Human colorectal carcinoma cell lines SW480 (ATCC^® CCL-228™) and HCT116 (ATCC^® CCL-247™) were purchased from the American Type Culture Collection (Manassas, VA, USA). The SW480 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HCT116 cells were cultured in McCoy’s 5A medium (Welgene, Inc., Gyeongsan, South Korea) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂. The pan-caspase inhibitor Z-VAD-FMK was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Inhibition of Fas and death receptor 5 (DR5) was performed using the small interfering RNA (siRNA). To transfect siRNA, SW480 and HCT116 cells were seeded into 6-well plates at a density of 3×10⁵ cells/well at 37°C such that they would be 40-50% confluent at the time of transfection. On the next day, 20 µM siRNA was transfected with 5 µl Lipofectamine^® RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocols. Further experiments were performed 24 h post transfection. Fas and DR5 siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Scrambled siRNA (AllStars Negative Control siRNA; Qiagen GmbH, Hilden, Germany) was used as a negative control. Reversine was purchased from Cayman Chemical (Ann Arbor, MI, USA).

SW480 and HCT116 cells were seeded onto a 96-well plate at a density of 1×10⁴ cells/well and were incubated for 24 h at 37°C. Cells were then treated with different concentrations of reversine (0, 0.5, 1, 5, 10, and 20 µM) with dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) for 24 and 48 h at 37°C. Subsequently, the cells were treated with WST-1 reagent (EZ-CYTOX; DoGen; Daeillab, Seoul, Korea) for 1 h at 37°C. After the treatment, absorbance at 450 nm was measured using a microplate reader (Infinite M200; Tecan Group Ltd., Mannedorf, Switzerland). Each experiment was conducted in triplicate wells and was repeated at least three times.

SW480 and HCT116 cells were seeded onto a 96-well plate at a density of 1×10⁴ cells/well and incubated for 24 h at 37°C. Cells were then treated with different concentrations of reversine (0, 0.5, 1, 5, 10 and 20 µM) with dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) for 24 at 37°C. After the reversine treatment, cells were stained with 1 µM Calcein-AM solution (Invitrogen; Thermo Fisher Scientific, Inc.) for 60 min at 37°C. The cells were observed and imaged using a fluorescence microscope (×200; DMI 3000B; Leica Microsystems GmbH, Wetzlar, Germany).

SW480 and HCT116 cells were seeded in a 6-well plate at a density of 5×10⁵ cells/well and were incubated for 24 h at 37°C prior to treatment with reversine. Subsequently, the cells were treated with reversine (0, 1, 5 and 10 µM) for 24 at 37°C, and were resuspended in 100 µl of 1X binding buffer (BD Biosciences, San Jose, CA, USA). These cells were incubated with 7-amino-actinomycin D and Annexin V-APC (BD Biosciences) for 20 min at room temperature. To analyze the number of apoptotic cells, a FACSCalibur flow cytometer (Becton-Dickinson and Company, Franklin Lakes, NJ, USA) and WinMDI version 2.9 (The Scripps Research Institute, San Diego, CA, USA) were used.

SW480 and HCT116 cells were seeded in a 6-well plate at a density of 5×10⁵ cells/well and were incubated for 24 h at 37°C. Subsequently, the cells were treated with reversine (0 and 1 µM) for 24 h at 37°C, and were fixed with 70% ethanol for 1 h at −20°C. These cells were resuspended in 100 µl of 1 U/ml RNase A (DNase free) for 20 min at room temperature and incubated with 10 µg/ml of propidium iodide (both from Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. To analyze the proportions of cell cycle, a FACSCalibur flow cytometer and WinMDI version 2.9 were used.

Reversine-treated cells were incubated with cell lysate buffer (1% NP-40 in 20 mM EDTA, and 50 mM Tris-HCl; pH 7.5) for 30 min on ice. The samples were then centrifuged at 12,000 × g for 30 min at 4°C. RNase A was added to the supernatant and incubated for 2 h at 56°C. Proteinase K was added to the supernatant and the mixture was incubated for 2 h at 37°C. An equal volume of isopropanol was added and the mixture was incubated at -80°C overnight to precipitate the genomic DNA. The genomic DNA was loaded onto 2% agarose gel and was stained with 0.1% ethidium bromide for 10 min at 37°C. The DNA was visualized under ultraviolet light transilluminator.

Reversine-treated cells were lysed in Radioimmunoprecipitation Assay extraction solution with Halt™ Phosphatase inhibitor and Halt™ Protease inhibitor cocktails (Thermo Fisher Scientific, Inc.) for 30 min on ice. The protein concentration was measured using a Bicinchoninic Acid protein assay (Thermo Fisher Scientific, Inc.). Equal amounts of 20 µg proteins were separated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes was blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies at 1:1,000 dilution. Primary antibodies against the following proteins were used: Cleaved poly(ADP-ribose) polymerase (PARP; cat. no. 5625), cleaved caspase-3 (cat. no. 9664), cleaved caspase-7 (cat. no. 8438), cleaved caspase-8 (cat. no. 9746), second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (SMAC/DIABLO; cat. no. 2954), B-cell lymphoma-extra large (Bcl-xL; cat. no. 2764), Fas (cat. no. 4233), DR5 (cat. no. 8074), p21 (cat. no. 2947), p27 (cat. no. 2552) and p57 (cat. no. 2557) (Cell Signaling Technology, Inc., Danvers, MA, USA), and GAPDH (cat. no. FL-335; Santa Cruz Biotechnology, Inc.). The membranes were washed four time with TBS-0.1% Tween-20 and were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody at 1:2,000 dilution for 1 h at room temperature. The HRP-conjugated anti-rabbit (cat. no. 7074) and anti-mouse (cat. no. 7076) secondary antibodies were purchased from Cell Signaling Technology, Inc. Protein bands were developed using an Enhanced Chemiluminescent reagent (GE Healthcare Life Sciences, Little Chalfont, UK) and the luminescent image analyzer LAS-4000 (Fujifilm, Tokyo, Japan) was used to analyze the bands.

The results are presented as the mean ± standard deviation. Differences between groups were determined by Student’s t-test. The statistical software program used was SPSS (Version 18.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Firstly, the effects of reversine on the growth of the human colorectal cancer cell lines SW480 and HCT116 were investigated. The human colorectal cancer cells were exposed to different concentrations of reversine (0-20 µM) for 24 and 48 h. The effects of reversine against the cells were determined with a cell proliferation assay, followed by observation under inverted microscopy. The cell proliferation assays and inverted microscopy demonstrated that reversine treatment significantly inhibited cell growth of SW480 and HCT116 cells, compared with the control cells (P<0.05) (Fig. 1A and B). These results indicated that reversine inhibits proliferation in human colorectal cancer cells.

In order to determine whether or not reversine induced apoptosis and cell cycle arrest in human colorectal cancer cells, flow cytometric analyses and a DNA fragmentation assay were performed. Human colorectal cancer cells were exposed to different concentrations of reversine (0-20 µM) for 24 h. Reversine treatment (0.5-10 µM) induced an increase in DNA fragmentation in SW480 and HCT116 cells, compared with non-treated control (Fig. 2A). The proportion of early and late apoptotic cells within the group treated with reversine was increased, compared with the non-treated control, in SW480 and HCT116 cells (Fig. 2B). To determine the activation of caspases, key enzymes of apoptosis, the caspase-specific activities were further investigated. The expression of cleaved PARP, caspase-3, -7 and -8 was upregulated in the SW480 and HCT116 cells following reversine treatment, in a dose-dependent manner (Fig. 2C). Additionally, whether reversine treatment resulted in modulation of apoptosis-regulatory proteins was further examined. As depicted in Fig. 2C, reversine treatment resulted in an increase in the levels of pro-apoptotic protein SMAC/DIABLO in SW480 and HCT116 cells, but the levels of the anti-apoptotic protein Bcl-xL were not altered in response to reversine treatment. In addition, the effect of reversine on death receptor signaling pathways was investigated, and it was observed that the expression of DR5 and Fas proteins was upregulated by reversine treatment in SW480 and HCT116 cells (Fig. 2C). Reversine treatment resulted in cell cycle arrest in the subG1 and G2/M phase of SW480 and HCT116 cells (Fig. 3A). Subsequently, the effects of reversine treatment on various CDK inhibitors (CDKIs) involved in cell cycle arrest in human colorectal cancer cells were evaluated. The p21, p27 and p57 protein levels were notably increased by reversine treatment in SW480 and HCT116 cells (Fig. 3B). Cyclins and CDKs were negatively regulated by CDKIs. Additionally, we examined the effects of reversine on the expression levels of cyclin D1 and CDK2. The cyclin D1 protein level was notably decreased by reversine treatment in SW480 and HCT116 cells. The CDK2 protein level was notably decreased in SW480 cells but was not altered in response to reversine treatment in HCT116 cells (Fig. 3B).

The pan-caspase inhibitor Z-VAD-FMK (20 µM) was used to determine whether cell growth was inhibited and apoptosis was induced by reversine treatment. The inhibition of cell growth by reversine treatment was reversed in SW480 and HCT116 cells by Z-VAD-FMK treatment (Fig. 4A). The increase in the number of early and late apoptotic cells by reversine treatment was also attenuated in SW480 and HCT116 cells by Z-VAD-FMK treatment (Fig. 4B). Furthermore, Z-VAD-FMK abrogated the reversine-induced PARP, and caspase-3 and -8 activation. However, Z-VAD-FMK did not notably affect reversine-induced DR5 and Fas protein expression (Fig. 4C). Therefore, reversine treatment exerts inhibition of cell growth and promotion of apoptosis via direct activation of caspases in human colorectal cancer cells.

To confirm whether Fas and DR5 signaling pathways are directly upregulated by reversine treatment, SW480 and HCT116 cells were transfected with Fas and DR5 siRNAs for 24 h, and then 5 µM reversine was added. To determine whether activation of Fas and DR5 is associated with reversine-induced cell apoptosis, flow cytometry was conducted. The increase in the number of early and late apoptotic cells by reversine treatment was attenuated following inhibition of Fas and DR5 by siRNA in SW480 and HCT116 cells (Fig. 5A). Inhibition of Fas and DR5 by siRNA suppressed reversine-induced cell cycle arrest of the subG1 and G2/M phase (Fig. 5B). Inhibition of Fas and DR5 by siRNAs suppressed reversine-induced upregulation of the Fas and DR5 proteins. Reversine-induced caspase-8 and -3, and PARP activation was attenuated following inhibition of Fas and DR5 by siRNA (Fig. 5C). These results indicated that reversine-induced apoptosis and cell cycle arrest in human colorectal cancer cells is mediated by activation of the Fas and DR5 signaling pathways.