CrossMark
In vitro antitumor activity of progesterone in human adrenocortical carcinoma
Martina Fragni1 . Chiara Fiorentini1 . Elisa Rossini1 . Simona Fisogni2 . Sara Vezzoli1 . Sara A. Bonini1 . Cristina Dalmiglio3 . Salvatore Grisanti3 . Guido A. M. Tiberio4 . Melanie Claps3 . Deborah Cosentini3 . Valentina Salvi5 . Daniela Bosisio5 . Massimo Terzolo6 . Cristina Missale1 . Fabio Facchetti2 . Maurizio Memo1 . Alfredo Berruti3 · Sandra Sigala1
Received: 2 July 2018 / Accepted: 15 October 2018 / Published online: 26 October 2018 @ Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Purpose The management of patients with adrenocortical carcinoma (ACC) is challenging. As mitotane and chemotherapy show limited efficacy, there is an urgent need to develop therapeutic approaches. The aim of this study was to investigate the antitumor activity of progesterone and explore the molecular mechanisms underlying its cytotoxic effects in the NCI-H295R cell line and primary cell cultures derived from ACC patients.
Methods Cell viability, cell cycle, and apoptosis were analyzed in untreated and progesterone-treated ACC cells. The ability of progesterone to affect the Wnt/ß-catenin pathway in NCI-H295R cells was investigated by immunofluorescence. Pro- gesterone and mitotane combination experiments were also performed to evaluate their interaction on NCI-H295R cell viability.
Results We demonstrated that progesterone exerted a concentration-dependent inhibition of ACC cell viability. Apoptosis was the main mechanism, as demonstrated by a significant increase of apoptosis and cleaved-Caspase-3 levels. Reduction of B-catenin nuclear translocation may contribute to the progesterone cytotoxic effect. The progesterone antineoplastic activity was synergically increased when mitotane was added to the cell culture medium.
Conclusions Our results show that progesterone has antineoplastic activity in ACC cells. The synergistic cytotoxic activity of progesterone with mitotane provides the rationale for testing this combination in a clinical study.
Keywords Adrenocortical carcinoma . Progesterone . Progesterone receptor . Cell viability
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12020-018-1795-x) contains supplementary material, which is available to authorized users.
☒ Alfredo Berruti alfredo.berruti@gmail.com
1 Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
2 Pathology Unit, Department of Molecular and Translational Medicine, University of Brescia and ASST Spedali Civili di Brescia, Brescia, Italy
3 Oncology Unit, Department of Medical and Surgical Specialties, Radiological Sciences, and Public Health, University of Brescia and ASST Spedali Civili di Brescia, Brescia, Italy
4 Surgical Clinic, Department of Clinical and Experimental Sciences, University of Brescia and ASST Spedali Civili di Brescia, Brescia, Italy
5 Section of Oncology and Experimental Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
6 Department of Clinical and Biological Sciences University of Turin, Internal Medicine 1, San Luigi Gonzaga Hospital, Orbassano, Italy
Abbreviations
ACC adrenocortical carcinoma
PgR progesterone receptor
IC Interval of Confidence
MTT 3-(4,5-Dimethyl-2-thiazol)-2,5-diphenyl-2H-tet- razolium bromide
mPR progesterone membrane receptor
PGRMC1 progesterone receptor membrane component 1
Introduction
Adrenocortical carcinoma (ACC) is a rare aggressive endocrine tumor [1] that in approximately 50% of adults is capable of hormone secretion [2]. Cushing’s syndrome is the most commonly associated endocrine disorder [3]. The systemic therapies have a limited efficacy [4-6]; thus, the prognosis of advanced ACC patients, not amenable to radical extirpation, is poor with a 5-year survival rate of 15% [7]; moreover, histological and molecular diagnostic parameters are not still completely shared [8]. Mitotane (o, p’-dichlorodiphe nyldichloroethane, o, p’-DDD) is the reference drug; however, toxicity and narrow therapeutic index limit its efficacy [9]. Therefore, there is an urgent need of new therapeutic approaches.
We have recently observed that abiraterone acetate (abiraterone) has both antisecretive and antitumor activ- ities in ACC cell lines [10]. The antisecretive effect of abiraterone is mediated by the inhibition of 17alpha- hydroxylase/17,20-lyase (CYP17A1), a key enzyme for steroid hormone synthesis [11, 12] leading to a rapid inhibition of cortisol secretion [10, 13]. Abiraterone mechanism of action may involve, at least in part, the Wnt/ß-catenin signaling pathway [10] that is con- stitutively active in approximatively 30% of ACC [14] and is a potential target for new molecular therapies. The cytotoxic effect of abiraterone remains to be fully eluci- dated, but evidence strongly indicates that it is directly associated with the drug-induced increase of progesterone levels, requiring the activation of the intracellular pro- gesterone receptors (PgRs). Interestingly, in addition to the well-known role of PgRs as nuclear transcription factors, different members of membrane progesterone receptors (mPRs) have been identified and the term “extranuclear” or “nongenomic” effects of progesterone was suggested to specifically defined mPR functions [15]. Another putative membrane-specific progesterone recep- tor, distinct from known mPRs and nuclear PgR, was isolated from different tissues and called Progesterone Receptor Membrane Component 1 (PGRMC1) [15, 16]. The extranuclear receptor activation leads to a rapid signaling, linked to various second messenger cascades,
including extracellular signal-regulated kinases (Erk 1/2, p42/44, p38 MAPKs) [15] and regulation of intracellular calcium mobilization [16] .* These effects are progesterone-dependent but independent of PgR tran- scriptional activity, and are integral part of progesterone cellular effects [17].
In this study, we investigated the cytotoxic effects of progesterone and the molecular mechanisms underlying its antitumor activity in NCI-H295R ACC cell line [18] and in ACC primary cell cultures derived from patients with either cortisol-secreting or nonsecreting ACC.
Materials and methods
Cell lines
NCI-H295R ACC cell line was obtained from the American Type Culture Collection (ATCC) and cultured as suggested by the manufacturer. Cells were authenticated by the AmpFISTR Identifiler PCR amplification kit (Applied Biosystems, Foster City, CA, USA). Media and supple- ments were supplied by Sigma Italia (Milano, Italy). SW13 cell line was obtained from ATCC and cultured as sug- gested by the manufacturer.
Primary cell cultures
Human ACC primary cells were derived from three patients with cortisol-secreting tumors (ACC01, ACC02, and ACC16) and from two patients with nonsecreting tumors (ACC03, ACC08). Clinical and histological features are reported in Table 1. After surgical removal, cells were enzymatically digested with (0.1 mg/mL) collagenase (Sigma Italia, Milano, Italy) and cultured in the same medium of NCI-H295R cells. The project was approved by the local Ethical Committee and written informed consent was obtained from all patients.
Immunohistochemistry
Immunohistochemistry for PgR was performed on 2 um sections from formalin fixed-paraffin-embedded ACC tis- sues. Ventana BenchMark Ultra platform was used according to the manufacturer’s recommended settings. Ultra Cell Conditioning 1 (CC1) solution was used for heat- induced epitope retrieval (95 ℃ for 64 min). Slides were incubated (36 °℃ for 16 min) with the ready-to-use anti-PgR antibody (monoclonal rabbit anti-human PR clone 1E2, Roche) [19] and followed by UltraView Universal DAB Detection Kit. Positive and negative controls from breast cancer tissue microarrays were included in the same slides.
| Primary culture identification | Tumor specimen | Histology | Disease stage | Hormone hypersecretion | PgR expression |
|---|---|---|---|---|---|
| ACC01 Female 66-year-old | Primary ACC | Weiss score 8 Mitotic index: >50/50 HPF Ki67 70% | Stage IV (hepatic metastases) | Cortisol (severe Cushing's syndrome) | 40% |
| ACC02 Female 63-year-old | Peritoneal metastases | Weiss score not available Mitotic index: >50/50 HPF Ki67 50% | Stage IV (peritoneal dissemination) | Cortisol (mild clinical signs of hypercortisolism) | 70% |
| ACC16 Male | Primary ACC | Weiss score not available | Stage IV (bone and multiple abdominal lymphonodal metastases) | Cortisol (severe Cushing's syndrome) | 40% |
| 55-year-old | Mitotic index: 10/50 HPF Ki67 50% | ||||
| ACC03 Male 59-year-old | Local relapse of ACC | Weiss score 8 Mitotic index: 25/50 HPF Ki67 20% | Stage IV (left hypochondrium soft tissue relapse and peritoneal dissemination) | No secretion | 3-5% |
| ACC08 Female 50-year-old | Lung metastases | Weiss score 8 Oncocytic features Mitotic index: 10/50 HPF Ki67 20% | Stage IV (lung and bone metastases) | No secretion | 1-2% |
Cell treatments
NCI-H295R cells and ACC primary cultures were seeded in 24-well plates and cultured in complete medium. Before treatment, culture medium was switched into charcoal- dextran-treated Nu-Serum (cNS)-medium with increasing concentrations of progesterone (0.1-160 uM) and/or mito- tane (25 nM-40 µM) for 4 days. Both progesterone and mitotane were dissolved in Dimethyl-Sulfoxide (DMSO). NCI-H295R cells were also exposed to mifepristone (0.1-500 nM) in combination with progesterone (25 µM) for 4 days. Mifepristone and mitotane were supplied by Selleckchem Chemicals (DBA Italia, Milano, Italy) and progesterone was purchased from Sigma Italia (Milano, Italy). SW13 cells were seeded in 24-well plates and cul- tured in complete medium. Before treatment, culture med- ium was switched into charcoal-dextran-treated Foetal Bovine Serum (FBS)-medium with increasing concentra- tions of progesterone (0.1-100 uM) for 3 days. Cell expo- sure to DMSO alone did not modify cell viability in any of the cell cultures used.
Cell viability assay
Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazol)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) dye reduction assay according to the manufacturer’s protocol (Sigma Ita- lia, Milan, Italy). Absorbance was measured by a spectro- photometer at 540/620 nm (GDV, Rome, Italy).
Drug combination experiments
Progesterone and mitotane combination experiments were performed to evaluate their interaction on NCI-H295R cell viability, according to the Chou and Talalay method [20].
Cells were treated for 4 days with progesterone (0.1-160 µM) and mitotane alone (25 nM-40 uM) or with progesterone in combination with mitotane at a fixed ratio (progesterone:mitotane = 4:1), as recommended for the most efficient data analysis [21, 22]. Cells were analyzed for cell viability using MTT. Data were then converted to Fraction affected (Fa, range from 0 to 1 where Fa=0 indicating 100% of cell viability and Fa = 1 indicating 0% of cell viability) and analyzed using the CompuSyn soft- ware (ComboSyn inc. Paramus, NJ, USA) to calculate the combination index (CI), being the CI value <0.9 an indi- cation of synergism, a CI = 0.9-1.1 an indication of addi- tive effect and CI> 1.1 and indication of antagonism.
Quantitative RT-PCR (qRT-PCR)
Gene expression was evaluated by qRT-PCR (ViiA7 Real- Time PCR System, ThermoFisher Scientific, Milan, Italy), using SYBR Green as fluorochrome, as described elsewhere [23]. The sequences of sense and antisense oligonucleotide primers are listed in Supplemental Table 1. Differences in the threshold cycle Ct values between the beta-actin housekeeping gene and the studied genes (ACt) were then calculated as an indicator of the amount of mRNA expressed.
Western blot
Whole cell lysates were prepared in ice-cold buffer with protease and phosphatase inhibitor cocktails (Roche, Milano, Italy) [24]. Equal amounts of protein were sepa- rated by electrophoresis on a 4-12% NuPAGE Bis-Tris Gel System (Life Technologies, Milan, Italy) and electroblotted to a nitrocellulose membrane. Rabbit monoclonal antibody against human Caspase-3 (Cell Signaling Technology,
Milan, Italy) [25] was used, both at a final concentration of 0.1 µg/mL. The Erk protein was detected using anti-total Erk and anti-phospho-Erk antibodies (Santa Cruz Bio- technologies, Heidelberg, Germany) [26] at a final con- centration of 0.7 ug/mL. Primary antibodies anti-mPR (0.5 µg/mL final concentration) and anti-PRGMC1 (1 µg/ mL final concentration) were purchased from Abcam (Cambridge, UK) [27] and Santa Cruz Biotechnologies (Heidelberg, Germany) [28]. A mouse monoclonal antibody directed against the N-terminal region of human «-Tubulin (Sigma Italia, Milano, Italy) was used to normalize the values. Secondary HRP-labeled anti-rabbit and anti-mouse antibodies (Santa Cruz Biotechnologies, Heidelberg, Ger- many) were used and the specific signal was visualized by the ECL-LiteAblot Extend Long (Euroclone, Milano, Italy). Densitometric analysis of the immunoblots was performed using the GelPro-Analyzer v 6.0 (MediaCybernetics, Bethesda, MD, USA).
Double staining AO/EtBr
NCI-H295R cells were treated with (progesterone (25 uM) for 4 days. A double staining with acridine orange (AO) and ethidium bromide (EtBr) was performed to visualize and quantify the number of viable, apoptotic, and necrotic cells, as previously described [10]. Cells were examined by a Zeiss LSM 510 META confocal laser-scanning microscope (Carl Zeiss AG, Germany). Several fields, randomly chosen, were digitalized and scored by using the NIH ImageJ software.
Cell cycle analyses
Flow cytometric cell cycle analysis was performed as described, with minor modifications [29]. Briefly, untreated and progesterone-treated NCI-H295R cells were fixed, treated with RNase A (12.5 µg/mL), stained with propidium iodide (40 µg/mL) (Sigma Italia, Milan, Italy) and analyzed by flow cytometry using an MACS Quant Analyzer (Mil- tenyi Biotec GmbH) for cell cycle status. Data were ana- lyzed using FlowJo (TreeStar).
Immunofluorescence
Cells were grown onto 12 mm poly-L-lysine-coated cover- slips and treated with IC50 value of progesterone for 3 days, with or without mifepristone (100 nM). Cells were then fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 1 h. Nonspecific bind- ing was blocked by incubation in PBS containing 0.2% Triton X-100 and 10% normal goat serum for 1 h. Cells were then incubated overnight at 4 ℃ with anti-ß catenin primary antibody (14.2 ng/ml, Cell Signaling Technolo- gies, Milan, Italy). After extensive washes, the Alexa
Fluor488 anti-rabbit secondary antibody (Life Technolo- gies, Milan, Italy) was applied for 1 h at room temperature, followed by counterstaining with Hoechst (Sigma Aldrich, Milan, Italy) for 5 min. After rinsing in PBS, coverslips were mounted using FluorPreserve™ Reagent and cell staining was detected using a Zeiss LSM 510 META con- focal laser-scanning microscope (Carl Zeiss AG, Oberko- chen, Germany). NIH-ImageJ software was used for image analysis and processing.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism software (version 5.02, GraphPad Software, La Jolla, CA, USA). One-way ANOVA with Bonferroni’s correction was used for multiple comparisons. Unless otherwise specified, data are expressed as mean ± SEM of at least three experi- ments run in triplicate. P values <0.05 were considered statistically significant.
Results
Progesterone effects on NCI-H295R cell viability
The predominant expression of the intracellular full-length PgR B isoforms was previously described in NCI-H295R cells [10]. The exposure of NCI-H295R cells to increasing concentrations of progesterone (0.1-160 uM) for 4 days led to a reduction in NCI-H295R cell viability in a concentration-dependent manner (Fig. 1a). Sigmoidal con- centration-response function was applied to calculate the IC50 value of progesterone in NCI-H295R cells, which was 25.5 µM (95% Interval of Confidence (IC), 19.9-32.9). Time-course experiments in NCI-H295R cells treated with progesterone at the IC50 value demonstrated that the reduction of cell viability reached its maximum at 4 days, with no significant change up to 6 days (IC50 value of 29.6 uM; 95% CI 23.6-37.0) (data not shown). Pretreat- ment of NCI-H295R cells with increasing concentration of the PgR antagonist mifepristone (0.1-500 nM) antagonized the cytotoxic effect elicited by progesterone at its IC50 for 4 days (Fig. 1b). This provides evidence that the anti- neoplastic activity of progesterone requires the stimulation of the PgRs. Mifepristone alone (0.1-500 nM; 4 days) did not affect NCI-H295R cells viability (data not shown).
Progesterone induces NCI-H295R cells apoptosis, without inducing changes in the cell cycle distribution
To provide explanation on the mechanism underlying the progesterone-induced NCI-H295R cell toxicity, cells were
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treated with progesterone (25 uM; 4 days) and stained with AO/EtBr. Progesterone deeply increased the number of apoptotic cells 39 ± 2%; while necrotic and living cells were 2 ±2% and 59±3% respectively (apoptotic cells: untreated
vs. treated cells: P < 0.001 Fig. 2a). Time-course experi- ments were conducted and our results demonstrated that after 2 days of treatment, the effect is as follows: 93 ±1% living cells, 7±1% apoptotic cells and no necrotic cells
(apoptotic cells: untreated vs. treated cells: P<0.05; Sup- plemental Fig. 1a). Progesterone-induced apoptotic cyto- toxicity reached its maximum after 4 days of treatment, as indicated above, and it was not modified if cells were exposed to progesterone up to 6 days: 47 ± 1% living cells, 43 ±3% apoptotic cells, 10±1% necrotic cells (apoptotic cells: untreated vs. treated cells: P<0.001; Supplemental Fig. 1b). We next examined the expression of total Caspase- 3 and the cleaved-Caspase-3, that play a central role in the execution phase of cell apoptosis [30], in progesterone- treated NCI-H295R cells in comparison to untreated cells (Fig. 2b). Progesterone exposure for 48 h significantly increased the expression of cleaved-Caspase-3 (% of increase: 23.9 ± 1.6) while total Caspase-3 levels were not affected. The analyses of the cell cycle progression by flow cytometry in untreated and progesterone-treated NCI- H295R cells did not show significant differences in cell distribution up to 4 days of treatment (Fig. 2c). However, we observed that treatment with progesterone for 4 days increased the proportion of cells in the sub-G0 phase: 29.7 ±4.6% untreated cells, 50.3±5.1% progesterone- treated cells (P<0.05), suggestive of DNA fragmentation. Taken together these observations suggest that apoptosis is
the main mechanism mediating the progesterone cytotoxicity.
Effect of progesterone on ß-catenin nuclear translocation in NCI-H295R cells
NCI-H295R cells were treated with progesterone and ana- lyzed for ß-catenin localization using immunofluorescence analyses. At baseline, ß-catenin was highly expressed in the nucleus (Fig. 3a), whereas the cell exposure to progesterone at its IC50 reduced ß-catenin nuclear localization and increased its retention into cytoplasm (Fig. 3b). The effect of progesterone in sequestering ß-catenin in cytoplasm was counteracted by 100 nM mifepristone (Fig. 3c). Immuno- fluorescence quantification using ImageJ software, reported in Table 2, demonstrated that progesterone significantly reduced ß-catenin nuclear localization. The progesterone- induced reduction of nuclear ß-catenin induced the decrease of mRNA expression of some of its target genes, namely MYC and survivin, while the mRNA expression level of another gene, CCND1, resulted unchanged by progesterone treatment (Supplemental Fig. 2).
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| % mean ± SEM | ||
|---|---|---|
| Untreated cells | Nucleus | 51.06 ±2.3 |
| Cytoplasm | 48.94 ±2.3 | |
| Progesterone-treated cells | Nucleus | 29.01 ±3.1 |
| Cytoplasm | 70.99 ± | |
| 3.9* | ||
| Progesterone-treated cells in the presence of mifepristone | Nucleus | 54.20 ±2.3 |
| Cytoplasm | 45.80 ±2.6 |
Cells were treated with progesterone (25 uM) alone or in combination with mifepristone (100 nM) for 3 days. Quantification was performed using the ImageJ software. Several cells in different fields, randomly chosen, were quantified
*P <0.001 vs. nuclear localization
mPR and PGRMC1
As underlined in the Introduction, accumulating evidence suggests that rapid progesterone responses are mediated by activation of mPRs [31]. In order to evaluate whether these receptors could contribute to the observed progesterone cytotoxic effect on NCI-H295R, we firstly evaluated their expression. As shown in Supplemental Fig. 3a, NCI-H295R expressed mPRs. These receptors were functionally active, as, when cells were treated with the IC50 value of proges- terone, we observed a reduction of phospho-Erk protein level (% of decrease: 30.39 ± 1.14) at a very early time, namely 15 min after progesterone exposure (Supplemental Fig. 3b). Finally, we demonstrated that NCI-H295R expressed as well the PGRMC1 (Supplemental Fig. 3a).
Progesterone enhanced NCI-H295R cytotoxicity induced by mitotane in drug-combination treatments
To evaluate whether progesterone treatment of NCI-H295R cells could enhance the cytotoxicity of mitotane, the com- bination index (CI) was calculated according to the Chou -Talalay method [21]. NCI-H295R cells were firstly exposed to increasing concentrations of mitotane (25 nM -40 uM) for 4 days and analyzed for cell viability by MTT assay. Sigmoidal concentration-response function was used to calculate the IC50 value, which was 3 uM (95% CI, 2.08-4.34) (Fig. 4a). The cytotoxic effect of progesterone in combination with mitotane was evaluated at the 1:4 fixed molar ratio for 4 days (Fig. 4b). We found that in NCI- H295R cells, the combination had a synergistic cytotoxic effect as compared to each single compound at a Fa = 0.09 -0.86 with range of CI, 0.08-0.88 (Fig. 4c).
Progesterone exerted cytotoxic effect in primary human ACC cells
Primary cultures derived from ACC patients were treated with increasing concentrations of progesterone for 4 days and analyzed for cell viability by MTT assay. In cortisol- secreting ACC cells (ACC01, ACC02, and ACC16), pro- gesterone exerted a concentration-dependent inhibition of cell viability with IC50 values of 18 uM (95% CI, 11.4 -31.7), 32.9 uM (95% CI, 26.5-40.9) and 39.2 uM (95% CI, 31.8-48.4) respectively. Immunohistochemical ana- lyses of PgR expression in ACC01, ACC02, and ACC16 tumors showed that at least 40% of neoplastic cells were positive for PgR (Table 1). By contrast, a lesser cytotoxic effect of progesterone was observed in the nonsecreting human ACC cells, ACC03 and ACC08, with IC50 values of 73.4 µM (95% CI, 46.1-116.8) and 80.8 µM (95% CI, 50.5 -129.5) respectively (Fig. 5). PgR expression in these cells was detected in less than 5% of ACC cells (Table 1).
Progesterone effect on SW13 cell line
Finally, as an internal control, we tested the effect of pro- gesterone in the nonsteroidogenic SW13 cell line, which can be found in adrenal small-cell carcinoma and of which the exact histopathological features are still under investi- gation [32]. We firstly analyzed the mRNA expression of PgRs and the results indicated that SW13 cells were devoid of PgR: indeed, q-RT-PCR analysis revealed a not- detectable PgR mRNA expression in this cell line com- pared to the ACt of 9.01 ± 0.25 in NCI-H295R cells, used as positive control. The western blot analysis of mPR and PGRMC1 expression in SW13 cell line indicated that these receptor proteins were expressed in this cell line (Supple- mental Fig. 4a). When exposed to increasing concentrations of progesterone within the same range of concentrations used for NCI-H295R cells, a cytotoxic effect could be observed, although it was non-concentration-dependent (Supplemental Fig. 4b).
Discussion
In the present study, we demonstrated that progesterone, through its receptors, exerted a concentration-dependent and time-dependent inhibition of ACC cell viability, and this effect was, at least in part, counteracted by the PgR antagonist mifepristone. The role of PgR is further supported by the data published by our group, where the PgR silencing induces the almost complete disappearance of the effect of abiraterone on cell viability [10]. The cytotoxic effect of progesterone was observed in the NCI-H295R cells, an ACC cell line that mainly express the full-length PgR B isoform [10, 33], and
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confirmed in primary cell cultures derived from cortisol- secreting ACC that are characterized by a marked expression of the PgR. Indeed, the cytotoxic effect of progesterone was less evident in nonsecreting ACC tumors in which PgR expression was low. Intriguingly, we demonstrated that NCI- H295R cells expressed also the mPR and PGRMC1 compo- nent, suggesting that the progesterone effects that we observed in ACC cells, both in cell line and primary cultures, could be a result of a multifactorial process involving both genomic and nongenomic actions, dependent on both mem- brane and intracellular progesterone receptor arrangement expressed by each ACC tumor. The scenario is even more complex than expected, in light of the evidence showing that the different effect of progesterone depends on many
ratio (progesterone: mitotane) for 4 days. Data are expressed as percent of viable cells vs. control (ctrl) cells. Data are the mean ± SEM of three experiments performed in triplicate. c Cell viability from b was con- verted to Fraction affected (Fa) values and resulting data were ana- lyzed with CompuSyn software to obtain combination index (CI) plot. Fa=0, 100% cell viability; Fa=1, 0% cell viability; CI value<0.9, synergism, CI = 0.9-1.1 additive effect and CI> 1.1 antagonism
variables, such as the type of cells, the genomic and/or non- genomic effects linked to the progesterone receptor expres- sion and the concentration of hormone present [34]. As a matter of fact, we observed that the nonsteroidogenic SW13 cell line, established from a small-cell carcinoma of the adrenal [35], was responsive to the cytotoxic effect of pro- gesterone (although without displaying a concentration- dependent curve) despite they expressed only mPR and PGRMC1. On the basis of these preliminary results the characterization of expression and function of all receptor components of progesterone pathway in our experimental models is now undergoing in our lab.
The present results confirm and extend our previous study [10] showing that the in vitro antineoplastic activity of abiraterone is mediated by the drug-induced increase in progesterone levels. PgRs, therefore, could represent a novel promising target in the management of ACC.
In PgR-positive breast cancer cell lines, progestins can induce a growth arrest due to decreased expression and activity of cyclin-dependent kinase (cdk) complexes [36]. In the present study, we showed that the progesterone-induced cytotoxicity of NCI-H295R ACC cells was not cell cycle mediated, but apoptosis represented the main molecular events, with a significant increase of the proapoptotic cleaved-Caspase-3 levels in the initial phase of the treatment. The ability of progesterone in modulating apoptotic events, both in vitro and in vivo, was previously demonstrated in several tumor cells [37-41]. In light of these results, we therefore explored the possible molecular mechanism reg- ulating the progesterone-induced apoptosis in ACC.
The Wnt/B-catenin pathway is frequently altered in ACC, which is characterized by CTNNB1 mutations leading to ß- catenin accumulation in the nucleus, where it binds with the T-cell factor (Tcf) and enhances its transcriptional activity.
In the NCI-H295R cell line, harboring the activating CTNNB1 p.S45P mutation, we found that progesterone treatment partially inhibited the ß-catenin translocation into the nucleus, thus suggesting the involvement of this path- way in the progesterone antineoplastic activity. These data are in line with our previous in vitro experiments showing that the increased levels of progesterone in NCI-H295R cell culture microenvironment, induced by the block of the CYP17A1 by abiraterone, significantly inhibited the ß- catenin migration into the nucleus. Further evidence comes from studies showing that progesterone is able to inhibit the Wnt/ß-catenin pathway in endometrial carcinoma [42]. The functional effect of the ß-catenin modification of translo- cation is the downregulation of the expression of some ß- catenin target genes, namely MYC and survivin, while CCND1 was not modified.
Taken together these data are suggestive of an involve- ment of ß-catenin inhibition in the progesterone-induced apoptosis of ACC cells. These data, however, are not exhaustive and the full evidence of the inhibitory effect would require the demonstration of a modulation of the expression of other specific ß-catenin target genes in NCI- H295R cells by progesterone treatment. These further experiments are outside the scope of the present paper and will be a matter of a future study.
Finally, the in vitro demonstration of the synergistic cytotoxic effect of the combination mitotane + progesterone could be of considerable interest for its possible clinical application, as progesterone and its derivatives are already part of the supportive approach in cancer patients. These preclinical data provide the rationale for a new trial, testing the efficacy of progesterone in association with current systemic therapies in the management of ACC patients.
In conclusion, the present study shows that progesterone exerts a cytotoxic activity in ACC cells, by inducing apoptosis via activation of the progesterone receptors. Both the genomic and nongenomic effects of progesterone seemed to mediate the cytotoxicity, although this point is still under investigation. The synergistic cytotoxic activity of progesterone with mitotane provides the rationale for testing this combination in a prospective clinical study.
Funding This work was supported by: AIRC project IG17678 (PI: M. T.); AIRC project IG14411 (PI: A.B.); Fondazione Camillo Golgi, Brescia; University of Brescia local grants; private donation of “gli Amici di Andrea” in memory of Andrea Gadeschi; private grant from the amateur dramatics group “Attori non per caso”, Parish church of Collio Valtrompia (Brescia). M.F. was supported by a grant from the Italian Society of Pharmacology.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
References
1. C.L. Ronchi, M. Kroiss, S. Sbiera, T. Deutschbein, M. Fassnacht, EJE prize 2014: current and evolving treatment options in adre- nocortical carcinoma: where do we stand and where do we want to go? Eur. J. Endocrinol. 171, R1-R11 (2014)
2. M. Terzolo, F. Daffara, A. Ardito, B. Zaggia, V. Basile, L. Ferrari, A. Berruti, Management of adrenal cancer: a 2013 update. J. Endocrinol. Invest. 37, 207-217 (2014)
3. A. Berruti, E. Baudin, H. Gelderblom, H.R. Haak, F. Porpiglia, M. Fassnacht, G. Pentheroudakis; ESMO Guidelines Working Group, Adrenal cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 23(Suppl 7), 131-138 (2012)
4. M. Fassnacht, M. Terzolo, B. Allolio, E. Baudin, H. Haak, A. Berruti, S. Welin, C. Schade-Brittinger, A. Lacroix, B. Jarzab, H. Sorbye, D.J. Torpy, V. Stepan et al. Combination chemotherapy in advanced adrenocortical carcinoma. N. Engl. J. Med. 366, 2189-2197 (2012)
5. A. Berruti, M. Terzolo, P. Sperone, A. Pia, S. Della Casa, D.J. Gross, C. Carnaghi, P. Casali, F. Porpiglia, F. Mantero, G. Reimondo, A. Angeli, L. Dogliotti, Etoposide, doxorubicin and cisplatin plus mitotane in the treatment of advanced adrenocortical carcinoma: a large prospective phase II trial. Endocr. Relat. Cancer 12, 657-666 (2005)
6. A. Berruti, M. Fassnacht, H. Haak, T. Else, E. Baudin, P. Sperone, M. Kroiss, T. Kerkhofs, A.R. Williams, A. Ardito, S. Leboulleux, M. Volante, T. Deutschbein, R. Feelders, C. Ronchi, S. Grisanti, H. Gelderblom, F. Porpiglia, M. Papotti, G.D. Hammer, B. Allolio, M. Terzolo, Prognostic role of overt hypercortisolism in completely operated patients with adrenocortical cancer. Eur. Urol. 65, 832-838 (2014)
7. R. Libé, I. Borget, C.L. Ronchi, B. Zaggia, M. Kroiss, T. Ker- khofs, J. Bertherat, M. Volante, M. Quinkler, O. Chabre, M. Tabarin, F. Beuschlein et al .. Prognostic factors in stage III-IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study. Ann. Oncol. 26, 2119-2125 (2015)
8. M. Volante, C. Buttigliero, E. Greco, A. Berruti, M. Papotti, Pathological and molecular features of adrenocortical carcinoma: an update. J. Clin. Pathol. 61, 787-793 (2008)
9. S. Puglisi, P. Perotti, D. Cosentini, E. Roca, V. Basile, A. Berruti, M. Terzolo, Decision-making for adrenocortical carcinoma: sur- gical, systemic, and endocrine management options. Expert. Rev. AntiCancer Ther. 8(11), 1-9 (2018)
10. C. Fiorentini, M. Fragni, P. Perego, S. Vezzoli, S.A. Bonini, M. Tortoreto, D. Galli, M. Claps, G.A. Tiberio, M. Terzolo, C. Missale, M. Memo, G. Procopio, N. Zaffaroni, A. Berruti, S. Sigala, Antisecretive and antitumor activity of abiraterone acetate in human adrenocortical cancer: a preclinical study. J. Clin. Endocrinol. Metab. 101, 4594-4602 (2016)
11. G. Attard, A.H. Reid, R.J. Auchus, B.A. Hughes, A.M. Cassidy, E. Thompson, N.B. Oommen, E. Folkerd, M. Dowsett, W. Arlt, J. S. de Bono, Clinical and biochemical consequences of CYP17A1 inhibition with abiraterone given with and without exogenous glucocorticoids in castrate men with advanced prostate cancer. J. Clin. Endocrinol. Metab. 97, 507-516 (2012)
12. A. Pia, F. Vignani, G. Attard, M. Tucci, P. Bironzo, G. Scagliotti, W. Arlt, M. Terzolo, A. Berruti, Strategies for managing ACTH dependent mineralocorticoid excess induced by abiraterone. Cancer Treat. Rev. 39, 966-973 (2013)
13. M. Claps, B. Lazzari, S. Grisanti, V. Ferrari, M. Terzolo, S. Sigala, S. Vezzoli, M. Memo, M. Castellano, A. Berruti, Man- agement of severe Cushing’s syndrome induced by adrenocortical
carcinoma with abiraterone acetate: a case report. AACE Clin. Rep. 2, 337-341 (2016)
14. A. Salomon, M. Keramidas, C. Maisin, M. Thomas, Loss of ß- catenin in adrenocortical cancer cells causes growth inhibition and reversal of epithelial-to-mesenchymal transition. Oncotarget 6, 11421-11433 (2015)
15. D. Garg, S.S.M. Ng, K.M. Baig, P. Driggers, J. Segars, Progesterone-mediated non-classical signaling. Trends Endocri- nol. Metab. 28, 656-668 (2017)
16. R.L. Ashley, C.M. Clay, T.A. Farmerie, G.D. Niswender, T.M. Nett, Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization. Endocrinology 147, 4151-4159 (2006)
17. V. Boonyaratanakornkit, N. Hamilton, D.C. Márquez-Garbán, P. Pateetin, E.M. McGowan, R.J. Pietras, Extranuclear signaling by sex steroid receptors and clinical implications in breast cancer. Mol. Cell. Endocrinol. 466, 51-72 (2018)
18. W.E. Rainey, K. Saner, B.P. Schimmer, Adrenocortical cell lines. Mol. Cell. Endocrinol. 228, 23-38 (2004)
19. E.N. Kornaga, A.C. Klimowicz, N. Guggisberg, T. Ogilvie, D.G. Morris, M. Webster, A.M. Magliocco, A systematic comparison of three commercial estrogen receptor assays in a single clinical outcome breast cancer cohort. Mod. Pathol. 29, 799-809 (2016)
20. T.C. Chou, P. Talalay, Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul. 22, 27-55 (1984)
21. T.C. Chou, Theoretical basis, experimental design, and compu- terized simulation of synergism and antagonism in drug combi- nation studies. Pharmacol. Rev. 58, 621-681 (2006)
22. J. Hofman, D. Ahmadimoghaddam, L. Hahnova, P. Pavek, M. Ceckova, F. Staud, Olomoucine II and purvalanol A inhibit ABCG2 transporter in vitro and in situ and synergistically potentiate cytostatic effect of mitoxantrone. Pharmacol. Res. 65, 312-319 (2012)
23. S. Sigala, S. Bodei, C. Missale, D. Zani, C. Simeone, S.C. Cunico, P.F. Spano, Gene expression profile of prostate cancer cell lines: effect of nerve growth factor treatment. Mol. Cell. Endocrinol. 284, 11-20 (2008)
24. C. Fiorentini, S. Bodei, F. Bedussi, M. Fragni, S.A. Bonini, C. Simeone, D. Zani, A. Berruti, C. Missale, M. Memo, P.F. Spano, S. Sigala, GPNMB/OA protein increases the invasiveness of human metastatic prostate cancer cell lines DU145 and PC3 through MMP-2 and MMP-9 activity. Exp. Cell Res. 323, 100-111 (2014)
25. V. Porrini, I. Sarnico, M. Benarese, C. Branca, M. Mota, A. Lanzillotta, A. Bellucci, E. Parrella, L. Faggi, P.F. Spano, B.P. Imbimbo, M. Pizzi, Neuroprotective and anti-apoptotic efects of CSP-1103 in primary cortical neurons exposed to oxygen and glucose deprivation. Int. J. Mol. Sci. 18, E184 (2017)
26. M. Babagana, S. Johnson, H. Slabodkin, W. Bshara, C. Morrison, E.S. Kandel, P21-activated kinase 1 regulates resistance to BRAF inhibition in human cancer cells. Mol. Carcinog. 56, 1515-1525 (2017)
27. N.M. Bashour, S. Wray, Progesterone directly and rapidly inhibits GnRH neuronal activity via progesterone receptor membrane component 1 endocrinology. Endocrinology 153, 4457-4469 (2012)
28. V. Lodde, J.J. Peluso, A novel role for progesterone and pro- gesterone receptor membrane component 1 in regulating spindle microtubule stability during rat and human ovarian cell mitosis. Biol. Reprod. 84, 715-722 (2011)
29. A. Chimento, R. Sirianni, I. Casaburi, F. Zolea, P. Rizza, P. Avena, R. Malivindi, A. De Luca, C. Campana, E. Martire, F. Domanico, F. Fallo, G. Carpinelli, L. Cerquetti, D. Amendola, A. Stigliano, V. Pezzi, GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo. Oncotarget 6, 19190-19203 (2015)
30. R.S.Y. Wong, Apoptosis in cancer: from pathogenesis to treat- ment. J. Exp. Clin. Cancer Res. 30, 87 (2011)
31. A.O. Mueck, X. Ruan, H. Seeger, T. Fehm, H. Neubauer, Genomic and non-genomic actions of progestogens in the breast. J. Steroid Biochem. Mol. Biol. 142, 62-67 (2014)
32. T. Wang, W.E. Rainey, Human adrenocortical carcinoma cell lines. Mol. Cell. Endocrinol. 351, 58-65 (2012)
33. K.M. Scarpin, J.D. Graham, P.A. Mote, C.L. Clarke, Progesterone action in human tissues: regulation by progesterone receptor (PR) isoform expression, nuclear positioning and coregulator expres- sion. Nucl. Recept. Signal. 7, e009 (2009)
34. J.H. Check, The role of progesterone and the progesterone receptor in cancer. Expert Rev. Endocrinol. Metab. 12, 187-197 (2017)
35. A. Leibovitz, W.M. McCombs 3rd, D. Johnston, C.E. McCoy, J. C. Stinson, New human cancer cell culture lines. I. SW-13, small- cell carcinoma of the adrenal cortex. J. Natl. Cancer Inst. 51, 691-697 (1973)
36. V. Boonyaratanakornkit, E. McGowan, L. Sherman, M.A. Man- cini, B.J. Cheskis, D.P. Edwards, The role of extranuclear sig- naling actions of progesterone receptor in mediating progesterone regulation of gene expression and the cell cycle. Mol. Endocrinol. 21, 359-375 (2007)
37. S.Z. Bu, D.L. Yin, X.H. Ren, L.Z. Jiang, Z.J. Wu, Q.R. Gao, G. Pei, Progesterone induces apoptosis and up-regulation of p53 expression in human ovarian carcinoma cell lines. Cancer 79, 1944-1950 (1997)
38. V. Syed, S.M. Ho, Progesterone-induced apoptosis in immorta- lized normal and malignant human ovarian surface epithelial cells involves enhanced expression of FasL. Oncogene 22, 6883-6890 (2003)
39. B. Formby, T.S. Wiley, Bcl-2, survivin and variant CD44 v7-v10 are downregulated and p53 is upregulated in breast cancer cells by progesterone: inhibition of cell growth and induction of apoptosis. Mol. Cell. Biochem. 202, 53-61 (1999)
40. K. Horita, N. Inase, S. Miyake, B. Formby, H. Toyoda, Y. Yoshizawa, Progesterone induces apoptosis in malignant meso- thelioma cells. Anticancer Res. 21, 3871-3874 (2001)
41. F. Atif, S. Yousuf, D.G. Stein, Anti-tumor effects of progesterone in human glioblastoma multiforme: role of PI3K/Akt/mTOR sig- naling. J. Steroid Biochem. Mol. Biol. 146, 62-73 (2015)
42. Y. Wang, P. Hanifi-Moghaddam, E.E. Hanekamp, H.J. Kloos- terboer, P. Franken, J. Veldscholte, H.C. van Doorn, P.C. Ewing, J.J. Kim, J.A. Grootegoed, C.W. Burger, R. Fodde, L.J. Blok, Progesterone inhibition of Wnt/beta-catenin signaling in normal endometrium and endometrial cancer. Clin. Cancer Res. 15, 5784-5793 (2009)