Revolving Door Action of Breast Cancer Resistance Protein (BCRP


Revolving Door Action of Breast Cancer Resistance Protein (BCRP...

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Article pubs.acs.org/molecularpharmaceutics

Revolving Door Action of Breast Cancer Resistance Protein (BCRP) Facilitates or Controls the Efflux of Flavone Glucuronides from UGT1A9-Overexpressing HeLa Cells Yingjie Wei,†,‡ Baojian Wu,‡ Wen Jiang,‡ Taijun Yin,‡ Xiaobin Jia,† Sumit Basu,‡ Guangyi Yang,‡,§ and Ming Hu*,‡ †

Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, 100 Shizi Street, Nanjing 210028, China ‡ Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 1441 Moursund Street, Houston, Texas 77030, United States § Taihe Hospital Affiliated with Hubei University of Medicine, 32 Remin South Road, Shiyan 420000, China S Supporting Information *

ABSTRACT: Cellular production of flavonoid glucuronides requires the action of both UDP-glucuronosyltransferases (UGT) and efflux transporters since glucuronides are too hydrophilic to diffuse across the cellular membrane. We determined the kinetics of efflux of 13 flavonoid glucuronides using the newly developed HeLa-UGT1A9 cells and correlated them with kinetic parameters derived using expressed UGT1A9. The results indicated that, among the seven monohydroxylflavones (HFs), there was moderately good correlation (r2 ≥ 0.65) between the fraction metabolized (f met) derived from HeLa-UGT1A9 cells and CLint derived from the UGT1A9-mediated metabolism. However, there was weak or no correlation between these two parameters for six dihydroxylflavones (DHFs). Furthermore, there was weak or no correlation between various kinetic parameters (Km, Vmax, or CLint) for the efflux and the metabolism regardless of whether we were using seven HFs, six DHFs, or a combination thereof. Instead, the cellular excretion of many flavonoid glucuronides appears to be controlled by the efflux transporter, and the poor affinity of glucuronide to the efflux transporter resulted in major intracellular accumulation of glucuronides to a level that is above the dosing concentration of its aglycone. Hence, the efflux transporters appear to act as the “Revolving Door” to control the cellular excretion of glucuronides. In conclusion, the determination of a flavonoid’s susceptibility to glucuronidation must be based on both its susceptibility to glucuronidation by the enzyme and resulting glucuronide’s affinity to the relevant efflux transporters, which act as the “Revolving Door(s)” to facilitate or control its removal from the cells. KEYWORDS: UGT, BCRP, flavonoid, glucuronide, excretion, structure−activity relationship



INTRODUCTION

bioavailability and drug interaction potentials are dependent on their metabolic susceptibility. Toward this predictive goal, investigators including ourselves have spent significant effort in understanding and determining the structure-metabolic activity relationship (SAR) between chemical structures and glucuronidation rates by a specific UGT isoform.2−8 In addition, a few reports have shown that glucuronidation rates in tissue microsomes (e.g., liver microsomes) is directly correlated with the expression patterns of various UGT isoforms and activity of each isoform.9−11 Therefore, investigators have made significant strides in predicting glucuronidation rates of compounds by one or several major UGT isoforms. Unlike the structural activity relationships demonstrated using various UGT isoforms such as UGT1A15 and UGT1A9,6

Glucuronidation is a significant phase II metabolic pathway responsible for the elimination of a variety of endogenous and exogenous chemicals including drugs, hormones, and dietary phytochemicals such as polyphenols and flavonoids.1 Cellular glucuronide production is a two-step process: the formation of glucuronides catalyzed by various UGT isoforms and excretion of glucuronides enabled by various anion transporting efflux transporters such as breast cancer resistance protein (BCRP) and multiresistance proteins (MRPs).1 Efflux transporters were thought to act as a “Revolving Door” for facilitating and/or controlling the cellular glucuronide excretion. Therefore, a thorough understanding of the cellular glucuronide production process usually involves delineation of the glucuronidation steps that involve the relevant UDP-glucuronosyltransferases (UGTs) and subsequent glucuronide efflux steps by various efflux transporters. This understanding is important for the development of predictive algorithm useful for selecting drug candidates based on their metabolic susceptibility, because their © 2013 American Chemical Society

Received: Revised: Accepted: Published: 1736

October 2, 2012 January 30, 2013 February 12, 2013 February 12, 2013 dx.doi.org/10.1021/mp300562q | Mol. Pharmaceutics 2013, 10, 1736−1750

Molecular Pharmaceutics

Article

We have chosen flavone glucuronides as the model compounds for the present studies because flavonoids appear to possess a wide variety of biological activities in the areas of anticancer, antiaging, and anti-inflammation, all of which may be related to their antioxidant effects and glucuronides retain antioxidant activities. However, their development into therapeutic agents is severely challenged by the lack of oral bioavailability, mainly due to extensive phase II metabolism via glucuronidation and sulfonation. Therefore, we hypothesized that a better understanding of flavonoid glucuronidation process will reveal novel means of improving their oral bioavailability. Here, we studied BCRP-mediated efflux of 13 flavone glucuronides (Figure 1), seven derived from monohydroxyflavones (HFs) and six dihydroxyflavones (DHFs) to understand how changes in flavone glucuronide structures affect BCRP-mediated glucuronide efflux.



MATERIALS AND METHODS Materials. Human UGT1A9-overexpressing HeLa cells (hereon referred to HeLa-UGT1A9 cells in this paper) were described previously.12 Human BCRP (Arg482) membrane vesicles and expressed human UGT1A9 were purchased from BD Biosciences (Woburn, MA). 2′-Hydroxyflavone (2′HF), 3′-hydroxyflavone (3′HF), 4′-hydroxyflavone (4′HF), 3-hydroxyflavone (3HF), 5-hydroxyflavone (5HF), 6-hydroxyflavone (6HF), 7-hydroxyflavone (7HF), 3,5-dihydroxyflavone (3,5DHF), 3,6-dihydroxyflavone (3,6DHF), 3,7-dihydroxyflavone (3,7DHF), 3,2′-dihydroxyflavone (3,2′DHF), 3,3′-dihydroxyflavone (3,3′DHF), and 3,4′-dihydroxyflavone (3,4′DHF) were purchased from Indofine Chemicals (Hillsborough, NJ). All other chemicals and solvents (analytical grade or better) were used as received. Cell Culture. Conditions for culture and maintenance of HeLa-UGT1A9 cells were described previously and used without alteration.12 Cells from 3 days postseeding were used for the glucuronide excretion experiments described below. Detailed molecular characterization of our UGT1A9 stably expressed Hela cells was performed previously.12 The numbers of cells were correlated to protein concentrations of harvested cells, which is determined using a protein assay kit (Bio-Rad, Hercules, CA) using bovine serum albumin as the standard. To determine the intracellular concentration of glucuronides based on amount of glucuronides present inside cells, the HeLa cell volume was estimated to be 4 μL/mg protein.12

Figure 1. Structures of seven monohydroxyflavones and six dihydroxyflavones. Shown in the scheme are structures of aglycone forms of hydroxyflavone analogues. Glucuronidated metabolites may be formed at the 5, 6, or 7 phenolic group on the A ring, or 2′-OH, 3′-OH, or 4′-OH phenolic group on the B ring, or 3-OH group on the C ring. 2′-Hydroxyflavone (2′HF), 3′-hydroxyflavone (3′HF), 4′-hydroxyflavone (4′HF), 3-hydroxyflavone (3HF), 5-hydroxyflavone (5HF), 6-hydroxyflavone (6HF), 7-hydroxyflavone (7HF), 3,2′-dihydroxyflavone (3,2′DHF), 3,3′-dihydroxyflavone (3,3′DHF), 3,4′-dihydroxyflavone (3,4′DHF), 3,5-dihydroxyflavone (3,5DHF), 3,6-dihydroxyflavone (3,6DHF), 3,7-dihydroxyflavone (3,7DHF).

very little is known about the structural activity relationship of glucuronide efflux by an efflux transporter. Although several types of membrane vesicles overexpressing a particular type of responsible efflux transporter (e.g., BCRP or MRPs) have been used to shown that these efflux transporters are capable of mediating the efflux of various organic anions, an actual structural activity relationship has not been established for glucuronides. This is largely because of the lack of commercially available glucuronide standards needed to conduct the aforementioned studies. Recently, we have developed a new tool: HeLa-UGT1A9 cells, which should allow us to determine the structure activity relationship without purified glucuronide standards.12 In these cells, we found that intracellular concentrations of glucuronides could rapidly reach the steady state (usually within 30 min), which in turn allows the determination of the steady state efflux rates.12 The latter will allow us to determine the kinetic parameters associated with the dominating efflux transporters, a necessary step to delineate the structure−activity relationship (i.e., glucuronide efflux SAR). We also found that HeLaUGT1A9 cells mainly used BCRP as its efflux transporter for flavonoid glucuronides using both molecular and kinetic characterization (using Ko143 as an inhibitor). Specifically, complete inhibition of this transporter decreases flavonoid glucuronide clearance by more than 95%. We also found that MRP2 and MRP3 did not make a significant contribution to the efflux of flavonoid glucuronides, using siRNAs and a chemical inhibitor of MRPs (LTC4).13 Therefore, these cells are used here to determine the SAR for BCRP mediated efflux of flavonoid glucuronides.



EXCRETION EXPERIMENTS Before experiments, the HeLa-UGT1A9 cells were washed twice with 37 °C HBSS buffer (Hank’s balanced salt solution, pH = 7.4). The cells were then incubated in HBSS buffer containing one of the seven monohydroxyflavones (2′-, 3′-, 4′-, 3-, 5-, 6-, and 7HF) or one of the six dihydroxyflavones (3,2′-, 3,3′-, 3,4′-, 3,5-, 3,6-, and 3,7DHF) (defined as the “loading solution”) for a predetermined time interval (shown in Table S1, Supporting Information) at 37 °C. In the loading solution, the final flavone concentration was derived by the dilution of a (100×) concentrated stock solution (DMSO/methanol = 1:4) to ensure constant organic solvent content in all transport experiments. The sampling times were selected to ensure that the amounts excreted vs time plots stay in the linear range. At each time point, 200 μL of incubating media from each well was collected as samples, and an equal volume of loading solutions 1737

dx.doi.org/10.1021/mp300562q | Mol. Pharmaceutics 2013, 10, 1736−1750

Molecular Pharmaceutics

Article

Determination of Concentration of Intracellular Glucuronides in HeLa-UGT1A9 Cells. At the end of an excretion experiment, HeLa−UGT1A9 cells were washed twice with an ice-cold HBSS buffer to remove the extracellular aglycone and conjugates. The cells were then removed and

was used to replenish each well. The collected samples were each mixed with a 50 μL “Stop Solution,” which consisted of 94% acetonitrile and 6% acetic acid. Supernatants were ready for ultra performance liquid chromatography (UPLC) analysis after centrifugation (15 min at 15 000 rpm).

Figure 2. continued 1738

dx.doi.org/10.1021/mp300562q | Mol. Pharmaceutics 2013, 10, 1736−1750

Molecular Pharmaceutics

Article

Figure 2. Kinetics profiles of BCRP-mediated efflux of seven HF glucuronides (A, 7HF-G; B, 2′HF-G; C, 3′HF-G; D, 3HF-G; E, 4′HF-G; F, 5HF-G; G, 6HF-G) (left) and corresponding Eadie−Hofstee plots for each kinetic profile (right). Solid lines (left) denote the excretion rate of flavone glucuronides. Each data point represents the average of three replicates, and error bars are the standard deviations of the mean (n = 3). “Conc.” is the abbreviation for concentration.

collected in 120 or 200 μL of HBSS buffer. In an ice-cold water bath (4 °C) collected cells were lyzed in an Aquasonic 150D sonicator (VWR Scientific, Bristol, CT) operating at the maximum power (135 average watts) for 30 min to ensure the release of aglycone and conjugates for measurement.14 In the lysate, no further metabolism is expected since UGT1A9 in cellular lysate will not function without the cofactor, UDPglucuronic acid. After centrifugation at 15 500 rpm for 20 min, supernatants (100 μL) were collected and mixed with the Stop Solution (25 μL). The treated samples are ready for UPLC analysis after centrifugation (15 min at 15 000 rpm) to remove larger particles and cellular debris. Vesicular Transport Studies for BCRP. Vesicular transport studies were performed with rapid filtration techniques as described previously.15−17 In brief, membrane fractions containing inside out efflux transporters were incubated in the presence or absence of 5 mM adenosine 5′-triphosphate (ATP) in a buffer containing 10 mM MgCl2 and 10 mM Tris/HCl (pH 7.4). The transport was stopped by addition of 1 mL of cold transport buffer to the membrane suspensions and then rapidly filtered through class F glass fiber filters (pore size, 0.45 μm). Filters were washed with 2 × 5 mL of ice-cold wash buffer, cut, and transferred to a 1.5 mL Eppendorf tube containing 400 μL of 50% methanol, followed by sonicating for 15 min. After centrifugation at 15 000 rpm for 15 min, the supernatant was subjected into UPLC quantitative analysis. ATP-dependent transport was calculated by subtracting the values obtained in the presence of AMP from those in the presence of ATP. Sample Analysis by UPLC. The conditions for UPLC analysis of HFs, DHFs, and their respective glucuronides were modified based on a previously published method.10 The conditions were: system, Waters Acquity UPLC with a binary pump, and a 2996 DPA diode array detector (DAD, Waters, Milford, MA); column, BEH C18 column (50 × 2.1 mm I.D. 1.7 μm, Waters, Milford, MA); mobile phase A, 2.5 mM

ammonium acetate (pH 7.4); mobile phase B, 100% acetonitrile; gradient, 0−2 min (10−20% B), 2−3 min (20−40% B), 3− 3.5 min (40−50% B), 3.5−4 min (50−90% B), 4−4.5 min (90% B), 4.5−5 min (90−10% B); injection volume, 10 μL; detection wavelength for HFs, DHFs, and their glucuronides were also shown in Table S1. The precision and accuracy were typically within acceptable range (