Effect of inflammation on the expression level of NRF-2, MDR-1, AND TET-2 in an in vitro model of inflammatory bowel disease

Abstract

Background and purpose: Inflammatory Bowel Disease (IBD) represents a group of complex diseases that are characterized by chronic inflammation of the gastrointestinal (GI) tract, attributed to cytokine storm and infiltrating immune cells. As part of the cell defensive mechanisms, a transcription factor called nuclear related factor 2 (NRF-2) activates genes encoding antioxidant enzymes. Additionally, NRF-2 has recently appeared as an important factor contributing to chemo-resistance in tumors including colon neoplasms, by upregulating a transmembrane protein known as P -glycoprotein or multi-drug resistant protein 1 (MDR-1). In addition, epigenetic factors have been shown to influence the onset of IBD. One of these epigenetic factors is the interplay between DNA hyper methylation and hypo methylation in regulating gene activity by two distinct classes of enzymes: DNA methyl transferases and DNA demethylation enzymes, coded by the ten- to-eleven translocation [Tet], yielding the methyl cytosine dioxygenases TET1, TET-2 and TET3. Recent studies have demonstrated a potential association between TET-2 and NRF-2 in IBD- associated colon cancer. This study aims to understand the alteration in the expression and activity of these markers under inflammatory conditions.Methods: NRF-2, MDR-1, and TET-2 expression level was assessed in HT-29 cells and in HT- 29shTET2 expressing low levels of TET 2; quantitative real-time polymerase chain reaction (qPCR) was performed in this study at transcriptional level while Western blotting and immunofluorescence attranslational level. Additionally, epithelial-to mesenchymal transition markers (EMT, E-cadherin and N- cadherin) were also evaluated by qPCR and Western blotting. For the production of inflammatory media, the human monocytic cell line (THP-1) is used.Results: Under inflammatory conditions, NRF-2, MDR-1, and TET-2 expression levels showed a remarkable increase. Moreover, NRF-2 and MDR-1 expression levels were downregulated in HT 29shTET-2 cells. Interestingly, N-cadherin levels were higher in HT-29shTET-2 exposed to inflammatory media compared to parental HT-29. Conclusion: NRF-2 and MDR-1 expression levels increase upon exposure of HT 29 cells to inflammation. On the other hand, their expression is downregulated when TET 2 is downregulated, suggesting the demethylation role of TET-2 in activating NRF-2 and MDR-1 gene expression.