TQ, PDTC, Tris, glycine, NaCl, sodium dodecyl sulfate (SDS), bovine serum albumin (BSA) and β-actin antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA). TQ was dissolved in dimethyl sulfoxide (DMSO; Merck Millipore, Darmstadt, Germany) to make a 50 mM stock solution and stored at −20°C until use in subsequent experiments. Additional dilutions were performed in cell culture medium (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) so that the final DMSO concentration was <0.1%. Primary antibodies, including rabbit polyclonal anti-human p65 (catalog no., sc-101749; 1:500), rabbit polyclonal anti-human B-cell lymphoma 2 (Bcl-2; catalog no., sc-492; 1:2,000), rabbit polyclonal anti-human vascular endothelial growth factor (VEGF; catalog no., sc-507; 1:1,000), mouse polyclonal anti-human c-Myc (catalog no., sc-40: 1:500), and secondary antibodies, including goat anti-rabbit horseradish peroxidase (HRP)-conjugated (catalog no., sc-2054: 1:10,000) and goat anti-mouse HRP-conjugated (catalog no., sc-2005 1:10,000) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Primary rabbit polyclonal anti-human β-actin (catalog no., sc-7210: 1:200) antibody was purchased from Sigma-Aldrich. The COLO205 and HCT116 colon cancer cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 2% penicillin/streptomycin (6 mg/ml penicillin, 10 mg/ml streptomycin; Sigma-Aldrich). The cells were cultured in tissue culture flasks (75 cm²; Corning Incorporated, New York, NY, USA) and incubated at 37°C in a humidified chamber containing 5% CO₂.

Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, 2×10⁴ cells per well were seeded into a 96-well plate (Corning Incorporated). Subsequent to 24 h culturing, cells were treated with cisplatin and TQ for 24 h or TQ alone for 48 h. Finally, 20 µl CCK-8 solution was added to each well, and was incubated for 2 h at 37°C. The optical density (OD) of each well at 450 nm was measured using a VICTOR™ X multi-label reader (PerkinElmer, Inc., Waltham, MA, USA). The percentage cell viability was calculated as follows: (OD_drug / OD_control) × 100. To analyze the role of nuclear factor-κB (NF-κB) in TQ activity, cells (2×10⁴ cells per well) were treated with 50 µm pyrrolidine dithiocarbamate (PDTC) in combination with TQ for 12, 24 and 48 h. The percentage cell growth inhibition was calculated as follows: (OD_control-OD_drug) × 100.

Nuclear extracts were prepared as previously described (25). Briefly, cells were harvested, washed twice with ice-cold phosphate buffered saline (PBS; Hyclone, Beijing, China) for 1 min and resuspended in 1 ml of ice-cold PBS. Cells were pelleted by centrifugation at 12,000 × g for 5 min, suspended in ice-cold buffer [10 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.5 mmol/l MgCl₂, 0.2 mmol/l KCl, 0.2 mmol/l phenylmethylsulphonyl fluoride, 0.5 mmol/l dithothreitol], mixed by vortexing for 10 sec and centrifuged at 12,000 × g for 5 min. The nuclear pellet was washed in 1 ml buffer (20 mmol/l HEPES, 25% glycerol, 0.42 mol/l NaCl, 1.5 mmol/l MgCl₂, 0.2 mmol/l ethylenediaminetetraacetic acid), resuspended in 30 ml buffer, mixed by rotation for 30 min at 4°C and centrifuged at 12,000 × g for 20 min. Finally, the supernatants were used as nuclear extracts.

Using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), whole cell lysates or nuclear extracts were electrophoresed, as previously described (26). The proteins were transferred onto a 0.4-µm polyvinylidene difluoride membrane (EMD Millipore, Bedford, MA, USA) in transfer buffer (25 mM Tris, pH 8.5, 0.2 M glycine and 20% methanol). The membranes were blocked by 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h, washed twice with TBST for 10 min each and incubated with primary β-actin, p65, Bcl-2, VEGF and c-Myc antibodies at 4°C overnight. Subsequent to three washes with TBST for 10 min each, the membranes were probed with secondary peroxidase-conjugated antibody (dilution, 1:10,000; Sigma-Aldrich). Finally, the immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (Immobilon WBKLS0500; Merck Millipore). β-actin was used as an internal control for all western blot analyses.

SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Values were expressed as the mean ± standard deviation. Numeric variables were compared by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Previous studies have shown the antitumor and anticarcinogenic activities of TQ in numerous types of cancer, including breast cancer, glioblastoma and lymphoma (27–29). In order to elucidate the molecular mechanisms underlying the function of TQ, the effect of TQ on colon cancer cells was examined in the present study. As shown in Fig. 1, 20 µM TQ treatment resulted in a significant decrease in cell viability in HCT1116 cells when compared with the control (P=0.013). Furthermore, 40 µM TQ treatment significantly decreased call viability in COLO205 cells compared with the control (P=0.007). Cytotoxicity assays indicated that TQ dose-dependently decreased cell viability of the COLO205 and HCT116 colon cancer cell lines (Fig. 1), which confirmed the antitumor activity of TQ, as previously reported (30,31).

The present study aimed to investigate whether TQ affected the sensitivity of colon cancer cells to chemotherapy. Therefore, TQ and CisPt were combined to treat COLO205 and HCT116 colon cancer cell lines. As shown in Fig. 2, treatment with cisplatin alone induced COLO205 and HCT1116 cell death in a dose-dependent manner. Combined treatment with cisplatin (0.2 µM) and TQ (20 or 40 µM) significantly decreased the cell viability of COLO205 (P=0.021, 20 µM TQ; P=0.003, 40 µM TQ) and HCT116 cells (P=0.038, 20 µM TQ; P=0.004, 40 µM TQ) when compared with 0.2 µM cisplatin treatment alone. These results revealed that cell death induced by CisPt was enhanced by TQ in a concentration-dependent manner (Fig. 2), which indicated that TQ may potentiate the chemosensitivity of colon cancer cells.

As demonstrated in the present results, TQ treatment may result in cell death and chemosensitizing of colon cancer cells. Therefore, the present study aimed to investigate the underlying mechanisms of TQ action, including the role of NF-κB in the process. Western blot analysis was used to determine the effect of TQ on NF-κB activation. The results demonstrated that 60 µM TQ significantly inhibited the phosphorylation of p65 protein, subunit of NF-κB (Fig. 3A). Furthermore, the expression levels of NF-κB-regulated genes that are involved in tumor angiogenesis, survival and apoptosis were measured. As demonstrated in Fig. 3B, protein levels of VEGF, c-Myc and Bcl-2 were markedly downregulated following 60 µM TQ treatment.

As TQ inhibited NF-κB activation, the effect of NF-κB on TQ function was assessed. The NF-κB inhibitor, PDTC, was used to investigate the activity of TQ. As demonstrated in Fig. 4, combined treatment with 10 µM cisplatin and 40 µM TQ treatment for 24 or 48 h significantly inhibited cell growth inhibition when compared with 40 µM TQ treatment alone (P=0.018 and P=0.009, respectively) in COLO205 cells. Treatment with 50 µM PDTC further potentiated the growth inhibition observed following 24 h (P=0.002) and 48 h (P=0.031) treatment when compared with 10 µM cisplatin and 40 µM TQ combined treatment. For HCT1116 cells, following 12, 24 and 48 h treatment with 50 µM PDTC, 10 µM cisplatin and 40 µM TQ significantly inhibited cell growth inhibition (P=0.014, P=0.043 and P=0.027, respectively) when compared with combined 10 µM cisplatin and 40 µM TQ treatment. Furthermore, a chemosensitization effect of TQ on colon cancer cells was enhanced by 50 µM PDTC treatment, which was demonstrated by increased cell growth inhibition (Fig. 4).