The CRC cell lines HCT116 and HT29 (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco-BRL), 10 mg/ml antibiotics (penicillin and streptomycin; Sigma-Aldrich, St Louis, MO, USA) and 2 mmol/l L-glutamine at 37°C under a humidified atmosphere with 5% CO₂. TSA (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) to a final concentration of 1.0 μM, which was used to treat the cells (3×10⁵ cells/well in a six-well plate). An equal volume of DMSO was used as vehicle control. The cells were incubated with TSA for 15 h for Bax detection, and for 24 h for the detection of the other proteins.

HCT116 and HT29 cells were cultured and treated with TSA as described above; cells were then digested using trypsin (Gibco-BRL), stained with 5 μl Annexin V-FITC (BD Pharmingen, San Diego, CA, USA) and 5 μl PI (BD Pharmingen) staining solution in the dark at room temperature (RT) for 15 min. The cell samples were analyzed by flow cytometry using a FACScan station with Cell Quest software (BD Biosciences, Franklin Lakes, NJ, USA) using the fluorescent resolution FL1 and FL2 ranges for Annexin V FITC and PI, respectively.

Cells were lysed in lysis buffer containing 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris (pH 8.0) (Sigma-Aldrich) and 1:25 protease inhibitor cocktail. For cellular fractionation, cells were washed with phosphate-buffered saline (PBS) and lysed in cytosolic lysis buffer containing 250 mM sucrose, 70 mM KCl, 137 mM NaCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄ (pH 7.2), 200 μg/ml digitonin, 100 mM phenylmethylsulfonyl fluoride and 1:25 protease inhibitor cocktail for 30 min on ice. Cells were centrifuged at 16,873 × g for 15 min at 4°C. The supernatants were stored as cytosolic protein extract and the pellets were further dissolved with lysis buffer containing 1% SDS as the mitochondrial fraction. Protein concentrations of the lysates were determined using the Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). In brief, equal amounts of protein (30 μg protein/lane) were separated using SDS-PAGE (5% stacking gel and 12% separation gel), transferred to nitrocellulose membranes (Hybond C, GE Healthcare, Little Chalfont, UK). Immunoblots were blocked with 5% skimmed milk in Tris-buffered saline/Tween 20 (0.05%, v/v) (Sigma-Aldrich) for 1 h at RT. The membranes were then incubated with the following primary antibodies overnight at 4°C: Mouse monoclonal anti-poly-adenyl-ribosyl polymerase (PARP; 1:2,000 dilution; cat. no. 556362; BD Pharmingen), rabbit monoclonal anti-cytochrome c (1:1,000 dilution; cat. no. 3895-1; Epitomics, Burlingame, CA, USA), rabbit polyclonal anti-cycloxygenase (COX) IV (1: 500 dilution; cat. no. GTX101499; GeneTex, San Antonio, TX, USA), rabbit monoclonal anti-Bax (1:1,000 dilution; cat. no. 1063-1; Epitomics), rabbit monoclonal anti-Ku70 (1:1,000; cat. no. 2829-1; Epitomics), rabbit polyclonal anti-active caspase 3 (1:800; cat. no. Ab2302; Abcam, Cambridge, MA, USA) and mouse monoclonal anti-actin (1:5,000 dilution; cat. no. A1978; Sigma-Aldrich). The membranes were then incubated with the corresponding secondary antibodies, including polyclonal goat anti-mouse IgG-H&L (1:7,000 dilution; cat. no. ab6787; Abcam) and polyclonal goat anti-rabbit IgG-H&L (1:7,000 dilution; cat. no. ab6721; Abcam) conjugated with horseradish peroxidase at RT for 1 h. Blots were developed using an enhanced chemiluminescence western blotting detection kit (GE Healthcare). For co-immunoprecipitation, HCT116 cells were treated with TSA (1 μM) and the cell lysates were precipitated with anti-Ku70 antibodies. The bound proteins were then subjected to SDS-PAGE and blotted for Bax, as previously described (10).

Ku70 siRNA and scrambled siRNA were purchased from Santa Cruz Biotechnology Inc. A siPORT NeoFX transfection agent (Ambion Life Techonolgies, Grand Island, NY, USA) was used for the siRNA transfection. At 24 h post-transfection, cells were treated with TSA for a further 24 h. Knockdown of Ku70 expression was confirmed by western blot analysis.

All values are presented as the mean ± standard deviation or a representative of ≥3 independent experiments. Statistical comparisons were performed using the Student’s t-test. SPSS version 11.0 (SPSS Inc., Chicago, IL, USA) was used to perfom all statistical analyses. P<0.05 was considered to indicate a statistically significant difference between values.

HCT116 and HT29 cells were exposed to various concentrations of TSA (0.1, 1.0 and 5.0 μM) for 24 h. Ku70 acetylation was detected by western blot and immunoprecipitation analysis. As shown in Fig. 1A, TSA induced the acetylation of Ku70 in HCT116 and HT29 cells. In addition, TSA was found to significantly enhance apoptosis, as measured using Annexin-V-FITC/PI staining (Fig. 1B), which was in parallel with changes in Ku70 acetylation levels. These results therefore suggested that Ku70 acetylation was involved in the mechanism of TSA-induced apoptosis in CRC cells.

As TSA was found to induce Ku70 acetylation, it was investigated whether TSA treatment impaired the interaction between Ku70 and Bax. Co-immunoprecipitation analysis was performed, the results of which revealed a weakened Bax band in TSA-treated cells following protein lysate immunoprecipitation by the Ku70 antibody compared with that of the untreated Bax band (Fig. 2). These results indicated that Bax was released from its association with Ku70 upon TSA-mediated Ku70 acetylation.

Bax translocation is an early event in Bax-dependent apoptosis, which results in damage of the mitochondrial outer membrane and the release of key apoptosis mediators, such as cytochrome c, subsequently leading to the activation of caspase-9 and the downstream cleavage of caspase-3 (11,12). In the present study, Bax localization in the cytosol and mitochondria was investigated following cell fractionation. This was performed using immunoblotting of lysates of HCT116 and HT29 cells following exposure to 1 μM TSA. As shown in Fig. 3A, Bax levels were high in the cytoplasm of control cells and declined following TSA treatment. By contrast, Bax levels in the mitochondria were increased when treated with TSA. In addition, cytochrome c levels in the cytoplasm were increased following TSA treatment, which was accompanied by a decrease in the mitochondrial fraction (Fig. 3A). Furthermore, western blot analysis revealed that active caspase 3 expression was increased following TSA treatment (Fig. 3B), indicating that TSA enhanced Bax translocation from the cytosol into the mitochondria, leading to the release of cytochrome c into the cytosol, which resulted in activation of caspase 3.

Ku70 has previously been reported to regulate apoptosis through interacting with cytosolic Bax and inhibiting its trans-location to the mitochondria (8). It was therefore hypothesized that decreased Ku70 may increase the release of Bax, which translocates to the mitochondria and initiates apoptosis upon TSA treatment. By contrast, the results of the present study showed that Ku70 siRNA impaired TSA-induced apoptosis in CRC cells, as determined by flow cytometric and western blot analysis (Fig. 4A–C). In addition, this decrease in apoptosis was accompanied with a decline in Bax expression, as detected by western blot analysis (Fig. 4D).

The decreased Bax protein levels may be due to proteasomal degradation, which increases the instability of the protein. MG132 is a potent proteasome inhibitor, which was found to induce apoptosis in CRC cells in a dose-dependent manner (Fig. 5A). A low dose of MG132 (0.5 μM), which was shown to have little effect on apoptosis, was selected for the pre-treatment of HCT116 cells prior to TSA treatment. MG132 pre-treatment rescued the Ku70 siRNA-induced decrease in apoptosis (Fig. 5B and C), as well as decreased the protein expression levels of activated caspase 3 (Fig. 5D) and Bax (Fig. 5E). These results demonstrated that Ku70 may have an important role in the protection of Bax against proteasomal degradation.