CYP1-mediated antiproliferative activity of dietary flavonoids in MDA-MB-468 breast cancer cells

Abstract

Among the different mechanisms proposed to explain the cancer-protecting effect of dietary flavonoids, substrate-like interactions with cytochrome P450 CYP1 enzymes have recently been explored. In the present study, the metabolism of the flavonoids chrysin, baicalein, scutellarein, sinensetin and genkwanin by recombinant CYP1A1, CYP1B1 and CYP1A2 enzymes, as well as their antiproliferative activity in MDA-MB-468 human breast adenocarcinoma and MCF-10A normal breast cell lines, were investigated. Baicalein and 6-hydroxyluteolin were the only conversion products of chrysin and scutellarein metabolism by CYP1 family enzymes, respectively, while baicalein itself was not metabolized further. Sinensetin and genkwanin produced a greater number of metabolites and were shown to inhibit strongly in vitro proliferation of MDA-MB-468 cells at submicromolar and micromolar concentrations, respectively, without essentially affecting the viability of MCF-10A cells. Cotreatment of the CYP1 family inhibitor acacetin reversed the antiproliferative activity noticed for the two flavones in MDA-MB-468 cells to 13 and 14 μM respectively. In contrast chrysin, baicalein and scutellarein inhibited proliferation of MDA-MB-468 cells to a lesser extent than sinensetin and genkwanin. The metabolism of genkwanin to apigenin and of chrysin to baicalein was favored by CYP1B1 and CYP1A1, respectively. Taken together the data suggests that CYP1 family enzymes enhance the antiproliferative activity of dietary flavonoids in breast cancer cells, through bioconversion to more active products.

Introduction

Cytochrome P450s are haem-containing enzymes, responsible for the metabolism of a vast number of xenobiotics as well as endogenous compounds. The first family consists of three members CYP1A1, CYP1A2 and CYP1B1. CYP1A2 is expressed predominantly in the liver, whereas CYP1B1 and CYP1A1 are found in extrahepatic tissues (Murray et al., 1997, McFadyen et al., 2004). Metabolism of xenobiotics by CYP1 family enzymes can elicit toxic responses, or contribute to the detoxification of potentially harmful compounds. For example it has been shown that CYP1A2 participates in the activation of the anti-androgenic drug Flutamide and the anthraquinone AQ4N, whereas recent evidence in Cyp1a1 (−/−) knockout mice, suggests that CYP1A1 contributes to the detoxification of the environmental carcinogen B[a]P (Shet et al., 1997, Patterson and Murray, 2002, Uno et al., 2004, Androutsopoulos et al., 2009c). With respect to environmental toxicology and chemoprevention the interactions of CYP1 family enzymes with flavonoids, a major class of natural products encountered frequently in the diet, have been well documented over the last decade. It has been proposed that flavonoids act either as CYP1 enzyme inhibitors and inhibitors of CYP1 gene-mediated transcriptional activation, thereby blocking any mutagenic effects produced by metabolism of environmental carcinogens, or as CYP1 substrates undergoing activation to antiproliferative agents, notably cell cycle inhibitors, in tissues or cells where CYP1 enzymes are expressed (Chun et al., 1999, Chun et al., 2001, Ciolino et al., 1998a, Ciolino et al., 1998b, Ciolino and Yeh, 1999a, Ciolino and Yeh, 1999b, Ciolino et al., 1999, Androutsopoulos et al., 2008, Androutsopoulos et al., 2009a, Androutsopoulos et al., 2009b, Androutsopoulos et al., in press, Atherton et al., 2006, Potter et al., 2002).

Baicalein (5,6,7-trihydroxyflavone), chrysin (5,7-dihydroxyflavone) and scutellarein (5,6,7,4′-tetrahydroxyflavone) (Fig. 1) are bioactive flavonoids, containing hydroxyl groups, present in SCUTELLARIA, a traditional herbal remedy with potential anticancer activity (Parajuli et al., 2009). Baicalein is also a major component of sho-saiko-to, which is a Chinese medicine administered in Japan for the chemoprevention of hepatic fibrosis and carcinoma, whereas chrysin, a major constituent of propolis found in the regions of Sonora in Mexico, exhibits antiproliferative activity in cancer cell lines (Shimizu, 2000, Hernandez et al., 2007). Genkwanin (7-methoxy,5,4′-dihydroxyflavone) and sinensetin (3′,4′,5,6,7-pentamethoxyflavone) (Fig. 1) are flavonoids containing one and five methoxy groups, respectively, which are present in dietary products, as well as medicinal plants. Genkwanin is found in its aglycone form in the oil droplets produced by the plant Origanum × intercedens, which is considered as a high quality oregano spice, as well as in the traditional medicinal herbs Artemisia afra and Combretum erythrophyllum (Combretaceae), used as antimalarial remedies and for the treatment of abdominal pains and venereal diseases respectively (Bosabalidis et al., 1998, Kraft et al., 2003, Martini et al., 2004). Sinensetin is found in small amounts in the juice of mandarins and related citrus fruits and in higher amounts in the corresponding peel extracts (Nogata et al., 2003, Lu et al., 2006, Wang et al., 2008). Genkwanin induces terminal differentiation of HL-60 leukemic cells, while sinensetin is capable of inhibiting production of TNF-α and suppress in vivo natural killer cell activity, as well as inhibit mutagenic activity caused by furylfuramide, a well-known mutagen (Suh et al., 1995, Delaney et al., 2001, Miyazawa et al., 1999). However little is known regarding the exact mechanism by which such flavonoids exert their cancer-protecting activity.

Recent studies in our lab have identified the dietary flavonoids eupatorin, cirsiliol and diosmetin as CYP1-activated prodrugs, i.e. metabolism of these compounds by CYP1 enzymes results in further activation and enhances their antiproliferative activity (Androutsopoulos et al., 2008, Androutsopoulos et al., 2009a, Androutsopoulos et al., 2009b, Androutsopoulos et al., in press). In the present study, the hypothesis that structurally similar flavonoids, present in the diet, act through the same mechanism was examined. The metabolism of the flavones genkwanin, sinensetin, chrysin, baicalein and scutellarein (Fig. 1A) by CYP1 enzymes was investigated. We suggest that all flavones with the exception of baicalein are CYP1 substrates and that they are activated to more antiproliferative conversion products in MDA-MB-468 cells, through CYP1-mediated metabolism. Together with our previous findings the first preliminary evidence for a tumor-suppressing role of CYP1 enzymes is presented.