ORIGINAL ARTICLE
Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells
Teresa Gagliano · Erica Gentilin · Katiuscia Benfini · Carmelina Di Pasquale · Martina Tassinari . Simona Falletta · Carlo Feo · Federico Tagliati ·
Ettore degli Uberti · Maria Chiara Zatelli
Received: 25 June 2014/ Accepted: 25 July 2014/Published online: 6 August 2014 @ Springer Science+Business Media New York 2014
Abstract Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocor- tical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotox- icity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at con- centrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits
T. Gagliano · E. Gentilin · K. Benfini · C. Di Pasquale ·
M. Tassinari · S. Falletta · F. Tagliati · E. d. Uberti · M. C. Zatelli
Section of Endocrinology, Department of Medical Sciences, University of Ferrara, Via A. Moro, 8, 44124 Ferrara, Italy e-mail: ztlmch@unife.it
C. Feo
Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Via A. Moro, 8, 44124 Ferrara, Italy
E. d. Uberti · M. C. Zatelli Laboratorio in Rete del Tecnopolo Tecnologie delle Terapie Avanzate (LTTA), University of Ferrara, Ferrara, Italy
P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater effi- cacy of combination therapy (mitotane plus chemothera- peutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.
Keywords Adrenocortical cancer . P-gp . Mitotane . Chemoresistance
Introduction
Adrenocortical carcinoma (ACC) is a rare malignancy generally characterized by a poor prognosis, despite extensive surgical approaches. The disease is often meta- static at diagnosis and relapses very frequently, with con- sequent very low survival rates at 5 years [1, 2]. The high ACC recurrence rates have prompted the use of adjuvant therapy by treatment with mitotane, an adrenolytic drug, that demonstrated a significant increase in recurrence-free survival rates [3]. Multiple cytotoxic approaches have also been attempted with limited results [1] due to the charac- teristic ACC chemoresistance. The latter has been ascribed to the presence of P-glycoprotein (P-gp), encoded by the MDR-1 gene, which mediates multidrug resistance by actively pumping out of the cell hydrophobic cytotoxic agents (doxorubicin, vinblastine, paclitaxel) [4, 5]. Previ- ous reports demonstrated that P-gp mediates multidrug resistance in several tumors, including those deriving from endocrine tissues [6-10]. It has been previously demon- strated that mitotane, at therapeutic concentrations, is capable of reverting chemoresistance in colon cancer cell lines [11], suggesting that this drug may be useful to overcome ACC chemoresistance, as well. In keeping with
this hypothesis, it has been shown that the combination of mitotane, at doses providing drug plasma levels within the ACC therapeutic window (14-20 µg/L), with three che- motherapeutic agents (etoposide, doxorubicin and cis- platin) provides important results on clinical grounds, with a significant improvement in progression-free survival [12]. The latter results indeed indicate that the combination of chemotherapeutic drugs with mitotane might be more effective in ACC treatment. However, this drug regiment has a high impact on patient’s quality of life, with a high burden in terms of side effects, also on the endocrine system [13-16] that may induce dosage reductions. The efficacy of mitotane doses lower than those used for ACC treatment in sensitizing ACC cells to chemotherapeutic agents has not been explored, so far. Therefore, the aim of our study was to evaluate in vitro whether mitotane at concentrations lower than 14-20 µg/L (corresponding to 44-62 uM) may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp.
Materials and methods
Compounds
Doxorubicin hydrochloride was obtained from Pharmacia (Milano, Italy). R(+)-verapamil hydrochloride was pur- chased from Sigma (Milano, Italy). All other reagents, if not otherwise specified, were purchased from Sigma.
Cell culture and tissues
The NCI-H295 and the SW13 human ACC cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). NCI-H295 cells were cultured as previously described [17, 18]. SW13 cells were maintained in D-MEM High Glucose, with 10 % FBS. Primary cul- tures from human adrenocortical tumors were obtained as previously described, with minor modifications [19]. Briefly, tissue samples were collected under sterile condi- tions in accordance with the guidelines of the local com- mittee on human research, and immediately minced in RPMI 1640 medium. Tissues were dissociated using 0.35 % collagenase and 1 % trypsin at 37 °℃ for 60 min. Cell suspensions were washed twice with serum-free RPMI (Euroclone Ltd., Wetherby, UK), and then passed through 18-gauge and then 20-gauge syringe needles. Tumor cells were resuspended in F-12 medium with 10 % FBS and antibiotics, seeded in 96-well culture plates (2 x 104 cells/ well) and incubated at 37 ℃ in a humidified atmosphere of 5 % CO2 and 95 % air. After ~18 h, cells were treated with test substances.
Viable cell number assessment
The number of viable cells was assessed by using the Cell Proliferation Kit (Roche, Basil, Switzerland), as previously described [20], after incubation with or without the test substances for the indicated time. After adding the revealing solution, the absorbance at 560 nm was recorded using the Wallac Victor 1420 Multilabel Counter (Perkin Elmer, Monza, Italy). Results are expressed as mean value ± SE percent viable cell number versus vehicle- treated control cells from three independent experiments in six replicates.
Caspase activity evaluation
Caspase activity was measured using Caspase-Glo 3/7 assay (Promega, Milano, Italy) as previously described [14]. Results are expressed as mean value ± SE percent relative light units versus vehicle-treated control cells from three independent experiments in six replicates.
Calcein uptake assay
In order to measure calcein uptake, Vybrant™M Multidrug Resistance Assay Kit (Invitrogen, Monza, Italy) was employed according to the manufacturer’s instructions. Briefly, 5 x 105/well NCI-H295 cells were seeded in 96-well plates and treated with 10 uM verapamil, a P-gp inhibitor, or 5 uM mitotane. Phosphate-buffered saline (PBS) was used as negative control. Plates were then mixed and incubated at 37 ℃ for 15 h. One micrometre calcein AM solution was then added to each microplate well at a final concentration of 0.25 uM. Plates were then mixed and incubated at 37 °℃ over night. The microplates were then centrifuged for 5 min at 200 x g, the supernatant was removed and the cells were washed with cold (4 ℃) tissue culture medium three times. Calcein retention was measured as calcein-specific fluorescence with appropriate filters for fluorescein (485 nm excitation/530 nm emission) by using the Victor3 1420 Multilabel Counter (Perkin Elmer).
P-gp ATPase activity assay
The changes in ATPase activity of P-gp were determined by using the P-gp-Glo TM assay system (Promega). Verapamil was used as positive control since it inhibits P-gp ATPase activity, which eventually leads to a reduced drug efflux. When P-gp ATPase activity is inhibited, ATP accumulates and becomes a substrate for the luciferase activity, with a consequent increase in luminescence. ATP levels therefore mirror the degree on ATPase activity inhibition. Mitotane at increasing concentrations (from 5 to 50 µM) was incubated with 5 mM MgATP and 25 µg
recombinant human P-gp membranes at 37 ℃ for 40 min. Luminescence emission was started by adding ATP detection buffer. After incubation at room temperature for 20 min to allow luminescent signal to develop, the samples were read in white opaque 96-well multiplates (Perkin Elmer) on the Envision luminometer (Perkin Elmer). The changes in relative light units (ARLU) were determined by comparing control samples to samples treated with verap- amil or mitotane.
Western blot analysis
For immunoblotting, cells and tissues were dissolved in RIPA Buffer (Thermolab inc, Waltham, MA, USA), as previously described [21]. Protein concentration was measured by BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA), as previously described [22]. Proteins were fractionated on 7.5 % SDS-PAGE, as previously described [9], and transferred by electrophoresis to nitro- cellulose transfer membrane (PROTRAN, Dassel, Ger- many). Membranes were incubated with a mouse anti-P-gp monoclonal antibody (Santa Cruz Biotechnology, Dallas, TE, USA). Horseradish peroxidase-conjugated anti-mouse antibody IgG (Dako, Cernusco sul Naviglio, Italy) was used to detect the protein, and binding was revealed using enhanced chemiluminescence (Pierce). The blots were then stripped and used for further blotting with anti-GAPDH antibody (Cell Signaling, Beverly, MA-USA).
Intracellular doxorubicin detection
NCI-H295 cells (3 × 104/well) were seeded in 8-well chamber slides (Lab-Tek Chamber Slide System, Nalge Nunc International, Roskilde, Denmark), incubated over- night in culture medium, and then treated for 24 h without or with 1 µM doxorubicin or 5 uM mitotane, alone or in combination. Subsequently, the culture medium was removed and cells were washed thrice with PBS. Cells were fixed in methanol and acetone (1:1) for 10 min at - 20 ℃ and washed thrice with PBS. Slides were then mounted with the ProLong Gold antifade reagent (Invit- rogen Molecular Probes) containing the nuclear stain 4’,6’-diamidino-2-phenylindole (DAPI) under glass cover- slips (Menzel-Glaser, Braunschweig, Germany). The cells were examined for natural doxorubicin fluorescence using the tetramethyl rhodamine-isothiocyanate (TRITC) filter. Nuclear staining with DAPI was detected with the UV filter. Images were visualized with the Nikon Eclipse TE2000-U fluorescent microscope (Nikon, Tokio, Japan) with a 60x objective magnification and acquired using the DS-5 M Nikon color charge coupled device (CCD) digital camera. All experiments were carried out at least thrice independently, analyzing 50 ± 10 individual cells.
Statistical analysis
Results are expressed as the mean ± SE. Student’s paired or unpaired t test was used to evaluate individual differ- ences between means, where applicable. P values < 0.05 were considered significant.
Results
Influence of doxorubicin and mitotane on cell viability and apoptosis in human ACC cells
Cell viability was evaluated in NCI-H295 and SW13 cells treated with increasing concentrations of mitotane (1.25-80 µM) or doxorubicin (1-100 nM) for 120 h. As shown in Fig. 1a, NCI-H295 cell viability was significantly reduced by treatment with 5-80 uM mitotane from 22 % (P < 0.05 vs. control) to 99 % (P <0.01 vs. control; IC50 = 21 µM) and by treatment with 50-100 nM doxo- rubicin by 34-49 % (P < 0.01 vs. control; IC50 = 82 nM). SW13 cell viability was significantly reduced by treatment with 40-80 µM mitotane by 32-68 % (P < 0.01 vs. control; IC50 = 59 µM) and by treatment with 100 nM doxorubicin by 25 % (P < 0.05 vs. control; IC50 = 1.2 µM).
To explore the hypothesis that mitotane may sensitize ACC cells to the cytotoxic effects of doxorubicin, the lowest effective concentrations of mitotane (5 uM) and doxorubicin (50 nM) on NCI-H295 cell viability reduction were employed in combination and then cell viability and caspase 3/7 activation were evaluated. In addition, the combined effects of 10 µM verapamil (a well-known P-gp inhibitor) were assessed as a control for Pg-p inhibition. As show in Fig. 1b, the inhibitory effects of doxorubicin on NCI-H295 cell viability were significantly enhanced by mitotane (+14 %; P < 0.05) and by verapamil (+25 %; P < 0.05). Similarly, the stimulatory effects of doxorubicin on caspase 3/7 activity were significantly enhanced by mitotane (+165 %; P < 0.05), while verapamil failed to influence this parameter. As show in Fig. 1c, neither mitotane nor verapamil were capable of increasing the sensitivity of SW13 cells to doxorubicin effects, both in terms of cell viability reduction and of caspase 3/7 activation.
Cell viability was also investigated in the same settings in four human adrenocortical neoplasia primary cultures. As shown in Fig. 1d, the effects of doxorubicin and mitotane on the different primary cultures were quite var- iable. Indeed, in primary culture #1 and #4 both mitotane and doxorubicin, when used alone, did not significantly affect cell viability, which was significantly reduced when the two drugs were used in combination (-10 % in primary culture #1 and -76 % in primary culture #4 as
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compared to doxorubicin alone; P < 0.05). On the con- trary, in primary culture #2 mitotane significantly reduced cell viability, but this effect was not enhanced by co- treatment with doxorubicin. In addition, primary culture #3 showed no variations in cell viability under treatment with either mitotane or doxorubicin, alone or in combination.
P-gp activity and expression
To verify whether mitotane could act as a P-gp inhibitor, calcein assay was performed using verapamil as a positive control [7, 23]. As shown in Fig. 2a, 10 uM verapamil was capable of increasing intracellular calcein by 81.8 %
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(P < 0.01). Similarly, 5 uM mitotane determined an increase in the amount of intracellular calcein by 47.7 % (P < 0.01), indicating that indeed mitotane inhibits P-gp activity as extrusion pump.
In order to assess whether mitotane interacts directly with P-gp, a P-gp-GLO assay was performed, using verapamil as a positive control. As shown in Fig. 2b, as expected, verapamil strongly induced ATP accumulation (corresponding to P-gp ATPase activity inhibition), that was dose-dependently enhanced also by mitotane, indicating that the latter indeed directly inhibits P-gp-mediated ATPase activity.
To verify whether mitotane effects are correlated with P-gp expression, the latter was assessed by Western blot. As shown in Fig. 2c, P-gp expression was detected in NCI- H295 cells but not in SW13 cells. As for the human adrenocortical neoplasia primary cultures, P-gp expression was detected in primary cultures #1 and #4 but not in primary cultures #2 and #3. These results show that P-gp was expressed in cell lines, and primary cultures in which the sensitizing effects of mitotane toward the antiprolifer- ative effects of doxorubicin were observed. On the con- trary, P-gp was not expressed in cell lines and primary cultures in which the sensitizing effects of mitotane toward the antiproliferative effects of doxorubicin were not observed.
In order to understand whether mitotane could influence P-gp expression levels, NCI-H295 cells were treated with or without either mitotane or verapamil and total proteins were analyzed by Western blot. As shown in Fig. 2d, P-gp protein levels did not change under treatment with mitotane or verapamil.
Effects of mitotane on doxorubicin intracellular retention
To assess whether mitotane might influence membrane permeability to doxorubicin, NCI-H295 cells were incu- bated with 1 µM doxorubicin without or with 5 uM mito- tane over night, fixed and then observed with the fluorescence microscope with the TRITC filter, taking advantage of doxorubicin natural red fluorescence. As shown in Fig. 3, doxorubicin fluorescence is more detect- able in cells co-treated with mitotane, indicating a greater intracellular retention of the chemotherapeutic drug.
Discussion
We here show that mitotane sensitizes P-gp expressing neoplastic adrenocortical cells to the cytotoxic effects of doxorubicin by inhibiting P-gp. Our findings indeed dem- onstrate that mitotane, at concentrations eightfold lower than those showing clinical efficacy in vivo, is capable of halving doxorubicin concentrations needed to exert cyto- toxic effects. However, these effects are detected only in adrenocortical cells expressing P-gp. Doxorubicin has shown limited clinical efficacy when used alone in ACC
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patients [24], while its combination with mitotane, etopo- side, doxorubicin and cisplatin (EDP) obtained better results [12], even if associated with severe side effects. Lately, liposomal variants of combined chemotherapeutic regimens have been demonstrated to be even more effec- tive in an ACC animal model [25]. Mitotane has been previously shown to sensitize the ACC cell line SW13 to the cytotoxic effects of doxorubicin, but very high con- centrations of both drugs were employed in those settings [26]. In addition, the mechanism by which combination
therapy exerts better effects has not been demonstrated so far.
To explore this issue, we employed as “in vitro” models two well established ACC cell lines, the NCI-H295 and the SW13 cells, that are widely accepted ACC preclinical models [11, 27-29]. In addition, we obtained human adrenocortical neoplasia primary cultures, which represent the closest available model to “in vivo” human neoplasia [30, 31].
Our results confirm previous studies showing that NCI- H295 cells are more sensitive to the antiproliferative
effects of mitotane as compared to SW13 cells [27, 28], since mitotane displays a 2.8-fold lower IC50 for cell viability reduction in NCI-H295 versus SW13 cells. In addition, we here demonstrate that SW13 cells are much more resistant to the cytotoxic effects of doxorubicin as compared to NCI-H295 cells, since doxorubicin displays a 14.6-fold greater IC-50 for cell viability reduction in SW13 versus NCI-H295 cells. Moreover, we found that P-gp is expressed in NCI-H295 but not in SW13 cells, indicating that the chemoresistance phenotype of the latter cell line is not due to a P-gp-dependent mechanism. Similarly, among the four of human adrenocortical neoplasia primary cul- tures analyzed, cell viability was significantly reduced by mitotane only in one sample (primary culture #2) and by doxorubicin in none of them. As outlined before, both drugs were employed at concentrations much lower than those reached in “vivo” (5 uM mitotane and 50 nM doxorubicin), chosen on the basis of the results obtained with ACC cell lines, due to the limited amount of primary cells.
Our results show that very low mitotane concentrations are capable of sensitizing NCI-H295 cells and two human adrenocortical neoplasia primary cultures (#1 and #4) to the cytotoxic effects of doxorubicin, which, in turn, was employed at much lower concentrations (50 nM) than those showing clinical efficacy in vivo (0.17-17 uM) [32]. Both the human adrenocortical neoplasia primary cultures and NCI-H295 cells express detectable P-gp levels, in keeping with previous studies [11, 33]. On the other hand, our results show the lack of mitotane-sensitizing effects toward doxo- rubicin cytotoxicity in SW13 cells and in the other two human adrenocortical neoplasia primary cultures (#2 and #3), all of which are devoid of P-gp expression. These data indicate that the sensitizing effects of mitotane toward the cytotoxic action of doxorubicin are mediated by P-gp.
P-gp, also known as multidrug resistance protein (MDR1), is a glycoprotein encoded by the ABCB1 gene [34]. P-gp is a well-characterized ABC-transporter which transports a wide variety of substrates, including doxoru- bicin, across extra- and intracellular membranes [35]. It has been previously demonstrated that the majority of ACC expresses P-gp at high levels [36, 37], providing the basis for including P-gp antagonists in the medical treatment of ACC. Previous studies have shown that, in vitro, P-gp inhibition is indeed effective in increasing intracellular doxorubicin retention with a consequent increase in its cytotoxic effects [8, 9]. These results have also been obtained employing mitotane in P-gp expressing cell lines different from ACC [11], supporting the hypothesis that mitotane directly interferes with P-gp function. Our data support this hypothesis, since mitotane increases calcein uptake in P-gp expressing NCI-H295 cells to the same extent of verapamil, a well-known P-gp inhibitor.
Treatment with Mitotane did not affect P-gp expression levels in NCI-H295 cells, suggesting that the observed reduction in P-gp activity under mitotane treatment is not due to changes in protein levels. In addition, the evidence that mitotane dose-dependently inhibits P-gp ATPase activity in a cell-free system demonstrates that mitotane directly interacts with P-gp. The reduced P-gp activity as trans-membrane efflux pump in the presence of mitotane is also mirrored by doxorubicin accumulation inside NCI- H295 cells, in keeping with previous evidence [9]. A pre- viously published study by Abraham et al. [38] reported data of a clinical trial using a combination of chemotherapy and mitotane, employed as P-gp antagonist, in patients with advanced ACC. The results did not show any superiority of mitotane/chemotherapy regimen as compared to mitotane as single agent. However, response to combination therapy was present also at low mitotane plasma levels and, in some patients, response occurred with mitotane levels <4 µg/ml (12.5 µM). This latter evidence is in agreement with our findings, since we show that 5 uM mitotane is able to enhance doxorubicin activity. In addition, the study by Abraham does not investigate P-gp expression levels in tumor cells, but only in circulating CD56-positive cells. Therefore, the provided data do not allow to correlate P-gp expression with mitotane activity on ACC cells.
The main finding of our study is represented by the demonstration that mitotane at very low concentrations sensitize P-gp-expressing cells to the cytotoxic effects of doxorubicin, providing an experimental support to a ther- apeutic strategy that may allow a reduction in the doses of both drugs, when used in combination. This approach may reduce the development of important side effects of both mitotane and doxorubicin that may limit the efficacy of the chemotherapeutic regimen [14-16, 39, 40]. In addition, our study confirms the efficacy of mitotane as adrenolytic drug, in keeping with previously reported evidence [41], also in a chemoresistant cell line, such as SW13 cells. On the other hand, in the absence of P-gp, mitotane does not have any chemosensitizing effect, clearly indicating that the latter is correlated to P-gp expression. These findings may account for the variable efficacy of the combination therapy with chemotherapeutic drugs and mitotane in vivo, since ACC express P-gp at variable high levels.
In conclusion, our data show that low-dose mitotane directly inhibits P-gp activity in ACC cells, allowing the activity of chemotherapeutic drugs, such as doxorubicin, at lower concentrations than those currently employed. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.
Acknowledgments This work was supported by grants from the Italian Ministry of Education, Research and University (FIRB
RBAP11884 M, RBAP1153LS, 2010TYCL9B_002), Fondazione Cassa di Risparmio di Ferrara, and Associazione Italiana per la Ric- erca sul Cancro (AIRC) in collaboration with Laboratorio in rete del Tecnopolo “Tecnologie delle terapie avanzate” (LTTA) of the Uni- versity of Ferrara.
Conflict of interest The authors declare that they have no conflict of interest.
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