Abstract
5-Fluorouracil (5-FU) is a widely used anticancer drug for the treatment of colorectal cancer (CRC). However, resistance to 5-FU often prevents the success of chemotherapy. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator and a possible target to overcome 5-FU resistance. The present study examined epigenetic changes associated with Nrf2 induction in a human CRC cell line (SNUC5) resistant to 5-FU (SNUC5/5-FUR). Nrf2 expression, nuclear translocation, and binding to promoter were higher in SNUC5/5-FUR cells than in SNUC5 cells. The activated Nrf2 in SNUC5/5-FUR cells led to an increase in the protein expression and activity of heme oxygenase-1 (HO-1), an Nrf2-regulated gene. SNUC5/5-FUR cells produced a larger amount of reactive oxygen species (ROS) than SNUC5 cells. The siRNA- or shRNA-mediated knockdown of Nrf2 or HO-1 significantly suppressed cancer cell viability and tumor growth in vitro and in vivo, resulting in enhanced 5-FU sensitivity. Methylation-specific (MS) or real-time quantitative MS-PCR data showed hypomethylation of the Nrf2 promoter CpG islands in SNUC5/5-FUR cells compared with SNUC5 cells. Expression of the DNA demethylase ten-eleven translocation (TET) was upregulated in SNUC5/5-FUR cells. ROS generated by 5-FU upregulated TET1 expression and function, whereas antioxidant had the opposite effect. These results suggested that the mechanism underlying the acquisition of 5-FU resistance in CRC involves the upregulation of Nrf2 and HO-1 expression via epigenetic modifications of DNA demethylation.
Highlights
Heme oxygenase-1 (HO-1) plays an important role in the maintenance of cellular redox homeostasis and prevents transformation of normal cells to precancer cancer cells by counteracting reactive oxygen species (ROS)-mediated carcinogenesis.[2]
DNMT1 expression is upregulated in tamoxifen-resistant breast cancer cells and it leads to aberrant methylation of the phosphatase and tensin homolog (PTEN) gene promoter, resulting in low expression of PTEN.[20]
This methylation process can be reversed by DNA demethylases, ten-eleven translocation enzymes (TETs): TET1, TET2, and TET3.22 These enzymes can convert 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), leading to cytosine.[22,23]
Summary
Sensitivity of SNUC5 and SNUC5/5-FUR cells to 5-FU. The cytotoxic effect of 5-FU in the SNUC5 cell line and its 5-FU-resistant variant, SNUC5/5-FUR, was compared. The concentration of 5-FU that yielded 50% growth inhibition (IC50) was 9 mM in SNUC5 cells and 2264 mM in SNUC5/5-FUR cells, in which resistance was induced by continuous culture in 140 mM 5-FU (Figure 1a) These results were confirmed by a colony formation assay; the colony numbers in both cell lines were decreased by IC50 concentration of 5-FU compared with the untreated group (Figure 1b). Flow cytometry data showed that ROS levels were higher in SNUC5/5-FUR cells (fluorescence intensity (FI) 342) than in SNUC5 cells (FI 132), and H2O2 treatment in SNUC5 cells (FI 640) was used as positive control (Figure 3a) These results were confirmed by confocal microscopic data; green FI generated by ROS was enhanced in SNUC5/5-FUR cells as compared with SNUC5 cells (Figure 3b). TET1 binding to the Nrf[2] promoter was
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