Abstract
Most hepatoma cell lines lack proper expression and induction of CYP3A4 enzyme, which limits their use for predicting drug metabolism and toxicity. Nuclear receptor pregnane X receptor (PXR) has been well recognized for its critical role in regulating expression of CYP3A4 gene. However, its physiological activity of binding to the particular site of promoter is significantly weakened in hepatic cell lines. To address this problem, we created “chimeric PXR” constructs by appending a strong activation domain (AD) from p53 subunit to either N- or C- termini of the human PXR (hPXR), that is, hPXR-p53 and p53-hPXR. C3A, a hepatoma cell line, was used as the cell model to test the regulation effect of chimeric hPXR over wild type (WT) hPXR on CYP3A4 expression at gene, protein, and metabolism levels, respectively. Compared with C3A cells transiently transfected with WT hPXR, the activity of CYP3A4.XREM.luc reporter gene in C3A cells transfected with hPXR-p53 or p53-hPXR increased 5- and 9-fold respectively, and the levels of CYP3A4 mRNA expression increased 3.5- and 2.6-fold, respectively. C3A cells stably transfected with hPXR-p53-AD exhibited an improved expression of CYP3A4 at both gene (2-fold) and protein (1.5-fold) levels compared to WT C3A cells. Testosterone, a CYP3A4-specific substrate, was used for detecting the metabolism activity of CYP3A4. No testosterone metabolite could be detected in microsomes from WT C3A cells and WT C3A cells-based array, while the formation of 6β-hydroxytestosterone metabolite in the transfected cells was 714 and 55 pmol/mg protein/min, respectively. In addition, all the above expression levels in the transfected cell models could be further induced with additional treatment of Rifampicin, a specific inducer for CYP3A4. In conclusion, our study established a proof-of-principle example that genetic modification with chimeric hPXR-p53-AD could improve CYP3A4 metabolism ability in hepatic cell line.
Highlights
Cytochromes P450 (P450s or CYPs) are a heme-thiolate monooxygenases that play an important role in the metabolism of drugs
CYP3A4 has a wide spectrum of metabolism substrates; its importance in drug metabolism is highlighted by the fact that it contributes to the metabolism of approximately 60% of marketed drugs [3]
The activation domain (AD) of p65 subunit of nuclear factor kB (p65-AD) has been applied to form a nuclear receptor-based regulator for in vitro cell model for drug and xenobiotic metabolism [22]. Based on these previous reports, we proposed that trans-activation effect of human PXR (hPXR) may be enhanced by fusion with a heterologous AD, and subsequently, the ability of these ‘‘chimeric hPXR’’ to activate CYP3A4 gene in hepatoma cells would be higher as compared to native hPXR; chimeric hPXRs might be effective in producing hepatoma cells with an increased expression of CYP3A4
Summary
Cytochromes P450 (P450s or CYPs) are a heme-thiolate monooxygenases that play an important role in the metabolism of drugs. Human CYP3A family consists of the subtypes CYP3A4, CYP3A5, CYP3A7, and CYP3A43 [1] These enzymes are ample in human liver, and CYP3A4 is the most important and abundant one [2]. In cell models for drug testing, primary human hepatocytes remain the standard method, even though they have well-known limitations including poor availability, batch-to-batch variability, non-proliferation in culture and severe phenotypic function dropoff, such as the rapid loss of CYPs activity, whatever systems or conditions are taken for in vitro culture [4,5,6]. Hepatoma cell lines are used with evident advantages with respect to their availability and relatively stable phenotype between appropriate generations; they express CYP enzymes at much lower levels compared to their primary counterpart [7]. Different strategies to up-regulate expression level of drug-metabolizing enzymes have been used with aim to generate primary hepatocyte-mimicing systems. The improved expression level of CYP genes initiated by these strategies only begins to approach that of primary hepatocytes, which are themselves significantly lower than fresh tissue [12]
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