THD, cisplatin, doxorubicin, paclitaxel and 5-fluorouracil (5-FU) were all purchased from Selleckchem (Houston, TX, USA), N-acetyl-L-cysteine (NAC) was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

CT26, HUVECs, SW480, SW620 and HCT116 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). CT26 and SW480 cells were grown in 100-cm² cell culture plates and maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. SW620 and HCT116 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) and HUVECs were maintained in DMEM-F12 (Gibco; Thermo Fisher Scientific, Inc.). DMEM and DMEM-F12 were also supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cells in the exponential growth phase were used for all experiments.

CT26 cells and HUVECs were plated into 24-well plates at a density of 1×10⁵ cells/well. THD dissolved in dimethyl sulfoxide was incubated with the cells at different concentrations (0, 12.5, 25, 50 and 100 µM) with three replicates being performed for each concentration. Following 24, 48 or 72 h incubation at 37°C, the cell proliferation was determined manually by cell number counting.

CT26 cells and HUVECs were seeded into 6-well plates at a density of 2×10⁵ cells/well and exposed to THD at various concentrations (0, 25 and 100 µM) at 37°C. Cells were harvested following 24-h incubation and washed twice with PBS. Subsequently, cells were stained using an Annexin V/PI double staining solution from an Annexin V-FITC apoptosis detection kit (BD Pharmingen; BD Biosciences; San Jose, CA, USA) at room temperature, according to the manufacturer's instructions. Following 15 min of incubation, the Annexin V/PI stained cells were detected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (version 5.1; BD Biosciences).

CT26 cells and HUVECs were seeded into 6-well plates at a density of 2×10⁵ cells/well and incubated with THD at various concentrations (0, 25, 50 and 100 µM) for 24 h at 37°C. Subsequently, cells were harvested and washed three times with cold PBS. Cells were fixed in 70% ice-cold ethanol overnight, washed twice with PBS, stained with PI/RNase staining buffer (BD Pharmingen; BD Biosciences) at room temperature for 15 min and detected using a flow cytometer. Data were analyzed using Modifit software (version 4.1; Verity Software House, Topsham, ME, USA).

CT26 cells and HUVECs were seeded into 6-well plates at a density of 2×10⁵ cells/well. Cells were exposed to THD (0, 25, 50 and 100 µM), cisplatin (0, 0.5, 0.8 and 1 µg/ml), paclitaxel (0, 10, 20 and 40 ng/ml), 5-FU (0, 1, 2 and 4 µM) and doxorubicin (0, 5 and 10 µM), irradiated with 2 Gy X-rays (29,30) or heated at 47°C for 3 min as previously described (31). To inhibit intracellular reactive oxygen species (ROS), the antioxidant NAC (2 and 4 mM, respectively) was added to the culture media, then they were exposed to 100 µM THD for 24 h at 37°C. In addition, SW480, SW620 and HCT116 cells were seeded at a density of 2×10⁵ cells/well and were exposed to THD at various concentrations (0, 25, 50 and 100 µM). Following 24-h incubation at 37°C, all cells were harvested by centrifugation (475 × g for 3 min at room temperature), washed twice using cold PBS, and subsequently lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice for 15 min for protein extraction.

Protein concentrations were determined using a bicinchoninic acid assay. Sample proteins (40 µg/sample) were separated on 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h. Membranes were subsequently incubated at 4°C overnight with the primary rabbit monoclonal antibodies against VEGFR-1 (cat. no. ab32125), cyclin dependent kinase (CDK)6 (cat. no. ab131439), cyclin D1 (cat. no. ab134175), C-MYC (cat. no. ab39688), B-cell lymphoma-2 (BCL-2; cat. no. ab59348), BCL-2-associated X protein (BAX; cat. no. ab182733) and caspase-3 (ab32351, all 1:1,000; all from Abcam, Cambridge, MA, USA). GAPDH protein was also determined using a specific antibody (dilution 1:1,000, cat. no. 2118; Cell Signaling Technology, Inc., Danvers, MA, USA) as a loading control. The membranes were washed with TBST three times and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721, 1:5,000; Abcam) at room temperature for 1 h. The membranes were washed with TBST three times and visualized using an enhanced chemiluminescence detection system (Beyotime Institute of Biotechnology).

CT26 cells and HUVECs were respectively seeded at 2×10⁵ cells/well in 6-well plates and incubated with THD at various concentrations (0, 25, 50 and 100 µM) for 6 h at 37°C. Cells were harvested and washed three times with cold PBS. Subsequently, cells were resuspended in diluted 2′,7′-dichlorofluorescein diacetate (DCFH-DA) at a cell concentration of 1×10⁶ to 20×10⁶ cells/ml according to the manufacturer's instructions and incubated at 37°C for 20 min. Cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that had not entered the cells (ROS kit, cat. no. s0033; Beyotime Institute of Biotechnology). Analysis was conducted using a flow cytometer and CellQuest software (version 5.1).

The present study was approved by the Animal Experimental Ethics Committee of the State Key Laboratory of Biotherapy, Sichuan University (Sichuan, China). A total of 20 female BALB/c mice (6–8 weeks old, 18–20 g) were obtained from the Vital River Laboratory Animal Technology (Beijing, China), and housed in a specific pathogen-free environment (temperature, 21±2°C; humidity, 40–70%; light cycle, 12/12 h; free access to food and water). A total of 5×10⁵ CT26 tumor cells were injected into the abdominal cavity of mice. Tumor-bearing mice were divided into two groups (n=10) and treatment began at 3 days post-inoculation. Mice in the THD group were administered 100 mg/kg THD every day by gavage. Mice in the control group received normal saline. The effect of THD on mice was evaluated at day 7 based on body weight, ascites volume and tumor weight.

Fresh tumor nodules were harvested, flash frozen and sliced into 4–8 µm sections. Frozen sections were treated with 3% hydrogen peroxide in methanol for 20 min to quench endogenous peroxidase activity. Subsequent to washing in PBS (5 min, 3 times; pH 7.6), sections were blocked with 10% normal goat serum (cat. no. s8080; Solarbio Science and Technology Co., Ltd., Beijing, China) for 10 min. Sections were subsequently incubated with rabbit monoclonal antibody against VEGFR1 (ab32152, 1:250 dilution; Abcam) overnight at 4°C, and then washed in PBS (5 min, 3 times; pH 7.6). The washed sections were incubated with biotinylated goat anti-rabbit IgG (1:100 dilution, cat. no. ZB-2301; OriGene Technologies, Inc., Beijing, China) for 30 min followed by a streptavidin-peroxidase conjugate for 30 min at room temperature. A solution of 0.02% diaminobenzidine hydrochloride containing 0.03% hydrogen peroxide was used as chromogen to visualize peroxidase activity at room temperature for 5–8 min. The preparations were counterstained with hematoxylin at room temperature for 30 sec to 2 min, dried, mounted and examined using light microscopy.

All statistical analyses were performed using GraphPad Prism 6.01 software (GraphPad Software, Inc., La Jolla, CA, USA). Data analysis was performed using the one-way analysis of variance and Dunnett's test for multiple groups. The Student's t-test was used for the analysis of two groups. All data were presented as the mean ± standard deviation of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

The effect of THD on murine CT26 colorectal tumor cells in vitro was evaluated. CT26 tumor cells were treated with various concentrations (0, 12.5, 25, 50 and 100 µM) of THD for 24, 48 and 72 h. The number of surviving tumor cells was determined. Results indicated (P<0.001; Fig. 1A).

As THD has antiangiogenic properties, and endothelial cells have a key role in angiogenesis (32,33), the effect of THD on HUVECs was evaluated. The results revealed that THD inhibited the proliferation of HUVECs compared with the control group (Fig. 1A).

The effect of THD on the apoptosis of CT26 cells and HUVECs was determined using an Annexin V/PI assay. The apoptosis rate was increased following exposure to THD for 24 h in a dose-dependent manner (Fig. 1B) and this difference was statistically significant (P<0.01; Fig. 1C). Collectively, these data revealed that THD had a growth-arresting and apoptosis-inducing effect on tumor cells and endothelial cells.

The effect of THD on cell cycle and apoptosis-associated molecules was investigated. CT26 tumor cells and HUVECs were exposed to different concentrations of THD for 24 h. Flow cytometry revealed that THD arrested the cell cycle at the G0/G1 phase in CT26 tumor cells, and at the S phase in HUVECs (Fig. 2A).

The protein expression levels of CDK6, cyclin D1, C-MYC, BCL-2, BAX and cleaved-caspase 3 were examined by western blot analysis. CDK6 is a protein kinase that activates cell proliferation and is associated with restriction in the cell cycle (34). Cyclin D1 protein required for progression through the G1 phase of the cell cycle; it is synthesized rapidly and accumulates in the nucleus during the G1 phase and is degraded as the cell enters the S phase (35). The protein product of the proto-oncogene C-MYC is a transcription factor that regulates a range of cellular processes that serve a key role in cell proliferation, most notably in the regulation of G1 specific cyclin dependent kinases (36). The results indicated that in CT26 cells the protein expression levels of CDK6 were markedly upregulated, whereas the protein expression levels of cyclin D1 exhibited no notable change following THD treatment compared with the control (Fig. 2B). However, exposure of HUVECs to THD downregulated CDK6 protein expression levels as the concentration increased. Conversely, the protein expression levels of cyclin D1 were only marginally increased as a result of THD exposure compared with the control group (Fig. 2B). Furthermore, the protein expression levels of C-MYC were markedly increased in the two cell lines, particularly in HUVECs, following THD treatment (Fig. 2B). BCL-2 is an anti-apoptotic protein, whereas BAX promotes apoptosis (37). In the present study, the protein expression levels of anti-apoptotic protein BCL-2 were downregulated with increased THD concentration, whereas the protein expression levels of pro-apoptotic protein BAX and cleaved-caspase-3 were upregulated compared with the control group (Fig. 2C). These findings suggested that THD alters the protein expression levels of cell cycle and apoptosis-associated molecules.

To determine whether THD influenced the protein expression levels of VEGR1 in endothelial cells, HUVECs were exposed to different concentrations (0, 25, 50 and 100 µM) of THD for 24 h. Results indicated that THD at a high concentration (100 µM) markedly upregulated the protein expression levels of VEGFR1 compared with the control group (Fig. 3A). Subsequently, this phenomenon was assessed in tumor cells. The CT26 tumor cells treated with THD exhibited clearly upregulated VEGFR1 expression levels compared with the control group (Fig. 3B). Furthermore, similar phenomena were observed in the human colorectal cell lines SW480 and HCT116. In the SW620 cell line, no obvious differences in VEGFR1 expression were observed between different dosages of THD. It may be that VEGFR1 expression is only affected by higher concentrations of THD (Fig. 3C). These data suggest that THD upregulates VEGFR1 protein expression levels in vitro.

Considering the cytotoxic effect of THD on CT26 tumor cells and HUVECs, and the upregulation of VEGFR1 observed in vitro, it was speculated that the upregulation of VEGFR1 may be induced by cellular stress caused by THD. The levels of intracellular ROS were subsequently analyzed using the probe DCFH-DA. ROS levels were significantly increased in the two cell types following exposure to THD (Fig. 4A and B). Notably, the ROS antagonist NAC reversed VEGFR1 elevation to some extent in CT26 cells and HUVECs (Fig. 4C). These results suggested that the elevation of VEGFR1 was associated with oxidative cellular stress.

To determine the effect of THD upregulation on VEGFR1 protein expression levels in vivo, CT26 cells were injected into the abdominal cavity of BALB/c mice. The antitumor effect of THD was evaluated based on body weight, ascites volume and tumor weight over the course of 7 days. There was no significant difference in body weight, ascites volume or tumor weight between the THD group and control group (P>0.05; Fig. 5A). Furthermore, the protein expression levels of VEGFR1 were increased in the THD-treated group compared with the control group according to immunohistochemical staining analysis (Fig. 5B). These data suggest that THD failed to inhibit CT26 murine tumor growth, but upregulated VEGFR1 protein expression levels in vivo.

In order to further determine whether VEGFR1 is a stress-inducible molecule, VEGFR1 expression levels were assessed in cells subjected to various stress-related situations. CT26 cells and HUVECs were exposed to chemotherapeutic drugs, including cisplatin, doxorubicin, paclitaxel and 5-FU, respectively. VEGFR1 expression levels were mostly upregulated following exposure to these chemotherapeutic agents compared with the respective control groups. However VEGFR1 in HUVECs for 20 and 40 ng/ml paclitaxel was lower than for 10 ng/ml. It was hypothesized that an appropriate dosage may produce a stress response and increase the expression of VEGFR1, whereas too high a dosage would increase cell apoptosis. VEGFR1 expression did not increase in HUVECs treated with 20 or 40 ng/ml paclitaxel. (Fig. 6A). Furthermore, stress was induced in CT26 cells and HUVECs by irradiation with 2 Gy X-rays. VEGFR1 expression levels were markedly upregulated following irradiation compared with the control group (Fig. 6B and C). Additionally, cellular stress was induced in CT26 tumor cells and HUVECs by heating the cells at 47°C for 3 min. VEGFR1 protein expression levels were increased in comparison with those in with the control group (Fig. 6B and C). Together, the data suggest VEGFR1 may be a stress-inducible molecule.