Promoter demethylation of nuclear factor-erythroid 2-related factor 2 gene in drug-resistant colon cancer cells

Abstract

The mechanisms underlying drug resistance in colorectal cancer (CRC) treatment remain to be fully elucidated. Therefore, the present study aimed to investigate the underlying mechanism resistance to a widely used anticancer drug, 5-Fluorouracil (5-FU). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important transcription factor involved in cellular protection. In the present study, it was hypothesized that the epigenetic modification of Nrf2 may be a potential target for 5-FU resistance in CRC treatment. Protein and messenger RNA levels of Nrf2, heme oxygenase-1 (HO-1), DNA methylases and DNA methyltransferases were determined and DNA methylation analysis for the Nrf2 promoter was performed in a human CRC control (SNU-C5) and resistant (SNU-C5R) cell line. The results demonstrated that Nrf2 expression levels, nuclear translocation and promoter binding were significantly increased in SNU-C5R cells compared with SNU-C5 cells. Elevated levels of activated Nrf2 in SNU-C5R cells resulted in the increased protein expression and activity of HO-1. In addition, increased production of reactive oxygen species (ROS) and upregulation of ten-eleven translocation (TET)1 were observed in SNU-C5R cells compared with SNU-C5 cells. Furthermore, methylation analysis revealed Nrf2 promoter cytosine-phosphate-guanine island hypomethylation in 5-FU-treated cells. In conclusion, the results indicated that 5-FU-induced ROS production resulted in the upregulation of TET1 expression and function. In addition, these results indicated that TET-dependent demethylation of the Nrf2 promoter upregulated Nrf2 and HO-1 expression, which induced cellular protection mechanisms, ultimately leading to drug resistance.

Keywords: 5-fluorouracil; SNU-C5 cells; colorectal cancer; cytosine-phosphate-guanine islands; epigenetics.

Figures

Figure 1.

Chemosensitivity of 5-FU-resistant human SNU-C5R colorectal cancer cells. SNU-C5 and SNU-C5R cells were exposed to the indicated concentration of 5-FU for 72 h. An MTT assay was then used to determine the cell viability of the two cell lines. Values are presented as the mean ± standard error of the mean for three independent experiments. *P

Figure 2.

Intracellular ROS levels in SNU-C5 and 5-fluorouracil-resistant SNU-C5R cells. (A) ROS levels were detected by flow cytometric analysis and (B) fluorescent microscopic imaging was performed following H2-dichlorodihydrofluorescein diacetate staining. Values are presented as the mean ± standard error of the mean for three independent experiments. *P<0.05 vs. SNU-C5 cells. ROS, reactive oxygen species.

Figure 3.

Antioxidant and DNA methylation-associated protein levels in SNU-C5 and 5-fluorouracil-resistant SNU-C5R cells. (A) Protein blots of antioxidants Nrf2 and HO-1. (B) Protein blots of DNA methylases DNMT1, DNMT3A and DNMT3B as well as DNA demethylases TET1, TET2 and TET3. β-actin and TBP were used as loading controls. Nrf2, nuclear factor-erythroid 2-related factor 2; HO-1, heme oxygenase-1; DNMT. DNA methyltransferase; TET, ten-eleven translocation enzyme; TBP, TATA box binding protein.

Figure 4.

Status of Nrf2 promoter DNA methylation and Nrf2 genes in SNU-C5 and 5-fluorouracil-resistant SNU-C5R cells. Bisulphite genomic DNA sequencing of SNU-C5 cells showing (A) average methylated CpG dinucleotides and (B) highly demethylated CpG dinucleotides in the region between −423 and −126 of Nrf2 promoter. A total of 11 individual clones of the bisulfite-converted DNA sequences were analyzed for DNA methylation, containing 20 CpG dinucleotides, of the Nrf2 promoter by Bisulfite Sequencing DNA Methylation Analysis software using default filtering threshold settings. Columns, CpG dinucleotide sites; red squares, methylated CpG dinucleotides; blue squares, unmethylated CpG dinucleotides. Color gradient bar indicates that red regions contain more methylated CpG dinucleotides and blue regions contain more unmethylated CpG dinucleotides. (C) Quantification of the percentage of Nrf2 demethylation. (D) Reverse transcription quantitative polymerase chain reaction analyses of SNU-C5R cells showing the increased expressions of Nrf2 messenger RNAs. Values are presented as the mean ± standard error of the mean for three independent experiments. *P