ORIGINAL ARTICLE
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The effects of mitotane and 1a,25-dihydroxyvitamin D3 on Wnt/ beta-catenin signaling in human adrenocortical carcinoma cells
B. Rubin1 . C. Pilon1 . R. Pezzani2 . A. Rebellato1 . F. Fallo1 D
Received: 2 July 2019 / Accepted: 30 September 2019 @ Italian Society of Endocrinology (SIE) 2019
Abstract
Purpose Mitotane is the only chemotherapeutic agent available for the treatment of adrenocortical carcinoma (ACC), how- ever, the anti-neoplastic efficacy is limited due to several side-effects in vivo. There is, therefore, a need of exploring for new anti-tumoral agents which can be used either alone or in combination with mitotane. The active vitamin D metabolite 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3) acts as an anti-proliferative agent in human cancer by inhibiting the Wnt/ beta-catenin pathway through the vitamin D receptor (VDR). The aim of this study was to study the effects of mitotane and 1a,25(OH)2D3, individually or in combination, in an in vitro model with H295R ACC cells, and to elucidate the molecular events behind their effects involving the Wnt/beta-catenin signaling.
Methods and results Multiple concentrations of mitotane and 1x,25(OH)2D3, individually or in combination, were tested on H295R cells for 24-96 h, and the effects analysed by MTT. A reduction in cell growth was observed in a dose/time- dependent manner for both mitotane and 1x,25(OH)2D3. In addition, a combination of clinically sub-therapeutic concen- trations of mitotane with 1x,25(OH)2D3, had an additive anti-proliferative effect (Combination Index = 1.02). In a wound healing assay, individual treatments of both mitotane and 1x,25(OH)2D3 reduced the migration ability of H295R cells, with the effect further enhanced on combining both the agents. Western blotting and qRT-PCR analysis showed a modulation of the Wnt/beta-catenin and VDR signaling pathways.
Conclusion Our results show an additive effect of mitotane and 1x,25(OH)2D3 on the inhibition of H295R ACC cell growth and viability, and suggest that molecular mechanisms of their effects involve a functional link between VDR and Wnt/beta- catenin pathways.
Keywords Adrenocortical cancer cells · Mitotane · 1x,25-Dihydroxyvitamin D3 · Wnt/beta-catenin
Introduction
Adrenocortical carcinoma (ACC) is a rare malignancy (0.5-2 cases per million/year) that carries a poor progno- sis due to its tendency to metastasize before diagnosis and has a high risk of relapse post radical surgery [1]. Mito- tane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p’DDD), associated with or without traditional chemotherapeutic agents, is the only referral drug used for
the treatment of ACC treatment. It inhibits cell growth and impairs steroidogenesis [2-4] through a modality of action that still remains unclear [5]. However, its efficacy is limited due to poor pharmacokinetic properties and dose-limiting toxicity. Furthermore, therapeutic serum levels of mitotane may take several months to be achieved [6]. Given the high mortality rate and aggressiveness of ACC, there is a need of exploring new anti-tumoral agents which can be used indi- vidually or in combination with mitotane.
Besides the classical role in calcium and bone homeo- stasis, 1x,25-dihydroxycholecalciferol D3 (1a,25(OH)2D3 or calcitriol), the active metabolite of vitamin D, is known to have “noncalcemic” effects in a variety of cells after bind- ing to vitamin D receptor (VDR) [7]. In spite of contro- versy, several clinical studies support the role of vitamin D either alone or in combination with other chemotherapeutic agents, for the prevention and treatment of different cancers
☒ F. Fallo
1 Endocrine-Metabolic Laboratory, Clinica Medica 3, Department of Medicine (DIMED), University of Padova, Via Giustiniani 2, 35128 Padua, Italy
2 Endocrinology Unit, Department of Medicine (DIMED), University of Padova, Padua, Italy
[8-10]. It has been recognized that 1x,25(OH)2D3 protects against tumor formation through several VDR-mediated effects, including the inhibition of cell growth, cell differ- entiation, migration, invasion, and apoptosis, making it an ideal candidate for cancer regulation [11-13]. The antipro- liferative effect of 1a,25(OH)2D3 in cancer was observed mainly through VDR mediated inhibition of the Wnt/beta- catenin pathway [14, 15]. A relationship between vitamin D system and adrenal pathophysiology and growth has been recently highlighted [16, 17]. We previously demonstrated an antiproliferative effect of 1a,25(OH)2D3 on the H295R ACC cells, and a decreased expression of VDR mRNA and protein in a series of human ACCs, which suggested the loss of a protective role of VDR against malignant adrenocortical cell growth in these cases [18, 19].
The aim of this study was to study the effects of mito- tane and 1x,25(OH)2D3, individually or in combination, in an in vitro model with H295R ACC cells, and to elucidate the molecular events behind their effects involving the Wnt/ beta-catenin signaling.
Materials and methods
Adrenocortical carcinoma cell line
The human ACC cell line H295R was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM-F12 (Gibco-ThermoFisher Scientific, Waltham, MA, USA) supplemented with 1% ITS Liquid Media Supplement (100 X; ThermoFisher Scientific), 2% of Fetal Bovine Serum (FBS) (Gibco-ThermoFisher Sci- entific), and 1% antibiotics (100 mg/ml streptomycin sulfate) (Gibco-ThermoFisher Scientific).
Chemical agents
Mitotane was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in ethanol at a stock concentra- tion of 10-1 M (stored a - 20 ℃) and diluted with DMEM- F12 to the desired final concentration for in vitro studies. 1a,25(OH)2D3 was dissolved in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich), preserved a - 20 ℃ and diluted in DMEM-F12 prior to use.
Cell viability analysis
H295R cells were plated in 96-well tissue culture plates at a density of 1 × 104 cells/well in supplemented medium and used for viability studies employing the 3-(4,5-dimeth- ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Briefly, cells maintained overnight in low serum medium (DMEM-F12 with 0.1% FBS) were
treated with various concentrations of mitotane (from 0.01 to 100 µM) and 1x,25(OH)2D3 (from 0.01 to 10 µM) for 24-96 h. MTT (2.5 mg/ml) was then added for an addi- tional 4 h and the percentage cell viability calculated from the absorbance values as follows: (A tested - A blank)/(A untreated control - A blank) × 100. All experiments were performed in quadruplicate.
Combination experiment analysis
To assess the interaction between mitotane and 1a,25(OH)2D3, the Combination Index (CI) according to the Chou-Talalay method [20] was calculated. Based on the dose-response curves and mean IC50 values, cell viability was measured by MTT after 96 h treatment with the indi- vidual agents or in combination at a constant dose ratio (mitotane: 1a,25(OH)2D3=1:1.42847), and a series of CI values was generated over a range of growth inhibition lev- els. Values of CI <1, =1, and> 1 indicate synergistic, addi- tive and antagonistic effects, respectively. The ‘CompuSyn’ software 3.0.1 (ComboSyn inc. Paramus, NJ, USA) was used for CI calculation.
Flow cytometry assessment of cell cycle and apoptosis
Cell cycle status was studied by propidium iodide (Sigma- Aldrich) staining. Briefly, H295R cells were plated in six-well plates at a density of 1 × 10° cells/well and were either untreated or treated for 96 h with mitotane and 1a,25(OH)2D3, individually or in combination. Apoptosis was then assessed by the Annexin V-FITC Apoptosis detec- tion Kit (Bender MedSystems, Vienna, Austria) in accord- ance with the manufacturer’s protocol. Cell cycle and apop- tosis were analyzed three times, using the CytoFLEX flow cytometer (Beckman Coulter, Milano, Italy) and data were analyzed with CytExpert flow cytometry analysis software (Beckman Coulter, Pasadena, CA, USA). Experiments were performed in triplicate.
Wound healing assay
Cells were plated in six-well plates at 2× 10° cells/well and were allowed to grow until confluence. A scratch wound was then applied with a 200 ul pipette tip before starting 96 h treatment with mitotane (10 µM) or 1a,25(OH)2D3 (3 M) individually and in combination. At the end of the treatment, the medium was replaced with fresh complete culture medium and the plates were incubated for a further 72 h without treatment (i.e., up to an overall time of 168 h). The average distance of migrating cells was measured under an inverted microscope (40 x) at 0 h and 168 h. Experi- ments were performed in triplicate. In accordance with our
and other previous studies [21-23], timing of experimental observations was based on considering the relatively long doubling time (~72 h) of H295R cell population in culture.
Western blotting
H295R cell lysates (10 µg of protein) were fractionated into cytoplasmic and nuclear fractions with NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (ThermoFisher Scien- tific) according to the manufacturer’s protocol. Briefly, pro- teins were extracted, loaded onto SDS/PAGE-GEL (Thermo Fisher Scientific), and electro-blotted onto nitrocellulose membranes. Membranes were then blocked with 5% BSA 0.1% (v/v) Tween20 in TBS for 1 h at room temperature, and incubated overnight at 4 ℃ with different primary antibod- ies. The antibodies applied are as follows: anti-beta-actin, Clone AC-15 (1/10,000, monoclonal) (Sigma-Aldrich), anti-beta-catenin (1/1000, monoclonal) (BD Transduction Laboratories, East Rutherford. NJ, USA), and VDR (1/500, polyclonal) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were then washed and incu- bated for 1 h at room temperature with 1/8000 dilution (anti- mouse) IRDye secondary antibody (Li-Cor Biosciences, Lincoln, Nebraska, USA). Immunoreactivity was detected by Odyssey CLX system (Li-Cor Biosciences, Lincoln, NE, USA) infrared scanner and the signal intensity quantified by ImageJ analytical software. Experiments were performed in triplicate.
Quantitative real-time PCR (qRT-PCR)
Total cellular RNA was extracted from H295R cells treated with or without mitotane 10 µM, with or without 1a,25(OH)2D3 3 µM, and with or without their combina- tion for 96 h, using a Qiagen RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) in accordance to the manufactur- er’s protocol. Gene expression levels of VDR, beta-catenin,
N-Cadherin (CDH-2), c-Myc, Dickkopf-related protein-1 (DKK-1) and beta-actin (as housekeeping gene) were eval- uated by quantitative real-time (qRT)-PCR using a Sybr Green Assay kit (Thermo Fisher Scientific). The primer sequences are summarized in Table 1.
Data were obtained as Ct values and used to determine AC, values (AC)=C, of the target gene minus Ct of the housekeeping gene). The equation 2-44Ct was applied to calculate the fold changes in gene expression between the categories of samples.
Statistical analysis
All statistical calculations were performed using GraphPad Prism, version 5.03 for Windows (GraphPad Software, San Diego, CA, USA) and Microsoft Excel software. Results are expressed as mean + SD. Data were tested for normal distribution using the Kolmogorov-Smirnov test, and dif- ferent groups were compared by ANOVA with Bonferroni correction. A P value <0.05 was considered statistically significant.
Results
Effects of mitotane and 1a,25(OH)2D3 on H295R cell viability and growth
The effects of mitotane and 1a,25(OH)2D3 on H295R cell viability were evaluated 24 and 96 h post treatment and compared with untreated control cells. Treatment of H295R cells with increasing concentrations of mitotane (0.1-100 µM) led to a reduction in cell growth in a dose and time-dependent manner (Fig. 1a) with an IC50 of 10 µM. Similarly, 1a,25(OH)2D3 inhibited H295R cell
| Gene | Primers pair sequence | Base pair |
|---|---|---|
| VDR | F: GAAGCCTTTGGGTCTGAAGTG R: CCGCCATTGCCTCCATCC | 96 |
| beta-catenin | F: CTTGCTCAGGACAAGGAAGC R: CATATGTCGCCACACCTTCA | 103 |
| DKK-1 | F: GATCATAGCACCTTGGATGGG R: GGCACAGTCTGATGACCGG | 226 |
| CDH-2 | F: TCCAGACCCCAATTCAATTAATATTAC R: AAAATCACCATTAAGCCGAGTGA | 139 |
| c-Myc | F: AATGAAAAGGCCCCCAAGGTAGTTATCC R: GTCGTTTCCGCAACAAGTCCTCTTC | 112 |
| beta-actin | F: GGGACGACATGGAGAAAATCTG R: CACGCAGCTCATTGTAGAAGGT | 51 |
A
120
100
% cell viability
80
60
24h
40
96h
20
0
0
0,001 μΜ 0,01 μ.Μ
0,1 μ.Μ
1 μΜ
10 μΜ
50 μ.Μ
100 μ.Μ
[Mitotane]
B
120
100
% cell viability
80
60
24h
96h
40
20
0
0
0,001μΜ
0,01μΜ
0,1μΜ
1μΜ
10μ.Μ
[1a,25(OH)2D3]
proliferation in a dose and time-dependent manner as well with an IC50 of 3 uM (Fig. 1b). On the basis of this information, we decided to use the IC50 concentration (10 µM for mitotane and 3 µM for 1x,25(OH)2D3 for both the agents individually or in combination in all subsequent experiments.
Effects of combination treatments on H295R cell viability
A dose-effect curve of H295R cells at 96 h showed an increase of fractional cell viability (Fa) with the use of increasing concentrations of both the agents individu- ally or in combination (Fig. 2a) in addition, five different mitotane-1a,25(OH)2D3 combination concentrations were tested with an additive effect on cell growth inhibition being observed at a mean CI of 1.02 (Fig. 2b).
Cell-cycle alteration and induction of apoptosis
Flow cytometry analysis was used to analyze the effects of mitotane, 1a,25(OH)2D3 and their combination, on cell- cycle phase distribution and apoptosis. We used the IC50 dose obtained from the MTT dose-response curves at 96 h.
Mitotane modified the cell cycle of H295R cells whereby 20% of the cells were observed in sub-G0 phase compared to control (10%). Furthermore, a concomitant reduction of cells in G0-G1, S and G2/M phases, from 53%, 13% and 24% (untreated cells) to 51%, 10% and 19% following treatment was also observed (Fig. 3a, b). Interestingly, 1a,25(OH)2D3 also led to a cell cycle growth arrest with an increase in GO-G1 phase, from 53% (untreated cells) to 64% post treat- ment. A concomitant reduction in S and G2/M phases, from 13 to 24% (untreated cells) to 8% and 16% following treat- ment was also observed (Fig. 3a, b). A combination treat- ment of mitotane and 1a,25(OH)2D3 also induced a cell
A
1
Fa
0.5
Mitotane
1a,25(OH), D3
Mitotane+1a,25(OH),D3
0
0
Dose
50
B
2
Mitotane+1a,25(OH),D3
CI
0
0
0.5
1
Fa
cycle modification, whereby a marked increase of cells in sub-G0 phase (23%) was observed compared to untreated cells (10%). In addition, a concomitant reduction in GO-G1, S and G2/M phases, from 53%, 13%, 24% (in untreated cells) to 51%, 7%, 19% was also observed following treatment (Fig. 3a, b).
Analysis of apoptosis showed no significant difference in necrotic cells or cells in early/late apoptosis between controls and cells treated with both the agents individually. However, a combination treatment was highly effective in increasing
necrotic cells (from 4% in untreated to 10% in treated cells) and reducing viable cells (from 94% in untreated to 87% in treated cells), while no change was observed in early/late apoptosis (Fig. 4a, b).
Inhibition of cell migration in wound healing assay
The ability of H295R cells to migrate after an exposure of 96 h to mitotane or 1a,25(OH)2D3 or their combination, followed by a further 72 h incubation without treatment, was tested by a wound-healing assay. Migration of H295R cells decreased from 63% (for untreated cells) to 24% with mitotane, and to 33% with 1a,25(OH)2D3, respectively. Fur- thermore, the reduction in migration ability was even greater (3%, P <0.05) following a combination treatment of mito- tane + 1a,25(OH)2D3 (Fig. 5a, b).
Modulation of Wnt/beta-catenin and VDR signaling pathways
Western blotting was used to assess the presence of beta- catenin and VDR proteins, and their intracellular localiza- tion, as well the effects of mitotane and 1x,25(OH)2D3 on Wnt/beta-catenin and VDR signaling. VDR and beta-catenin were both detected in H295R cells with the former more evident in nuclear extracts while the latter was expressed at both the cytoplasmic and nuclear level (Fig. 6a).
As shown in Fig. 6b-d, treatment with mitotane was unable to induce significant changes, in beta-catenin (cyto- plasmic/nuclear) or VDR (nuclear) expression. Interestingly, 1a,25(OH)2D3 had no effect on cytoplasmic beta-catenin, but a reduction in the nuclear expression of beta-catenin (P <0.05) and an increase in nuclear VDR expression (P <0.05) was observed. A combination treatment of mito- tane with 1a,25(OH)2D3 had no significant effect on beta- catenin (nuclear/cytoplasm) and VDR expression in com- parison with control.
We further examined the effects of the two agents and their combination treatment on H295R cell gene expres- sion of VDR, beta-catenin and beta-catenin related factors (Table 1), by qRT-PCR. As shown in Fig. 7a, b, treatment with mitotane had no effect on VDR and beta-catenin mRNA levels. At variance, la,25(OH)2D3 induced a slight decrease in beta-catenin mRNA levels and a significant increase of VDR mRNA levels (P<0.01). In addition, a combined treatment caused a further reduction in beta-catenin mRNA levels as well as a further increase of VDR mRNA lev- els. mRNA expression levels of N-Cadherin (CDH-2), a calcium-sensitive cell adhesion molecule regulating beta- catenin and found to be down-regulated in ACCs [24], and c-Myc, a downstream effector of Wnt/beta-catenin sign- aling, were unchanged by each single treatment or their combination (data not showed). mRNA levels of DKK-1,
A
100%
24
19
16
19
80%
10
8
7
Cell population (%)
13
60%
G2/M
51
51
OS
40%
64
53
GO-G1
O sub GO
20%
20
23
10
12
0%
Control
Mitotane 10uM
1a,25(OH)2D33uM
Mitotane 10uM +
1a,25(OH)2D33uM
B
(x 101)
100
(x101)
100
Sub00(8,91%)
Sub00(22,00%)
00-01(51,23%)
00-01(46,58%)
Court
H
4
Court
A
02-M(20,00%)
8
G2-M(16,92%)
$(11,42%)
S(8,45%)
0
.
0
50
ECD-A
100
0
50
(x10)
ECD-A
100
(x10)
Control
Mitotane 10uM
(x101)
100
(x101)
100
Sub00(8,80%)
00-01(63,62%)
Sub00(20,60%)
00-01(54,35%)
Court
Court
8
02-M(14,89%)
8
02-M(11,42%)
S(7,42%)
S(9.20%)
.
o
0
50
ECD-A
100
(x10)
0
50
ECD-A
100
(x 10)
1a,25(OH)2D3 3uM
Mitotane 10uM +
1a,25(OH), D3 3uM
B
3
Necrosis(0,02%)
Late apoptosis(0,01%)
2
Necrosis(7,76%)
Labe apoptosis(1,81%)
5
3
A
PIECO-A
O
PIECD-A
105
100%
4
4
5
1
1
NH
4
HH
10
10*
HN
3
80%
Necrosis
·
Early apoptosis(0.01%)
0
Early apoptosis(1,53%)
0
10ª
10º
100
0
104
10º
10ª
Annexin V FITO.A
Annexin V FITO-A
Cell population (%)
Late apoptosis
Control
Mitotane 10uM
60%
Early apoptosis
3
Necrosis(6,8%)
Lice apoptosis(1,37%)
94
3
Necrosis(17,51%)
Late apoptosis(3,23%)
93
94
40%
87
Viable cells
O
3
PIECO-A
105
PIECO-A
10º
20%
5
104
0%
Control
Mitotane 10uM
1a, 25(OH)2D33uM
Mitotane 10uM+
0
Mable cells(90 83%)
Early apoptosis(0,97%)
104
105
10º
o
Mable cells(78,69%)
Early apoptosis(0,58%)
1a,25(OH)2D33uM
0
Annexin V FITO-A
0
104
10ª
Annexin V FITC.A
10ª
1a,25(OH)2 D3 3uM
Mitotane 10uM +
1a,25(OH)2 D3 3uM
A
B
T=0h
T=168h
100
Control
80
Occupied cell area (%)
Occupied cell area (9%)
Mitotane 10uM
60
**
*
40
T
1a,25(OH), D3 3uM
20
63
24
33
3
Mitotane 10uM + 1a,25(OH), D3 3uM
0
0
Mitotane 10uM
1a,25(OH)2D33uM
Mitotane 10uM +
1a,25(OH)2D33uM
a downstream inhibitor of Wnt/beta-catenin pathway, were also analyzed. We detected the presence of DKK-1 mRNA in H295R cells, where it was observed that treatment with the drugs individually or in combination increased its expression significantly (P<0.05) (Fig. 7c), with a more marked effect with the combined treatment.
Discussion
H295R ACC cells provide the most appropriate preclinical model for testing drug-mediated effects on adrenal prolif- eration and steroidogenesis [25]. 1a,25(OH)2D3 elicits anti- tumor effects mainly through the induction of cancer cell apoptosis, cell cycle arrest, differentiation, angiogenesis and inhibition of cell invasiveness, and is known to potentiate the anti-tumor activities of multiple chemotherapeutics agents [8, 26-28]. As reported previously [18], we confirmed a (strong) negative effect of 1a,25(OH)2D3 on H295R cell viability using MTT. This effect was potentiated in combi- nation with mitotane at a concentration (10 uM) lower than the therapeutic range (i.e. 14-20 mg/l) in vivo in humans. Reduction of cell growth was dose- and time-dependent for either mitotane or 1a,25(OH)2D3, and the Combination Index indicated that the effect was of an additive type. This additive effect was also observed in a wound healing study, whereby both mitotane and 1a,25(OH)2D3 decreased the ability of H295R cells to migrate. Furthermore, the effect was more marked when using both the drugs in combination.
of nine measurements, with experiments performed in triplicate. (treatment vs control, *P<0.05); b representative image of wound healing assay in H295R cells after 96 treatment with mitotane, or 1a,25(OH)2D3 3 uM or their combination, and a further 72 h incuba- tion without treatment (i.e., overall time of 168 h)
Analyses of flow cytometry data revealed that mitotane as an individual treatment was able to moderately increase necro- sis of H295R cells, in line with other studies [29]. The dif- ference in terms of a lesser percentage of cells death using MTT in comparison with flow cytometry was apparently strong, since the latter procedure implies multiple cell wash- ing, leading per se to a loss of a relevant number of necrotic cells. Furthermore, 1a,25(OH)2D3 induced a cell cycle arrest in G1 phase, confirming our previous data [18]. Mitotane has been shown to potentiate the effect of chemotherapeutic or other agents in human ACC cell lines [30-32]. This seems to apply to our combination study, suggesting its use in a clinical setting. In fact, combination modality of treatment might be useful when mitotane is at the minimal effective doses, i.e. in the initial titration time or in patients intolerant to this drug at the therapeutic range.
A major reason for the lack of an effective targeted treat- ment strategy for ACC is an inadequate understanding of the molecular pathogenesis of the disease [33, 34]. Genetic and epigenetic dysregulations of Wnt/beta-catenin signaling appear to dominate various cancer-driving abnormalities in majority of ACCs [35-37]. Activating mutations of exon 3 of the beta-catenin gene (CTNNB1), that encodes a specific serine/threonine rich domain which is phosphorylated by GSK-3beta and is essential for the targeted degradation of beta-catenin, are found in human cancers including ACCs [38-40]. Of note, the H295R cells harbor the S45P CTNNB1 activating mutation, leading to constitutive beta-catenin- dependent transcription [41]. Treatment of this cell line
A
Cytoplasm
Nucleus
1
2
3
4
1
2
3
4
VDR
beta-actin
Fig. 6 Western blotting. a Representative cytoplasmic and nuclear Western blots showing the expression of beta-catenin, VDR and beta-actin proteins in H295R cells after 96 h treatment with mito- tane, or la,25(OH)2D3 or their combination; b histogram quantifica- tion of cytoplasmic beta-catenin expression; c histogram quantifica-
with the Tcf/beta-catenin antagonist PKF115-584 inhibits the transcriptional effect of beta-catenin and decreases cell proliferation [41]. Silencing mutated beta-catenin inhibits cell proliferation and stimulates apoptosis [42]. Altogether, these findings establish the aberrant Wnt/beta-catenin path- way activation as a potential therapeutic target in ACC.
VDR activation has been also shown to antagonize the Wnt/beta-catenin pathway through several mechanisms in human and murine colon cancer [43]. In cell lines derived from this cancer, VDR activation promotes transcriptional upregulation of E-cadherin, which inhibits beta-catenin nuclear localization and induces translocation to adherens junction on the plasma membrane [44]. In addition, ligand- dependent VDR-signaling increases the mRNA expression of DKK-1, an inhibitor of the canonical Wnt signaling path- way, preventing activation of beta-catenin [45]. Western blot data on H295R cells showed that mitotane alone did not induce relevant alterations of beta-catenin and VDR expres- sion, while 1a,25(OH)2D3 caused a significant reduction of nuclear beta-catenin expression and a parallel elevation of nuclear VDR expression. On the other hand, a combination
beta-catenin
B
cytoplasmic beta-catenin
Mean band density (arbitrary unit)
1,2
1
T
0,8
T
T
T
0,6
0,4
0,2
0
1
2
3
4
nuclear beta-catenin
1,2
Mean band density (arbitrary unit)
1
0,8
0,6
0,4
T
*
0,2
0
1
2
3
4
nuclear VDR
Mean band density (arbitrary unit
4
*
3
2
1
0
1
2
3
4
treatment was unable to confirm the same reciprocal changes in nuclear beta-catenin and VDR expression as seen using 1a,25(OH)2D3 alone. At variance, at the molecular level, a combination of the two agents downregulated beta-catenin mRNA levels and upregulated VDR mRNA levels, greater than those obtained on applying 1a,25(OH)2D3 as an indi- vidual treatment. With regard to this, we speculate a negative interference of the combined presence of the two agents on antibody binding during blotting.
Regarding other key components of Wnt signaling, either mitotane or 1a,25(OH)2D3 as single agents, or their combination treatment, markedly increased the expression of DKK-1, which was associated with a strong reduction in nuclear beta-catenin expression. Tumor-suppressor like properties of DKK-1 are activated by vitamin D in cancer [45], and this may occur in ACC. mRNA levels of N-Cad- herin and c-Myc were not modified by any treatment in H295R cells. These results are in line with some reports [46] ], and in contrast with others [47].
The concentration of 1a,25(OH)2D3 at which an anti- proliferative effect on H295R cells in combination with
A
Relative gene expression
2
beta-catenin
1,5
1
0,5
0
Control
Mitotane 10 UM
1a,25(OH)2D33uM
Mitotane 10uM+
1a,25(OH)2D33 uM
B
VDR
Relative gene expression
4
3,5
3
2,5
2
T
1,5
1
0,5
0
Control
Mitotane 10 μ.Μ
1a,25(OH)2D33uM
Mitotane 10uM+
1a,25(OH)2D33uM
C
DKK-1
Relative gene expression
4
3,5
3
2,5
2
1,5
1
0,5
0
Control
Mitotane 10 μ.Μ
1a,25(OH)2D33uM
Mitotane 10uM+
1a,25(OH)2D33uM
mitotane was observed, were supra-physiological when com- pared with those normally circulating in humans. In a recent study among patients with advanced or metastatic colorectal cancer, the addition of a high-dose of vitamin D3 together with chemotherapy resulted in a significantly improved sup- portive hazard ratio [48]. 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 vitamin D analogs of 1a,25(OH)2D3 that dissociate the anti-prolif- erative and calcemic effects, raising the possibility of using vitamin D analogs with anti-proliferative potency at much lower concentrations [49]. Many vitamin D analogues have been in fact synthesized in search for VDR agonists with bet- ter ratio of activity: side effects at high therapeutic doses and some of them show a promising profile in preclinical settings [49, 50]. The main limitation of our study remains indeed
the unknown pharmacodynamic and metabolic interaction between mitotane and bioactive vitamin D in vivo. In fact, mitotane stimulates CYP3A4 expression, which potentially leads to reduced 25(OH)D3 and 1a,25(OH)2D3 bioavailabil- ity [51], limiting an in vivo application. Therefore, caution has to be maintained before translating the results into a clinical perspective.
In conclusion, our results show an additive effect of 1a,25(OH)2D3 and mitotane on the inhibition of adreno- cortical H295R cell growth and viability, and suggest that molecular mechanisms of their effects may involve a func- tional link between VDR and Wnt/beta-catenin pathways.
Acknowledgements This study was partially supported by HRA Pharma (Grant no. 266) (Paris, France).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent No informed consent was needed since no human participants are included in the manuscript.
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