Although CPA and IFO have long been used as anti-cancer alkylating agents in clinical practice with their pharmacology, metabolism and pharmacokinetic/pharmacodynamic profiles well elucidated, there have been rising concerns regarding drug-drug interactions, and autoinduction along with the increase of combinatory therapeutic strategies involving these drugs (35
). Accumulating evidence thus far, revealed that hepatic metabolism of CPA and IFO is auto-inducible and repeated administration of CPA and IFO was associated with elevated 4-hydroxylation of the oxazaphosphorines and expression of several CYP enzymes (15
). However, the molecular mechanisms behind the induction are not fully understood. In this report, we demonstrated that in addition to the promiscuous xenobiotic receptor PXR, CAR plays an important role in the enzymatic autoinduction of these oxazaphosphorines. More importantly, although PXR appears to be equally involved in the inductive activity of CPA and IFO, CAR functions as a preferential mediator of CPA- rather than IFO-mediated induction of hepatic DMEs. This evidence suggests that co-administration of drugs that selectively disturbing the expression and function of CAR may differentially affect the autoinduction and drug-drug interactions of CPA rather than IFO.
In order to accommodate their own metabolism and clearance, xenobiotics including drugs and environmental chemicals can alter the expression of DMEs and transporters through the transactivation of a group of xenobiotic receptors. Previously, PXR was reported as the mediator of CPA and IFO autoinduction by transcriptional up-regulation of CYP2B6 and CYP3A4 (18
). Both CPA and IFO increased PXR activity in luciferase reporter assays at the concentrations that significantly induced CYP2B6 and CYP3A4 expression in HPH cultures (18
). Interestingly, we showed that CPA and IFO treatment resulted in greater increase in CYP2B6 mRNA than that of CYP3A4, which is in agreement with data from a previous report (19
). Given that CYP2B6 is a favorable target gene of CAR over PXR and potent induction of CYP2B6 by CPA and IFO was observed, activation of PXR alone by these oxazaphosphorines may not be able to sufficiently accommodate the extent of DME induction in hepatocytes. Accordingly, it is conceivable that CAR, the closest relative of PXR, may mediate a compensatory role in CPA and IFO associated induction. However, unlike that of PXR, investigation of CAR activation in vitro has been hindered by several specific features of this receptor including that: 1) CAR is constitutively activated and spontaneously nuclear localized in immortalized cell lines; and 2) it can be activated by both direct ligand binding and indirect ligand-independent mechanisms (32
). Recently, we have generated a chimerical construct namely hCAR1+A with an alanine insertion at 271, which converts the constitutive wide-type hCAR into a chemically responsive xenobiotic sensor (23
). In cell-based reporter assays utilizing this chimerical vector, our results revealed that CPA and IFO dose-dependently enhanced CYP2B6 luciferase expression through the activation of hCAR1+A. Given that over 90% of known hCAR activators positively response to this chimerical constructs (23
), this initial experiment supports the involvement of CAR in the inductive activity of CPA and IFO.
Alternatively, CAR exhibits significant nuclear translocation from cytoplasm of primary cultured hepatocytes in the presence of CAR activators such as PB and CITCO (25
). This initial step of CAR activation in primary hepatocytes has been successfully established as an efficient approach to identify hCAR activators in vitro (25
). Remarkably, both direct activators (e. g. CITCO, and artemicinin) and indirect activators (e. g. PB, and phenytoin) are effective in the nuclear accumulation of CAR in hepatocyte cultures (25
). Intriguingly, our results showed that CPA but not IFO displayed marked effects on nuclear accumulation of CAR in HPHs. Although activation of CAR is a multistep process, the lack of initial nuclear translocation of CAR by IFO suggests that IFO is less likely to be an effective activator of hCAR.
In contrast to other nuclear receptors, activation of CAR doesn't require ligand binding (indirect activation), and as a matter of fact the majority of known hCAR activators identified thus far activate CAR through PB-like indirect machinery rather than CITCO-like ligand binding (25
). Recent reports from this laboratory demonstrated that in HepG2 cells, the PK11195, a prototypical peripheral benzodiazepine receptor and potent hCAR deactivator, repressed hCAR activity can be efficiently reactivated by direct activator CITCO but not the prototypical indirect activator PB (34
). Akin to PB, CPA was unable to reactive PK11195 inhibited CAR activity, suggesting CPA may function as a PB-type indirect CAR activator.
To date, mounting evidence suggests that CAR and PXR share their target genes through recognizing of, and binding to, xenobiotic responsive elements located in the upstream of these genes (37
). An asymmetrical cross-regulation of CYP2B6 and CYP3A4 by hCAR but not hPXR has been established in that hCAR exhibits preferential induction of CYP2B6 over CYP3A4, while hPXR exerts less-selective induction of both genes (39
). In an effort to determine the relative involvement of CAR in CPA- and IFO-mediated CYP2B6 induction, experiments in HPHs revealed that SFN, a selective inhibitor of hPXR, dramatically repressed IFO induced expression of CYP2B6 mRNA, while to a lesser extent in response to the CPA-mediated induction. In concert with aforementioned luciferase reporter and nuclear translocation data, these results strongly suggest that CPA is a stronger activator of hCAR compared to IFO.