RPMI-1640 medium, fetal bovine serum (FBS) and trypsin were purchased from Gibco; Thermo Fisher Scientific, Inc. The Cell Counting Kit-8 (CCK-8) was provided by Dojindo Co., Ltd. The Hoechst 33258 staining kit, caspase activity assay kit, ROS assay kit, Z-VAD-FMK, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and N-acetyl-L-cysteine (NAC), were purchased from Beyotime Institute of Biotechnology. Antibodies against cyclin D1 (CCND1; cat no. BS1741), cyclin-dependent kinase (CDK)4 (cat no. MB0027), MMP-2 (cat no. BS1236), MMP-9 (cat. no. bs1241) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat no. AP0063) were purchased from Bioworld Technology, Inc. The Cell cycle and FITC Annexin V Apoptosis Detection kits were obtained from BD Pharmingen; BD Biosciences. The CytoSelect™ 48-Well Cell Adhesion Assay and CytoSelect™ 24-Well Cell Invasion Assay kits were manufactured by Cell Biolabs, Inc. α-Solanine was purchased from Santa Cruz Biotechnology, Inc. (CAS no. 20562-02-1, cat no. sc-252340, lot no. E1619) and its purity (as assessed by high-performance liquid chromatography) was >95%. The chemical structure of α-solanine is shown in Fig. 1. α-Solanine (10 mM) was dissolved in dimethyl sulfoxide (DMSO) and the same volume of DMSO was used as control.

Human colorectal cancer RKO and HCT-116 cells were obtained from and authenticated with short-tandem repeat analysis by The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium with 10% FBS and 1% streptomycin--penicillin, and maintained at 37°C in a humidified atmosphere containing 5% CO₂.

In this study, 1×10⁴ colorectal cancer cells were inoculated into 96-well plates, and different concentrations of α-solanine with the same volume of DMSO were added after 24 h; an equal volume of DMSO was used as control. At different time points during treatment, 10 µl CCK-8 solution was added to the wells and incubated at 37°C for 2 h, and the optical density (OD) was measured by a plate reader (SpectraMax M5; Molecular Devices LLC) at the read mode of absorbance (450 nm). The cell survival rate was calculated as follows: Cell survival rate (%)=(experimental OD value/control OD value) ×100%. The IC₅₀ of α-solanine was calculated using IBM^® SPSS^® Statistics 25 (IBM Corp.).

RKO cells (3×10⁵) were inoculated in 6-well plates. At 24 h after inoculation, the cells were treated with 0–25 µM α-solanine (final concentration) with the same volume of DMSO. After 48 h of treatment, the cells were collected, in with 75% ethanol, washed with phosphate-buffered saline (PBS), stained with 400 µl propidium iodide (PI) solution in the dark at room temperature for 15 min, and the cell cycle distribution was detected by flow cytometry.

A total of 5×10³ colorectal cancer cells were inoculated into 24-well plates and treated with different doses of α-solanine with the same volume of DMSO after 24 h of adherence. After 48 h of treatment, the cells were fixed by the fixative solution provided with the kit according to the manufacturer's instructions, stained by Hoechst 33258 for 20 min at room temperature in the dark, washed with PBS, and the cell morphology was observed under a fluorescence microscope (magnification, ×200).

Apoptosis was detected according to the manufacturer's instructions. Briefly, 3×10⁵ colorectal cancer cells were inoculated into 6-well plates and treated with different concentrations of α-solanine with the same volume of DMSO after 24 h of adherence. After 48 h of treatment, the cells were harvested, washed with PBS, resuspended in binding buffer, labeled by Annexin V-FITC, incubated with PI, and apoptosis was detected by flow cytometry.

Caspase activity was detected according to the manufacturer's protocol. In brief, colorectal cancer cells were treated with different doses of α-solanine with the same volume of DMSO for 48 h, collected, lysed and quantified. The activities of caspase-3, −8 and −9 were detected using Ac-DEVD-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA as substrates, respectively. For inhibition of caspases, RKO cells were pre-incubated with Z-VAD-FMK (100 µM) for 2 h, and then treated with different concentrations of α-solanine for 48 h.

RKO cells (3×10⁵) were inoculated into 6-well plates and, after 24 h of adherence, the cells were treated with 0–25 µM α-solanine (final concentration) with the same volume of DMSO. After 48 h of treatment, the cells were stained with 1 ml DCFH-DA (1:1,000) at 37°C for 20 min and washed with serum-free RPMI-1640 medium. A fluorescence microscope was utilized for visualization, and fluorescence was detected by a plate reader (excitation wavelength at 488 nm and emission wavelength at 525 nm). For ROS inhibition, RKO cells were incubated with NAC (250 µM) for 2 h, and were then treated with different concentrations of α-solanine for 48 h.

Cell adhesion was measured according to the manufacturer's protocol. Briefly, 5×10⁵ RKO cells were inoculated into 6-well plates and treated with 0–12 µM α-solanine (final concentration) with same volume of DMSO. After 48 h of treatment, cells were collected and inoculated in extracellular matrix (ECM) pre-coated 24-well plates (1.5×10⁵), and cultured for 90 min. The cells were stained with Cell Stain Solution for 10 min at room temperature, and extracted with Extraction Solution for 10 min on an orbital shaker. The OD values (560 nm) of the wells were detected by a plate reader.

Cell migration was measured using the scratch assay (17,18). Briefly, 8×10⁵ serum-starved colorectal cancer cells were inoculated into 6-well plates and cultured until the cell confluence was >95%. The cell layer was scratched on the bottom of the well with a sterile 20-µl pipette tip, washed with PBS, fresh culture medium was added, and the cells were treated with different doses of α-solanine with the same volume of DMSO for 48 h. Cell migration to the center of the scratch was observed under a phase-contrast microscope (magnification, ×50) and measured by Adobe Photoshop CC 2014.

The cell invasion assay was performed according to the manufacturer's instructions. Briefly, 3×10⁵ colorectal cancer cells were added into the inner side of the insert and different doses of α-solanine with the same volume of DMSO was added to the wells. In addition, 500 µl RPMI-1640 supplemented with 10% FBS was added to the lower well of the invasion plate. After 48 h of treatment, the invading cells were stained with Cell Stain Solution for 10 min at room temperature, washed with PBS, and observed under a phase-contrast microscope (magnification, ×200). For quantification, the invading cells were extracted with the Extraction Solution provided by manufacturer for 10 min on an orbital shaker and measured at an OD value of 560 nm by a plate reader.

Western blot analysis was performed as described previously (19,20). In brief, RKO cells were treated with 0–25 µM α-solanine (final concentration) with the same volume of DMSO for 48 h, collected, lysed with RIPA buffer (Beyotime Institute of Biotechnology), quantified by BCA Protein Assay Kit (Beyotime Institute of Biotechnology), and subjected to 8–10% SDS-PAGE. The proteins (40 µg/lane) were transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat milk for 2 h at room temperature, probed with antibodies against CNCD1 (1:1,000), CDK4 (1:1,000), MMP-2 (1:1,000), MMP-9 (1:1,000) and GAPDH (1:1,000) at 4°C overnight and washed with PBST (0.1% Tween-20). The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized by an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.).

The activities of MMP-2 and MMP-9 were determined by gelatin zymography. Briefly, α-solanine-treated cell culture supernatant was collected, quantified, and subjected to 10% SDS-PAGE containing 1 mg/ml gelatin without prior denaturation. The gels were washed with 2.5% Triton X-100 for 2 h at room temperature to remove SDS, washed with ddH₂O, incubated with developing buffer overnight at 37°C, stained with Coomassie brilliant blue for 3 h at room temperature and de-stained in methanol/acetic acid. The activities of MMP-2 and MMP-9 were detected as unstained bands against a blue background.

A total of 10 male 6–7-week-old BALB/c nude mice were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. The mice were acclimatized for 1 week under pathogen-free conditions at the animal laboratory of Longhua Hospital with a 12-h light/dark cycle, regulated temperature of 24±2°C and humidity of 50±10%, with ad libitum access to water and animal chow. The mice were injected with 2×10⁶ RKO cells in 0.1 ml PBS in the right flank. When the tumors were palpable, the mice were randomly divided into two groups (5 mice/group) that did or did not receive 5 mg/kg α-solanine once per day. The tumor length (L) and width (W) were measured every 2 days by calipers and the tumor volume (Tv) was calculated according to the formula: Tv=π/6 × L × W². After 12 days of treatment (the diameter of the largest tumor was 17.68 mm), the mice were sacrificed under anesthesia and the tumors were removed, weighed and subjected to further experiments. All studies involving mice were approved by the Longhua Hospital Animal Care and Use Committee.

Apoptotic cells in tumor tissue were identified by TUNEL assay following the instructions of the manufacturer (Roche Diagnostics). The slides were examined under an Olympus light microscope (Olympus Corporation). Three fields at a magnification of ×200 were randomly selected from each slide and apoptotic cells were counted by Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

The tumor tissues were fixed, embedded in paraffin, cut into 4-µm sections, blocked with 3% hydrogen peroxide, probed with Ki-67 antibody (1:100) at 4°C overnight, followed by incubation with HRP-conjugated anti-rabbit/anti-mouse IgG secondary antibody (1:4,000) at 37°C for 30 min and visualized using 3,3-diaminobenzidine at room temperature for 3 min. The sections were then counterstained with hematoxylin for 45 sec at room temperature and mounted with glass coverslips. The slides were examined under an Olympus light microscope (Olympus Corporation). Three fields at a magnification of ×200 were randomly selected from each slide and analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

Data are expressed as mean ± standard deviation, and were analyzed by one-way analysis of variance and Dunnett's, Student-Newman-Keuls or least significance difference test. P<0.05 was considered to indicate statistically significant differences.

We first investigated the effects of α-solanine on cell growth. As shown in Fig. 2, 13–32 µM α-solanine significantly inhibited the proliferation of RKO cells in a dose-dependent manner, and the proliferation of RKO cells was fully inhibited following treatment with 32 µM α-solanine for 72 h (P<0.01). The IC₅₀ of α-solanine was 20.84 µM. Furthermore, 19–25 µM α-solanine inhibited the proliferation of RKO cells in both a dose- and time-dependent manner (P<0.05). Following treatment with α-solanine, the RKO cells became larger, exhibited a lower confluence, and 7.03, 12.52 and 29.78% of the cells died and floated in the 19, 22 and 25 µM groups, respectively.

In the present study, the effects of α-solanine on the cell cycle distribution of RKO cells were examined by flow cytometry. As displayed in Fig. 3, treatment with α-solanine increased the number of RKO cells in the G0/G1 phase and decreased the number of RKO cells in the S phase and G2/M phase in a dose-dependent manner (P<0.01), indicating that α-solanine caused cell cycle arrest in the G0/G1 phase. Furthermore, the results of western blotting revealed that 19–25 µM α-solanine inhibited the expression levels of CCND1 and CDK4 in a dose-dependent manner.

The effects of α-solanine on apoptosis were investigated. Hoechst 33258 staining revealed that, after treatment with α-solanine, the number of RKO cells was decreased, and their distribution was sparse. In addition, the nuclei of some cells were densely stained or fragmented (9.81, 17.43 and 24.17% of the cells in the 19, 22 and 25 µM groups, respectively), indicating the occurrence of apoptosis. Furthermore, Annexin V-FITC/PI labeling and flow cytometry demonstrated that α-solanine at 19–25 µM promoted apoptosis of RKO cells in a dose-dependent manner (P<0.01; Fig. 4).

Apoptosis is a caspase-mediated programmed cell death (21). The effects of α-solanine on caspase activity were detected by an enzyme-substrate system. The results indicated that the activities of caspase-3, −8 and −9 were significantly increased following treatment with α-solanine (P<0.01). Z-VAD-FMK, a caspase inhibitor, was able to antagonize the effects of α-solanine on of RKO cell apoptosis (P<0.05), which suggested that caspases contributed to α-solanine-induced apoptosis (Fig. 4).

High levels of ROS could promote apoptosis of tumor cells (22). It has been reported that α-solanine could induce the production of ROS in hepatocellular carcinoma cells (16). In the present study, ROS levels were detected by DCFH-DA staining. The findings demonstrated that, following treatment with α-solanine, the number of RKO cells emitting green fluorescence was increased, and the fluorescence density was markedly increased in a dose-dependent manner (P<0.01), indicating that α-solanine increased the ROS level in RKO cells. In addition, NAC, as a ROS scavenger, antagonized the effects of α-solanine on caspase-3, −8 and −9 (P<0.05) and apoptosis (P<0.01), suggesting that ROS contributed to α-solanine-induced activation of caspase-3, −8 and −9 and promoted apoptosis of RKO cells (Fig. 5).

The scratch assay is a common method for detecting cell migration, which is widely used in various cell models (17,18). The results of the scratch assay revealed that the migration of RKO cells to the center of the scratch was reduced following treatment with low-dose α-solanine, which did not significantly affect cell proliferation, demonstrating that α-solanine inhibited the migration of RKO cells. Further studies revealed that low-dose α-solanine also inhibited the invasion and adhesion of RKO cells in a dose-dependent manner (P<0.01). In addition, low-dose α-solanine inhibited the expression and activities of MMP-2 and MMP-9 in RKO cells (Fig. 6).

The effects of α-solanine on HCT-116 cells were also evaluated. As shown in Fig. 7, 11–28 µM α-solanine significantly inhibited the proliferation of HCT-116 cells in a dose-dependent manner (P<0.05). The IC₅₀ of α-solanine was 20.32 µM. α-Solanine induced apoptosis as indicated by Hoechst 33258 staining and flow cytometry analysis (P<0.01). α-Solanine also activated caspase-3, −8 and −9 (P<0.01). In addition, α-solanine inhibited the migration and invasion of HCT-116 cells (P<0.01).

To observed the effects of α-solanine on tumor growth, an RKO tumor was established in BALB/c nude mice. When the tumors became palpable, the mice were treated with α-solanine. As shown in Fig. 8, the tumors grew progressively in control group, whereas α-solanine significantly inhibited tumor growth as demonstrated by tumor volume and tumor weight (P<0.05). The expression of Ki-67, a proliferation marker, was detected by immunohistochemistry. α-Solanine inhibited Ki-67 expression, suggesting that α-solanine can inhibit cell proliferation in vivo (P<0.01). TUNEL assay was further used to identify apoptosis. Consistent with the in vitro observations, α-solanine induced apoptosis in vivo (P<0.01). The potential toxicity of α-solanine was also evaluated. Following treatment with α-solanine, no significant adverse consequences were observed, such as weight loss (Fig. 8C), ruffling of the fur, or changes in behavior and feeding (data not shown).