Inhibition of human cytochrome CYP1 enzymes by flavonoids of St. John's wort

Abstract

CYP1B1 is involved in metabolizing both polycyclic aromatic hydrocarbons and estradiol to potentially carcinogenic intermediates, and it is also over-expressed in human cancer cells. In order to investigate whether flavonoids could specifically inhibit CYP1B1, seven flavonoids in St. John's wort and apigenin were screened for their inhibition of recombinant human CYP1B1 and CYP1A1. While seven flavonoids (myricetin, apigenin, kaempferol, quercetin, amentoflavone, quercitrin and rutin) were slightly more selective for CYP1B1 EROD inhibition (K_is 0.06–5.96 μM) compared to CYP1A1 (K_is 0.20–1.6 μM) the difference in K_is for the P450s were not significantly different. Rutin did not inhibit CYP1A1 at concentrations up to 10 μM. Kinetic analyses determined that apigenin and amentoflavone were competitive inhibitors of CYP1B1, while quercetin showed mixed type inhibition. To characterize the inhibition potential of these flavonoids, five were studied further for their ability to inhibit TCDD-induced EROD activity in 22Rv1 human prostate cancer cells. 22Rv1 cells express constitutive and TCDD-inducible CYP1A1 and CYP1B1 mRNA. In the cells, the IC₅₀s were similar to those measured for the recombinant CYP1A1 except for amentoflavone. Quercetin (IC₅₀: 4.1 μM), kaempferol (3.8 μM), myricetin (3.0 μM) and apigenin (3.1 μM) caused significant inhibition of EROD activity whereas amentoflavone did not cause inhibition. Depending on their bioavailability, flavonoids that can selectively inhibit CYP1 enzymes may be useful as chemoprotective agents in prostate cancer prevention.

Introduction

Our research focus is the potential for bioflavonoids to act as therapeutic and/or preventative agents in prostate cancer via inhibition of the CYP1 cytochrome P450 enzymes, which have been implicated in carcinogenesis (Chun and Kim, 2003, McFadyen et al., 2004). The P450 superfamily of genes is involved in the oxidation and excretion of both endogenous and exogenous nonpolar compounds in the body. The human CYP1 gene family is comprised of CYP1A1, CYP1A2, and CYP1B1. CYP1A2 is expressed primarily in liver, CYP1A1 is expressed in both liver and extrahepatic tissues, whereas CYP1B1 is primarily expressed extrahepatically. The CYP1s are involved in bioactivating polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines, and estradiol to mutagenic and carcinogenic intermediates (Murray et al., 2001). In the prostate, CYP1B1 message has been detected in normal (Finnstrom et al., 2001) and benign prostatic hyperplasia (Luo et al., 2002) and is up-regulated in malignant prostate tissue (Chaib et al., 2001, Carnell et al., 2004). In a Japanese population a CYP1B1 polymorphism at codon 119 has been associated with prostatic carcinogenesis (Tanaka et al., 2002). This polymorphism has also been associated with the highest catalytic bioactivation of estradiol (Hanna et al., 2000). More recently, Chang et al. (2003) identified a frequent haplotype in CYP1B1 that was associated with an increased risk for prostate cancer. Together these studies, implicate CYP1B1 as a potential therapeutic target for reducing development of prostate cancer.

Inhibiting CYP1B1 selectively could be considered chemoprotective for several reasons. First, Shimada et al. (1999) found that recombinant human CYP1B1 was more active than CYP1A1 in metabolizing benzo(a)pyrene (BaP) to the proximate toxicant BaP-7,8-diol. In addition, human CYP1B1 is an estradiol hydroxylase, acting primarily at the C-4 position. CYP1A1, in contrast, has activity at the C-2, C-6α and C-15α positions of estradiol (Hayes et al., 1996). In rodent tissues, including the Noble rat prostate, estrogen induces tumors when estradiol is primarily converted to the 4-hydroxyestradiol metabolite, yet tumors fail to develop where 2-hydroxylation predominates (Cavalieri et al., 2002). Lastly, CYP1B1 can detoxify cancer drugs, such as flutamide and possibly docetaxel, which reduces their cytotoxic potential and effectiveness (Rochat et al., 2001, McFadyen et al., 2001, Bournique and Lemarie, 2002). This is particularly significant because CYP1B1 is over-expressed in a variety of human tumors compared to adjacent noncancerous cells (McFadyen et al., 2001). Therefore, the hypothesis guiding this study was that a bioflavonoid could be potentially anti-carcinogenic if it could inhibit CYP1B1's ability to metabolize PAHs and estrogen to toxic intermediates and/or decrease CYP1B1's ability to detoxify cancer drugs. Because prostate cancer typically has a long latency period and develops in older men, this disease may be particularly amenable to chemopreventive approaches (Saleem et al., 2003).

St. John's wort (Hypericium perforatum) is an herbal product widely used as an over the counter remedy for mild to moderate depression. It contains a number of classes of pharmacologically active compounds including napthodianthrones, flavonoids, phloroglucinols, procyanidins, tannins, essential oils, amino acids, phenylpropanes, xanthones and other water-soluble compounds (Greeson et al., 2001). It is well known that some of the flavonoids in St. John's wort exert a modulating role on the metabolism of drugs and xenobiotics by acting as inducers or inhibitors of cytochrome P450s. Evidence is prevalent that St. John's wort induces CYP3A, one of the most important human drug metabolizing enzymes and the cause of adverse drug reactions in some patients (Vogel, 2001). Other commercially available St. John's wort extracts and individual components have been investigated for their effects on CYP1A1, CYP2D6, CYP2C9, CYP3A4, CYP1A2 and CYP2C19 (Obach, 2000). Prior to our work, little has been reported about the effects of flavonoids found in St. John's wort on CYP1B1 specifically.

In this present study, we characterized eight flavonoids: quercetin, amentoflavone, kaempferol, myricetin, quercitrin, isoquercetin, rutin, and apigenin (Fig. 1) for their inhibition of recombinant CYP1B1 and CYP1A1. Apigenin, while not a component of St. John's wort, is a monomer of amentoflavone and was included in these experiments to determine if the effects of amentoflavone would reflect its degradation into monomers. While none of the flavonoids were significantly selective for CYP1B1 compared to CYP1A1, apigenin especially was a relatively potent CYP1B1 inhibitor. In addition, we showed that myricetin, apigenin, kaempferol, and quercetin can inhibit EROD activity, a conventional assay for assessment of CYP1 enzyme activity, in the 22Rv1 human prostate cancer cell line. By distinguishing the relative roles of cytochrome P450s (specifically CYP1s) in human prostate cancer cell lines, new therapies or chemoprotective strategies against prostate cancer formation may emerge.