Primary mouse monoclonal anti-GSTP-1, anti-DNMT1, anti-MBD2 antibodies and primary rabbit polyclonal anti-histone H3 (tri-methyl K4) and anti-histone H3 (tri-methyl K9) antibodies were purchased from Abcam, Inc. (Cambridge, MA, USA). Primary rabbit anti-acetyl-histone H3 (K9) and anti-c-Jun antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Primary mouse monoclonal anti-HDAC1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide (MTT) and primary rabbit anti-β-actin antibody were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade.

The biospecimens and data used in this study were provided by the Biobank of Jeju National University Hospital, a member of Korea Biobank Network (Jeju, Korea) (A03-2). This study was approved by the institutional review board for ethics of Jeju National University Hospital (IRB 2011-38), and informed written consent was obtained from all the patients.

The human colon cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 were obtained from the Korean Cell Line Bank (Seoul, Korea), and the normal human colon cell line FHC was purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained at 37°C in an incubator with a humidified atmosphere containing 5% CO. HCT-116, HT-29, SNU-407 and SNU-1033 cells were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin (100 U/ml). Caco-2 cells were cultured in MEM medium containing 10% heat-inactivated FBS, streptomycin (100 μg/ml) and penicillin (100 U/ml). FHC cells were cultured in a 1:1 mixture of Ham’s F12 and DMEM containing HEPES (25 mM), cholera toxin (10 ng/ml; Calbiochem-Novabiochem Corp., La Jolla, CA, USA), insulin (5 μg/ml), transferrin (5 μg/ml), hydrocortisone (100 ng/ml) and 10% FBS.

Two samples were pooled from 10 colon cancer and normal tissue, respectively. The extracted proteins from tissues were digested using modified gel-assisted digestion protocol (30). The gel pieces containing proteins were reduced and alkylated. Proteolytic digestion was conducted with 100 ng/μl trypsin and incubated at 37°C overnight. The peptides were extracted from the gel. The nanoscale LC separation of tryptic peptides was performed by using a nanoAcquity system (Waters Corp., Milford, MA, USA), equipped with a Symmetry C18 5 μm, 5 mm × 300 μm precolumn and an BEH C18 1.7 μm, 25 cm × 75 μm analytical reversed phase column (Waters Corp.). The samples were initially transferred, with an aqueous 0.1% formic acid solution, to the precolumn at a flow rate of 10 μl/min for 3 min. The mobile phase A was composed of water with 0.1% formic acid, and the mobile phase B was composed of 0.1% formic acid in acetonitrile. The peptides were separated with a gradient of 3–40% mobile phase B over 180 min at a flow rate of 300 nl/min, followed by a 25 min rinse with 90% of mobile phase B. The analysis of tryptic peptides was performed using a Q-Tof Premier mass spectrometer (Waters Corp.). Tryptic peptide was detected within the retention time range of approximately 0.12 min and at mass precision of approximately 3.1 ppm from three independent runs. Accurate mass LC-MS data were collected in an alternating, low energy and elevated energy mode of acquisition (31). Continuum LC-MS data were processed for generating peaklist and searched, using ProteinLynx GlobalServer v2.3 IdentityE (Waters Corp.). All MS/MS data were searched against the IPI human protein database (version 3.36; containing 69,012 sequences and 29,002,682 residues) using the Proteinlynx Global Server v2.3 IdentityE software. All database searches allowed for fixed modification in regard to the cysteine residue and variable modification in regard to the methionine. The relative quantitation was performed using the precursor intensity measurements available in the clustered exact mass and retention time. The redundant quantitative measurements provided by the multiple tryptic peptides from each protein were used to determine an average relative fold-change. A 95% confidence interval was determined for each average fold-change from the standard deviation of the observed quantitative measurements and the total number of observed tryptic peptides.

Cells were lysed on ice for 30 min in 100 μl lysis buffer [120 mM NaCl, 40 mM Tris (pH 8.0), 0.1% NP-40] and centrifuged at 13,000 × g for 15 min. Supernatants were collected and the protein concentrations were determined. Aliquots containing 40 μg of protein were boiled for 5 min and electrophoresed on 10% SDS-polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes, which were subsequently incubated with anti-GSTP-1 antibody overnight at 4°C. The membranes were further incubated with secondary horseradish peroxidase-conjugated anti-immunoglobulin-G (Pierce, Rockford, IL, USA). Protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK).

For detection of GSTP1 in tissue specimens, tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. The same paraffin-embedded tissues as those used for the original hematoxylin and eosin stained sections were used for immunohistochemistry. Tissue blocks were cut into 3 μm thick slices and mounted on SuperFrost^® Plus coated slides. Sections were then deparaffinized in xylene and rehydrated through a graded ethanol series. A standard immunohistochemical technique was performed using a Ventana BenchMark XT immunostainer with the anti-GSTP-1 antibody at a dilution of 1:100. Antigen retrieval on the immunostainer was carried out for 30 min. The anti-GSTP-1 antibody was incubated at 37°C for 60 min, and 3,3′-diaminobenzidine was used as a chromogen; slides were counterstained with hematoxylin prior to mounting. All staining procedures were performed according to the manufacturer’s recommendations. Intramucosal mononuclear cells in the tissue samples, which stain strongly, served as internal positive controls. Negative controls for non-specific binding were obtained by omitting the primary antibody. The immunohistochemical slides were evaluated and interpreted by a pathologist. For detection of GSTP-1 in cells, cells plated on coverslips were fixed with 4% paraformaldehyde for 30 min and permeabilized with PBS containing 0.1% Triton X-100 for 2.5 min. Cells were then treated with blocking solution (PBS containing 3% bovine serum albumin) for 1 h, and then incubated for 2 h with anti-GSTP-1 antibody diluted in blocking solution. Immunoreacted primary anti-GSTP-1 antibody was detected by incubating samples for 1 h with a FITC-conjugated secondary antibody at a dilution of 1:200 (Jackson Immuno Research Laboratories, West Grove, PA, USA). After washing with PBS, stained cells were mounted onto microscope slides in mounting medium. Microscopic images were collected on a confocal microscope using the Laser Scanning Microscope 5 PASCAL software (Carl Zeiss, Jena, Germany).

GST enzyme activity in colon tissues or cells was measured using the GST activity assay kit (Abcam, Inc.). The GST substrate 1-chloro-2,4-dinitrobenzene (CDNB) was used to assess GST-related enzyme activity. Briefly, colon tissues or cells were homogenized in 0.5 ml GST assay buffer, and homogenates were centrifuged at 10,000 × g for 15 min at 4°C. The supernatants were collected and the protein concentrations were determined. Samples were prepared with GST assay buffer in a total volume of 50 μl, including a negative control (50 μl GST assay buffer alone) and a positive control (10 μl of GST positive control diluted 1:50 and 40 μl GST assay buffer). To each well containing sample or control, 5 μl of glutathione was added, followed by 50 μl of substrate mixture (45 μl of GST assay buffer and 5 μl of CDNB). After addition of the substrate mixture, plates were shaken carefully to start the reaction, and the absorbance at 340 nm was read once every minute to obtain at least 5 time points. The change in absorbance (ΔA) per minute was determined by plotting the absorbance values as a function of time to obtain the slope of the linear portion of the curve. The reaction rate was then determined using the CDNB extinction coefficient at 340 nm, 0.0053 μM⁻¹. GST activity is expressed as nmol/min/ml.

Total RNA was isolated from cells using the easy-BLUE™ total RNA extraction kit (iNtRON Biotechnology, Inc., Korea), and cDNA was amplified using 1 μl of the reverse transcription reaction buffer, primers, dNTPs and 0.5 U of Taq DNA polymerase in a final volume of 25 μl. The PCR conditions were: initial denaturation for 5 min at 94°C; 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec; and a final elongation step at 72°C for 7 min. The following primers were used to amplify GSTP1 cDNA: forward, 5′-ATGCCGCCCTACACCGTG-3′; reverse, 5′-CCAGGTGACGCAGGATGG-3′. The amplified products were resolved by electrophoresis on a 1% agarose gel, stained with RedSafe™ nucleic acid staining solution (iNtRON Biotechnology, Inc.) and photographed under UV light using the Image Quant™ TL analysis software (Amersham Bioscience, Sweden).

DNA bisulfate modification was carried out using the Methylamp™ DNA modification kit (Epigentek, Pittsburgh, PA, USA), according to the manufacturer’s instructions. To analyze GSTP-1 DNA methylation, MS-PCR was performed using an EpiTect MSP kit (Qiagen, Valencia, CA, USA). Primers to perform MS-PCR on the GSTP-1 promoter were designed using the Methyl Primer Express^® software v1.0 (Applied Biosystems). The methylated and unmethylated GSTP1 primer sets used for PCR were as follows: unmethylated GSTP-1 forward, 5′-GGAGTTTTGTTGTTGTAGTTTTT-3′, and reverse, 5′-CCACTAACAACACTAAAAACATC-3′; methylated GSTP-1 forward, 5′-GTTTCGTCGTCGTAGTTTTC-3′, and reverse, 5′-CCGCTAACGACACTAAAAAC-3′. The amplified products were resolved by electrophoresis on a 3% agarose gel, stained with RedSafe nucleic acid staining solution and photographed under UV light using Image Quant™ TL analysis software.

Cells were processed using the Simple ChIP™ enzymatic chromatin IP kit (Cell Signaling Technology, Inc.), according to the manufacturer’s instructions. Briefly, the procedure was initiated by cross-linking the proteins to DNA by adding 1% formaldehyde to the culture dishes and rocking on a platform for 10 min at room temperature. The cross-linking was stopped by the addition of glycine solution. Cells were washed twice with ice-cold PBS, pelleted by centrifugation and resuspended in 1 ml cell lysis buffer containing protease inhibitor. The soluble chromatin was sheared by sonication followed by centrifugation at 15,000 × g. Diluted supernatants were pre-cleared and blocked with protein A/G agarose, and the sonicated chromatin-DNA complex was precipitated overnight with the antibodies of interest. Bound DNA was eluted by incubating the beads in elution buffer, purified and amplified using primers flanking the SP-1 and AP-1 binding sites within the human GSTP1 proximal promoter. The oligonucleotide containing the transcription factor binding site in the GSTP-1 promoter was obtained from Bioneer (Seoul, Korea). The ChIP procedure was analyzed by PCR using human GSTP-1 promoter-specific primers as follows (32): forward, 5′-GCAGCGGTCTTAGGGAATTT-3′; reverse, 5′-CTTTCCCTCTTTCCCAGGTC-3′. The cycle parameters were as follows: 95°C for 5 min; 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec; and a final extension at 72°C for 7 min. The amplified products were resolved by electrophoresis on a 3% agarose gel, stained with RedSafe nucleic acid staining solution and photographed under UV light using Image Quant™ TL analysis software.

SNU-407 cells were seeded at a density of 1.5×10⁵ cells/well in 24-well plates and allowed to reach approximately 50% confluency on the day of transfection. The siRNA constructs were mismatched siRNA Control and siRNA GSTP-1 (Santa Cruz Biotechnology). Cells were transfected with 10–50 nM siRNA using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions. At 24 h after transfection, cells were treated with 5-fluorouracil (5-FU) for 48 h, and then examined by the MTT assay.

The effect of GSTP-1 on the viability of human colon cancer cells was determined using the MTT assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase in viable cells (33). Cells were treated with 5-FU at a concentration of 800 μM. MTT (50 μl of a 2 mg/ml stock solution) was added to each well to obtain a total reaction volume of 200 μl. After incubation for 4 h, the supernatants were aspirated. Formazan crystals present in each well were dissolved in 150 μl of dimethyl sulfoxide, and the absorbance at 540 nm was measured on a scanning multi-well spectrophotometer.

The results were subjected to analysis of variance using Tukey’s test to analyze differences. P<0.05 was considered statistically significant.

To compare the fold-changes of expressed proteins from normal and colon carcinoma tissues, shotgun proteomic approach based on label-free quantification was applied. A total of 403 non-redundant proteins were identified from the analysis of normal and colon carcinoma tissues. Among them, 146 proteins were identified in common. The numbers of unique proteins were 135 and 122 proteins in normal and cancer tissue, respectively (Fig. 1A). To show disease-specific proteins, we listed up some proteins based on score of protein recovery quantification which is proportional to accuracy of quantification (Fig. 1B). Among them, we focused on glutathione S-transferase which was overexpressed in the cancer group. To confirm this proteomic result, GSTP-1 protein level and enzyme activity were assessed in colon cancer tissues from 10 patients by western blotting and an enzyme activity assay, respectively. The expression level of GSTP-1 protein that showed >1.5-fold higher than in corresponding normal tissues was detected in 70% (7/10) of tumor tissues from colon cancer patients (Fig. 2A). Immunohistochemical analysis revealed faint expression of GSTP-1 protein in normal mucosa (Fig. 2Ba). The carcinoma cells exhibited increased GSTP-1 expression in the cytoplasm, which was distributed in a diffuse pattern (Fig. 2Bb–d), and some cells were strongly positive (Fig. 2Bd). Neuroendocrine carcinoma cells in specimens exhibited no GSTP-1 expression (Fig. 2Be). GST enzyme activity was higher in tumor tissues than in normal tissues from colon cancer patients, consistent with the western blotting data (Fig. 2C). These results demonstrate that GSTP-1 is highly expressed in tumor tissues from Korean colon cancer patients, suggesting that this protein might be used as a biomarker for cancer detection.

The assessment of GSTP-1 expression in colon cancer cell lines by RT-PCR and western blotting revealed that GSTP-1 mRNA (Fig. 3A) and protein levels (Fig. 3B) were higher in the colon cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 than in the normal colon cell line FHC. The protein expression and cytoplasmic localization of GSTP-1 was assessed by confocal microscopy (Fig. 3C), yielding data consistent with the western blotting results. GST enzyme activity was also higher in cancer cell lines, consistent with the pattern of GSTP-1 protein expression (Fig. 3D), although both GSTP-1 expression and activity levels differed among the cell lines.

We next investigated why the expression of GSTP-1 is higher in colorectal cancers than in normal tissues. To this end, the methylation status of GSTP-1 regulatory regions was determined by MS-PCR to elucidate the relationship between methylation status and protein expression. The methylation status of GSTP-1 in patient tissues was determined. While the relationship between methylation degree and protein expression in tissues was not close enough (Fig. 4A). Intriguingly, GSTP-1 was methylated to a lesser extent in the colon cancer cell lines Caco-2, HCT-116, SNU-407 and SNU-1033, but not HT-29 (Fig. 4B), than in the FHC cell line, almost concordant with GSTP-1 protein expression. These results suggest that overexpression of GSTP-1 in human colon cancers is only partially due to decreased methylation of the GSTP-1 regulatory region.

We next performed ChIP assays to investigate the epigenetic-related proteins associated with the GSTP-1 promoter region. The levels of DNA methylation- and heterochromatin-associated proteins such as DNMT1, MBD2 and HDAC1 were lower in the HCT-116 and SNU-407 cell lines than in the FHC cell line. In addition, the GSTP-1 promoter in HCT-116 and SNU-407 cells was highly enriched for trimethylation of lysine 4 of histone H3 (H3K4me3) and for acetylation of histone H3 (AcH3), which induce active transcription (Fig. 5A). Whereas the level of H3K9me3, which represses transcription, was lower in two cancer cell lines than in the FHC line (Fig. 5A). The GSTP-1 promoter region contains binding sites for the transcription factors SP-1 and AP-1. We performed ChIP assays to determine the roles of c-Jun (a component of AP-1) and SP-1 in the regulation of GSTP-1 expression. The c-Jun and SP-1 were associated with the GSTP-1 promoter region in the HCT-116 and SNU-407 cell lines (Fig. 5B). These data reveal that the upregulation of GSTP-1 in human colorectal cancers is probably due to reduced methylation of the GSTP-1 promoter and active form of histone modification and resulting in the increased activity of specific transcription factors.

To investigate whether GSTP-1 is involved in drug resistance, SNU-407 cells transfected with GSTP-1 siRNA were treated with the anticancer agent 5-FU, at the IC value of 800 μM, for 48 h. siRNA-mediated suppression of GSTP-1 expression increased the sensitivity of SNU-407 cells to 5-FU (Fig. 6). These results indicate that knockdown of GSTP-1 in SNU-407 cells increases sensitivity to the anticancer drug.