Food Chemistry 138 (2013) 2267–2274

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Food Chemistry

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Identification of novel dietary phytochemicals inhibiting the efflux transporter breast cancer resistance protein (BCRP/ABCG2) ⇑ Kee W. Tan a,b,c, , Yan Li b,1, James W. Paxton b, Nigel P. Birch c,d, Arjan Scheepens a a Food Innovation, The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, New Zealand b Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand c School of Biological Sciences, Faculty of Science, The University of Auckland, Private Bag 92019, Auckland, New Zealand d Centre for Brain Research, The University of Auckland, Private Bag 92019, Auckland, New Zealand article info abstract

Article history: Breast cancer resistance protein (BCRP/ABCG2) plays an important role in determining the absorption Received 31 October 2012 and disposition of consumed xenobiotics including various drugs and dietary phytochemicals and is also Received in revised form 2 December 2012 one of the prominent efflux transporters involved in multidrug resistance (MDR). In this study, we have Accepted 3 December 2012 investigated the interactions between ABCG2 and 56 naturally-occurring phytochemicals including Available online 27 December 2012 phenolic acids, flavonoids, triterpenes and other common dietary phytochemicals, as well as two non plant-based compounds (hippuric acid and propyl gallate) using cell- and membrane-based transport Keywords: inhibition assays. Of the non-flavonoid phytochemicals tested, berberine, celastrol, ellagic acid, limonin, ABC transporters oleanolic acid, propyl gallate, sinapic acid and ursolic acid demonstrated significant inhibition of ABCG2- Bioavailability 0 0 0 Food–drug interactions mediated transport. Chrysoeriol, laricitrin, 3 ,4 ,5 -trimethylether, pinocembrin, , 0 0 0 Multidrug resistance , tricetin and tricetin 3 ,4 ,5 -trimethylether were also identified as novel flavonoid ABCG2 Polyphenols inhibitors. The identified inhibitory activity of dietary phytochemicals on ABCG2 provides a framework for further investigation of ABCG2-modulated phytochemical bioavailability, MDR, and possible food– drug interactions. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction agents of various classes such as antivirals (e.g. abacavir and lamivudine), antibiotics (e.g. ciprofloxacin and nitrofurantoin), Breast cancer resistance protein (BCRP/ABCG2) is a member of HMG-CoA reductase inhibitors (e.g. atorvastatin), calcium channel the evolutionarily-conserved family of ATP-binding cassette blockers (e.g. azidopine) and anti-neoplastics (e.g. methotrexate, (ABC) efflux transporters. It is primarily expressed at the apical gefitinib and topotecan) (Meyer zu Schwabedissen & Kroemer, membrane of epithelial cells of organs such as the gastrointestinal 2011). ABCG2 can influence the absorption and disposition of tract, liver and kidney, as well as physiological barriers such as the consumed substrates by limiting their net uptake from the gastro- blood–brain barrier, blood–testis barrier and maternal–foetal bar- intestinal tract and distribution to target organs such as the central rier (Fetsch et al., 2006). Consistent with its tissue localization, nervous system. In addition, ABCG2 has been attributed as a accumulating evidence suggests that ABCG2 functions as a cellular prominent molecular cause of multidrug resistance (MDR) in many efflux pump, limiting foetal exposure, brain penetration and intes- human cancers through its active efflux of chemotherapeutic tinal absorption of substrate xenobiotics, and also facilitating their agents out of neoplastic cells (Natarajan, Xie, Baer, & Ross, 2012). mammary, biliary and kidney secretion. Endogenous substrates of In light of the clinical importance of ABCG2 in drug disposition ABCG2 include estradiol-17b glucuronide, folic acid (vitamin B9), and MDR, the discovery of ABCG2 inhibitors has attracted consid- riboflavin (vitamins B2), vitamin K3, uric acid and protoporphyrin erable scientific interest as one of the possible options in tackling IX, while exogenous substrates of ABCG2 encompass therapeutic the issues of low drug bioavailability and MDR. Daily dietary intake of fruits and vegetables exposes the body to various types of plant-derived compounds known as phytochemi- ⇑ Corresponding author at: Food Innovation, The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland, New Zealand. Tel.: +64 9 925 cals. Phytochemicals are secondary metabolites of plants which 7116; fax: +64 9 925 7001. cover a broad range of structurally diverse compounds including E-mail addresses: [email protected], [email protected] alkaloids, organosulfur compounds, phenolic compounds and (K.W. Tan). terpenes. Unlike essential micronutrients such as vitamins and 1 Current address: School of Interprofessional Health Studies, Faculty of Health and minerals, phytochemicals are not associated with deficiency Environmental Sciences, Auckland University of Technology, Auckland 0627, New Zealand. diseases resulting from insufficient dietary intake. They appear to

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.12.021 2268 K.W. Tan et al. / Food Chemistry 138 (2013) 2267–2274 contribute to long-term health maintenance, preventing and atten- against harmful ABCG2-modulated food-drug interactions. On the uating the development of degenerative diseases when regularly other hand, phytochemical–ABCG2 interactions may potentially consumed. This is supported by numerous epidemiological studies be applied for the development of beneficial phytochemical–drug that have indicated an inverse relationship between the consump- substrate combinations that allow increased drug efficacy and tion of various fruits and vegetables and the development of safety at lower doses or phytochemical–phytochemical combina- chronic diseases such as cancer, coronary heart disease, hyperten- tions that enhance phytochemical bioavailability and/or bioactivi- sion, stroke, certain eye diseases, dementia and osteoporosis ties (Scheepens, Tan, & Paxton, 2010). (Boeing et al., 2012). In addition, specific and beneficial bioactivi- In the present study, we explored the ABCG2 inhibitory activi- ties of phytochemicals are being actively studied and have been ties of 56 dietary phytochemicals and two non plant-based but demonstrated in various areas of medicine including cancer, car- diet-relevant compounds (hippuric acid and propyl gallate) using diovascular diseases, neurodegenerative diseases, eye health and both cell- and membrane-based methods. In the cell-based skin health (Krzyzanowska, Czubacka, & Oleszek, 2010). Despite mitoxantrone accumulation assay, the effect of test compounds the epidemiological association and many putative health effects on ABCG2-mediated efflux of the fluorescent substrate, mitoxan- derived from in vitro and animal studies of phytochemicals, very trone, was measured in HEK293 (human embryonic kidney) cells few randomised controlled human clinical trials have confirmed overexpressing ABCG2. In the membrane-based assay, the effect their clinical efficacy. This is likely due to limited bioavailability of test compounds on the transport of 3H-methotrexate into in- at the systemic level and more so at target organs. For instance, side-out Sf9 (Spodoptera frugiperda) insect cell membrane vesicles maximal plasma concentrations of total anthocyanins after overexpressing ABCG2 was investigated. consumption of anthocyanin-rich foods are typically in the range of 1–100 nM (Yang, Koo, Song, & Chun, 2011), which is about a 2. Materials and methods thousand fold lower than the concentration range commonly used to achieve in vitro bioactivity. 2.1. Materials Orally consumed phytochemicals display different absorption, distribution, metabolism and excretion (ADME) profiles that dic- The following compounds were procured from the commercial tate their exposure at the systemic level as well as their target sources indicated: 2,4-dihydroxybenzoic acid, 3,4-dihydroxyben- sites. Phenolic acids, for example, are generally bioavailable be- zoic acid, apigenin, berberine, (+)-catechin, caffeic acid, chrysin, cause of their high absorption from the upper part of the gastroin- p-coumaric acid, coumarin, ellagic acid, ()-epicatechin, ferulic testinal tract (Lafay & Gil-Izquierdo, 2008). Flavonoids, on the other acid, gallic acid, hesperidin, hippuric acid, indole-3-carbinol, limo- hand, undergo extensive phase II metabolism in the intestine (and nin, luteolin, malic acid, methotrexate, mitoxantrone, myricetin, liver) to form conjugates, such as glucuronides and sulfates, which naringenin, naringin, phloridzin, phloroglucinol, propyl gallate, are then subjected to phase III efflux by transporters. In terms of , quinic acid, salicylic acid, sinapic acid, sinigrin, syringic phase III efflux of phytochemicals in the gastrointestinal tract, cur- acid and vanillin (Sigma–Aldrich, Auckland, NZ); acacetin, chrysoe- rent evidence from animal studies suggests that ABCG2 plays a ma- riol, cyanidin, cyanidin-3-glucoside, cyanidin-3-rutinoside, del- jor role in apical efflux of phytochemicals and their phase II phinidin, diomestin, , , laricitrin, malvidin, metabolites back to gut lumen and consequently limits their bio- , myricetin 30,40,50-trimethylether, peonidin, pinocembrin, availability (Enokizono, Kusuhara, & Sugiyama, 2007; Sesink quercitrin, sinensetin, tamarixetin, tangeritin, tricetin and tricetin et al., 2005; van de Wetering et al., 2009). For instance, knocking 30,40,50-trimethylether (Extrasysnthese, Genay Cedex, France); del- out mouse Abcg2 led to significant increases in the oral bioavail- phinidin-3-glucoside and delphinidin-3-rutinoside (Polyphenols ability of daidzen (3.7-fold) and genistein (1.8-fold) compared with Laboratories AS, Sandnes, Norway); celastrol, oleanolic acid and wild-type mice (Enokizono et al., 2007). A higher plasma level and ursolic acid (Cayman Chemical, MI, USA); Ko143 (Tocris Biosci- an increased urinary excretion of resveratrol phase II metabolites ences, Bristol, UK); 3H-methotrexate (American Radiolabeled were also observed in Abcg2 knock-out mice (van de Wetering Chemicals, MO, US). et al., 2009). In addition, in vitro studies have also indicated that a variety of flavonoids interact with ABCG2 as inhibitors (Cooray, Janvilisri, van Veen, Hladky, & Barrand, 2004; Dreiseitel et al., 2.2. Cell culture 2009; Zhang, Yang, & Morris, 2004) and/or substrates (An, Gallegos, & Morris, 2011; An & Morris, 2011). Wild-type and ABCG2-overexpressing HEK293 (human embry- The interplay between ABCG2 and dietary phytochemicals onic kidney) cell lines (HEK293/WT and HEK293/ABCG2) were raises potential issues concerning food–drug interactions that kindly provided by Prof. P. Borst (Netherlands Cancer Institute, 0 may occur when ABCG2 drug substrates are taken concomitantly the Netherlands). Cells were cultured in Dulbecco s Modified Eagle with phytochemical-containing foods or dietary supplements. En- Medium (Cat. No. 11995-065, Life Technologies, Auckland, NZ) hanced exposure to ABC transporter substrates, such as anticancer supplemented with 10% foetal bovine serum (FBS) in a humidified agents, calcium-channel blockers and immuno-suppressants has incubator maintaining 5% CO2 at 37 °C. To maintain selection pres- been reported in humans and animals after substrate drugs are sure on the transfected ABCG2 gene, HEK293/ABCG2 cells were administered orally together with phytochemical-rich foods and grown in the presence of 200 lg/mL G418 (Sigma–Aldrich, Auck- drinks (Li, Revalde, Reid, & Paxton, 2010). In contrast, certain phy- land, NZ). Sf9 insect cells were cultured in SF-900 III medium (Life tochemicals may induce efflux transporter expression through Technologies, Auckland, NZ) supplemented with 10% FBS. Cells interactions with nuclear receptors, resulting in sub-optimal drug were maintained at 28 °C in a non-humidified and ambient air-reg- concentration and therapeutic failure (Tirona & Bailey, 2006). With ulated incubator, on an orbital shaker platform rotating at the increasing production and the widespread use of herbal and 120 rpm. dietary supplements, the risk of harmful interactions increases considerably as dietary supplements commonly provide pure phy- 2.3. Expression of ABCG2 in Sf9 insect cells tochemicals or phytochemical-containing herbal extracts at con- centrations many fold higher than the levels found in foods. The human ABCG2 coding sequence (kindly provided by Prof. P. Hence, understanding the interactions between ABCG2 and dietary Borst) was inserted into the pFastBac1 plasmid and the MultiBac phytochemicals may serve as a cautionary and preventive step bacmid (Berger, Fitzgerald, & Richmond, 2004) following the man- K.W. Tan et al. / Food Chemistry 138 (2013) 2267–2274 2269 ufacturer’s guidelines (Bac-to-BacÒ Baculovirus Expression System, nm, respectively. The fluorescence level was expressed as the mean Life Technologies, Auckland, NZ). Recombinant baculovirus was value of 10000 events. used to transfect exponentially growing Sf9 cells (2 106 cells/ ml) at a multiplicity of infection of 0.001. After incubation at 2.7. Membrane vesicular transport assay 28 °C for 72 h, cells were pooled by centrifugation and washed with ice-cold phosphate-buffered saline. Harvested cells were The membrane vesicular transport assay was performed using a stored at 80 °C until use. rapid filtration technique. Frozen membrane vesicles prepared from wild-type (Sf9/WT) or ABCG2-expressing (Sf9/ABCG2) Sf9 2.4. Preparation of Sf9 membrane vesicles cells were quickly thawed at room temperature and diluted with suspension buffer (0.25 M sucrose, 10 mM Tris-HEPES (pH 7.4)) Frozen cell pellets derived from 100 ml culture were quickly to the required concentrations (50 lg protein per reaction for stan- thawed and diluted with 25 ml of hypotonic buffer (0.5 mM Tris- dard screening). Individual test compounds were added to vesicle HEPES (pH 7.4), 0.1 mM EGTA) supplemented with 1 Complete preparations to a volume of 19 ll per reaction. The reaction was Protease Inhibitor Cocktail (Roche, Auckland, NZ). The resuspended started by adding pre-warmed (37 °C) reaction mixture (250 mM cells were homogenised with a Potter–Elvehjem homogenizer and sucrose, 10 mM Tris-HEPES (pH 7.4), 10 mM MgCl2, 4 mM ATP, centrifuged at 2000g at 4 °C for 10 min. The supernatant was col- 10 mM creatine phosphate, 100 lg/ml creatine phosphokinase, lected and the cell pellet re-extracted. The pooled supernatant 100 mM methotrexate, 0.05 lM 3H-methotrexate) to the vesicle was subjected to ultracentrifugation at 27,000 rpm for 40 min at preparation to a final volume of 50 ll. The final reaction mix typi- 4 °C in a SW28 rotor. The resulting pellets were resuspended in cally contained test compounds at 5 or 50 lM with a final concen- suspension buffer (0.25 M sucrose, 10 mM Tris-HEPES (pH 7.4)) tration of 0.5% DMSO. DMSO up to 1% was found to have no effect and homogenised with a Potter–Elvehjem homogenizer before on the assay (data not shown). After incubation at 37 °C for 20 min, repeating the ultracentrifugation step. The pellet was resuspended the reaction was stopped by adding 150 ll of ice-cold suspension in 5–8 ml of suspension buffer and passed through a 27-gauge nee- buffer. The resulting mixture was transferred to a 96-well Multi- dle. The resulting membrane vesicles were snap-frozen using li- ScreenHTS-FB plate (Merck-Millipore, MA, USA) pre-wet with ice- quid nitrogen and stored at 80 °C until use. cold suspension buffer. Under vacuum aspiration, each well was rinsed four times with 200 ll of ice-cold suspension buffer. Radio- 2.5. Western blot analysis of ABCG2 expression activity retained on the membranes was measured using a LKB Wallac 1214 RackBeta liquid scintillation counter. HEK293/WT and HEK293/ABCG2 cell lysates were prepared in lysis buffer (50 mM Tris–Cl (pH 7.5), 150 mM NaCl, 1% Nonidet 2.8. Data analysis P-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1 Complete Protease Inhibitor Cocktail (Roche, Auckland, NZ). For the mitoxantrone accumulation assay, treatment with vehi- HEK293 cell lysates and Sf9 membrane vesicles (20 and 5 lg, cle (DMSO) and mitoxantrone were defined as background fluores- respectively, as determined using the DC Protein Assay (Bio-Rad cence (BG1) and 100% mitoxantrone accumulation, respectively. Laboratories, Auckland, NZ)) were electrophoresed on 10% SDS– The relative mitoxantrone accumulation (%) in the presence of a polyacrylamide gels and transferred onto Hybond ECL nitrocellu- test compound was calculated as follows: lose membranes (GE Healthcare, Auckland, NZ). Membranes were blocked for 1 h at room temperature in Tris-buffered saline con- ðTest compound þ MitoxantroneÞBG1 100 taining 0.2% (v/v) Tween 20 and 5% (w/v) low fat milk powder Mitoxantrone BG1 and were incubated overnight at 4 °C with anti-ABCG2 BXP-21 For the membrane vesicular transport assay, treatment with antibody (Abcam, Cambridge, UK) at 1:5000 dilution. Membranes methotrexate in the absence and presence of ATP were defined were then incubated with horseradish peroxidase-conjugated goat as background (BG2) and 100% transport activity, respectively. anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories, The relative methotrexate transport (%) in the presence of a test UK) secondary antibody for 1 h at room temperature. Immunoreac- compound was calculated as follows: tive proteins were detected using the SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, Auckland, ðTest compound þ Methotrexate þ ATPÞBG2 100 NZ) and a Fuji LAS 4000 Imaging System (Fuji Film Science Labora- ðMethotrexate þ ATPÞBG2 tory, Tokyo, Japan). IC50 (half-maximal inhibitory concentration) was defined as the 2.6. Mitoxantrone accumulation assay test compound concentration required for inhibiting transport of the reporter substrate by 50%. SigmaPlot 10.0 (Systat Software HEK293/WT or HEK293/ABCG2 cells grown to 95% confluence Inc., CA, USA) was used for curve fitting and IC50 calculation. in 75 cm2 flasks were detached, washed in Hank’s balanced salt Statistical analyses were performed using SPSS 20 (IBM New solution (HBSS) and then resuspended in HBSS to a density of Zealand Limited, Wellington, NZ). The differences between controls 5 and treatments were tested for significance using a linear mixed 7 10 cells/ml. The assay was performed by incubating 1 ml of cells with test compounds (typically 50 lM), or Ko143 (5 lM) as model. Logarithm transformation was applied to data before anal- a reference inhibitor, or dimethyl sulfoxide (DMSO) for non-inhib- ysis to improve homogeneity of variance. The Sidak correction was applied for multiple comparisons to adjust for multiple testing. All ited controls, at 37 °C for 15 min before the addition of 5 lMof mitoxantrone. The final level of DMSO was 0.2% (v/v). DMSO up statistical testing was carried out at the 5% level of significance. to 2% was found to have no effect on the assay (data not shown). After incubation for 90 min, the reaction was stopped by adding 3. Results 3 ml of ice-cold PBS buffer. The cells were collected and washed again with 3 ml of ice-cold PBS. Cells were resuspended in ice-cold The expression of ABCG2 in cell lines and membrane vesicles PBS with 1% paraformaldehyde. The intracellular level of mitoxan- used in this study was confirmed by Western blot analysis. As trone was detected by a BD LSR II flow cytometer (BD Biosciences) shown in Fig. 1A, ABCG2 was detected as a 72 kDa immunoreac- at excitation and emission wavelengths of 635 and 670 (bandpass) tive band in HEK293/ABCG2 cells (a significant amount of immu- 2270 K.W. Tan et al. / Food Chemistry 138 (2013) 2267–2274

In HEK293/WT cells, treatment with 5 lM Ko143 resulted in an 18.2% increase (87.9% in HEK293/ABCG2 cells) of mitoxantrone accumulation. The majority of compounds tested positive in HEK293/ABCG2 cells showed no significant effects in HEK293/WT cells. Notably, a decrease in mitoxantrone accumulation was again observed in treatment with delphinidin-3-rutinoside, ellagic acid, myricetin and tricetin, as well as quercetin and kaempferol. Limo- nin, sinensetin and tamarixetin caused a significant increase in HEK293/WT cells, although this was not significantly different from the effect of Ko143 treatment. A second approach to identify and verify ABCG2-modulating properties of the test compounds was also used. Effects on the transport of methotrexate (a standard ABCG2 substrate) into Sf9/ ABCG2 membrane vesicles were measured at 5 and 50 lM for each compound (Table 1). For the phenolic acids, significant inhibition of methotrexate transport was observed only with ellagic and sin- apic acid at 50 lM. In agreement with the results of the mitoxan- trone accumulation data, most flavonoids induced a significant inhibition of methotrexate transport at both concentrations tested. (+)-Catechin is the only flavonoid that showed no effect on meth- otrexate transport at both concentrations, while ()-epicatechin and anthocyanins inhibited methotrexate transport only at 50 lM. Many flavonoids at 50 lM restricted the transport of meth- otrexate to below 10%, which is comparable to the effect of 1 lM Fig. 1. (A) Western blot analysis of ABCG2 expression in (1) HEK293/ABCG2 cells, Ko143. For compounds other than the phenolic acids and flavo- (2) HEK293/WT cells, (3) Sf9/ABCG2 membrane vesicles, and (4) Sf9/WT membrane noids, berberine, celastrol, limonin, oleanolic acid, propyl gallate vesicles. Protein amounts are 20 lg for HEK293/WT and HEK293/ABCG2 cell and and ursolic acid significantly inhibited the vesicular transport of 5 lg for Sf9/WT and Sf9/ABCG2 membrane vesicles; (B) transport of methotrexate methotrexate, at least at 50 lM. No ABCG2-inhibitory activity in Sf9/WT and Sf9/ABCG2 membrane vesicles in the absence () and presence (+) of 4 mM ATP and 5 lM Ko143. Reactions were performed with 50 lg protein for was observed for coumarin, indole 3-carbinol, malic acid, phloro- 20 min at 37 °C. Data are expressed as mean ± S.D., n =3. glucinol, quinic acid, sinigrin, or vanillin at either 5 or 50 lM in this assay. Selected compounds of interest that had exhibited the potential noreactive protein was also detected as an aggregate above the to inhibit ABCG2-mediated methotrexate transport were further highest marker, data not shown) while no detectable expression investigated for their IC50 values as shown in Table 2. Seven flavo- was observed in HEK293/WT cells. In Sf9/ABCG2 membrane vesi- noids (apigenin, chrysoeriol, diosmetin, kaempferol, myricetin cles, ABCG2 was detected as a 65 kDa immunoreactive protein. 30,40,50-trimethylether, tamarixetin, tricetin 30,40,50-trimethylether) Likewise, ABCG2 was not detected in Sf9/WT membrane vesicles. exhibited IC50 values of less than 0.1 lM and of similar magnitude The functionality of Sf9/ABCG2 membrane vesicles was demon- to Ko143 (0.026 ± 0.003 lM). A further eight (acacetin, chrysin, strated in the membrane vesicular transport assay (Fig. 1B). No sig- kaempferide, laricitrin, luteolin, myricetin 30,40,50-trimethylether, nificant ATP-dependent transport of methotrexate was detected in quercetin, tricetin) had IC50 values of less than 0.5 lM. Of the Sf9/WT membrane vesicles (Fig. 1B). non-flavonoid inhibitors, highest potencies were observed with In the mitoxantrone accumulation assay, test compounds were celastrol (8.7 ± 1.1 lM) and ellagic acid (20.7 ± 2.7 lM). Because first investigated using HEK293/ABCG2 cells for their ability to of the limited solubility of ellagic acid, high concentration stocks attenuate the efflux of mitoxantrone, which resulted in an in- were prepared as suspension in DMSO. Assuming homogeneity of creased intracellular accumulation of mitoxantrone. Compounds suspension, ellagic acid was soluble in subsequent dilutions and that tested positive were then evaluated in HEK293/WT cells to an IC50 value was able to be determined. We were however unable confirm that the observed effects were ABCG2-specific. Anthocy- to determine the IC50 values for oleanolic acid and ursolic acid be- anidins (cyanidin, delphinidin, malvidin and peonidin) were ex- cause of the low solubility of both compounds in aqueous solution. cluded from the data set owing to significant intrinsic Chemical structures of identified non-flavonoid ABCG2 inhibitors fluorescence (data not shown). As shown in Table 1, none of the and selected flavonoids are shown in Fig. 2. tested phenolic acids at 50 lM resulted in a significant increase in mitoxantrone accumulation. In contrast, most of the tested flavonoids significantly enhanced the accumulation of mitoxan- 4. Discussion trone, except for anthocyanins (cyanidin 3-glucoside, cyanidin 3- rutinoside, delphinidin 3-glucoside and delphinidin 3-rutinoside), Two different approaches have been used to identify and verify flavanols ((+)-catechin and ()-epicatechin), hesperidin, morin, the ABCG2-inhibitory effects of 56 naturally-occurring phyto- myricetin and tricetin. For phytochemicals other than the phenolic chemicals and two non plant-based compounds, most of which acids and flavonoids, a significant increase in mitoxantrone can be found in common diets. The non plant-based compounds, accumulation was observed for berberine, celastrol, limonin, ole- propyl gallate (E310) and hippuric acid, were included because of anolic acid and ursolic acid. On the other hand, indole 3-carbinol, their relevance as a common food additive and a major polyphenol coumarin, malic acid, phloroglucinol, propyl gallate, quinic acid, metabolite, respectively. The two assay systems probed the inter- sinigrin and vanillin showed no significant effect on mitoxantrone actions between ABCG2 and test compounds in different microen- accumulation at 50 lM. Among the compounds tested, delphini- vironments. The cell-based mitoxantrone efflux assay better din-3-rutinoside, ellagic acid, myricetin and tricetin resulted in a mimics the cellular environment in vivo. However, endogenous significant decrease in mitoxantrone accumulation in HEK293/ transporters and metabolising enzymes present in the HEK293 ABCG2 cells. cells may interfere with the interactions. ABCG2-overexpressing K.W. Tan et al. / Food Chemistry 138 (2013) 2267–2274 2271

Table 1 Reaction parameters derived from mitoxantrone accumulation assay and membrane vesicular transport assay. Values are expressed as percentage of control, mean ± SEM, n P 4 for mitoxantrone accumulation, n P 6 for methotrexate membrane vesicular transport.

Test compound Mitoxantrone accumulation (%) Methotrexate membrane vesicular transport (%) HEK293/ABCG2 HEK293/WT Sf9/ABCG2 Control Mitoxantrone (5 lM) 100.0 ± 0.4 100.0 ± 0.3 Methotrexate (100 lM) 100.0 ± 0.5 Ko143 187.9 ± 4.4* (5 lM) 118.2 ± 3.9* (5 lM) 4.0 ± 0.9* (1 lM) Concentration (lM)a 50 50 5 50 Phenolic acid 2,4-Dihydroxybenzoic acid 101.0 ± 0.2 102.4 ± 4.9 101.4 ± 3.2 3,4-Dihydroxybenzoic acid 106.5 ± 10.1 91.7 ± 3.6 98.2 ± 1.9 Caffeic acid 97.3 ± 1.9 98.7 ± 1.6 98.1 ± 1.7 Chlorogenic acid 97.8 ± 1.4 104.6 ± 4.5 96.2 ± 1.2 p-Coumaric acid 102.5 ± 1.8 94.9 ± 3.1 93.2 ± 3.6 Ellagic acid 41.0 ± 3.7* (10 lM) 46.9 ± 1.7* (10 lM) 84.1 ± 5.7 16.9 ± 0.8* Ferulic acid 103.5 ± 1.2 99.1 ± 1.3 94.1 ± 1.4 Gallic acid 108.4 ± 3.4 106.7 ± 4.6 93.5 ± 1.7 Hippuric acid 106.5 ± 8.7 101.1 ± 1.9 109.9 ± 4.1 Salicylic acid 117.8 ± 13.4 107.5 ± 4.9 105.5 ± 4.9 Sinapic aid 107.1 ± 4.4 89.4 ± 2.1 45.1 ± 1.8* Syringic acid 98.9 ± 3.3 98.0 ± 1.1 100.7 ± 1.9 Flavonoid Acacetin 159.8 ± 4.0* (10 lM) 100.2 ± 3.4 (10 lM) 18.5 ± 1.5* 8.0 ± 1.3* Apigenin 175.2 ± 15.0* 90.4 ± 2.1 9.0 ± 1.3* 6.5 ± 1.2* (+)-Catechin 100.4 ± 1.6 97.4 ± 1.4 93.6 ± 3.1 Chrysin 186.9 ± 2.5* 111.3 ± 2.9 27.5 ± 1.8* 9.0 ± 0.4* Chrysoeriol 207.4 ± 12.3* 100.0 ± 2.5 7.7 ± 0.4* 5.7 ± 0.2* Cyanidin - 78.6 ± 3.3* 13.2 ± 0.8* Cyanidin-3-glucoside 85.5 ± 1.9 104.2 ± 3.4 78.3 ± 1.3* Cyanidin-3-rutinoside 87.0 ± 1.9 107.1 ± 2.7 62.3 ± 2.0* Delphinidin - 73.7 ± 1.6* 9.7 ± 0.4* Delphinidin-3-glucoside 95.9 ± 7.2 101.7 ± 1.8 72.4 ± 3.1* Delphinidin-rutinoside 74.8 ± 1.5* 73.6 ± 1.1* 104.6 ± 1.7 53.6 ± 1.4* Diosmetin 191.8 ± 15.4* 106.5 ± 1.7 7.4 ± 0.7* 5.2 ± 0.4* ()-Epicatechin 101.5 ± 1.7 91.3 ± 2.2 47.4 ± 1.1* Hesperidin 122.6 ± 8.5 95.6 ± 2.7 63.7 ± 3.4* 22.2 ± 0.6* Kaempferide 143.3 ± 3.6* (10 lM) 84.0 ± 0.8* (10 lM) 11.1 ± 1.1* 6.8 ± 0.3* Kaempferol 189.6 ± 13.3* 107.0 ± 3.4 5.1 ± 0.6* 2.3 ± 0.7* Laricitrin 146.6 ± 5.0* 94.5 ± 0.5 4.3 ± 0.5* 4.8 ± 0.6* Luteolin 173.1 ± 8.9* 93.0 ± 3.1 5.7 ± 0.2* 4.9 ± 0.2* Malvidin - 49.4 ± 3.5* 3.5 ± 0.5* Morin 92.5 ± 6.5 15.5 ± 0.8* 2.0 ± 0.2* Myricetin 59.7 ± 6.9* 45.4 ± 0.3* 19.4 ± 0.4* 1.7 ± 0.4* Myricetin 30,40,50-trimethylether 148.2 ± 14.6* 99.9 ± 5.3 10.2 ± 0.3* 5.4 ± 0.5* Naringenin 177.0 ± 2.4* 99.8 ± 0.6 11.8 ± 0.5* 5.6 ± 0.4* Naringin 133.6 ± 7.1* 98.9 ± 1.0 31.8 ± 0.3* 6.9 ± 0.2* Peonidin – 67.4 ± 1.9* 9.5 ± 0.5* Pinocembrin 160.5 ± 7.5* 93.4 ± 1.5 31.6 ± 0.6* 11.3 ± 0.3* Quercetin 148.8 ± 9.0* 86.4 ± 1.6* 6.2 ± 0.4* 3.9 ± 0.3* Quercitrin 135.2 ± 7.0* 92.9 ± 1.5 10.8 ± 0.3* 2.7 ± 0.1* Sinensetin 181.7 ± 3.7* (10 lM) 114.9 ± 2.6*,b (10 lM) 8.7 ± 0.3* 6.9 ± 0.8* Tamarixetin 181.2 ± 8.1* 117.6 ± 3.7*,b 8.0 ± 0.3* 5.6 ± 0.2* Tangeretin 148.1 ± 2.3* (1 lM) 101.3 ± 0.7 (1 lM) 13.3 ± 0.8* 5.1 ± 0.3* Tricetin 68.7 ± 4.2* 64.3 ± 0.7* 10.9 ± 0.6* 5.5 ± 0.3* Tricetin 30,40,50-trimethylether 147.5 ± 3.5* 96.0 ± 1.1 8.2 ± 0.5* 6.5 ± 0.4* Other Berberine 164.3 ± 13.0* 108.9 ± 0.8 83.2 ± 2.2 28.4 ± 0.6* Celastrol 137.1 ± 3.0* (10 lM) 111.8 ± 6.9 (10 lM) 74.2 ± 2.7* 9.4 ± 0.8* Coumarin 114.7 ± 4.6 109.7 ± 3.5 104.9 ± 1.8 Indole 3-carbinol 103.0 ± 2.7 97.7 ± 1.8 92.9 ± 3.7 Limonin 138.1 ± 1.9* 121.2 ± 1.6*,b 93.5 ± 0.7 66.6 ± 1.0* Malic acid 99.2 ± 2.1 103.4 ± 1.5 101.5 ± 2.4 Oleanolic acid 153.6 ± 0.9* 110.5 ± 2.5 84.8 ± 2.2 (2 lM) 43.5 ± 1.9* (20 lM) Phloroglucinol 112.1 ± 9.0 102.4 ± 1.6 103.7 ± 1.6 Propyl gallate 117.2 ± 2.0 106.1 ± 3.1 69.1 ± 2.8* Quinic acid 109.9 ± 11.2 99.1 ± 2.5 104.2 ± 4.4 Sinigrin 109.6 ± 8.0 99.2 ± 2.1 101.0 ± 1.4 Ursolic acid 136.5 ± 12.2* 108.7 ± 2.9 76.9 ± 3.6*(2 lM) 59.5 ± 3.9*(20 lM) Vanillin 95.7 ± 4.7 98.2 ± 4.2 99.3 ± 2.7

* Pairwise comparisons with control, p < 0.05. a Concentrations may differ as indicated in brackets. b Significantly different from mitoxantrone control but not significantly different from Ko143 control. 2272 K.W. Tan et al. / Food Chemistry 138 (2013) 2267–2274

Table 2 ferent cell line (Pan, Winter, Roberts, Fairbanks, & Elmquist, 2010). IC50 values of selected test compounds derived from membrane In addition, although no ABCG2 expression was detected in vesicular transport assay. Data are expressed as mean ± SEM, n =3 HEK293/WT cells in the Western blot analysis (Fig. 1A), an 18.2% curve fittings; each curve was generated from 7–8 concentrations at duplicate or triplicate. increase of mitoxantrone accumulation was detected in HEK293/ WT cells when co-incubated with Ko143 (Table 1). The presence Test compound IC (lM) 50 of endogenous ABCG2 undetected by immunostaining or the Ko143 0.026 ± 0.002 involvement of an unidentified endogenous Ko143-sensitive Non-flavonoid mitoxantrone efflux transporter(s) may account for this Berberine 39.3 ± 4.5 observation. Celastrol 8.7 ± 0.7 The phenolic acids are important dietary phytochemicals com- Ellagic acid 20.7 ± 1.6 Limonin 93.3 ± 8.4 monly found in foods and beverages like apples, berries, cereals, Propyl gallate 87.1 ± 2.7 coffee and wine (see Supplementary table). Among the 11 phenolic Sinapic acid 117.5 ± 33.5 acids tested, none displayed significant inhibition of mitoxantrone Flavonoid efflux in the mitoxantrone accumulation assay. In the membrane Acacetin 0.145 ± 0.027 vesicular transport assay, only sinapic acid and ellagic acid exhib- Apigenin 0.055 ± 0.003 ited significant inhibitory activities against ABCG2-mediated trans- Chrysin 0.202 ± 0.031 Chrysoeriol 0.015 ± 0.002 port of methotrexate, with IC50 values of 117.5 and 20.7 lM, Diosmetin 0.065 ± 0.005 respectively, values which are considerably less potent than those Kaempferide 0.286 ± 0.054 of flavonoids (Table 2). The greater affinity for ABCG2 displayed by Kaempferol 0.086 ± 0.015 ellagic acid may be explained by its structural difference from the Laricitrin 0.102 ± 0.033 other phenolic acids tested, as it was the only dimer (of gallic acid) Luteolin 0.142 ± 0.017 Myricetin 1.547 ± 0.119 used in the present work. Studies in rats and intestinal Caco-2 cell Myricetin 30,40,50-trimethylether 0.076 ± 0.007 monolayers have suggested that the major phenolic acids are ab- Quercetin 0.213 ± 0.009 sorbed from the upper part of the GI tract, either via paracellular Tamarixetin 0.040 ± 0.014 diffusion or by the monocarboxylate transporters (MCTs) (Lafay Tricetin 0.407 ± 0.006 Tricetin 30,40,50-trimethylether 0.025 ± 0.002 & Gil-Izquierdo, 2008). The lack of interaction between phenolic acids and ABCG2 reported in the present study suggests that ABCG2 is largely not involved in the efflux of phenolic acids, which is in agreement with the rapid absorption and high oral bioavail- ability observed for phenolic acids. membrane vesicles therefore provide a relatively ‘clean’ environ- The flavonoids are well-characterised as inhibitors of ABCG2 ment for monitoring the direct interaction between ABCG2 and (Cooray et al., 2004; Dreiseitel et al., 2009; Zhang et al., 2004). A test compounds. It is notable that ABCG2 expressed in the Sf9 in- few flavonoids and their conjugated metabolites have also been sect cells displayed a lower molecular weight (65 kDa) as com- demonstrated to be substrates of ABCG2 (An et al., 2011; An & pared with ABCG2 expressed in human cell lines (72 kDa), Morris, 2011; Enokizono et al., 2007). In the present study, we have (Fig. 1A). The molecular weight difference has been reported previ- additionally identified chrysoeriol, laricitrin, myricetin trimethyle- ously and shown to be the result of a lack of post-translational gly- ther, pinocembrin, quercitrin, tamarixetin, tricetin and tricetin tri- cosylation of ABCG2 in the insect cells (Ishikawa et al., 2003). It has methylether as novel flavonoid ABCG2 inhibitors. In terms of also been demonstrated that the glycosylation difference does not structure–activity relationships (SAR) of flavonoid-mediated affect the expression and functionality of ABCG2 (Diop & Hrycyna, ABCG2 inhibition, 2D and 3D SAR studies have been reported based 2005). on inhibition potency derived from cell-based efflux inhibition as- For the cell-based mitoxantrone accumulation assay, it is gener- says (Pick et al., 2011; Zhang, Yang, Coburn, & Morris, 2005). IC50 ally accepted that permeability of test compounds into cells either values from our membrane vesicular transport studies are appre- by passive diffusion or active influx transport is a pre-requisite for ciably lower than in previous reports and this may relate to the dif- interaction with ABC transporters such as P-gp and ABCG2 (Hom- ferences in assay procedure (membrane-based versus cell-based) olya, Orban, Csanady, & Sarkadi, 2011). Compounds that are inher- and ABCG2 substrates used. The inside-out orientation of ABCG2 ently less permeable or lack facilitation of influx transporters may in the membrane vesicles is likely to provide better access for test therefore escape interaction with ABCG2, resulting in false nega- compounds to interact with ABCG2, without the pre-requisite of tives in the mitoxantrone accumulation assay. This may explain permeability across the plasma membrane, thereby allowing the lack of significant inhibitory activity displayed by anthocya- greater inhibition potency on ABCG2-mediated transport. Further- nins, hesperidin and morin (all of which are glycosides and have more, it has been suggested that multiple binding sites exist in lower lipophilicity compared with their respective aglycons) in ABCG2, which could mean that substrates bound to different sites the mitoxantrone accumulation assay, but not in the membrane might be inhibited by different inhibitors (Giri et al., 2009). Among vesicular transport assay. This observation is also in agreement the eight flavones and seven flavonols tested for inhibition po- with previous reports on interactions between flavonoid glycosides tency, four from each subclass are O-methylated, either singly or and ABCG2 (Dreiseitel et al., 2009; Pick et al., 2011). Another pos- triply at the 30,40,or50 position (Fig. 2). The tested flavones and sible explanation for negative results is interaction with an endog- flavonols with O-methylation at the 30,40,or50 position generally enous influx transporter of mitoxantrone, inhibition of which can exhibited greater ABCG2 inhibition potencies compared with their lead to reduced entry of mitoxantrone into the cells. This explana- hydroxylated counterparts, except for acacetin and kaempferide tion may account for the decreased mitoxantrone accumulation in which displayed lower potencies than apigenin and kaempferol, HEK293/ABCG2 and HEK293/WT cells following treatment with respectively. Notably, both acacetin and kaempferide are O-meth- delphinidin-3-rutinoside, ellagic acid, myricetin and tricetin, as ylated at the 40 position. This is contrary to the previous report by well as the reduced accumulation in HEK293/WT cells treated with Zhang et al. that showed an increased ABCG2-inhibitory effect of kaempferide and quercetin, (Table 1). The organic cation trans- methylation at the 40-hydroxy group (Zhang et al., 2005), although porter II (OCTII) is a possible candidate as it has been proposed direct comparison may not be appropriate because of the above- to be responsible for mitoxantrone influx transport, albeit in a dif- mentioned assay differences. K.W. Tan et al. / Food Chemistry 138 (2013) 2267–2274 2273

OH O OH O O OH O HO H3C OH

O HO HO CH3 O HO O O O CH3 OH Sinapic acid Ellagic acid Propyl gallate

CH3 O

H3C CH3 OH H3C H3C

CH OH 3 OH CH3 CH3 H CH3 CH3 H H C H CH O 3 3 O O H H CH3 H H CH3 CH H 3 H H3C CH3 H3C CH3 HO

Oleanolic acid Ursolic acid CH3 Celastrol

O

O O CH3

N O O O CH3 CH3 H O O O H O

CH3 H3C O O H H3C Berberine Limonin

R 3' R3' 3' 3' 2' 4' R 4' 2' 4' R4'

HO O 1' 5' HO O 1' 5' 6' R5' 6' R 5'

OH

OH O OH O Flavone 1-8 Flavonol 9-15

No. Compound name R3’ R4’ R5’ 1 Acacetin H OCH3 H 2 Apigenin H OH H 3 Chrysin H H H 4 Chrysoeriol OCH3 OH H 5 Diosmetin OH OCH3 H 6 Luteolin OH OH H 7 Tricetin OH OH OH 8 Tricetin 3',4',5'-trimethylether OCH3 OCH3 OCH3 9 Kaempferide H OCH3 H 10 Kaempferol H OH H 11 Laricitrin OH OH OCH3 12 Myricetin OH OH OH 13 Myricetin 3',4',5'-trimethylether OCH3 OCH3 OCH3 14 Quercetin OH OH H 15 Tamarixetin OH OCH3 H

Fig. 2. Chemical structures of identified non-flavonoid ABCG2 inhibitors and selected flavonoids.

The triterpenes are another group of phytochemicals commonly This is in agreement with the reported anticancer effect observed found in foods and herbal medicine and are represented by ursolic with oleanolic acid and ursolic acid (Sultana, 2011; Sultana & acid, oleanolic acid and celastrol in this study. All three triterpenes Ata, 2008). A few other of the novel ABCG2 inhibitors identified showed significant ABCG2-inhibitory activity in both assays. Celas- in the present study also harbour interesting potential health ben- trol, in particular, displayed the highest inhibition potency among efits. Berberine, for instance, has been tested in therapy for dis- the non-flavonoid inhibitors tested. The present study suggests eases such as Type 2 diabetes mellitus and cardiovascular disease that triterpenes may be another class of phytochemicals, like flavo- (Derosa, Maffioli, & Cicero, 2012). P-glycoprotein (P-gp/ABCB1), a noids, that exhibit common inhibitory activities against ABCG2. close relative of ABCG2, has previously been described to mediate 2274 K.W. Tan et al. / Food Chemistry 138 (2013) 2267–2274 intestinal transport of berberine (Pan, Wang, Liu, Fawcett, & Xie, activity, or trafficking to the plasma membrane. Biochemistry, 44(14), 2002). Our data indicate the possible involvement of ABCG2 in ber- 5420–5429. Dreiseitel, A., Oosterhuis, B., Vukman, K. V., Schreier, P., Oehme, A., Locher, S., et al. berine oral bioavailability, in addition to ABCB1. (2009). Berry anthocyanins and anthocyanidins exhibit distinct affinities for the Phytochemicals identified as ABCG2 inhibitors in the present efflux transporters BCRP and MDR1. British Journal of Pharmacology, 158(8), study could also be further investigated as potential reversal 1942–1950. Enokizono, J., Kusuhara, H., & Sugiyama, Y. (2007). Effect of breast cancer resistance agents for multidrug resistance (MDR). An ideal MDR-reversal protein (Bcrp/Abcg2) on the disposition of phytoestrogens. Molecular agent should have a broad spectrum and potent ABC-transporter Pharmacology, 72(4), 967–975. inhibitory activity with low or no toxicity. The high potency, low Fetsch, P. A., Abati, A., Litman, T., Morisaki, K., Honjo, Y., Mittal, K., et al. (2006). Localization of the ABCG2 mitoxantrone resistance-associated protein in toxicity and intrinsic anticancer properties of many phytochemi- normal tissues. Cancer Letters, 235(1), 84–92. cals make them attractive candidates for the reversal of MDR in Fletcher, J. I., Haber, M., Henderson, M. J., & Norris, M. D. (2010). ABC transporters in chemotherapy. To date, whereas many cytotoxic compounds are cancer: More than just drug efflux pumps. Nature Reviews Cancer, 10(2), 147–156. known to be effluxed by ABCG2 and/or ABCB1, most inhibitors Giri, N., Agarwal, S., Shaik, N., Pan, G., Chen, Y., & Elmquist, W. F. (2009). Substrate- developed for clinical trials target only ABCB1 (Fletcher, Haber, dependent breast cancer resistance protein (Bcrp1/Abcg2)-mediated Henderson, & Norris, 2010). To overcome MDR in clinical cancer interactions: Consideration of multiple binding sites in in vitro assay design. chemotherapies, there has thus far been little success in the devel- Drug Metabolism and Disposition, 37(3), 560–570. Homolya, L., Orban, T. I., Csanady, L., & Sarkadi, B. (2011). Mitoxantrone is expelled opment of an ABCB1 inhibitor (Shukla, Wu, & Ambudkar, 2008; by the ABCG2 multidrug transporter directly from the plasma membrane. Szakacs, Paterson, Ludwig, Booth-Genthe, & Gottesman, 2006), Biochimica et Biophysica Acta, Biomembranes, 1808(1), 154–163. leaving the field open to other avenues for investigation, including Ishikawa, T., Kasamatsu, S., Hagiwara, Y., Mitomo, H., Kato, R., & Sumino, Y. (2003). 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N-Linked glycosylation of the human ABC transporter ABCG2 on asparagine 596 is not essential for expression, transport