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Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor
Drug Metabolism and Pharmacokinetics xxx (2016) 1e7
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Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor Takeshi Kumagai a, *, Yusuke Aratsu a, b, Ryosuke Sugawara a, Takamitsu Sasaki a, c, Shinichi Miyairi d, Kiyoshi Nagata a a
Department of Environmental Health Science, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan Drug Metabolism and Pharmacokinetics Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan d Laboratory of Organic Chemistry, School of Pharmacy, Nihon University, 7-7-1 Narashino, Funabashi, Chiba 274-8555, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2015 Received in revised form 14 January 2016 Accepted 14 January 2016 Available online xxx
Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an antiinflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-LanGen, activated CYP3A4 gene transcription through the activation of the human PXR.
Keywords: AhR activator Indirubin CYP3A4 induction PXR Herbedrug interactions
Copyright © 2016, Published by Elsevier Ltd on behalf of The Japanese Society for the Study of Xenobiotics.
1. Introduction Chinese herbal medicines are an integral part of health care in Asia, and many herbal medicines are extensively used as alternative and/or complementary therapies. However, the clinical efficacy, mechanisms of action, and safety profiles of most herbal medicines
Abbreviations: AdCont, b-galactosidase-expressing adenovirus; AdPXR, human PXR-expressing adenovirus; AdhPXR-shRNA, AdhPXR-short hairpin RNA; AhR, aryl hydrocarbon receptor; Ct, threshold PCR cycle; CAR, constitutive androstane receptor; CYP, cytochrome P450; DMEM, Dulbecco's modified Eagle's medium; dNR1, distal nuclear receptor-binding protein 1; DMSO, dimethyl sulfoxide; D-PBS, Dulbecco's phosphate buffered saline; DR3, direct repeats spaced by 3 bases; eNR3A4, essential distal nuclear receptor-binding element; ER6, everted repeats separated by 6 bases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBE, Ginkgo biloba extract; GRa, glucocorticoid receptor-a; MOI, multiplicity of infection; PAHs, polycyclic aromatic hydrocarbons; prER6, ER6 in the CYP3A4 proximal promoter; PXR, pregnane X receptor; RXRa, retinoid X receptor a; SJW, St. John's wort; TCID50, 50% titer culture infectious dose; VDR, vitamin D receptor. * Corresponding author. E-mail address: [email protected] (T. Kumagai).
are not well characterized. Moreover, herbal medicines are often co-administered with therapeutic drugs, creating the potential for herbedrug interactions [1,2]. While the underlying mechanism of most herbedrug interactions is not known, induction and inhibition of drug-metabolizing enzymes or drug transporters have been implicated as potential mechanisms. Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Indigo plants have been an important source of dyes in Asia since ancient times, and today, they are commonly used as therapeutics in traditional Chinese medicine. Ban-Lan-Gen is frequently used as an anti-inflammatory and antiviral for the treatment of hepatitis, influenza, and various kinds of inflammation [3,4]. Several biologically active ingredients have been identified in Ban-Lan-Gen, including indigoid alkaloids (e.g., indigo and indirubin) and quinazolinone alkaloids (e.g., tryptanthrin) [5]. In fact, indigo, indirubin, and tryptanthrin are three marker compounds found in Ban-Lan-Gen [6]. Among these marker compounds, it has been reported that indirubin causes transactivation of cytochrome P450 1A1 (CYP1A1) and CYP1A2 genes via
http://dx.doi.org/10.1016/j.dmpk.2016.01.002 1347-4367/Copyright © 2016, Published by Elsevier Ltd on behalf of The Japanese Society for the Study of Xenobiotics.
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
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the aryl hydrocarbon receptor (AhR) [7]. In addition, a content of indirubin have been reported in the roots of I. indigotica as main raw materials of Ban-Lan-Gen (0.224e34.4 mg/g) [5,6]. Members of the CYP supergene family of monooxygenases play an important role in detoxification by converting pollutants, plant toxins, carcinogens, and drugs to products that are then excreted in to urine or bile [8,9]. Human CYP3A4 is of particular significance in this respect, because it is involved in the metabolism of approximately two-thirds of clinically relevant drugs [10]. A number of compounds, including pesticides, herbal supplements, vitamins, and drugs, activate CYP3A4 gene transcription both in the liver and in the small intestine [11,12]. This induction process is the molecular basis for a number of important drug interactions that occur in patients taking multiple medications. The pregnane X receptor (PXR; NR1I2) is the principal regulator of CYP3A4 gene expression and binds as a heterodimer with retinoid X receptor a (RXRa) to regulatory DNA sequences. These sequences include: (1) AG(G/T)TCA-like direct repeats spaced by 3 bases (DR3) located 8 kb upstream from the transcription start point and identified as distal nuclear receptor-binding element 1 (dNR1), and (2) everted repeats separated by 6 bases (ER6) located in the CYP3A4 proximal promoter (prER6) [13]. Recently, Toriyabe et al. identified a distinct PXR response element as an essential distal nuclear receptor-binding element (eNR3A4) for CYP3A4 gene induction [14]. PXR is activated by a number of structurally and chemically diverse ligands, such as drugs [rifampicin (RIF), clotrimazole] [15], pesticides (pyributicarb, endosulfan) [16,17], natural and synthetic steroids (dexamethasone) [15], bile acids (lithocholic acid) [18], and herbal medicines (St. John's wort [SJW], Ginkgo biloba, and Sophora flavescens) [19e21]. CYP3A4 induction by these PXR activators leads to accelerated metabolism of the activators themselves and concomitantly-administered drugs that are metabolized by CYP3A4. Induction and inhibition of CYP3A4 by Ban-Lan-Gen are not fully understood. In this study, we investigated the effect of Ban-LanGen on transcriptional activation of the CYP3A4 gene. 2. Materials and methods 2.1. Materials Ban-Lan-Gen (lot. 9115476) was purchased from Beijing Tong Ren Group Co., Ltd. (Beijing, China). SJW was purchased from ChromaDex (Irvine, CA). Indirubin and tryptanthrin were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). Indican, isatin, and indigo were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ginkgo biloba extract (GBE) was purchased from Tama Biochemicals Co., Ltd (Tokyo, Japan). RIF was purchased from SigmaeAldrich (St. Louis, MO). The chemicals used for this study were dissolved in dimethyl sulfoxide (DMSO). Ban-Lan-Gen, SJW, and GBE were extracted with culture medium used for the cell culture in this study, for 2 h at 37 C. These extract solutions were centrifuged at 2,000 g for 15 min at 4 C and the supernatants were used for this study. All other reagents used were of the highest quality available. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Hyclone Laboratories (Logan, UT), respectively. MEM non-essential amino acids and antibiotic-antimycotic were purchased from Invitrogen (Paisley, UK). 2.2. Cell culture HepG2 cells were obtained from RIKEN cell bank (Tsukuba, Japan). The HepG2-derived cell line stably expressing the CYP3A4-
luciferase reporter gene, clone 3-1-20, was maintained as reported previously [22]. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, MEM non-essential amino acids, and antibiotic-antimycotic. The cells were seeded at 3 104 cells per well onto 48-well tissue culture plate (BD Biosciences, Heidelberg, Germany) for luciferase gene reporter assay, and 5 104 cells per well onto 24-well tissue culture plate (BD Biosciences) for real-time polymerase chain reaction (PCR). After 24 h, the cell medium was changed with the extract solutions or various subjects dissolved in DMSO (final concentration, 0.1%) and cultured for 48 h. 2.3. Luciferase gene reporter assay 3-1-20 cells were washed with Dulbecco's phosphate buffered saline (D-PBS) and suspended in passive lysis buffer (Promega, Madison, WI) in a microcentrifuge tube. The cell suspension was centrifuged at 12,000 g for 5 min at 4 C, and the cell extract was used for the luciferase assay. The luciferase assay was performed using the Luciferase Assay System and a GloMax™ 96 Microplate Luminometer (Promega) according to the manufacturer's instructions. The resulting data are presented as the ratio of luminescence of treated cell samples to that of control. The luminescence of each sample was normalized by its protein concentration as determined with the Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). 2.4. Reporter gene constructs and transient transfection The luciferase reporter plasmids, pGL3-Basic and pGL4.70, were purchased from Promega. Preparation of the CYP3A4 luciferase reporter gene constructs, including pCYP3A4-362-7.7k, pCYP3A4362-7.7km, pCYP3A4-362m-7.7k, pCYP3A4-362m-7.7km, and pCYP3A4-362-7.7kmDa, was described previously [14]. One day before transfection, cells were seeded in 48-well plates. Each reporter plasmid and pGL4.70 was transfected using Targefect F-1 (Targeting System, El Cajon, CA) according to the manufacturer's protocol. pGL4.70 was used for the normalization of transfection efficiency. After transfection, cells were cultured in medium in the presence of various chemicals for 48 h. Control cells were cultured with vehicle (0.1% DMSO) alone. Subsequently, the cells were harvested and suspended in passive lysis buffer (Promega). Luciferase activities were determined with Dual-Luciferase® Reporter Assay System (Promega). 2.5. Construction of recombinant adenovirus and infection Construction of the human PXR-expressing adenovirus (AdhPXR) and AdhPXR-short hairpin RNA (AdhPXR-shRNA) was described previously [17]. Control adenovirus, a b-galactosidaseexpressing adenovirus (AdCont; AxCALacZ), was provided by Dr. Izumi Saito (Tokyo University, Japan) [23]. The titer of the adenoviruses, 50% titer culture infectious dose (TCID50), was determined as reported previously [17]. Multiplicity of infection (MOI) was calculated by dividing the TCID50 by the number of cells. One day before transfection, cells were seeded in 24-well plates. Adenovirus infection was carried out as described previously [24]. 2.6. Isolation of RNA and analysis of quantitative real-time polymerase chain reaction Total RNA was isolated from HepG2 cells using TRI Reagent (Molecular Research Center, Inc., Montgomery, AL) according to the manufacturer's protocol. cDNA was prepared from 2.0 mg of total RNA with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega) using oligo(dT)20 primer (Greiner Japan, Japan)
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
T. Kumagai et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e7
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and porcine RNase inhibitor (TaKaRa Bio, Japan). Quantitative realtime PCR was performed using Permix Ex Taq (Perfect Real Time, TaKaRa Bio) in a Thermal Cycler Dice Real Time System (TaKaRa Bio). All samples were quantified using a comparative threshold PCR cycle (Ct) method for relative quantification of gene expression, normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers used are shown in Table 1. 2.7. Statistical analysis Data are presented as mean ± standard deviation (S.D.) and were evaluated by paired Student's t-test. A P value < 0.05 was considered to be significant. 3. Results 3.1. Ban-Lan-Gen increases CYP3A4 gene reporter activity The effects of Ban-Lan-Gen, SJW, and GBE on CYP3A4 gene reporter activity were confirmed in HepG2-derived cells stably expressing the CYP3A4 gene reporter (3-1-20 cells) as reported previously [22]. SJW and GBE served as positive controls for CYP3A4 induction. Similar to SJW and GBE, Ban-Lan-Gen extract increased CYP3A4 gene reporter activity, increasing luminescence 11-fold over control levels (Fig. 1). Furthermore, Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner (Fig. 2). 3.2. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription We next examined the effect of components of Ban-Lan-Gen on CYP3A4 gene reporter activity. RIF was used as a positive control. 31-20 cells were treated with RIF (5 mM) or various Ban-Lan-Gen components (5 mM), such as indirubin, indigo, isatin, indicant, and tryptanthrin, for 48 h. The CYP3A4 gene reporter activity was significantly elevated in response to indirubin, and the induction level reached the same level as that of RIF (Fig. 3). Indirubin increased reporter activity in a dose-dependent manner (Fig. 4). We further investigated the effect of indirubin on intrinsic CYP3A4 mRNA expression in HepG2 cells. In these cells, indirubin increased CYP3A4 mRNA expression to a similar magnitude as that observed with RIF (Fig. 5). Previous studies reported that indirubin is a prototypical AhR ligand [25]. In this study, indirubin also resulted in an increase in CYP1A1 mRNA expression. Furthermore, we investigated the effect of indirubin on CYP3A4 mRNA expression in human hepatocyte. When human cryopreserved hepatocyte was incubated with 5 mM of RIF and indirubin, CYP3A4 mRNA expression was increased 6.3-fold by RIF compared with untreated cells, but not by indirubin (data not shown).
Fig. 1. Effects of SJW, GBE, and Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-120 cells. Clone 3-1-20 was seeded at 3 104 cells in 24-well tissue culture plates with 0.3 mL of DMEM, 24 h before SJW, GBE, and Ban-Lan-Gen extract treatment. The cells were treated with SJW (0.1 mg/mL), GBE (0.5 mg/mL), and Ban-Lan-Gen extract (10 mg/mL) for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicletreated cells. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, difference from the vehicle-treated cells.
3.3. Transcriptional activation of CYP3A4 reporter genes by indirubin To elucidate the molecular mechanism of CYP3A4 gene transcriptional activation by indirubin, we performed transient transfection assays with reporter gene constructs containing mutations in the hPXR binding elements. As shown in Fig. 6, treatment of HepG2 cells with 5 mM indirubin or RIF resulted in an increase in luciferase activity (18.1-fold and 32.9-fold, respectively) after transfection of pCYP3A4-362-7.7k, which included the CYP3A4 50 flanking regions dNR1, eNR3A4, and prER6, all known to be highly involved in the transcriptional regulation of the CYP3A4 gene [14]. Mutation of either dNR1 (pCYP3A4-362-7.7km) or prER6 (pCYP3A4-362m-7.7k) of pCYP3A4-362-7.7k did not abolish the indirubin response completely (6.8- and 13.9-fold, respectively).
Table 1 Sequences of the oligonucleotide primers used for PCR amplification. Primer sequence Human CYP3A4 Sense Antisense Human CYP1A1 Sense Antisense Human GAPDH Sense Antisense
50 -CTGTGTGTTTCCAAGAGAAGTTAC-30 50 -TGCATCAATTTCCTCCTGCAG-30 50 -CATCCCCCACAGCACAACA-30 50 -CAGGGGTGAGAAACCGTTCA-30 50 -CATGGGTGTGAACCATGAGAA-30 50 -GGTCATGAGTCCTTCCACGAT-30
Fig. 2. Effect of various concentrations of Ban-Lan-Gen on the CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3 104 cells in 24-well tissue culture plates with 0.3 mL of DMEM 24 h before Ban-Lan-Gen treatment. The cells were treated with 1e30 mg/mL Ban-Lan-Gen for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments.
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
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Fig. 3. Effect of various components of Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3 104 cells in 48-well tissue culture plates with 0.3 mL DMEM, 24 h before Ban-Lan-Gen treatment. The cells were treated with each 5 mM of indirubin, indigo, isatin, indicant, tryptanthrin, and RIF for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, **P < 0.05, difference from the vehicle-treated cells.
However, the response was significantly decreased by the introduction of a mutation into the a half-site of eNR3A4 (pCYP3A4-3627.7kmDa) of pCYP3A4-362-7.7k (2.5-fold). RIF, in contrast, significantly decreased the response in cells transfected with the mutated pCYP3A4-362-7.7ks in dNR1, prER6, or eNR3A4 motifs (3.4-, 9.4and 0.8-fold, respectively). 3.4. Indirubin increases CYP3A4 gene reporter activity through the PXR pathway PXR is widely known as a major transcription factor mediating CYP3A4 induction. To clarify whether indirubin activates the CYP3A4 reporter gene via this pathway, we utilized knockdown of PXR using AdhPXR-shRNA and overexpression of PXR using AdhPXR (hPXR-expressing adenovirus). Indirubin-induced CYP3A4 gene reporter activities were significantly increased by the introduction of AdhPXR (Fig. 7). In addition, CYP3A4 gene reporter activation after treatment with indirubin, as well as RIF, was significantly decreased by the introduction of AdhPXR-shRNA in 31-20 cells (Fig. 8).
the CYP3A4 reporter gene (Figs. 1 and 2). These results indicate that Ban-Lan-Gen extract induces CYP3A4 gene expression. In addition, indirubin, a biological ingredient found in Ban-Lan-Gen, increased CYP3A4 gene reporter activity in a dose-dependent manner (Fig. 4) and increased CYP3A4 mRNA expression (Fig. 5). Although indirubin activated CYP3A4 gene transcription in HepG2 cells, it did not increase CYP3A4 mRNA expression in human hepatocytes. A major difference between these two cells types is cell proliferation. HepG2 cells grow without limitation, but human hepatocytes dose not. It is known that indirubin is a potent inhibitor of cyclindependent kinase 2 (CDK2), a modulator of cell cycle [28]. In addition, Sivertsson et al. reported that the CYP3A4 induction in confluent Huh 7 cells was regulated by the endogenous activation of PXR as a result of decreased CDK2 activity, which was linked to reduced cell proliferation in the confluent cell culture [29]. Therefore, the difference in CYP3A4 induction by indirubin between HepG2 cells and the human hepatocytes may be due to CDK2 activity. Further studies are needed to resolve this issue. CYP3A4 induction by xenobiotics and hormones is mediated by the PXR [30e32], the constitutive androstane receptor (CAR, NR1I3) [33], the vitamin D receptor (VDR, NR1I1) [34], and the
4. Discussion Herbal medicines and supplements are used worldwide [26]. Because the safety and efficacy of herbedrug combinations are largely unknown, research on herbedrug interactions is urgently needed to guide the therapeutic use of herbs [27]. Metabolismmediated herbedrug interactions are very common and can decrease the efficacy or increase the toxicity of the drug. Although various studies have described the actions of Ban-Lan-Gen [3,4], there are few reports on Ban-Lan-Genedrug interactions. In this study, we investigated the effect of Ban-Lan-Gen on transactivation of the CYP3A4 gene using 3-1-20 cells, which have a CYP3A4 reporter gene incorporated into their chromosomes. In general, there is a plurality of the extraction method of herbal medicine such as Ban-Lan-Gen. Thus, it is considered that the difference of the extraction method of Ban-Lan-Gen might be reflected on the CYP3A4 induction activity. However, to mimic extraction of components from herbal medicine and their absorption in human intestine, we used the extraction from the Ban-LanGen product extracted with culture medium for CYP3A4 induction study. As a result, Ban-Lan-Gen extract dose-dependently activated
Fig. 4. Dose effect of indirubin on the CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3 104 cells in 24-well tissue culture plates with 0.3 mL of DMEM 1 day before indirubin treatment. The cells were treated with 1e20 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments.
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
T. Kumagai et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e7
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Fig. 5. Effect of indirubin on CYP mRNA expression in HepG2 cells. HepG2 cells were seeded at 5 104 cells in 24-well tissue culture plates with 0.5 mL DMEM, 24 h before RIF and indirubin treatment. The cells were treated with 5 mM RIF and 5 mM indirubin for 48 h and then assayed using quantitative polymerase chain reaction (qPCR). CYP3A4 mRNA or CYP1A1 mRNA expression was normalized by expression of GAPDH housekeeping gene and presented as fold increase compared with that of vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments. (A) CYP3A4 mRNA level, (B) CYP1A1 mRNA level. *P < 0.005, **P < 0.01, difference from the vehicle-treated cells.
glucocorticoid receptor-a (GRa, NR3C1) [35] in the liver. Chemically-induced activation of the CYP3A4 gene is mainly mediated by PXR binding to the CYP3A4 50 -flanking region [30,32,36]. Therefore, we knocked down hPXR expression using AdhPXR-shRNA. When AdhPXR-shRNA was introduced into the 31-20 cells, the activation of the CYP3A4 reporter gene by indirubin, as well as by RIF, was significantly decreased (Fig. 8). Furthermore, the overexpression of PXR using AdhPXR increased CYP3A4 reporter activity by indirubin in an infection dose-dependent manner (Fig. 7). These results strongly suggest that indirubin enhanced the transactivation of the CYP3A4 gene through PXR activation.
Since indirubin is a natural AhR ligand isolated from human urine [7], it is possible that indirubin activation of the CYP3A4 gene is through the AhR pathway. We previously demonstrated that polycyclic aromatic hydrocarbons (PAHs) enhance the transactivation of the CYP3A4 gene through PXR activation but not AhR activation [24]. Recently, Luckert et al. also reported that PAHs and their metabolites induce CYP3A4 gene expression via PXR [37]. Thus, indirubin and other PAHs activate CYP3A4 gene transcription through the PXR pathway but not the AhR pathway. A number of independent studies have examined the relative contribution of the individual functional PXR-binding motifs (dNR1,
Fig. 6. Mutational analysis of putative human PXR responsive elements in the CYP3A4 gene. Schematic structures of reporter plasmids used are shown in the middle. Closed boxes represent mutated PXR binding elements. HepG2 cells seeded at 3 104 cells in 48-well tissue culture plates pre-incubated for 24 h before transfection. Transfection into HepG2 cells was performed as described in the Materials and methods section. The cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, **P < 0.05, difference from the indirubin- or RIF-treated cells transfected with the pCYP3A4-362-7.7k.
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
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(dNR1, prER6, and eNR3A4). Notably, the introduction of a mutation into eNR3A4 significantly decreased the indirubin-induced transcriptional activation, whereas mutations of dNR1 and prER6 did not (Fig. 6). These results indicate that eNR3A4 might be involved in the activation of CYP3A4 gene through PXR by indirubin. In conclusion, we have demonstrated that Ban-Lan-Gen activated CYP3A4 gene transcription and that indirubin, a component of Ban-Lan-Gen, played an important role in CYP3A4 gene transcription. In addition, we have shown that indirubin activated CYP3A4 gene through the activation of PXR in HepG2 cells. More detailed studies on the molecular mechanism of CYP3A4 induction by indirubin may provide important information on the herbedrug and drugedrug interactions.
Fig. 7. Effect of PXR on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 2 104 cells in 24-well tissue culture plates pre-incubated for 24 h and then treated with AdhPXR (MOI of 0, 1, 3, and 10). Forty-eight hours after infection, these cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells uninfected with AdhPXR. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, difference from the corresponding AdLacZ-infected cells.
prER6, and eNR3A4) on PXR-mediated transactivation of the CYP3A4 gene. In particular, a novel functional cis-acting PXRbinding element designated eNR3A4 has been discovered to be essential for RIF-inducible CYP3A4 transactivation in human liver [14]. This element is located approximately 7.6 kb upstream from the transcription initiation site of the CYP3A4 gene to which PXR binds as a heterodimer with RXRa. Furthermore, Pavek et al. reported that eNR3A4 has negligible or no effect on CYP3A4 transactivation through VDR [38]. These findings strongly suggest that eNR3A4 is a key regulatory element for the xenobiotic induction of CYP3A4 through PXR in the liver. Thus, we investigated the involvement of eNR3A4 in the transactivation of the CYP3A4 through PXR activation by indirubin with the CYP3A4 reporter gene constructs that have a mutation in three PXR response elements
Fig. 8. Effects of PXR-shRNA on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 31-20 was seeded at 2 104 cells in 24-well tissue culture plates pre-incubated for 24 h and then treated with AdhPXR-shRNA (MOI of 0, 0.3, 1, and 3). Forty-eight hours after infection, these cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells uninfected with AdhPXRshRNA. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, **P < 0.01, difference from the corresponding AdLacZ-infected cells.
Conflict of interest None. Acknowledgments This work was supported by a Health and Labour Sciences Research Grant for Research on Food Safety (No. 22230301) from the Ministry of Health, Labour and Welfare, Japan. We would like to thank Ms. Machi Miura for the excellent technical assistance provided. References [1] Fugh-Berman A. Herbedrug interactions. Lancet 2000;355:134e8. [2] Hu Z, Yang X, Ho PC, Chan SY, Heng PW, Chan E, et al. Herbedrug interaction: a literature review. Drugs 2005;65:1239e82. [3] Hamburger M. Isatis tinctoria e from the rediscovery of an ancient medical plant towards a novel anti-inflammatory phytopharmaceutical. Phytochem Rev 2002;1:333e44. [4] Ho YL, Chang YS. Studies on the antinociceptive, anti-inflammatory and anti pyretic effects of Isatis indigotica root. Phytomedicine 2002;9:419e24. [5] Honda G, Tosirisuk V, Tabata M. Isolation of an antidermatophytic, tryptanthrin, from indigo plants, Polygonum tinctorium and Isatis tinctoria. Planta Med 1980;38:275e6. [6] Liau BC, Jong TT, Lee MR, Chen SS. LCeAPCI-MS method for detection and analysis of trypthanthrin, indigo, and indirubin in daqingye and banlangen. J Pharm Biomed Anal 2007;43:346e51. [7] Adachi J, Mori Y, Matsui S, Takigami H, Fujino J, Kitagawa H, et al. Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. J Biol Chem 2001;276:31475e8. [8] Gonzalez FJ. Human cytochromes P450: problem and prospects. Trends Pharmacol Sci 1992;13:346e52. [9] Nerbert DW, Russell DW. Clinical importance of cytochromes P450. Lancet 2002;360:1155e62. [10] Thummel KE, Wilkinson GR. In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Phamacol Toxicol 1998;38:389e430. [11] Quattrochi LC, Guzelian PS. CYP3A regulation: from pharmacology to nuclear receptors. Drug Metab Dispos 2001;29:615e22. [12] Thelen K, Dressman JB. Cytochrome P450-mediated metabolism in the human gut wall. J Pharm Pharmacol 2009;61:541e58. [13] Tolson AH, Wang H. Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Adv Drug Deliv Rev 2010;62:1238e49. [14] Toriyabe T, Nagata K, Takada T, Aratsu Y, Matsubara T, Yoshinari K, et al. Unveiling a new essential cis element for the transactivation of the CYP3A4 gene by xenobiotics. Mol Pharmacol 2009;75:677e84. [15] Luo G, Cunningham M, Kim S, Burn T, Lin J, Sinz M, et al. CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. Drug Metab Dispos 2002;30: 795e804. [16] Casbar RC, Das PC, DeKrey GK, Gardiner CS, Cao Y, Rose R, et al. Endosulfan induce CYP2B6 and CYP3A4 by activating the pregnane X receptor. Toxicol Appl Pharmacol 2010;245:335e43. [17] Matsubara T, Noracharttiyapot W, Toriyabe T, Yoshinari K, Nagata K, Yamazoe Y. Assessment of human pregnane X receptor involvement in pesticide mediated activation of CYP3A4 gene. Drug Metab Dispos 2007;35: 728e33. [18] Matsubara T, Yoshinari K, Aoyama K, Sugawara M, Sekiya Y, Nagata K, et al. Role of vitamin D receptor in the lithocholic acid-mediated CYP3A induction in vitro and in vivo. Drug Metab Dispos 2008;36:2058e63.
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Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor Takeshi Kumagai a, *, Yusuke Aratsu a, b, Ryosuke Sugawara a, Takamitsu Sasaki a, c, Shinichi Miyairi d, Kiyoshi Nagata a a
Department of Environmental Health Science, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan Drug Metabolism and Pharmacokinetics Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan d Laboratory of Organic Chemistry, School of Pharmacy, Nihon University, 7-7-1 Narashino, Funabashi, Chiba 274-8555, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2015 Received in revised form 14 January 2016 Accepted 14 January 2016 Available online xxx
Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an antiinflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-LanGen, activated CYP3A4 gene transcription through the activation of the human PXR.
Keywords: AhR activator Indirubin CYP3A4 induction PXR Herbedrug interactions
Copyright © 2016, Published by Elsevier Ltd on behalf of The Japanese Society for the Study of Xenobiotics.
1. Introduction Chinese herbal medicines are an integral part of health care in Asia, and many herbal medicines are extensively used as alternative and/or complementary therapies. However, the clinical efficacy, mechanisms of action, and safety profiles of most herbal medicines
Abbreviations: AdCont, b-galactosidase-expressing adenovirus; AdPXR, human PXR-expressing adenovirus; AdhPXR-shRNA, AdhPXR-short hairpin RNA; AhR, aryl hydrocarbon receptor; Ct, threshold PCR cycle; CAR, constitutive androstane receptor; CYP, cytochrome P450; DMEM, Dulbecco's modified Eagle's medium; dNR1, distal nuclear receptor-binding protein 1; DMSO, dimethyl sulfoxide; D-PBS, Dulbecco's phosphate buffered saline; DR3, direct repeats spaced by 3 bases; eNR3A4, essential distal nuclear receptor-binding element; ER6, everted repeats separated by 6 bases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBE, Ginkgo biloba extract; GRa, glucocorticoid receptor-a; MOI, multiplicity of infection; PAHs, polycyclic aromatic hydrocarbons; prER6, ER6 in the CYP3A4 proximal promoter; PXR, pregnane X receptor; RXRa, retinoid X receptor a; SJW, St. John's wort; TCID50, 50% titer culture infectious dose; VDR, vitamin D receptor. * Corresponding author. E-mail address: [email protected] (T. Kumagai).
are not well characterized. Moreover, herbal medicines are often co-administered with therapeutic drugs, creating the potential for herbedrug interactions [1,2]. While the underlying mechanism of most herbedrug interactions is not known, induction and inhibition of drug-metabolizing enzymes or drug transporters have been implicated as potential mechanisms. Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Indigo plants have been an important source of dyes in Asia since ancient times, and today, they are commonly used as therapeutics in traditional Chinese medicine. Ban-Lan-Gen is frequently used as an anti-inflammatory and antiviral for the treatment of hepatitis, influenza, and various kinds of inflammation [3,4]. Several biologically active ingredients have been identified in Ban-Lan-Gen, including indigoid alkaloids (e.g., indigo and indirubin) and quinazolinone alkaloids (e.g., tryptanthrin) [5]. In fact, indigo, indirubin, and tryptanthrin are three marker compounds found in Ban-Lan-Gen [6]. Among these marker compounds, it has been reported that indirubin causes transactivation of cytochrome P450 1A1 (CYP1A1) and CYP1A2 genes via
http://dx.doi.org/10.1016/j.dmpk.2016.01.002 1347-4367/Copyright © 2016, Published by Elsevier Ltd on behalf of The Japanese Society for the Study of Xenobiotics.
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the aryl hydrocarbon receptor (AhR) [7]. In addition, a content of indirubin have been reported in the roots of I. indigotica as main raw materials of Ban-Lan-Gen (0.224e34.4 mg/g) [5,6]. Members of the CYP supergene family of monooxygenases play an important role in detoxification by converting pollutants, plant toxins, carcinogens, and drugs to products that are then excreted in to urine or bile [8,9]. Human CYP3A4 is of particular significance in this respect, because it is involved in the metabolism of approximately two-thirds of clinically relevant drugs [10]. A number of compounds, including pesticides, herbal supplements, vitamins, and drugs, activate CYP3A4 gene transcription both in the liver and in the small intestine [11,12]. This induction process is the molecular basis for a number of important drug interactions that occur in patients taking multiple medications. The pregnane X receptor (PXR; NR1I2) is the principal regulator of CYP3A4 gene expression and binds as a heterodimer with retinoid X receptor a (RXRa) to regulatory DNA sequences. These sequences include: (1) AG(G/T)TCA-like direct repeats spaced by 3 bases (DR3) located 8 kb upstream from the transcription start point and identified as distal nuclear receptor-binding element 1 (dNR1), and (2) everted repeats separated by 6 bases (ER6) located in the CYP3A4 proximal promoter (prER6) [13]. Recently, Toriyabe et al. identified a distinct PXR response element as an essential distal nuclear receptor-binding element (eNR3A4) for CYP3A4 gene induction [14]. PXR is activated by a number of structurally and chemically diverse ligands, such as drugs [rifampicin (RIF), clotrimazole] [15], pesticides (pyributicarb, endosulfan) [16,17], natural and synthetic steroids (dexamethasone) [15], bile acids (lithocholic acid) [18], and herbal medicines (St. John's wort [SJW], Ginkgo biloba, and Sophora flavescens) [19e21]. CYP3A4 induction by these PXR activators leads to accelerated metabolism of the activators themselves and concomitantly-administered drugs that are metabolized by CYP3A4. Induction and inhibition of CYP3A4 by Ban-Lan-Gen are not fully understood. In this study, we investigated the effect of Ban-LanGen on transcriptional activation of the CYP3A4 gene. 2. Materials and methods 2.1. Materials Ban-Lan-Gen (lot. 9115476) was purchased from Beijing Tong Ren Group Co., Ltd. (Beijing, China). SJW was purchased from ChromaDex (Irvine, CA). Indirubin and tryptanthrin were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY). Indican, isatin, and indigo were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ginkgo biloba extract (GBE) was purchased from Tama Biochemicals Co., Ltd (Tokyo, Japan). RIF was purchased from SigmaeAldrich (St. Louis, MO). The chemicals used for this study were dissolved in dimethyl sulfoxide (DMSO). Ban-Lan-Gen, SJW, and GBE were extracted with culture medium used for the cell culture in this study, for 2 h at 37 C. These extract solutions were centrifuged at 2,000 g for 15 min at 4 C and the supernatants were used for this study. All other reagents used were of the highest quality available. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Hyclone Laboratories (Logan, UT), respectively. MEM non-essential amino acids and antibiotic-antimycotic were purchased from Invitrogen (Paisley, UK). 2.2. Cell culture HepG2 cells were obtained from RIKEN cell bank (Tsukuba, Japan). The HepG2-derived cell line stably expressing the CYP3A4-
luciferase reporter gene, clone 3-1-20, was maintained as reported previously [22]. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, MEM non-essential amino acids, and antibiotic-antimycotic. The cells were seeded at 3 104 cells per well onto 48-well tissue culture plate (BD Biosciences, Heidelberg, Germany) for luciferase gene reporter assay, and 5 104 cells per well onto 24-well tissue culture plate (BD Biosciences) for real-time polymerase chain reaction (PCR). After 24 h, the cell medium was changed with the extract solutions or various subjects dissolved in DMSO (final concentration, 0.1%) and cultured for 48 h. 2.3. Luciferase gene reporter assay 3-1-20 cells were washed with Dulbecco's phosphate buffered saline (D-PBS) and suspended in passive lysis buffer (Promega, Madison, WI) in a microcentrifuge tube. The cell suspension was centrifuged at 12,000 g for 5 min at 4 C, and the cell extract was used for the luciferase assay. The luciferase assay was performed using the Luciferase Assay System and a GloMax™ 96 Microplate Luminometer (Promega) according to the manufacturer's instructions. The resulting data are presented as the ratio of luminescence of treated cell samples to that of control. The luminescence of each sample was normalized by its protein concentration as determined with the Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). 2.4. Reporter gene constructs and transient transfection The luciferase reporter plasmids, pGL3-Basic and pGL4.70, were purchased from Promega. Preparation of the CYP3A4 luciferase reporter gene constructs, including pCYP3A4-362-7.7k, pCYP3A4362-7.7km, pCYP3A4-362m-7.7k, pCYP3A4-362m-7.7km, and pCYP3A4-362-7.7kmDa, was described previously [14]. One day before transfection, cells were seeded in 48-well plates. Each reporter plasmid and pGL4.70 was transfected using Targefect F-1 (Targeting System, El Cajon, CA) according to the manufacturer's protocol. pGL4.70 was used for the normalization of transfection efficiency. After transfection, cells were cultured in medium in the presence of various chemicals for 48 h. Control cells were cultured with vehicle (0.1% DMSO) alone. Subsequently, the cells were harvested and suspended in passive lysis buffer (Promega). Luciferase activities were determined with Dual-Luciferase® Reporter Assay System (Promega). 2.5. Construction of recombinant adenovirus and infection Construction of the human PXR-expressing adenovirus (AdhPXR) and AdhPXR-short hairpin RNA (AdhPXR-shRNA) was described previously [17]. Control adenovirus, a b-galactosidaseexpressing adenovirus (AdCont; AxCALacZ), was provided by Dr. Izumi Saito (Tokyo University, Japan) [23]. The titer of the adenoviruses, 50% titer culture infectious dose (TCID50), was determined as reported previously [17]. Multiplicity of infection (MOI) was calculated by dividing the TCID50 by the number of cells. One day before transfection, cells were seeded in 24-well plates. Adenovirus infection was carried out as described previously [24]. 2.6. Isolation of RNA and analysis of quantitative real-time polymerase chain reaction Total RNA was isolated from HepG2 cells using TRI Reagent (Molecular Research Center, Inc., Montgomery, AL) according to the manufacturer's protocol. cDNA was prepared from 2.0 mg of total RNA with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega) using oligo(dT)20 primer (Greiner Japan, Japan)
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
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and porcine RNase inhibitor (TaKaRa Bio, Japan). Quantitative realtime PCR was performed using Permix Ex Taq (Perfect Real Time, TaKaRa Bio) in a Thermal Cycler Dice Real Time System (TaKaRa Bio). All samples were quantified using a comparative threshold PCR cycle (Ct) method for relative quantification of gene expression, normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers used are shown in Table 1. 2.7. Statistical analysis Data are presented as mean ± standard deviation (S.D.) and were evaluated by paired Student's t-test. A P value < 0.05 was considered to be significant. 3. Results 3.1. Ban-Lan-Gen increases CYP3A4 gene reporter activity The effects of Ban-Lan-Gen, SJW, and GBE on CYP3A4 gene reporter activity were confirmed in HepG2-derived cells stably expressing the CYP3A4 gene reporter (3-1-20 cells) as reported previously [22]. SJW and GBE served as positive controls for CYP3A4 induction. Similar to SJW and GBE, Ban-Lan-Gen extract increased CYP3A4 gene reporter activity, increasing luminescence 11-fold over control levels (Fig. 1). Furthermore, Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner (Fig. 2). 3.2. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription We next examined the effect of components of Ban-Lan-Gen on CYP3A4 gene reporter activity. RIF was used as a positive control. 31-20 cells were treated with RIF (5 mM) or various Ban-Lan-Gen components (5 mM), such as indirubin, indigo, isatin, indicant, and tryptanthrin, for 48 h. The CYP3A4 gene reporter activity was significantly elevated in response to indirubin, and the induction level reached the same level as that of RIF (Fig. 3). Indirubin increased reporter activity in a dose-dependent manner (Fig. 4). We further investigated the effect of indirubin on intrinsic CYP3A4 mRNA expression in HepG2 cells. In these cells, indirubin increased CYP3A4 mRNA expression to a similar magnitude as that observed with RIF (Fig. 5). Previous studies reported that indirubin is a prototypical AhR ligand [25]. In this study, indirubin also resulted in an increase in CYP1A1 mRNA expression. Furthermore, we investigated the effect of indirubin on CYP3A4 mRNA expression in human hepatocyte. When human cryopreserved hepatocyte was incubated with 5 mM of RIF and indirubin, CYP3A4 mRNA expression was increased 6.3-fold by RIF compared with untreated cells, but not by indirubin (data not shown).
Fig. 1. Effects of SJW, GBE, and Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-120 cells. Clone 3-1-20 was seeded at 3 104 cells in 24-well tissue culture plates with 0.3 mL of DMEM, 24 h before SJW, GBE, and Ban-Lan-Gen extract treatment. The cells were treated with SJW (0.1 mg/mL), GBE (0.5 mg/mL), and Ban-Lan-Gen extract (10 mg/mL) for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicletreated cells. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, difference from the vehicle-treated cells.
3.3. Transcriptional activation of CYP3A4 reporter genes by indirubin To elucidate the molecular mechanism of CYP3A4 gene transcriptional activation by indirubin, we performed transient transfection assays with reporter gene constructs containing mutations in the hPXR binding elements. As shown in Fig. 6, treatment of HepG2 cells with 5 mM indirubin or RIF resulted in an increase in luciferase activity (18.1-fold and 32.9-fold, respectively) after transfection of pCYP3A4-362-7.7k, which included the CYP3A4 50 flanking regions dNR1, eNR3A4, and prER6, all known to be highly involved in the transcriptional regulation of the CYP3A4 gene [14]. Mutation of either dNR1 (pCYP3A4-362-7.7km) or prER6 (pCYP3A4-362m-7.7k) of pCYP3A4-362-7.7k did not abolish the indirubin response completely (6.8- and 13.9-fold, respectively).
Table 1 Sequences of the oligonucleotide primers used for PCR amplification. Primer sequence Human CYP3A4 Sense Antisense Human CYP1A1 Sense Antisense Human GAPDH Sense Antisense
50 -CTGTGTGTTTCCAAGAGAAGTTAC-30 50 -TGCATCAATTTCCTCCTGCAG-30 50 -CATCCCCCACAGCACAACA-30 50 -CAGGGGTGAGAAACCGTTCA-30 50 -CATGGGTGTGAACCATGAGAA-30 50 -GGTCATGAGTCCTTCCACGAT-30
Fig. 2. Effect of various concentrations of Ban-Lan-Gen on the CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3 104 cells in 24-well tissue culture plates with 0.3 mL of DMEM 24 h before Ban-Lan-Gen treatment. The cells were treated with 1e30 mg/mL Ban-Lan-Gen for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments.
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Fig. 3. Effect of various components of Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3 104 cells in 48-well tissue culture plates with 0.3 mL DMEM, 24 h before Ban-Lan-Gen treatment. The cells were treated with each 5 mM of indirubin, indigo, isatin, indicant, tryptanthrin, and RIF for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, **P < 0.05, difference from the vehicle-treated cells.
However, the response was significantly decreased by the introduction of a mutation into the a half-site of eNR3A4 (pCYP3A4-3627.7kmDa) of pCYP3A4-362-7.7k (2.5-fold). RIF, in contrast, significantly decreased the response in cells transfected with the mutated pCYP3A4-362-7.7ks in dNR1, prER6, or eNR3A4 motifs (3.4-, 9.4and 0.8-fold, respectively). 3.4. Indirubin increases CYP3A4 gene reporter activity through the PXR pathway PXR is widely known as a major transcription factor mediating CYP3A4 induction. To clarify whether indirubin activates the CYP3A4 reporter gene via this pathway, we utilized knockdown of PXR using AdhPXR-shRNA and overexpression of PXR using AdhPXR (hPXR-expressing adenovirus). Indirubin-induced CYP3A4 gene reporter activities were significantly increased by the introduction of AdhPXR (Fig. 7). In addition, CYP3A4 gene reporter activation after treatment with indirubin, as well as RIF, was significantly decreased by the introduction of AdhPXR-shRNA in 31-20 cells (Fig. 8).
the CYP3A4 reporter gene (Figs. 1 and 2). These results indicate that Ban-Lan-Gen extract induces CYP3A4 gene expression. In addition, indirubin, a biological ingredient found in Ban-Lan-Gen, increased CYP3A4 gene reporter activity in a dose-dependent manner (Fig. 4) and increased CYP3A4 mRNA expression (Fig. 5). Although indirubin activated CYP3A4 gene transcription in HepG2 cells, it did not increase CYP3A4 mRNA expression in human hepatocytes. A major difference between these two cells types is cell proliferation. HepG2 cells grow without limitation, but human hepatocytes dose not. It is known that indirubin is a potent inhibitor of cyclindependent kinase 2 (CDK2), a modulator of cell cycle [28]. In addition, Sivertsson et al. reported that the CYP3A4 induction in confluent Huh 7 cells was regulated by the endogenous activation of PXR as a result of decreased CDK2 activity, which was linked to reduced cell proliferation in the confluent cell culture [29]. Therefore, the difference in CYP3A4 induction by indirubin between HepG2 cells and the human hepatocytes may be due to CDK2 activity. Further studies are needed to resolve this issue. CYP3A4 induction by xenobiotics and hormones is mediated by the PXR [30e32], the constitutive androstane receptor (CAR, NR1I3) [33], the vitamin D receptor (VDR, NR1I1) [34], and the
4. Discussion Herbal medicines and supplements are used worldwide [26]. Because the safety and efficacy of herbedrug combinations are largely unknown, research on herbedrug interactions is urgently needed to guide the therapeutic use of herbs [27]. Metabolismmediated herbedrug interactions are very common and can decrease the efficacy or increase the toxicity of the drug. Although various studies have described the actions of Ban-Lan-Gen [3,4], there are few reports on Ban-Lan-Genedrug interactions. In this study, we investigated the effect of Ban-Lan-Gen on transactivation of the CYP3A4 gene using 3-1-20 cells, which have a CYP3A4 reporter gene incorporated into their chromosomes. In general, there is a plurality of the extraction method of herbal medicine such as Ban-Lan-Gen. Thus, it is considered that the difference of the extraction method of Ban-Lan-Gen might be reflected on the CYP3A4 induction activity. However, to mimic extraction of components from herbal medicine and their absorption in human intestine, we used the extraction from the Ban-LanGen product extracted with culture medium for CYP3A4 induction study. As a result, Ban-Lan-Gen extract dose-dependently activated
Fig. 4. Dose effect of indirubin on the CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 3 104 cells in 24-well tissue culture plates with 0.3 mL of DMEM 1 day before indirubin treatment. The cells were treated with 1e20 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments.
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
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Fig. 5. Effect of indirubin on CYP mRNA expression in HepG2 cells. HepG2 cells were seeded at 5 104 cells in 24-well tissue culture plates with 0.5 mL DMEM, 24 h before RIF and indirubin treatment. The cells were treated with 5 mM RIF and 5 mM indirubin for 48 h and then assayed using quantitative polymerase chain reaction (qPCR). CYP3A4 mRNA or CYP1A1 mRNA expression was normalized by expression of GAPDH housekeeping gene and presented as fold increase compared with that of vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments. (A) CYP3A4 mRNA level, (B) CYP1A1 mRNA level. *P < 0.005, **P < 0.01, difference from the vehicle-treated cells.
glucocorticoid receptor-a (GRa, NR3C1) [35] in the liver. Chemically-induced activation of the CYP3A4 gene is mainly mediated by PXR binding to the CYP3A4 50 -flanking region [30,32,36]. Therefore, we knocked down hPXR expression using AdhPXR-shRNA. When AdhPXR-shRNA was introduced into the 31-20 cells, the activation of the CYP3A4 reporter gene by indirubin, as well as by RIF, was significantly decreased (Fig. 8). Furthermore, the overexpression of PXR using AdhPXR increased CYP3A4 reporter activity by indirubin in an infection dose-dependent manner (Fig. 7). These results strongly suggest that indirubin enhanced the transactivation of the CYP3A4 gene through PXR activation.
Since indirubin is a natural AhR ligand isolated from human urine [7], it is possible that indirubin activation of the CYP3A4 gene is through the AhR pathway. We previously demonstrated that polycyclic aromatic hydrocarbons (PAHs) enhance the transactivation of the CYP3A4 gene through PXR activation but not AhR activation [24]. Recently, Luckert et al. also reported that PAHs and their metabolites induce CYP3A4 gene expression via PXR [37]. Thus, indirubin and other PAHs activate CYP3A4 gene transcription through the PXR pathway but not the AhR pathway. A number of independent studies have examined the relative contribution of the individual functional PXR-binding motifs (dNR1,
Fig. 6. Mutational analysis of putative human PXR responsive elements in the CYP3A4 gene. Schematic structures of reporter plasmids used are shown in the middle. Closed boxes represent mutated PXR binding elements. HepG2 cells seeded at 3 104 cells in 48-well tissue culture plates pre-incubated for 24 h before transfection. Transfection into HepG2 cells was performed as described in the Materials and methods section. The cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, **P < 0.05, difference from the indirubin- or RIF-treated cells transfected with the pCYP3A4-362-7.7k.
Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002
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T. Kumagai et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e7
(dNR1, prER6, and eNR3A4). Notably, the introduction of a mutation into eNR3A4 significantly decreased the indirubin-induced transcriptional activation, whereas mutations of dNR1 and prER6 did not (Fig. 6). These results indicate that eNR3A4 might be involved in the activation of CYP3A4 gene through PXR by indirubin. In conclusion, we have demonstrated that Ban-Lan-Gen activated CYP3A4 gene transcription and that indirubin, a component of Ban-Lan-Gen, played an important role in CYP3A4 gene transcription. In addition, we have shown that indirubin activated CYP3A4 gene through the activation of PXR in HepG2 cells. More detailed studies on the molecular mechanism of CYP3A4 induction by indirubin may provide important information on the herbedrug and drugedrug interactions.
Fig. 7. Effect of PXR on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 3-1-20 was seeded at 2 104 cells in 24-well tissue culture plates pre-incubated for 24 h and then treated with AdhPXR (MOI of 0, 1, 3, and 10). Forty-eight hours after infection, these cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells uninfected with AdhPXR. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, difference from the corresponding AdLacZ-infected cells.
prER6, and eNR3A4) on PXR-mediated transactivation of the CYP3A4 gene. In particular, a novel functional cis-acting PXRbinding element designated eNR3A4 has been discovered to be essential for RIF-inducible CYP3A4 transactivation in human liver [14]. This element is located approximately 7.6 kb upstream from the transcription initiation site of the CYP3A4 gene to which PXR binds as a heterodimer with RXRa. Furthermore, Pavek et al. reported that eNR3A4 has negligible or no effect on CYP3A4 transactivation through VDR [38]. These findings strongly suggest that eNR3A4 is a key regulatory element for the xenobiotic induction of CYP3A4 through PXR in the liver. Thus, we investigated the involvement of eNR3A4 in the transactivation of the CYP3A4 through PXR activation by indirubin with the CYP3A4 reporter gene constructs that have a mutation in three PXR response elements
Fig. 8. Effects of PXR-shRNA on CYP3A4 gene reporter activity in 3-1-20 cells. Clone 31-20 was seeded at 2 104 cells in 24-well tissue culture plates pre-incubated for 24 h and then treated with AdhPXR-shRNA (MOI of 0, 0.3, 1, and 3). Forty-eight hours after infection, these cells were treated with 5 mM RIF and 5 mM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells uninfected with AdhPXRshRNA. Results represent the mean ± S.D. of three separate experiments. *P < 0.005, **P < 0.01, difference from the corresponding AdLacZ-infected cells.
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Please cite this article in press as: Kumagai T, et al., Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor, Drug Metabolism and Pharmacokinetics (2016), http://dx.doi.org/10.1016/j.dmpk.2016.01.002