Two human CRC cell lines, SW1116 and HT29, were used in this study and cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) and McCoy’s 5A medium (Sigma, St. Louis, MO, USA), respectively. Both media were supplemented with 10% foetal bovine serum and maintained at 37°C in a humidified 5% CO₂ atmosphere.

The DNA MTI, 5-aza-2′-deoxycytidine (5-aza-dc) (Sigma-Aldrich, St. Louis, MO, USA), was dissolved in acetic acid and stored at –20°C until used. Final concentrations of 5-aza-dc used throughout this study included 0, 1 and 5 μM, and 5-aza-dc in fresh medium was added to cell cultures daily to maintain the concentration needed. MG132, a pharmacological proteasome inhibitor, was dissolved in dimethyl sulfoxide and stored at –20°C. At the time of the experiment, an aliquot of MG132 was thawed and diluted with tissue culture media to a final concentration of 10 μM.

Quantitative PCR was performed using FastStart SYBR Green Master (Roche, Mannheim, Germany) in the Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The real-time PCR primers used for each gene are as follows: for SOCS1, 5′-GACCTGAACTCGCACCTCCTA-3′ (forward), 5′-CCCCCAACCCCTGGTTT-3′ (reverse); for SOCS3, 5′-GACCAGCGCCACTTCTTCTTCAC-3′ (forward), 5′-CTGGATGCGCAGGTTCTTG -3′ (reverse); for SHP1, 5′-CAGCACTTGGCTC CTTAGGAA-3′ (forward), 5′-CCAAACCAAGGAAGTCCAATG -3′ (reverse); for JAK2, 5′-GATGAGAATAGCCAAAGAAAACG-3′ (forward), 5′-TTGCTGAATAA ATCTGCGAAAT-3′ (reverse); for STAT3, 5′-GCTTTTGTCAGCGATGGAGT-3′ (forward), 5′-ATTTGTTGACGGGTCTGAAGTT-3′ (reverse); for STAT5a, 5′-AATGAGAACACCCGCAACG-3′ (forward), 5′-TTCCTGAAGTGGGCACTGA G-3′ (reverse); and for STAT5b, 5′-ACTGCTAAAGCTGTTGATGGATAC-3′ (forward), 5′-TGAGTCAGGGTTCTGTGGGTA-3′ (reverse). PCR cycles included steps of 95°C for 10 min., 40 cycles of 95°C for 15 sec. and 60°C for 1 min. Each reaction was performed in triplicate, analysed individually relative to GAPDH (a normalization control) and analysed using the 2^−ΔΔCt method [30]. Thereafter, data for transcript expression levels were expressed as fold difference relative to that of untreated control cells.

DNA was extracted from CRC cells cultured in the absence or presence of 5 μM 5-aza-dc for 96 hrs and then treated with bisulphite as previously described [31]. The primers used for SHP1, SOCS1 and SOCS3, annealing temperatures and expected PCR products sizes are summarized in Table 1. PCR products were gel-extracted (Qiagen, Valencia, CA, USA) and ligated into the TA cloning vector (pMD18-T, Takara, Dalian, China). Plasmid-transformed DH5α bacterial cells were cultured overnight and at least 10 separate clones were selected for isolation of plasmid DNA (Qiagen) and sequence analysis.

Full-length SHP1 cDNA was synthesized and cloned into pEGFP-N1 (Clontech, Mountain View, CA, USA). The sequence and orientation of the SHP1 insert was confirmed by DNA sequencing using an ABI 3100 gene sequencer (ABI, San Jose, CA, USA). For transfection, 8 μg of vector were transfected into 2 × 10⁶ cells using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. All pEGFP-N1- or pEGFP-N1-SHP1-transfected cells used throughout this study were sorted using the EPICS ALTRA (Beckman-Coulter, Mississauga, ON, Canada) to isolate GFP-expressing cells.

Western blot analysis was performed using standard techniques. Briefly, the cells were lysed in M-PER Reagent (Pierce, Rockford, IL, USA) containing protease inhibitors. Equal amounts of protein (50–200 μg/lane) from whole cell lysates were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose (Amersham, Amersham, UK), probed with specific primary antibodies, and incubated with the appropriate horseradish peroxidase conjugated secondary antibodies (Pierce). Antibody binding was detected using an enhanced chemiluminescence detection kit (SuperSignal West Femto Substrate, Pierce). As a loading control, detection of GAPDH was performed.

Antibodies used in this study were purchased from Cell Signaling Technology (CST, Boston, MA, USA) except for JAK2 (Santa Cruz, CA, USA), Bcl-2 (R&D Systems, Minneapolis, MN, USA) and GAPDH (Kangchen, China). All primary antibodies were used at a 1:1000 dilution.

Cells (1 × 10⁵) were cultured in 24-well plates to 90% confluence. Cells were washed three times before the addition of serum-free medium containing 5-aza-dc or an equivalent volume of acetic acid (vehicle control). After 24 hrs, the medium was collected and the concentration of vascular endothelial growth factor (VEGF), matrix metalloproteinases 2 (MMP-2) and matrix MMP-9 were determined using an ELISA kit (R&D Systems) according to the manufacturer’s instruction.

DNA was extracted from CRC cells cultured in the absence or presence of 5 μM 5-aza-dc for 72 hrs, and the bisulphite-treated DNA was amplified by MSP. The primers, annealing temperatures and expected PCR products sizes are summarized in Table 2. The PCR product was directly loaded onto 2% agarose gels and electrophoresed, followed by direct visualization under UV illumination.

Cell viability was assessed using a tetrazolium salt (WST-8)-based colorimetric assay provided by the Cell Counting Kit 8 (CCK-8, Dojindo, Japan) [32, 33]. Briefly, treated and untreated CRC cells (5 × 10³ cells/well) were seeded into 96-well plates. At specific time-points, 10 μl of CCK-8 solution was added to wells and the plates were incubated for 1 hr. Cell viability was determined from absorbance readings at 450 nm. Data are expressed as relative viability (%) calculated from: [A₄₅₀(treated) – A₄₅₀(blank)]/[A₄₅₀ (control) – A₄₅₀(blank)]× 100%.

Approximately 1 × 10⁶ cells were removed at specific time-points, washed twice with PBS and fixed in cold ethanol for 30 min. Samples were then incubated with propidium iodide (PI) for 30 min. and analysed by flow cytometry (BD, San Diego, CA, USA).

Apoptosis was analysed using an annexin-V FITC/PI double-stain assay (performed in accordance with the manufacturer’s protocol) and analysed by flow cytometry (Biovision, Mountain View, CA, USA). Briefly, both floating and trypsinized adherent cells (5 × 10⁵) were collected and resuspended in 500 μl binding buffer containing 5 μl of annexin-V FITC and 5 μl of PI. After 5 min. in the dark at room temperature, samples were subjected to flow cytometry analysis (BD, USA). For detection of caspase-3 activity, cleaved caspase-3 was measured in 100 μg of cell lysate using a PathScan cleaved caspase-3 sandwich ELISA kit (Cell Signaling) and absorbance at 450 nm was measured with a microplate reader.

Cell invasion assays were performed as described by Hecht et al.[34]. Briefly, 8-μm-pored polycarbonate membranes were coated with Matrigel on the upper side (Becton-Dickinson, San Diego, CA, USA). Cells (1 × 10⁵) in serum-free medium were seeded into the upper chamber and medium supplemented with 10% foetal bovine serum was applied to the lower chamber. After 24 hrs, cells remaining on the upper chamber of the filter were removed with a cotton swab. Cells that had migrated to the bottom surface of the filter were fixed, stained and counted. For a subset of assays, the upper chamber was filled with 5 μM 5-aza-dc or acetic acid (as a vehicle control) at an equivalent volume, respectively.

Results were expressed as the mean ± S.D. The data were analysed for significance by ANOVA and results were considered significant if P < 0.05.

Western blot analysis was used to establish the protein levels of SHP1 and SOCSs induced by 5-aza-dc in CRC cells. Levels of SHP1 increased with time in both CRC cell lines and the highest levels of SHP1 expression were detected on day 5. In contrast, no detectable changes in the protein levels of SOCS1 and SOCS3 were detected following 5-aza-dc treatment in either the SW1116 or HT29 cell lines (Fig. 1A).

To measure mRNA levels, quantitative real-time PCR assays were performed. After 72 hrs of incubation with 5 μM 5-aza-dc, SHP1 mRNA in SW1116 cells was ∼4.73-fold higher than that in untreated SW1116 cells (Fig. 1B). No significant change in SOCS1 and SOCS3 mRNAs were detected. These results were consistent with data from the Western blot analyses and demonstrate that SHP1, but not SOCS1 or SOCS3, is up-regulated in response to 5-aza-dc.

We examined the methylation status of the SHP1 and SOCSs promoters using bisulphite sequencing. CpGs of the SHP1 promoter in untreated SW1116 and HT29 cells showed multiple cytosines. However, after cells were treated with 5-aza-dc for 96 hrs, numerous thymidines were detected as a result of the conversion of the unmethylated cytosines to uracil by sodium bisulphite. For example, in HT29 cells treated with 5 μM 5-aza-dc for 96 hrs, one of the 10 individual clones analysed contained 11 demethylated CG pairs within the SHP1 promoter (at positions −330, −314, −310, −289, −273, −265, −236, −203, −195, −185 and −179) (Fig. 2A). For the 10 sequenced clones, the proportion of methylated residues detected ranged from 18.2% (2/11) to 100% (11/11) (Fig. 2B). In contrast, no significant changes in methylation status of the SOCS1 and SOCS3 promoters were detected (Fig. 2B). These findings provide a correlation between hypermethylation of the SHP1 promoter and changes in SHP1 expression in CRC cells.

To determine whether up-regulation of SHP1 mediates down-regulation of JAK/STAT signalling in CRC cells, SW1116 and HT29 cell lines were transfected with pEGFP-N1 or pEGFP-N1-SHP1. Transfection efficiency was determined from the expression of GFP detected by flow cytometry, and was estimated to vary between 40% and 70%, with a median of 50% for both vectors. When transfected cells were sorted, 98% of the isolated GFP-expressing cells were obtained and analysed by Western blot. As shown in Fig. 3, SHP1 expression levels were higher in both CRC cell lines transfected with pEGFP-N1-SHP1 relative to CRC cell lines transfected with pEGFP-N1. Additionally, expression of SHP1 was also associated with substantial decreases in the protein levels of phosphorylated JAK2 (pJAK2), pSTAT3 and pSTAT5, as well as levels of unphosphorylated JAK2. However, decreases in JAK2 were less than that for pJAK2. For unphosphorylated STAT3 and STAT5 protein levels, no significant changes were observed following exogenous expression of SHP1. These data demonstrate that the JAK2/STAT3/STAT5 pathway is subject to negative regulation by SHP1 in CRC cells, and negative regulation of JAK2 by SHP1 may include both dephosphorylation and others, such as protein degradation.

We further evaluated whether up-regulated SHP1 expression in response to 5-aza-dc was associated with any changes in JAK/STAT signalling. Western blot analysis detected substantial decreases in the protein levels of JAK2, pJAK2, pSTAT3 and pSTAT5 in CRC cells following 5-aza-dc treatment. For example, in SW1116 cells treated with 5-aza-dc for 24 hrs, no significant changes in the protein levels of JAK2, STAT3, or STAT5 were detected. However, at later time-points, higher levels of SHP1 expression were associated with decreases in JAK2 and pJAK2 levels that were followed by decreases in pSTAT3 and pSTAT5. A decrease in STAT3 was detected in cells exposed to 5-aza-dc; however no appreciable changes in STAT5 protein levels were detected following 5-aza-dc treatment (Fig. 4A).

Quantitative real-time PCR assays were also performed to analyse levels of JAK2, STAT3 and STAT5 mRNA. In SW1116 cells, after 72 hrs of treatment with 5 μM 5-aza-dc, JAK2 and STAT3 mRNA levels decreased to 0.373- and 0.428-fold, respectively, compared to untreated SW1116 cells (Fig. 4B). In contrast, no significant changes in STAT5 mRNA levels were detected in SW1116 cells. The results of the PCR assays were consistent with the analysis of protein levels by Western blot.

To determine whether the proteasome pathway has a role in SHP1-mediated degradation of JAK2, CRC cells were treated with 10 μM MG132, a pharmacological proteasome inhibitor, 48 hrs after transfection with pEGFP-N1-SHP1 and cell sorting for the GFP-positive cell population. As shown in Fig. 5, the basal level of JAK2 in pEGFP-N1-SHP1 transfected SW1116 cells was much lower than that in pEGFP-N1 transfected cells. However, after 10 hrs in the presence of 10 μM MG132, the level of JAK2 protein was approximately equal between the pEGFP-N1-SHP1- and pEGFP-N1-transfected cells. A similar protein expression pattern was seen in HT29 cells, indicating that MG132 can prevent SHP1-mediated down-regulation of JAK2 through the proteasome pathway.

To test if alterations in the JAK2/STAT3/STAT5 pathway by 5-aza-dc lead to significant changes in downstream targets, we examined the expression of various proteins involved in apoptosis, cell cycle progression, invasion and migration. As illustrated in Fig. 6A, p16^ink4a, p21^waf1/cip1 and p27^kip1 showed time-dependent increases in their expression levels, concomitant with decreases in levels of Bcl-2 and focal adhesion kinase (FAK) in SW1116 cells. We were unable to detect changes in expression of survivin and E-cadherin. Furthermore, ELISA assays detected reduced secretion of VEGF following treatment with 5-aza-dc, but no significant changes in the secretion of MMP-2 and MMP-9 (Fig. 6B). Similar changes in protein expression were observed for HT29 cells treated with 5-aza-dc. Additionally, we chose p27^kip1 as an example to detect the methylation status. As shown in Fig. 6C, methylation did not seem to be involved in the regulation of p27^kip1 in CRC cells, demonstrating that the 5-aza-dc-induced change in p27^kip1 expression is not confounded by methylation of the p27^kip1 gene. Therefore, our results indicate that disruption of JAK2/STAT3/STAT5 signalling by 5-aza-dc may be at least one of the causes of modulation of these target genes.

Using a CCK-8 assay (Fig. 7A), concentration- and time-dependent decreases in cell viability for SW1116 and HT29 cells following 5-aza-dc treatment were identified relative to untreated cells. To explore the mechanism responsible for the observed decrease in cell viability, the effects of 5-aza-dc on cell cycle progression and apoptosis were examined. As illustrated in Fig. 7B, pre-treatment of CRC cells with 5-aza-dc for 72 hrs blocked cells in the G2 phase. In SW1116 cells, the percentage of cells in the G2 phase increased from 4.9% (untreated) to 19.7% following treatment with 5 μM 5-aza-dc (Fig. 7B). These observations were consistent with the up-regulation of p16^ink4a, p21^waf1/cip1 and p27^kip1 detected by Western blot following treatment of CRC cells with 5-aza-dc.

To evaluate the role of apoptosis in the decreased cell viability exhibited following 5-aza-dc treatment, flow cytometry analysis of annexin V binding was performed. After 96 hrs of treatment with 5 μM 5-aza-dc, annexin V binding to the surface of CRC cells increased (Fig. 7C), corresponding to a 3.80- and 3.12-fold increase in apoptosis for the SW1116 and HT29 cell lines, respectively. In addition, as shown in Fig. 7D, pre-treatment of CRC cells with 5 μM 5-aza-dc for 96 hrs stimulated cleavage of caspase-3, suggesting that caspase-3 activation may play a significant role in 5-aza-dc induced apoptosis. Cell invasion assays were also performed, and no significant inhibition of invasion was observed following cell treatment with 5-aza-dc (Fig. 7E).