Extending the Good Diet, Good Health Paradigm: Modulation of Breast Cancer Resistance Protein (BCRP) by Flavonoids
Department of Metabolism and Pharmacokinetics, Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Pharmaceutical Research Institute, Pennington, NJ 08534
1 For correspondence via fax: (609) 818-7130. E-mail: mengmeng.wang{at}bms.com.
Received February 1, 2007; accepted February 1, 2007
Flavonoids, important constituents of the human diet, are found in a variety of fruits, vegetables, and beverages including wine and teas. As a class, flavonoids are recognized to have numerous biological activities, and a healthy diet rich in fruit and vegetables is linked directly to a reduced risk of cancer (Lee et al., 1991
; Middleton et al., 2000) or coronary heart disease (Hertog et al., 1993; Kelly, 2001). With increasing recognition of the potential long-term benefits of these components, they are also being used more frequently as dietary supplements. The biological effects ascribed to the anticancer actions of flavonoids are diverse and include their ability to modulate (1) signal transduction pathways involved in cell proliferation and angiogenesis, (2) enzymes required for metabolic activation of procarcinogens and the detoxification of carcinogens, (3) enzymes involved in estrogen biosynthesis, and (4) potent antioxidant activities (Morris and Zhang, 2006).
Recently, drug efflux transporters such as P-glycoprotein (PgpABCB1) and breast cancer resistance protein (BCRP/ABCG2) were found to transport a range of flavonoids and other nonnutritive food compounds. In this issue, Ebert et al. (2006) report for the first time that these phytochemicals modulate BCRP at the transcriptional level, most likely acting through the aryl hydrocarbon receptor (AhR) pathway. The authors also properly connect the two critical aspects of induction and function by showing that phytochemicals can induce BCRP and link this induction to the observation that phase II metabolites of the widely recognized carcinogen, benzo[a]pyrene (BP), are substrates of BCRP. These data also provide yet another example of the central role of xenobiotic transporters in regulating the disposition and subsequent toxicity of xenobiotics. Specifically, phytochemicals might protect against BP-related cancer by inducing the expression of BCRP and ultimately, decreasing exposure BP and its promutagenic metabolites.
The regulation of the enzymes and transporters that contribute to xenobiotic disposition has been extensively studied, and numerous transcription factors are known to function as master regulators of xenobiotic metabolism, disposition, toxicity, and drug-drug interactions. For example, nuclear hormone receptors pregnane X receptor, liver X receptor, or constitutive androstane receptor are known to regulate cytochrome P450s, phase II metabolism enzymes, and transporters including Pgp or members of the multidrug resistance protein (MRP) family (Chan et al., 2004), but surprisingly, not BCRP (Assem et al., 2004; Klucken et al., 2000). Regulation of BCRP is not completely understood but was found to be affected by other pathways such as hypoxia-inducible factor (Krishnamurthy et al., 2004) and sex hormones (Ee et al., 2004). Only recently did Ebert et al. (2005) identify that activation of AhR could induce the expression of BCRP. Interestingly, AhR appears to regulate the induction of the CYP1 family, BCRP, and MRPs but does not induce Pgp (Colombo et al., 2003). Thus, it appears that, although BCRP is localized on the apical surface of polarized cells like Pgp and shares a wide range of substrate specificity with Pgp, the regulation of BCRP and Pgp is distinguished by the activation of different transcription factors. This distinction may provide a compensatory mechanism in the cellular defense system, such that, when one pathway is blocked, the other can compensate.
It is well established that food components like flavonoids can induce gene expression through transcription factors including AhR (Hankinson, 1995). With the recent finding that AhR regulates BCRP expression, the authors wisely speculated and carried out additional experiments to evaluate the induction of BCRP by a wide range of phytochemicals. Flavonoids such as chrysin, flavone, quercetin and genistein, silymarin, curcumin, and dibenzoylmethane are known AhR agonists. Tert-butyl hydroquinone (TBHQ), which is a synthetic antioxidant, quercetin, diethylmaleate, and isothiocyanate sulforaphane were used to differentiate the AhR-xenobiotic response element induction pathway from nuclear factor-erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) induction pathway because all these compounds are known to induce the Nrf2-ARE pathway. Resveratrol from red wine or grapes, an AhR-antagonist, and the green tea component (–)-epicatechin, later also found to be an antagonist of AhR (Mandlekar et al., 2006), were also used. In addition to using the antagonists as negative controls and Nrf2 inducers such as TBHQ for differentiation, the authors utilized a "superinduction" method, in which an inhibitor of proteasomal degradation was included with the AhR inducer. The ligand-activated AhR protein complex is ubiquitinated and degraded in the cytosol (Ma and Baldwin, 2000). Further increased expression of BCRP as a result of adding inhibiting proteasomal function served to provide additional confirmation that the activation is through AhR pathway.
An extremely important observation in the highlighted paper is the observation that mRNA and protein levels of BCRP were not always correlated. While compounds such as quercetin increased both mRNA and protein expression of BCRP, some compounds such as chrysin increased only the mRNA. Unlike flavonoid induction of cytochrome P450 1A enzymes, whose mRNA and protein levels are generally consistent (Netsch et al., 2006), there is an increasing body of evidence that the regulation of xenobiotic transporter mRNA and protein is not always straightforward (Nakanishi et al., 2006). The correlation between mRNA and protein expression may be regulated by posttranscriptional factors such as translational efficiency and/or half-life of the protein (Wang et al., 2006). In addition, Nakanishi et al. (2006) recently found that mRNA 5' untranslated region variants on BCRP are differentially expressed in drug-selected cancer cells and in normal human tissues. Thus, the present work provides compelling evidence that, when evaluating the regulation of BCRP expression, both mRNA and protein levels should be measured.
BP is ubiquitous in the environment, but BP-contaminated food (from grilled and broiled meat) is thought to be the main source of exposure (Ebert et al., 2005). Accordingly, the intestine functions as the first barrier to protect against BP exposure. In the intestine, BP can be metabolized, and the hydrophilicity of the generated phase-2 metabolites makes them impermeable without the facility of transporters. Combining the findings that phytochemicals can induce functional BCRP and that sulfated BP is a substrate of BCRP, it is possible that flavonoids can help detoxification of BP by inducing BCRP. Furthermore, the authors also found that neither Pgp nor MRP2 can transport these BP conjugates, thereby implicating BCRP as the major elimination pathway for sulfated BP.
With the promising in vitro result described in this paper, the next important step is to investigate the in vivo effect of phytochemicals in the disposition and toxicity of BP. These will not be trivial or simple experiments, as flavonoids are not only the inducers but can also be substrates or inhibitors of BCRP (Morris and Zhang, 2006). With the complicity of the interaction of phytochemicals and other drug metabolizing enzymes, it may be hard to predict and interpret the effect of the compounds in vivo. However, such work will be important to advancing our understanding of the characteristics of flavonoids in general. The work by Ebert et al. (2006) in this issue provides yet another compelling argument for ongoing studies of the effects of flavonoids on xenobiotic dispostion, and the evidence that phytochemicals can induce BCRP, which in turn may play an important role in detoxification of carcinogens such as BP, is an important finding relative to understanding mechanisms of toxicity. Additional evidence from in vivo studies will help to establish the role for BCRP and other xenobiotic transporters in both disposition and toxic mechanisms. Until such data are available, keep eating those vegetables.
ACKNOWLEDGMENTS
Conflict of interest: None declared.
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