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Carrier-mediated transport of quercetin conjugates: Involvement of organic anion transporters and organic anion transporting polypeptides
Biochemical Pharmacology 84 (2012) 564–570
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Carrier-mediated transport of quercetin conjugates: Involvement of organic anion transporters and organic anion transporting polypeptides Chi Chun Wong a, Yasutoshi Akiyama b, Takaaki Abe b, Jonathan D. Lippiat c, Caroline Orfila a, Gary Williamson a,d,* a
School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT, UK Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK d Nestle Research Center, Vers Chez les Blanc, 1000 Lausanne 26, Switzerland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 March 2012 Accepted 14 May 2012 Available online 23 May 2012
Flavonoids modulate cell signaling and inhibit oxidative enzymes. After oral consumption, they circulate in human plasma as amphiphilic glucuronide or sulfate conjugates, but it is unknown how these physiological metabolites permeate into cells. We examined the mechanisms of uptake of these conjugates into hepatocellular carcinoma (HepG2) cells, and found that uptake of quercetin-30 -O-sulfate was saturable and temperature-dependent, indicating the involvement of carrier-mediated transport. Quercetin-3-O-glucuronide was taken up predominantly via passive diffusion in these cells. Quantitative real-time PCR analysis showed high expression of OATP4C1, followed by OAT2, OAT4 and low expression of OATP1B1 in HepG2 cells, and addition of inhibitors of OATs and OATPs resulted in a significant reduction in quercetin-30 -O-sulfate uptake. The accumulation of quercetin-30 -O-sulfate was further evaluated in HEK293 cells expressing OAT2, OAT4 and OATP4C1. Uptake of quercetin-30 -O-sulfate was 2.3- and 1.4-fold higher in cells expressing OAT4 and OATP4C1 at pH 6.0, respectively, than in control HEK293 cells. siRNA knockdown of OATP4C1 expression in HepG2 cells reduced uptake of quercetin-30 O-sulfate by 40%. This study highlights a role for OATs and OATPs in the cellular uptake of biologically active flavonoid conjugates. ß 2012 Elsevier Inc. All rights reserved.
Keywords: Quercetin HepG2 Carrier-mediated transport Organic anion transporter Organic anion transporting polypeptide
1. Introduction Quercetin is a biologically active flavonoid that influences cell signaling pathways [1], inhibits the sodium-dependent vitamin C transporter [2], and modulates oxidative enzymes such as COX-2 [3] and lipoxygenase [4], thereby reducing the formation of intracellular superoxide and other reactive oxygen species. The biological activity is likely derived from the predominant forms of quercetin found in blood in vivo, the 30 -O-sulfated, 3-O-glucuronidated and 30 or 40 methylated derivatives. Quercetin glucuronides reduce oxidative stress via inhibition of xanthine oxidase and myeloperoxidase, enzymes that participate in free radical generation [5,6]. Some quercetin conjugates down-regulate the expression of cyclooxygenase-2 (COX-2), a key enzyme in the production of pro-inflammatory
Abbreviations: COX2, cyclooxygenase-2; HBSS, hank’s balanced salt solution; OATs, organic anion transporters; OATPs, organic anion transporting polypeptides. * Corresponding author at: School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT, UK. Tel.: +44 0 113 343 8380; fax: +44 0 113 343 2982. E-mail address: [email protected] (G. Williamson). 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.05.011
eicosanoids. Quercetin-30 -O-sulfate directly inhibited COX-2 activity, whereas quercetin-3-O-glucuronide did not [7]. Emerging evidence supports a role for quercetin metabolites in vascular health. The sulfated and glucuronidated conjugates of quercetin prevent oxidative stress-induced endothelial dysfunction in rat aorta [8]. Quercetin-3-O-glucuronide was also found to exert an antiatherosclerotic effect by suppressing the expression of scavenger receptors in activated macrophages [9]. In humans, quercetin is extensively metabolized via glucuronidation, sulfation, or methylation, resulting in the formation of numerous conjugated metabolites [10]. Phase II conjugates, such as quercetin-30 -O-sulfate, are the predominant circulating forms in plasma (<10 mM), and the aglycone is not found in vivo [10,11]. Contribution of the bile to the secretion of quercetin conjugates was estimated to be 10–30% and their concentration in the bile could reach in excess of 300 mM in mice [12]. Quercetin conjugates are markedly more hydrophilic compared to quercetin [13]. Transport of hydrophilic and ionized substances from the circulation into bile or urine involves interplay of uptake transporters on the basolateral membrane and efflux transporters on the apical side, which are present in hepatocytes and proximal
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tubular cells. Basolateral uptake of organic anions, such as glucuronide and sulfate conjugates, is thought to involve organic anion transporters (OATs) and/or organic anion-transporting polypeptides (OATPs) [14]. They are multispecific and mediate the transport of various amphiphilic organic anions, including endogenous compounds, structurally diverse drugs and their conjugated metabolites. Thus, OATs and OATPs are important determinants for excretion of xenobiotics. Human liver showed a high expression of OATP1B1 and OATP1B3, as well as moderate expression of OATP1A2, OATP4C1 and OATP2B1 [15,16]. OAT2, OAT4 and OAT7 mRNA have also been detected [16,17]. OAT1, OAT3, and OATP4C1 are the major uptake transporters found in the human kidneys. Active efflux is carried out by ATP-binding cassette transporters including P-glycoprotein, multi-drug resistance protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) [18]. OATs/OATPs and ABC transporters are believed to act in concert to affect vectorial transport of various organic anions, and thus facilitate their elimination from the body. Recently, it has been shown that the green tea flavonoids epicatechin gallate and epigallocatechin gallate are substrates of OATP1A2 and OATP1B3 [19], and that several flavonoid and their conjugated forms can interact with OAT1, OATP1A2 and OATP1B1 [20–26]. However, no physiologically relevant flavonoid conjugates have been shown to interact with members of the OAT or OATP families. Hence, we hypothesized that conjugates of flavonoids such as quercetin may be potential substrates for OATs and OATPs. Here, we demonstrate a carrier-mediated uptake mechanism for quercetin-30 -O-sulfate into HepG2 cells, but not for quercetin glucuronides, and identified potential OAT and OATP transporters involved in the uptake in HepG2 cells. 2. Materials and methods 2.1. Chemicals Sulfobromophthalein was from Acros Organics (Geel, Belgium). Quercetin-7-O-glucuronide, quercetin-3-O-glucuronide and quercetin-30 -O-glucuronide were synthesized enzymatically and then purified by HPLC [27]. Chemical synthesis of quercetin-30 -O-sulfate was performed as described [28]. The identity of the compounds was further confirmed by comparing the retention time and absorption spectra of quercetin-3-O-glucuronide and quercetin-30 O-sulfate kindly provided by Dr. Paul Kroon, Institute of Food Research, UK. The purities were checked by HPLC to be over 95%. HepG2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human embryonic kidney cells HEK-293 were purchased from Invitrogen (Carlsbad, CA). OAT1 (SLC22A6 transcript variant 2, Genebank accession number: NM_153276), OAT2 (SLC22A7 transcript variant 1, Genebank accession number: NM_006672.2), OAT3 (SLC22A8 transcript variant 1, Genebank accession number: NM_004254) and OAT4 (Genebank accession number: NM_018484.2) expression plasmids were obtained from Origene (Rockville, MD). Full length OATP4C1 cDNA (Image clone 100016240) (Geneservice, Cambridge, UK) was re-cloned into pcDNA6/V5-His (forward primer: 50 -ggatccatgaagagcgccaaaggtatt-30 ; reverse primer: 50 -gcggccgcacttacccttcttttactattttgttgag-30 ) for transfection into mammalian cell lines. Fugene HD was purchased from Roche. Other chemicals, unless otherwise stated, were purchased from Sigma–Aldrich (St. Louis, MO). 2.2. Cell culture HepG2 cells were routinely cultured in 75 cm2 cell culture flasks (Corning Costar Corp., Cambridge, MA) at 37 8C under a humidified 5% CO2/O2 atmosphere. The culture media consisted of
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Eagle’s Minimum Essential Medium media supplemented with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin. All experiments were performed with cells between passages 80 and 95. HEK-293 cells were cultured in Dulbecco’s modified Minimum Essential Medium high-glucose media supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 50 U/mL penicillin–streptomycin, in 5% CO2 at 37 8C. Cells were used between passages 4 and 20. 2.3. Confocal microscopy 5 104 HepG2 cells were plated in 12 mm poly-L-lysine-coated coverslips (BD Bioscience, Bedford, MA) within six-well plates. Cells were then cultured for 96 h before the experiment. Incubation medium used was Hank’s Balanced Salt Solution (HBSS) buffer supplemented with 1.8 mM CaCl2, adjusted to pH 6 with 1 M HCl. Various concentrations of quercetin, quercetin-7O-glucuronide, quercetin-3-O-glucuronide, quercetin-30 -O-glucuronide and quercetin-30 -O-sulfate were added to the cells and then incubated for 30 min at 37 8C. Uptake was stopped by washing with ice-cold HBSS three times and then cells were fixed with 4% formaldehyde for 5 min. Coverslips were washed with water and mounted upside-down on glass slides. The images were obtained with a Leica TCS SP2 confocal laser scanning microscope (CLSM) equipped with a Linkam PE 94 heating/cooling stage. CLSM was operated in reflection mode with an Ar/ArKr laser source (488 nm) and a 40 oil-immersion objective lens [29]. 2.4. Uptake of quercetin conjugates into HepG2 cells HepG2 cells were seeded into 12-well plates (Corning Costar Corp., Cambridge, MA) at a cell density of 2 105 per well. After 72–96 h, uptake experiments were carried out using HBSS buffer supplemented with 1.8 mM CaCl2 and adjusted to pH 6 or pH 7.4 with 1 M HCl. After removal of media, cells were washed twice with 0.4 mL transport buffer and incubated for 15 min. It was replaced with 0.4 mL transport buffer containing quercetin metabolites. Quercetin glucuronides were dissolved in DMSO (final concentration < 0.5%); whereas quercetin-30 -O-sulfate was dissolved in water. Digoxin and inhibitors were dissolved in DMSO (final concentration < 0.1%) and controls with identical concentrations of DMSO were used. After 10 min, during which uptake was linear, 2 mL ice-cold transport buffer containing 0.2% bovine serum albumin (BSA) was added. This was quickly aspirated and further washed twice with 0.5 mL ice-cold transport buffer with 0.2% BSA. Finally, the cells were rinsed with 0.5 mL ice-cold transport buffer. 2.5. Uptake of quercetin-30 -O-sulfate into OAT2, OAT4 and OATP4C1 expressing cells HEK-293 cells were seeded into poly-L-lysine coated 24-well plates at a density of 1.2 105 cells/per well. 2 mg of OAT2, OAT4 or OATP4C1 plasmids, or the empty vector was mixed with 3 mL Fugene HD reagent, in 100 mL Opti-MEM (Invitrogen). After incubation for 18 min at room temperature, 25 mL of transfection complex was added to each well. Uptake assays were performed 22–24 h after transfection. Each was carried out in HBSS containing 1.8 mM CaCl2 and 1.8 mM MgCl2 at pH 7.4. Media was removed and the monolayer was washed twice with 0.25 mL transport buffer. After 10 min, buffer was replaced with transport buffer containing the test compounds. After the incubation period, uptake was stopped by adding 1 mL ice-cold transport buffer containing 0.2% BSA. This was removed and washed twice with 0.5 mL ice-cold transport buffer with 0.2% BSA, and finally washed with 1 mL icecold transport buffer.
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2.6. OATP4C1-knockdown in HepG2 cells OATP4C1-siRNA and the control-siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HepG2 cells were transfected with siPORTTM NeoFXTM Transfection Agent (Ambion, Austin, TX). Briefly, 3 mL of siPORTTM NeoFXTM was mixed with 47 mL of Opti-MEM and incubated at room temperature for 10 min. Five mL of OATP4C1 siRNA or control siRNA was diluted with 45 mL Opti-MEM. Then, 50 mL of diluted siPORTTM NeoFXTM were mixed with 50 mL of the diluted siRNA and further incubated for 10 min at room temperature. Transfection complexes (100 mL per well) were pipetted into 12-well plates and 900 mL of suspended HepG2 cells in complete media without antibiotics was added (1 105 cells/ mL). At 24 h post-transfection, media was replaced with fresh complete media without antibiotics. Transfected cells were used for uptake studies at 72 h post-transfection. 2.7. Extraction of intracellular quercetin metabolites To quantify intracellular quercetin metabolites and digoxin, cells were collected with 0.4 mL lysis solution (50% methanol containing 10 mM hesperetin as internal standard) and stored at 80 8C. Extraction was performed by sonication for 5 min followed by the addition of 1 mL of ice-cold acetone. Samples were placed in a 20 8C freezer for 1 h and centrifuged at 17,000 g for 5 min. The supernatant was collected and evaporated to dryness in vacuo at 30 8C and stored at 20 8C until analysis. The protein pellet was redissolved in 0.1 N NaOH and the protein content determined by the Bradford assay. All the uptake values were corrected against protein content. Recovery of samples spiked with quercetin metabolites was found to be over 95%. 2.8. RNA isolation and Quantitative RT-PCR HepG2 or HEK-293 cells were harvested in lysis buffer and total RNA was prepared using an RNeasy mini kit (Qiagen, Valencia, CA). cDNA was synthesized from RNA using a High Capacity RNA-tocDNA Master Mix (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed in a StepOneTM real-time PCR System (Applied Biosystems) and TaqMan1 Gene Expression Master Mix (Applied Biosystems). The specific primers for OAT1, OAT2, OAT3, OAT4, OATP4C1, OATP1B1 and GADPH were obtained from Applied Biosystems. A four-step PCR programme was used for the quantitative RT-PCR, first holding at 50 8C for 5 min and then at 95 8C for 10 min, followed by 40 cycles of 95 8C for 15 s and 60 8C for 1 min. Levels of mRNA expression of the transporter genes was expressed relative to expression of GAPDH. 2.9. Western-blot analysis of OATP4C1 Whole cell extracts (20 mg protein) were mixed with an equal volume of 2 times concentrated sample buffer (2% SDS, 125 mmol Tris–HCl, pH 7.4, 20% glycerol, and 2% 2-mercaptoethanol) and subjected to SDS-PAGE (samples were not boiled) using standard protocols. After electrophoresis, proteins were transferred to a PVDF membrane (Bio-Rad) using standard protocols. After transfer, membranes were blocked with 5% dried milk in PBS-T (PBS containing 0.1% Tween 20) at room temperature for 1 h. Blocked membranes were incubated with anti-human SLCO4C1 polyclonal antibody (1.5 mg/mL) in PBS-T containing 1% dried milk at 4 8C overnight as described previously [30]. After washing with PBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (10 ng/mL) (Pierce, Rockford, IL) for 1 h at room temperature. Signals were detected by chemiluminescence with a Super Signal West Dura Extended Duration Substrate (Pierce).
2.10. HPLC analysis of quercetin metabolites and LC–MS analysis of digoxin The dried samples were re-constituted in 50 mL of 30% methanol. HPLC analyses were carried out on an Agilent 1200 series liquid chromatography system equipped with a diode array detector. The analyses were performed with a Zorbax XDB-C18 column (4.6 mm 50 mm, 1.8 mm) with 20 mM ammonium formate, pH 4.5 (A) and methanol (B) as the mobile phase. Injection volume was 25 mL. Elution was performed at 1 mL/min with gradient started at 30% (B) and increased linearly to 50% in 10 min, then equilibrated at 30% for 2.5 min. The limit of detection for the quantification of quercetin was 0.02 mM. Quantification of quercetin metabolites was based on the peak area of quercetin at 370 nm, assuming similar response factors for flavonoids and their conjugates [5,31]. For the analysis of digoxin, the HPLC system comprised an Agilent 1200 series (Santa Clara, CA) coupled to an Agilent 6410 triple-quad MS with an electrospray source. Chromatography was performed with a Zorbax Eclipse SB-C18 column (2.1 mm 10 mm, 1.8 mm), with a mobile phase of 0.2% formic acid (A) and methanol (B). The gradient started at 15% for 2 min, followed by a linear increase to 80% in 8 min, and returned to 15% in 2 min. Flow rate was 0.5 mL/min. The MS conditions were as follows: negative ionization mode (ESI-), capillary potential was 4 kV, and the fragmentor potential was 120 V. Detection was in multiple reaction monitoring (MRM) mode m/z 798.3 ! 651.3 with collision cell potential set at 10 V. Quantification was provided by the integrated peak area of the plot of total product ion abundance over time. 2.11. Statistical analysis All results are expressed as means S.D. Apparent enzyme kinetic data were fitted using the OriginPro 8 software. Non-linear regression analysis was performed based on the Michaelis–Menten equation. 3. Results 3.1. Uptake of quercetin conjugates by HepG2 cells The uptake of quercetin conjugates was analyzed qualitatively by fluorescence microscopy and quantitatively by HPLC. Incubation of HepG2 cells with quercetin (25 mM) for 30 min resulted in a strong fluorescent signal inside the cells when observed under UV light, in agreement with previous literature [32]. Fluorescent staining was also observed after incubation with quercetin glucuronides and quercetin-30 -O-sulfate (Fig. 1), although to a weaker extent compared to quercetin. This indicates a significant qualitative intracellular accumulation of these compounds in HepG2 cells. Uptake of quercetin-7-O-glucuronide, quercetin-3-O-glucuronide, quercetin-30 -O-glucuronide and quercetin-30 -O-sulfate (50 mM) was also measured by HPLC analysis of cell extracts. Uptake was linear over at least 10 min. Uptake of quercetin-30 -O-sulfate was much higher (8-fold) than that of quercetin glucuronides (Fig. 2A). The rate of uptake of quercetin glucuronides was in the order of 30 > 3 > 7-glucuronides. In order to investigate the possibility of the presence of a carrier-mediated uptake mechanism, the uptake at 37 8C was compared to that at 4 8C. There was no significant difference in the uptake of quercetin-7-O-glucuronide and quercetin3-O-glucuronide at 4 8C compared to 37 8C, indicating that passive diffusion might account for their uptake into HepG2 cells. On the other hand, uptake of quercetin-30 -O-glucuronide and quercetin-30 O-sulfate showed temperature-dependence, with the uptake at 37 8C approximately 2- and 8-fold greater than at 4 8C respectively. Kinetics of the uptake of quercetin-30 -O-sulfate into HepG2 cells was further characterized. The uptake of quercetin-30 -O-sulfate was examined
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Fig. 1. Fluorescence microscopy of HepG2 cells. HepG2 cells were treated with buffer (0.1% DMSO) (A), 25 mM quercetin (B), 25 mM quercetin-7-O-glucuronide (C) and 25 mM quercetin-30 -O-sulfate (D) for 30 min.
over the concentration range of 2.5–200 mM (Fig. 2B). We found that the uptake was both temperature-dependent and saturable, with an apparent Km and Vmax value of 59 18 mM and 150 18 pmol/ (min mg), respectively. 3.2. Effect of pH and of OATs/OATPs inhibitors on the uptake of quercetin-30 -O-sulfate by HepG2 cells Due to the anionic and amphiphilic nature of quercetin-30 -Osulfate, we investigated the possibility of involvement of organic anion transporters (OATs) and/or organic anion transporting polypeptides (OATPs) in the transporter-mediated uptake of the sulfate conjugate into HepG2 cells (Fig. 3). Uptake of quercetin-30 O-sulfate by HepG2 cell was compared between pH 6 and pH 7.4. Transport of quercetin-30 -O-sulfate (25 mM) was significantly higher at pH 6 (30.7 2.5 pmol/(min mg)) than at pH 7.4 (3.87 1.70 pmol/(min mg)). Next we tested whether substrates/ inhibitors of OATs and OATPs would affect the uptake of quercetin-30 O-sulfate. Estrone-3-sulfate and sulfobromophthalein (BSP), inhibitors for both OATs and OATPs, resulted in a significant decrease in the uptake (Fig. 3). OATP substrates taurocholate and digoxin also significantly inhibited quercetin-30 -O-sulfate into HepG2 cells. Digoxin is a substrate of OATP4C1 [33], an OATP transporter that is highly expressed in HepG2 cells [16]. 3.3. Uptake of quercetin-30 -O-sulfate into OAT2, OAT4 and OATP4C1expressing cells To investigate the potential roles of OATs and OATPs in the uptake of quercetin-30 -O-sulfate into HepG2 cells, OAT2, OAT4 and
OATP4C1 were over-expressed in HEK-293 cells. Real-time PCR analysis revealed highly effective induction in mRNA expression in the transfected HEK-293 cells (data now shown). OAT2- and OAT4expressing cells were able to specifically accumulate model substrates, p-aminohippuric acid and carboxyfluorescein, respectively. Quercetin-30 -O-sulfate uptake was measured at pH 6 and pH 7.4 (Fig. 4). At pH 7.4, the uptake of quercetin-30 -O-sulfate by OAT4 expressing cells was 7.9-fold higher compared to the control, while OAT2 and OATP4C1 over-expression had no effect. At pH 6, the basal uptake in control cells was enhanced, which may be attributed to the increased hydrophobicity of quercetin-30 -Osulfate at lower pH. Similarly, the uptake by OAT2, OAT4 and OATP4C1 expressing cells was higher at pH 6 than at pH 7.4. OAT4 and OATP4C1-overexpressing cells showed significant (2.3- and 1.4- fold, respectively) increase in the uptake of quercetin-30 -Osulfate compared to the control; while there was no difference between the control cells and OAT2-expressing cells. 3.4. siRNA knockdown of OATP4C1 in HepG2 cells and the effect on the uptake of quercetin-30 -O-sulfate and digoxin Real-time PCR analysis of OAT and OATP gene expression in HepG2 cells revealed high expression of OATP4C1, and significant expression of OAT2 and OAT4, but low expression of OATP1B1 (data not shown), in good agreement with previous studies [16,15,34]. Examination of OATP4C1 expression in OATP4C1 siRNA transfected cells by real-time PCR showed effective knockdown of OATP4C1 mRNA compared to control cells (Fig. 5A). The decrease in OATP4C1 gene expression was around 80–90% at 24–72 h posttransfection. To verify knockdown specificity, we examined
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Fig. 4. Uptake of quercetin-30 -O-sulfate (50 mM, 10 min) into control, OAT2, OAT4 and OATP4C1-expressing cells. HEK293 cells were transiently transfected with OAT2, OAT4 or OATP4C1 and the uptake of quercetin 30 -O-sulfate was measured at pH 6 and pH 7.4. (*) Uptake statistically different from control (p < 0.05).
uptake of 50 mM hesperetin, a hydrophobic compound that is expected to enter cells via passive diffusion, was not affected. None of the quercetin glucuronides showed uptake in OATP4C1 siRNA treated cells that were significantly different from control (data not shown). 4. Discussion
Fig. 2. Accumulation of quercetin metabolites in HepG2 cells. (A) Quercetin glucuronides or quercetin-30 -O-sulfate (50 mM) in HBSS (pH 6) was incubated with HepG2 cells for 10 min and the cell extract was analyzed by HPLC. The uptake of quercetin-30 -O-sulfate appeared to be highly active and temperature dependent compared to the quercetin glucuronides tested. Uptake at 37 8C was significantly different from at 4 8C (*p < 0.05; **p < 0.01, n = 3). Quercetin-3-O-glucuronide: Q3G; quercetin-7-O-glucuronide: Q7G; quercetin-30 -O-glucuronide: Q30 G; quercetin-30 -O-sulfate: Q30 S. (B) Kinetic analysis of quercetin-30 -O-sulfate uptake in HepG2 cells (pH 6). Uptake of quercetin-30 -O-sulfate was concentrationdependent and saturable.
Dietary quercetin is conjugated by phase II metabolism following absorption in the intestinal tissues, and these conjugates are predominant in human plasma. Previous research has shown that quercetin conjugates inhibit oxidative enzymes such as COX-2 and also suppress the expression of COX-2 in Caco-2 cells [7,35]; however, little is known how these physiological metabolites interact with or enter into cells, essential for their biological activity. Disposition of these hydrophilic conjugates relies, at least in part, on transporters mediating their influx and efflux out of cells, for example, how quercetin glucuronides are taken up by liver cells and further metabolized. In this study, we found that the
OATP1B1 expression in control-siRNA and OATP4C1-siRNA treated cells. OATP4C1-siRNA did not reduce the gene expression of OATP1B1. In agreement with RT-PCR, transfection with OATP4C1siRNA resulted in decreased OATP4C1 protein levels (Fig. 5B). The uptake of quercetin-30 -O-sulfate (50 mM) was significantly decreased by about 40% in OATP4C1-siRNA transfected cells compared to control-siRNA transfected cells (Fig. 6A). Uptake of digoxin, a model substrate for OATP4C1, was also significantly reduced with OATP4C1 knockdown (Fig. 6B). On the other hand,
Fig. 3. Effect of OAT/OATP inhibitors on the uptake of quercetin-30 -O-sulfate in HepG2 cells. Cellular uptake of quercetin-30 -O-sulfate (50 mM) in HepG2 cells in HBSS (pH 6) for 10 min was measured in the presence of substrates and inhibitors of OATPs (500 mM). (*) Uptake statistically different from control (p < 0.05, n = 3).
Fig. 5. Effect on OATP4C1-siRNA on the expression of OATP4C1 in HepG2 cells. HepG2 cells were transfected with control-siRNA or OATP4C1-siRNA and the mRNA (A) and protein expression (B) was measured using real-time PCR and western blots (72 h time point), respectively (n = 3).
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Fig. 6. Effect of OATP4C1-knockdown on the uptake of quercetin-30 -O-sulfate and hesperetin (A) and digoxin (B) (all at 50 mM for 10 min). HepG2 cells were transfected with control-siRNA or OATP4C1-siRNA, and the uptake experiments were performed 72 h post-transfection. (*) Uptake statistically different from control (p < 0.05, n = 5).
uptake of quercetin-30 -O-sulfate into HepG2 cells was pH-, temperature, concentration-dependent and saturable, suggesting a carrier mediated mechanism. The Km value obtained for the uptake of quercetin-30 -O-sulfate in HepG2 cells (58.7 17.8 mM) was higher than physiological concentrations (<10 mM) achieved with dietary doses of quercetin [36]. Nevertheless, we also observed active uptake at lower concentrations (<5 mM) in HepG2 cells. Hence, active uptake transporters may play an important role in hepatic uptake and clearance of quercetin conjugates in humans. In the liver and kidneys, OATs and OATPs are expressed on the basolateral membrane and mediate uptake of organic anions, including glucuronides and sulfate conjugates of steroids and xenobiotics. Typical inhibitors of OATs and OATPs potently inhibit the initial uptake of quercetin-30 -O-sulfate into HepG2 cells. Uptake of quercetin-30 -O-sulfate was stimulated by a lower buffer pH, consistent with reports of higher transport activity of OATPs at lower extracellular pH [37]. HepG2 cells express several OATs and OATPs found in the human liver, although there are quantitative differences in their levels of expression. Several OATs/OATPs have been detected in HepG2 cells, including OAT2, OAT4, OATP1B1, OATP1B3, OATP4A1 and OATP4C1 [16]. Consistent with previous studies, we found high expression of OAT2, OAT4 and OATP4C1. We hypothesized that these transporters may be involved in the uptake of quercetin-30 -O-sulfate into HepG2 cells. OAT4- and OATP4C1 expression enhanced quercetin-30 -O-sulfate uptake compared to control cells. In addition, siRNA-mediated knockdown of OATP4C1 in HepG2 cells resulted in 40% reduction in the uptake of quercetin-30 -O-sulfate. Uptake of quercetin glucuronides was not affected. OAT4 and OATP4C1 were first isolated from human kidney cDNA [33,38], and their transcripts are also detected in human liver [15,16]. OAT4 mediates the uptake of sulfated hormones, such as estrone-3-sulfate [38], and drugs such as antibiotics, antivirals, methotrexate and NSAIDs [39,40]. OATP4C1 has narrow substrate specificity and it transports digoxin, thyroid hormone, methotrexate and estrone-3-sulfate [33,37,41]. Both OAT4 and OATP4C1 showed poor transport activity toward glucuronide conjugates such as estradiol-3-glucuronide [33]. This apparent preference of OAT4 and OATP4C1 for sulfates over glucuronides conjugates may explain a lack of active uptake of quercetin glucuronides into HepG2 cells. These results provide evidence that OATs and OATPs may be involved in the active uptake of flavonoid metabolites in human hepatoma cells. In the human liver, other OATP isoforms, including OATP1B1 and OAT1B3, are highly expressed. These OATP transporters may also participate in the uptake of quercetin metabolites in vivo.
Vectorial transport of organic anions in hepatocytes involved the interplay of basolateral OATs/OATPs and apical MRPs. Conjugates of quercetin are known substrates for MRP2 [42,43] and BCRP [44]. MRP2, in particular, is involved in apical efflux of quercetin glucuronides and quercetin-30 -O-sulfate from HepG2 cells [42]. MRP2 and BCRP are likely to act in concert with OATs and OATPs to result in the removal of quercetin-30 -O-sulfate from blood into bile, leading to reduced bioavailability of quercetin. Due to the poor passive permeability of the hydrophilic glucuronide or sulfate conjugates to cell membranes, transport by OATs and OATPs may play an important role affecting their distribution into target tissues. Studies in rats and pigs showed that there is minimal accumulation in body tissues outside the gastrointestinal tract, liver and kidney [45–47]. However, there is biological activity since, for example, several animal studies showed that orally administered quercetin was protective against liver injury induced by various agents such as ethanol [48] and microcystin [49]. Quercetin also reduced liver damage in biliaryobstructed rats [50]. Selective uptake by OATs and OATPs expressed in the liver and kidney may account for beneficial effects of quercetin in these drug-metabolizing tissues. In summary we showed a potential role for OAT4 and OATP4C1 in quercetin-30 -O-sulfate uptake in HepG2 cells. In humans, the OATs and OATPs may act in concert with efflux transporters to result in the excretion of quercetin metabolites generated from the small intestine, and play a role in limiting the bioavailability of quercetin. Acknowledgment We thank Dr. Hassan Firoozmand for providing assistance in confocal microscopy and the Nestle Research Center, Switzerland, for funding. References [1] Lee ER, Kang YJ, Kim JH, Lee HT, Cho SG. Modulation of apoptosis in HaCaT keratinocytes via differential regulation of ERK signaling pathway by flavonoids. J Biol Chem 2005;280:31498–507. [2] Song J, Kwon O, Chen S, Daruwala R, Eck P, Park JB, et al. Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2 (GLUT2), intestinal transporters for vitamin C and Glucose. J Biol Chem 2002;277:15252–60. [3] Garcia-Mediavilla V, Crespo I, Collado PS, Esteller A, Sanchez-Campos S, Tunon MJ, et al. The anti-inflammatory flavones quercetin and kaempferol cause inhibition of inducible nitric oxide synthase, cyclooxygenase-2 and reactive Cprotein, and down-regulation of the nuclear factor kappaB pathway in Chang Liver cells. Eur J Pharmacol 2007;557:221–9.
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Carrier-mediated transport of quercetin conjugates: Involvement of organic anion transporters and organic anion transporting polypeptides Chi Chun Wong a, Yasutoshi Akiyama b, Takaaki Abe b, Jonathan D. Lippiat c, Caroline Orfila a, Gary Williamson a,d,* a
School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT, UK Division of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK d Nestle Research Center, Vers Chez les Blanc, 1000 Lausanne 26, Switzerland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 March 2012 Accepted 14 May 2012 Available online 23 May 2012
Flavonoids modulate cell signaling and inhibit oxidative enzymes. After oral consumption, they circulate in human plasma as amphiphilic glucuronide or sulfate conjugates, but it is unknown how these physiological metabolites permeate into cells. We examined the mechanisms of uptake of these conjugates into hepatocellular carcinoma (HepG2) cells, and found that uptake of quercetin-30 -O-sulfate was saturable and temperature-dependent, indicating the involvement of carrier-mediated transport. Quercetin-3-O-glucuronide was taken up predominantly via passive diffusion in these cells. Quantitative real-time PCR analysis showed high expression of OATP4C1, followed by OAT2, OAT4 and low expression of OATP1B1 in HepG2 cells, and addition of inhibitors of OATs and OATPs resulted in a significant reduction in quercetin-30 -O-sulfate uptake. The accumulation of quercetin-30 -O-sulfate was further evaluated in HEK293 cells expressing OAT2, OAT4 and OATP4C1. Uptake of quercetin-30 -O-sulfate was 2.3- and 1.4-fold higher in cells expressing OAT4 and OATP4C1 at pH 6.0, respectively, than in control HEK293 cells. siRNA knockdown of OATP4C1 expression in HepG2 cells reduced uptake of quercetin-30 O-sulfate by 40%. This study highlights a role for OATs and OATPs in the cellular uptake of biologically active flavonoid conjugates. ß 2012 Elsevier Inc. All rights reserved.
Keywords: Quercetin HepG2 Carrier-mediated transport Organic anion transporter Organic anion transporting polypeptide
1. Introduction Quercetin is a biologically active flavonoid that influences cell signaling pathways [1], inhibits the sodium-dependent vitamin C transporter [2], and modulates oxidative enzymes such as COX-2 [3] and lipoxygenase [4], thereby reducing the formation of intracellular superoxide and other reactive oxygen species. The biological activity is likely derived from the predominant forms of quercetin found in blood in vivo, the 30 -O-sulfated, 3-O-glucuronidated and 30 or 40 methylated derivatives. Quercetin glucuronides reduce oxidative stress via inhibition of xanthine oxidase and myeloperoxidase, enzymes that participate in free radical generation [5,6]. Some quercetin conjugates down-regulate the expression of cyclooxygenase-2 (COX-2), a key enzyme in the production of pro-inflammatory
Abbreviations: COX2, cyclooxygenase-2; HBSS, hank’s balanced salt solution; OATs, organic anion transporters; OATPs, organic anion transporting polypeptides. * Corresponding author at: School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT, UK. Tel.: +44 0 113 343 8380; fax: +44 0 113 343 2982. E-mail address: [email protected] (G. Williamson). 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.05.011
eicosanoids. Quercetin-30 -O-sulfate directly inhibited COX-2 activity, whereas quercetin-3-O-glucuronide did not [7]. Emerging evidence supports a role for quercetin metabolites in vascular health. The sulfated and glucuronidated conjugates of quercetin prevent oxidative stress-induced endothelial dysfunction in rat aorta [8]. Quercetin-3-O-glucuronide was also found to exert an antiatherosclerotic effect by suppressing the expression of scavenger receptors in activated macrophages [9]. In humans, quercetin is extensively metabolized via glucuronidation, sulfation, or methylation, resulting in the formation of numerous conjugated metabolites [10]. Phase II conjugates, such as quercetin-30 -O-sulfate, are the predominant circulating forms in plasma (<10 mM), and the aglycone is not found in vivo [10,11]. Contribution of the bile to the secretion of quercetin conjugates was estimated to be 10–30% and their concentration in the bile could reach in excess of 300 mM in mice [12]. Quercetin conjugates are markedly more hydrophilic compared to quercetin [13]. Transport of hydrophilic and ionized substances from the circulation into bile or urine involves interplay of uptake transporters on the basolateral membrane and efflux transporters on the apical side, which are present in hepatocytes and proximal
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tubular cells. Basolateral uptake of organic anions, such as glucuronide and sulfate conjugates, is thought to involve organic anion transporters (OATs) and/or organic anion-transporting polypeptides (OATPs) [14]. They are multispecific and mediate the transport of various amphiphilic organic anions, including endogenous compounds, structurally diverse drugs and their conjugated metabolites. Thus, OATs and OATPs are important determinants for excretion of xenobiotics. Human liver showed a high expression of OATP1B1 and OATP1B3, as well as moderate expression of OATP1A2, OATP4C1 and OATP2B1 [15,16]. OAT2, OAT4 and OAT7 mRNA have also been detected [16,17]. OAT1, OAT3, and OATP4C1 are the major uptake transporters found in the human kidneys. Active efflux is carried out by ATP-binding cassette transporters including P-glycoprotein, multi-drug resistance protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) [18]. OATs/OATPs and ABC transporters are believed to act in concert to affect vectorial transport of various organic anions, and thus facilitate their elimination from the body. Recently, it has been shown that the green tea flavonoids epicatechin gallate and epigallocatechin gallate are substrates of OATP1A2 and OATP1B3 [19], and that several flavonoid and their conjugated forms can interact with OAT1, OATP1A2 and OATP1B1 [20–26]. However, no physiologically relevant flavonoid conjugates have been shown to interact with members of the OAT or OATP families. Hence, we hypothesized that conjugates of flavonoids such as quercetin may be potential substrates for OATs and OATPs. Here, we demonstrate a carrier-mediated uptake mechanism for quercetin-30 -O-sulfate into HepG2 cells, but not for quercetin glucuronides, and identified potential OAT and OATP transporters involved in the uptake in HepG2 cells. 2. Materials and methods 2.1. Chemicals Sulfobromophthalein was from Acros Organics (Geel, Belgium). Quercetin-7-O-glucuronide, quercetin-3-O-glucuronide and quercetin-30 -O-glucuronide were synthesized enzymatically and then purified by HPLC [27]. Chemical synthesis of quercetin-30 -O-sulfate was performed as described [28]. The identity of the compounds was further confirmed by comparing the retention time and absorption spectra of quercetin-3-O-glucuronide and quercetin-30 O-sulfate kindly provided by Dr. Paul Kroon, Institute of Food Research, UK. The purities were checked by HPLC to be over 95%. HepG2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human embryonic kidney cells HEK-293 were purchased from Invitrogen (Carlsbad, CA). OAT1 (SLC22A6 transcript variant 2, Genebank accession number: NM_153276), OAT2 (SLC22A7 transcript variant 1, Genebank accession number: NM_006672.2), OAT3 (SLC22A8 transcript variant 1, Genebank accession number: NM_004254) and OAT4 (Genebank accession number: NM_018484.2) expression plasmids were obtained from Origene (Rockville, MD). Full length OATP4C1 cDNA (Image clone 100016240) (Geneservice, Cambridge, UK) was re-cloned into pcDNA6/V5-His (forward primer: 50 -ggatccatgaagagcgccaaaggtatt-30 ; reverse primer: 50 -gcggccgcacttacccttcttttactattttgttgag-30 ) for transfection into mammalian cell lines. Fugene HD was purchased from Roche. Other chemicals, unless otherwise stated, were purchased from Sigma–Aldrich (St. Louis, MO). 2.2. Cell culture HepG2 cells were routinely cultured in 75 cm2 cell culture flasks (Corning Costar Corp., Cambridge, MA) at 37 8C under a humidified 5% CO2/O2 atmosphere. The culture media consisted of
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Eagle’s Minimum Essential Medium media supplemented with 10% fetal bovine serum and 100 U/mL penicillin–streptomycin. All experiments were performed with cells between passages 80 and 95. HEK-293 cells were cultured in Dulbecco’s modified Minimum Essential Medium high-glucose media supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 50 U/mL penicillin–streptomycin, in 5% CO2 at 37 8C. Cells were used between passages 4 and 20. 2.3. Confocal microscopy 5 104 HepG2 cells were plated in 12 mm poly-L-lysine-coated coverslips (BD Bioscience, Bedford, MA) within six-well plates. Cells were then cultured for 96 h before the experiment. Incubation medium used was Hank’s Balanced Salt Solution (HBSS) buffer supplemented with 1.8 mM CaCl2, adjusted to pH 6 with 1 M HCl. Various concentrations of quercetin, quercetin-7O-glucuronide, quercetin-3-O-glucuronide, quercetin-30 -O-glucuronide and quercetin-30 -O-sulfate were added to the cells and then incubated for 30 min at 37 8C. Uptake was stopped by washing with ice-cold HBSS three times and then cells were fixed with 4% formaldehyde for 5 min. Coverslips were washed with water and mounted upside-down on glass slides. The images were obtained with a Leica TCS SP2 confocal laser scanning microscope (CLSM) equipped with a Linkam PE 94 heating/cooling stage. CLSM was operated in reflection mode with an Ar/ArKr laser source (488 nm) and a 40 oil-immersion objective lens [29]. 2.4. Uptake of quercetin conjugates into HepG2 cells HepG2 cells were seeded into 12-well plates (Corning Costar Corp., Cambridge, MA) at a cell density of 2 105 per well. After 72–96 h, uptake experiments were carried out using HBSS buffer supplemented with 1.8 mM CaCl2 and adjusted to pH 6 or pH 7.4 with 1 M HCl. After removal of media, cells were washed twice with 0.4 mL transport buffer and incubated for 15 min. It was replaced with 0.4 mL transport buffer containing quercetin metabolites. Quercetin glucuronides were dissolved in DMSO (final concentration < 0.5%); whereas quercetin-30 -O-sulfate was dissolved in water. Digoxin and inhibitors were dissolved in DMSO (final concentration < 0.1%) and controls with identical concentrations of DMSO were used. After 10 min, during which uptake was linear, 2 mL ice-cold transport buffer containing 0.2% bovine serum albumin (BSA) was added. This was quickly aspirated and further washed twice with 0.5 mL ice-cold transport buffer with 0.2% BSA. Finally, the cells were rinsed with 0.5 mL ice-cold transport buffer. 2.5. Uptake of quercetin-30 -O-sulfate into OAT2, OAT4 and OATP4C1 expressing cells HEK-293 cells were seeded into poly-L-lysine coated 24-well plates at a density of 1.2 105 cells/per well. 2 mg of OAT2, OAT4 or OATP4C1 plasmids, or the empty vector was mixed with 3 mL Fugene HD reagent, in 100 mL Opti-MEM (Invitrogen). After incubation for 18 min at room temperature, 25 mL of transfection complex was added to each well. Uptake assays were performed 22–24 h after transfection. Each was carried out in HBSS containing 1.8 mM CaCl2 and 1.8 mM MgCl2 at pH 7.4. Media was removed and the monolayer was washed twice with 0.25 mL transport buffer. After 10 min, buffer was replaced with transport buffer containing the test compounds. After the incubation period, uptake was stopped by adding 1 mL ice-cold transport buffer containing 0.2% BSA. This was removed and washed twice with 0.5 mL ice-cold transport buffer with 0.2% BSA, and finally washed with 1 mL icecold transport buffer.
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2.6. OATP4C1-knockdown in HepG2 cells OATP4C1-siRNA and the control-siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HepG2 cells were transfected with siPORTTM NeoFXTM Transfection Agent (Ambion, Austin, TX). Briefly, 3 mL of siPORTTM NeoFXTM was mixed with 47 mL of Opti-MEM and incubated at room temperature for 10 min. Five mL of OATP4C1 siRNA or control siRNA was diluted with 45 mL Opti-MEM. Then, 50 mL of diluted siPORTTM NeoFXTM were mixed with 50 mL of the diluted siRNA and further incubated for 10 min at room temperature. Transfection complexes (100 mL per well) were pipetted into 12-well plates and 900 mL of suspended HepG2 cells in complete media without antibiotics was added (1 105 cells/ mL). At 24 h post-transfection, media was replaced with fresh complete media without antibiotics. Transfected cells were used for uptake studies at 72 h post-transfection. 2.7. Extraction of intracellular quercetin metabolites To quantify intracellular quercetin metabolites and digoxin, cells were collected with 0.4 mL lysis solution (50% methanol containing 10 mM hesperetin as internal standard) and stored at 80 8C. Extraction was performed by sonication for 5 min followed by the addition of 1 mL of ice-cold acetone. Samples were placed in a 20 8C freezer for 1 h and centrifuged at 17,000 g for 5 min. The supernatant was collected and evaporated to dryness in vacuo at 30 8C and stored at 20 8C until analysis. The protein pellet was redissolved in 0.1 N NaOH and the protein content determined by the Bradford assay. All the uptake values were corrected against protein content. Recovery of samples spiked with quercetin metabolites was found to be over 95%. 2.8. RNA isolation and Quantitative RT-PCR HepG2 or HEK-293 cells were harvested in lysis buffer and total RNA was prepared using an RNeasy mini kit (Qiagen, Valencia, CA). cDNA was synthesized from RNA using a High Capacity RNA-tocDNA Master Mix (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed in a StepOneTM real-time PCR System (Applied Biosystems) and TaqMan1 Gene Expression Master Mix (Applied Biosystems). The specific primers for OAT1, OAT2, OAT3, OAT4, OATP4C1, OATP1B1 and GADPH were obtained from Applied Biosystems. A four-step PCR programme was used for the quantitative RT-PCR, first holding at 50 8C for 5 min and then at 95 8C for 10 min, followed by 40 cycles of 95 8C for 15 s and 60 8C for 1 min. Levels of mRNA expression of the transporter genes was expressed relative to expression of GAPDH. 2.9. Western-blot analysis of OATP4C1 Whole cell extracts (20 mg protein) were mixed with an equal volume of 2 times concentrated sample buffer (2% SDS, 125 mmol Tris–HCl, pH 7.4, 20% glycerol, and 2% 2-mercaptoethanol) and subjected to SDS-PAGE (samples were not boiled) using standard protocols. After electrophoresis, proteins were transferred to a PVDF membrane (Bio-Rad) using standard protocols. After transfer, membranes were blocked with 5% dried milk in PBS-T (PBS containing 0.1% Tween 20) at room temperature for 1 h. Blocked membranes were incubated with anti-human SLCO4C1 polyclonal antibody (1.5 mg/mL) in PBS-T containing 1% dried milk at 4 8C overnight as described previously [30]. After washing with PBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (10 ng/mL) (Pierce, Rockford, IL) for 1 h at room temperature. Signals were detected by chemiluminescence with a Super Signal West Dura Extended Duration Substrate (Pierce).
2.10. HPLC analysis of quercetin metabolites and LC–MS analysis of digoxin The dried samples were re-constituted in 50 mL of 30% methanol. HPLC analyses were carried out on an Agilent 1200 series liquid chromatography system equipped with a diode array detector. The analyses were performed with a Zorbax XDB-C18 column (4.6 mm 50 mm, 1.8 mm) with 20 mM ammonium formate, pH 4.5 (A) and methanol (B) as the mobile phase. Injection volume was 25 mL. Elution was performed at 1 mL/min with gradient started at 30% (B) and increased linearly to 50% in 10 min, then equilibrated at 30% for 2.5 min. The limit of detection for the quantification of quercetin was 0.02 mM. Quantification of quercetin metabolites was based on the peak area of quercetin at 370 nm, assuming similar response factors for flavonoids and their conjugates [5,31]. For the analysis of digoxin, the HPLC system comprised an Agilent 1200 series (Santa Clara, CA) coupled to an Agilent 6410 triple-quad MS with an electrospray source. Chromatography was performed with a Zorbax Eclipse SB-C18 column (2.1 mm 10 mm, 1.8 mm), with a mobile phase of 0.2% formic acid (A) and methanol (B). The gradient started at 15% for 2 min, followed by a linear increase to 80% in 8 min, and returned to 15% in 2 min. Flow rate was 0.5 mL/min. The MS conditions were as follows: negative ionization mode (ESI-), capillary potential was 4 kV, and the fragmentor potential was 120 V. Detection was in multiple reaction monitoring (MRM) mode m/z 798.3 ! 651.3 with collision cell potential set at 10 V. Quantification was provided by the integrated peak area of the plot of total product ion abundance over time. 2.11. Statistical analysis All results are expressed as means S.D. Apparent enzyme kinetic data were fitted using the OriginPro 8 software. Non-linear regression analysis was performed based on the Michaelis–Menten equation. 3. Results 3.1. Uptake of quercetin conjugates by HepG2 cells The uptake of quercetin conjugates was analyzed qualitatively by fluorescence microscopy and quantitatively by HPLC. Incubation of HepG2 cells with quercetin (25 mM) for 30 min resulted in a strong fluorescent signal inside the cells when observed under UV light, in agreement with previous literature [32]. Fluorescent staining was also observed after incubation with quercetin glucuronides and quercetin-30 -O-sulfate (Fig. 1), although to a weaker extent compared to quercetin. This indicates a significant qualitative intracellular accumulation of these compounds in HepG2 cells. Uptake of quercetin-7-O-glucuronide, quercetin-3-O-glucuronide, quercetin-30 -O-glucuronide and quercetin-30 -O-sulfate (50 mM) was also measured by HPLC analysis of cell extracts. Uptake was linear over at least 10 min. Uptake of quercetin-30 -O-sulfate was much higher (8-fold) than that of quercetin glucuronides (Fig. 2A). The rate of uptake of quercetin glucuronides was in the order of 30 > 3 > 7-glucuronides. In order to investigate the possibility of the presence of a carrier-mediated uptake mechanism, the uptake at 37 8C was compared to that at 4 8C. There was no significant difference in the uptake of quercetin-7-O-glucuronide and quercetin3-O-glucuronide at 4 8C compared to 37 8C, indicating that passive diffusion might account for their uptake into HepG2 cells. On the other hand, uptake of quercetin-30 -O-glucuronide and quercetin-30 O-sulfate showed temperature-dependence, with the uptake at 37 8C approximately 2- and 8-fold greater than at 4 8C respectively. Kinetics of the uptake of quercetin-30 -O-sulfate into HepG2 cells was further characterized. The uptake of quercetin-30 -O-sulfate was examined
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Fig. 1. Fluorescence microscopy of HepG2 cells. HepG2 cells were treated with buffer (0.1% DMSO) (A), 25 mM quercetin (B), 25 mM quercetin-7-O-glucuronide (C) and 25 mM quercetin-30 -O-sulfate (D) for 30 min.
over the concentration range of 2.5–200 mM (Fig. 2B). We found that the uptake was both temperature-dependent and saturable, with an apparent Km and Vmax value of 59 18 mM and 150 18 pmol/ (min mg), respectively. 3.2. Effect of pH and of OATs/OATPs inhibitors on the uptake of quercetin-30 -O-sulfate by HepG2 cells Due to the anionic and amphiphilic nature of quercetin-30 -Osulfate, we investigated the possibility of involvement of organic anion transporters (OATs) and/or organic anion transporting polypeptides (OATPs) in the transporter-mediated uptake of the sulfate conjugate into HepG2 cells (Fig. 3). Uptake of quercetin-30 O-sulfate by HepG2 cell was compared between pH 6 and pH 7.4. Transport of quercetin-30 -O-sulfate (25 mM) was significantly higher at pH 6 (30.7 2.5 pmol/(min mg)) than at pH 7.4 (3.87 1.70 pmol/(min mg)). Next we tested whether substrates/ inhibitors of OATs and OATPs would affect the uptake of quercetin-30 O-sulfate. Estrone-3-sulfate and sulfobromophthalein (BSP), inhibitors for both OATs and OATPs, resulted in a significant decrease in the uptake (Fig. 3). OATP substrates taurocholate and digoxin also significantly inhibited quercetin-30 -O-sulfate into HepG2 cells. Digoxin is a substrate of OATP4C1 [33], an OATP transporter that is highly expressed in HepG2 cells [16]. 3.3. Uptake of quercetin-30 -O-sulfate into OAT2, OAT4 and OATP4C1expressing cells To investigate the potential roles of OATs and OATPs in the uptake of quercetin-30 -O-sulfate into HepG2 cells, OAT2, OAT4 and
OATP4C1 were over-expressed in HEK-293 cells. Real-time PCR analysis revealed highly effective induction in mRNA expression in the transfected HEK-293 cells (data now shown). OAT2- and OAT4expressing cells were able to specifically accumulate model substrates, p-aminohippuric acid and carboxyfluorescein, respectively. Quercetin-30 -O-sulfate uptake was measured at pH 6 and pH 7.4 (Fig. 4). At pH 7.4, the uptake of quercetin-30 -O-sulfate by OAT4 expressing cells was 7.9-fold higher compared to the control, while OAT2 and OATP4C1 over-expression had no effect. At pH 6, the basal uptake in control cells was enhanced, which may be attributed to the increased hydrophobicity of quercetin-30 -Osulfate at lower pH. Similarly, the uptake by OAT2, OAT4 and OATP4C1 expressing cells was higher at pH 6 than at pH 7.4. OAT4 and OATP4C1-overexpressing cells showed significant (2.3- and 1.4- fold, respectively) increase in the uptake of quercetin-30 -Osulfate compared to the control; while there was no difference between the control cells and OAT2-expressing cells. 3.4. siRNA knockdown of OATP4C1 in HepG2 cells and the effect on the uptake of quercetin-30 -O-sulfate and digoxin Real-time PCR analysis of OAT and OATP gene expression in HepG2 cells revealed high expression of OATP4C1, and significant expression of OAT2 and OAT4, but low expression of OATP1B1 (data not shown), in good agreement with previous studies [16,15,34]. Examination of OATP4C1 expression in OATP4C1 siRNA transfected cells by real-time PCR showed effective knockdown of OATP4C1 mRNA compared to control cells (Fig. 5A). The decrease in OATP4C1 gene expression was around 80–90% at 24–72 h posttransfection. To verify knockdown specificity, we examined
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Fig. 4. Uptake of quercetin-30 -O-sulfate (50 mM, 10 min) into control, OAT2, OAT4 and OATP4C1-expressing cells. HEK293 cells were transiently transfected with OAT2, OAT4 or OATP4C1 and the uptake of quercetin 30 -O-sulfate was measured at pH 6 and pH 7.4. (*) Uptake statistically different from control (p < 0.05).
uptake of 50 mM hesperetin, a hydrophobic compound that is expected to enter cells via passive diffusion, was not affected. None of the quercetin glucuronides showed uptake in OATP4C1 siRNA treated cells that were significantly different from control (data not shown). 4. Discussion
Fig. 2. Accumulation of quercetin metabolites in HepG2 cells. (A) Quercetin glucuronides or quercetin-30 -O-sulfate (50 mM) in HBSS (pH 6) was incubated with HepG2 cells for 10 min and the cell extract was analyzed by HPLC. The uptake of quercetin-30 -O-sulfate appeared to be highly active and temperature dependent compared to the quercetin glucuronides tested. Uptake at 37 8C was significantly different from at 4 8C (*p < 0.05; **p < 0.01, n = 3). Quercetin-3-O-glucuronide: Q3G; quercetin-7-O-glucuronide: Q7G; quercetin-30 -O-glucuronide: Q30 G; quercetin-30 -O-sulfate: Q30 S. (B) Kinetic analysis of quercetin-30 -O-sulfate uptake in HepG2 cells (pH 6). Uptake of quercetin-30 -O-sulfate was concentrationdependent and saturable.
Dietary quercetin is conjugated by phase II metabolism following absorption in the intestinal tissues, and these conjugates are predominant in human plasma. Previous research has shown that quercetin conjugates inhibit oxidative enzymes such as COX-2 and also suppress the expression of COX-2 in Caco-2 cells [7,35]; however, little is known how these physiological metabolites interact with or enter into cells, essential for their biological activity. Disposition of these hydrophilic conjugates relies, at least in part, on transporters mediating their influx and efflux out of cells, for example, how quercetin glucuronides are taken up by liver cells and further metabolized. In this study, we found that the
OATP1B1 expression in control-siRNA and OATP4C1-siRNA treated cells. OATP4C1-siRNA did not reduce the gene expression of OATP1B1. In agreement with RT-PCR, transfection with OATP4C1siRNA resulted in decreased OATP4C1 protein levels (Fig. 5B). The uptake of quercetin-30 -O-sulfate (50 mM) was significantly decreased by about 40% in OATP4C1-siRNA transfected cells compared to control-siRNA transfected cells (Fig. 6A). Uptake of digoxin, a model substrate for OATP4C1, was also significantly reduced with OATP4C1 knockdown (Fig. 6B). On the other hand,
Fig. 3. Effect of OAT/OATP inhibitors on the uptake of quercetin-30 -O-sulfate in HepG2 cells. Cellular uptake of quercetin-30 -O-sulfate (50 mM) in HepG2 cells in HBSS (pH 6) for 10 min was measured in the presence of substrates and inhibitors of OATPs (500 mM). (*) Uptake statistically different from control (p < 0.05, n = 3).
Fig. 5. Effect on OATP4C1-siRNA on the expression of OATP4C1 in HepG2 cells. HepG2 cells were transfected with control-siRNA or OATP4C1-siRNA and the mRNA (A) and protein expression (B) was measured using real-time PCR and western blots (72 h time point), respectively (n = 3).
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Fig. 6. Effect of OATP4C1-knockdown on the uptake of quercetin-30 -O-sulfate and hesperetin (A) and digoxin (B) (all at 50 mM for 10 min). HepG2 cells were transfected with control-siRNA or OATP4C1-siRNA, and the uptake experiments were performed 72 h post-transfection. (*) Uptake statistically different from control (p < 0.05, n = 5).
uptake of quercetin-30 -O-sulfate into HepG2 cells was pH-, temperature, concentration-dependent and saturable, suggesting a carrier mediated mechanism. The Km value obtained for the uptake of quercetin-30 -O-sulfate in HepG2 cells (58.7 17.8 mM) was higher than physiological concentrations (<10 mM) achieved with dietary doses of quercetin [36]. Nevertheless, we also observed active uptake at lower concentrations (<5 mM) in HepG2 cells. Hence, active uptake transporters may play an important role in hepatic uptake and clearance of quercetin conjugates in humans. In the liver and kidneys, OATs and OATPs are expressed on the basolateral membrane and mediate uptake of organic anions, including glucuronides and sulfate conjugates of steroids and xenobiotics. Typical inhibitors of OATs and OATPs potently inhibit the initial uptake of quercetin-30 -O-sulfate into HepG2 cells. Uptake of quercetin-30 -O-sulfate was stimulated by a lower buffer pH, consistent with reports of higher transport activity of OATPs at lower extracellular pH [37]. HepG2 cells express several OATs and OATPs found in the human liver, although there are quantitative differences in their levels of expression. Several OATs/OATPs have been detected in HepG2 cells, including OAT2, OAT4, OATP1B1, OATP1B3, OATP4A1 and OATP4C1 [16]. Consistent with previous studies, we found high expression of OAT2, OAT4 and OATP4C1. We hypothesized that these transporters may be involved in the uptake of quercetin-30 -O-sulfate into HepG2 cells. OAT4- and OATP4C1 expression enhanced quercetin-30 -O-sulfate uptake compared to control cells. In addition, siRNA-mediated knockdown of OATP4C1 in HepG2 cells resulted in 40% reduction in the uptake of quercetin-30 -O-sulfate. Uptake of quercetin glucuronides was not affected. OAT4 and OATP4C1 were first isolated from human kidney cDNA [33,38], and their transcripts are also detected in human liver [15,16]. OAT4 mediates the uptake of sulfated hormones, such as estrone-3-sulfate [38], and drugs such as antibiotics, antivirals, methotrexate and NSAIDs [39,40]. OATP4C1 has narrow substrate specificity and it transports digoxin, thyroid hormone, methotrexate and estrone-3-sulfate [33,37,41]. Both OAT4 and OATP4C1 showed poor transport activity toward glucuronide conjugates such as estradiol-3-glucuronide [33]. This apparent preference of OAT4 and OATP4C1 for sulfates over glucuronides conjugates may explain a lack of active uptake of quercetin glucuronides into HepG2 cells. These results provide evidence that OATs and OATPs may be involved in the active uptake of flavonoid metabolites in human hepatoma cells. In the human liver, other OATP isoforms, including OATP1B1 and OAT1B3, are highly expressed. These OATP transporters may also participate in the uptake of quercetin metabolites in vivo.
Vectorial transport of organic anions in hepatocytes involved the interplay of basolateral OATs/OATPs and apical MRPs. Conjugates of quercetin are known substrates for MRP2 [42,43] and BCRP [44]. MRP2, in particular, is involved in apical efflux of quercetin glucuronides and quercetin-30 -O-sulfate from HepG2 cells [42]. MRP2 and BCRP are likely to act in concert with OATs and OATPs to result in the removal of quercetin-30 -O-sulfate from blood into bile, leading to reduced bioavailability of quercetin. Due to the poor passive permeability of the hydrophilic glucuronide or sulfate conjugates to cell membranes, transport by OATs and OATPs may play an important role affecting their distribution into target tissues. Studies in rats and pigs showed that there is minimal accumulation in body tissues outside the gastrointestinal tract, liver and kidney [45–47]. However, there is biological activity since, for example, several animal studies showed that orally administered quercetin was protective against liver injury induced by various agents such as ethanol [48] and microcystin [49]. Quercetin also reduced liver damage in biliaryobstructed rats [50]. Selective uptake by OATs and OATPs expressed in the liver and kidney may account for beneficial effects of quercetin in these drug-metabolizing tissues. In summary we showed a potential role for OAT4 and OATP4C1 in quercetin-30 -O-sulfate uptake in HepG2 cells. In humans, the OATs and OATPs may act in concert with efflux transporters to result in the excretion of quercetin metabolites generated from the small intestine, and play a role in limiting the bioavailability of quercetin. Acknowledgment We thank Dr. Hassan Firoozmand for providing assistance in confocal microscopy and the Nestle Research Center, Switzerland, for funding. References [1] Lee ER, Kang YJ, Kim JH, Lee HT, Cho SG. Modulation of apoptosis in HaCaT keratinocytes via differential regulation of ERK signaling pathway by flavonoids. J Biol Chem 2005;280:31498–507. [2] Song J, Kwon O, Chen S, Daruwala R, Eck P, Park JB, et al. Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2 (GLUT2), intestinal transporters for vitamin C and Glucose. J Biol Chem 2002;277:15252–60. [3] Garcia-Mediavilla V, Crespo I, Collado PS, Esteller A, Sanchez-Campos S, Tunon MJ, et al. The anti-inflammatory flavones quercetin and kaempferol cause inhibition of inducible nitric oxide synthase, cyclooxygenase-2 and reactive Cprotein, and down-regulation of the nuclear factor kappaB pathway in Chang Liver cells. Eur J Pharmacol 2007;557:221–9.
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