HCT-116 cells were treated with DA and the cell viability was determined by MTT assay. The treatment of HCT-116 cells for 24–72 h with 5, 10 and 15 μg/mL of DA resulted in cell growth inhibition in a dose- and time-dependent manner (Figure 2). To confirm cell growth inhibition, we also conducted a cell proliferation assay using the BrdU labeling and Detection Kit (Roche, Indianapolis, IN); we found similar results as in the MTT assay using this method (data not shown).

To investigate the effect of DA on cell cycle distribution of HCT-116 cells, the DNA content was analyzed by flow cytometry. As shown in Figure 3, it indicated that DA causes a significant increase in the percentage cells in the G⁰/G¹ phase of the cell cycle in a dose-dependent manner.

HCT-116 cells were treated with 5, 10 and 15 μg/mL of DA for 48 h. After treatment, the degree of apoptosis was measured. The induction of apoptosis was found to be dose-dependent (Figure 4). These results provided convincing data showing that DA could induce apoptosis in HCT-116 cells.

The Bcl-2 family plays a major role in the regulation of apoptosis by functioning as promoters or inhibitors of cell death. We therefore examined the expression of Bcl-2 family proteins in DA-treated HCT-116 cells in a dose-dependent manner. As shown in Figure 5A, DA suppresses the expression of anti-apoptotic proteins such as Bcl-2 and Bcl-xl and moderately increases the expression levels of pro-apoptotic proteins such as Bax and Bid. In addition, the ratio of Bax and Bcl-2 was measured by quantification of bands. The results indicated that DA treatment induces a dose-dependent increase in the Bax/Bcl-2 ratio in HCT-116 cells (Figure 5B).

To determine whether systemic therapy with DA could stunt tumor growth in animals, we established HCT-116 xenografts in nude mice. As shown in Figure 6C, DA treatment significantly inhibited tumor growth compared with untreated control. In addition, no toxicity judged by parallel monitoring of the body weight was observed in DA-treated mice. Immunohistochemical staining showed that the expression of Bcl-2 was down-regulated in the DA-treated group, whereas, the expression of Bax was markedly higher in the DA-treated group (Figure 6D).

DA was obtained from Huakang Pharmaceutical Company (Deyang, China) and the purity was determined by high-performance liquid chromatography.

Human CRC cell lines HCT-116 were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Minhai Biotech, Lanzhou, China) and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin). All cells were grown in 5% CO² at 37 °C and were sub cultured at an initial density of 1 × 10⁵ cells/mL every three days. All experiments were performed with cells in the logarithmic phase of growth.

The cells (5 × 10³) were seeded in a 96-well plate and subsequently treated with DA (2.5, 5, 10 μg/mL) for 12, 24 and 48 h. Twenty micro liters of MTT (5 mg/mL) solution (Sigma, St Louis, MO) were added to each well at 37 °C for four hours. After removing the media, 150 μL of dimethylsulfoxide (DMSO) were added and left for 20 min at room temperature to dissolve the formazan crystals in a shaker. The absorbance at 570 nm was measured in a micro plate reader (Bio-Rad, Hercules CA).

To estimate the proportion of cells in the different phases of the cell cycle, the cell DNA contents were measured using flow cytometry. HCT-116 cells plated on 20-cm² tissue culture flasks were collected at 24 h after the incubation with vehicle and DA. Then, the cells were fixed gently with 70% ethanol in ice and were then resuspended in PBS containing 50 μg/mL PI (Sigma, St Louis, MO) and 0.1 mg/mL RNase (Sigma, St Louis, MO). After 30 min at 37 °C, the cells were analyzed with a flow-cytometer and the cell cycle was determined. Experiments were performed in triplicate and repeated three times.

The cell death detection ELISA kit (Roche, Indianapolis, IN) was used for assessing apoptosis according to the manufacturer’s instructions. Briefly, cells were treated with DA for 48 h. After treatment, the cells were lysed and the cell lysates were overlaid and incubated in a micro titer plate modules coated with anti-histone antibody for detection of apoptosis.

Western blotting was performed as we have previously described [6]. Briefly, equal amounts of protein were separated by 12% SDS-PAGE and transferred into a PVDF membrane (Bio-Rad, Hercules, CA). After blocking with 5% non-fat milk at 4 °C overnight, the membranes were incubated with specific primary antibodies (Santa Cruz, Calif, USA) at room temperature for 2 h. Washing the membranes with TBST three times, and then incubated with horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz, Calif, USA) at room temperature for 1 h. The signal was detected using an enhanced chemiluminescence kit (Boster, Wuhan, China).

Male nude mice (4- to 5-week old) were obtained from the Sichuan University Animal Center. Mice were housed in temperature-controlled rooms with a 12-h alternating light-dark cycle. HCT-116 cells (1 × 10⁷ cells/mL, 0.2 mL/mouse) were injected subcutaneously into the upper left flank region of mice. The mice were randomly divided into four groups (n = 6 per group). Mice in each group were treated daily with DA (0.3, 0.6, 1.2 mg/kg) or the same volume of saline as a negative control group by intraperitoneal administration every alternate day from day one. The dose of DA was based on our previous work that did not show any cytotoxic effect in normal mice. All the mice were sacrificed on day 13 after inoculation with HCT-116 cells. The tumor was measured for largest (a) and smallest (b) diameters, and the tumor volume was calculated as V = a × b²/2. Tumor inhibitory rates were calculated with the following formula: tumor inhibitory rate (%) = 1 − (tumor weight of treated group/tumor weight of control group) × 100%. Guidelines for the humane treatment of animals were followed as approved by the Sichuan University.

DA-embedded nude mouse xenograft tissues were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked using hydrogen peroxide for 20 min. The slides were incubated with primary antibody of Bcl-2 and Bax overnight at 4 °C, and then incubated with a second antibody for 2 h at room temperature. Binding streptavidin-HRP was 20 min at RT. Staining time of DAB (Boster, Wuhan, China) solution depended on the sample condition. The stained slides were visualized on a microscope. Images were captured with an attached camera linked to a computer.

The results are expressed as the mean ± SD. Multiple comparisons were performed by one-way ANOVA followed by Duncan’s post-hoc test using SPSS 18.0 (Chicago, IL). A value of p < 0.05 was considered statistically significant.