Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 found no marked difference in the levels of the PXR (exon 5-6) and PXR (exon 1a-2) transcripts in the colon cancer cells. Therefore, a tissue-specific DNA methyla- tion profile is most likely involved in the transcriptional regulation of the PXR gene. DNA methylation of the PXR promoter was detected in only 1 of the 18 colorectal cancer tissue samples. The results reflect the genuine DNA methylation status, because we examined pure cancerous and normal epithelia using crypt isolation and directly compared the DNA methylation status between paired epithelia. Therefore, a low level of PXR promoter methylation, which was observed in the high expression cells, appears to be a common feature of colorectal cancers. We also demonstrated that the level of PXR promoter methyla- tion is decreased during carcinogenesis, since paired adjacent normal tissues mostly showed higher levels of PXR promoter methylation. We could not directly com- pare DNA methylation status with the PXR mRNA expression, because crypt isolation provided ethanol- fixed epithelia and it was difficult to obtain fresh mRNA samples. However, most cancer tissues exhibited a pat- tern of promoter methylation quite similar to that observed in cultured cells with high expression (LS180 and LoVo) (Figures 5 and 6c). Therefore, the association between promoter methylation and transcriptional silen- cing of the PXR gene is most likely applicable to pri- mary colorectal cancers. As observed in the colon cancer cell lines, the decreased level of PXR promoter methylation most likely led to increased expression of PXR mRNA in the colorectal cancer tissues. These results are consistent with a recent study that showed strong expression of PXR mRNA in colon cancers, with great variability [24]. In contrast, Ouyang et al. found that PXR expression was lost or greatly diminished in many colon cancers using histochemical analysis [25]. Although the role of the altered PXR expression in col- orectal carcinogenesis remains to be clarified, Zhou et al. demonstrated that PXR plays an antiapoptotic role in colon carcinogenesis by induction of multiple antia- poptotic genes [26]. We cannot rule out the possibility that alterations of the PXR methylation levels play direct roles in tumori- genesis, because certain oncogenes or tumor suppressor genes may be trascriptionally regulated by PXR. Partial methylation of the PXR observed in adjacent normal mucosa may be associated with “field defect” for carcino- genesis. However, numerous studies have demonstrated that ligand-binding activation or siRNA-mediated silen- cing of the PXR can affect the activity of metabolic enzymes including CYP3A4, without changes in the cell proliferation capacity. Therefore, we think that altered level of the PXR methylation does not provide a selective growth advantage during colorectal cancer progression. Page 9 of 10 Interestingly, overexpression of PXR in the colorectal cancer tissue samples was correlated with an increase in UDP glucuronosyl transferases UGT1A1, UGT1A9 and UGT1A10, and led to a marked chemoresistance to the active metabolite of irinotecan (CPT-11) [24]. In addi- tion, CYP3A4 and p-glycoprotein, which are transcrip- tionally activated by PXR, play important roles in intestinal first-pass metabolism and determine a drug’s bioavailability. We hypothesized that PXR may play a key role in the colon cancer cell response to anticancer drugs by modulating expression of drug metabolizing enzymes and transporters including UGT1A, CYP3A4 and p-glycoprotein. Therefore, DNA methylation of the PXR promoter might be a good predictor of chemother- apy outcome and toxicity in colorectal cancers. Conclusions PXR promoter methylation is involved in the regulation of intestinal PXR and CYP3A4 expression. This methy- lation might be associated with the inter-individual variability of the drug response of colon cancer cells. Abbreviations PXR: pregnane X receptor; CYPs: cytochrome P450s; VDR: vitamin D receptor; PRMT1: protein arginine methyltrasferase 1; 5-aza-dC: 5-aza-2’-deoxycytidine; COBRA: combined bisulfite restriction analysis. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) (21590402) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Author details 1Department of Pharmacodynamics and Molecular Genetics, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwa- Gun 028-3694, Japan. 2Division of Molecular Diagnostic Pathology, Department of Pathology, School of Medicine, Iwate Medical University, Iwate Medical University, 19-1 Uchimaru, Morioka 020-8505, Japan. 3Department of Surgery, School of Medicine, Iwate Medical University, Iwate. Authors’ contributions WH designed this study and carried out the cell culture, molecular genetic studies and drafted the manuscript. GT and JT participated in the data analysis. TS performed crypt isolation and pathological diagnosis. KO and GW performed the surgeries and obtained informed consent from the patients. SO participated in the design of this study and helped to draft the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Received: 26 July 2010 Accepted: 22 February 2011 Published: 22 February 2011 References 1. Zhang B, Xie W, Krasowski MD: PXR: a xenobiotic receptor of diverse function implicated in pharmacogenetics. Pharmacogenomics 2008, 9:1695-1709. 2. Zhou C, Verma S, Blumberg B: The steroid and xenobiotic receptor (SXR), beyond xenobiotic metabolism. Nucl Recept Signal 2009, 7:e001. 3. 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