The HCT-116 human CRC cell line (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) was cultured with McCoy's 5A (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 IU penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C in 5% CO₂. The HF-91 human fibroblast cell line (Type Culture Collection of the Chinese Academy of Sciences) was cultured in DEME/H (Sigma-Aldrich; Merck KGaA) supplemented with 10% (vol/vol) heat-inactivated FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 IU penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C in 5% CO₂. HCT-116 and HF-91 cells were plated in 96-well cell culture plates at 5×10³ cells/well, and incubated for 24 h. Triplicate wells were then treated with GSPs at various concentrations (25, 50 or 100 µg/ml), 25 µg/ml 5-fluorouracil (5-Fu) as a positive control or PBS as the negative solvent control for 48 h prior to the analyses described below.

GSPs were purchased from Sigma-Aldrich (Merck KGaA; 20315-25-7). The antibodies used for western blotting were as follows: Mouse anti-caspase-2 (sc-5292), mouse anti-caspase-3 (sc-7272), mouse anti-caspase-9 (sc-56073), mouse anti-Bcl-2 (sc-7382), mouse anti-Bax (sc-20067), mouse anti-β-actin (sc-47778), rabbit anti-Bak (sc-7873), rabbit anti-mouse IgG-horseradish peroxidase (HRP; sc-358914) and mouse anti-rabbit IgG-HRP (sc-2357). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

HCT-116 and HF-91 cells were plated in 96-well cell culture plates at 5×10³ cells/well and incubated for 24 h at 37°C. Triplicate wells were then treated with GSPs at various concentrations (25, 50 or 100 µg/ml), 25 µg/ml 5-Fu as a positive control or PBS as the negative solvent control. Cell viability was determined after 48 h by adding 10 µl (5 mg/ml) cell proliferation reagent (MTT; Sigma-Aldrich; Merck KGaA) and incubating for 24 h at 37°C until a purple precipitate was visible. The precipitate was then solubilized by adding dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA). The absorbance was evaluated at 570 nm using a microplate reader. The percentage inhibition of cell viability was determined using the following formula: Percentage inhibition=1-optical density (OD)_{treatment group}/OD_{solvent control} ×100%.

Annexin V and fluorescein isothiocyanate (FITC) staining was performed using a commercial Annexin V-FITC kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells were treated with GSPs (25, 50 or 100 µg/ml), 25 µg/ml 5-Fu as a positive control or PBS as a solvent control for 48 h. The cells were then harvested, washed twice with cold PBS and resuspended in binding buffer at a concentration of 5×10⁵ cells/ml. A 100-µl aliquot containing 5×10⁴ cells was incubated in the dark with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) at room temperature for 15 min. Subsequently, 400 µl binding buffer was added and the cells were viewed (5 fields for each group) using a fluorescence microscope (Olympus Corporation, Tokyo, Japan) at magnification ×400. Viable cells were stained green and apoptotic cells were stained red. This experiment was repeated three times.

JC-1 analysis was performed using a commercial Mitochondrial Membrane Potential Detection JC-1 kit (BD Biosciences). Briefly, cells were treated with GSPs (25, 50 or 100 µg/ml), 25 µg/ml 5-Fu as a positive control or PBS as a solvent control for 48 h prior to harvesting, washing twice with cold PBS and resuspending in binding buffer at a concentration of 1×10⁶ cells/ml. A 100-µl aliquot was incubated with 5 µl JC-1 and 5 µl PI in the dark at room temperature for 15 min. Subsequently, 400 µl binding buffer was added and the cells were analyzed using a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). Cells negative for JC-1 and PI were considered to be viable; JC-1+/PI+ cells were in early apoptosis; JC-1+/PI- cells were necrotic or in late apoptosis. This experiment was repeated three times.

Total RNA was isolated from treated cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). First-strand cDNA was reverse transcribed using the PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan), in accordance with the manufacturer's protocols. Relative expression levels of mRNA were determined by qPCR using the primer sequences presented in Table I and a Thermo^® PikoReal 96 system (Thermo Fisher Scientific., Inc.). GAPDH was used as the endogenous reference gene. cDNA was subjected to 40 PCR cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec using FastStart Universal SYBR Green Master (Roche Diagnostics, Basel, Switzerland). Experiments were performed in triplicate and data were analyzed using Pikoreal software version 2.1. The average cycle threshold (Cq) values of the target genes were normalized to GAPDH gene expression level as 2^−ΔΔCq (35). Changes in expression levels were presented either as fold increases or as the ratio of the target gene expression in the treated cells to its expression level in the control cells.

Six proteins involved in apoptosis (caspase-2, caspase-3, caspase-9, Bcl-2, Bak and Bax) were analyzed by western blotting. HCT-116 cells were exposed to GSPs (25, 50 or 100 µg/ml) at a concentration of 5×10⁵ cells/well for 48 h in 6-well plates, harvested and lysed in RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.). Following a 30-min incubation on ice, the cell lysate was centrifuged at 10,000 × g at 4°C for 5 min and the supernatants were stored at −80°C until use. The protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (30 µg) were separated by 12% SDS-PAGE, transferred to polyvinyl difluoride membranes (EMD Millipore, Billerica, MA, USA) and blocked using 5% non-fat dry milk in Tris-buffered saline (TBS) for 2 h at room temperature. The membranes were then incubated with 1:1,000 dilutions of the aforementioned primary antibodies in TBS overnight at 4°C, washed three times with TBS-Tween-20 and incubated with 1:5,000 dilutions of the appropriate secondary antibodies conjugated with HRP in TBS for 1 h at room temperature. Membranes were washed three times in TBS-Tween-20 at room temperature. Protein bands were visualized on X-ray film using an enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) detection system. Western blotting bands were quantified using the Odyssey infrared imaging system version 1.2 (LI-COR Biosciences, Lincoln, NE, USA).

Data are presented as the mean ± standard deviations and all analyses were performed using Origin8 version 8.1.10.86 SRO (Origin8 Technologies Ltd., London, UK). Statistical significance was evaluated using two-way analysis of variance and Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

MTT assays of HCT-116 and HF-91 cells treated with GSPs (25, 50 or 100 µg/ml), 5-Fu (25 µg/ml) or solvent control indicated that GSPs reduced HCT-116 cell viability in a concentration-dependent manner, whereas the inhibition of GSPs in HF-91 cells was significantly lower compared with that in 5-Fu-treated cells (P<0.01; Fig. 1A). HCT-116 cells exposed to GSPs demonstrated cell shrinkage and membrane blebbing (Fig. 1B). Annexin V/PI double staining of the treated and untreated cells revealed GSP concentration-dependent formation of apoptotic bodies in HCT-116 cells (Fig. 2).

In the present study, the expression levels of p53 and cytochrome c increased in GSP-treated cells. Significant overexpression was observed in HCT-116 cells exposed to 100 µg/ml GSPs compared with 5-Fu, but not in those treated with lower concentrations of GSPs or with PBS (negative control; Fig. 3).

Considering the significance of caspases to apoptosis, the present study determines the mRNA and protein expression levels of initiator caspases (caspase-2 and −9) and an effector caspase (caspase-3). Upregulation of the mRNAs encoding caspase-2, caspase-3 and caspase-9 were observed in HCT-116 cells exposed to GSPs for 48 h (Fig. 4A). Furthermore, the expression levels of cleaved caspase-2, caspase-3 and caspase-9 protein were also significantly increased in cells exposed to 100 µg/ml GSPs compared with 5-Fu for 48 h (Fig. 4B and C).

To further investigate the mechanism underlying GSP-induced apoptosis, the present study evaluated the mRNA and protein expression levels of Bcl-2, Bax and Bak by qPCR, and western blotting. The Bcl-2 expression level in 100 µg/ml GSPs group was significantly reduced compared with 5-Fu, whereas the expression levels of Bax and Bak were upregulated in HCT-116 cells exposed to GSPs for 48 h (Fig. 5A-C).

Caspase-9 is involved in the mitochondrial apoptosis pathway and an increased expression level of cleaved caspase-9 therefore implied mitochondrial involvement in GSPs-induced apoptosis. JC-1 staining revealed that the percentage of cells with a loss of mitochondrial membrane potential increased from 18.7% in the negative control cells to 80.1% in the HCT-116 cells exposed to 100 µg/ml GSPs (Fig. 6A). The percentage of apoptotic cells observed in Fig. 6A is summarized in Fig. 6B. This loss of mitochondrial membrane potential, coupled with the observed increase in cleaved caspase-9, suggested that GSPs induced HCT-116 cell death via the mitochondrial apoptosis pathway.