Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells
Ségolène Hescot1,2, Abdelhamid Slama3, Anne Lombes4, Angelo Paci5, Hervé Remy5, Sophie Leboulleux6, Rita Chadarevian7, Séverine Trabado1,2,8, Larbi Amazit1,2, Jacques Young1,2,9, Eric Baudin1,2,5,* and Marc Lombès1,2,9,*
1INSERM U693, Fac Med Paris Sud, Rue Gabriel Péri, Le Kremlin-Bicêtre F-94276, France 2UMR-S693, Univ Paris-Sud, Fac Med Paris-Sud, Le Kremlin Bicêtre F-94276, France 3 Assistance Publique-Hôpitaux de Paris, Service de Biochimie, Hôpital de Bicêtre, Le Kremlin Bicêtre F-94275, France 4INSERM UMRS 1016, Institut Cochin, Paris F-75014, France 5Service Interdépartemental de Pharmacologie et d’Analyse du Médicament, Institut Gustave Roussy, Villejuif F-94805, France 6Oncologie Endocrinienne, Institut Gustave Roussy, Villejuif F-94805, France 7HRA Pharma, Paris F-75003 France 8Assistance Publique-Hôpitaux de Paris, Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpital de Bicêtre, Le Kremlin Bicêtre F-94275, France 9Assistance Publique-Hôpitaux de Paris, Service d’Endocrinologie et des Maladies de la Reproduction, Hôpital de Bicêtre, Le Kremlin Bicêtre F-94275, France *(E Baudin and M Lombès contributed equally to this work)
Correspondence should be addressed to M Lombès Email marc.lombes@u-psud.fr
Abstract
Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane is the most effective medical therapy for adrenocortical carcinoma, but its molecular mechanism of action remains poorly understood. Although mitotane is known to have mitochondrial (mt) effects, a direct link to mt dysfunction has never been established. We examined the functional consequences of mitotane exposure on proliferation, steroidogenesis, and mt respiratory chain, biogenesis and morphology, in two human adrenocortical cell lines, the steroid-secreting H295R line and the non-secreting SW13 line. Mitotane inhibited cell proliferation in a dose- and a time-dependent manner. At the concentration of 50 uM (14 mg/l), which corresponds to the threshold for therapeutic efficacy, mitotane drastically reduced cortisol and 17-hydroxyprogesterone secretions by 70%. This was accompanied by significant decreases in the expression of genes encoding mt proteins involved in steroidogenesis (STAR, CYP11B1, and CYP11B2). In both H295R and SW13 cells, 50 uM mitotane significantly inhibited (50%) the maximum velocity of the activity of the respiratory chain complex IV (cytochrome c oxidase (COX)). This effect was associated with a drastic reduction in steady-state levels of the whole COX complex as revealed by blue native PAGE and reduced mRNA expression of both mtDNA-encoded COX2 (MT-CO2) and nuclear DNA-encoded COX4 (COX4I1) subunits. In contrast, the activity and expression of respiratory chain complexes II and III were unaffected by mitotane treatment. Lastly, mitotane exposure enhanced mt biogenesis (increase in mtDNA content and PGC1x (PPARGC1A) expression) and triggered fragmentation of the mt network. Altogether, our results provide first evidence that mitotane induced a mt respiratory chain defect in human adrenocortical cells.
Key Words
adrenocortical carcinoma
mitotane
o,p’-DDD
mitochondria
cytochrome c oxidase
Endocrine-Related Cancer (2013) 20, 371-381
Introduction
Adrenocortical carcinoma (ACC) is a rare disease affecting two patients per million people per year, representing <0.1% of all cancer cases. ACC prognosis is poor with <15% of patients surviving 5 years or more once metastases are diagnosed (Icard et al. 2001, Assie et al. 2007, Fassnacht & Allolio 2009, Lughezzani et al. 2010).
Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlo- rophenyl)ethane (o,p’-DDD), is a synthetic derivative of an insecticide. It acts selectively on the adrenal cortex where it has a cytotoxic effect and impairs steroidogenesis (Bergenstal & Dao 1953). Mitotane is a part of the reference treatment of advanced ACC (Berruti 2012, Fassnacht et al. 2012). Indeed, it remains the single most effective drug, inducing a partial response in up to one third of the treated patients (Baudin et al. 2011). Several retrospective studies have shown that plasma mitotane levels above 14 mg/l are associated with a higher partial response rate and improve overall survival (Haak et al. 1994, Baudin et al. 2001, Malandrino et al. 2010, Wangberg et al. 2010, Hermsen et al. 2011). The current recommendation to achieve optimal benefit over risk ratio in patients with unresectable ACC is to maintain plasma mitotane levels between 14 and 20 mg/l (Berruti 2012).
Mitotane’s molecular mechanisms of action remain largely unknown, although mitochondrial (mt) effects have been reported. Kaminsky et al. (1962) observed swollen mitochondria in the adrenal cortex of mitotane- treated dogs by electron microscopy. Subsequently, Martz & Straw (1977) suggested that metabolic transformation of o,p’-DDD into the active metabolite o,p’-DDA occurs in mitochondria and is catalyzed by an unknown cyto- chrome P450. Mitotane metabolism seems to involve two successive reactions of ß-hydroxylation and dehydro- chlorination, leading to production of free radicals that could potentially result in apoptosis (Cai et al. 1995). Critical steps of mitotane’s inhibitory effects on steroido- genesis may occur in mitochondria possibly involving CYP11A1, a mt enzyme that catalyzes the transformation of cholesterol to pregnenolone (Cai et al. 1997). Elevated levels of 11-deoxycortisol and 11-deoxycorticosterone in mitotane-treated patients suggest that mitotane may affect CYP11B1, which is responsible for cortisol synthesis (Asp et al. 2012). More recently, Stigliano et al. (2008) showed by proteomic analysis of H295R cells that expression of proteins involved in stress response, energy metabolism, and tumorigenesis was greatly altered by mitotane exposure. Interestingly, some of these regulated proteins were mt components, even though a direct
impact on their synthesis and/or stability has not been clearly demonstrated. The functional consequences of mitotane on respiratory chain expression and activity have not yet been examined. The respiratory chain consists of four multienzymatic complexes located in the mt inner membrane. Together with the ATP synthase complex, it performs an essential mt function, generating the vast majority of cellular ATP synthesis, while reducing molecular oxygen into water. It is a major source of free radicals in most cells and its function is tightly linked to apoptosis balance. The respiratory chain has been shown to be the target of several pharmacological compounds including non-steroidal anti-inflammatory drugs, antire- trovirals, and chemotherapy agents (Viengchareun et al. 2007, Fedeles et al. 2011, Scatena 2012).
The aim of this study was to evaluate the functional consequences of mitotane exposure on mt oxidative phosphorylation (OXPHOS) in human adrenocortical steroid-secreting H295R and non-secreting SW13 cells, both derived from human ACC. We used complementary experimental approaches including spectrophotometric assays, western blot, quantitative PCR, and mt morpho- logical analysis to explore how mitotane affects mediators of steroidogenesis and respiratory chain activity.
Materials and methods
Cell culture and treatment
H295R and SW13 cells were cultured in DMEM/HAM’S F-12 (PAA, Les Mureaux, France) supplemented with 20 mM HEPES (Invitrogen, Life Technologies), antibiotics (penicillin 100 IU/ml and streptomycin 100 µg/ml), and 2 mM glutamine. The medium for H295R cell culture was enriched with 10% fetal bovine serum and a mixture of insulin/transferrin/selenium. Both cell lines (from pass- ages 2-15) were cultured at 37 °℃ in a humidified incubator with 5% CO2. Mitotane (supplied by HRA Pharma, Paris, France) dissolved in DMSO was added to cell cultures at final concentrations of 10-100 uM; the therapeutic plasma mitotane level is 50 uM (approxi- mately 14 mg/l).
Cell proliferation analysis
Cell proliferation was studied in Celltiter 96 assays (Promega) according to the manufacturer’s recommen- dations. Cells were cultured in 96-well plates and treated
with 10-100 uM mitotane for 24, 48, or 72 h. Absorbance was measured by photometry (Viktor, Perkin Elmer, Courtaboeuf, France) 1 h after addition of 20 ul Celltiter solution per well.
Cortisol and 17-hydroxyprogesterone secretion
The cortisol and 17-hydroxyprogesterone (17-OH- progesterone) concentrations in H295R culture super- natants were determined by radioimmunometric assays using polyclonal antibodies (anti-cortisol: Orion Diagnos- tica, Spectria, Espoo, Finland; anti-17-OH-progesterone: MP Biomedical, Solon, OH, USA). The intra- and interassay coefficients of variation of the cortisol were respectively 4.5 and 5.5% at 22 µg/1, and 4.2 and 4.3% at 269 µg/1, with a detection limit of 5 µg/1 while those of the 17-OH- progesterone assay were 7.8 and 12% at 0.92 ng/ml, and 8.3 and 9.8% at 4.3 ng/ml with a detection limit of 0.02 ng/ml.
Reverse transcriptase-PCR and quantitative real-time PCR
Total RNA was extracted from tissues or cells with the RNeasy Kit (Qiagen) according to the manufacturer’s recommendations. RNA was thereafter processed for reverse transcriptase-PCR (RT-PCR) as described pre- viously (Martinerie et al. 2011). Quantitative real-time PCR (qRT-PCR) was performed using the Fast SYBR Green Master Mix (ABI, Applied Biosystems) and carried out on a StepOnePlus Real-Time PCR System (Applied Biosystems) as described previously (Martinerie et al. 2011). Standards and samples were amplified in dupli- cate and analyzed from three independent experiments. The internal control for data normalization was the ribosomal 18S rRNA. The relative expression of each gene is expressed as the ratio of attomoles of the specific gene to femtomoles of 18S rRNA. The primer sequences of the genes analyzed by qRT-PCR are shown in the Supplementary Table 1, see section on supplementary data given at the end of this article.
mtDNA quantification
mtDNA quantification was performed on total DNA extracted from tissues or cells using standard techniques. DNA was quantified by qPCR using the cytochrome c oxidase 2 (COX2 (MT-CO2)) gene on the mtDNA as a target gene as described previously (Viengchareun et al. 2007). Results were expressed as relative expression of COX2 normalized with the nuclear 18S gene.
Respiratory chain analysis
Respiratory chain activities were measured using spectro- photometric assays. H295R and SW13 cells were treated with mitotane or vehicle (DMSO) alone for various periods, 24, 48, or 72 h, and the activity of four mt respiratory complexes - complex I (NADH-ubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), complex III (ubiquinone-cytochrome c oxidoreductase), and complex IV (COX) - were measured in a Cary 50 Spectrophoto- meter (Rustin et al. 1994). Assays of complexes II, III, and IV were performed on cell homogenates, and their activities normalized to citrate synthase activity, as an index of mt mass. Complex I assays were performed on purified mt fractions and prepared from permeabilized cells as described previously (Chretien et al. 2003).
BN-PAGE analysis
Mitochondria and OXPHOS complexes were isolated from cultured cells using 2% (W/V) digitonin and analyzed as described (Nijtmans et al. 2002a,b). Fifteen micrograms of solubilized OXPHOS proteins were loaded on a 4-16% gradient acrylamide non-denaturing gel (Invitrogen). After electrophoresis, proteins were transferred to a PVDF membrane. Immunoblotting was performed with MABs (Mitosciences, Mundolsheim, France) raised against the complex I subunit GRIM19, the 70 kDa complex II subunit, the complex III subunit core2, and the complex IV subunit COX1. Peroxidase-conjugated anti-mouse IgG secondary antibodies were added and the signal was generated using ECL (Pierce, Rockford, IL, USA). Mem- branes were scanned using the Odyssey infrared imaging system and images were processed with the Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA).
mt morphology
Cells were seeded at subconfluence on a glass coverslip and incubated for 24-48 h in the presence or absence of 50 uM mitotane, briefly rinsed with warm PBS, and then fixed in 3% paraformaldehyde in PBS. Mitochondria were labeled with antibodies against COX2 subunit as described (Agier et al. 2012).
Statistical analysis
Results are expressed as means±S.E.M. of n independent replicates performed in the same experiment or from n separated experiments. Differences between groups were
analyzed using nonparametric Kruskal-Wallis ANOVA followed by Dunn’s multiple comparison test or nonpara- metric Mann-Whitney U test as appropriate. The signi- ficance level was P<0.05.
Results
Mitotane treatment reduces human adrenocortical H295R and SW13 cell proliferation
Proliferation index was calculated using the colorimetric solution Celltiter 96. Exposure to mitotane for 48 h inhibited the proliferation of H295R and SW13 cells in a dose-dependent manner, 100 µM o,p’-DDD significantly reducing the proliferation rate of H295R by 45% and that of SW13 cells by 30% (Fig. 1A and B). The anti-proliferative effect of mitotane was also time dependent, 100 uM mitotane inhibiting the proliferation of H295R cells by 18% after 24 h and by 70% after 72 h. Subsequent experiments were performed using 50 uM mitotane to minimize the drug’s potential cytotoxic effects.
Effect of mitotane on steroidogenesis in H295R cells
To confirm the ability of mitotane to inhibit hormone secretion, we measured several steroid hormone concen- trations in the culture supernatant of H295R cells. Exposure to 50 uM mitotane for 48 h significantly reduced the secretion of both cortisol and 17-OH-progesterone about 80% by H295R cells (Fig. 1C). Other steroid hormones such as aldosterone were undetectable in culture super- natants under these experimental conditions.
To address the mechanisms underlying this decreased steroid secretion, we analyzed the expression of genes that encode mt effectors of steroidogenesis by qRT-PCR. Mitotane significantly decreased the expression of such genes: STAR, which encodes the STAR that transports cholesterol into mitochondria, the first rate-limiting step for the intra-mt steroidogenic pathway (80% inhi- bition after 48 h; Fig. 2A); cholesterol desmolase (CYP11A (CYP11A1)), 3ß-hydroxysteroid dehydrogenase (HSD3B2); 11ß-hydroxylase (CYP11B1), which catalyzes 11-deoxy- corticosterone and 11-deoxycortisol transformation into corticosterone and cortisol respectively (75% inhibition; Fig. 2B); and aldosterone synthase (CYP11B2), the last intra-mt enzymatic step in aldosterone synthesis (97%, inhibition; Fig. 2C). The mitotane-induced inhibition of steroid secretion observed in H295R cells therefore appeared to be due to decreased expression of the steroidogenic enzymes.
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Effect of mitotane on the respiratory chain
The impact of mitotane on respiratory chain activity was evaluated by spectrophotometric assays of the activities of the four mt respiratory complexes in H295R and SW13 cells treated with vehicle (DMSO) or 50 uM mitotane during 48 h (Table 1). Citrate synthase activity, belonging to the mt citric acid cycle, was used as an index of the mt mass. Its activity was very high in H295R cells (299± 22 nmol/min per mg protein; n=12) but lower in SW13 cells (159±11 nmol/min per mg protein; n=8), suggesting that H295R cells have a greater mt population than SW13 cells consistent with their important steroido- genic capacity. However, citrate synthase activity was not affected by mitotane exposure (260±31 nmol/min per mg protein in H295R and 135 +7 nmol/min per mg protein in SW13 cells).
Both H295R and SW13 mitotane-treated cells exhib- ited a significant COX (or complex IV) defect of ~50% after 48 h while complex II (succinate-ubiquinone oxido- reductase) appeared unaffected (Table 1). Complex III (ubiquinol-cytochrome c oxidoreductase) activity
remained unchanged in H295R cells and was slightly reduced in SW13 cells after mitotane treatment (Table 1), but this decrease was not confirmed after normalization to citrate synthase activity (Table 1). Complex I (NADH- ubiquinone oxidoreductase) activity can only be reliably
| Conditions | H295R cells | SW13 cells | ||
|---|---|---|---|---|
| Vehicle | 50 μ.Μ mitotane | Vehicle | 50 μ.Μ mitotane | |
| Citrate synthase | 299±22 | 260±31 | 159±11 | 135±7 |
| Complex I | 8.3±0.7 | 3.6±1.1* | 14.4 | 7.5 |
| Complex II | 55±8 | 43±9 | 37±3 | 32±1 |
| Complex III | 95±14 | 86±26 | 89±6 | 55±10* |
| Complex IV | 276±15 | 153±19+ | 283±53 | 127 ±37+ |
| CII/CS | 0.28±0.03 | 0.21±0.03 | 0.26±0.02 | 0.30±0.04 |
| CIII/CS | 0.29±0.03 | 0.36±0.07 | 0.68±0.07 | 0.68±0.10 |
| CIV/CS | 1.05±0.08 | 0.52±0.02 | 0.78±0.08 | 0.56±0.01* |
Enzymatic activities were measured in cell homogenates with the exception of complex I, which was measured on purified mitochondrial fractions; values are mean+s.E.M. of 6-12 independent experiments, expressed as nmol/min per mg protein. Ratio between complex Il or complex III or complex IV:citrate synthase (CS) activities is also presented. * P<0.05, +P<0.01, and +P<0.001 with nonparametric Mann-Whitney U test.
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measured on purified mt fractions due to the presence of numerous non-mt NADH oxidase activities in cell homo- genates but its activity was greatly decreased after exposure to mitotane in both human adrenocortical cell lines after exposure to mitotane (Table 1). Altogether, our results demonstrate that mitotane selectively inhibits some but not all respiratory chain complexes.
The effect of mitotane on complex IV was concen- tration dependent, as shown in Fig. 3, with an IC50 calculated at ~67 uM mitotane (linear regression test; y=92.862-0.583x, 12=0.97). This mitotane concen- tration corresponds to the therapeutic plasma threshold predictive of efficacy in clinical practice (Haak et al. 1994, Baudin et al. 2001).
To examine whether o,p’-DDD might directly affect the enzymatic activity of complex IV, we measured COX activity on cell homogenates incubated with increasing concentrations of mitotane. Under these conditions, we demonstrated that mitotane dose dependently decreased complex IV activity with an IC50 of ~133 uM (linear regression test; y=100.2-0.3749x, 12=0.96; Fig. 4). This IC50 in the cell homogenate system is twice as high as the IC50 observed when whole cells were treated for 48 h, indicating that mitotane exerts both direct and indirect inhibitory effects on COX activity. Our results strongly suggested that mitotane inhibits enzymatic activity directly but presumably inhibits the expression of the enzyme. We therefore studied the expression of COX at
both the mRNA and protein levels. The COX complex consists of 13 subunits, three of which, including COX2, are encoded by the mt genome while the remaining ten subunits, including COX4 (COX4I1), are encoded by nuclear genes. We observed that the steady-state levels of mt and nuclear DNA-encoded COX2 and COX4 transcripts in both H295R and SW13 cells were drastically decreased (by 70%) in H295R cells after exposure to 50 uM mitotane for 48 h (Fig. 5A and B). Similar results were obtained in SW13 cells (data not shown).
We analyzed the whole respiratory chain complexes by blue native PAGE (BN-PAGE). Immunoblotting with antibodies directed against a component of each mt complexes revealed that mitotane exposure for 48 h induced a 45-70% decrease in the steady-state expression of complex IV and complex I proteins while the abundance of complexes II and III appeared unchanged (Fig. 5C and D). These data were fully consistent with the decreased enzymatic activities described earlier (Table 1). Altogether, our results demonstrate that mitotane has deleterious consequences by acting at the mRNA and protein level to impair respiratory chain expression and function.
To evaluate the possibility that mitotane has direct toxic effects on the mtDNA, we quantified mtDNA by qPCR. As illustrated in Fig. 6A, the mt:nuclear DNA ratio was unaffected by exposure of low or moderate doses of
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mitotane for 48 h. However, this ratio increased sig- nificantly after treatment with 100 uM mitotane and with longer exposure times (e.g. 50 uM mitotane for 72 h), suggesting the presence of a compensatory response of mt biogenesis (Fig. 6B). To further explore this
hypothesis, we quantified the expression of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC1a (PPARGC1A)), a transcriptional coactivator considered a key regulator of mt biogenesis. PGC1a mRNA expression was slightly but significantly induced
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by 50 uM mitotane treatment for 48 h, suggesting acti- vation of transcriptional response (Fig. 6C). Furthermore, no mtDNA mutations or deletions were found by sequencing mtDNA from mitotane-treated cells.
Effect of mitotane on mt morphology
Finally, to get an integrated evaluation of the mitotane- induced mt respiratory chain defect, we analyzed the mt morphology by immunocytochemistry using an antibody against COX2. Treatment with 50 uM mitotane induced drastic morphological alterations in the mitochondria of adrenocortical cells. In the absence of treatment, the mt compartment appeared as a highly interconnected tubular network with a filamentous appearance. However, after exposure to the drug the compartment exhibited a more punctiform pattern, consistent with mt fragmenta- tion (Fig. 7).
Discussion
Even though combination of mitotane and cisplatin-based chemotherapy has been recently shown to clinically improve the overall survival in advanced ACC (Fassnacht et al. 2012), mitotane remains the single most active pharmacological option for the management of ACC, as recognized in recent recommendations (Berruti 2012). However, its mechanism of action still remains unclear. In this study, we addressed the question on the mt effects of mitotane on two different human adrenocortical cell lines derived from human ACC aiming at identifying potential molecular targets of the drug. Attempts to perform similar experiments on primary human ACC cells have been so far unsuccessful.
We found that at optimal therapeutic concentrations (50 µM, i.e. 14-20 mg/l), mitotane drastically altered mt function in both steroid-secreting and non-secreting adrenocortical cell lines derived for human ACC. Mitotane inhibited steroid hormone production and secretion, which was accompanied by a reduction in steady-state mRNA levels of genes encoding mt proteins involved in steroidogenesis pathways. More importantly, we demon- strated for the first time that exposure to 50 uM mitotane significantly impairs the mt respiratory chain. Mitotane exposure also stimulated mt biogenesis and altered mt morphology in adrenocortical cells.
It is well established that the in vivo anti-proliferative efficacy of mitotane depends on its circulating plasma level (Baudin et al. 2001). However, its pharmacokinetic profile with an unmet need for improved bioavailability
COX2
DAPI
DMSO
Mitotane
and its metabolic conversion constitute potential limi- tations (Schteingart 2007). It has been suggested that the metabolic transformation of o,p’-DDD is carried out in the adrenal mitochondria, the first enzymatic step being catalyzed by an unknown P450 cytochrome-mediated hydroxylase leading to an adrenolytic effect (Martz & Straw 1977, Cai et al. 1995). In accordance with previous studies, we confirmed that mitotane inhibits steroido- genesis reducing cortisol and 17-OH-progesterone secretions by 70% (Schteingart et al. 1993, Stigliano et al. 2008). Mitotane exposure also decreased mRNA levels of STAR, the cholesterol carrier into the mitochondria, as well as CYP11A, CYP11B1, and CYP11B2, three mt enzymes involved in cortisol and aldosterone biosynthesis respectively. However, the degree and extent of mitotane- induced repression of genes involved in steroidogenesis seem to vary greatly between studies (Asp et al. 2012, Lin et al. 2012, Zsippai et al. 2012), supporting mitochondria as a main target of the drug’s action.
The mitotane transformation into active acylchlorine metabolites that takes place in the mitochondria of adrenal gland is believed to be responsible for cell toxicity and may explain the selective adrenolytic effect of the drug (Cai et al. 1995, Lindhe et al. 2002). This hypothesis awaits further confirmation at the clinical level (Hermsen et al. 2011). At variance with the hepatic microsomal transformation of mitotane by CYP3A4 (van Erp et al. 2011, Kroiss et al. 2011), which is likely responsible for the pharmacokinetic interaction whereby mitotane reduces plasma levels of sunitinib (Fassnacht et al. 2012, Kroiss et al. 2012), it has been suggested that CYPc11 or CYP11B1 could be involved in tissue-specific and compartment-selective mitotane metabolism (Lund & Lund 1995, Lindhe et al. 2002). Although CYP11B1 may catalyze the initial hydroxylation step of mitotane (Cai et al. 1995, Lund & Lund 1995, Lindhe et al. 2002), its direct involvement in mt dysfunction is very unlikely given that SW13 cells, which do no express CYP11B1, were similarly affected by mitotane treatment. In any case, the relationship between the potential hepatic metabolism of mitotane and its adrenal effect remains currently unknown. For instance, it remains to be estab- lished whether intra-mt transformation of mitotane into o,p’-DDA and o,p’-DDE compounds has deleterious con- sequences on OXPHOS. However, preliminary results from our laboratory reveal the presence of active mitotane uptake into H295R cells, suggesting that intracellular accumulation of mitotane and/or one of its metabolites may account for its cytotoxic effects.
Given that most enzymatic steps of steroid hormone biosynthesis take place in the mitochondria and that mitotane inhibits steroidogenesis, we examined whether mitotane impedes mt respiratory chain function. Interest- ingly, in both H295R and SW13 cells, OXPHOS analyses indicated that mitotane induced a significant and selective decrease in the maximum velocity of COX activity, whereas complex II and III activities were unaltered. Mitotane has both direct and indirect inhibitory effects on COX: direct inhibition of the enzymatic activity was revealed in our experiments on cell homogenate incubation with o,p’-DDD but the drug also inhibited expression of the enzyme at both the mRNA and protein levels. Inhibition of gene expression was observed for both the mtDNA-encoded COX2 and the nuclear DNA-encoded COX4 subunits. Immunoblotting provided additional support for a reduction in steady-state COX protein expression. Concomitantly, normal activity and expression of respiratory chain complexes II and III or of citrate synthase, a Krebs cycle enzyme, suggest that mitotane caused selective enzymatic disruption rather than global mt damage, as initially proposed (Kaminsky et al. 1962).
Herein, we confirm the adrenolytic effect of mitotane by showing that mitotane exposure leads to a time- and concentration-dependent reduction of adrenocortical cell numbers. Interestingly, this was accompanied by enhanced mt biogenesis, as demonstrated by increased mtDNA content and PGC1« expression, reminiscent of a cellular compensation mechanism in response to the respiratory chain defect. This adaptive pathway, combining increased mt mass, increased mtDNA copy level and impaired OXPHOS, which has already been reported in mt myopa- thies caused by mtDNA mutations (Srivastava et al. 2009). However, no mtDNA mutations or deletions were found by sequencing mtDNA from mitotane-treated cells. Of particu- lar interest, mitotane exposure also triggered morphologic fragmentation of the mt network, which could be related to disequilibrium between mt fission and fusion (Chen & Chan 2010). It is well established that the integrity of mt outer and inner membranes is required for respiratory chain activity (Liesa et al. 2009, Chen et al. 2010) and presumably steroidogenesis (Duarte et al. 2012). It is not known, however, whether mt fragmentation has a direct relation- ship with or a causal role in genotoxic stress and apoptosis.
In summary, our results show that mitotane alters mt respiratory chain activity in human adrenocortical cells, notably by inducing a COX defect. Further studies are needed to examine whether and how such mitotane- induced mt dysfunction translates into adrenolytic and antitumor effects on human ACC (Costa et al. 2011).
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/ ERC-12-0368.
Declaration of interest
All authors have no disclosure except Dr Rita Chadarevian who is an employee of HRA Pharma.
Funding
This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Faculté de Médecine Paris-Sud, and a grant from HRA Pharma Laboratories. S H was a recipient of a fellowship from the French Endocrine Society (Société Française d’Endocrinologie (SFE)).
Author contribution statement
S H, A S, E B, and M L designed the study; S H, A S, A L, S T, and M L performed the experiments and analyzed the results; A L, A P, H R, R C, S B, and J Y helped interpret the data and participated in discussions; and S H, E B, and M L wrote the paper; all the authors have read, revised, and approved the manuscript.
Acknowledgements
The authors would like to thank C Clemenson (Institut Gustave Roussy, Villejuif, France) for providing cell lines and Dr Say Viengchareun for his help in preparing the figures.
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Received in final form 19 March 2013 Accepted 3 April 2013 Made available online as an Accepted Preprint 5 April 2013