THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
JCEM
EARLY RELEASE:
SOCIETY ENDOCRINE
Lipoprotein-free mitotane exerts high cytotoxic activity in adrenocortical carcinoma
Ségolène Hescot1,2, Atmane Seck3, Maryse Guerin4, Florence Cockenpot3, Thierry Huby4, Sophie Broutin3, Jacques Young1,5, Angelo Paci3, Eric Baudin1,2, and Marc Lombès1,5
1 INSERM UMR S 1185, Fac Med Paris Sud, Le Kremlin-Bicêtre F-94276, France; 2 Gustave Roussy, Nuclear Medicine and Endocrine Oncology Department, Villejuif F-94805, France; 3 Gustave Roussy, Pharmacology Department, Villejuif F-94805, France; 4 INSERM UMR S ICAN/1166, LA Pitié-Salpetrière Hospital, Paris, F-75013, France; 5 CHU Bicêtre, Department of Endocrinology, Le Kremlin-Bicêtre F- 94276, France
Context: Mitotane (o,p’-DDD), the only approved drug for advanced adrenocortical carcinoma (ACC), is a lipophilic agent that accumulates into circulating lipoprotein fractions and high lipid- containing tissues.
Objective: The aim of our study was to evaluate the in vivo and in vitro biological implication of serum lipoproteins on pharmacological action of mitotane. Distribution and concentration of mitotane were studied in plasma and adrenal tissue samples from mitotane-treated patients. The impact of lipoprotein-bound or free (LP-F) mitotane was analyzed on proliferation and apoptosis of human adrenocortical H295R cells. A retrospective study of ACC patients treated or not with statins was also performed.
Results: o,p’-DDD distribution among VLDL, LDL, HDL and lipoprotein-free (LP-F) fractions ob- tained after ultracentrifugation of 23 plasmas of mitotane-treated patients was widely distributed in each subfraction. A positive correlation was observed between mitotane levels in plasma and in LDL, HDL but also LP-F compartment. Intra-tumor o,p’-DDD concentrations in 5 ACC samples of mitotane-treated patients were found independent of cholesterol transporter expression, scav- enger receptors (SrB1) and LDL-Receptors. In vitro studies showed significant higher anti-prolif- erative and pro-apoptotic effects and higher cell and mitochondrial uptake of mitotane when H295R cells were grown in LP-F medium. Finally, retrospective study of an ACC cohort of 26 mi- totane-treated patients revealed that statin therapy was significantly associated with a higher rate of tumor control.
Conclusions: Altogether, our in vitro and in vivo studies provided compelling evidence for a greater efficacy of lipoprotein-free mitotane. ACC patients may thus benefit from therapeutic strategies that aim to increase LP-F mitotane fraction.
M itotane is the only drug approved in advanced ad- renocortical carcinoma (ACC) (1). The antitumor clinical impact of mitotane has been shown on both pro- spective and retrospective studies that found partial re- sponse rates in 10 to 33% of patients treated with mito- tane alone but also improved overall survival (2, 3). Based on these results, mitotane is also recommended as an ad-
juvant therapy in ACC patients at high risk of recurrence (4-6). In both indications, plasma mitotane monitoring is recommended to look for a therapeutic window of 14-20 mg/L (1). Indeed, several studies have reported higher re- sponse rate and/or a prolonged survival in patients with plasma mitotane levels above 14 mg/L (7-11). In addition,
Abbreviations:
Copyright @ 2015 by the Endocrine Society
Received April 24, 2015. Accepted June 23, 2015.
doi: 10.1210/JC.2015-2080
neurological toxicities have been described with plasma level above 20 mg/L (11, 12).
Mitotane is also known as o,p’-DDD, an insecticide- derivative lipophilic drug that accumulates in lipoproteins (13). Dyslipidemia have been observed in mitotane- treated patients but the mitotane-induced dyslipidemic profile differs from one study to another (14-19). The mechanism of mitotane-induced hypercholesterolemia is not fully understood but could be related to an activation of HMGCoA reductase (20) and to an increase in choles- terol and lipoproteins synthesis (14). Little is known about the influence of dyslipidemia on mitotane distribution among lipoproteins and about the influence of this distri- bution on its antitumor efficacy.
Mechanism of mitotane action was poorly understood until recently. Two mitotane metabolites are described: o,p’-DDE and o,p’-DDA, the latter being described as the main urinary metabolite of o,p’-DDD (21). We recently reported evidence that o,p’-DDA is unlikely an active me- tabolite of mitotane (22). Several studies suggested that mitotane could have a mitochondrial effect (23, 24), and more specifically, our group demonstrated a mitotane- induced defect in cytochrome c oxidase (complex IV of the mitochondrial respiratory chain) (25).
In the present study, we explored the biologic implica- tion of serum lipoproteins on mitotane pharmacological action using human plasma samples, tissues of mitotane- treated patients and finally in vitro on human adrenocor- tical H295R cells. Altogether, our results showed that li- poprotein-free mitotane appeared to be the most efficient form. Based on these findings, we retrospectively exam- ined the disease control rate of 26 consecutive stage IV- ACC patients treated with mitotane, according to the con- current use of statins.
Patients, material and methods
Patients
Medical files of 70 metastatic ACC patients treated with mitotane, followed between 2007 and 2014 at Gustave Roussy were retrospectively reviewed to study the correlation between occurrence of dyslipidemia or statin therapy (Rosuvastatin not metabolized by Cyp3A4) within the first 3 months of mitotane therapy and neuro- logical toxicity or tumor response. Inclusion criteria were patients with stage IV ACC treated with mitotane after 2007 and the exclusion criteria was the absence of lipid profile available within the first 3 months of mitotane therapy. In each file, the following criteria were recorded: mitotane plasma level, HDL, LDL triglycerides levels (high HDL, high LDL or high triglycerides defined as above 1.5 time the upper value, concurrent use of statins given during at least 3 months of mitotane therapy, pres-
ence of neurological toxicities and disease control rate (stabilization and partial response) at 6 months according to RECIST 1.1 (26). An informed consent was obtained from all patients.
Human plasma samples and human adrenal tissues
Twenty-five plasma samples from 20 mitotane-treated ACC patients were taken at Gustave Roussy hospital and then made available for this study from the central repos- itory of HRA Pharma (Paris, France) to evaluate lipopro- tein partitioning in normal and dyslipidemic samples. To analyze the correlation between mitotane concentrations and cholesterol transporter expression, human adrenal tissues were obtained from 6 mitotane-treated patients followed at Gustave Roussy or Bicêtre Hospital. All were ACC but one who underwent bilateral adrenalectomy for an ectopic Cushing syndrome. Tissues were collected while patients underwent surgery for therapeutic reasons. Tissues were lysed in H2O using a TissueLyser apparatus (Qiagen, Courtaboeuf, France). All patients signed an in- formed consent.
Human adrenocortical cells and mitochondria isolation
For in vitro studies, H295R cells (from passage 2 to 15) were cultured as previously described (25). Media were enriched with 10% fetal calf serum (FCS, control medium) or lipoprotein deficient FCS (LP-F medium) enriched or not with LDL or HDL subfractions obtained from ultra- centrifugation (with a final cholesterol concentration of 30 mg/L, similar as in all media). O,p’-DDD (HRA Pharma) and BLT1 (Sigma-Aldrich, St. Louis, MO) were solubilized in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and used at indicated concentrations. The percentage of DMSO in culture medium never exceeded 0.1%. Choles- terol (Sigma-Aldrich) and Bovine serum albumin (BSA, Euromedex, Mundelsheim, France) were solubilized in culture medium and used at final concentrations of 9.6 mg/L (low) and 38.4 mg/L (high) for cholesterol and 1.8 mg/L (low) and 3.6 mg/L (high) for BSA.
Mitochondrial fractions were purified and prepared from permeabilized cells using digitonin and percoll as previously described (27).
Lipoprotein isolation from plasma and fetal calf serum by ultracentrifugation
Density gradient ultracentrifugation using iodaxinol (Optiprep®, Sigma-Aldrich) was used for the isolation of lipoprotein fractions in plasma samples. A saline solution with HEPES buffer was added to a mixed solution of 60% (m/v) iodixanol in water (d = 1.32 g/mL) and plasma in Optiseal® vials. This final solution was ultracentrifuged
at 350 000 g at 16℃ for 3 hours. Each lipoprotein fraction was collected with syringe and needle systems.
Individual lipoprotein subfractions were isolated from fetal calf serum by isopycnic density gradient ultracentrif- ugation for 48 hours at 288 000 g using a Beckman XL70 centrifuge and a SW41 rotor as previously described (28). After centrifugation, gradients were collected from the top of the tubes with an Eppendorf precision pipette in frac- tions corresponding to LDL subfractions (density < 1.063 g/mL) and HDL subfractions (density from 1.063 g/mL to 1.179 g/mL).
Measurements of o,p’DDD in plasma, tissues, cells and mitochondria
Analyses of plasma samples were conducted by high performance liquid chromatography combined to an ul- traviolet (UV) detection (HPLC-UV) as previously de- scribed (22) and analyses of cells and mitochondria sam- ples were conducted by gas chromatography combined to a mass spectrometry (GCMS). All samples were spiked with known amounts of p,p’-DDE used as an internal standards (IS) of o,p’-DDD measurements. O,p’-DDD concentrations were determined through the ratio of their peak surface area to the peak surface of known concen- trations of IS. Concentrations of o,p’-DDD in cells and mitochondria are expressed in ng per femtomoles of nu- clear or mitochondrial DNA. Nuclear and mtDNA were extracted from samples using standard techniques and quantified by real-time quantitative PCR using the 18S gene and the cytochrome c oxidase 2 (COX2) gene as nuclear and mitochondrial specific genes, respectively as previously described (25).
Cell proliferation and apoptosis analysis
Cell proliferation tests were performed by using the WST1 assay (Roche, Meylan, France) and apoptosis tests were performed by using the Caspase-Glo 3/7 assay (Pro- mega, Madison, WI) according to the manufacturer’s rec- ommendations. Cells were cultured in 96-well plates and treated with 0 to 150 AM o,p’-DDD for 24 or 48 hours. Optical densities were measured 4 hours after addition of WST1 solution (10 ul per well) by spectrophotometry (Viktor, Perkin Elmer, Courtaboeuf, France). Lumines- cence was measured 1h after addition of Caspase-Glo 3/7 solution (equal volume) by luminometry (Viktor, Perkin Elmer).
Reverse Transcriptase-PCR (RT-PCR) and Quantitative real-time PCR (RT-qPCR)
Total RNAs were extracted from cells with the RNeasy kit (Qiagen, Courtaboeuf, France) according to the man- ufacturer’s recommendations. RNA was thereafter pro-
cessed for RT-PCR as previously described (25). Quanti- tative real-time PCR (RT-qPCR) was performed using the Fast SYBR® Green Master Mix (Life Technologies) and carried out on a StepOnePlus™M Real-Time PCR System (Life Technologies) as previously described (25). The rel- ative expression of each gene was expressed as the ratio of attomoles of specific gene to femtomoles of 18S rRNA.
Western Blot analysis
Total protein extracts were prepared as previously de- scribed (22). Western Blot analyses were performed as described (22). Antibodies used were a rabbit anti-BCL2 antibody (1:500 dilution, Cell Signaling, Saint Quentin en Yvelines, France) with a mouse anti-«-Tubulin antibody (1:10,000 dilution, Sigma-Aldrich) or a rabbit anti- CYP11A1 antibody (1:500 dilution, Sigma-Aldrich) with a mouse anti-GAPDH antibody (1:10,000 dilution, Sig- ma-Aldrich). Proteins were visualized with an Odyssey-Fc apparatus (LI-COR).
Statistical Analysis
Results are expressed as means ± SEM of n indepen- dent replicates performed in the same experiment or from separated experiments (n). Correlation was tested with a Pearson test. Nonparametric Mann Whitney tests were used when appropriate and differences between groups were analyzed using nonparametric Kruskall-Wallis mul- tiple comparison test followed by a post-test of Dunn’s (Prism software, GraphPad, CA). Difference between groups of patients was assessed using the Fisher’s exact test. A P value of 0.05 was considered as statistically sig- nificant (*P <. 05; ** P <. 01; *** P <. 001).
Results
Distribution of o,p’-DDD and its metabolites among lipoproteins and lipoprotein-free subfractions in ACC patients plasma samples
The distribution of mitotane and its two major metabo- lites, o,p’-DDE and o,p’-DDA, in lipoproteins was evalu- ated in 20 ACC patients. O,p’-DDD, o,p’-DDA and o,p’- DDE were thus measured with HPLC-UV after ultracentrifugation of plasma samples in VLDL, LDL, HDL and lipoprotein-free (LP-F) subfractions. O,p’-DDD was widely distributed among lipoprotein fractions as fol- lows : 34.6 ± 9.9% LP-F (including proteins-bound and free mitotane), 26.3 ± 5.8% HDL, 26 ± 4.6% LDL and 13 ± 4.2% VLDL (Figure 1A). The distribution of o,p’- DDE among these subfractions favoring LP-F, was as fol- lows (72.9 ± 15.3% LP-F, 17.7 + 12.2% HDL, 8.2 + 1 0.5% LDL and 1.3 + 2.7% VLDL) while, in sharp con- trast, o,p’-DDA was almost exclusively recovered in LP-F
fractions (94.6 ± 3.1%, Suppl. Figure S1A and B). No significant difference in o,p’-DDD, o,p’-DDA or o,p’- DDE distribution was observed according to the presence and the degree of dyslipidemia or the plasma mitotane level (data not shown). Of interest, plasma mitotane level
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correlated with o,p’-DDD measured in LP-F fractions (Figure 1B, r2=0.41; P < . 001) but also in those of HDL and LDL (Figure 1C, r2=0.76; P < . 001).
Intra-tumor o,p’-DDD concentrations and cholesterol transporters expression.
To further explore which LP subfraction might account for mitotane uptake in human ACC tissue, we measured the relative expression of genes encoding for SrB1 (Scav- enger B1 receptor, HDL receptor) and LDL-R in six sam- ples of human adrenal tissue collected from one ectopic Cushing’s disease and 5 ACC mitotane-treated patients (Table 1). On a case by case analysis, no association was found between SrB1 or LDL-R expression and intra-tu- mor o,p’-DDD concentrations.
Influence of lipoprotein-binding on mitotane efficiency in vitro
Impact on cell proliferation and apoptosis
To evaluate the influence of lipoprotein binding on mi- totane efficacy in terms of cell proliferation and apoptosis, H295R cells were incubated in different culture condi- tions containing either HDL lipoproteins (HDL), LDL li- poproteins (LDL) or lacking lipoprotein fractions (lipo- protein-free, LP-F) and compared to control (FCS). Cell proliferation index was measured at baseline and after incubation with various mitotane concentrations for 48h. Basal cell proliferation was not different between these conditions (Suppl. Figure S2A). Mitotane exerts a dose- dependent antiproliferative effect in all conditions but was more efficient when cells were cultured in LP-F medium with an IC50 of approximately 40 µM compared to 140 uM under control conditions (Figure 2A) with a left-shift of dose-dependent curve. Apoptosis index as measured by caspase 3/7 assays was significantly higher after a 24h treatment with 100 µM mitotane in LP-F condition com- pared to LDL, HDL or control conditions (Figure 2B). Furthermore, expression of the antiapoptotic protein BCL2 was reduced by 100 µM mitotane in LP-F condition but not in others (Suppl. Figure S2B). Altogether, our re- sults clearly indicate that the cytotoxic effects of mitotane are more pronounced in the absence of lipoproteins.
Intracellular uptake of mitotane and mitochondrial impact
To examine whether the nature of LP fractions affects mitotane uptake, o,p’-DDD concentrations were mea- sured in cell pellets after 48h of 50 µM exposure in dif- ferent conditions. Intracellular o,p’-DDD concentrations, measured by the sensitive GC-MS technique and normal- ized to nuclear DNA were at least 3 fold higher in cells cultured in LP-F medium than in other media (Figure 3A).
| Patients | Clinical presentation | SrB1 | LDL-R | o,p'DDD | Plasma mitotane level (mg/liter) |
|---|---|---|---|---|---|
| (#) | (mRNA) | (mRNA) | Tissue | ||
| 1 | Ectopic Cushing | 33.86 | 1.46 | 53.75 | 3.3* |
| 2 | ACC | 2.62 | 0.03 | 2.47 | 20.03 ** |
| 3 | ACC | 0.33 | 0.02 | 0.66 | 18.5 ** |
| 4 | ACC | 3.61 | 0.18 | 1.48 | 23.7 ** |
| 5 | ACC | 20.42 | 1.31 | 2.01 | 14.7 ** |
| 6 | ACC | 4.50 | 0.21 | 12.81 | 4* |
An informed consent was obtained for each patient. SrB1 and LDL-R expression in adrenal tissue samples was measured by RT-qPCR after DNA extraction and are expressed as attomol per attomol of GAPDH as described in material and methods section. O,p’DDD concentration was measured in tissue homogenates and plasma using HPLC-UV analysis and are expressed in nmol per mg of tissue or in mg/liter respectively.
* Plasma mitotane level measured two weeks before surgery
** Mean of 4 to 6 plasma mitotane levels assessed during treatment
The intracellular mitotane was mostly recovered in the mitochondrial fraction (89.2 ± 3.6% of o,p’-DDD) whereas only 10.8 ± 3.6% of the measured intracellular o,p’-DDD was in the cytosolic fraction (data not shown).
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Intra-mitochondrial o,p’-DDD concentration was 15 higher (90.26 ± 7.66 ng/ atomol of mt DNA) in cells grown in LP-F condition compared to other culture con- ditions, highly suggestive of a better cellular and thus mi- tochondrial uptake of mitotane in the absence of lipopro- teins (Figure 3B). We next studied the expression of genes encoding proteins involved in oxidative phosphorylation or steroidogenesis by RT-qPCR in o,p’-DDD treated cells under HDL, LDL, LP-F and control conditions. In LP-F medium, mitotane strongly inhibited COX2 expression (encoded by the mitochondrial mtDNA for the subunit 2 of cytochrome c oxidase or respiratory chain complex IV, Figure 3C) but also StAR and CYP11A1 involved in ste- roidogenesis (Suppl. Figure S3A and B). LP-F condition also led to a drastic reduction in CYP11A1 protein ex- pression in H295R cells after a 48h treatment with 50 p.M mitotane (Suppl. Figure S3C). Collectively, our findings provide additional evidence that lipoprotein-free mitotane was the most efficient leading to alter cellular functions.
Effect of BLT1 treatment and cholesterol saturation
BLT1 is a powerful but not fully specific inhibitor of SrB1 and thus a pharmacological inhibitor of cellular li- poprotein uptake. SrB1 also participates to cellular efflux of lipophilic molecules such as cholesterol or vitamin E and very likely mitotane. O,p’-DDD displays a more po- tent antiproliferative activity on H295R cells with a left- shift dose-dependent in the presence of BLT1 and a pro- liferation index at 7.5 ± 3.8% in BLT1-treated cells compared to 69.5 ± 4.3% in control cells (P <. 01) at 100 M mitotane concentration for 48h. Likewise, mitotane- treated cells exhibited a higher apoptotic capacity in the presence of BLT1, as revealed by the reduced expression of the antiapoptotic protein BCL2 (Figure 4B). This was ac-
companied by an significant increase in intracellular o,p’- DDD concentration when lipoproteins receptors were in- hibited with BLT1 (Figure 4C). Free cholesterol was added into the culture medium to saturate lipoproteins in an at- tempt to reduce lipoprotein-bound mitotane through cho- lesterol exchange, thus potentially enhancing LP-F mito- tane bioavailability. Cholesterol supplementation in the medium dose-dependently enhanced mitotane cytotoxic
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effects, confirming the key role of LP-F mitotane in vitro (Figure 4D).
Efficiency of unbound mitotane in vitro
To examine the relative contribution of free and pro- tein-bound mitotane on H295R cell proliferation or ap- optosis, the impact of mitotane was compared in FCS-free medium, devoid of protein and lipoproteins, supple- mented with increasing concentrations of bovine-serum albumin (BSA) and in LP-F media. Consistently, o,p’- DDD was more efficient in inhibiting cell proliferation in protein-free medium while BSA supplementation dose-de- pendently impaired mitotane efficiency (Figure 4E). Moreover, relative caspase activity was 4-fold higher in mitotane treated cells when incubated in the absence of protein than in the control medium (Figure 4F). Alto- gether, our results demonstrate that the most potent ac- tivity in vitro was achieved with free mitotane.
Statin therapy and disease control rate in stage IV-ACC patients
To translate these observations into the clinic, we ret- rospectively collected data from ACC patients followed at our institution. Stage IV-ACC patients under mitotane therapy initiated between September 2007 and January 2014 were included. Twenty-six patients had a mean plasma mitotane level of 16.7 ± 9.2 mg/L (13.2 ± 9.4 mg/L at 1 month, 17.8 ± 12.2 mg/L at 3 month and 18.4 + 8.5 mg/L at 6 month). Among them, 16 (61.5%) experi- enced hypertriglyceridemia, 15 (57.7%) an increase in HDL cholesterol or 8 (30.8%) an increase in LDL choles- terol level under mitotane. Eleven patients (42.3%) were treated with statins (introduced at least 3 months before RECIST evaluation). Neurological toxicity was reported in 8 out of 26 patients (30.8%) at 1, 3 and/or 6 months. According to RECIST criteria, (Response Evaluation Cri- teria in Solid Tumors) disease control rate (DCR) at 6 months was 46.2% (12 out of 26 patients) including 4 patients that experienced a partial response. A significant positive association was found between the use of statin therapy and DCR at 6 months: 67% or 33% DCR was observed in patients treated or not with statins (P < . 05, Fisher’s exact test, Figure 5). No association was found between dyslipidemia and neurological toxicity or tumor control or statins therapy and neurological toxicity.
Discussion
As mitotane remains the most effective treatment of ad- vanced ACC, many efforts are done to better understand its mechanism of action (2). Its lipophilic properties lead to a distribution of o,p’-DDD in lipoproteins (13) and a
storage in adipose tissue (29). Given that mitotane content in lipoprotein fractions has been assumed to play a role in drug distribution in tissues (30), we therefore evaluate the potential role in its antitumor activity using different ma- terials: ACC patients’ plasma samples, human adrenal cortex tissues and human adrenocortical H295R cells.
The partitioning of mitotane and its metabolites in li- poproteins was found to differ strongly. Indeed, whereas o,p’-DDD and o,p’DDE were equally distributed among different fractions (vLDL, LDL, HDL and LP-F subfrac- tions), o,p’-DDA was entirely recovered with protein frac- tions, consistent with its hydrophilic properties. In our 23
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plasma samples originating from 20 ACC patients, we found a correlation between plasma o,p’-DDD levels and its corresponding lipoprotein contents but more impor- tantly between circulating mitotane concentrations and its distribution in LP-F subfraction. These findings raise the question of the relative contribution of mitotane-free vs bound lipoprotein fractions in the pharmacological action of o,p’-DDD (30). To further explore this question, ex- pression of genes encoding for lipoprotein receptors (SrB1 and LDLR) were studied and compared to intra-tissue o,p’-DDD concentrations of mitotane-treated patients’ adrenals. In this small number of tissue samples owing to the low incidence of ACC, no relationship was observed between adrenal cortex o,p’-DDD content and lipoprotein receptor expression, suggesting no predominant impact of mitotane-bound lipoproteins on tissue mitotane uptake.
We then explored the role of mitotane binding to each lipoprotein subfraction or lipoprotein-free mitotane on its cytotoxic effect in human adrenocortical H295R cells. We provided evidence that mitotane exerts a more efficient antiproliferative and proapoptotic action when cells are grown in LP-F medium suggesting that lipoprotein-bound mitotane is not the most potent pharmacological vehicle.
In the present study, we also confirm that mitochon- drion is a critical target of mitotane action since most in- tracellular mitotane was recovered within the mitochon- drial compartment, consistent with our previous results (25). Herein, we further demonstrated that LP-F mitotane was more effectively captured by mitochondria and more efficient in inhibiting the respiratory chain activity. Ex- periments using BLT1, an SrB1 receptor inhibitor, or cho- lesterol saturation, that both favor mitotane action through its LP-F fraction, comfort the prominent phar- macological role of free mitotane by showing an increased efficiency of mitotane when added to the culture medium. These results do not exclude that SrB1 could be involved
*
15
statins
no statin
patients (N)
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0
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in mitotane efflux. Van Slooten first suggested that albu- min-bound mitotane might be responsible for at least the neurological side effects observed in patients (12). We fur- ther evaluated the impact of albumin on o,p’-DDD tox- icity in H295R cells, and unambiguously demonstrated that free mitotane exerts the most efficient pharmacolog- ical properties.
Altogether, our results indicate that free mitotane in- duces the most potent cytotoxic effects, questioning the precise molecular mechanism of its transmembrane trans- port and yet excluding that intracellular transport of li- poproteins might play a major role in the adrenal speci- ficity of mitotane action.
In ACC patients, a variable delay of several weeks be- tween mitotane initiation and antitumorigenic effect is well described in the literature (2). Consistent with our results demonstrating that free mitotane might constitute the active form of the drug, this delay may correspond to the time required to fully saturate circulating lipoproteins in patients’ plasma. To further explore the implication of our in vitro results in humans, and based on clinical and biochemical data collected from a cohort of ACC, mito- tane-treated patients, we examined mitotane plasma lev- els, lipid profile as well as neurological toxicity and clinical responses according to RECIST at 1, 3 and 6 months after mitotane initiation. Among the 26 patients included, 20 (76.9%) had dyslipidemia under mitotane therapy, in- cluding isolated hypertriglyceridemia, hypercholesterol- emia (HDL and/or LDL) or both, in accordance to previ- ous reports (16-19). More interestingly, we found that patients who received statins (42.3%), presented with a better tumor control including stable disease and partial responses. We hypothesize that statins, through a reduc- tion of plasma lipoproteins levels could lead to an in- creased free mitotane ratio. Despite the hypothesis of van Slooten who suggested that albumin-bound mitotane could be responsible for neurological toxicity, we didn’t find any association between LDL, HDL or statins and neurological side effects. Our study has clear limitation including the small sample size, the heterogeneity in pa- tients’ follow-up and plasma collections and the uncon- trolled nature of the design inherent to a the retrospective data collection. These preliminary data should be further confirmed and the question on whether patients could benefit from statins is presently being addressed through an ongoing prospective study (MITOLIPO Study).
In sum, we provide strong evidence that o,p’-DDD un- bound to lipoprotein fractions is more efficient in vitro and that patients could benefit strategies that aim to in- crease LP-F mitotane fraction. The potential role of dys- lipidemia and statin therapy on mitotane effects will be further explored in a prospective study.
Acknowledgments
We thank Rita Chadarevian and Michele Resche-Rigon for the helpful discussion
Address all correspondence and requests for reprints to: Marc Lombès, email: marc.lombes@u-psud.fr; phone: +33149596702; fax: +33149596732.
This work was supported by.
Disclosure summary: S.H. is recipient of a fellowship from HRA Pharma Laboratories (Bourse CIFRE).
The authors declare that they have no conflict of interest.
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