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Oleanolic Acid-Mediated Inhibition of Pregnane X Receptor and Constitutive Androstane Receptor Attenuates Rifampin-Isoniazid Cytotoxicity Yen-Ning Lin,†,# Chao-Jung Chen,‡,¶,# Hsiao-Yun Chang,⊥,# Wai-Kok Cheng,† Ying-Ray Lee,§ Jih-Jung Chen,∥ and Yun-Ping Lim*,†,○,× †

Department of Pharmacy, College of Pharmacy, China Medical University, Taichung 40402, Taiwan Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan ¶ School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan ⊥ Department of Biotechnology, Asia University, Taichung 41354, Taiwan § Translational Medicine Research Center, Chia-Yi Christian Hospital, Chiayi 60002, Taiwan ∥ Faculty of Pharmacy, School of Pharmaceutical Sciences, National Yang Ming University, Taipei, Taiwan ○ Department of Internal Medicine, China Medical University Hospital, Taichung 40402, Taiwan × Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan ‡

S Supporting Information *

ABSTRACT: Interactions between transcriptional inducers of cytochrome P450 (CYP450) and pharmacological agents might decrease drug efficacy and induce side effects. Such interactions could be prevented using an antagonist of the pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Here, we aimed to determine the antagonistic effect of oleanolic acid (OA) on PXR and CAR. OA attenuated the promoter activities, expressions, and enzyme catalytic activities of CYP3A4 and CYP2B6 mediated by rifampin (RIF) and CITCO. Moreover, OA displayed species specificity for rodent PXR. Interaction of coregulators with PXR and transcriptional complexes on the CYP3A4 promoter was disrupted by OA. Additionally, OA reversed the cytotoxic effects of isoniazid induced by RIF. These data demonstrate that OA inhibited the transactivation of PXR and CAR, reduced the expression and function of CYP3A4 and CYP2B6, and may therefore serve as an effective agent for reducing probability adverse interactions between transcriptional inducers of CYP450 and therapeutic drugs. KEYWORDS: oleanolic acid, cytochrome P450, pregnane X receptor, constitutive androstane receptor, rifampin, isoniazid

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INTRODUCTION Cytochrome P450 (CYP450) enzymes are a group of hemethiolate isoenzymes that are expressed at high levels in the liver and are responsible for the metabolism of many endogenous compounds, xenobiotics, and environmental pollutants.1,2 Among these, CYP3A4 and CYP2B6 are involved in the metabolism of approximately 50% and 15% of all prescribed medications, respectively.1 As a consequence, coadministration of CYP inducers or inhibitors could influence drug metabolism, potentially altering drug efficacy or causing toxicity.2 Therefore, the potential for drug−drug interactions and drug-induced adverse reactions associated with alterations in CYP450 expression should be investigated. Transcriptional expression of CYP450s is largely under the control of the nuclear receptor (NR) superfamily. The pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3) are two ligand-activated transcription factors that primarily control the expression of CYP3A4 and CYP2B6, respectively.2 Upon activation by specific ligands, PXR and CAR heterodimerize with 9-cis retinoic acid receptor α (RXRα, NR2B1), translocate from the cytosol to the nucleus, and then bind to specific xenobiotic response elements of CYP3A4 and CYP2B6 promoter regions.3,4 Among other © XXXX American Chemical Society

NRs, CYP3A4 is also reported to be under the control of glucocorticoid receptor (GR, NR3C1) and vitamin D receptor (VDR, NR1I1).1 Activation of PXR and CAR results in increased expression of genes, including the CYP3A4, CYP2C8/ 9/19, and CYP2B6 subfamilies, involved in the metabolism in the liver and intestine.3 Apart from phase 1 drug metabolizing enzymes (DMEs), PXR is also involved in the control of phase 2 and 3 DMEs.4,5 Thus, activation of PXR and CAR could lead to the alterations of xenobiotics biotransformation and disposition through transcriptional control, and these factors could represent the molecular foundation for drug−drug interactions (DDIs) in clinical settings. PXR ligands are highly diverse, including xenobiotics [such as rifampin (RIF) and clotrimazole],6 endogenous substances (bile acids),3 and environment-derived toxicants.7 Although PXR may improve the metabolism, transportation, and elimination of potential compounds, it has been reported that certain types of drug-induced cytotoxicity are associated with Received: June 10, 2017 Revised: September 6, 2017 Accepted: September 8, 2017

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Assessment of Cell Cytotoxicity. Cell viability was measured using an acid-phosphatase (ACP) assay with p-nitrophenyl phosphate (PNPP) disodium salt as a substrate. In brief, after removal of the culture medium, treated cells were washed with 1× phosphate-buffered saline (PBS). Cells were then treated with 100 μL of freshly prepared ACP reagent (contained 10 mM PNPP, dissolved in 0.1 M sodium acetate [pH 5.5], and 0.1% Triton X-100) for 1 h at 37 °C. Reaction activity was terminated via the addition of 1 N NaOH (10 μL), and the absorbance of each well was measured photometrically at 405 nm. Plasmid Constructs. The expression plasmids for human PXR (pcDNA3-PXR), rat PXR (pcDNA3-rPXR), mouse CAR (pCMXmCAR), human HNF4α (pcDNA3-HNF4α), human SRC-1 (pCR3.1SRC-1), CYP3A4 reporter plasmid (pGL3B-CYP3A4), and mouse cyp2b10 reporter plasmid (pGL3-cyp2b10) were constructed as described previously.15 The human CAR expression construct (pCR3-CAR3) and CYP2B6 reporter plasmid (pGL3-CYP2B6-2.2 kb) were constructed as previously described16 and were kindly provided by Dr. Hongbing Wang (Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD). Full-length human RXRα (pGEM-3Z-RXRα) and PGC-1α (pTARGET-PGC-1α) expression plasmids were previously constructed17 and were kindly provided by Dr. Tsuyoshi Yokoi (Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakumamachi, Kanazawa, Japan). The transfection control plasmid pRCCMV-β-galactosidase was obtained from Invitrogen (Waltham, MA). Transient Transfection and Reporter Assays. HepG2 cells (1.8 × 104 cells/well) were seeded in 96-well plates (Greiner Bio-One International GmbH, Kremsmunster, Austria) overnight before transfection. Transient transfection was then performed using the PolyJET transfection reagent (SignaGen Laboratories, Rockville, MD), according to the manufacturer’s instructions. Reporter constructs (0.15 μg), each of the expression plasmids (0.04 μg), and 0.02 μg of βgalactosidase plasmid were cotransfected simultaneously in each well. Transfected cells were subjected to drug treatment after 6 h and incubated for an additional 20 h. After incubation, the cell culture media were aspirated, and cells were washed with 1× PBS and lysed with 80 μL of cell culture lysis reagent (Promega Corporation, Fitchburg, WI). Thirty microliters of cleared supernatant was used to determine the activity of β-galactosidase, and the residual aliquot of the lysate was subjected to luciferase assay analysis using 80 μL of luciferase assay reagent (Promega). Signals were detected using a multimode microplate reader. Luciferase activity was corrected by βgalactosidase activity. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Total RNA was extracted from differentiated HepaRG cells under various treatment conditions using a Direct-zol RNA MiniPrep kit (ZYMO Research, Irvine, CA), according to the manufacturer’s instructions. To ensure sample quality, only RNA samples with A260/A280 absorbance ratios ≥1.8 were used in this study. Total RNA (1 μg) was utilized for first-strand cDNA synthesis using a MultiScribe reverse transcriptase kit (Thermo Fisher Scientific). The mRNA expression levels of CYP3A4, CYP2B6, and GAPDH were then analyzed by quantitative PCR using Luminaris Color HiGreen qPCR master mix (Thermo Fisher Scientific) in a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with standard procedures. The primers used were as follows: CYP3A4 Forward, 5′-TGA GGC GGG AAG CAG AGA-3′; CYP3A4 Reverse, 5′-CAT GCT GTA GGC CCC AAA GA-3′; CYP2B6 Forward, 5′CAC TCA TCA GCT CTG TAT TCG G-3′; CYP2B6 Reverse, 5′GTA TGG CAT TTT GGC TCG G-3′; GAPDH Forward, 5′-ACC CAG AAG ACT GTG GAT GG-3′; GAPDH Reverse, 5′-TTC AGC TCA GGG ATG ACC TT-3′. Relative mRNA expression levels of each gene were normalized to that of GAPDH by determining a fractional PCR threshold cycle number (Ct value). Data were processed according to the calculation based on the 2−(Ct CYP−Ct GAPDH) method. Western Blotting. CYP3A4 and CYP2B6 protein expression levels were measured via Western blot analysis. Differentiated HepaRG cells were treated with various concentrations of OA, either alone or in

promoting CYP3A4 by PXR activation. Examples of compounds reported to exert this effect include amiodarone (AMD), acetaminophen (APAP), and isoniazid (INH).8,9 Only a limited number of PXR antagonists have been characterized.10 CAR is abundantly expressed in the liver and greatly influences the expression of CYP2B6.2 Therefore, antagonists of PXR and CAR might protect against PXR/ CAR-induced adverse effects and enhance the efficacy of coadministrated drugs. Oleanolic acid (OA, 3β-hydroxyolean-12-en-28-oic acid) is a common pentacyclic triterpenoid compound predominantly found in a variety of plant sources and fruits, such as olives and olive oil (Olea europaea L.), Hawthorn berries (Crataegus sp.), apples, and cranberries.11 OA has been reported to exert several pharmacological effects, including anticancer, chemoprevention, anti-inflammatory, antimicrobial, antioxidation, antiobesity, and immunomodulatory activities, through multiple signaling pathways, both in vitro and in vivo.12 RIF and INH, or their combination, are common medications used as first-line therapeutics for tuberculosis.13 These agents have been reported to cause adverse off-target effects associated with hepatotoxicity, including hepatocellular carcinoma (HCC).13 Inclusion of RIF, a strong CYP3A4 inducer, in combination with INH, may increase the formation of the toxic INH metabolites, acetylhydrazine and hydrazine, which in turn could induce reactive oxygen species production and oxidative stress, leading to mitochondrial dysfunction and cell death.14 Elevation of serum transaminase and liver injury in treated animals were also described in a previous report.14 Thus, RIF and INH cotreatment may aggravate hepatic toxicity. Since PXR-CYP3A4 and CAR-CYP2B6 are important pathways that influence drug efficacy, there is a need for modulators of these NRs that can inhibit unwanted druginduced interactions and toxicity. To address this issue, in this study, we investigated the effect of OA on the transcriptional activity of PXR and CAR using reporter assays. The mRNA and protein expression, as well as the enzyme catalytic activities, of CYP3A4 and CYP2B6 were evaluated using a differentiated hepatocarcinoma cell line, HepaRG. Furthermore, the effect of OA on the transcriptional machinery associated with the CYP3A4 promoter was characterized in the presence of multiple coactivators. Lastly, we investigated the effect of OA on cytotoxicity and protein thiol group content caused by INH on the induction of RIF.

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MATERIALS AND METHODS

Chemicals and Cell Culture. All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO), with the highest pure grade available. Chemicals were dissolved in either DMSO or ultrapure water at appropriate concentrations. The human hepatocarcinoma cell line, HepG2, was purchased from Taiwan Food Industry Research and Development Institute and maintained in minimum essential medium α, supplemented with 10% fetal bovine serum without antibiotics. Cells were cultured in a humidified CO2 atmosphere of 5% and at 37 °C. Passages >20 were not used in our experiments. HepaRG cells were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA). Frozen cells were thawed and directly seeded and maintained in William’s E medium (Sigma-Aldrich) supplemented with 10% FetalClone II serum (Hyclone, GE Healthcare, Chicago, IL), 5 μg/mL human insulin, and 50 μM hydrocortisone hemisuccinate for 2 weeks. Subsequently, cells were cultivated in differentiation medium (above-mentioned medium plus 2% DMSO) for two more weeks to induce differentiated hepatocytelike properties. B

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Journal of Agricultural and Food Chemistry combination with RIF or CITCO, for 24 h. After incubation, the culture medium was aspirated and rinsed twice with 1× ice-cold PBS; cells were then scraped into 200 μL of ice-cold RIPA buffer with protease inhibitors. Cleared lysates were obtained by centrifugation at 14 000g for 30 min at 4 °C. For each sample, 50 μg of total protein were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a nitrocellulose membrane (BioTrace NT Nitrocellulose Transfer Membrane; PALL Corporation, Port Washington, NY). Membranes were probed with anti-CYP3A4, 2B6, and β-actin primary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX) and developed after incubation with corresponding secondary antibodies. Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA) and an ImageQuant LAS 4000 densitometer (GE Healthcare). Enzyme Catalytic Activity of CYP3A4 and CYP2B6 in Differentiated HepaRG Cells. Differentiated HepaRG cells were treated with various concentrations of OA alone or in combination with RIF or CITCO for 72 h. After 24 h of the 72 h incubation period, 3 μM midazolam (CYP3A4 probe substrate, for the RIF-treated group) or 200 μM bupropion HCl (CYP2B6 probe substrate, for the CITCO-treated group) were added to the cells. Next, cells were lysed using a hypotonic extraction buffer (contained 10 mM HEPES-KOH, 1.5 mM MgCl2, and 10 mM KCl), and cleared lysates were obtained via high-speed centrifugation, after which total protein contents were determined using a BCA assay kit (Pierce, Thermo Fisher Scientific Inc.) for normalization. Metabolite contents were determined using an LC−MS/MS method, as described previously.18 Measurement of Free Thiol Group Levels among Total Protein Samples. Differentiated HepaRG cells were treated with OA alone or in combination with RIF for 72 h. For the last 24 h of the 72 h culture period, INH was added to the culture medium. Cell lysates were harvested using RIPA buffer, and the total protein free thiol group content within each cell supernatant was determined using a 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) assay, as described previously.19 Preparation of Nuclear Extracts. Differentiated HepaRG cells were treated with OA alone or in combination with RIF or CITCO (for CAR detection) for 24 h, washed twice with 1× PBS, scraped into 500 μL of PBS, and pelleted by centrifugation at 7500g for 20−30 s. The resulting supernatants were removed and each pellet was resuspended in 400 μL of buffer A [contained 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, and 10 mM KCl] and incubated at 4 °C for 10 min. After centrifugation at 7500g for 20−30 s, pelleted nuclei were resuspended in 100 μL of buffer C [contained 20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 0.2 mM EDTA, 420 mM NaCl, and 25% glycerol] and incubated at 4 °C for 20 min. Nuclear extracts were collected after centrifugation at 7500g for 2 min and stored at −80 °C. Both buffer A and C contained 0.5 mM dithiothreitol, 2 μg/mL leupeptin, 1 mM orthovanadate, 2 μg/mL pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride. DNA Affinity Precipitation Assay (DAPA). Nuclear extracts from treated differentiated HepaRG cells were prepared after exposure to OA alone or in combination with RIF/CITCO for 24 h. A mixture contained: 500 μg of nuclear extract protein, 2 μg of biotinylated CYP3A4 ER6 oligonucleotides (ER6 sequence, 5′-biotin-TAG AAT ATG AAC TCA AAG GAG GTC AGT GAG T-3′), and 20 μL of a 50% streptavidin-agarose bead slurry (final 4% of slurry bead) (SigmaAldrich). The mixture was rotated at room temperature for 1 h. Beads were repeated washed with 1 × 4 °C PBS and pelleted for three times. At the end, 1× SDS-PAGE sampling buffer was used to boil the samples and the bound proteins were eluted. The samples were separated by 10% SDS-PAGE, followed by Western blot analysis using specific antibodies against PXR, CAR, RXRα, HNF4α, SRC-1, and PGC-1α. Statistical Analysis. Data shown with error bars indicate the mean ± standard error (SE). The p value for each group comparison was determined via analysis of variance (ANOVA) followed by Tukey’s test while for multiple groups comparison. All p values were determined relative to the mock control group or ligand-treated group, as pointed out in the figures. All statistical calculations were

analyzed using SPSS, version 20.0 (for Windows, SPSS Statistics, Inc., Chicago, IL). A p value less than 0.05 was considered statistically significant.

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RESULTS Viability of HepG2 and HepaRGCells with OA Alone or in Combination with RIF. OA was reported to inhibit cell viability in several cancer cell lines.11 Here, we intended to study the functional effects of OA rather than potential cytotoxic effects. Thus, we performed a cell viability assay to rule out possible cytotoxic effects from OA. As shown in Figure 1, treatment with 10 and 20 μM OA for 24 h, alone or in combination with 20 μM RIF, resulted in no significant cytotoxic effects in either HepG2 or HepaRG cell lines.

Figure 1. Viability of HepG2 and HepaRG cells following exposure to oleanolic acid (OA) in the presence or absence of rifampin (RIF). HepG2 and HepaRG cells were treated with OA (10−20 μM), RIF (20 μM), or both RIF (20 μM) and OA (10−20 μM) for 24 h. Viability of the cells was monitored by screening for cellular acid phosphatase activity. Data are presented as means ± SE (n = 3).

Effects of OA on PXR Transactivation of CYP3A4 Promoter Activity and CYP3A4 Expression in a Differentiated Hepatoma Cell Line. RIF, a well-known human PXR ligand, was used to induce PXR transcriptional activity and evaluate the effects of OA on CYP3A4 promoter activity. For these analyses, a full-length human PXR expression plasmid was transiently transfected into HepG2 cells to assess the inhibitory effects on CYP3A4 promoter activity in the presence of OA alone or in combination with RIF. As shown in Figure 2A, 20 μM RIF enhanced CYP3A4 promoter activity up to 25-fold in the presence of the PXR plasmid. However, 10 and 20 μM OA attenuated this transactivation effect in a concentrationdependent manner by 36% and 81%, respectively. The activation of PXR demonstrated species-specific differences in a previous study.20 Likewise, a separate study found that while rodent PXR was insensitive to RIF, this receptor could be activated by pregnenolone-16α-carbonitrile (PCN), which is not a human PXR ligand.21 Thus, we tested the inhibition of rat PXR activation using OA in HepG2 cells in the presence of rat PXR and PCN. As shown in Figure 2B, OA did not attenuate rat PXR-mediated CYP3A4 promoter activity, neither with 10 nor 20 μM OA under the induction of 20 μM PCN. These results suggest that OA inhibited human but not rat PXR activity. We further examined whether OA could inhibit the expression of CYP3A4 in a differentiated hepatocarcinoma C

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Journal of Agricultural and Food Chemistry Figure 2. continued

transfected to HepG2 cells. The transfected cells were then treated with OA, with or without PCN for 24 h. Luciferase activity was measured and corrected by β-galactosidase activity. Data are presented as means ± SE (n = 4). (C) Differentiated HepaRG cells were treated with OA and RIF, either individually or in combination, for 24 h, total mRNA was collected, the expression levels of CYP3A4 and GAPDH were analyzed via qRT-PCR. Values were corrected by GAPDH expression, and CYP3A4 expression in mock control was set at 1. Data are presented as means ± SE (n = 3) (***p < 0.001). (D) Treated differentiated HepaRG cells by OA and RIF, either individually or in combination, for 24 h were harvested and total lysates were collected. Protein of CYP3A4 and β-actin (internal control) expression levels were analyzed via Western blot. Quantitation of the CYP3A4 protein bands was corrected by β-actin expression. The representative blot shown was quantified with ImageJ software.

cell line, HepaRG, which is derived from a human hepatic progenitor cell line and retains many features of primary human hepatocytes.22 PXR activation is known to induce CYP3A4 mRNA and protein expression.23 The phenomenon was consistent with PXR-mediated transactivation of CYP3A4 promoter activity. OA effectively inhibited RIF-induced CYP3A4 mRNA expression in HepaRG cells (Figure 2C) by 60% (10 μM OA) and 71% (20 μM OA). We also examined the effect of OA on CYP3A4 protein expression. As shown in Figure 2D, RIF induced CYP3A4 protein expression up to 83%, and this induction attenuated by 51% and 55% in the presence of 10 and 20 μM OA, respectively. Notably, HepaRG cells exhibit endogenous PXR expression, and treatment with OA alone resulted in inhibition of CYP3A4 expression, suggesting that the observed effect was transcriptionally dependent. Effects of OA on CAR-Mediated Transactivation of CYP2B6 Promoter Activity and CYP2B6 Expression in a Differentiated Hepatoma Cell Line. Expression of human CYP3A4 is also under the control of CAR, through the same response element as PXR at the CYP3A4 promoter.24 Thus, we transfected HepG2 cells with a human CAR expression plasmid and the CYP3A4 promoter construct to evaluate the influence of OA on human CAR activation using a promoter luciferase assay. As shown in Figure 3, a human CAR-specific ligand, CITCO, enhanced CYP3A4 promoter activity up to 11.2-fold, compared to that observed in transfected cells without CITCO induction. However, this induction was significantly attenuated by 27% and 60.7%, upon treatment with 10 and 20 μM of OA, respectively. Induction of CYP2B6 expression by clinically used drugs or xenobiotics is also predominantly mediated by the transactivation of CAR through the phenobarbital responsive element module (PBREM) located in the upstream promoter region of CYP2B6.25 To further assess the effect of OA on CAR-mediated CYP2B6 promoter activity, a CYP2B6 reporter construct and a CAR3 expression plasmid were cotransfected into HepG2 cells and a luciferase assay was performed. As shown in Figure 4A, cells treated with CITCO displayed greater CAR transactivation activity on CYP2B6 than CYP3A4 promoter activity (11.2-fold on CYP3A4 and 23.5-fold on CYP2B6). Again, however, this induction was significantly reduced in the presence of 10 and 20 μM OA (by 33% and 61.2%, respectively). Our previous study showed that OA did not inhibit rodent PXR. However, we intended to examine the effect of OA on mouse CAR transactivation. Using TCPOBOP as a mouse CAR-specific ligand, treatment with 10 and 20 μM

Figure 2. Effects of oleanolic acid (OA) on pregnane X receptor (PXR) transactivation and CYP3A4 mRNA and protein expression. (A) Reporter assays to define the effects of OA and PXR ligandmediated activation on CYP3A4 promoter activity in HepG2 cells. HepG2 cells were cotransfected with pcDNA3 or pcDNA3-PXR, CYP3A4-promoter construct, and a β-galactosidase vector. The transfected cells were then treated with OA with or without RIF for 20−24 h. Luciferase activity was measured and corrected by βgalactosidase activity. Data are presented as means ± SE (n = 4) (***p < 0.001). (B) A rat PXR expression plasmid (pcDNA3-rat PXR), a CYP3A4 promoter construct, and a β-galactosidase vector were D

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Figure 3. Transactivation of CYP3A4 promoter activity by human CAR under the treatment of oleanolic acid (OA), by the presence or absence of CITCO. HepG2 cells were cotransfected with pCR3 or pCR3-CAR3, a CYP3A4 promoter construct, and a β-galactosidase vector. The transfected cells were then exposed to OA and/or the human CAR-specific ligand (CITCO) for 20−24 h, after which luciferase activity was measured. Data are presented as means ± SE (n = 4) (***p < 0.001).

OA effectively inhibited cyp2b10 promoter activity by 43.4% and 57.2%, respectively (Figure 4B). To further investigate the inhibitory effect of OA on CYP2B6 gene and protein expression levels, differentiated HepaRG cells were treated with OA individually or in combination with CITCO for 24 h. While this treatment induced CYP2B6 mRNA expression by 34%, compared with control cells (Figure 4C), this effect was abrogated upon treatment with 10 and 20 μM OA (decreased by 30% and 65.5%, respectively). Likewise, the 18% increase in CYP2B6 protein expression observed in these cells was reduced by 35% and 62.7% in the presence of 10 and 20 μM OA, respectively (Figure 4D). Together, these data demonstrate that OA inhibits CYP3A4 and CYP2B6 expression through transcriptional control, mediated by PXR and CAR. In addition, we determined that the inhibition of PXR, but not that of CAR, was species-specific. Inhibition of CYP3A4 and CYP2B6 Enzyme Catalytic Activities in Differentiated HepaRG Cells with OA under the Induction of RIF and CITCO. To further investigate the potential inhibition of OA on CYP3A4 and CYP2B6 enzyme catalytic activity, we evaluated probe substrate metabolism in differentiated HepaRG cells by measuring parent compounds and metabolites (midazolam and 1′-hydroxymidazolam for CYP3A4; bupropion hydrochloride and ±-hydroxybupropion for CYP2B6). After 48 h of pretreatment with 20 μM RIF or 10 μM CITCO alone or in combination with 10 and 20 μM OA, the cells were treated with probe substrates for further 24 h. Under RIF induction, CYP3A4 catalytic activity increased to 3.55-fold that of the control cells; however, this induction was attenuated 2.87- and 0.58-fold upon treatment with 10 and 20 μM OA, respectively. Since bupropion acts as CYP2B6 specific substrate, we evaluated the effect of OA on CYP2B6 activity by measuring ±-hydroxybupropion levels in lysates generated from differentiated HepaRG cells. Similar to the metabolic results from CYP3A4, ±-hydroxybupropion levels were significantly increased by 30% after pretreatment with 10 μM CITCO, and this induction was significantly attenuated by treatment with 10 and 20 μM OA (Figure 5A,B).

Figure 4. Effects of oleanolic acid (OA) on constitutive androstane receptor (CAR) transactivation and CYP2B6 mRNA and protein expression. (A) HepG2 cells were cotransfected with pCR3 or pCR3CAR3, a CYP2B6 promoter construct, and the β-galactosidase plasmid. The transfected cells were then subjected to OA and/or CITCO for 20−24 h, after which luciferase activity was measured. Data are presented as means ± SE (n = 4) (*p < 0.05; ***p < 0.001). (B) HepG2 cells were cotransfected with pCMX-mCAR, a cyp2b10 promoter construct, and β-galactosidase vector. The transfected cells were then exposed to OA and TCPOBOP (mouse CAR-specific ligand) for 24 h, after which luciferase activity was measured. Data are E

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constructs into HepG2 cells. Luciferase assays were then performed after 20 h of drug exposure time. Cotransfection of PXR with each coactivator resulted in significantly increased of CYP3A4 promoter activity in the stimulation of RIF (Figure 6A−D). OA strongly attenuated this activated promoter activity, especially in RIF-treated cells. These results indicate that OA disrupted the coregulation effects between PXR and coactivators on CYP3A4 promoter activity. We also evaluated the basal expression of these NRs and coactivators in the presence of OA alone or in combination with ligands, as these effects could have originated from a reduction of protein expression due to drug exposure. As shown in Figure 6E, no significant change in the protein expression of PXR, CAR, RXRα, HNF4α, SRC-1, and PGC-1α was observed following treatment with OA alone or the presence of RIF/CITCO (for CAR detection only). We subsequently applied DAPA, which provides a quantitative measure of transactivator binding, to examine whether NRs and coregulators bound to the promoter CYP3A4 ER6 sequence, and to evaluate the effects of RIF/CITCO and OA on this binding. For these experiments, oligonucleotides covering the CYP3A4 ER6 site, which acts as a PXR and CAR recognition site in gel shift assays,3 were synthesized and biotinylation as probes before used. Nuclear extracts from HepaRG cells treated with 20 μM RIF or 10 μM CITCO and OA for 24 h were incubated with a biotinylated probe specific to the CYP3A4 ER6 site and streptavidin-agarose beads. The levels of PXR, CAR, RXRα, HNF4α, SRC-1, and PGC-1α present in the complexes were then analyzed via Western blot (Figure 6F). We observed increased binding of PXR, CAR, RXRα, HNF4α, SRC-1, and PGC-1α to the 5′-biotinylated CYP3A4 ER6 sequence in cells treated with RIF or CITCO (CITCO, for CAR detection only) (1.37-, 1.57-, 1.53-, 1.80-, 1.50-, and 1.39fold, respectively). Again, however, these increases were abrogated upon exposure to different concentrations of OA. OA Attenuated RIF-Mediated INH-Induced Cytotoxicity and Glutathione Content in HepaRG Cells. Increased cytotoxicity of INH in the presence of RIF was reported in previous studies.9 INH is a CYP3A4 substrate, and its metabolism generates toxic metabolites that cause cytotoxicity.9 Cell viability was assessed in the presence of OA to investigate its potential for protecting cells from the toxic effects produced by RIF-INH exposure. Differentiated HepaRG cells were treated with 20 μM RIF alone or in combination with 10 μM OA for 72 h, followed by addition of 25 and 50 mM INH to the media. After 20 h incubation, cytotoxicity assays were performed (Figure 7A). A concentration-dependent increase in cell death was observed upon the addition of INH in cells pretreated with RIF alone (22% and 36% at 25 and 50 mM INH, respectively). Conversely, cells treated with 10 μM OA showed higher viability than those treated with RIF alone, suggesting that OA protected cells from the RIF-induced cytotoxic effects of INH at the doses and time points analyzed. Similarly, while the levels of intracellular total protein thiol groups decreased after treatment with 25 and 50 mM INH, these levels were enhanced by treatment with OA (Figure 7B).

Figure 4. continued presented as means ± SE (n = 4) (***p < 0.001). (C) Differentiated HepaRG cells were treated with OA alone or in combination with CITCO for 24 h, after which total mRNA was collected and the expression levels of CYP2B6 and GAPDH were analyzed via real timePCR. CYP2B6 expression was corrected to that of GAPDH. Data are presented as means ± SE (n = 3) (***p < 0.001). (D) Differentiated HepaRG cells were subjected to OA and CITCO, either alone or in combination, for 24 h. Total lysates were harvested, and the protein expression levels of CYP2B6 and β-actin were analyzed via Western blot. CYP2B6 protein expression was corrected to that of β-actin. A representative blot, which was quantified using ImageJ software, is shown.

Figure 5. Enzyme catalytic activity of CYP3A4 and CYP2B6 in the presence of oleanolic acid (OA) individually or in combination with rifampin (RIF) or CITCO. Differentiated HepaRG cells were pretreated with OA individually or in combination with (A) RIF or (B) CITCO for 72 h. For the last 24 h of incubation period, the cells were then treated with (A) 3 μM midazolam or (B) 200 μM bupropion HCl. Whole cell lysates were then prepared, and the metabolites were prepared. Midazolam and bupropion HCl metabolites levels were determined as described in the Materials and Methods. Data are presented as means ± SE (n = 3) (***p < 0.001).

OA-Mediated Inhibition of Coregulation of Human RXRα, HNF4α, SRC-1, and PGC-1α by PXR. Previous studies showed that full activation of PXR under RIF induction was dependent on the coregulation of RXRα, HNF4α, SRC-1, and PGC-1α.26 Thus, we assessed the coregulation of these coactivators by PXR on CYP3A4 promoter activity in the presence of RIF and OA. Full-length human RXRα, HNF4α, SRC-1, and PGC-1α expression constructs were cotransfected with human PXR/vector control and CYP3A4 promoter

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DISCUSSION DDIs are an important clinical issue, representing approximately 7% of hospitalization cases each year;27 such interactions can increase the risk of adverse drug reactions and even lead to fatality. An important aspect of DDIs is the metabolic framework containing the CYP450s. The predomF

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Figure 6. Co-regulation of RXRα, HNF4α, SRC-1, and PGC-1α with pregnane X receptor (PXR) under oleanolic acid (OA) treatment on CYP3A4 reporter assays, the effect of OA on basal and induced expression of PXR, CAR, RXRα, HNF4α, SRC-1, and PGC-1α, and DNA binding affinity assay (DAPA) analysis at the CYP3A4 response element. HepG2 cells were cotransfected with pcDNA3 or pcDNA3-PXR, along with (A) pGEM3Z-RXRα; (B) pcDNA3-HNF4α; (C) pCR3.1-SRC-1; and (D) pTARGET-PGC-1α, in combination with the CYP3A4 promoter construct and the β-galactosidase vector. The transfected cells were then subjected to OA and/or rifampin for 20−24 h, after which luciferase activity was measured and corrected by β-galactosidase activity. Data are presented as means ± SE (n = 4) (***p < 0.001). (E) Differentiated HepaRG cells were exposed to various concentrations of OA, individually or in combination with 20 μM RIF (for PXR, RXRα, HNF4α, SRC-1, and PGC-1α detection) or 10 μM CITCO (for CAR detection), for 24 h. Total lysates were then collected, and the expression levels of indicated proteins were evaluated via Western blot. (F) 500 μg of nuclear extracts, from OA treatment in combination with RIF or CITCO (CITCO group was only for CAR detection), proceeded the DAPA experiment as mentioned in the Materials and Methods. A representative blot was presented, and the protein expression in mock control was set at 1. G

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sex, and comorbidities, long-term cumulative use (>12 months) of INH, RIF, and RIF + INH was associated with an increased risk of HCC, with adjusted odds ratios of 3.51, 4.17, and 7.17, respectively. These HCC risk values increased in a dosedependent manner according to annual average dosage while using RIF + INH. Here, we found that OA showed a protective effect against cell damage induced by RIF-INH in the HCCderived cell line HepaRG, suggesting a potentially effective combination of OA with these agents. RIF is the most potent human PXR activator and affects the therapeutic efficacy of CYP3A4 substrate medications. Indeed, failure of cancer treatment and bone marrow transplantation was observed in some patients,43 and the antihypertensive effect of verapamil was reduced when coadministrated with RIF.44 Additionally, increased frequency of pregnancy was reported among women who had taken RIF while also administered oral contraceptives,45 and a loss of analgesic therapeutic effect was reported in patients that were cotreated with RIF and analgesic drugs.46 A naturally derived product, hyperforin, significantly reduced the anticancer effect of irinotecan,47 while two traditional Chinese Medicines, Wu Wei Zi and Gan Cao, were shown to activate PXR and increase the metabolism of warfarin when coadministered in rats.48 The aforementioned ineffectiveness of therapies was largely due to induction of CYP450s through PXR activation. The identification and development of PXR antagonists could therefore comprise a useful approach for preventing deleterious DDIs, thereby enhancing the efficacy and safety of pharmacological agents. It could also be useful for studying fundamental receptor functions at the molecular level. However, a specific PXR antagonist that is effective at micromolar dosages has yet to be identified.18 Here, we provided the first evidence that OA reduces the RIF-inducible expression of CYP3A4 under control of PXR. As OA is found in many herbs and fruits and is also used as a health supplement, the period and amount of OA supplement utilization should be monitored in patients using multiple therapeutic regimens that are PXR ligands. The probable increase in CYP3A4-mediated drug metabolism by known PXR ligands could possibly be antagonized by OA. Earlier studies revealed that OA affected the activity of CYP3A4 without exploring the underlying mechanism.49 Here, we identified a crucial role for PXR in suppressing the RIFinducible CYP3A4 expression by OA. Remarkable differences in the transactivation of PXR and CAR orthologs among diverse species have been reported.50 RIF and omeprazole are potent inducers of CYP3A and CYP1A, respectively, in human but not rodent hepatocytes.51 Additionally, RIF and CITCO are potent ligands for PXR and CAR in humans, respectively, but not in rodents. In contrast, PCN and TCPOBOP activate PXR and CAR in rodents but not humans.52 Here, the species-specific differences observed in our CYP3A4 reporter data indicated that OA only inhibited human PXR but not rodent PXR. Conversely, OA inhibited both human and rodent CAR activity. Thus, OA acted as an effective antagonist of CAR function in both humans and rodents and of PXR function in humans but not rodents. To clarify the possibility of the observed suppressive effect by OA on PXR-mediated CYP3A4 induction, additional studies should be conducted. The fully activation of PXR functions depends on the ligands binding to the ligand-binding domain which located in the receptor, interaction with coregulators such as RXRα, HNF4α, SRC-1, and PGC-1α, and binding to the specific responsive element within the promoters of target

Figure 7. Effects of oleanolic acid (OA) on rifampin (RIF)-induced isoniazid (INH) cell viability and on total protein thiol group contents in HepaRG cells. Pretreated differentiated HepaRG cells with RIF, either alone or in combination with OA, for 72 h. For the final 24 h of treatment, the cells were further subjected to INH. (A) Cell viability was monitored by assessing cellular acid phosphatase activity. (B) Total protein thiol group levels were measured by 5,5′-dithiobis(2nitrobenzoic acid) (DTNB) assay analysis. Data are presented as means ± SE (n = 3) (***p < 0.001).

inant NR, PXR, serves as the main molecular controller for CYP450-mediated DDIs.20 If one drug activates or antagonizes PXR, it could either enhance or reduce the metabolism and elimination of another coadministrated xenobiotics, which also metabolized and cleared by these PXR-targeted CYP450s. In this manner, the viability of many treatments may be lessened or improved in patients administered combination treatments and could consequently influence the efficacy of the second drug or cause adverse effects. Additionally, PXR has been shown to mediate the accumulation of potentially dangerous drug metabolites due to CYP3A4 induction.8,9 These kinds of interactions can lead to significant, potentially life-threatening, clinical events.28 OA is a triterpenoid compound that exists in natural plants in the form of free acid or aglycones for triterpenoid saponins12 and is ubiquitously obtained from several kinds of plants (Supplemental Table 1).29−35 Several studies revealed that OA possesses multiple biological and pharmacological benefits, including hepatoprotector effects.11,36−40 Excessive dosing of therapeutic drugs is a common cause of drug-induced liver injury (DILI) and can lead to severe outcomes, from liver cirrhosis to HCC.41 For example, hepatotoxic effects have been observed in patients receiving the first-line antituberculosis agents RIF and INH. In our previous work, we conducted a large-scale case-control study to address the risk of the longterm use and effects of INH and RIF, alone and in combination (RIF + INH), in cirrhotic patients.42 After adjusting for age, H

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Journal of Agricultural and Food Chemistry genes.26 When no ligand binding, PXR forms a complex with corepressors such as NCoR and inhibits transcriptional activity. Meanwhile, ligand binding results in dissociation of PXR from the corepressor and recruitment of coactivator proteins. It has been shown that ketoconazole disrupts the interaction of PXR with SRC-1.53 Therefore, it is of particular interest to determine whether OA interferes with the binding of PXR and coregulators to the CYP3A4 promoter response element (ER6). In our study, we found that basal protein expression of PXR, CAR, and their coregulators were not significantly modified by OA treatment. OA significantly decreased inducible forms of CYP3A4 and CYP2B6 mRNA and protein expression levels without changing the basal expression of these control elements. Results from our DAPA showed that OA functions as a receptor antagonist of human PXR/CAR and that its presence could disrupt the recruitment of transcriptional complexes of PXR, thereby inhibiting PXR transactivation ability. Thus, the disruption of these transcriptional complexes could inhibit target gene expression. The search for agents effective in preventing the risk of hepatotoxicity in tuberculosis patients receiving antituberculosis drug therapy is an increasingly important issue. Some synthetic or naturally occurring compounds, such as N-acetylcysteine, curcumin, silymarin, hepatoplus, reamberine, remaxol, and ademethionine, have been reported to exert hepatoprotective effects.54−56 The search for hepatoprotective agents that avoid antituberculosis DILI is undergoing validation. In a randomized, controlled clinical trial, addition of Curcuma longa and Tinospora cordifolia to the standard antituberculosis regimen significantly alleviated the duration and severity of hepatotoxic episodes in patients with tuberculosis.57 Therefore, whereas metabolic activation of CYP3A4 or PXR increases toxic effects, the administration of CYP3A4 inhibitors or PXR deactivators could protect against RIF-INH-mediated liver toxicity. In particular, PXR ligand-mediated hepatotoxicity could be enhanced by the induction of CYP3A4, which produces a saquinone metabolite of the parent drug. Thus, development of PXR antagonists could have substantial clinical potential for reducing adverse events resulting from overdoses of otherwise benign drugs. Our present study showed that OA attenuated cell toxicity via inhibition of a PXR-CYP3A4-mediated metabolic pathway and enhancement of cellular glutathione regeneration capacity. Taken together, we identified a common and a naturally occurring compound, OA, which acts as a potent partial agonist of human PXR and CAR. We demonstrated that OA reduces the induction of CYP3A4 and CYP2B6 by inhibiting agonistactivated PXR and CAR. Additionally, OA reduces the enzyme catalytic activities of CYP3A4 and CYP2B6 in differentiated HepaRG cells and disrupts the coregulation of PXR with RXRα/SRC-1/HNF4α/PGC-1α. These results suggest a complementary effect for OA, which could be useful as an adjuvant therapy to reduce the frequency of adverse drugassociated reactions. Thus, our findings provide a new avenue for PXR antagonism by preventing harmful inducer-drug interactions and improving the therapeutic efficacy of existing medications.

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Oleanolic acid distribution within various dry plant materials (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: + 886-4-2205-3366 ext. 5802. Fax: +886-4-2207-8083. E-mail: [email protected], [email protected]. ORCID

Yun-Ping Lim: 0000-0001-9312-048X Author Contributions #

Y.-N.L., C.-J.C., and H.-Y.C. contributed equally to this work.

Funding

This study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (Grant MOST105-2320-B-039031) and China Medical University, Taichung, Taiwan (Grant CMU105-ASIA-22), and in part by the Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (Grant MOHW106-TDU-B-212-113004). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Hongbing Wang (Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA) and Dr. Tsuyoshi Yokoi (Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa, Japan) for kindly providing constructs used in this study.

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ABBREVIATIONS CAR, constitutive androstane receptor; CITCO, 6-(4chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O(3,4-dichlorobenzyl)-oxime; CYP450, cytochrome P450; DMEs, drug metabolizing enzymes; DMSO, dimethyl sulfoxide; DR, direct repeat; ER, everted repeat; HNF4α, hepatocyte nuclear factor 4α; OA, oleanolic acid; PCN, pregnenolone 16αcarbonitrile; PGC-1α, Peroxisome proliferator-activated receptor-gamma coactivator 1α; PXR, pregnane X receptor; RXRα, retinoic X receptor α; SRC-1, steroid receptor coactivator-1; TB, tuberculosis; XREM, xenobiotic response element

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02696. I

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