BIOPHARMACEUTICS & DRUG DISPOSITION Biopharm. Drug Dispos. 36: 232–244 (2015) Published online 11 February 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/bdd.1936
The molecular mechanism underlying the induction of hepatic MRP3 expression and function by omeprazole‡ Yu-Qin Pana,†, Qiong-Yu Mia,†, Bang-Shun Hea, Shu-Li Zhaoa, Ting Taia, and Hong-Guang Xiea,b,* a
General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China Department of Pharmacology, Nanjing Medical University School of Pharmacy, Nanjing 210034, China
b
ABSTRACT: Previous work has indicated that there is increased protein expression of multidrug resistance-associated protein 3 (MRP3) in the liver samples of patients treated with omeprazole compared with those who were not. However, evidence is still lacking to show the mechanisms underlying that induction. This study aimed to assess changes in the fold-induction of MRP3 mRNA and protein expression over controls in omeprazole-treated HepG2 cells after transient transfection of human MRP3 siRNA, or after pretreatment with actinomycin D (Act-D). Furthermore, MRP3 siRNA knock-down or MRP-specific inhibition (indomethacin) was used to determine whether the MRP3 protein induced by omeprazole possessed an enhanced efflux transport. The results demonstrated that omeprazole induced MRP3 mRNA and protein expression in a concentration- and timedependent manner. Moreover, that induction was almost completely abolished by the addition of human MRP3 siRNA and also by pretreatment with Act-D, respectively. In addition, the decay rate of MRP3 mRNA in vehicle- and omeprazole-treated cells was similar in the presence of Act-D, suggesting transcriptional up-regulation of MRP3 mRNA expression by omeprazole. Most importantly, omeprazole induced MRP3 efflux transport activity, as measured by the 5-carboxyfluorescein assay in the absence and presence of human MRP3 siRNA or indomethacin. It is concluded that omeprazole can induce MRP3 mRNA and protein expression and enhance MRP3 efflux transport activity through transcriptional up-regulation, and that omeprazole can also induce other MRP transporters. Copyright © 2015 John Wiley & Sons, Ltd. Key words: ABCC3; hepatic efflux transporter; MRP3; omeprazole; transcriptional regulation
Introduction Multidrug resistance-associated protein 3 (also known as MRP3), an ATP-dependent efflux transporter, is encoded by the ABCC3 gene [1,2]. Human MRP3 is predominantly expressed in the *Correspondence to: General Clinical Research Center, Nanjing First Hospital, 68 Changle Road, Nanjing 210006, China. E-mail: [email protected] † These authors contributed equally to this work. ‡ Part of this work was presented at the 114th Annual Meeting of the American Society for Clinical Pharmacology and Therapeutics, 5–9 March 2013, Indianpolis, Indian, USA, and the abstract was published in Clinical Pharmacology and Therapeutics 2013; 93 (suppl 1): S97 (abstract # PIII-29).
Copyright © 2015 John Wiley & Sons, Ltd.
major organs responsible for the disposition of conjugated substances, such as the liver, small intestine and kidney [1–3]. In polarized cells, MRP3 is expressed exclusively in the basolateral membrane of hepatocytes [4,5]. Because hepatic sinusoidal blood is found across the basolateral membrane of hepatocytes, MRP3 mediates the export of drugs and/or their glucuronide metabolites from hepatocytes to blood, consequently increasing their systemic exposure and bioavailability [6–9]. Clearly, MRP3 functions as an important sinusoidal efflux transporter in the liver, and thus plays an important role in drug disposition and response in humans. Received 28 September 2014 Revised 21 December 2014 Accepted 8 January 2015
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An earlier clinical observational study indicated that the expression of hepatic MRP3 is highly variable in humans [10]. Although the amount of hepatic MRP3 is relatively low under physiological conditions [11], a large number of studies have demonstrated that human MRP3 expression is increased markedly in patients with certain diseases [12–16], or in response to some chemicals and drugs [4,13,17–19]. Emerging evidence strongly suggests that MRP3 is highly inducible. Increased expression of hepatic MRP3 mRNA and protein was initially found in 62 liver samples from patients treated with omeprazole and also in omeprazole-treated HepG2 cells compared with those without omeprazole treatment [20]. Although MRP3 mRNA and protein were induced 2.4- and 1.8-fold, respectively, in the HepG2 cells treated with omeprazole [20], no data are available to suggest whether that induction was concentration- and time-dependent, and whether the efflux transport functionality of MRP3 was enhanced, as determined by the use of a subtype-specific transporter probe or RNAi knock-down. Because the potential effects of some diseases and drugs on that induction cannot be excluded in the clinical setting, it is difficult to characterize the specificity and functionality of MRP3 protein induction in patients treated with omeprazole. This work was designed to further determine the specificity and functionality of hepatic MRP3 induction as measured by levels of its mRNA, protein and transport activity in HepG2 cells treated with omeprazole alone or in combination with a transcription inhibitor, MRP-specific inhibitor, MRP-specific fluorescent probe or human MRP3 siRNA, respectively.
Materials and Methods Chemicals and reagents Omeprazole (Ome), indomethacin (Indo), actinomycin D (Act-D), carboxyfluorescein diacetate (5CFDA) and dimethyl sulfoxide (DMSO) were purchased from Sigma (Sigma-Aldrich Co., LLC, Shanghai, China). Lipofectamine 2000 was obtained from Invitrogen-Life Technologies (Grand Island, NY, USA). Human MRP3 siRNA (sc40748) and control siRNA (sc-37007) were provided by Santa Cruz Biotechnology, Inc. (Santa Copyright © 2015 John Wiley & Sons, Ltd.
Cruz, CA, USA). All other chemicals and reagents were of analytical grade or higher and were readily available from commercial sources.
HepG2 cell culture and treatment Human hepatoma HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% FBS (Hyclone, UT, USA), 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin, and kept at 37 °C in 5% CO2 and 95% humidity.
Cell proliferation assay Cell proliferation in vitro was analysed using a cell counting kit-8 (also known as CCK-8) (Sigma-Aldrich Chemicals, St Louis, MO, USA) according to the manufacturer’s protocol. In brief, 1 × 104 cells were plated per well in 96-well microplates in 100 μl medium. After 12, 24, 48 and 72 h of culture of the cells treated with omeprazole at a concentration of 25, 50, 100, and 200 μM, respectively, the CCK-8 solution (10 μl) was added to each well, and the plates were put back in a standardized tissue incubator for another 1.5 h. The absorbance was measured at 450 nm using a microplate reader Bio-Rad 680 (OEM Systems Co. Ltd, Kyoto, Japan). The inhibition rate was calculated as [1 (OD value of the treated cells/OD value of untreated cells)] × 100%. Each experiment was done in triplicate.
Omeprazole induction assay The HepG2 cells were seeded at 1 × 105 cells per 6well cluster tray for omeprazole induction studies. They were grown up to > 60% confluent, and exposed to omeprazole at a concentration of 25, 50, 100 and 200 μM for 12, 24 and 48 h, respectively. Omeprazole was dissolved in DMSO, and a final concentration of 0.1% DMSO (v/v) was used as a vehicle control in the cell culture.
Transient transfection assay The HepG2 cells were seeded at 1 × 106 cells per 6well cluster tray, and incubated at 37 °C in a CO2 incubator until the cells were 60–80% confluent. The cells were transiently transfected with 1 μg per plate of human MRP3 siRNA in the presence Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
234 of lipofectamine 2000 reagent according to the manufacturer’s instructions, and treated with 100 μM omeprazole or 0.1% DMSO at 24 h after transfection.
Actinomycin D inhibition assay To confirm whether induction of MRP3 by omeprazole could be through increased de novo synthesis of mRNA, the transcription inhibitor Act-D (10 μg/ml) was added to the medium 10 min prior to omeprazole treatment (100 μM) or vehicle control (0.1% DMSO) as described elsewhere [21]. The total RNA was isolated after treatment for 6 or 12 h, respectively. Moreover, the biologic half-life (t1/2) of MRP3 mRNA was determined by an Act-D chase assay as described elsewhere [22,23]. In brief, the HepG2 cells were pretreated with omeprazole (100 μM) or vehicle control for 24 h. Thereafter, Act-D was added into the medium at a final concentration of 5 μg/ml for another 24 h to suppress further RNA synthesis in the absence and presence of omeprazole (100 μM). The cells were harvested at 0, 3, 6, 12 and 24 h after the addition of 5 μg/ml Act-D, respectively, the total RNA was isolated and the MRP3 mRNA levels were quantified by quantitative real-time PCR (qRT-PCR) as described elsewhere [24]. The data are represented as the percentage decrease in MRP3 mRNA levels versus time using a semilogarithmic scale. The t1/2 values of mRNA were determined with the use of regression curves as described elsewhere [22,23].
Quantitative real-time PCR assay The total cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After treatment with DNase I (TaKaRa Biotechnology Co., Ltd, Dalian, China) at 37 °C for 30 min, the RNA was quantified using spectrophotometry (Eppendorf BioPhotometer Plus, Hamburg, Germany). The RNA (1 μg) was subsequently incubated with 1 μl Oligo dT primer (50 μM), 1 μl Random 6-mers (100 μM), 1 μl PrimeScript TM RT Enzyme Mix, 4 μl 5 × PrimeScript TM buffer and RNase-free dH2O, and the synthesis of firststrand cDNA was conducted in a total volume of 20 μl. The primer sequences used for the cDNA synthesis of MRP3 and GAPDH are shown elsewhere [24]. The PCR amplification was Copyright © 2015 John Wiley & Sons, Ltd.
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undertaken in a LightCycler apparatus (ABI 7500, USA) using SYBR Premix Ex TaqTM (TaKaRa Biotech Co., Ltd, Dalian, China). Thermocycling was done in a final volume of 20 μl, consisting of 1 μl cDNA sample, 0.8 μl forward primer (10 μM), 0.8 μl reverse primer (10 μM), 10 μl SYBR Premix Ex TaqTM, 0.4 μl ROX Reference Dye and 7 μl dH2O. After 30 s at 95 °C to denature the cDNA and to activate Taq DNA polymerase, the cycling conditions were as follows: 40 cycles consisting of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. The Ct used in the real-time PCR quantification was defined as the PCR cycle number that crossed an arbitrarily chosen signal threshold in the log phase of the amplification curve. To verify accurately the fold-change of MRP3 mRNA expression, the calculated Ct values were normalized against that of β-actin amplified from the same sample (ΔCt = CtMRP3 – CtGAPDH), and the 2-ΔΔCt method was used to calculate the fold change over control [25]. The amplification was monitored on an ABI prism 7500 real-time PCR apparatus (Applied Biosystems, Carlsbad, CA, USA). Each sample was run in triplicate, and all reactions were repeated three times independently to ensure the reproducibility of the results.
Western blotting analysis Following incubations performed as mentioned above, the HepG2 cells were scraped from the 6-well plates and lysed on ice with a lysis buffer (25 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 1 mM Na orthovanadate, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM phenylmethylsulphonyl fluoride; pH 7.6), and thereafter centrifuged at 12 000 × g for 15 min at 4 °C. Whole-cell extracts containing 80 μg of proteins were separated by SDS-PAGE (8.0% acrylamide), electroblotted onto polyvinylidene fluoride membranes and probed overnight at 4 °C with anti-MRP3 antibody (1:500 dilution; Santa Cruz, CA, USA) or anti-β-actin antibody (1:1000 dilution; Santa Cruz, CA, USA). After incubation with peroxidase-conjugated secondary antibody (1:2000 dilution for donkey anti-goat and goat anti-mouse; Santa Cruz, CA, USA) for 2 h at ambient temperature, the membranes were developed using enhanced chemiluminescence (ECL) substrate Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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(Amersham Life Science Ltd, Little Chalfont, Buckinghamshire, UK). Band intensity was analysed using Image J 1.25s software (National Institutes of Health, Bethesda, MD, USA).
MRP3 transporter activity assay 5-Carboxyfluorescein diacetate (5-CFDA) is a non-fluorescent probe that can be hydrolysed by the intracellular esterase to release the fluorescent anion 5-carboxyfluorescein (5-CF), which does not distinguish the activity of MRP3 from that of other MRP transporters expressed in HepG2 cells, although it is well known to be a specific substrate of the MRP transporter family [27,28]. To further verify whether induction of MRP3 by omeprazole could result in an increase in MRP3 transporter activity, the 5-CFDA/5-CF efflux assay [17,26–28] was performed to measure the magnitude of omeprazole induction in the presence of either human MRP3 siRNA or indomethacin (a well-characterized MRP-specific inhibitor) [17,27,29]. In brief, the HepG2 cells were seeded onto 6-well cell culture clusters and pre-incubated for 24 h at 37 °C/5% CO2, and then transiently transfected with human MRP3 siRNA, and treated with omeprazole (100 μM) 24 h after transfection. After 24 h, the control and treated cells were washed and preincubated with 5-CFDA (5 μM) for 30 min, the medium removed and the cells incubated with 5-CFDA-free medium in the absence and presence of 200 mM indomethacin for 30 min. Subsequently, the cells were washed with PBS, harvested and kept on ice, and analysed by a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The results are presented as the mean ± standard deviation (SD) of arbitrary units (a.u.) of mean fluorescence intensity (MFI) of triplicate measurements as shown by representative histograms.
Statistical analysis All data are expressed as mean ± SD, and analysed using Student’s t-test or repeated measures ANOVA as appropriate, by the GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, USA). A value of p < 0.05 (two-tailed) was considered statistically significant. Copyright © 2015 John Wiley & Sons, Ltd.
Results Effects of omeprazole on cell viability This study was designed to determine subtoxic concentrations of omeprazole, which would be used in the subsequent HepG2 cell studies. The cytotoxicity of omeprazole to the HepG2 cells was examined using the CCK-8 assay. The concentration- and time-dependent changes in the viability of HepG2 cells treated with omeprazole for 12, 24, 48 or 72 h, respectively, are presented in Figure 1. The results showed that omeprazole did not result in a significant change in the viability of the treated cells at 12 and 24 h, compared with the respective vehicle control (n = 3 each; p > 0.05) (Figure 1a); however, significant cytotoxicity was seen when treated with 200
Figure 1. Cytotoxicity of omeprazole in HepG2 cells as measured by CCK-8 assay. Cell viability was determined at 12, 24, 48 and 72 h in HepG2 cells treated with omeprazole at concentrations of 25, 50, 100 and 200 μM, respectively, as measured by CCK-8 assay. (a) Concentration-dependent cytotoxicity of # omeprazole at a given time. p < 0.001 vs. Ctrl; n = 3 each. (b) Time-dependent cytotoxicity of omeprazole at a given concen# tration. *p < 0.01, p < 0.001 vs. 12 h; n = 3 each Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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μM omeprazole for 48 h (n = 3 each; p < 0.001 vs. control). Furthermore, the omeprazole-induced cytotoxicity increased over time, with a significant decrease in cell viability seen at 72 h for all tested omeprazole concentrations (25–200 μM) compared with the vehicle control (n = 3 each; p < 0.001 vs. control). Therefore, a 72 h exposure to omeprazole was not considered in all cell studies. On the other hand, sub-analysis was carried out at the given concentration of omeprazole used as shown in Figure 1b. However, at the given concentration of 200 μM omeprazole, no significant difference in cell viability was seen at 12 and 24 h, respectively, when compared with the respective baseline value at 0 μM (i.e. vehicle control), but cell viability was decreased over time, with the worst seen at 72 h. These results demonstrated that omeprazole induced cytotoxic effects in a concentration- and time-dependent manner, with significantly attenuated cell viability seen at 48 h after omeprazole treatment of 200 μM.
Concentration- and time-dependent induction of MRP3 mRNA expression by omeprazole To determine whether omeprazole could induce MRP3 expression, the concentration- and timedependent response of MRP3 mRNA to omeprazole exposure was examined first. The effects of omeprazole treatment were analysed as a function of time on the expression of MRP3 mRNA in HepG2 cells. The cells were exposed to omeprazole at a concentration of 25, 50, 100 and 200 μM for 12, 24 and 48 h, respectively, and the MRP3 mRNA levels were measured by qRTPCR. As shown in Figure 2a, there was a marked concentration-dependent induction of MRP3 mRNA expression by omeprazole at a given time of 24–48 h. The MRP3 mRNA levels were increased significantly in response to exposure to 200 μM omeprazole as early as 12 h, or of 100 μM at 24 h (1.73-fold, p < 0.01) and 48 h (2.07fold, p < 0.01). Moreover, the MRP3 mRNA levels were maximally up-regulated after the 24 and 48 h treatment with 200 μM omeprazole (2.03-fold and 2.91-fold over control, p < 0.01, p < 0.001, respectively). On the other hand, subanalysis was conducted at a given concentration of omeprazole as demonstrated in Figure 2b. There was a marked time-dependent induction Copyright © 2015 John Wiley & Sons, Ltd.
Figure 2. Omeprazole induces MRP3 mRNA expression concentration- and time-dependently. HepG2 cells were treated with vehicle or omeprazole at a concentration of 25, 50, 100 and 200 μM for 12, 24 and 48 h, respectively. The MRP3 mRNA levels were measured by qRT-PCR analysis, and are presented as fold-induction over the respective vehicle control (100%). (a) Concentration-dependent induction of MRP3 mRNA at a given time. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl; n = 3 each. (b) Time-dependent induction of MRP3 mRNA at a given concentration. *p < 0.05, **p < 0.01, ***p < 0.001 vs. 12 h; n = 3 each
of MRP3 mRNA expression by a given concentration of omeprazole (50–200 μM). Notably, omeprazole induced a clear concentration- and time-dependent up-regulation of MRP3 mRNA levels.
Concentration- and time-dependent induction of MRP3 protein expression by omeprazole Earlier work indicated that a 48 h treatment with 100 μM omeprazole significantly induced MRP3 protein expression in HepG2 cells [20]. However, no data are currently available to show the Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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concentration- and time-dependent induction of MRP3 protein by omeprazole. In the present study, MRP3 protein expression in the treated HepG2 cells was determined by Western blotting analysis after the cells were treated with a given concentration of omeprazole (25–200 μM) for 12, 24 or 48 h, respectively. As illustrated in Figure 3, the MRP3 protein expression did not differ in cells exposed to 25–100 μM omeprazole for 12 h (Figure 3a), nor in the cells treated with 25 μM omeprazole for 24 h (Figure 3b) compared with the vehicle-treated cells. By contrast, 12 h exposure to 200 μM omeprazole led to an increased expression of MRP3 protein (Figure 3a; p < 0.001). When treated with omeprazole for 24 or 48 h, respectively, the cells showed increased MRP3 protein expression in a concentration-dependent manner (Figures 3b and 3c). A significant induction was observed after 24 h exposure when treated with over 50 μM omeprazole (all p < 0.05 or less), with
237 a 5.2-fold induction seen at 48 h when treated with 200 μM omeprazole (p < 0.001). Furthermore, it was also found that 100 μM omeprazole caused a clear time-dependent increase in MRP3 protein expression as shown in Figure 3d.
The MRP3 siRNA caused effective and specific down-regulation of increased MRP3 mRNA and protein expression induced by omeprazole For a direct comparison of the changes in MRP3 mRNA and protein expression, a non-targeting control siRNA that does not show significant homology to any sequence in the human genome was used as a negative control in all RNAi studies. After the HepG2 cells had been transiently transfected with human MRP3 siRNA or the negative-control siRNA for 24 h, the cells were treated with 100 μM omeprazole for another 24 or 48 h induction, respectively. The knock-down
Figure 3. Omeprazole induces MRP3 protein expression in a concentration- and time-dependent manner. (a–c) HepG2 cells were exposed to vehicle (Ctrl) or different concentrations of omeprazole for 12, 24 and 48 h, respectively. (d) HepG2 cells were treated with 100 μM omeprazole for 12, 24 and 48 h, respectively. The MRP3 protein levels were evaluated by Western blotting analysis, quantified by densitometry, and presented as relative changes to vehicle (100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl; n = 3 each Copyright © 2015 John Wiley & Sons, Ltd.
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efficiency of human MRP3 siRNA in HepG2 cells was evaluated using qRT-PCR. The relative levels of MRP3 mRNA expression in transiently transfected cells were normalized to that of GAPDH (an internal control gene). As shown in Figure 4a (left panel), the cells transfected with human MRP3 siRNA showed a significantly reduced transcription of MRP3 mRNA compared with that of omeprazole alone (p < 0.01) or in combination with the negative-control siRNA (p < 0.01), respectively. As expected, a 48 h induction of omeprazole led to a much greater knock-down efficiency of MRP3 mRNA expression than the 24 h induction as shown in Figure 4a (right panel). In order to further confirm the specificity of the induction effect of omeprazole on MRP3 expression, a transient transfection assay of human MRP3 siRNA was done to examine the relative levels of MRP3 protein expression. Western blotting analysis revealed that a 24 h incubation with 100 μM omeprazole caused a marked up-regulation of MRP3 protein expression compared with the vehicle control (p < 0.05), which was almost completely abolished by pretreatment with human MRP3 siRNA (p < 0.01) as shown in Figure 4b. In addition, the levels of MRP3 mRNA and protein expression in response to omeprazole alone or in combination with the negativecontrol siRNA were all similar to each other, indicating that the siRNA negative-control had little or no effect on MRP3 mRNA and protein expression, as expected. All the data have demonstrated well that omeprazole can specifically induce MRP3 expression at the mRNA and protein levels.
Effects of omeprazole on MRP3 mRNA synthesis and stability To further confirm whether that induction could act through increased de novo synthesis of mRNA, Act-D (10 μg/ml) was added to the medium 10 min prior to the addition of the inducer omeprazole. The results showed that induction of MRP3 mRNA expression by omeprazole was completely abrogated by Act-D pretreatment (Figure 5). Moreover, the t1/2 values of MRP3 mRNA in vehicle- and omeprazole-treated cells were examined by use of an Act-D chase Copyright © 2015 John Wiley & Sons, Ltd.
Figure 4. Transfection of HepG2 cells with human MRP3 siRNA abolished omeprazole-induced MRP3 expression. (a) HepG2 cells were transfected by human MRP3 siRNA or negative-control siRNA, followed by the addition of 100 μM omeprazole for a further 24 or 48 h, respectively. The MRP3 mRNA expression was evaluated by qRT-PCR analysis. *p < 0.05 Δ ΔΔ vs. Ctrl; p < 0.05 vs. Ome treatment; p < 0.01 vs. Ome treatment; # ## p < 0.05 vs. Ome + control siRNA; p < 0.01 vs. Ome + control siRNA; n = 3 each. (b) Cells were transfected by human MRP3 siRNA or negative-control siRNA, followed by addition of 100 μM omeprazole for a further 24 h. The MRP3 protein expression was evaluated by Western blotting analysis, and is presented as relative changes to that of ΔΔ Ctrl (100%). *p < 0.05 vs. Ctrl; p < 0.01 vs. Ome treatment; ## p < 0.01 vs. Ome + control siRNA; n = 3 each Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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Figure 5. Inhibition of omeprazole induction by actinomycin D. The HepG2 cells were pretreated with Act-D (10 μg/ml) or DMSO for 10 min, followed by the addition of omeprazole (100 μM), and incubated for another 6 or 12 h, respectively. The MRP3 mRNA expression was evaluated by qRT-PCR anal# ysis. *p < 0.05 vs. Ctrl; p < 0.05 vs. Ome treatment; n = 3 each
experiment. The HepG2 cells were stimulated with omeprazole for 24 h to induce MRP3 mRNA expression, and then the transcription inhibitor Act-D was added in the presence of omeprazole for another 24 h. Steady-state levels of MRP3 mRNA were determined at a series of prespecified time points after the addition of Act-D. As shown in Figure 6, there was no significant change in the decline rate of MRP3 mRNA in vehicle- versus omeprazole-treated cells. The t1/2 of MRP3 mRNA was estimated to be around 26 h, regardless of the absence and presence of the inducer omeprazole.
Modulation of MRP3 transport activity by omeprazole It is well known that cells with a high expression of the MRP proteins can actively export 5-CF (a fluorescent anion generated after the cleavage of 5-CFDA by intracellular esterases) out of the cell, and that intracellular retention of 5-CF can be used as a highly efficient and specific indicator of MRP pump activity [17,26–28]. The study examined whether omeprazole could also enhance MRP3 transport activity in HepG2 cells after omeprazole induced MRP3 mRNA and protein expression. However, an altered MFI (derived from intracellular 5-CF) cannot differentiate individual MRP members, only reflecting the efflux Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 6. Effects of omeprazole on MRP3 mRNA decay after actinomycin D. The HepG2 cells were treated with omeprazole (100 μM) for 24 h, and Act-D was used at a final concentration of 5 μg/ml for the following 24 h, in the absence and presence of omeprazole 100 μM. Cells were harvested at 0, 3, 6, 12 and 24 h, respectively, after addition of 5 μg/ml Act-D, the total RNA was isolated and the MRP3 mRNA levels were quantified by qRT-PCR analysis. The mRNA level at 0 (or just before the Act-D treatment) was considered as the baseline (100%). The half-life (t1/2) of mRNA was determined with the use of regression curves
activity of the overall MRP transporter family [28]. To overcome this limitation, pretreatment of HepG2 cells with human MRP3 siRNA was used to specifically quantify the MRP3-mediated export function in omeprazole induction. As shown in Figure 7, the MFI value of 5-CF retained in cells treated with omeprazole was lower than that in vehicle-treated cells (p < 0.05), indicating an enhanced MRP-mediated export transport activity after omeprazole treatment. When compared with omeprazole alone, pretreatment with human MRP3 siRNA led to an increased MFI in the cells (p < 0.01), suggesting that increased retention of intracellular 5-CF was the result of a marked knock-down of MRP3 protein expression by human MRP3 siRNA, and that specific induction of the human MRP3 efflux pumping activity by omeprazole is consistent with specific induction of its MRP3 mRNA and protein in cells treated with omeprazole. As expected, in omeprazole-treated cells, indomethacin caused a significantly greater MFI value than human MRP3 siRNA as shown in Figure 7, revealing that omeprazole significantly induces other MRP efflux pumping activity in HepG2 cells in addition to MRP3. Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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Figure 7. Omeprazole induces MRP3 efflux activity in HepG2 cells. The activity of the transporter proteins was evaluated in HepG2 cells as measured by the intracellular retention of a fluorescence probe used. The HepG2 cells were incubated with medium or 5 μM 5-CFDA in the absence and presence of 100 μM omeprazole for 30 min, followed by treatment with 5-CFDA-free medium in the absence and presence of 200 mM indomethacin (Indo) for another 30 min, and the cell fluorescence was measured by flow cytometry. (a) The MRP3 efflux activity in the HepG2 cells was measured using flow cytometry in cells incubated with 5 μM 5-CFDA. The results are presented as arbitrary units (a.u.) of the MFI from three independent experiments. (b) Results are expressed as the mean ± SD of the mean fluorescence intensity (MIF) obtained in three different experiments. *p < 0.05 vs. # ## Ctrl; p < 0.05 vs. Ome treatment; p < 0.001 vs. Ome treatment, **p < 0.001 vs. Ome + MRP3 siRNA; n = 3 each. Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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Discussion The new findings in this study are summarized as follows: (1) omeprazole specifically induces MRP3 expression at the mRNA and protein level as determined by the human MRP3 siRNA knockdown assay; (2) induction of MRP3 protein expression by omeprazole is the result of increased mRNA transcription rather than stabilization, as measured by Act-D inhibition; and (3) omeprazole-induced hepatic MRP3 efflux pump activity is abolished by human MRP3 siRNA pretreatment, confirming an enhanced MRP3 transport activity that is induced by omeprazole. In addition, omeprazole can also induce other MRP transporter family members as evidenced by the pronounced difference in the MFI mean value between MRP3 siRNA- versus indomethacin-treated HepG2 cells. An earlier pilot study indicated that a 48 h treatment with 100 μM omeprazole significantly increased the MRP3 protein expression in HepG2 cells [20]. In contrast, this work demonstrated a definitely clear concentration- and timedependent induction of MRP3 mRNA and protein expression in the HepG2 cells when treated with a wide range of omeprazole concentrations (25–200 μM) over time (12 – 48 h) as shown in Figures 2–6, respectively. This provides substantial pharmacological evidence strongly suggesting that MRP3 mRNA and protein expression can be induced by omeprazole in a concentration- and time-dependent manner. Furthermore, as shown in Figure 4, compared with omeprazole alone or in combination with the negative-control siRNA, human MRP3 siRNA significantly suppressed omeprazole-stimulated transcription of MRP3 mRNA. In addition, the specificity of that induction at the protein level was strengthened further, because pretreatment with human MRP3 siRNA could almost completely abrogate omeprazole-induced MRP3 protein expression. Because there was no significant difference in the levels of MRP3 mRNA and protein expression between omeprazole alone and in combination with the negativecontrol siRNA, respectively, this indicates that the negative-control siRNA had little or no effects on omeprazole-induced MRP3 mRNA and protein expression, as anticipated. The above Copyright © 2015 John Wiley & Sons, Ltd.
241 molecular biology evidence has documented the specificity of the induction effect of omeprazole on MRP3 mRNA and protein expression. Omeprazole can induce MRP3 mRNA and protein expression, and thus an enhanced MRP3 efflux transport activity after omeprazole treatment can be deduced. Because there is a lack of substrates and/or inhibitors that are specific for each MRP family member, it is difficult to differentiate individual MRP members. In this study, 5-CFDA was used to measure the total MRP efflux transport activity as described elsewhere [17,26–28]. MRP3-mediated transport activity was estimated according to the difference in intracellular accumulation of 5-CF with or without human MRP3 siRNA knock-down. In addition, an MRP-specific inhibitor indomethacin was also used to determine the relative contribution of MRP3 in the overall MRP-mediated efflux transport activity, as described elsewhere [27]. As shown in Figure 7, indomethacin had a greater intracellular retention of 5-CF than MRP3 siRNA in the omeprazole-treated cells, demonstrating that indomethacin also inhibited the functionality of other efflux transporters besides MRP3. After omeprazole treatment, enhanced MRP-mediated efflux transport activity as measured by the 5-CF assay was consistent with the induction of MRP3 mRNA and protein expression. However, induction of MRP3 mRNA, protein and transport functionality by omeprazole were all abolished by human MRP3 siRNA knock-down or indomethacin. These results provide direct evidence that omeprazole-induced MRP3 is functional, although the involvement of other MRP transporters cannot be ruled out on the basis of these functional data alone. Transcriptional up-regulation of MRP3 mRNA expression by omeprazole was proven by inhibition of induction with Act-D pretreatment as shown in Figure 5. Moreover, studies with ActD revealed that a decrease in MRP3 mRNA decay after omeprazole induction was prevented by treatment of the cells with Act-D, indicating an active transcription-dependent mechanism of MRP3 mRNA induction after the use of omeprazole. As shown in Figure 6, the t1/2 values of human MRP3 mRNA were consistent with one another between vehicle- and omeprazole-treated cells, also suggesting a transcriptional mode of Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
242 omeprazole regulation, rather than a posttranscriptional mode (characterized by increased mRNA stabilization). This study demonstrates for the first time that omeprazole induces MRP3 expression through transcriptional mRNA upregulation. After careful evaluation of the time course of MRP3 mRNA and protein expression, it was observed that treatment with 25 μM omeprazole for 48 h in HepG2 cells induced MRP3 protein expression significantly (Figure 3c), but failed to induce significantly its mRNA expression (Figure 2b). The conclusion that MRP3 protein is induced by omeprazole more rapidly and at a lower concentration compared with its mRNA needs to be explained with caution. The t1/2 of MRP3 mRNA was estimated to be 26 h (Figure 6). This means that approximately 75% of the MRP3 mRNA synthesized was degraded after the passing of two t1/2 long (i.e. approx. 52 h), although mRNA synthesis is a dynamic and consecutive process. In contrast, the synthesized MRP3 protein is relatively stable, being accumulated gradually. This could explain why MRP3 protein, rather than mRNA expression, was increased in the cells treated with 25 μM omeprazole for 48 h. In addition, omeprazole-mediated transcriptional regulation and activation of mRNA translation could be involved. As mentioned above, the fluorescent anion 5CF, a hydrolysed product of the non-fluorescent probe 5-CFDA by the intracellular esterase, is an MRP-specific substrate [27,28], but it cannot be used to distinguish between MRP3 and other MRP transporters expressed in the omeprazoletreated HepG2 cells. As shown in Figure 7, omeprazole increased the MFI value significantly, indicating that omeprazole can induce MRP expression and transport. Compared with MRP3 siRNA-treated cells, a significantly increased MFI value in indomethacin-treated cells further demonstrated that omeprazole can also induce expression and transport activity of other MRP family members, since indomethacin is a MRP-specific, not MRP-subtype-specific, inhibitor. In general, in vitro studies cannot accurately simulate or predict in vivo or clinical situations. The major limitations of this study were that the result obtained from HepG2 cells may not be extrapolated completely into the observations either Copyright © 2015 John Wiley & Sons, Ltd.
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from primary cultured human hepatocytes or the liver, because there may be some differences with respect to transporter regulatory pathways between human hepatocytes and human hepatoma cells [30]. In this study, a relatively high concentration of omeprazole (100 μM), which had been used in the literature [20], was used to produce a measurable induction of MRP3 mRNA, protein and transport function within a short-time period of 24–48 h, although it is substantially higher than what can be achieved in patient care. In healthy subjects, the maximal steady-state plasma concentrations of omeprazole range from 1 to 5 μM after ingestion of recommended daily doses, dependent on the levels of CYP2C19 activity [31]. In HepG2 cells treated with 25 μM omeprazole for 48 h, MRP3 protein expression was induced significantly (Figure 3c). By contrast, the plasma concentration of omeprazole required to achieve a marked induction of MRP3 could be lower than that observed in the HepG2 cell line studies, because MRP3 protein expression would accumulate gradually in the liver to a greater extent after patients have taken omeprazole consecutively for a long-term period in the clinical setting. Therefore, such an induction in HepG2 cells should be repeated and confirmed in primary cultured human hepatocytes or in future clinical research studies. In summary, this study has documented well that omeprazole can induce MRP3 mRNA and protein expression in a concentration- and timedependent manner, that omeprazole can induce MRP3 transport functionality, that omeprazole promotes the transcription of MRP3 mRNA, and that omeprazole can also induce other MRP transporters besides MRP3.
Acknowledgements This work was supported by a launching research grant (No. 31010300010339), funded by Nanjing First Hospital, a grant BK2012525, by the Jiangsu (Province) Natural Science Foundation (JSNSF), and a grant BL2013001, by the Department of Science and Technology of Jiangsu Province, China (all to Dr Xie), and in part by a grant from the Technology Development Program of Nanjing Medical University, China (2011NJMU200, to Dr Mi), and grants QRX11255 and QRX11247 Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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supported by Nanjing Medical Science and Technology Development Foundation (to Pan and Mi). The HepG2 cell line was a generous gift from Dr Xiang-Rong Cao, Nanjing Normal University College of Life Sciences, Nanjing, China. The authors thank Dr Zhao-Tao Duan, Division of Digestive Diseases, the First People’s Hospital of Yangzhou City, Yangzhou, Jiangsu, China, for his help with improving the quality of all the Figures.
11.
12.
13.
Conflict of Interest The authors have declared that there is no conflict of interest. 14.
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Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
(wileyonlinelibrary.com) DOI: 10.1002/bdd.1936
The molecular mechanism underlying the induction of hepatic MRP3 expression and function by omeprazole‡ Yu-Qin Pana,†, Qiong-Yu Mia,†, Bang-Shun Hea, Shu-Li Zhaoa, Ting Taia, and Hong-Guang Xiea,b,* a
General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China Department of Pharmacology, Nanjing Medical University School of Pharmacy, Nanjing 210034, China
b
ABSTRACT: Previous work has indicated that there is increased protein expression of multidrug resistance-associated protein 3 (MRP3) in the liver samples of patients treated with omeprazole compared with those who were not. However, evidence is still lacking to show the mechanisms underlying that induction. This study aimed to assess changes in the fold-induction of MRP3 mRNA and protein expression over controls in omeprazole-treated HepG2 cells after transient transfection of human MRP3 siRNA, or after pretreatment with actinomycin D (Act-D). Furthermore, MRP3 siRNA knock-down or MRP-specific inhibition (indomethacin) was used to determine whether the MRP3 protein induced by omeprazole possessed an enhanced efflux transport. The results demonstrated that omeprazole induced MRP3 mRNA and protein expression in a concentration- and timedependent manner. Moreover, that induction was almost completely abolished by the addition of human MRP3 siRNA and also by pretreatment with Act-D, respectively. In addition, the decay rate of MRP3 mRNA in vehicle- and omeprazole-treated cells was similar in the presence of Act-D, suggesting transcriptional up-regulation of MRP3 mRNA expression by omeprazole. Most importantly, omeprazole induced MRP3 efflux transport activity, as measured by the 5-carboxyfluorescein assay in the absence and presence of human MRP3 siRNA or indomethacin. It is concluded that omeprazole can induce MRP3 mRNA and protein expression and enhance MRP3 efflux transport activity through transcriptional up-regulation, and that omeprazole can also induce other MRP transporters. Copyright © 2015 John Wiley & Sons, Ltd. Key words: ABCC3; hepatic efflux transporter; MRP3; omeprazole; transcriptional regulation
Introduction Multidrug resistance-associated protein 3 (also known as MRP3), an ATP-dependent efflux transporter, is encoded by the ABCC3 gene [1,2]. Human MRP3 is predominantly expressed in the *Correspondence to: General Clinical Research Center, Nanjing First Hospital, 68 Changle Road, Nanjing 210006, China. E-mail: [email protected] † These authors contributed equally to this work. ‡ Part of this work was presented at the 114th Annual Meeting of the American Society for Clinical Pharmacology and Therapeutics, 5–9 March 2013, Indianpolis, Indian, USA, and the abstract was published in Clinical Pharmacology and Therapeutics 2013; 93 (suppl 1): S97 (abstract # PIII-29).
Copyright © 2015 John Wiley & Sons, Ltd.
major organs responsible for the disposition of conjugated substances, such as the liver, small intestine and kidney [1–3]. In polarized cells, MRP3 is expressed exclusively in the basolateral membrane of hepatocytes [4,5]. Because hepatic sinusoidal blood is found across the basolateral membrane of hepatocytes, MRP3 mediates the export of drugs and/or their glucuronide metabolites from hepatocytes to blood, consequently increasing their systemic exposure and bioavailability [6–9]. Clearly, MRP3 functions as an important sinusoidal efflux transporter in the liver, and thus plays an important role in drug disposition and response in humans. Received 28 September 2014 Revised 21 December 2014 Accepted 8 January 2015
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An earlier clinical observational study indicated that the expression of hepatic MRP3 is highly variable in humans [10]. Although the amount of hepatic MRP3 is relatively low under physiological conditions [11], a large number of studies have demonstrated that human MRP3 expression is increased markedly in patients with certain diseases [12–16], or in response to some chemicals and drugs [4,13,17–19]. Emerging evidence strongly suggests that MRP3 is highly inducible. Increased expression of hepatic MRP3 mRNA and protein was initially found in 62 liver samples from patients treated with omeprazole and also in omeprazole-treated HepG2 cells compared with those without omeprazole treatment [20]. Although MRP3 mRNA and protein were induced 2.4- and 1.8-fold, respectively, in the HepG2 cells treated with omeprazole [20], no data are available to suggest whether that induction was concentration- and time-dependent, and whether the efflux transport functionality of MRP3 was enhanced, as determined by the use of a subtype-specific transporter probe or RNAi knock-down. Because the potential effects of some diseases and drugs on that induction cannot be excluded in the clinical setting, it is difficult to characterize the specificity and functionality of MRP3 protein induction in patients treated with omeprazole. This work was designed to further determine the specificity and functionality of hepatic MRP3 induction as measured by levels of its mRNA, protein and transport activity in HepG2 cells treated with omeprazole alone or in combination with a transcription inhibitor, MRP-specific inhibitor, MRP-specific fluorescent probe or human MRP3 siRNA, respectively.
Materials and Methods Chemicals and reagents Omeprazole (Ome), indomethacin (Indo), actinomycin D (Act-D), carboxyfluorescein diacetate (5CFDA) and dimethyl sulfoxide (DMSO) were purchased from Sigma (Sigma-Aldrich Co., LLC, Shanghai, China). Lipofectamine 2000 was obtained from Invitrogen-Life Technologies (Grand Island, NY, USA). Human MRP3 siRNA (sc40748) and control siRNA (sc-37007) were provided by Santa Cruz Biotechnology, Inc. (Santa Copyright © 2015 John Wiley & Sons, Ltd.
Cruz, CA, USA). All other chemicals and reagents were of analytical grade or higher and were readily available from commercial sources.
HepG2 cell culture and treatment Human hepatoma HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% FBS (Hyclone, UT, USA), 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin, and kept at 37 °C in 5% CO2 and 95% humidity.
Cell proliferation assay Cell proliferation in vitro was analysed using a cell counting kit-8 (also known as CCK-8) (Sigma-Aldrich Chemicals, St Louis, MO, USA) according to the manufacturer’s protocol. In brief, 1 × 104 cells were plated per well in 96-well microplates in 100 μl medium. After 12, 24, 48 and 72 h of culture of the cells treated with omeprazole at a concentration of 25, 50, 100, and 200 μM, respectively, the CCK-8 solution (10 μl) was added to each well, and the plates were put back in a standardized tissue incubator for another 1.5 h. The absorbance was measured at 450 nm using a microplate reader Bio-Rad 680 (OEM Systems Co. Ltd, Kyoto, Japan). The inhibition rate was calculated as [1 (OD value of the treated cells/OD value of untreated cells)] × 100%. Each experiment was done in triplicate.
Omeprazole induction assay The HepG2 cells were seeded at 1 × 105 cells per 6well cluster tray for omeprazole induction studies. They were grown up to > 60% confluent, and exposed to omeprazole at a concentration of 25, 50, 100 and 200 μM for 12, 24 and 48 h, respectively. Omeprazole was dissolved in DMSO, and a final concentration of 0.1% DMSO (v/v) was used as a vehicle control in the cell culture.
Transient transfection assay The HepG2 cells were seeded at 1 × 106 cells per 6well cluster tray, and incubated at 37 °C in a CO2 incubator until the cells were 60–80% confluent. The cells were transiently transfected with 1 μg per plate of human MRP3 siRNA in the presence Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
234 of lipofectamine 2000 reagent according to the manufacturer’s instructions, and treated with 100 μM omeprazole or 0.1% DMSO at 24 h after transfection.
Actinomycin D inhibition assay To confirm whether induction of MRP3 by omeprazole could be through increased de novo synthesis of mRNA, the transcription inhibitor Act-D (10 μg/ml) was added to the medium 10 min prior to omeprazole treatment (100 μM) or vehicle control (0.1% DMSO) as described elsewhere [21]. The total RNA was isolated after treatment for 6 or 12 h, respectively. Moreover, the biologic half-life (t1/2) of MRP3 mRNA was determined by an Act-D chase assay as described elsewhere [22,23]. In brief, the HepG2 cells were pretreated with omeprazole (100 μM) or vehicle control for 24 h. Thereafter, Act-D was added into the medium at a final concentration of 5 μg/ml for another 24 h to suppress further RNA synthesis in the absence and presence of omeprazole (100 μM). The cells were harvested at 0, 3, 6, 12 and 24 h after the addition of 5 μg/ml Act-D, respectively, the total RNA was isolated and the MRP3 mRNA levels were quantified by quantitative real-time PCR (qRT-PCR) as described elsewhere [24]. The data are represented as the percentage decrease in MRP3 mRNA levels versus time using a semilogarithmic scale. The t1/2 values of mRNA were determined with the use of regression curves as described elsewhere [22,23].
Quantitative real-time PCR assay The total cellular RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After treatment with DNase I (TaKaRa Biotechnology Co., Ltd, Dalian, China) at 37 °C for 30 min, the RNA was quantified using spectrophotometry (Eppendorf BioPhotometer Plus, Hamburg, Germany). The RNA (1 μg) was subsequently incubated with 1 μl Oligo dT primer (50 μM), 1 μl Random 6-mers (100 μM), 1 μl PrimeScript TM RT Enzyme Mix, 4 μl 5 × PrimeScript TM buffer and RNase-free dH2O, and the synthesis of firststrand cDNA was conducted in a total volume of 20 μl. The primer sequences used for the cDNA synthesis of MRP3 and GAPDH are shown elsewhere [24]. The PCR amplification was Copyright © 2015 John Wiley & Sons, Ltd.
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undertaken in a LightCycler apparatus (ABI 7500, USA) using SYBR Premix Ex TaqTM (TaKaRa Biotech Co., Ltd, Dalian, China). Thermocycling was done in a final volume of 20 μl, consisting of 1 μl cDNA sample, 0.8 μl forward primer (10 μM), 0.8 μl reverse primer (10 μM), 10 μl SYBR Premix Ex TaqTM, 0.4 μl ROX Reference Dye and 7 μl dH2O. After 30 s at 95 °C to denature the cDNA and to activate Taq DNA polymerase, the cycling conditions were as follows: 40 cycles consisting of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. The Ct used in the real-time PCR quantification was defined as the PCR cycle number that crossed an arbitrarily chosen signal threshold in the log phase of the amplification curve. To verify accurately the fold-change of MRP3 mRNA expression, the calculated Ct values were normalized against that of β-actin amplified from the same sample (ΔCt = CtMRP3 – CtGAPDH), and the 2-ΔΔCt method was used to calculate the fold change over control [25]. The amplification was monitored on an ABI prism 7500 real-time PCR apparatus (Applied Biosystems, Carlsbad, CA, USA). Each sample was run in triplicate, and all reactions were repeated three times independently to ensure the reproducibility of the results.
Western blotting analysis Following incubations performed as mentioned above, the HepG2 cells were scraped from the 6-well plates and lysed on ice with a lysis buffer (25 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 1 mM Na orthovanadate, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM phenylmethylsulphonyl fluoride; pH 7.6), and thereafter centrifuged at 12 000 × g for 15 min at 4 °C. Whole-cell extracts containing 80 μg of proteins were separated by SDS-PAGE (8.0% acrylamide), electroblotted onto polyvinylidene fluoride membranes and probed overnight at 4 °C with anti-MRP3 antibody (1:500 dilution; Santa Cruz, CA, USA) or anti-β-actin antibody (1:1000 dilution; Santa Cruz, CA, USA). After incubation with peroxidase-conjugated secondary antibody (1:2000 dilution for donkey anti-goat and goat anti-mouse; Santa Cruz, CA, USA) for 2 h at ambient temperature, the membranes were developed using enhanced chemiluminescence (ECL) substrate Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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(Amersham Life Science Ltd, Little Chalfont, Buckinghamshire, UK). Band intensity was analysed using Image J 1.25s software (National Institutes of Health, Bethesda, MD, USA).
MRP3 transporter activity assay 5-Carboxyfluorescein diacetate (5-CFDA) is a non-fluorescent probe that can be hydrolysed by the intracellular esterase to release the fluorescent anion 5-carboxyfluorescein (5-CF), which does not distinguish the activity of MRP3 from that of other MRP transporters expressed in HepG2 cells, although it is well known to be a specific substrate of the MRP transporter family [27,28]. To further verify whether induction of MRP3 by omeprazole could result in an increase in MRP3 transporter activity, the 5-CFDA/5-CF efflux assay [17,26–28] was performed to measure the magnitude of omeprazole induction in the presence of either human MRP3 siRNA or indomethacin (a well-characterized MRP-specific inhibitor) [17,27,29]. In brief, the HepG2 cells were seeded onto 6-well cell culture clusters and pre-incubated for 24 h at 37 °C/5% CO2, and then transiently transfected with human MRP3 siRNA, and treated with omeprazole (100 μM) 24 h after transfection. After 24 h, the control and treated cells were washed and preincubated with 5-CFDA (5 μM) for 30 min, the medium removed and the cells incubated with 5-CFDA-free medium in the absence and presence of 200 mM indomethacin for 30 min. Subsequently, the cells were washed with PBS, harvested and kept on ice, and analysed by a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The results are presented as the mean ± standard deviation (SD) of arbitrary units (a.u.) of mean fluorescence intensity (MFI) of triplicate measurements as shown by representative histograms.
Statistical analysis All data are expressed as mean ± SD, and analysed using Student’s t-test or repeated measures ANOVA as appropriate, by the GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA, USA). A value of p < 0.05 (two-tailed) was considered statistically significant. Copyright © 2015 John Wiley & Sons, Ltd.
Results Effects of omeprazole on cell viability This study was designed to determine subtoxic concentrations of omeprazole, which would be used in the subsequent HepG2 cell studies. The cytotoxicity of omeprazole to the HepG2 cells was examined using the CCK-8 assay. The concentration- and time-dependent changes in the viability of HepG2 cells treated with omeprazole for 12, 24, 48 or 72 h, respectively, are presented in Figure 1. The results showed that omeprazole did not result in a significant change in the viability of the treated cells at 12 and 24 h, compared with the respective vehicle control (n = 3 each; p > 0.05) (Figure 1a); however, significant cytotoxicity was seen when treated with 200
Figure 1. Cytotoxicity of omeprazole in HepG2 cells as measured by CCK-8 assay. Cell viability was determined at 12, 24, 48 and 72 h in HepG2 cells treated with omeprazole at concentrations of 25, 50, 100 and 200 μM, respectively, as measured by CCK-8 assay. (a) Concentration-dependent cytotoxicity of # omeprazole at a given time. p < 0.001 vs. Ctrl; n = 3 each. (b) Time-dependent cytotoxicity of omeprazole at a given concen# tration. *p < 0.01, p < 0.001 vs. 12 h; n = 3 each Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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μM omeprazole for 48 h (n = 3 each; p < 0.001 vs. control). Furthermore, the omeprazole-induced cytotoxicity increased over time, with a significant decrease in cell viability seen at 72 h for all tested omeprazole concentrations (25–200 μM) compared with the vehicle control (n = 3 each; p < 0.001 vs. control). Therefore, a 72 h exposure to omeprazole was not considered in all cell studies. On the other hand, sub-analysis was carried out at the given concentration of omeprazole used as shown in Figure 1b. However, at the given concentration of 200 μM omeprazole, no significant difference in cell viability was seen at 12 and 24 h, respectively, when compared with the respective baseline value at 0 μM (i.e. vehicle control), but cell viability was decreased over time, with the worst seen at 72 h. These results demonstrated that omeprazole induced cytotoxic effects in a concentration- and time-dependent manner, with significantly attenuated cell viability seen at 48 h after omeprazole treatment of 200 μM.
Concentration- and time-dependent induction of MRP3 mRNA expression by omeprazole To determine whether omeprazole could induce MRP3 expression, the concentration- and timedependent response of MRP3 mRNA to omeprazole exposure was examined first. The effects of omeprazole treatment were analysed as a function of time on the expression of MRP3 mRNA in HepG2 cells. The cells were exposed to omeprazole at a concentration of 25, 50, 100 and 200 μM for 12, 24 and 48 h, respectively, and the MRP3 mRNA levels were measured by qRTPCR. As shown in Figure 2a, there was a marked concentration-dependent induction of MRP3 mRNA expression by omeprazole at a given time of 24–48 h. The MRP3 mRNA levels were increased significantly in response to exposure to 200 μM omeprazole as early as 12 h, or of 100 μM at 24 h (1.73-fold, p < 0.01) and 48 h (2.07fold, p < 0.01). Moreover, the MRP3 mRNA levels were maximally up-regulated after the 24 and 48 h treatment with 200 μM omeprazole (2.03-fold and 2.91-fold over control, p < 0.01, p < 0.001, respectively). On the other hand, subanalysis was conducted at a given concentration of omeprazole as demonstrated in Figure 2b. There was a marked time-dependent induction Copyright © 2015 John Wiley & Sons, Ltd.
Figure 2. Omeprazole induces MRP3 mRNA expression concentration- and time-dependently. HepG2 cells were treated with vehicle or omeprazole at a concentration of 25, 50, 100 and 200 μM for 12, 24 and 48 h, respectively. The MRP3 mRNA levels were measured by qRT-PCR analysis, and are presented as fold-induction over the respective vehicle control (100%). (a) Concentration-dependent induction of MRP3 mRNA at a given time. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl; n = 3 each. (b) Time-dependent induction of MRP3 mRNA at a given concentration. *p < 0.05, **p < 0.01, ***p < 0.001 vs. 12 h; n = 3 each
of MRP3 mRNA expression by a given concentration of omeprazole (50–200 μM). Notably, omeprazole induced a clear concentration- and time-dependent up-regulation of MRP3 mRNA levels.
Concentration- and time-dependent induction of MRP3 protein expression by omeprazole Earlier work indicated that a 48 h treatment with 100 μM omeprazole significantly induced MRP3 protein expression in HepG2 cells [20]. However, no data are currently available to show the Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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concentration- and time-dependent induction of MRP3 protein by omeprazole. In the present study, MRP3 protein expression in the treated HepG2 cells was determined by Western blotting analysis after the cells were treated with a given concentration of omeprazole (25–200 μM) for 12, 24 or 48 h, respectively. As illustrated in Figure 3, the MRP3 protein expression did not differ in cells exposed to 25–100 μM omeprazole for 12 h (Figure 3a), nor in the cells treated with 25 μM omeprazole for 24 h (Figure 3b) compared with the vehicle-treated cells. By contrast, 12 h exposure to 200 μM omeprazole led to an increased expression of MRP3 protein (Figure 3a; p < 0.001). When treated with omeprazole for 24 or 48 h, respectively, the cells showed increased MRP3 protein expression in a concentration-dependent manner (Figures 3b and 3c). A significant induction was observed after 24 h exposure when treated with over 50 μM omeprazole (all p < 0.05 or less), with
237 a 5.2-fold induction seen at 48 h when treated with 200 μM omeprazole (p < 0.001). Furthermore, it was also found that 100 μM omeprazole caused a clear time-dependent increase in MRP3 protein expression as shown in Figure 3d.
The MRP3 siRNA caused effective and specific down-regulation of increased MRP3 mRNA and protein expression induced by omeprazole For a direct comparison of the changes in MRP3 mRNA and protein expression, a non-targeting control siRNA that does not show significant homology to any sequence in the human genome was used as a negative control in all RNAi studies. After the HepG2 cells had been transiently transfected with human MRP3 siRNA or the negative-control siRNA for 24 h, the cells were treated with 100 μM omeprazole for another 24 or 48 h induction, respectively. The knock-down
Figure 3. Omeprazole induces MRP3 protein expression in a concentration- and time-dependent manner. (a–c) HepG2 cells were exposed to vehicle (Ctrl) or different concentrations of omeprazole for 12, 24 and 48 h, respectively. (d) HepG2 cells were treated with 100 μM omeprazole for 12, 24 and 48 h, respectively. The MRP3 protein levels were evaluated by Western blotting analysis, quantified by densitometry, and presented as relative changes to vehicle (100%). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl; n = 3 each Copyright © 2015 John Wiley & Sons, Ltd.
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efficiency of human MRP3 siRNA in HepG2 cells was evaluated using qRT-PCR. The relative levels of MRP3 mRNA expression in transiently transfected cells were normalized to that of GAPDH (an internal control gene). As shown in Figure 4a (left panel), the cells transfected with human MRP3 siRNA showed a significantly reduced transcription of MRP3 mRNA compared with that of omeprazole alone (p < 0.01) or in combination with the negative-control siRNA (p < 0.01), respectively. As expected, a 48 h induction of omeprazole led to a much greater knock-down efficiency of MRP3 mRNA expression than the 24 h induction as shown in Figure 4a (right panel). In order to further confirm the specificity of the induction effect of omeprazole on MRP3 expression, a transient transfection assay of human MRP3 siRNA was done to examine the relative levels of MRP3 protein expression. Western blotting analysis revealed that a 24 h incubation with 100 μM omeprazole caused a marked up-regulation of MRP3 protein expression compared with the vehicle control (p < 0.05), which was almost completely abolished by pretreatment with human MRP3 siRNA (p < 0.01) as shown in Figure 4b. In addition, the levels of MRP3 mRNA and protein expression in response to omeprazole alone or in combination with the negativecontrol siRNA were all similar to each other, indicating that the siRNA negative-control had little or no effect on MRP3 mRNA and protein expression, as expected. All the data have demonstrated well that omeprazole can specifically induce MRP3 expression at the mRNA and protein levels.
Effects of omeprazole on MRP3 mRNA synthesis and stability To further confirm whether that induction could act through increased de novo synthesis of mRNA, Act-D (10 μg/ml) was added to the medium 10 min prior to the addition of the inducer omeprazole. The results showed that induction of MRP3 mRNA expression by omeprazole was completely abrogated by Act-D pretreatment (Figure 5). Moreover, the t1/2 values of MRP3 mRNA in vehicle- and omeprazole-treated cells were examined by use of an Act-D chase Copyright © 2015 John Wiley & Sons, Ltd.
Figure 4. Transfection of HepG2 cells with human MRP3 siRNA abolished omeprazole-induced MRP3 expression. (a) HepG2 cells were transfected by human MRP3 siRNA or negative-control siRNA, followed by the addition of 100 μM omeprazole for a further 24 or 48 h, respectively. The MRP3 mRNA expression was evaluated by qRT-PCR analysis. *p < 0.05 Δ ΔΔ vs. Ctrl; p < 0.05 vs. Ome treatment; p < 0.01 vs. Ome treatment; # ## p < 0.05 vs. Ome + control siRNA; p < 0.01 vs. Ome + control siRNA; n = 3 each. (b) Cells were transfected by human MRP3 siRNA or negative-control siRNA, followed by addition of 100 μM omeprazole for a further 24 h. The MRP3 protein expression was evaluated by Western blotting analysis, and is presented as relative changes to that of ΔΔ Ctrl (100%). *p < 0.05 vs. Ctrl; p < 0.01 vs. Ome treatment; ## p < 0.01 vs. Ome + control siRNA; n = 3 each Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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Figure 5. Inhibition of omeprazole induction by actinomycin D. The HepG2 cells were pretreated with Act-D (10 μg/ml) or DMSO for 10 min, followed by the addition of omeprazole (100 μM), and incubated for another 6 or 12 h, respectively. The MRP3 mRNA expression was evaluated by qRT-PCR anal# ysis. *p < 0.05 vs. Ctrl; p < 0.05 vs. Ome treatment; n = 3 each
experiment. The HepG2 cells were stimulated with omeprazole for 24 h to induce MRP3 mRNA expression, and then the transcription inhibitor Act-D was added in the presence of omeprazole for another 24 h. Steady-state levels of MRP3 mRNA were determined at a series of prespecified time points after the addition of Act-D. As shown in Figure 6, there was no significant change in the decline rate of MRP3 mRNA in vehicle- versus omeprazole-treated cells. The t1/2 of MRP3 mRNA was estimated to be around 26 h, regardless of the absence and presence of the inducer omeprazole.
Modulation of MRP3 transport activity by omeprazole It is well known that cells with a high expression of the MRP proteins can actively export 5-CF (a fluorescent anion generated after the cleavage of 5-CFDA by intracellular esterases) out of the cell, and that intracellular retention of 5-CF can be used as a highly efficient and specific indicator of MRP pump activity [17,26–28]. The study examined whether omeprazole could also enhance MRP3 transport activity in HepG2 cells after omeprazole induced MRP3 mRNA and protein expression. However, an altered MFI (derived from intracellular 5-CF) cannot differentiate individual MRP members, only reflecting the efflux Copyright © 2015 John Wiley & Sons, Ltd.
239
Figure 6. Effects of omeprazole on MRP3 mRNA decay after actinomycin D. The HepG2 cells were treated with omeprazole (100 μM) for 24 h, and Act-D was used at a final concentration of 5 μg/ml for the following 24 h, in the absence and presence of omeprazole 100 μM. Cells were harvested at 0, 3, 6, 12 and 24 h, respectively, after addition of 5 μg/ml Act-D, the total RNA was isolated and the MRP3 mRNA levels were quantified by qRT-PCR analysis. The mRNA level at 0 (or just before the Act-D treatment) was considered as the baseline (100%). The half-life (t1/2) of mRNA was determined with the use of regression curves
activity of the overall MRP transporter family [28]. To overcome this limitation, pretreatment of HepG2 cells with human MRP3 siRNA was used to specifically quantify the MRP3-mediated export function in omeprazole induction. As shown in Figure 7, the MFI value of 5-CF retained in cells treated with omeprazole was lower than that in vehicle-treated cells (p < 0.05), indicating an enhanced MRP-mediated export transport activity after omeprazole treatment. When compared with omeprazole alone, pretreatment with human MRP3 siRNA led to an increased MFI in the cells (p < 0.01), suggesting that increased retention of intracellular 5-CF was the result of a marked knock-down of MRP3 protein expression by human MRP3 siRNA, and that specific induction of the human MRP3 efflux pumping activity by omeprazole is consistent with specific induction of its MRP3 mRNA and protein in cells treated with omeprazole. As expected, in omeprazole-treated cells, indomethacin caused a significantly greater MFI value than human MRP3 siRNA as shown in Figure 7, revealing that omeprazole significantly induces other MRP efflux pumping activity in HepG2 cells in addition to MRP3. Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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Figure 7. Omeprazole induces MRP3 efflux activity in HepG2 cells. The activity of the transporter proteins was evaluated in HepG2 cells as measured by the intracellular retention of a fluorescence probe used. The HepG2 cells were incubated with medium or 5 μM 5-CFDA in the absence and presence of 100 μM omeprazole for 30 min, followed by treatment with 5-CFDA-free medium in the absence and presence of 200 mM indomethacin (Indo) for another 30 min, and the cell fluorescence was measured by flow cytometry. (a) The MRP3 efflux activity in the HepG2 cells was measured using flow cytometry in cells incubated with 5 μM 5-CFDA. The results are presented as arbitrary units (a.u.) of the MFI from three independent experiments. (b) Results are expressed as the mean ± SD of the mean fluorescence intensity (MIF) obtained in three different experiments. *p < 0.05 vs. # ## Ctrl; p < 0.05 vs. Ome treatment; p < 0.001 vs. Ome treatment, **p < 0.001 vs. Ome + MRP3 siRNA; n = 3 each. Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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Discussion The new findings in this study are summarized as follows: (1) omeprazole specifically induces MRP3 expression at the mRNA and protein level as determined by the human MRP3 siRNA knockdown assay; (2) induction of MRP3 protein expression by omeprazole is the result of increased mRNA transcription rather than stabilization, as measured by Act-D inhibition; and (3) omeprazole-induced hepatic MRP3 efflux pump activity is abolished by human MRP3 siRNA pretreatment, confirming an enhanced MRP3 transport activity that is induced by omeprazole. In addition, omeprazole can also induce other MRP transporter family members as evidenced by the pronounced difference in the MFI mean value between MRP3 siRNA- versus indomethacin-treated HepG2 cells. An earlier pilot study indicated that a 48 h treatment with 100 μM omeprazole significantly increased the MRP3 protein expression in HepG2 cells [20]. In contrast, this work demonstrated a definitely clear concentration- and timedependent induction of MRP3 mRNA and protein expression in the HepG2 cells when treated with a wide range of omeprazole concentrations (25–200 μM) over time (12 – 48 h) as shown in Figures 2–6, respectively. This provides substantial pharmacological evidence strongly suggesting that MRP3 mRNA and protein expression can be induced by omeprazole in a concentration- and time-dependent manner. Furthermore, as shown in Figure 4, compared with omeprazole alone or in combination with the negative-control siRNA, human MRP3 siRNA significantly suppressed omeprazole-stimulated transcription of MRP3 mRNA. In addition, the specificity of that induction at the protein level was strengthened further, because pretreatment with human MRP3 siRNA could almost completely abrogate omeprazole-induced MRP3 protein expression. Because there was no significant difference in the levels of MRP3 mRNA and protein expression between omeprazole alone and in combination with the negativecontrol siRNA, respectively, this indicates that the negative-control siRNA had little or no effects on omeprazole-induced MRP3 mRNA and protein expression, as anticipated. The above Copyright © 2015 John Wiley & Sons, Ltd.
241 molecular biology evidence has documented the specificity of the induction effect of omeprazole on MRP3 mRNA and protein expression. Omeprazole can induce MRP3 mRNA and protein expression, and thus an enhanced MRP3 efflux transport activity after omeprazole treatment can be deduced. Because there is a lack of substrates and/or inhibitors that are specific for each MRP family member, it is difficult to differentiate individual MRP members. In this study, 5-CFDA was used to measure the total MRP efflux transport activity as described elsewhere [17,26–28]. MRP3-mediated transport activity was estimated according to the difference in intracellular accumulation of 5-CF with or without human MRP3 siRNA knock-down. In addition, an MRP-specific inhibitor indomethacin was also used to determine the relative contribution of MRP3 in the overall MRP-mediated efflux transport activity, as described elsewhere [27]. As shown in Figure 7, indomethacin had a greater intracellular retention of 5-CF than MRP3 siRNA in the omeprazole-treated cells, demonstrating that indomethacin also inhibited the functionality of other efflux transporters besides MRP3. After omeprazole treatment, enhanced MRP-mediated efflux transport activity as measured by the 5-CF assay was consistent with the induction of MRP3 mRNA and protein expression. However, induction of MRP3 mRNA, protein and transport functionality by omeprazole were all abolished by human MRP3 siRNA knock-down or indomethacin. These results provide direct evidence that omeprazole-induced MRP3 is functional, although the involvement of other MRP transporters cannot be ruled out on the basis of these functional data alone. Transcriptional up-regulation of MRP3 mRNA expression by omeprazole was proven by inhibition of induction with Act-D pretreatment as shown in Figure 5. Moreover, studies with ActD revealed that a decrease in MRP3 mRNA decay after omeprazole induction was prevented by treatment of the cells with Act-D, indicating an active transcription-dependent mechanism of MRP3 mRNA induction after the use of omeprazole. As shown in Figure 6, the t1/2 values of human MRP3 mRNA were consistent with one another between vehicle- and omeprazole-treated cells, also suggesting a transcriptional mode of Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
242 omeprazole regulation, rather than a posttranscriptional mode (characterized by increased mRNA stabilization). This study demonstrates for the first time that omeprazole induces MRP3 expression through transcriptional mRNA upregulation. After careful evaluation of the time course of MRP3 mRNA and protein expression, it was observed that treatment with 25 μM omeprazole for 48 h in HepG2 cells induced MRP3 protein expression significantly (Figure 3c), but failed to induce significantly its mRNA expression (Figure 2b). The conclusion that MRP3 protein is induced by omeprazole more rapidly and at a lower concentration compared with its mRNA needs to be explained with caution. The t1/2 of MRP3 mRNA was estimated to be 26 h (Figure 6). This means that approximately 75% of the MRP3 mRNA synthesized was degraded after the passing of two t1/2 long (i.e. approx. 52 h), although mRNA synthesis is a dynamic and consecutive process. In contrast, the synthesized MRP3 protein is relatively stable, being accumulated gradually. This could explain why MRP3 protein, rather than mRNA expression, was increased in the cells treated with 25 μM omeprazole for 48 h. In addition, omeprazole-mediated transcriptional regulation and activation of mRNA translation could be involved. As mentioned above, the fluorescent anion 5CF, a hydrolysed product of the non-fluorescent probe 5-CFDA by the intracellular esterase, is an MRP-specific substrate [27,28], but it cannot be used to distinguish between MRP3 and other MRP transporters expressed in the omeprazoletreated HepG2 cells. As shown in Figure 7, omeprazole increased the MFI value significantly, indicating that omeprazole can induce MRP expression and transport. Compared with MRP3 siRNA-treated cells, a significantly increased MFI value in indomethacin-treated cells further demonstrated that omeprazole can also induce expression and transport activity of other MRP family members, since indomethacin is a MRP-specific, not MRP-subtype-specific, inhibitor. In general, in vitro studies cannot accurately simulate or predict in vivo or clinical situations. The major limitations of this study were that the result obtained from HepG2 cells may not be extrapolated completely into the observations either Copyright © 2015 John Wiley & Sons, Ltd.
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from primary cultured human hepatocytes or the liver, because there may be some differences with respect to transporter regulatory pathways between human hepatocytes and human hepatoma cells [30]. In this study, a relatively high concentration of omeprazole (100 μM), which had been used in the literature [20], was used to produce a measurable induction of MRP3 mRNA, protein and transport function within a short-time period of 24–48 h, although it is substantially higher than what can be achieved in patient care. In healthy subjects, the maximal steady-state plasma concentrations of omeprazole range from 1 to 5 μM after ingestion of recommended daily doses, dependent on the levels of CYP2C19 activity [31]. In HepG2 cells treated with 25 μM omeprazole for 48 h, MRP3 protein expression was induced significantly (Figure 3c). By contrast, the plasma concentration of omeprazole required to achieve a marked induction of MRP3 could be lower than that observed in the HepG2 cell line studies, because MRP3 protein expression would accumulate gradually in the liver to a greater extent after patients have taken omeprazole consecutively for a long-term period in the clinical setting. Therefore, such an induction in HepG2 cells should be repeated and confirmed in primary cultured human hepatocytes or in future clinical research studies. In summary, this study has documented well that omeprazole can induce MRP3 mRNA and protein expression in a concentration- and timedependent manner, that omeprazole can induce MRP3 transport functionality, that omeprazole promotes the transcription of MRP3 mRNA, and that omeprazole can also induce other MRP transporters besides MRP3.
Acknowledgements This work was supported by a launching research grant (No. 31010300010339), funded by Nanjing First Hospital, a grant BK2012525, by the Jiangsu (Province) Natural Science Foundation (JSNSF), and a grant BL2013001, by the Department of Science and Technology of Jiangsu Province, China (all to Dr Xie), and in part by a grant from the Technology Development Program of Nanjing Medical University, China (2011NJMU200, to Dr Mi), and grants QRX11255 and QRX11247 Biopharm. Drug Dispos. 36: 232–244 (2015) DOI: 10.1002/bdd
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supported by Nanjing Medical Science and Technology Development Foundation (to Pan and Mi). The HepG2 cell line was a generous gift from Dr Xiang-Rong Cao, Nanjing Normal University College of Life Sciences, Nanjing, China. The authors thank Dr Zhao-Tao Duan, Division of Digestive Diseases, the First People’s Hospital of Yangzhou City, Yangzhou, Jiangsu, China, for his help with improving the quality of all the Figures.
11.
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Conflict of Interest The authors have declared that there is no conflict of interest. 14.
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