The human SNU-C5 CRC cell line and the 5-FU-resistant SNU-C5R cell line were purchased from the cell bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a 5% CO₂ incubator. A total of 1×10⁴ SNU-C5R cells/well were then subcultured at 37°C in 140 mM 5-FU twice per week for >6 months in order to establish stable drug-resistant cell lines (26).

Cell viability was assessed using a colorimetric assay with the tetrazolium salt, MTT. Transfection with short interference (si)RNA was performed using the JetSI Transfection Reagent for siRNA (Polyplus-Transfection, Illkirch, France) at 50 nM, according to the manufacturer's instructions. After 24 h of transfection with siRNA, the cells were exposed to different concentrations of 5-FU (0, 10 or 100 µM; Sigma-Aldrich, St. Louis, MO, USA) for 72 h at room temperature. MTT (0.5 mg/ml; Sigma-Aldrich) was then added to each well and incubated for 3 h at room temperature. Plates were centrifuged at 4,500 × g for 5 min at room temperature, the medium was then removed and 100 µl acidic isopropanol (40 mM) was added to solubilize the crystals. The absorbance was measured at 570 mm using a microplate reader (Victor 3; Perkin Elmer, Turku, Finland).

Cells (3×10⁵/well) were seeded onto six-well plates and administered 25 mM dichlorodihydrofluorescein diacetate (DCF-DA; 30 µl). Next, 2,7-dichlorofluorescein (DCF) fluorescence levels were then detected using a flow cytometer (BD FACSCanto™; BD Bioscience, Franklin Lakes, NJ, USA). CellQuest software (version 6.0; BD Biosciences) was used to analyze the flow cytometry results. In order to evaluate the production of intracellular ROS, image analysis was performed through seeding cells (2×10⁵/well) onto a coverslip-loaded six-well plate. Cells were then stained with 1 µM H₂-DCFH-DA (Invitrogen Life Technologies) in phosphate-buffered saline (PBS) for 30 min at room temperature. Cells were then washed twice with PBS and visualized using an Eclipse TE2000-U fluorescent microscope (Nikon Corp., Tokyo, Japan) using a green filter (450–490 nm).

Radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) was used to lyse cells. A total of 10–20 µg/µl soluble proteins were separated using 12% SDS-PAGE and then blotted onto polyvinylidene fluoride membranes. Membranes were then blocked using 5% non-fat dry milk for 1 h at room temperature prior to incubation overnight at 4°C with the following primary antibodies for: Heme oxygenase-1 (HO-1; cat. no. sc-10789), Nrf2 (cat. no. sc-722;), TET1 (cat. no. HPA019032), TET2 (cat. no. sc-136926), TET3 (cat. no. sc-139186), DNMT3B (cat. no. sc-130740) and β-actin (cat. no. sc-130657), which were all rabbit polyclonal IgG antibodies, used at a dilution of 1:200 and purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), with the exception of TET1 that was obtained from Sigma-Aldrich. In addition, DNMT1 (cat. no. ab19905; rabbit polyclonal IgG), phospho-Nrf2 (cat. no. ab76026; rabbit monoclonal IgG), TATA box binding protein (TBP; cat. no. ab52701; mouse polyclonal IgG) and DNMT3A (cat. no. ab23565; mouse polyclonal IgG) were purchased from Abcam (Cambridge, MA, USA) and used at a dilution of 1:200. Membranes were washed with PBS and then incubated with secondary antibodies (goat anti-rabbit polyclonal IgG; cat. no. SAB3700843; dilution, 1:500; Sigma-Aldrich) for 1 h at room temperature. Membranes were incubated with luminol reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA) and the bands were visualized using a Bio-Rad XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total RNA was isolated from SNU-C5 and SNU-C5R cells using Quick-RNA™MicroPrep solution (Zymo Research Corp., Irvine, CA, USA). iScript™ Reverse Transcription Supermix for real-time PCR (Bio-Rad Laboratories, Inc.) was used to reverse transcribe column purified total RNA, which was analyzed using a MiniOpticon™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) using SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories, Inc.) as previously described (27). ProbeFinder software (Roche, Basel, Switzerland) was used to design the primers, which were then commercially synthesized by Shanghai Generay Biotech Co. (Shanghai, China; http://www.generay.com.cn/). The primers used were as follows: Nrf2 forward, 5′-GAGAGCCCAGTCTTCATTGC-3′, and reverse, 5′-TTGGCTTCTGGACTTGGAAC-3′; GAPDH forward, 5′-AACGTGTCAGTGGTGGACCTG-3′, and reverse, 5′-AGTGGGTGTCGCTGTTGAAGT-3′. The PCR conditions were as follows: denaturation program (95°C for 10 min), followed by an amplification and quantification program repeated 40 times (95°C for 15 sec, 60°C for 10 sec, 72°C for 60 sec). Agarose gel electrophoresis (2%; Sigma-Aldrich) was then used to visualize the PCR products of the primers, which were verified by DNA sequencing that was performed by Shanghai Generay Biotech Co. All reactions were performed in triplicate and three independent experiments were run. Serial dilutions of a reference sample were used to construct standard curves for determining the individual PCR amplification efficiencies; this was included in each quantitative run in order to correct for variations in product amplification. Standard curves were used to obtain relative copy numbers, which were normalized to the values obtained for Gapdh, the internal control. CFX manager 3.1 software (Bio-Rad Laboratories, Inc.) was employed for data acquisition, analysis and determining PCR efficiencies.

The genomic DNA of SNU-C5 and SNU-C5R cells was subjected to bisulfite conversion using an EZ DNA Methylation-Direct™ kit (Zymo Research Corp.). Amplification of the bisulfite-modified DNA was then performed through bisulfite sequencing PCR (Bio-Rad T100; Bio-Rad Laboratories, Inc.) using Platinum® PCR SuperMix High Fidelity (Invitrogen Life Technologies) with human Nrf2 promoter-specific primers: Nrf2 forward, 5′-TGAGATATTTTGCACATCCGATA-3′ and reverse, 5′-ACTCTCAGGGTTCCTTTACACG-3′. Subsequently, the PCR amplification products were purified through gel extraction with Zymoclean™ Gel DNA recovery kit (Zymo Research Corp.). A TOPO TA Cloning® kit (Invitrogen Life Technologies) was then used to clone the purified products into pCR®4-TOPO vectors. The recombinant plasmids were transformed into One Shot® TOP10 chemically competent E. coli (Invitrogen Life Technologies) using the calcium chloride transformation method (28). The plasmid DNA of ~10 independent clones of each amplicon was isolated using a PureLink™Quick PlasmidMiniprep kit (Invitrogen Life Technologies) and then sequenced in order to determine the cytosine-phosphate-guanine methylation status. Clones with an insert with N 99.5% bisulfite conversion, i.e. non-methylated cytosine residues to thymine, were included in the present study and the remaining were excluded. Bisulfite Sequencing DNA Methylation Analysis software (http://biochem.jacobs-university.de/BDPC/BISMA/) was then used to analyze the sequenced data of each clone for DNA methylation in the Nrf2 promoter, using default filtering threshold settings.

All statistical analyses were performed with the SPSS 20.0 software (IBM SPSS, Armonk, NY, USA). Quantitative data are expressed as the mean ± standard deviation. The statistically significant differences between patient and control groups were tested using t-test and ANOVA test. P<0.05 was considered to indicate a statistically significant difference.

The relative chemosensitivity of the SNU-C5R 5-FU-resistant cell line was confirmed using an MTT cell viability assay. As shown in Fig. 1, 5-FU treatment for 72 h resulted in a dose-dependent suppression of cell growth in SNU-C5 cells, which was significantly decreased compared with that of the SNI-C5R cells (Fig. 1). The 5-FU concentrations causing a 50% growth inhibition as compared with control cells (IC₅₀) were calculated by a modified Kärbers method (29). The IC₅₀ value for 5-FU in SNU-C5R cells was 118.7±4.9 µM, whereas the corresponding value for their parental SNU-C5 cells was 23.2±3.4 µM (P<0.05).

Flow cytometric analysis data revealed that ROS levels were significantly elevated in SNU-C5R cells [fluorescence intensity (FI), 340] compared with SNU-C5 cells (FI, 130; P<0.05) (Fig. 2A). Fluorescent microscopic imaging confirmed these results, as it demonstrated that the ROS green FI was markedly enhanced in SNU-C5R cells compared with SNU-C5 cells (Fig. 2B). This therefore suggested that SNU-C5R cells were exposed to an increased level of oxidative stress conditions compared with SNU-C5 cells.

HO-1 and Nrf2 protein levels were observed to be increased in SNU-C5R cells compared with SNU-C5 cells (Fig. 3A). In addition, the expression levels of the epigenetic modification-associated proteins, in terms of those for DNA methylation, was investigated by assessing the protein levels of DNMTs DNMT1, DNMT3A and DNMT3B as well as the DNA demethylases, including TET1, TET2 and TET3. The results revealed that DNMT expression was not significantly different between SNU-C5 and SNU-C5R cells, whereas TET expression was elevated in SNU-CR cells compared with SNU-C5 cells (Fig. 3B).

The bisulphate DNA sequencing of the Nrf2 promoter region revealed significant demethylation in SNU-C5R cells compared with that of SNU-C5 cells (P<0.05) (Fig. 4A and B). The overall percentage of Nrf2 promoter demethylation was ~40% in SNU-C5 cells and ~70% in SNU-C5R cells (P<0.05) (Fig. 4C). In addition, messenger (m)RNA levels of Nrf2 in SNU-C5R cells exhibited a 3.5-fold increase, which was significantly increased compared with the 1.5-fold increase observed in SNU-C5 cells (P<0.05); increases were compared to that of the control house keeping gene (Fig. 4D).