects cell tumor invasion and angiogenesis through various molecular mechanisms [9-11], thereby making it a candidate agent for cancer regulation.
Adrenocortical carcinoma (ACC) is a rare tumor with a very poor prognosis [12-14]. Currently, in localized ACC, only surgery provides a chance for long-term cure [15]. Addition- ally, recurrence rates as high as 60-80%, after radical resection, have been reported indicating a need for adjuvant treatment [13]. To date, the adrenolytic drug mitotane, 1,1-dichloro-2-(o- chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p’-DDD), alone or in combination with cytotoxic drugs, remains the treatment of choice because of its ability to impair adrenal steroidogenesis [13]. The lack of specific treatment comes from limited knowledge of molecular mechanisms underlying ACC development. Recently, a 1x,25(OH)2D3-mediated effect on hormone production and steroidogenic genes has been reported in human adrenocortical carcinoma H295R cells [16]. These cells provide an adrenocortical model that can secrete steroids characteristic of the three adreno- cortical zones, and thus appear pluripotent [17]. Adrenocortical cell strains other than H295R derived cell lines, i.e. steroid non- secreting small carcinoma SW13 cells, are less appropriate models for ACC study [18].
In the present study, using the H295R cells as a model system, we analyzed the possible role of 1a,25(OH)2D3-VDR axis in the growth of adrenocortical cancer, and the underlying mechanisms. The presence of VDR in adrenocortical tissues, including ACC, was also investigated.
2. Methods
2.1. Cell culture and tissues
H295R cells, a cell line established from a human adrenocor- tical carcinoma, was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modi- fied Eagle’s medium/Ham’s F12 (DMEM/F12; 1:1; Gibco®, Carlsbad, CA, USA) supplemented with 1% ITS Liquid Media Supplement (100x; Invitrogen Co., Eugene, OR, USA), 2% Fetal Bovine Serum and antibiotics (Gibco®), at 37 ℃ in an atmosphere of humidified air containing 5% CO2. Cell monolayers were subcultured onto 60-mm dishes for protein extraction (1 x 106 cells/well), cell-cycle analysis and cell transfection. Cell monolayers were subcultured in 24- well culture dishes for proliferation experiments, RNA extraction, steroid secretion (2 x 105 cells/well). Before experiments, after 2 days of cell growth, cells were starved overnight in DMEM/F-12 serum free medium containing antibiotics.
Fresh-frozen samples of adrenocortical tumors, and normal adrenal cortex, macroscopically dissected from adrenal glands of kidney donors, were collected at surgery. Adrenocortical samples included 3 normal adult adrenal glands, 5 non-functioning ade- nomas, 5 cortisol-producing adenomas, 4 aldosterone-producing adenomas and 6 cortisol-producing carcinomas. Diagnosis of malignancy was performed according to the histopathological criteria proposed by Weiss et al. [19] and the modification proposed by Aubert et al. [20], as well using the Reticulin Score proposed by Volante et al. [21]. All patients with ACC were treated with mitotane before surgery.
Tissue samples were obtained with the approval of the institu- tional review board of the University Hospital of Padova and with written informed consent from patients, in accordance with the Declaration of Helsinki guidelines as revised in 1983.
2.2. RNA isolation/quantitative real-time PCR (qRT-PCR)
Total cellular RNA was extracted from H295R cells or adrenal tissues with a Qiagen RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the protocol provided by the manu- facturer. One µg RNA was treated with DNase and removal reagents (Ambion, Austin, TX, USA) and reverse transcribed for 1h at 37° C in a 50ml reaction volume containing 1x RT buffer, 150ng random hexamers, 0.5 mM deoxynucleotide triphosphates, 20U RNAsin ribonuclease inhibitor, and 200U Moloney murine leukemia virus RT (Promega Corp., Madison, WI, USA). Evaluation of gene expression was performed by quan- titative RT-PCR. For VDR and HMBS (housekeeping gene) the primers were the following: 5’-GAAGCCTTTGGGTCTGAAGTG- 3’ (VDR forward), 5’-CCGCCATTGCCTCCATCC-3’ (VDR reverse), 5’-GGCAATGCGGCTGCAA-3’ (HMBS forward), 5’- GGGTACCCACGCGAATCAC-3’ (HMBS reverse). The annealing temperature was 60℃ for all genes. PCR was carried out using a DNA Engine (Opticon 2 continuous fluorescence detection system; MJ Research, Waltham, MA, USA). Reactions were performed three times with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and 1 ng/ml cDNA as previously described [22]. Standard curves for rRNA VDR and HMBS, were obtained using cDNAs from H295R cell line. For each sample, results were normalized with the housekeeping gene rRNA HMBS. The PCR products were analyzed by 2% agarose gel electrophoresis with ethidium bromide. Kidney was used as positive control.
2.3. Western blot and densitometric analysis
Total cell or adrenal tissue lysates (35 µg of protein) were sub- jected to western blot analyses by 10% Tris-HCI polyacrylamide gel electrophoresis (PAGE) (Invitrogen Co., Eugene, OR, USA) in run- ning buffer (Tris/Glycine/SDS). Proteins were transferred for 1 h at room temperature to nitrocellulose membranes in transfer buffer (Tris/Glycine/Methanol). The non-specific binding was blocked by immersing the membranes into 5% non-fat dried milk, 0.1% (v/v) Tween 20 in TBS for 1 h at room temperature. After several washes with washing buffer (TBS Tween 0.1%), membranes were incubated with primary antibodies overnight at 4℃, monoclonal antibodies were as follows: (a) ß-actin, Clone AC-15 (1:10,000) (Sigma-Aldrich, St. Louis, MO, USA), and (b) cyclin-dependent kinase 4 (CDK4) (1:500) (Cell Signaling Technology, Inc., Danvers, MA, USA). Polyclonal antibody was used for VDR (1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The washed mem- branes were incubated for 1 h at room temperature with 1:2000 dilution (anti-mouse) of secondary antibody linked to horseradish peroxidase (Jackson Laboratory, Bar Harbor, USA). Probed blots were incubated with Immobilon® Western HRP substrate (Milli- pore Corporate, Billerica, MA, USA) and exposed to Hyperfilm ECL film (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The films were developed with GBX Kodak developmental solutions (Sigma-Aldrich). Densitometric analysis of the immunoreactive protein bands was performed using Image] software [23,24].
2.4. Cell-proliferation assay
The effect of 1x,25(OH)2D3 on the proliferation of H295R cells was determined by [3H]thymidine incorporation assay. Briefly, the cells were plated in 24-well plate at a density of 2 x 105 cells/well in DMEM F-12 medium. The cells were then treated with
various concentrations of 1x,25(OH)2D3 (0.1 nM-1 (M) and incu- bated at 37 ℃ in a 5% CO2 environment for 96 h. Control cells were treated with the same amount of vehicle alone (ethanol 95%) that never exceeded the concentration of 0.01% (v/v). [3H]thymidine incorporation was evaluated after a 24h incubation 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 ℃ for 30 min. The total suspension was added to 4ml optifluor fluid and was counted in a scintillation counter.
2.5. Cell cycle analysis
Alterations in cell cycle were determined by flow cytometry. Cells were seeded at a density of 1 x 106 cells/well in six-well plates and synchronized by plating in DMEM F-12 without FBS for 24 h. The cells were incubated in 2 ml fresh DMEM F-12 with 10 nM of 1a,25(OH)2D3 (Sigma-Aldrich) for 96 h. After treatment, the cells were harvested with trypsin, washed once with PBS, and then fixed in 70% ethanol overnight at 4℃. Before flow cytometry, cells were stained with 200 µg/ml Ribonuclease A and 50 µg/ml propidium iodide (Sigma-Aldrich) for 30 min at room temperature away from light. 1 x 104 cells per sample were analyzed using a FC500 flow cytometer (Beckman Coulter Inc., Carlsbad, CA, USA). Percentage of apoptotic cells was measured before and after expo- sure to 1a,25(OH)2D3. Data were evaluated using Kulaza Software version 1.2.
2.6. Cell transfection and gene silencing
To confirm the specific effect of 1x,25(OH)2D3 on cell prolif- eration through its ligand, H295R cells (1 x 106) were transfected
by Amaxa nucleofection in 100 ML of Nucleofector R solu- tion (Program P20 Amaxa Biosystems, Cologne, Germany), as described previously [25]. VDR gene expression was silenced using small-interfering RNA (siRNA) by transfection of 1 x 106 cells resus- pended in 100 ML of nucleofector R solution containing 10 L of a 20 MM solution of siRNA (Dharmacon, Denver, CO, USA) [26]. Control silencing transfections were performed using the silencer negative control siRNA (Dharmacon). Following nucleofection, cells were transferred directly to prepared 6-well plates containing 5 mL of complete medium at 37 ℃. Transfected cells were treated with 10 nM 1a,25(OH)2D3 for 96 h in serum-free medium and incubated in a humidified incubator at 37 ℃ with 5% CO2. The cells were then lysed in ice-cold RIPA buffer (50 (L) and solubilized proteins were analyzed by western blot. The cell lysate obtained before and after electroporation was also subjected to western blot analysis for ana- lyzing VDR expression and comparing CDK4 expression, a protein which is a positive regulator of the G1 to S progression, before and after VDR silencing.
2.7. Immunofluorescence
The semi-confluent H295R cells from the flasks were seeded on sterile 1-cm round glass coverslips and placed in 24-well culture plates 48 h before the start of experiment. For immunofluores- cence, the cells were fixed with 4% paraformaldehyde for 30 min, and VDR was detected using a mouse polyclonal primary antihu- man VDR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,USA) diluted 1:50, and an ALEXA 488-conjugated goat sec- ondary antibody (Molecular Probes, Invitrogen Co.), diluted 1:200. The nuclei were highlighted by DAPI staining. The fluorescence was examined and photographed with an Leica EL 6000 fluorescence
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PCR amplification products (A) and Western blot (B) of VDR from normal human adrenal, representative adrenococortical tumors and H295R cells. 1 = positive control; 2 =normal adrenal; 3 = cortisol-producing adrenocortical carcinoma (ACC); 4=aldosterone-producing adenoma (APA); 5 =non-functioning adenoma (NFA); 6 =cortisol- producing adenoma (CPA); 7 =H295R cells; (C) expression levels of VDR mRNA in normal human adrenals (n=3), benign adrenocortical tumors (n=14), and 6 ACCs (n =6). Individual values and means (horizontal bars) are shown.
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The signal for VDR (green) displayed a diffuse nuclear location (blue DAPI) of H295R cells. Merged images indicate the extent of overlap between VDR and other nuclear proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
microscope using software Leica LAS/AF 3.1 (Leica Microsystems GmbH, Germany).
2.8. Immunohistochemistry
Rat monoclonal antibody for VDR (9A7yE101.4) was purchased from Calbiochem (Darmstadt, Germany). Immunohistochemical analysis was performed in a group of archive adrenocortical samples, including 2 aldosterone-producing adenomas, 3 cortisol- producing adenomas, 3 non-functioning adrenal adenomas, and 4 cortisol-producing ACCs. Each case was analyzed for VDR protein expression on 10% formalin-fixed and paraffin-embedded sam- ples. Immunostaining was performed by the streptavidin-biotin amplification method using a Histfine Kit (Nichirei Co. Ltd., Tokyo, Japan). Antigen retrieval was performed by heating the slides in an autoclave for 5 min in citric acid buffer (2mM citric acid and 9 mM trisodium citrate dehydrate, pH 6.0). The dilution of the primary antibodies was 1:50. The antigen-antibody complex was visualized with 3,3’-diaminobenzidine solution [1 mM 3,3’- diaminobenzidine, 50 mM Tris-HCl buffer (pH 7.6), and 0.006% H2O2] and counterstained with hematoxylin. A human breast cancer specimen was used as a positive control. Negative con- trols were incubated with normal mouse antiserum instead of the primary antibody, which uniformly demonstrated no reaction (not shown).
In the same cases (4 ACCs and 8 benign tumors), we per- formed immunohistochemistry for Ki67/MIB1, a cellular marker of all active phases of the cell cycle (G1, S, G2, and mitosis) used as a guidance for clinical management of ACC [12,27], on 4 pm slices from paraffin-embedded adrenal tissue specimens. The monoclonal mouse antibody against Ki67 (MIB1) (Dako, Glostrup, Denmark) was diluted 1:100. Phosphate buffered saline (0.01 M, pH 7.4) or normal mouse IgG was used instead of the primary antibody as negative controls, and no immunoreactivities were observed in control sections. Before counting, the areas for analysis were assessed by the same observer (A. F.). Five to 10 high power fields (400-folds) were selected, and at least 1000 cells were evaluated. The number of Ki67/MIB1-positive cells per 100 adrenocortical cells was designated as labeling index.
2.9. Steroid determination
Subconfluent H295R cells, seeded in 24-well plates at a con- centration of 2 x 105 cells/well, were washed twice with PBS and grown in medium without serum. After 24h, cells were washed again with PBS and grown in 0.5 ml/well fresh serum-free medium. H295R cells were exposed for 96h to 1 nM and 10 nM 1,25(OH)2D3. The experiment was performed three times in trip- licate. Aldosterone, cortisol and dehydroepiandrosterone-sulfate
(DHEA-S) were determined in aliquots (1 ml) of conditioned media by RIA kits (Diagnostic System Laboratories, Webster, TX, USA) fol- lowing the manufacturer’s instructions. Results were normalized to the cellular protein content of each well and compared with medium from control cells without 1@,25(OH)2D3.
2.10. Statistical analysis
Multiple comparisons between groups were assessed by one- way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. The Mann-Whitney U test was used for two-group com- parisons. Values are expressed are means ± SD. A Pvalue <0.05 was considered statistically significant. Analyses were performed with GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA).
3. Results
3.1. Expression of VDR in H295R cells and adrenocortical tissues
The presence of VDR was demonstrated by qRT-PCR, and the amplified cDNA fragments were visualized in agarose gels in H295R cells, in normal adrenals and in pathological adrenocortical tissues (Fig. 1A). Western blotting confirmed the presence of VDR related protein (Fig. 1B). VDR mRNA showed a tendency, although not sig- nificant, to be expressed at higher levels in benign adrenocortical tumors than in ACC (1.77±0.9 vs. 0.7±0.6 arbitrary units) (Fig. 1C).
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Values expressed as percentages of control cells (100%). Mean ±SD from five independent experiments each performed with triplicate sample. *** P<0.001 and **** P < 0.0001 compared with control cells (vehicle).
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In H295R cells, the immunofluorescent signal for VDR displayed a diffuse nuclear localization (Fig. 2).
3.2. Effect of 1a,25(OH)2D3 on H295R cell proliferation and cell-cycle phase
Incubation of H295R cells for 96 h with 1a,25(OH)2D3 at a con- centration ranging from 1 nM to 1 µM induced a moderate but significant decrease (P<0.01) of cell proliferation, as measured by [3H]thymidine incorporation (Fig. 3).
Flow cytometry analysis showed that a 96h treatment with 10nM 1a,25(OH)2D3 increased the percentage of H295R cells in G0/G1 phase from 44.08 to 62.30%, while the percentage of cells in S phase decreased from 19.56 to 14.76%, (Fig. 4). There was no apoptotic effect after treatment with 10,25(OH)2D3, since the percentage of H295R apoptotic cells (APO) did not change (2.96-2.71%). Silencing of VDR in H295R cells by transfection with a siRNA to specifically interfere with VDR gene expression resulted in a strong reduction of VDR protein expression as shown by western blotting (Fig. 5A). Protein expression of the cell-cycle
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(A) Western blot analysis of VDR expression in H295R cells: 1 = untreated cells; 2 = cells treated with 10,25(OH)2D3; 3 = untreated cells after VDR silencing; 4 = cells treated with 1a,25(OH)2D3 after VDR silencing; (B) western blot analysis of CDK4 expression in H295R cells: control (crl) untreated cells and cells treated with 1,25(OH)2 D3; (C) western blot analysis of CDK4 expression in H295R cells after VDR silencing: control (crl) untreated cells and cells treated with 1x,25(OH)2D3. Values are mean ± SD from three independent experiments. * P<0.01.
regulator CDK4 was reduced using the same 1x,25(OH)2D3 dose and time treatment (Fig. 5B). CDK4 expression was not affected by 1a,25(OH)2D3 in H295R cells after VDR silencing with specific siRNA-transfection (Fig. 5C).
3.3. Effect of 1a,25(OH)2D3 on adrenocortical steroid production
To study whether 1a,25(OH)2 D3 has any effect on the secretion of steroid hormones, H295R cells were treated with 1@,25(OH)2D3 at two different concentrations (1 nM and 10nM), corresponding to those able to induce a significant reduction in cell number (see above). In comparison with untreated control cells, cells treated with 1a,25(OH)2D3 showed around a 20% decrease of aldosterone, cortisol and DHEA-S in the culture medium. Values expressed as a percentage of steroid production by control cells are shown in Fig. 6.
3.4. Immunohistochemical findings
Immunohistochemical staining of two representative cases of adrenal tumorous tissues was reported in Fig. 7. VDR immunore- activity was clearly detected in a cortisol-producing adenoma, predominantly in the cytoplasm, with occasional nuclear staining. VDR immunoreactivity was present in a cortisol-producing carci- noma, but it was weak and limited to only scattered tumor cells. The immunohistochemical pattern was similarly different between benign and malignant tumors in all other tissue specimens exam- ined (see Section 2.8).
Ki67/MIB1 labeling index was higher in the 4 ACCs than in the 8 benign adrenocortical tissue samples examined at immuno- histochemistry (14.50±4.79 vs 1.87±0.83%, P=0.002). Three out of 8 benign tumors (2 incidentally found non-hormonally active adrenal masses and one cortisol-producing adenomas) showed a VDR mRNA level below the highest VDR mRNA single value of ACC samples (Fig. 1). In all cases, gross examination of the surgical specimens revealed a cortical nodule (12, 20 and 25 mm diame- ter, respectively) located in the adrenal gland, with quite sharp out-linings from the normal cortex and yellow-colored homoge- neous cut surface. All the criteria for adrenocortical adenoma were fulfilled at histological analysis, showing an overall nodular pat- tern growth and two type of cells based on cytoplasm characters, i.e., clear and eosinophilic, with marked prevalence of clear zona- fasciculata like cells. Tumor cells were aggregated in nests and/or cords lined by thin fibrovascular endocrine type-stroma. Mitoses, necroses and hemorrages were absent. The tissue adjacent to the tumor was normal. Ki67/MIB 1 labeling index ranged from 1 to 3%.
4. Discussion
Previous clinical studies have reported an association between primary aldosteronism or cortisol excess and a vitamin D deficient state, suggesting a causal role of adrenal steroid over-production in inducing vitamin D deficiency, through various mechanisms [28-30]. However, scarce data are available on the effects of vita- min D deficiency on adrenal growth and function either in vivo or in vitro. Early reports have shown the presence of VDR in the human adrenal [31], but its function has remained elusive. In our study, we first confirmed the presence of VDR in H295R cells by mRNA expression, western blot and immunofluorescence, with VDR dis- playing a diffuse nuclear localization. Lundqvist et al. [16] observed that 24h treatment with 1x,25(OH)2D3 concentrations of 1 nM significantly decreased the production of several adrenocortical steroid hormones in the H295R cell line. H295R cells transfected by high-efficiency nucleofection, leading to VDR overexpression, showed an increase of angiotensin II-stimulated steroidogenesis [32]. However, the role of 1a,25(OH)2D3 in tumor cell proliferation
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potentially associated with these effects was not investigated. Our findings are in accordance with previous data that 1x,25(OH)2D3 reduces the secretion of steroid hormones, but also indicate that it affects cell proliferation in the human H295R cell line model. Treatment of these cells with 1x,25(OH)2D3 at a concentration of 1 nM, i.e., a dose corresponding to the high-normal levels found in the human circulation and shown to be effective in several other types of tumor cell lines [8,33], exerted in fact a moderate anti- proliferative effect (approximately 20%). Due to the long doubling time of H295R cells (i.e., >72 h) we tested the effect 1a,25(OH)2D3
Adenoma
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using a 96 h time-treatment [34]. A significant decrease of aldos- terone, cortisol and DHEA-S paralleled cell growth inhibition after exposure to 1a,25(OH)2D3. Percentage inhibition of cell prolifer- ation was similar to the percentage reduction in adrenocortical steroid production using the same 1x,25(OH)2D3 treatment con- centrations. This would suggest that the inhibitory effect of adrenal steroid production in vitro was mainly due to suppression of adrenal cell growth per se. Lundqvist et al. [16] observed in H295R cells a significant decrease in corticosterone and androgen production using 24h treatment (a time span not interfering on cell prolif- eration doubling time of this cell line) with 1 nM 1x,25(OH)2D3; aldosterone decrease was slight. This was attributed to suppres- sion of 21-hydroxylase activity. We cannot exclude that in our experiments 1a,25(OH)2D3, in addition to cell growth inhibition, has affected directly steroidogenesis.
Exploring the mechanisms underlying the antitumor effect of 1a,25(OH)2D3, we found that the decrease in H295R cell prolifer- ation was accompanied by no change in apoptotic cell proportion, as assessed by fluorescence propidium iodide-stained flow cytom- etry. The anti-proliferative effect of 1x,25(OH)2D3 was mainly due to the block of transition from G1 to S cell phase. In fact, cell cycle arrest in G1 was confirmed by reduced CDK4 expression, a molec- ular marker of G1-S cell cycle transition phase, in contrast to its unaltered expression when VDR gene expression was specifically silenced. This indicates VDR as the key effector for proliferation in this human ACC cell line. The absence of apoptosis after expo- sure to 1a,25(OH)2D3 has been observed in other cell models, and studies have shown that apoptosis is not always the predominant mechanism induced by vitamin D for cell growth inhibition [35,36].
VDR was also demonstrated in all our adrenocortical tissues by western blot, and mRNA was quantified by qRT-PCR; in addition, VDR was detected at immunohistochemistry. Expression of VDR mRNA was lower in ACC than in benign adrenocortical tumors and, in terms of protein immunostaining, VDR intensity was evident in benign tumors but it was weak in cortisol-producing ACC. This may reflect the loss of a protective role of VDR against malignant
transformation of adrenocortical cells, as reported in many other cancer types [10]. Whether this event is a primary phenomenon leading cells to malignancy or is a simple association with abnor- mally high cell proliferation (as shown by high Ki67/MIB1 labeling index), is unclear. Moreover, it remains to be clarified by VDR bind- ing studies in H295R cells compared with cell lines derived from benign adrenocortical tumors, whether a reduced activation of VDR in ACC would have been due to decreased number of VDRs or to reduced affinity of 1x,25(OH)2D3 for its receptor. An alterna- tive explanation could be that mitotane, the drug used in all our patients with ACC, stimulates CYP3A4 expression, potentially lead- ing to reduced 25(OH)D3 and 1x,25(OH)2D3 bioavailability [37,38]. Although actual values of these hormones were not available in our patients, it cannot be excluded that mitotane could have deac- tivated VDR in their adrenals. A blocking effect of mitotane on adrenocortical cancer cell cycle, which could potentially involve VDR, is still controversial [39]. Furthermore, VDR tissue expres- sion was detected both in the nucleus and in the cytoplasm of adrenocortical cells of both adenomas and ACC, consistent with translocation of the VDR from the cytoplasm to the nucleus after ligand binding [40,41].
4.1. Conclusions
In summary, (1) VDR was demonstrated in H295R cells by mRNA expression, western blot and immunofluorescence; (2) slightly supra-physiological concentrations of 1x,25(OH)2D3 had a moder- ate anti-proliferative effect on these cells, leading to a concomitant decrease in cortisol, aldosterone and DHEA-S; (3) anti-proliferative effect of 1,25(OH)2D3 was due to cell cycle arrest, promoting accumulation of cells in G1 phase, without inducing apoptosis; (4) the low mRNA expression levels at qRT-PCR as well as the weak immunohistochemical expression of VDR in ACC tissues suggests a protective role of VDR against malignant adrenocortical growth. The potential use of 1a,25(OH)2D3 in the prevention and/or treat- ment of patients has been in fact proposed for other types of human
malignant tumors [8,10]. In this regard, hypercalcemia is the dose- limiting factor for the application of 1x,25(OH)2D3 in the clinic, particularly when continuous dosing schedules are employed. Efforts are currently underway to develop analogs of 1a,25(OH)2 D3 that dissociate the anti-proliferative and calcemic effects, raising the possibility of using bioactive vitamin D analogs with anti- proliferative potency at much lower concentrations [9,42].
Authors disclosure summary
The authors have nothing to declare.
Acknowledgements
This work was supported by Fondo Investimenti Ricerca di Base (FIRB) Accordi di Programma 2011, RBAP1153LS-02 from the Ministry of Education, University, and Research, Rome, Italy and by Grant IG 10344 from Associazione Italiana Ricerca sul Cancro (AIRC), Milan, Italy.
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