Mitotane Induces CYP3A4 Expression Via Activation of the Steroid and Xenobiotic Receptor

A TAKESHITA and others Mitotane causes drug–drug 216:3 297–305 Research interactions

Mitotane induces CYP3A4 expression via activation of the steroid and xenobiotic receptor

Akira Takeshita, Junko Igarashi-Migitaka1, Noriyuki Koibuchi2 and Yasuhiro Takeuchi

Endocrine Center, Toranomon Hospital and Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato, Tokyo 105-8470, Japan Correspondence 1Department of Anatomy and Cell Biology, St Marianna University School of Medicine, Kawasaki, Kanagawa should be addressed 216-8511, Japan to A Takeshita 2Department of Integrative Physiology, Gunma University Graduate School of Medicine, Maebashi, Email Gunma 371-8511, Japan [email protected]

Abstract

Adrenocortical carcinoma (ACC) is a rare disease with an extremely poor prognosis. Mitotane Key Words alone or in combination with other cytotoxic drugs is a common therapeutic option for ACC. " mitotane In addition to its adrenolytic function, mitotane has been known for decades to increase the " CYP3A4 metabolic clearance of glucocorticoids. It was recently shown that the tyrosine kinase " SXR inhibitor sunitinib is also rapidly metabolized in patients treated with mitotane, indicating " adrenocortical carcinoma that mitotane engages in clinically relevant drug interactions. Although the precise mechanism of these interactions is not well understood, cytochrome P450 mono-oxygenase 3A4 (CYP3A4) is a key enzyme to inactivate both glucocorticoids and sunitinib. The nuclear receptor steroid and xenobiotic receptor (SXR (NR1I2)) is one of the key transcriptional Journal of Endocrinology regulators of CYP3A4 gene expression in the liver and intestine. A variety of xenobiotics bind to SXR and stimulate transcription of xenobiotic-response elements (XREs) located in the CYP3A4 gene promoter. In this study, we evaluated the effects of mitotane on SXR-mediated transcription in vitro by luciferase reporter analysis, SXR–steroid receptor coactivator 1 (SRC1) interactions, quantitative real-time PCR analysis of CYP3A4 expression, SXR knockdown, and CYP3A4 enzyme activity assays using human hepatocyte-derived cells. We found that mitotane activated SXR-mediated transcription of the XREs. Mitotane recruited SRC1 to the ligand-binding domain of SXR. Mitotane increased CYP3A4 mRNA levels, which was attenuated by SXR knockdown. Finally, we showed that mitotane increased CYP3A4 enzyme activity. We conclude that mitotane can induce CYP3A4 gene expression and suggest

that mitotane is used cautiously due to its drug–drug interactions. Journal of Endocrinology (2013) 216, 297–305

Introduction

Adrenocortical carcinoma (ACC) is a rare disease resection has been performed, eventual local recurrence or with an extremely poor prognosis. The annual incidence distant metastases are often recognized (reviewed in is w1–2 per million individuals worldwide. At the time of Allolio & Fassnacht (2006)). diagnosis, w40–70% of the tumors have already under- Mitotane,1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro- gone metastatic spread. Even when an apparently radical phenyl) ethane (o,p0-DDD), is a compound derived from the

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insecticidedichlorodiphenyltrichloroethane (DDT),which SXR agonist that reduces plasma concentrations of specifically destroys the adrenal cortex and is able to block coadministered drugs metabolized by CYP3A4, such as cortisol synthesis by inhibiting 11b-hydroxylation and glucocorticoid derivatives, calcium channel blockers, oral cholesterol side chain cleavage (Hague et al. 1989, Allolio contraceptives, and cyclosporine. In this study, we tested & Fassnacht 2006). In patients with metastatic or pro- whether mitotane induces CYP3A4 gene expression via gressive disease, medical treatment with mitotane is usually SXR activation. begun. In addition to its action on the adrenal cortex, mitotane also affects hepatic microsomal enzyme induction. High-dose glucocorticoid replacement, a daily Materials and methods dose of 50 mg hydrocortisone or greater, is typically Materials required to prevent adrenal insufficiency (Allolio & Fassnacht 2006). The plasma half-life of dexamethasone is RFP was obtained from Sigma–Aldrich. Mitotane was from shortened among patients who are treated with mitotane Wako Pure Chemical Industries, Ltd (Osaka, Japan), and (Robinson et al. 1987). Urinary excretion of 6b-hydro- 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbal- xycortisol, which is an indicator of hepatic microsomal dehyde O-3,4-dichlorobenzyl) oxime (CITCO) was enzyme induction, is increased in patients after mitotane obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, administration (Fukushima et al. 1971). USA). Cryopreserved HepaRG cells and cryopreserved The hepatic cytochrome P450 enzymes (CYPs) human hepatocytes of donor A (batch number catalyze the 6b-hydroxylation of steroid hormones. HEP187186, 58-year-old man) were purchased from CYP mono-oxygenase 3A4 (CYP3A4) is the most abundant Biopredic International (Rennes, France). Cryopreserved CYP expressed in human liver and is involved in the human hepatocytes of donor B (lot LMP, 38-year-old metabolism of approximately one-half of the drugs in female Caucasian) were purchased from In Vitro clinical use today. Recently, van Erp et al. (2011) reported Technologies, Inc. (Baltimore, MD, USA). that mitotane-treated ACC patients exhibited high levels of induced CYP3A4 activity. These authors used orally Plasmids administered midazolam as a phenotypic probe to Human SXR, GAL4 steroid receptor coactivator 1 (SRC1)- determine the activity of CYP3A4. In their pharmacoki- receptor-interacting domain (RID), VP16 SXR-ligand- netic study using time-course measurement of plasma Journal of Endocrinology binding domain (LBD), and VP16 SXR-DAF2 LBD concentrations, mitotane-treated patients showed an expression plasmids were described previously (Takeshita w18-fold reduced exposure to midazolam (AUC ) 0–12 h et al. 2006, 2011). The luciferase (LUC) reporter construct, but an w12-fold increased exposure to its metabolite, 5X upstream activating sequence (UAS)-thymidine 1-hydroxy midazolam. Furthermore, they observed that kinase minimum promoter (TK)-LUC (Cohen et al. the tyrosine kinase inhibitor sunitinib, which is a substrate 2000), and xenobiotic-responsive enhancer module of CYP3A4, was rapidly metabolized in patients treated (XREM)–CYP3A4–LUC (Goodwin et al. 1999) were with mitotane. However, the precise mechanism of described previously. Plasmid phRL-TK, which contains CYP3A4 induction by mitotane is not well understood. the RenillaLUC gene with the TK promoter, was purchased The orphan nuclear receptor (NR), steroid and from Promega. xenobiotic receptor (SXR, also called the pregnane X receptor, NR1I2), is highly expressed in the liver and Transient co-transfection experiments intestine where it regulates genes that control xenobiotic and endogenous steroid hormone metabolism. SXR binds HepG2 cells were grown in DMEM containing 10% FCS. to xenobiotic-response elements (XREs) located in the The serum was stripped of hormones by constant mixing promoters of genes that encode phase I CYP enzymes (e.g. with 10% (w/v) AG1-X8 resin (Bio-Rad) and powdered CYP3A4), phase II conjugating enzymes (e.g. UDP glucur- charcoal before ultrafiltration. Cells were maintained onosyltransferase 1A1 (UGT1A1)), and phase III drug without antibiotics and were transiently transfected transporters (e.g. P-glycoprotein/multidrug resistance using the calcium phosphate coprecipitation method in protein 1 (MDR1)) (Kliewer et al. 2002, Zhang et al. 2008, 24-well plates with 0.5 mg reporter plasmid containing Zhou et al. 2009). Induction of such drug-metabolizing either XREM–CYP3A4–LUC or 5XUAS–TK–LUC cDNA and genes by SXR can cause drug–drug interactions. For the 0.1 mg human SXR expression plasmid. One hundred example, the antibiotic rifampicin (RFP) is a well-known nanograms of phRL-TK were used as an internal control.

In some samples, empty mock vectors were added to TTCCACTACCAAG-30, reverse primer 50-AGAAGCCAGA- equalize the concentration of total transfected plasmid. GAAGAGCTCAAA-30;forUGT1A1:forwardprimer Cells were grown for 24 h in the absence or presence of 50-GAATCAACTGCCTTCACCAAA-30, reverse primer compounds and then harvested. Cell extracts were 50-ACCACAATTCCATGTTCTCCA-30;forSXR:forward analyzed for luciferase activity using the Dual-Luciferase primer 50-ACATGTTCAAAGGCATCATCAG-30, reverse Reporter Assay System (Promega). The firefly luciferase primer 50-ATCTCAGTTGACACAGCTCGAA-30;forCAR activity of the reporter plasmid was corrected by the (NR1I3): forward primer 50-TGATCAGCTGCAAGAG- Renilla luciferase activity of the control plasmid. The GAGA-30, reverse primer 50-AGGCCTAGCAACTTCG- corrected luciferase activities of untreated samples were CATA-30; and for glyceraldehyde 3-phosphate normalized to the luciferase activities of samples as dehydrogenase (GAPDH): forward primer 50-GGCCTCC- described in the figure legends. All transfection studies AAGGAGTAAGACC-30, reverse primer 50-AGGGGAGATT- were repeated in triplicate. The results shown are the CAGTGTGGTG-30. Results were normalized with GAPDH KDDCt meanGS.D.(nZ3). using the 2 method and expressed relative to the untreated control. Representative results are expressed Cell culture as the meanGS.D.(nZ3 wells per treatment) of two independent experiments. Differentiated HepaRG cells were seeded into collagen I-coated 96-well plates using HepaRG Thawing and Seeding Medium 670 (Biopredic International), according RNAi to the manufacturer’s instructions. After 3 days, the HepaRG cells or human hepatocytes from donor A were culture medium was changed to HepaRG Induction cultured in collagen I-coated 96-well plates. The cells were Medium 640 (Biopredic International) containing either transfected with 5 nM Silencer Select predesigned siRNA mitotane or RFP dissolved in DMSO (final concentration (Ambion, Austin, TX, USA) against human SXR (s16909) 0.1% (v/v)). The control group received 0.1% DMSO only. or human CAR (s19369)) or with the negative control Human hepatocytes from donor A were seeded into siRNA (control No. 1). Introduction of siRNA was carried collagen I-coated 96-well plates using Hepatocyte Seeding out using Lipofectamine RNAiMAX (Invitrogen) and the Medium (Biopredic International), according to the reverse transfection method according to the manufac- manufacturer’s instructions. After 24 h, the medium was turer’s instructions. After 24 h, cells were treated with Journal of Endocrinology changed to Hepatocyte Culture Medium (Biopredic the test compounds. After 48 h, CYP3A4, CYP2B6, and International) containing mitotane, RFP, or CITCO UGT1A1 expressions were determined by qRT-PCR. dissolved in 0.1% DMSO. Human hepatocytes from donor B were cultured in collagen-coated 12-well plates withLanfordmedium. After a24-hincubation, the cells were CYP3A4 activity treatedwitheither mitotaneorRFP dissolved in0.1%DMSO. To determine the effects of test compounds on CYP3A4 activity in HepaRG cells, we used the P450-Glo CYP3A4 Quantitative real-time PCR (qRT-PCR) Assay with Luciferin-IPA (Promega) according to the HepaRG cells and human hepatocytes from donor A were manufacturer’s instructions. Briefly, HepaRG cells were analyzed with Power SYBR Green Cells-to-Ct (Applied grown in HepaRG Induction Medium 640 containing Biosystems) using a PE-Applied Biosystems Prism 7700 mitotane or RFP dissolved in 0.1% DMSO. Cell culture instrument. For human hepatocytes from donor B, total medium without cells served as the background control. RNA was prepared using the TRIzol reagent (Invitrogen) After a 48-h treatment, cells were incubated with according to the manufacturer’s protocol. RNA was reverse 3 mM Luciferin-IPA in HepaRG Induction Medium 640 transcribed using random hexamers and TaqMan reagents for an additional 60 min. An aliquot of the medium was (Applied Biosystems). With the resulting cDNA as a then combined with an equal volume of the luciferin template, qRT-PCR was performed using Power SYBR detection reagent in a white luminometer plate, and the Green PCR Master Mix (Applied Biosystems) and gene- luminescence was read. The remaining cells were assessed specific primers. The following primers were used for using the Cell Titer-Glo Luminescent Cell Viability Assay CYP3A4: forward primer 50-CATTCCTCATCCCAATTCTT- (Promega) to estimate the number of cells in each well, GAAGT-30, reverse primer 50-CCACTCGGTGCTTTTGTG- and CYP3A4 activity was normalized to cell number. 0 0 TATCT-3 ; for CYP2B6: forward primer 5 -GGAAAACGA Results are expressed as the meanGS.D.(nZ3).

Statistical analysis 70 Groups were compared by the Mann–Whitney U test; Control P!0.05 was considered signiﬁcant in all cases. Statistical 60 RFP 1, 3, 10, 30 µM analysis was carried out with StatView 5.0 software for Mitotane 1, 3, 10, 30 µM Macintosh (SAS Institute, Cary, NC, USA). 50

40 Results 30 To determine whether mitotane stimulates SXR-mediated transcription of the CYP3A4 gene, transient transfection 20

assays were performed with a reporter plasmid, XREM– activity luciferase Relative CYP3A4–LUC, containing the enhancer (nucleotides K 10 7836 to K7208) and promoter (nucleotides K362 to C53) regions of CYP3A4 to drive luciferase gene expression 0 (Goodwin et al. 1999) in HepG2 cells. A previous study identified four putative SXR-response elements within the Mock plasmid hSXR plasmid XREM; three of the four elements cooperatively promote RFP induction by SXR (Goodwin et al. 1999). As shown in Figure 1 Fig. 1, mitotane, as well as RFP, increased transcription of Mitotane activates SXR-mediated transcription of the CYP3A4 promoter in HepG2 cells. HepG2 cells were co-transfected with the reporter plasmid, the CYP3A4 gene in a dose-dependent manner, although 0.5 mg XREM–CYP3A4–LUC, 0.1 mg human SXR (hSXR) expression plasmid the magnitude of the activation by mitotane was less than (right), or empty mock plasmid (left), and the phRL-TK control vector that of RFP. Co-transfection of the human SXR plasmid (0.1 mg). Cells were treated with different doses of RFP or mitotane for 24 h and analyzed for luciferase activity. The firefly luciferase activity from the further enhanced transcriptional activation, consistent reporter plasmid was normalized to Renilla luciferase activity from the with previous reports of the relative low expression of control plasmid with onefold basal activity defined as luciferase activity in endogenous SXR in this cell line (Zucchini et al. 2005, the absence of ligand and exogenous SXR. The results are expressed as meanGS.D.(nZ3). Naspinski et al. 2008). This reporter assay suggests that mitotane binds to SXR as a ligand and then stimulates Journal of Endocrinology CYP3A4 transcription. ligand-bound receptors interact with coactivators (Smith Transcriptional activation by NRs is mediated by & O’Malley 2004). When the C-terminal truncated AF2 ligand-dependent interactions with coactivators, includ- mutant VP16 SXR-DAF2 was used, neither RFP nor ing p160 of the NR coactivator family (SRC1, also called mitotane increased luciferase activity, suggesting NCOA1; TIF2, also called SRC2, GRIP1, or NCOA2; and that mitotane-bound SXR recruits coactivators in an TRAM1, also called SRC3, p/CIP, AIB1, ACTR, RAC3, or AF2-dependent manner. NCOA3) (Smith & O’Malley 2004). To examine the ligand- To determine whether mitotane induces CYP3A4 induced interactions between SXR and the p160 coactiva- expression in the liver, HepaRG cells (Fig. 3A, B, and C; tors, we employed one representative of p160, SRC1, in Kanebratt & Andersson 2008) and cryopreserved human mammalian two-hybrid assays. The RID of SRC1 was fused hepatocytes of two donors (donor A: Fig. 3D, E, and F; to the DNA-binding domain of GAL4 (GAL4 SRC1-RID), donor B: G, H, and I) were treated with either RFP or and the LBD of human SXR was fused to the transactiva- mitotane for 24 or 48 h. CYP3A4 mRNA levels were then tion domain of VP16 (VP16 SXR-LBD). These constructs determined by qRT-PCR. As shown in Fig. 3A, D, and G, were co-transfected with a reporter plasmid containing both mitotane and RFP increased CYP3A4 mRNA levels in five copies of a GAL4 UAS in HepG2 cells (Fig. 2). GAL4 these human hepatocyte-derived cells. Although mitotane SRC1-RID interacted with VP16 SXR-LBD in the presence had a smaller effect than RFP, apparent CYP3A4 induction of mitotane as well as RFP in a dose-dependent manner. by mitotane was observed at concentrations exceeding The C-terminal region of the LBD (AF-2) is conserved 10 mM. In addition, both RFP and mitotane increased among NRs, and deletion or point mutations in this region mRNA levels of CYP2B6 (Fig. 3B, E, and H) and UGT1A1 impair transcriptional activation without changing either (Fig. 3C, F, and I). Interestingly, the effect of mitotane on ligand- or DNA-binding affinities. Ligands of NRs induce CYP2B6 mRNA levels was higher than that of RFP in major conformational changes in the AF-2 region so that HepaRG cells (Fig. 3B) and donor A hepatocytes (Fig. 3E).

shares a number of the same targets. CAR also has distinct target genes and many chemical activators (Gao & Xie 35 Control 2010, Tolson & Wang 2010). As shown in Fig. 4D, E, and F, µ 30 RFP 1, 10 M donor A hepatocytes were transfected with siRNAs against 1, 10 µM Mitotane CAR and SXR. In this experiment, 1 mM CITCO was used 25 as a positive control ligand for human CAR (Maglich et al. 2003). Transfection of gene-speciﬁc siRNAs reduced SXR 20 mRNA levels by 73% and CAR mRNA levels by 74% (data

15 not shown). Similar to results observed in HepaRG cells, SXR knockdown in donor A hepatocytes markedly 10 reduced mitotane-induced expression of CYP3A4 (Fig. 4D)

Relative luciferase activity luciferase Relative but not CYP2B6 (Fig. 4E), whereas RFP-induced expression 5 of both genes was suppressed. In agreement with the previous reports, CITCO demonstrated preferential 0 induction of CYP2B6 (Fig. 4E, control siRNA) over CYP3A4 (Fig. 4D, control siRNA). As expected, CAR GAL4 SRC1 SRC1 SRC1 + + + siRNA but not SXR siRNA abrogated CYP2B6 induction VP16 Mock SXR-LBD SXR∆AF2-LBD by CITCO (Fig. 4E). Similarly, CAR knockdown significantly reduced mitotane-induced CYP2B6 expression Figure 2 (from 5.3- to 1.6-fold) (Fig. 4E), indicating that CAR is a Mitotane recruits SRC1 to the LBD of SXR in HepG2 cells. The expression key regulator of mitotane-induced CYP2B6 expression. plasmids encoding GAL4 SRC1-RID (0.1 mg) and VP16 mock (left), VP16 SXR- LBD (center), or VP16 SXRDAF2-LBD (right) (0.5 mg) were co-transfected Both SXR and CAR knockdown reduced mitotane-induced with the 5X UAS–TK–LUC reporter plasmid (0.5 mg) and the phRL-TK control UGT1A1 expression (Fig. 4F). Taken together, our results vector (0.1 mg) in HepG2 cells. Cells were treated with 1 or 10 mMRFPor suggest that mitotane induction of CYP3A4 is primarily mitotane for 24 h. The corrected luciferase activity was calculated as fold luciferase activity with onefold basal activity defined as luciferase activity mediated by SXR, mitotane induction of CYP2B6 is of GAL4 SRC1-RID and VP16 mock plasmids in the absence of ligand. primarily mediated by CAR, and mitotane induction of G Z The results are expressed as mean S.D.(n 3). UGT1A1 is mediated by both SXR and CAR. To determine the effects of mitotane on CYP3A4 Journal of Endocrinology To confirm that mitotane induction of CYP3A4 in enzymatic activity in HepaRG cells, we used an assay kit human hepatocyte-derived cells requires SXR, we evalu- containing Luciferin-IPA. This substrate is metabolized ated the effects of SXR knockdown on mitotane-induced specifically by CYP3A4 to release luciferin, which can be gene expression in HepaRG cells (Fig. 4A). After a 24-h quantified by luminescence through its reaction with ATP- transfection with control or SXR-specific siRNA, the cells luciferase. HepaRG cells were treated for 48 h with were treated with 10 mM RFP, 10 mM mitotane, or DMSO different concentrations of RFP or mitotane. Mitotane (vehicle) for 48 h, and gene expression was analyzed by increased CYP3A4 activity in a dose-dependent manner, qRT-PCR. Transfection of gene-specific siRNA reduced SXR although the magnitude of mitotane-induced CYP3A4 mRNA levels by 67% (data not shown). CYP3A4 mRNA activity was lower than that induced by RFP (Fig. 5). levels increased in cells transfected with control siRNA after treatment with RFP or mitotane. However, SXR Discussion knockdown suppressed CYP3A4 induction by RFP (from 105.1- to 9.8-fold) and mitotane (from 30.5- to 4.2-fold), In patients with ACC in whom surgical cure is not suggesting that SXR is a key regulator of mitotane-induced possible, mitotane, either as a single agent or in CYP3A4 expression. By contrast, knockdown of SXR only combination with other traditional cytotoxic chemo- partially suppressed mitotane induction of CYP2B6 therapies, is generally administered. Adverse effects of (Fig. 4B) and UGT1A1 (Fig. 4C), whereas RFP induction mitotane, such as gastrointestinal distress, neurological of these genes was markedly reduced. This finding suggests disturbances, hepatotoxicity, and adrenal insufficiency, that mitotane induction of CYP2B6 and UGT1A1 in arewellknown.Althoughithasbeenknownfor HepaRG cells can be mediated by a pathway other than decades that mitotane increases the metabolic clearance SXR. The orphan NR constitutive androstane receptor of glucocorticoids, drug–drug interactions had not (CAR; also called NR1I3) is a sister receptor of SXR and been clearly documented until the recent report by

A B C HepaRG 120 16 6 100 DMSO 14 5 mRNA mRNA RFP mRNA 12 80 Mitotane 10 4 60 8 3 40 6 2 4 20 2 1 0 0 0 CYP3A4/GAPDH CYP2B6/GAPDH UGT1A1/GAPDH µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM 1 1 1 1 1 1 Control0.1 10 20 0.1 10 20 30 40 Control0.1 10 20 0.1 10 20 30 40 Control0.1 10 20 0.1 10 20 30 40

D E F

30 Donor A 10 DMSO 15 RFP 25 8 mRNA mRNA mRNA Mitotane 20 6 10 15 4 10 5 5 2 0 0 0 CYP3A4/GAPDH CYP2B6/GAPDH UGT1A1/GAPDH µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM µM 1 1 1 1 1 1 Control0.1 10 20 0.1 10 20 30 40 Control0.1 10 20 0.1 10 20 30 40 Control0.1 10 20 0.1 10 20 30 40

G H I Donor B 20 6 5 * * DMSO * mRNA mRNA RFP mRNA 5 15 4 Mitotane 4 3 10 3 * 2 * * 2 5 1 1 0 0 0 CYP3A4/GAPDH CYP2B6/GAPDH UGT1A1/GAPDH µM µM µM µM µM µM

Journal of Endocrinology Control 10 20 Control 10 20 Control 10 20

Figure 3 Mitotane induces drug metabolism genes in human liver. HepaRG cells or 0.1% DMSO (vehicle control) for 24 h. Expression levels of CYP3A4 (A, B, and C) and human hepatocytes from donor A (D, E, and F) were (A, D, and G), CYP2B6 (B, E, and H), and UGT1A1 (C, F, and I) were analyzed treated with RFP (0.1, 1, 10, or 20 mM), mitotane (0.1, 1, 10, 20, 30, or by qRT-PCR, normalized to GAPDH, and expressed relative to the DMSO 40 mM), or 0.1% DMSO (vehicle control) for 48 h. (G, H, and I) Human control. Results are expressed as meanGS.D.(nZ3); *P!0.05 vs control. hepatocytes from donor B were treated with 10 mM RFP, 20 mM mitotane,

van Erp et al. (2011). Our results indicate that mitotane a potent activator of human SXR as well (Medina-Diaz & can activate SXR and possibly contribute to the increase Elizondo 2005, Medina-Diaz et al. 2007). Thus, mitotane- in CYP3A4. bound SXR recruits p160 coactivators to induce CYP3A4 A wide variety of xenobiotics can bind to SXR as gene expression. ligands and stimulate SXR-mediated transcription. X-ray In clinical usage, it is recommended that mitotane crystallographic studies of the LBD of human SXR revealed blood levels be maintained between 14 and 20 mg/ml that SXR contains a relatively large ligand-binding pocket (i.e. 43.7–62.5 mM) to achieve tumor regression and avoid compared with other NRs (Watkins et al. 2001). In adverse effects. Mitotane, at all measured endpoint levels, addition, the crystal structure of SXR with one of its significantly increased CYP3A4 activity. Although our ligands, SR12813, revealed that a single drug molecule in vitro assays may not appropriately reflect the in vivo could be bound in the LBD in three distinct orientations situations of patients treated with mitotane, the concen- (Watkins et al. 2001). The flexibility of the LBD likely tration of mitotane used in our in vitro studies is certainly enables SXR to recognize a wide range of xenobiotics, achievable pharmacologically in vivo. including mitotane. It is noteworthy that DDT, from CYP3A4 is not only a liver enzyme induced by which mitotane is derived, has been reported to be mitotane. A case report previously described the

A B HepaRG C 120 5 DMSO 20 100 RFP 10 µM Mitotane 10 µM 4 mRNA mRNA 80 15 mRNA 3 60 10 2 40 5 20 1

CYP3A4/GAPDH CYP2B6/GAPDH UGT1A1/GAPDH 0 0 0 Control SXR Control SXR Control SXR siRNA siRNA siRNA siRNA siRNA siRNA

D E Donor A F 10 20 5 DMSO RFP 10 µM 8 4 Mitotane 10 µM 15 mRNA mRNA mRNA CITCO 1 µM 6 3 10 4 2 5 2 1

CYP3A4/GAPDH CYP2B6/GAPDH UGT1A1/GAPDH 0 0 0 Control SXR CAR Control SXR CAR Control SXR CAR siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA CYP3A4 CYP2B6 UGT1A1

Figure 4 Journal of Endocrinology Mitotane induces CYP3A4 expression in human liver cells by activating SXR. with 10 mM RFP, 10 mM mitotane, 1 mM CITCO, or 0.1% DMSO (vehicle HepaRG cells (A, B, and C) were transfected with control siRNA or SXR control) for 48 h. Expression levels of CYP3A4 (A and D), CYP2B6 (B and E), siRNA and treated with 10 mM RFP, 10 mM mitotane, or 0.1% DMSO (vehicle and UGT1A1 (C and F) were analyzed by qRT-PCR, normalized to GAPDH, control) for 48 h. Human hepatocytes from donor A (D, E, and F) were and expressed relative to the DMSO control. Results are expressed as transfected with control siRNA, SXR siRNA, or CAR siRNA and then treated meanGS.D.(nZ3).

hypoprothrombinemic effects of warfarin, a substrate hormones, and glycolipids) into water-soluble, excretable of CYP2C9, during mitotane treatment (Cuddy & Loftus metabolites (Tolson & Wang 2010). Therefore, CYP2B6 1986). In this study, we showed that mitotane induces and UGT1A1 possess important functions in human CYP2B6 and UGT1A1 as well as CYP3A4 in human liver- drug metabolism. derived cells. CYP2B6 contributes 2–10% of total hepatic Results of our knockdown experiments suggest that CYP content and 3–12% of drug metabolism capacity CAR participates in mitotane-induced CYP2B6 and (Wang & Tompkins 2008). Known substrates of CYP2B6 UGT1A1 activation, whereas mitotane-induced CYP3A4 include the anticancer drugs cyclophosphamide and activation appears to be largely explained by SXR. CAR ifosfamide, antiretroviral drugs nevirapine and efavirenz, and SXR regulate an overlapping set of xenobiotic- anesthetic drugs propofol and ketamine, the synthetic metabolizing genes, including genes that encode several opioid methadone, and the anti-Parkinson drug selegiline CYP enzymes (i.e. CYP3A4, CYP2B6, CYP2Cs, and (Wang & Tompkins 2008). UGT1A1, one of the most CYP2A6), UGTs (i.e. UGT1A1, UGT1A6, and UGT1A9), extensively characterized UGT isoforms, is an enzyme of and the drug transporter MDR1 (Tolson & Wang 2010). the glucuronidation pathway, which transforms a diverse By contrast to most NRs, CAR displays high constitutive range of lipophilic xenobiotics and endogenous activity in the absence of agonist binding but can also be compounds (e.g. drugs, environmental chemicals, carci- directly activated by ligands (e.g. CITCO) or indirectly nogens, vitamins, bilirubin, steroid hormones, thyroid activated (e.g. by phenobarbital). In liver cells, CAR is

advanced and metastatic ACC treatment (the FIRM-ACT 20 study) showed that the combination regimen of mitotane DMSO and etoposide, doxorubicin, and platinum (EDP) had RFP a higher response rate than that of the combination of Mitotane 15 mitotane and streptozocin, even though both etoposide and doxorubicin are substrates of CYP3A4 (Fassnacht et al. 2012). Additionally, no one has shown that EDP 10 toxicity is decreased when used in combination with mitotane. Therefore, therapeutic drug monitoring is needed to clarify this issue, which may in turn improve

Fold induction (CYP3A4) Fold 5 chemotherapy outcomes for patients with ACC (Kroiss et al. 2011). In conclusion, our in vitro study shows that mitotane 0 induces CYP3A4 gene expression via the activation of µM µM µM µM µM µM µM µM µM µM µM 1 3 1 3 SXR, explaining the many clinical observations of the Control 0.3 10 20 0.3 10 20 40 drug–drug interactions caused by mitotane. Careful therapeutic drug monitoring may therefore lead to Figure 5 Mitotane induces CYP3A4 enzymatic activity in HepaRG cells. HepaRG cells improved outcomes and prognoses for patients with ACC. were treated with different doses of RFP, mitotane, or 0.1% DMSO (vehicle control). After 48 h, the medium was replaced with medium containing 4 mM Luciferin-IPA, and cells were incubated for an additional 60 min. An aliquot of the medium was combined with an equal volume of P450-Glo Declaration of interest Luciferin Detection Reagent, and luminescence was read on a Lumat The authors declare that there are no conflicts of interest that could be LB9507 luminometer. CYP3A4 activity was normalized to the number of perceived as prejudicing the impartiality of the research reported. cells and expressed relative to the DMSO control. Results are presented as the meanGS.D.(nZ3). Funding This work was supported by Grants-in-Aid for Scientific Research (C: grant found predominantly in the cytoplasm in the absence of number 22591020 to A T and B: grant number 80234681 to N K) from the CAR activators but translocates into the nucleus after Japan Society for the Promotion of Science and the Okinaka Memorial Journal of Endocrinology exposure to phenobarbital-type compounds (Tolson & Institute for Medical Research to A T. Wang 2010). Therefore, it will be interesting to know whether mitotane activates CAR by direct ligand binding or nuclear translocation. Further study is necessary to Acknowledgements The authors thank Dr Paul M Yen (Duke-NUS Graduate Medical School, address this issue. Singapore) for helpful discussion. This study suggests that the concomitant administration of mitotane with anticancer drugs metabolized by CYP3A4 and CYP2B6 may result in subtherapeutic plasma References concentrations of these drugs due to the clinically relevant Allolio B & Fassnacht M 2006 Clinical review: adrenocortical carcinoma: acceleration of their clearance. In fact, a pharmacokinetic clinical update. Journal of Clinical Endocrinology and Metabolism 91 study of sunitinib, a substrate of CYP3A4, in mitotane- 2027–2037. (doi:10.1210/jc.2005-2639) treated patients showed its rapid clearance (van Erp et al. Cohen RN, Putney A, Wondisford FE & Hollenberg AN 2000 The nuclear 2011). As discussed in a recent review of mitotane drug corepressors recognize distinct nuclear receptor complexes. Molecular Endocrinology 14 900–914. (doi:10.1210/me.14.6.900) interactions (Kroiss et al. 2011), clinical trials of several Cuddy PG & Loftus LS 1986 Influence of mitotane on the hypopro- small-molecule antineoplastic agents, including two thrombinemic effect of warfarin. Southern Medical Journal 79 387–388. epidermal growth factor inhibitors (gefitinib and erloti- (doi:10.1097/00007611-198603000-00037) van Erp NP, Guchelaar HJ, Ploeger BA, Romijn JA, Hartigh J & nib) and the tyrosine kinase inhibitor imatinib (all three Gelderblom H 2011 Mitotane has a strong and a durable are substrates of CYP3A4), resulted in either no or only inducing effect on CYP3A4 activity. European Journal of Endocrinology minor responses to metastases. Importantly, the majority 164 621–626. (doi:10.1530/EJE-10-0956) of these patients were treated either previously or Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, Jarzab B et al. 2012 Combination concomitantly with mitotane. On the other hand, recent chemotherapy in advanced adrenocortical carcinoma. New England results of the first international randomized trial of locally Journal of Medicine 366 2189–2197. (doi:10.1056/NEJMoa1200966)

Fukushima DK, Bradlow HL & Hellman L 1971 Effects of o,p0-DDD on Naspinski C, Gu X, Zhou GD, Mertens-Talcott SU, Donnelly KC & Tian Y cortisol and 6-beta-hydroxycortisol secretion and metabolism in man. 2008 Pregnane X receptor protects HepG2 cells from BaP-induced DNA Journal of Clinical Endocrinology and Metabolism 32 192–200. damage. Toxicological Sciences 104 67–73. (doi:10.1093/toxsci/kfn058) (doi:10.1210/jcem-32-2-192) Robinson BG, Hales IB, Henniker AJ, Ho K, Luttrell BM, Smee IR & Stiel JN Gao J & Xie W 2010 Pregnane X receptor and constitutive androstane 1987 The effect of o,p0-DDD on adrenal steroid replacement therapy receptor at the crossroads of drug metabolism and energy metabolism. requirements. Clinical Endocrinology 27 437–444. (doi:10.1111/j.1365- Drug Metabolism and Disposition 38 2091–2095. (doi:10.1124/dmd.110. 2265.1987.tb01171.x) 035568) Smith CL & O’Malley BW 2004 Coregulator function: a key to under- Goodwin B, Hodgson E & Liddle C 1999 The orphan human pregnane standing tissue specificity of selective receptor modulators. Endocrine X receptor mediates the transcriptional activation of CYP3A4 by Reviews 25 45–71. (doi:10.1210/er.2003-0023) rifampicin through a distal enhancer module. Molecular Pharmacology Takeshita A, Inagaki K, Igarashi-Migitaka J, Ozawa Y & Koibuchi N 2006 56 1329–1339. The endocrine disrupting chemical, diethylhexyl phthalate, activates Hague RV, May W & Cullen DR 1989 Hepatic microsomal enzyme MDR1 gene expression in human colon cancer LS174T cells. Journal induction and adrenal crisis due to o,p’DDD therapy for meta- of Endocrinology 190 897–902. (doi:10.1677/joe.1.06664) static adrenocortical carcinoma. Clinical Endocrinology 31 51–57. Takeshita A, Igarashi-Migitaka J, Nishiyama K, Takahashi H, Takeuchi Y & (doi:10.1111/j.1365-2265.1989.tb00453.x) Koibuchi N 2011 Acetyl tributyl citrate, the most widely used phthalate Kanebratt KP & Andersson TB 2008 HepaRG cells as an in vitro model for substitute plasticizer, induces cytochrome p450 3a through steroid and evaluation of cytochrome P450 induction in humans. Drug Metabolism xenobiotic receptor. Toxicological Sciences 123 460–470. (doi:10.1093/ and Disposition 36 137–145. (doi:10.1124/dmd.107.017418) toxsci/kfr178) Kliewer SA, Goodwin B & Willson TM 2002 The nuclear pregnane Tolson AH & Wang H 2010 Regulation of drug-metabolizing enzymes X receptor: a key regulator of xenobiotic metabolism. Endocrine Reviews by xenobiotic receptors: PXR and CAR. Advanced Drug Delivery Reviews 23 687–702. (doi:10.1210/er.2001-0038) 62 1238–1249. (doi:10.1016/j.addr.2010.08.006) Kroiss M, Quinkler M, Lutz WK, Allolio B & Fassnacht M 2011 Drug Wang H & Tompkins LM 2008 CYP2B6: new insights into a historically interactions with mitotane by induction of CYP3A4 metabolism overlooked cytochrome P450 isozyme. Current Drug Metabolism 9 in the clinical management of adrenocortical carcinoma. Clinical 598–610. (doi:10.2174/138920008785821710) Endocrinology 75 585–591. (doi:10.1111/j.1365-2265.2011.04214.x) Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, Williams SP, Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, Willson TM, Kliewer SA & Redinbo MR 2001 The human nuclear Stoltz CA, Kliewer SA, Lambert MH, Willson TM et al. 2003 xenobiotic receptor PXR: structural determinants of directed prom- Identification of a novel human constitutive androstane receptor iscuity. Science 292 2329–2333. (doi:10.1126/science.1060762) (CAR) agonist and its use in the identification of CAR target genes. Zhang B, Xie W & Krasowski MD 2008 PXR: a xenobiotic receptor of diverse Journal of Biological Chemistry 278 17277–17283. (doi:10.1074/jbc. function implicated in pharmacogenetics. Pharmacogenomics 9 M300138200) 1695–1709. (doi:10.2217/14622416.9.11.1695) Medina-Diaz IM & Elizondo G 2005 Transcriptional induction of CYP3A4 Zhou C, Verma S & Blumberg B 2009 The steroid and xenobiotic receptor by o,p0-DDT in HepG2 cells. Toxicology Letters 157 41–47. (doi:10.1016/ (SXR), beyond xenobiotic metabolism. Nuclear Receptor Signaling 7 e001. j.toxlet.2005.01.003) (doi:10.1621/nrs.07001) Medina-Diaz IM, Arteaga-Illan G, de Leon MB, Cisneros B, Sierra-Santoyo A, Zucchini N, de Sousa G, Bailly-Maitre B, Gugenheim J, Bars R, Lemaire G & Vega L, Gonzalez FJ & Elizondo G 2007 Pregnane X receptor-dependent Rahmani R 2005 Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein Journal of Endocrinology induction of the CYP3A4 gene by o,p0-1,1,1,-trichloro-2,2-bis expression by nuclear receptor PXR in primary cultures of human and (p-chlorophenyl)ethane. Drug Metabolism and Disposition 35 95–102. rat hepatocytes. Biochimica et Biophysica Acta 1745 48–58. (doi:10.1016/ (doi:10.1124/dmd.106.011759) j.bbamcr.2005.02.005)

Received in ﬁnal form 31 October 2012 Accepted 22 November 2012 Accepted Preprint published online 22 November 2012