Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 R E S E A R C H A R T I C L E Open Access Involvement of promoter methylation in the regulation of Pregnane X receptor in colon cancer cells Wataru Habano1*, Toshie Gamo1, Jun Terashima1, Tamotsu Sugai2, Koki Otsuka3, Go Wakabayashi3, Shogo Ozawa1 Abstract Background: Pregnane X receptor (PXR) is a key transcription factor that regulates drug metabolizing enzymes such as cytochrome P450 (CYP) 3A4, and plays important roles in intestinal first-pass metabolism. Although there is a large inter-individual heterogeneity with intestinal CYP3A4 expression and activity, the mechanism driving these differences is not sufficiently explained by genetic variability of PXR or CYP3A4. We examined whether epigenetic mechanisms are involved in the regulation of PXR/CYP3A4 pathways in colon cancer cells. Methods: mRNA levels of PXR, CYP3A4 and vitamin D receptor (VDR) were evaluated by quantitative real-time PCR on 6 colon cancer cell lines (Caco-2, HT29, HCT116, SW48, LS180, and LoVo). DNA methylation status was also examined by bisulfite sequencing of the 6 cell lines and 18 colorectal cancer tissue samples. DNA methylation was reversed by the treatment of these cell lines with 5-aza-2’-deoxycytidine (5-aza-dC). Results: The 6 colon cancer cell lines were classified into two groups (high or low expression cells) based on the basal level of PXR/CYP3A4 mRNA. DNA methylation of the CpG-rich sequence of the PXR promoter was more densely detected in the low expression cells (Caco-2, HT29, HCT116, and SW48) than in the high expression cells (LS180 and LoVo). This methylation was reversed by treatment with 5-aza-dC, in association with re-expression of PXR and CYP3A4 mRNA, but not VDR mRNA. Therefore, PXR transcription was silenced by promoter methylation in the low expression cells, which most likely led to downregulation of CYP3A4 transactivation. Moreover, a lower level of PXR promoter methylation was observed in colorectal cancer tissues compared with adjacent normal mucosa, suggesting upregulation of the PXR/CYP3A4 mRNAs during carcinogenesis. Conclusions: PXR promoter methylation is involved in the regulation of intestinal PXR and CYP3A4 mRNA expression and might be associated with the inter-individual variability of the drug responses of colon cancer cells. Background Nuclear receptor families play a pivotal role in regulating genes involved in drug metabolism and disposition. Preg- nane X receptor (PXR, also termed SXR, PAR, and NR1I2 as its gene name) is a crucial regulator of various phase I and phase II drug metabolizing enzymes and drug transporters. PXR is expressed in liver, small intes- tine and other organs. PXR, with a number of therapeutic drugs and other xenobiotics as its ligands, dimerizes with retinoid X receptor a (RXRa). The ligand-PXR-RXRa * Correspondence: whabano@iwate-med.ac.jp 1Department of Pharmacodynamics and Molecular Genetics, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwa- Gun 028-3694, Japan Full list of author information is available at the end of the article complex binds to promoter and enhancer elements located upstream of cytochrome P450s (CYPs) 3A and 2C family members, UDP-glucuronosyltrans- ferases (UGTs), sulfotransferases (SULTs), glutathione S-transferases (GSTs), and ATP binding cassette (ABC) drug transporters (reviewed in [1-3]). Wide inter-individual variability has been documented in the expression of hepatic CYP3A4 with respect to basal and PXR-inducible activities. The genetic variability of PXR and CYP3A4 is not sufficiently frequent to explain the apparent inter-individual variability [4]. The inter-individual variability in basal hepatic CYP3A4 expression may include variability in PXR expression, as PXR is activated by endogenous steroid hormones and bile acids. Inter-individual variability of CYP3A4 © 2011 Habano et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.