Chen Feng, Rao Xiao-Hui, Yang Jin-Lian, Pan Ming-Xing, Gao Yi, Li Zhen-Lin, Li Yang, Zhu You-Fu, Wang Yan
Institute of Regenerative Medicine, Southern Medical University Zhujiang Hospital, Guangzhou, China.
Department of Hepatobiliary Surgery, Huizhou Municipal Central Hospital, Huizhou, China.
PLoS One. 2014 May 1;9(5):e95752. doi: 10.1371/journal.pone.0095752. eCollection 2014.
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.
大多数肝癌细胞系缺乏细胞色素P450 3A4（CYP3A4）酶的正常表达和诱导，这限制了它们在预测药物代谢和毒性方面的应用。核受体孕烷X受体（PXR）因其在调节CYP3A4基因表达中的关键作用而被广泛认可。然而，其与启动子特定位点结合的生理活性在肝癌细胞系中显著减弱。为了解决这个问题，我们通过将来自p53亚基的强激活域（AD）附加到人PXR（hPXR）的N端或C端来构建“嵌合PXR”构建体，即hPXR-p53和p53-hPXR。肝癌细胞系C3A被用作细胞模型，分别在基因、蛋白质和代谢水平上测试嵌合hPXR对野生型（WT）hPXR调节CYP3A4表达的影响。与瞬时转染WT hPXR的C3A细胞相比，转染hPXR-p53或p53-hPXR的C3A细胞中CYP3A4.XREM.luc报告基因的活性分别增加了5倍和9倍，CYP3A4 mRNA表达水平分别增加了3.5倍和2.6倍。与WT C3A细胞相比，稳定转染hPXR-p53-AD的C3A细胞在基因（2倍）和蛋白质（1.5倍）水平上均表现出CYP3A4表达的改善。睾酮是一种CYP3A4特异性底物，用于检测CYP3A4的代谢活性。在WT C3A细胞和基于WT C3A细胞的阵列的微粒体中未检测到睾酮代谢物，而在转染细胞中6β-羟基睾酮代谢物的形成分别为714和55 pmol/mg蛋白质/分钟。此外，用CYP3A4的特异性诱导剂利福平进一步处理后，转染细胞模型中的所有上述表达水平均可被进一步诱导。总之，我们的研究建立了一个原理验证实例，即嵌合hPXR-p53-AD的基因修饰可以提高肝癌细胞系中CYP3A4的代谢能力。
