Identification of novel dietary phytochemicals inhibiting the efflux transporter breast cancer resistance protein (BCRP/ABCG2)
Highlights
► We examined ABCG2-inhibitory activity of dietary phytochemicals. ► Novel non-flavonoid inhibitors: berberine, celastrol, ellagic acid, limonin, oleanolic acid, propyl gallate, sinapic acid, ursolic acid. ► Novel flavonoid inhibitors: chrysoeriol, laricitrin, myricetin 3′,4′,5′ trimethylether, pinocembrin, quercitrin, tamarixetin, tricetin, tricetin 3′,4′,5′ trimethylether.
Introduction
Breast cancer resistance protein (BCRP/ABCG2) is a member of the evolutionarily-conserved family of ATP-binding cassette (ABC) efflux transporters. It is primarily expressed at the apical membrane of epithelial cells of organs such as the gastrointestinal tract, liver and kidney, as well as physiological barriers such as the blood–brain barrier, blood–testis barrier and maternal–foetal barrier (Fetsch et al., 2006). Consistent with its tissue localization, accumulating evidence suggests that ABCG2 functions as a cellular efflux pump, limiting foetal exposure, brain penetration and intestinal absorption of substrate xenobiotics, and also facilitating their mammary, biliary and kidney secretion. Endogenous substrates of ABCG2 include estradiol-17β glucuronide, folic acid (vitamin B9), riboflavin (vitamins B2), vitamin K3, uric acid and protoporphyrin IX, while exogenous substrates of ABCG2 encompass therapeutic agents of various classes such as antivirals (e.g. abacavir and lamivudine), antibiotics (e.g. ciprofloxacin and nitrofurantoin), HMG-CoA reductase inhibitors (e.g. atorvastatin), calcium channel blockers (e.g. azidopine) and anti-neoplastics (e.g. methotrexate, gefitinib and topotecan) (Meyer zu Schwabedissen & Kroemer, 2011). ABCG2 can influence the absorption and disposition of consumed substrates by limiting their net uptake from the gastrointestinal tract and distribution to target organs such as the central nervous system. In addition, ABCG2 has been attributed as a prominent molecular cause of multidrug resistance (MDR) in many human cancers through its active efflux of chemotherapeutic agents out of neoplastic cells (Natarajan, Xie, Baer, & Ross, 2012). In light of the clinical importance of ABCG2 in drug disposition and MDR, the discovery of ABCG2 inhibitors has attracted considerable scientific interest as one of the possible options in tackling 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 phytochemicals. Phytochemicals are secondary metabolites of plants which cover a broad range of structurally diverse compounds including alkaloids, organosulfur compounds, phenolic compounds and terpenes. Unlike essential micronutrients such as vitamins and minerals, phytochemicals are not associated with deficiency diseases resulting from insufficient dietary intake. They appear to contribute to long-term health maintenance, preventing and attenuating the development of degenerative diseases when regularly consumed. This is supported by numerous epidemiological studies that have indicated an inverse relationship between the consumption of various fruits and vegetables and the development of chronic diseases such as cancer, coronary heart disease, hypertension, stroke, certain eye diseases, dementia and osteoporosis (Boeing et al., 2012). In addition, specific and beneficial bioactivities of phytochemicals are being actively studied and have been demonstrated in various areas of medicine including cancer, cardiovascular diseases, neurodegenerative diseases, eye health and skin health (Krzyzanowska, Czubacka, & Oleszek, 2010). Despite the epidemiological association and many putative health effects derived from in vitro and animal studies of phytochemicals, very few randomised controlled human clinical trials have confirmed their clinical efficacy. This is likely due to limited bioavailability at the systemic level and more so at target organs. For instance, maximal plasma concentrations of total anthocyanins after consumption of anthocyanin-rich foods are typically in the range of 1–100 nM (Yang, Koo, Song, & Chun, 2011), which is about a thousand fold lower than the concentration range commonly used to achieve in vitro bioactivity.
Orally consumed phytochemicals display different absorption, distribution, metabolism and excretion (ADME) profiles that dictate their exposure at the systemic level as well as their target sites. Phenolic acids, for example, are generally bioavailable because of their high absorption from the upper part of the gastrointestinal tract (Lafay & Gil-Izquierdo, 2008). Flavonoids, on the other hand, undergo extensive phase II metabolism in the intestine (and liver) to form conjugates, such as glucuronides and sulfates, which are then subjected to phase III efflux by transporters. In terms of phase III efflux of phytochemicals in the gastrointestinal tract, current evidence from animal studies suggests that ABCG2 plays a major role in apical efflux of phytochemicals and their phase II metabolites back to gut lumen and consequently limits their bioavailability (Enokizono et al., 2007, Sesink et al., 2005, van de Wetering et al., 2009). For instance, knocking out mouse Abcg2 led to significant increases in the oral bioavailability of daidzen (3.7-fold) and genistein (1.8-fold) compared with wild-type mice (Enokizono et al., 2007). A higher plasma level and an increased urinary excretion of resveratrol phase II metabolites were also observed in Abcg2 knock-out mice (van de Wetering et al., 2009). In addition, in vitro studies have also indicated that a variety of flavonoids interact with ABCG2 as inhibitors (Cooray et al., 2004, Dreiseitel et al., 2009; Zhang, Yang, & Morris, 2004) and/or substrates (An, Gallegos, & Morris, 2011; An & Morris, 2011).
The interplay between ABCG2 and dietary phytochemicals raises potential issues concerning food–drug interactions that may occur when ABCG2 drug substrates are taken concomitantly with phytochemical-containing foods or dietary supplements. Enhanced exposure to ABC transporter substrates, such as anticancer agents, calcium-channel blockers and immuno-suppressants has been reported in humans and animals after substrate drugs are administered orally together with phytochemical-rich foods and drinks (Li, Revalde, Reid, & Paxton, 2010). In contrast, certain phytochemicals may induce efflux transporter expression through interactions with nuclear receptors, resulting in sub-optimal drug concentration and therapeutic failure (Tirona & Bailey, 2006). With the increasing production and the widespread use of herbal and dietary supplements, the risk of harmful interactions increases considerably as dietary supplements commonly provide pure phytochemicals or phytochemical-containing herbal extracts at concentrations many fold higher than the levels found in foods. Hence, understanding the interactions between ABCG2 and dietary phytochemicals may serve as a cautionary and preventive step against harmful ABCG2-modulated food-drug interactions. On the other hand, phytochemical–ABCG2 interactions may potentially be applied for the development of beneficial phytochemical–drug substrate combinations that allow increased drug efficacy and safety at lower doses or phytochemical–phytochemical combinations that enhance phytochemical bioavailability and/or bioactivities (Scheepens, Tan, & Paxton, 2010).
In the present study, we explored the ABCG2 inhibitory activities of 56 dietary phytochemicals and two non plant-based but diet-relevant compounds (hippuric acid and propyl gallate) using both cell- and membrane-based methods. In the cell-based mitoxantrone accumulation assay, the effect of test compounds on ABCG2-mediated efflux of the fluorescent substrate, mitoxantrone, was measured in HEK293 (human embryonic kidney) cells overexpressing ABCG2. In the membrane-based assay, the effect of test compounds on the transport of 3H-methotrexate into inside-out Sf9 (Spodoptera frugiperda) insect cell membrane vesicles overexpressing ABCG2 was investigated.
Section snippets
Materials
The following compounds were procured from the commercial sources indicated: 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, apigenin, berberine, (+)-catechin, caffeic acid, chrysin, p-coumaric acid, coumarin, ellagic acid, (−)-epicatechin, ferulic acid, gallic acid, hesperidin, hippuric acid, indole-3-carbinol, limonin, luteolin, malic acid, methotrexate, mitoxantrone, myricetin, naringenin, naringin, phloridzin, phloroglucinol, propyl gallate, quercetin, quinic acid, salicylic acid,
Results
The expression of ABCG2 in cell lines and membrane vesicles used in this study was confirmed by Western blot analysis. As shown in Fig. 1A, ABCG2 was detected as a ∼72 kDa immunoreactive band in HEK293/ABCG2 cells (a significant amount of immunoreactive protein was also detected as an aggregate above the highest marker, data not shown) while no detectable expression was observed in HEK293/WT cells. In Sf9/ABCG2 membrane vesicles, ABCG2 was detected as a ∼65 kDa immunoreactive protein. Likewise,
Discussion
Two different approaches have been used to identify and verify the ABCG2-inhibitory effects of 56 naturally-occurring phytochemicals and two non plant-based compounds, most of which can be found in common diets. The non plant-based compounds, propyl gallate (E310) and hippuric acid, were included because of their relevance as a common food additive and a major polyphenol metabolite, respectively. The two assay systems probed the interactions between ABCG2 and test compounds in different
Acknowledgements
K.T. and A.S. acknowledge funding from the New Zealand Institute for Plant & Food Research Limited (Excellence Programme Ph.D. studentship and the PFR capability fund CF10-22). We thank James Dickson (the University of Auckland) and Stephen Edgar (the University of Auckland) for technical assistance in protein expression and flow cytometry, respectively. We thank Mark Wohler for assistance with statistical analysis. We also thank David Stevenson, Jeffrey Greenwood, Nayden Koon, and Roger Hurst
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Current address: School of Interprofessional Health Studies, Faculty of Health and Environmental Sciences, Auckland University of Technology, Auckland 0627, New Zealand.