The pregnane × receptor (PXR) is a key xenobiotic receptor that regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and drug transporters [1, 2]. Expression of PXR target gene is regulated by binding of PXR to its promoter region, such as that of cytochrome P450 3A4 (CYP3A4), a key enzyme that catalyzes the metabolism of more than 50% of all clinically prescribed drugs . Changes in the expression of CYP3A4 affect drug metabolism and alter the therapeutic and toxicologic responses to drugs, which may in turn lead to adverse drug interactions.
The activity of PXR is regulated not only by direct ligand binding  but also by various cell signaling pathways , such as those mediated by protein kinase C (PKC) , protein kinase A (PKA) [7, 8], cyclin-dependent kinase 2 (Cdk2) , 70kDa form of ribosomal protein S6 kinase (p70 S6K) , forkhead in rhabdomyosarcoma (FKHR) , and nuclear factor κB (NF-κB) [12–14].
Flavonoids - secondary metabolites found ubiquitously in plants - are the most common group of polyphenolic compounds consumed by humans as dietary constituents . Thousands of naturally occurring flavonoids, such as flavones and isoflavones, have been characterized . Flavonoids have been reported to have anti-allergic, anti-inflammatory, anti-microbial and anti-cancer activities [17, 18]. The widespread use of flavonoids, coupled with their potentially beneficial effects, has triggered studies on the mechanism by which they modulate signaling pathways.
Natural flavonoids have been shown to inhibit Cdk1, Cdk2 , and Cdk5 . Most Cdks, including Cdk1 and Cdk2, are involved in cell cycle regulation and require the binding of cyclins for their activation. However, the activation of Cdk5 requires one of the two non-cyclin regulatory subunits p35 or p39, which have 57% amino acid homology. p35 can be converted in a Ca2+-dependent manner to p25, a highly active and stable proteolytic product [21, 22]. The protease calpain catalyzes the cleavage of p35, and this reaction can be effectively inhibited by specific inhibitors of calpain such as calpeptin [23, 24]. Cdk5 is not involved in cell cycle progression, and is expressed in all tissues, but its levels of expression and activity are highest in the nervous system [21, 25]. The expressions of p35 and p39 are also highest in the nervous system. Although Cdk5 has been mainly implicated in early development of the central nervous system (CNS) and maintenance of neuronal architecture [21, 26], the expression and regulatory activity of Cdk5/p35 have also been reported in several non-CNS tissues such as lens epithelia , muscle tissues , hepatoma cells , adipose tissues , and male reproductive system .
The widespread use of flavonoids has triggered studies to investigate their effects on drug metabolism and herbal-drug interactions. Recently, flavonoids have been shown to induce CYP expression through PXR [31–36], but the mechanism of flavonoids-mediated PXR activation and CYP induction remain unknown.
Because the function of PXR can be modulated by cellular signaling pathways, we used a cell-based screening approach in this study to identify compounds with known bioactivities that activate PXR-mediated gene expression. By screening a library of known bioactive compounds, we identified a series of flavonoids that are PXR activators. Since these flavonoids did not directly bind to PXR, and flavonoids might inhibit Cdk5, we studied the effect of flavonoids on the activity of Cdk5/p35 and the regulation of PXR by Cdk5 in order to determine the possible role of flavonoids in regulating PXR-mediated gene expression of CYP3A4.