Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium and fetal bovine serum (FBS) were supplied by Hyclone; GE Healthcare Life Sciences (Logan, UT, USA). MTT, SFN and Cisplatin (CPDD; cat. no. P4394) were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The ROS, phosphorylated (p)-p38 and Erk inhibitors [ammonium pyrrolidinedithiocarbamate (APDC), SB203580 and SCH772984], the caspase 3, 7, 8 and 9 activity assay kits (cat. nos. C1116, C1136, C1151 and C1157), ROS Assay kit (cat. no. S0033), Lipid Peroxidation Malondialdehyde (MDA) Assay kit (cat. no. S0131), Total Superoxide Dismutase (SOD) Assay kit with water-soluble tetrazolium salt (WST-8; cat. no. S0101) and Cellular Glutathione Peroxidase (GSH-Px) Assay kit (cat. no. S0056), were all supplied by Beyotime Institute of Biotechnology (Nanjing, China). Protein isolation and bicinchoninic acid (BCA) protein quantification kits were supplied by Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit was obtained from Multi Sciences (Lianke) Biotech Co, Ltd. (Hangzhou, China). Primary antibodies against B-cell lymphoma (Bcl-2; cat. no. BS4024), Bcl-2-associated X protein (Bax; cat. no. MB9013), p73 (cat. no. BS2452), p53 upregulated modulator of apoptosis (PUMA; cat. no. BS2922), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA; cat. no. BS6214) and γH2AX (cat. no. BS4760) were obtained from Bioworld Technology, Inc. (St. Louis Park, MN, USA). Primary antibodies against p-Erk 1/2 (cat. no. sc-23759-R), p-p38 (cat. no. sc-7973) and p-JNK (cat. no. sc-135642) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The primary antibody against β-actin (cat. no. BA2359) was supplied by Wuhan Boster Biological Technology, Ltd. (Wuhan, China). IRDye-conjugated anti-rabbit (cat. no. 611-645-122) and anti-mouse (cat. no. 610-142-002) secondary antibodies were obtained from Rockland Immunochemicals, Inc. (Limerick, PA, USA).

The human colon adenocarcinoma cell line, SW480, and the human colon cancer cell line, HCT-116, were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). SW480 and HCT-116 cells were cultured in DMEM and RPMI-1640 medium, respectively, supplemented with 10% (v/v) FBS, and incubated at 37°C in a humidified 5% CO₂ incubator.

Cell viability was determined using an MTT assay. Briefly, SW480 or HCT-116 cells were plated into 96-well culture plates at an optimal density of 5×10³ cells/well. SW480 cells were incubated for 24 h until cells had reached 90% confluence, and were pretreated with or without 0.1 µM of the specific inhibitors against p38, Erk and ROS for 1 h, before they were treated with 0, 1, 5, 10, 15 and 20 µM SFN for 3, 6, 12, 24 or 48 h, respectively. HCT-116 cells were incubated for 24 h until cells had reached 90% confluence before they were treated with 0, 1, 5, 10 or 15 µM SFN, together with 0 or 10 µM CPDD, for 24 h. The supernatant was replaced with 100 µl (500 µg/ml) MTT solution and incubated for a further 4 h. The supernatant was removed and 150 µl dimethyl sulfoxide was added to dissolve the formazan crystals. The optical density was measured at 570 nm for each sample using a microtiter plate reader (BioTek Instruments, Inc, Winooski, VT, USA). The results are expressed as the percentage viability of the control at each time point (100%).

Cell apoptosis was analyzed using an annexin V-FITC and PI double-staining method. Briefly, SW480 cells were seeded in 6-well plates at 3×10⁵ cells/well and incubated for 24 h until they had reached 90% confluence. Cells were then treated with 0, 1, 5, 10, 15 and 20 µM SFN for 24 h, and adherent cells were washed with PBS, collected and re-suspended in a binding buffer containing 1% (v:v) annexin V-FITC and 2% (v:v) PI. Cells were then incubated in the dark at room temperature for 20 min. Apoptotic cells were quantified using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). A minimum of 1×10⁵ cells for each sample were collected for flow cytometry analysis, recorded on a dot plot, and the percentage of apoptotic cells in the population was calculated using ModFit software (version 2.0.0; Verity Software House, Inc, Topsham, ME, USA).

The protein expression levels of p73, PUMA, NOXA, p-p38, p-Erk, p-JNK, Bcl-2 and Bax were measured in SW480 cells, and the expression levels of γH2AX were measured in HCT-116 cells by western blot analysis. β-actin was used as a loading control for these cell lines. Briefly, SW480 or HCT-116 cells were seeded in 25 cm² cell culture dishes (1×10⁶ cells/dish) and incubated for 24 h until they reached 90% confluence. SW480 and HCT-116 cells were treated with 0, 1, 5, 10, 15 and/or 20 µM SFN, together with 0 or 10 µM CPDD, for 24 h, and the adherent cells were washed with PBS, collected and lysed using a protein isolation kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. Protein concentrations were quantified using a BCA protein quantification kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. Whole cell lysates were mixed with a loading buffer (cat. no. P0015; Beyotime Institute of Biotechnology), and a total of 40 µg cell lysate was boiled at 95°C for 5 min and then separated by 8–12% SDS-PAGE. The protein bands were transferred to polyvinylidene fluoride (PVDF) membranes according to manufacturer's instructions (EMD Millipore, Billerica, MA, USA). The PVDF membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% non-fat skim milk at 4°Cfor 1 h, rinsed with TBST and probed with primary antibodies at 4°Covernight (dilution, 1:500). Membranes were then incubated with the corresponding IRDye-conjugated secondary antibodies (dilution, 1:2,000) at room temperature for 1 h. Membranes were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The relative optical densities of the target proteins were analyzed using Quantity One software (v4.62, Bio-Rad Laboratories, Inc.). The expression levels of target proteins were normalized to that of β-actin internal controls.

The activities of caspase-3, −7, −8 and −9 were determined using caspase activity kits (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, SW480 cells were seeded in 25 cm² cell culture dishes (1×10⁶/dish) and incubated for 24 h until they reached 90% confluence. Cells were then treated with 0, 1, 5, 10, 15 and 20 µM SFN for 24 h. Adherent cells were washed with PBS, collected and lysed with Radioimmunoprecipitation (RIPA) Lysis Buffer (cat. no. P0013C; Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA protein quantification kit (Bio-Rad Laboratories, Inc.). Equal quantities (4 µg/ml) of whole cell lysates were then incubated with the corresponding caspase substrates to a final concentration of 2 mM at 37°Cfor 2 h. Samples were measured using a EL800 Microplate Reader (BioTek Instruments, Inc.) at 488 nm excitation and 525 nm emission.

The MMP was analyzed by staining SW480 cells using a JC-1 probe (C2005, Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, SW480 cells were seeded in 96-well plates at 3×10³ cells/well and incubated for 24 h until reaching 90% confluence. The cells were subsequently treated with 0, 1, 5, 10, 15 and 20 µM SFN for 24 h. The cells were then incubated with 100 µl JC-1 staining solution (5 µg/ml) at 37°C for 20 min, before cells were rinsed twice with phosphate-buffered saline. The MMP was measured by determining the relative quantity of dual emissions from mitochondrial JC-1 monomers or aggregates using a fluorescence EL800 Microplate Reader (BioTek Instruments, Inc.) at excitation and emission wavelengths of 488 and 525 nm, respectively. Mitochondrial depolarization is expressed as the red/green fluorescence intensity ratio, according to manufacturer's recommendations.

The production of ROS was analyzed by staining SW480 cells with a dichlorodihydrofluorescein diacetate (H₂DCF-DA) probe. Briefly, the SW480 cells were seeded in 96-well plates at 3×10³ cells/well and incubated for 24 h until they reached 90% confluence. The cells were then treated with 0, 1, 5, 10, 15 and 20 µM SFN for 24 h, before the supernatant was removed. Cells were subsequently incubated with H₂DCF-DA solution (2 µM) for 30 min at room temperature and rinsed three times using FBS-free medium. The fluorescence intensity was measured using a fluorescent ELISA reader (BioTek Instruments, Inc.) at excitation and emission wavelengths of 488 and 525 nm, respectively.

MDA generation was detected using a lipid peroxidation MDA assay kit based on thiobarbituric acid (TBA) method. SW480 cells were seeded, cultured and treated with SFN using the same aforementioned methods, before the supernatant was subsequently collected and incubated with TBA (at a dilution of 1:200) at 100°C for 15 min. The remaining procedures were performed according to the manufacturer's instructions (Beyotime Institute of Biotechnology). The MDA content in the supernatant was measured at 532 nm using an EL800 Microplate Reader (BioTek Instruments, Inc.).

SOD activities in total cell lysates were examined using a total SOD assay kit based on the WST-8 method (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, SW480 cells were seeded in 25 cm² cell culture dishes (1×10⁶/dish) and incubated for 24 h until they reached 90% confluence. Cells were then treated with 0, 1, 5, 10, 15 and 20 µM SFN for 24 h. Subsequently, the adherent cells were washed with PBS, collected and lysed with RIPA Lysis Buffer (Beyotime Institute of Biotechnology) according to the buffer manufacturer's instructions. The protein concentration was determined using a BCA protein quantification kit (Bio-Rad Laboratories, Inc.). Equal quantities of whole cell lysates (4 µg/µl) were incubated with WST-8 solution (at a dilution of 1:200) at 37°C for 30 min, and the remaining procedures were performed according to the manufacturer's instructions (Beyotime Institute of Biotechnology). SOD activity in total cell lysates was measured at 550 nm using the EL800 Microplate Reader (BioTek Instruments, Inc.).

GSH-Px activities in total cell lysates were examined using a cellular GSH-Px assay kit. Briefly, SW480 cells were seeded in 25 cm² cell culture dishes and incubated for 24 h until they reached 90% confluence. The cells were then treated with 0, 1, 5, 10, 15 and 20 µM SFN for 24 h before the adherent cells were washed with PBS, collected and lysed with the RIPA Lysis Buffer (Beyotime Institute of Biotechnology), according to the buffer manufacturer's instructions. Protein concentrations were quantified using a BCA protein quantification kit. Equal quantities of whole cell lysates (2 µg/µl) were incubated with the assay kit buffer (GPx detection working buffer, at a 1:1 dilution) at 37°C for 30 min, and the remaining procedures were performed according to the manufacturer's instructions. The GSH-Px activity in total cell lysates was measured at 412 nm using the EL800 Microplate Reader (BioTek Instruments, Inc.).

The results are presented as the mean ± standard deviation of three independent experiments and the statistical significance was analyzed by one-way analysis of variance followed by a post-hoc Dunnett's test. Analysis was performed using SPSS 16.0 (SPSS, Inc, Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of SFN on SW480 cell growth, the cells were treated with increasing concentrations (0, 1, 5, 10, 15 and 20 µM) of SFN for 3, 6, 12, 24 and 48 h, and an MTT assay was performed to assess cell viability. As shown in Fig. 1A, SFN inhibited SW480 cell proliferation in a dose- and time-dependent manner. When compared with the untreated control group at each time point, the viability of cells treated with 5, 10, 15 and 20 µM SFN were significantly inhibited (P<0.05 and P<0.01). In addition, the viability of cells treated with 20 µM SFN at 3, 6, 12, 24 and 48 h were significantly reduced when compared with controls, with 70.2, 66.3, 51.5, 38.6 and 20.1% viabilities, respectively (P<0.01; Fig. 1A). However, compared with the untreated controls, no significant differences in the viability of cells treated with 1 µM SFN for 3, 6, 12, 24 and 48 h were observed. Therefore, for all subsequent assays, cells were exposed to SFN for 24 h as it exhibited an appropriate inhibiting ratio for cell viability.

In order to determine whether the SFN-induced inhibition of SW480 cell viability was due to the induction of apoptosis, the proportion of apoptotic cells in the population was analyzed by flow cytometry using FITC and PI double-staining. As shown in Fig. 1B and summarized in Fig. 1C, exposure of SW480 cells to 5, 10, 15 and 20 µM SFN for 24 h, significantly increased the percentage of apoptotic cells when compared with the untreated controls (P<0.01); the percentage of apoptotic cells in the 20 µM SFN-treated group reached 71.9%. However, 1 µM SFN failed to induce a significant difference in the proportion of apoptotic cells (Fig. 1B and C). These results indicated that SW480 cells may undergo apoptosis following treatment with SFN, and suggested an association between the induction of apoptosis and the inhibition of cell viability.

To determine whether SFN-induced SW480 cell apoptosis is p73-dependent, the protein expression levels of p73, as well as its downstream effectors, PUMA and NOXA, were detected. As shown in Fig. 2A, p73 protein was not detected in the untreated control group nor in the SFN-treated groups. By contrast, PUMA and NOXA were expressed in the SW480 cells; however, no significant differences were observed between the untreated and SFN-treated groups (Fig. 2A).

The present study further investigated the role of caspase-3, −7, −8, and −9 in SFN-induced SW480 cell apoptosis. As shown in Fig. 2B, the activity of the intrinsic apoptotic executioners, caspase-3, −7 and −9, were significantly increased in the 5, 10, 15 and 20 µM SFN-treated groups when compared with the untreated control group (P<0.01). By contrast, the activity of the extrinsic apoptotic executioner, caspase 8, was not significantly induced under the same conditions (Fig. 2B).

In addition to p53 and p73, the MAPK signaling pathway regulates cellular proliferation and apoptosis under cell stress conditions (30). Therefore, the present study investigated whether SFN-induced apoptosis was regulated by the activation of Erk, JNK and p38. As shown in Fig. 3A, the protein expression levels of p-Erk and p-p38 were significantly increased in 5, 10, 15 and 20 µM SFN-treated groups when compared with the untreated control group (P<0.05 and P<0.01).

A previous study indicated that the anti-apoptotic functions of Bcl-2 and the pro-apoptotic functions of Bax are regulated by MAPKs (37). Therefore, the present study examined the protein expression levels of Bax and Bcl-2 in SFN-treated SW480 cells. As shown in Fig. 3B, the protein expression levels of Bcl-2 were significantly decreased in the 5, 10, 15 and 20 µM SFN-treated groups when compared with the untreated control group (P<0.05 and P<0.01); however, no significant differences in the level of Bax protein were detected in the SFN-treated groups compared with the controls. Accordingly, a significant increase in the Bax/Bcl-2 ratio was observed when compared with the untreated controls (P<0.05 and P<0.01; Fig. 3C). Previous studies have demonstrated that an increased Bax/Bcl-2 ratio induces alterations in the mitochondrial permeability transition; an event which subsequently leads to disruption of the MMP (38,39). Therefore, the present study investigated whether SFN exposure altered the MMAs shown in Fig. 3D, exposure to 5, 10, 15 and 20 µM SFN induced significant mitochondrial depolarization when compared with the untreated group, as indicated by the decreased red/green fluorescence ratio (P<0.01).

ROS are known to function as messengers that activate MAPKs (26). Therefore, the present study investigated whether SFN induced SW480 cell apoptosis via the generation of ROS. As shown in Fig. 4A, exposure to 5, 10, 15 and 20 µM SFN significantly increased ROS production when compared with the untreated group (P<0.01). This result was further confirmed by the significant increase in MDA production following exposure of cells to 1, 5, 10, 15 and 20 µM SFN when compared with untreated controls (Fig. 4B). MDA production is the direct consequence of ROS production (40). The activity of the anti-oxidative enzymes, SOD and GSH-Px, were not significantly altered following exposure to increasing concentrations of SFN (Fig. 4C and D).

To discriminate between the different roles of ROS, p38 and Erk in SFN-induced apoptosis, SFN-treated SW480 cells were treated with specific inhibitors against p38, Erk and ROS. First, the effects of the inhibitors on ROS generation were investigated. As shown in Fig. 5A, the ROS inhibitor, APDC, completely attenuated the SFN-induced production of ROS. By contrast, treatment with the p38 and Erk inhibitors (SB203580 and SCH772984, respectively), demonstrated no marked effects on the SFN-induced production of ROS. The effects of the inhibitors on cell viability were then evaluated. As presented in Fig. 5B, treatment of cells with the ROS inhibitor, APDC, completely attenuated the SFN-mediated inhibition of SW480 cell viability. By contrast, treatment with the p38 inhibitor (SB203580), and the Erk inhibitor (SCH772984) partially attenuated the SFN-mediated inhibition of SW480 cell proliferation. These results indicated that SFN increased ROS generation and induced cell apoptosis via the p38 and Erk cell signaling pathways.

Finally, the present study evaluated the ability of SFN to potentiate the inhibitory action of CPDD against the proliferation of p53-proficient HCT-116 colon cancer cells. The effects of SFN on CPDD-mediated inhibition of HCT-116 cell viability were first determined. As shown in Fig. 6A, treatment with 10 µM CPDD alone, and 10 µM CPDD combined with 5, 10 and 15 µM SFN, significantly inhibited HCT-116 cell viability when compared with the untreated control group (P<0.01). In addition, when compared with 10 µM CPDD treatment alone, combined SFN and CDDP treatment significantly reduced the viability of HCT-116 cells (P<0.05 and P<0.01; Fig. 6A). The anti-cancer activity of CPDD is based on its interactions with DNA and the subsequent inhibition of DNA replication, which leads to DNA damage (41). Therefore, the present study investigated the effect of SFN on CPDD-induced DNA damage by detecting the levels of γH2AX, a biomarker of DNA damage (42). As shown in Fig. 6B, treatment with 10 µM CPDD alone, or 10 µM CPDD combined with 5, 10 and 15 µM SFN, significantly increased the level of γH2AX when compared with the untreated group (P<0.05 and P<0.01). In addition, combined treatment with SFN and CDDP induced a significant increase in γH2AX expression when compared with CPDD treatment alone (P<0.05 and P<0.01; Fig. 6B), which suggested that SFN may increase the cytotoxic effects of CDDP by increasing the production of γH2AX.