Aryl hydrocarbon receptor as a DNA methylation reader in the stress response pathway.

The aryl hydrocarbon receptor (AhR) mediates various cellular responses upon exposure to exogenous and endogenous stress factors. In these responses, AhR plays a dual role as a stress sensor for detecting various AhR ligands and as a transcription factor that upregulates the expression of downstream effector genes, such as those encoding drug-metabolizing enzymes. As a transcription factor, it selectively binds to the unmethylated form of a specific sequence called the xenobiotic responsive element (XRE). We suggest that AhR is a novel DNA methylation reader, unlike classical methylation readers, such as methyl-CpG-binding protein 2, which binds to methylated sequences. Under physiological conditions of continuous exposure to endogenous AhR ligands, such as kynurenine, methylation states of the individual target XREs must be strictly regulated to select and coordinate the expression of downstream genes responsible for maintaining homeostasis in the body. In contrast, long-term exposure to AhR ligands frequently leads to changes in the methylation patterns around the XRE sequence. These data indicate that AhR may contribute to the adaptive cellular response to various stresses by modulating DNA methylation. Thus, the DNA methylation profile of AhR target genes should be dynamically controlled through a balance between robustness and flexibility under both physiological and stress conditions. AhR is a pivotal player in the regulation of stress response as it shows versatility by functioning as a stress sensor, methylation reader, and putative methylation modulator.

Cellular irinotecan resistance in colorectal cancer and overcoming irinotecan refractoriness through various combination trials including DNA methyltransferase inhibitors: a review.

Treatment with pharmacological drugs for colorectal cancer (CRC) remains unsatisfactory. A major cause of failure in pharmacotherapy is the resistance of colon cancer cells to the drugs, creating an urgent issue. In this review, we summarize previous studies on the resistance of CRC cells to irinotecan and discuss possible reasons for refractoriness. Our review presents the following five major causes of irinotecan resistance in human CRC: (1) cellular irinotecan resistance is induced mainly through the increased expression of the drug efflux transporter, ABCG2; (2) cellular irinotecan resistance is also induced in association with a nuclear receptor, pregnane/steroid X receptor (PXR/SXR), which is enriched in the CYP3A4 gene enhancer region in CRC cells by exposing the cells to SN-38; (3) irinotecan-resistant cells possess either reduced DNA topoisomerase I (Top1) expression at both the mRNA and protein levels or Top1 missense mutations; (4) alterations in the tumor microenvironment lead to drug resistance through intercellular vesicle-mediated transmission of miRNAs; and (5) CRC stem cells are the most difficult targets to successfully treat CRC. In the clinical setting, CRC gradually develops resistance to initially effective irinotecan-based therapy. To solve this problem, several clinical trials, such as irinotecan plus cetuximab vs. cetuximab monotherapy, have been conducted. Another clinical trial on irinotecan plus guadecitabine, a DNA-methyltransferase inhibitor, has also been conducted.

ß-naphthoflavone-induced upregulation of CYP1B1 expression is mediated by the preferential binding of aryl hydrocarbon receptor to unmethylated xenobiotic responsive elements.

Human cytochrome P450 1 (CYP1) enzymes are transcriptionally induced by specific xenobiotics through a mechanism that involves the binding of aryl hydrocarbon receptors (AhR) to target xenobiotic responsive element (XRE) sequences. To examine the effect of DNA methylation on the AhR-mediated pathway, reverse transcription-quantitative PCR analysis was performed. ß-naphthoflavone (ßNF)-induced CYP1B1 expression was found to be potentiated by pre-treatment of human HepG2 liver cancer cells with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, but not HuH7 cells. It was hypothesized that this increase is mediated by the demethylation of CpG sites within XRE2/XRE3 sequences, suggesting that methylation of these sequences inhibits gene expression by interfering with the binding of AhR to the target sequences. To test this hypothesis, a novel method combining the modified chromatin immunoprecipitation of AhR-XRE complexes with subsequent DNA methylation analysis of the XRE regions targeted by activated AhR was applied to both liver cancer cell lines treated with ßNF. XRE2/XRE3 methylation was found to be exclusively observed in the input DNA from HepG2 cells but not in the precipitated AhR-bound DNA. Furthermore, sub-cloning and sequencing analysis revealed that the two XRE sites were unmethylated in the samples from the AhR-bound DNA even though the neighboring CpG sites were frequently methylated. To the best of our knowledge, the present study provides the first direct evidence that ligand-activated AhR preferentially binds to unmethylated XRE sequences in the context of natural chromatin. In addition, this approach can also be applied to assess the effects of DNA methylation on target sequence binding by transcription factors other than AhR.

Recent Progress in Prediction Systems for Drug-induced Liver Injury Using In vitro Cell Culture.

BACKGROUND: In order to avoid drug-induced liver injury (DILI), in vitro assays, which enable the assessment of both metabolic activation and immune reaction processes that ultimately result in DILI, are needed. OBJECTIVE: In this study, recent progress in the application of in vitro assays using cell culture systems is reviewed for potential DILI-causing drugs/xenobiotics and a mechanistic study on DILI, as well as on the limitations of in vitro cell culture systems for DILI research, was carried out. METHODS: Information related to DILI was collected through a literature search of the PubMed database. RESULTS: The initial biological event for the onset of DILI is the formation of cellular protein adducts after drugs have been metabolically activated by drug metabolizing enzymes. The damaged peptides derived from protein adducts lead to the activation of CD4+ helper T lymphocytes and recognition by CD8+ cytotoxic T lymphocytes, which destroy hepatocytes through immunological reactions. Because DILI is a major cause of drug attrition and drug withdrawal, numerous in vitro systems consisting of hepatocytes and immune/inflammatory cells or spheroids of human primary hepatocytes containing non-parenchymal cells have been developed. These cellular-based systems have identified DILI-inducing drugs, with approximately 50% sensitivity and 90% specificity. CONCLUSION: Different co-culture systems consisting of human hepatocyte-derived cells and other immune/inflammatory cells have enabled the identification of DILI-causing drugs and of the actual mechanisms of action.