HCT1116 and COLO205 colon cancer cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (Gibco-BRL, Gasthersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL) and 2% penicillin/streptomycin (penicillin, 10,000 U/ml; streptomycin, 10 mg/ml). The cells were placed into tissue culture flasks (75 cm², 250 ml) and grown at 37°C in humidified atmosphere consisting of 5% CO₂ and 95% air. TSA was purchased from Dasherb Corp. (Shenyang, China). Aprotinin and leupeptin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide was purchased from EMD Millipore Corporation (Darmstadt, Germany). The antibodies used in the present study were the following: Mouse monoclonal anti-actin (1:1,000; cat. no. sc-8432; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit polyclonal anti-vascular endothelial growth factor (VEGF; 1:500; cat. no. ab46154; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-c-Myc (1:1,000; cat. no. 9402S; Cell Signaling Technology, Inc., Denvers, MA USA), mouse monoclonal anti-cyclooxygenase-2 (COX2; 1:1,000; cat. no. sc-376861) and rabbit polyclonal anti-B-cell lymphoma-2 (Bcl-2; 1:2,000; cat. no. sc-492) (both from Santa Cruz Biotechnology Inc.).

Cell viability was measured using a Cell Counting kit (CCK)-8 kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, cells were seeded in a 94-well plate at a density of 2×10⁴ cells per well in 200 µl culture medium. The following day the cells were incubated with drug for 48 h. At the end of the experiment, 20 µl CCK-8 solution was added to the cells. The cells were then further incubated at 37°C for 2 h. Subsequently, the optical density (OD) at 450 nm was measured using a VICTOR™ × Multi-Label reader (PerkinElmer, Inc., Waltham, MA, USA) and the percentage of cell viability was calculated as OD_drug/OD_control × 100%.

Following treatment, the cells were harvested and washed twice with ice-cold phosphate-buffered saline (PBS). Next, the cell samples were resuspended in 1 ml PBS and nuclear extracts were prepared on ice as described in a previous study (19). Following centrifugation at 15,000 × g for 10 min at 4°C, the cell pellet was suspended in an ice-cold buffer (consisting of 10 mmol/l HEPES, 1.5 mmol/l MgCl₂, 0.5 mmol/l dithiothreitol, 0.2 mmol/l phenylmethylsulphonylfluoride and 0.2 mmol/l KCl), vortexed for 10 sec and centrifuged at 15,000 × g for 5 min at 4°C. Subsequently, the nuclear pellet was washed in 1 ml buffer (consisting of 20 mmol/l HEPES, 25% glycerol, 0.2 mmol/l ethylenediaminetetraacetic acid, 1.5 mmol/l MgCl₂ and 0.42 mol/l hypertonic saline), resuspended in 30 ml buffer (20 mmol/l HEPES, 25% glycerol, 0.42 mol/l NaCl, 1.5 mmol/l MgCl₂, 0.2 mmol/l EDTA), rotated for 30 min at 4°C and centrifuged at 14,500 × g for 20 min at 4°C. Finally, the supernatants were used as nuclear extracts.

Whole cell lysates or nuclear extracts were separated using 12% SDS-PAGE, as described previously (20). Transfer buffer [25 mM Tris (pH 8.5), 20% methanol and 0.2 M glycine] was used to equilibrate the separated proteins, which were then transferred onto a 0.4-µm polyvinylidene fluoride membrane (EMD Millipore Corporation, Bedford, MA, USA). The membranes were incubated with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, followed by washing and then incubation with appropriate dilutions of specific primary antibodies at 4°C overnight. Following incubation with secondary anti-mouse peroxidase-conjugated antibody (dilution, 1:15,000; Sigma-Aldrich), the immune-reactive bands were visualized using an enhanced chemiluminescence detection kit (EMD Millipore Corporation, Darmstadt, Germany). β-actin was used as an internal control for western blotting.

The statistical analysis was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) and are presented as the mean ± standard deviation. One-way analysis of variance was performed to compare numeric variables between groups. P<0.05 was considered to indicate a statistically significant difference.

TSA has been demonstrated to exert an antitumor activity in numerous types of human cancer, including breast, lung, liver, prostate and ovarian cancer and leukemia (15,21–25). The data of the present study showed that the exposure of the HCT1116 and COLO205 colon cancer cell lines to TSA greatly decreased their cell viability in a concentration-dependent manner (P<0.05; Fig. 1), which suggested an antitumor effect of TSA in colon cancer.

Drug resistance is a clinical challenge that can result in the failure of colon cancer therapy (26). In order to examine how TSA influences the sensitivity of colon cancer cells to chemotherapy, 5-FU chemotherapy was combined with TSA treatment. As shown in Fig. 2, 5-FU-induced cell death in HCT1116 and COLO205 cells was considerably enhanced by 20 µM TSA treatment (P<0.05), which suggested that TSA was able to potentiate the chemosensitivity of colon cancer cells.

As shown by the aforementioned data, TSA can decrease cell viability and increase the chemosensitivity of colon cancer cells; however, the molecular mechanisms of TSA action remain to be elucidated. In order to further investigate the mechanisms of TSA action, the influence of TSA on the activation of NF-κB was determined, since it has been suggested that NF-κB is involved in the development and progression of various malignant neoplasms (27–33), which made it a target for tumor therapeutics (34,35). The present data revealed that 20 µM TSA treatment significantly decreased the level of phosphorylation of p65, a subunit of NF-κB (Fig. 3A), which indicated the inhibitory effect of TSA on NF-κB activation. In addition, the expression of NF-κB regulated genes was examined. As illustrated in Fig. 3B, 20 µM TSA was able to considerably decrease VEGF, c-Myc, COX-2 and Bcl-2 expression.

As indicated in Fig. 4, the inhibition of NF-κB signaling with specific inhibitor PDTC significantly potentiated the suppression of TSA-induced cell growth in HCT1116 and COLO205 cells. Furthermore, the chemosensitizing effect of TSA on colon cancer cells was also enhanced by 50 µM PDTC treatment (P<0.05), which is illustrated as increased cell growth inhibition in Fig. 4.