Colorectal cancer HCT116 and CT26 cell lines were purchased from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/high glucose medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 12% heat-inactivated fetal bovine serum (Minhai Biological Engineering Co., Ltd., Lanzhou, China), and 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and cultured at 37°C in a humidified atmosphere of 5% CO₂. When the concentration of cells reached 1×10⁵ cells/ml for subculture the treatments were performed and all experiments were performed with cells in the logarithmic phase of growth. OSR was extracted from Sophoraalopecuroides L. (purity ≥98%; Chengdu Herbpurify Co., Ltd., Chengdu, China) and dissolved in normal saline (NS) at an initial concentration of 200 mg/ml and stored at −20°C. Fluorouracil (5-Fu) was obtained from Shanghai Xudong Haipu Pharmaceutical Co., Ltd., Shanghai, China. Primary antibodies against caspase-3, cytochrome c, Bcl-2, Bax and poly (adenosine 5′-diphosphate-ribose) polymerase 1 (PARP-1) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Trypsin and MTT were obtained from Sigma-Aldrich; Merck KGaA.

To assess cell growth inhibition ratio, HCT116 cells (5×10³) were seeded into 96-well plates (Guangzhou Jet Bio-Filtration, Co., Ltd., Guangzhou, China). Following overnight incubation at 37°C, different concentrations of OSR (3, 6, 12, 24, 48, 96 and 192 mg/l) were used to treat the cells, and the cells were incubated at 37°C for 48 h (6 wells for each concentration). Briefly, 20 µl MTT solutions (5 mg/ml) was added to each well and incubated for 4 h at 37°C, then 150 µl dimethyl sulfoxide was added to each well to dissolve MTT formazan, and the plate was shaken at room temperature for 15 min. The absorbance was measured at 490 nm with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The half maximal inhibitory concentration (IC₅₀) was calculated using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA).

To evaluate the cell growth curve, HCT116 cells (1×10³ cells/well) were inoculated into 96-well plates. After 24 h, the cells were treated with OSR at different concentrations (100, 50, and 25 mg/l) for 1 to 7 days. The control group was incubated in a drug-free medium, and the positive group was exposed to 5-Fu (20 mg/l) (21). The number of surviving cells from each group was counted daily using Trypan Blue staining assay, according to the directions below. A total of 10 µl 0.4% Trypan Blue solution was added to 90 µl of cell-suspension with sufficient mixing, and 10 µl solution was added on to the cell-count boards. Cells were then counted to assess the number of viable cells, under a light microscope (Olympus CX31) at ×10 magnification.

For the colony formation assay, 200 cells were seeded into 6-well plates and exposed to various concentrations of OSR (100, 50 and 25 mg/l) for 14 days at 37°C in 5% CO₂ atmosphere with a relative humidity of 95%. The colonies were fixed in pure methanol solution at room temperature for 15 min and then stained with Giemsa (3%) at room temperature for 30 min and washed 3 times with PBS, 5 min/time. Images of the colonies were captured and counted under a light microscope, and the colony formation inhibitory rate was calculated as follows: (1-mean number of colonies in the OSR group/mean number of colonies in the negative control group) ×100.

The HCT116 cells were seeded at a density of 5×10³ cells/ml into a 24-well plate. After 24 h, the cells were treated with OSR at various concentrations (100, 50, and 25 mg/l) for 48 h. The cells were washed with PBS three times, fixed with 4% neutral paraformaldehyde at room temperature for 30 min, washed with PBS three times and stained with 2 mg/l Hoechst for 30 min at room temperature. The chromatin structure of the cells was observed by fluorescence microscopy at ×200 magnification, and the apoptosis rate was calculated as follows: Apoptosis rate (%) = apoptosis cell number/(normal cell number + apoptosis cell number) ×100.

Total RNA was isolated from treated cells or tissues with TRIzol^® reagent (Bio Basic, Inc., Markham, ON, Canada), according to the manufacturer's protocol. Total RNA was reverse transcribed with the RevertAid™ First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. A total of 2 µl RNA samples were diluted by DEPC water, and the absorbance value (A) of RNA solution was detected by UV spectrophotometer at 260 and 280 nm respectively. The purity of RNA samples was determined according to the ratio of A260 nm/A280 nm. A ratio between 1.8–2.0, indicates that the purity of the RNA samples are high, whereas <1.8 demonstrates that the samples are contaminated by protein, and >2.0 demonstrated that the samples have RNA degradation. Primer sequences of all the genes are listed in Table I. A total reaction volume of 25 µl was used, composed of 2 µl cDNA, and 12.5 µl Taq Master mix (Beyotime Institute of Biotechnology, Haimen, China) and 8.5 µl nuclease-free water. The reaction conditions were as follows: 30 sec at 95°C for denaturation (1 cycle), annealing at 58°C for 30 sec and extension at 72°C for 30 sec, for a total of 35 cycles. The PCR products (10 µl) were visualized using electrophoresis on 2.0% agarose gel. The expression levels of the target mRNAs were normalized to the reference gene β-actin (22).

The cells were collected and lysed with lysis buffer (150 mM NaCl, 1.0 mM EDTA, 1% NP-40, 50 Mm Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml Leupeptin, 1 µg/ml aprotinin and 1 g/ml pepstatin) by incubating for 30 min at 4°C. The lysates were centrifuged at 13,500 × g for 15 min at 4°C, and the protein concentrations were determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc.). Equal amounts of total proteins (50 µg) were mixed with loading buffer, at 95°C for 5 min, and separated on 12% SDS-PAGE gels, and then blotted onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1 h, and incubated with specific primary antibodies at room temperature for 2 h. The primary antibodies used in this study were as follows: PARP-1 antibody (1:400; sc-8007), cytochrome c antibody (1:400; sc-514435), Bcl-2 antibody (1:400; sc-509), Bax antibody (1:400; sc-4239), caspase-3 antibody (1:400; sc-136219; all from Santa Cruz Biotechnology, Inc.). All of these antibodies were rabbit monoclonal antibodies. After washing with TBST (TBS containing 0.05% Tween-20, pH 7.6) three times, the membranes were incubated with goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (1:1,000; cat. no. Ba1055; Boster Biological Technology, Pleasanton, CA, USA) for 1 h at room temperature. Following washing with TBST three times, the proteins signal was detected using an electrochemiluminescence kit (Beyotime Institute of Biotechnology). Densitometric analyses of resultant western blots were performed with ImageJ software (ImageJ version 1.47 public domain software; National Institutes of Health, Bethesda, MD, USA).

Five-week old ICR male mice (weight, 18–22 g) were obtained from the Experimental Animal Center of Sichuan University (Chengdu, China). The mice were housed in an air-conditioned room, which was maintained at 23±2°C, and the relatively humidity of the house was kept at 55±5% CO₂ with a 12:12 h light/dark cycle, with free access to food and water. The CT26 cells were homogenized with NS at a ratio of (cell solution, NS=1:4; cell concentration, ~1×10⁶/ml). The mice were injected with the cell suspension (0.2 ml/mouse) subcutaneously at the right front axilla. Once transplanted, the tumor (~50 mg) was palpable. The mice were randomly divided into different treatment groups, with 10 mice in each group. The mice in the three OSR groups were administered daily with OSR at 300, 150 and 75 mg/kg of body weight via intraperitoneal injection (i.p). The mice in the positive control group were injected with (30 mg/kg) 5-Fu once a day, and the mice in the negative control group were administered daily with an equal volume of NS. From the third day, tumor volume (V) was measured using calipers on alternate days and calculated using the standard formula: V (mm³) = AB²/2, where A is the longest superficial diameter, and B is the smallest superficial diameter. After 13 days, all mice were sacrificed by dislocation of cervical vertebra, and the tumor tissues were removed and weighed. Then, the tumor tissues were used to detect the expression levels of caspase-3, Bax, Bcl-2, cytochrome c and PARP-1 by RT-PCR and western blotting. The animal experiments were approved by the Animal Ethics Committee of the Luohe Medical College (Luohe, China).

The data are presented as the mean ± standard deviation. Data were analyzed by one-way analysis of variance followed by Duncan's post-hoc test using the SPSS software package version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To detect the growth inhibition effects of OSR in HCT116 cells, cell growth inhibition rate, growth curve and colony-forming assays were performed. The HCT116 cells were treated with serial concentrations of OSR (3, 6, 12, 24, 48, 96 and 192 mg/l) for 48 h. The results demonstrate that the cell growth inhibition rate significantly increased in a dose-dependent manner (Fig. 2A), and that the IC₅₀ value was 59.28 mg/l. The cell growth curve assay indicated that OSR may inhibit HCT116 cell growth in a time and dose-dependent manner (Fig. 2B). Compared with the negative group, the colony formation assay demonstrated that OSR may inhibit the proliferation of HCT116 cells markedly. The colony formation inhibition rate significantly increased from 51.1% for 25 mg/l OSR to 93.9% for 100 mg/l OSR (P<0.01; Fig. 3).

The changes in the levels of apoptosis in OSR-treated HCT116 cells were detected via Hoechst 33258 nucleus staining. Compared with the negative group, the cells exhibited a marked increase in apoptosis following treatment with OSR for 48 h, exhibiting nuclear condensation, DNA fragmentation and the formation of apoptotic bodies. The cell apoptosis rate of the negative control group was 5.21%. Treatment with 25, 50 and 100 mg/l OSR significantly upregulated the rate of apoptosis from 46.37% (25 mg/1) to 87.62% (100 mg/l; P<0.01; Fig. 4).

To determine whether OSR is able to inhibit tumor growth in vivo, CT26-xenografts were established in ICR mice. It was identified that the tumor volume of the negative control group was higher compared with the tumor volume of the OSR-treated (150 and 300 mg/kg) mice from day 9 to 13. The growth curve assay indicated that OSR was able to inhibit tumor tissue growth significantly (P<0.05, P<0.01; Fig. 5).

The levels of caspase-3, Bax, Bcl-2, cytochrome c and PARP-1expression in HCT116 cells were analyzed by RT-qPCR and western blotting. The HCT116 cells were treated with different concentrations of OSR (25, 50 and 100 mg/l). Compared with the control group, treatment with 50 and 100 mg/l OSR downregulated the expression of Bcl-2 and PARP-1, whereas the levels of caspase-3, Bax and cytochrome c were upregulated (Fig. 6).

The caspase-3, Bax, Bcl-2, cytochrome c and PARP-1 expression levels in the transplanted CT26CRC tissues were also detected by RT-PCR and western blot analysis. Compared with the negative control group, the levels of Bcl-2 and PAPR-1 were significantly decreased in cells treated with 150 and 300 mg/kg OSR, and the levels of caspase-3, Bax and cytochrome c were increased in the OSR groups (150 and 300 mg/kg; Fig. 7).