The human hepatocellular carcinoma HepG2 cell line was provided by the Shanghai Cancer Institute (Shanghai, China). The HepG2 cells were cultured in HyClone™ Dulbecco's modified Eagle's medium (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) in a 5% CO₂ Forma™ humidified incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C. The cells were seeded onto a 6-well plate (4×10⁴ cells/well), allowed to adhere overnight, and subsequently treated with solanine (95% mass fraction; Sigma-Aldrich) with a final concentration of 2 mg/ml. The cells were incubated for 24–48 h. A negative control [dimethyl sulfoxide (DMSO); Sangon Biotech Co., Ltd., Shanghai, China] and a positive control (camptothecin, 0.5 mg/ml; Sigma-Aldrich) were used.

Various concentrations of hydrogen peroxide (H₂O₂; 0.5 mM; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added to each well, followed by an incubation period of 30 min prior to uniformly blending the cells in each well. The cells were washed twice with phosphate-buffered saline (PBS) and the Invitrogen™ fluorescent probes (Thermo Fisher Scientific, Inc.), 2′,7′-dichlorofluorescin diacetate (DCFDA), dihydrorhodamine 123 (DHR 123), dihydroethidium (DHE) and MitoSOX™ (MITSOX; a mitochondrial superoxide probe), were added to the cells according to the manufacturer's protocols. The cells were incubated at 37°C for 30 min in the dark. Subsequently, the cells were washed twice with PBS and analyzed using flow cytometry (BD LSR II Flow Cytometer; BD Biosciences, San Jose, CA, USA).

In total, 48 h after solanine treatment, total protein was extracted from the HepG2 cells using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Inc.), which contained proteinase and phosphatase inhibitors, at 4°C for 30 min. The cell lysates were centrifuged at 12,000 × g for 20 min at 4°C. The protein concentrations of the resulting supernatants were determined using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The supernatants were mixed with 5X sodium dodecyl sulfate (SDS) loading buffer (Thermo Fisher Scientific, Inc.) and heated at 95°C for 5 min. In total, 40 µg of protein was loaded onto and separated by 12% Tris-Glycine SDS gel (Sangon Biotech Co., Ltd.), which was subsequently transferred onto polyvinylidene fluoride membranes (pore size, 0.22 µm; Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk in Tris-buffered saline and Tween 20 (Sangon Biotech Co., Ltd.), and incubated overnight with primary antibodies. The primary antibodies were as follows: Anti-apoptosis signal-regulating kinase 1 (ASK1; polyclonal rabbit anti-human; dilution, 1:1,000; catalog no., 3762; Cell Signaling Technology Inc., Danvers, MA, USA), anti-thioredoxin binding protein 2 (TBP-2; monoclonal mouse anti-human; dilution, 1:1,000; catalog no., K02053; MBL International Co., Woburn, MA, USA) and anti-histone deacetylase 1 (HDAC1; polyclonal rabbit anti-human; dilution, 1:1,000; catalog no., 2062; Cell Signaling Technology Inc.). Subsequently, the membranes were incubated with a horseradish peroxidase-labeled secondary antibody (dilution, 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, Inc.) was used as an endogenous control. The results were analyzed by AlphaView software version 3.3.0.0 (ProteinSimple, Santa Clara, CA, USA). The integral optical density (IOD) of each band was determined. The relative protein levels were used to evaluate the differences between the solanine-treated group and the control group, using the following equation: The relative protein level = IOD ratio between the target gene product bands and the β-actin protein bands in the solanine-treated group/IOD ratio between the target gene product bands and the β-actin protein bands in the control group.

The HepG2 cells were harvested 48 h after solanine treatment. The percentage of cells undergoing apoptosis was detected using a FITC Annexin V Apoptosis Detection kit II (BD Pharmingen, San Diego, CA, USA) and flow cytometry (BD LSR II Flow Cytometer).

The cells were harvested at the logarithmic growth phase and counted using a Countess II FL Automated Cell Counter (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 4×10⁴ cells were added to each well of a 6-well plate containing 2 ml medium, and the plate was placed into a 5% CO₂ humidified incubator at 37°C overnight (16–24 h). Subsequently, DMSO, 2 mg/ml solanine or 0.5 mg/ml camptothecin was added to the cells and incubated for an additional 24–48 h. The morphological alterations in the cells were observed using an optical microscope (Olympus IX70 Inverted Microscope; Olympus Corporation, Tokyo, Japan) following staining with Wright's stain (Sangon Biotech Co., Ltd.).

All experiments were repeated three times independently. The results are presented as the mean ± standard deviation. One-way analysis of variance was performed using SPSS version 11.0 software (SPSS, Inc., Chicago, IL, USA) in order to detect significant differences in measured variables between groups. P<0.05 was considered to indicate a statistically significant difference.

The HepG2 cells were treated with various concentrations of H₂O₂ for the same amount of time (30 min), and separately with the same concentration (0.5 mmol/l) for various time periods. The cells were incubated with DCFDA for 30 min. The results demonstrated that the optimal concentration and effective time for H₂O₂ stimulation of the cells were 0.5 mM and 30 min, respectively.

Following solanine pretreatment, the HepG2 cells were stimulated by H₂O₂ according to the pretested parameters, and various probes were used that were specific to certain ROS. The results revealed that ROS probes DCFDA and DHR 123 detected abundant ROS, including hydroxyl radical (OH⁻) and H₂O₂, in the cytoplasm and mitochondria of the HepG2 cells pretreated with solanine compared with the control group [DCFDA (n=5), P=0.0389; DHR 123 (n=5), P=0.0215]. The amount of ROS produced by the solanine-treated cells was decreased compared with the ROS produced by camptothecin-treated cells, which was observed using the DCFDA probe. By contrast, the amount of ROS produced by the solanine-treated cells was increased compared with the ROS produced by the camptothecin-treated cells, as determined using the DHR123 probe. This suggests that ROS production in various cellular locations may be by differential mechanisms. Increased levels of ROS production were also observed post-H₂O₂ stimulation, as detected by the DHR123 probe (n=5; P=0.0043). Superoxide anion (O₂⁻) ROS, which was generated by solanine pretreatment, was also detected by O₂⁻-specific DHE and MITSOX probes. However, there was no significant difference between the solanine-treated and control groups [DHE (n=5), P=0.606; MITSOX (n=5), P=0.107] (Fig. 1).

It has previously been suggested that ASK1 is an important protein involved in regulating apoptosis (22). Another apoptosis-associated protein is TBP-2, also termed vitamin D-upregulated protein 1, which effectively combines with thioredoxin, eventually leading to cellular apoptosis (23). The increased expression of HDAC1 increases the proliferation of tumor cells and alters the extracellular matrix; therefore, significantly promoting tumor cell migration and aggression (24). In addition, HDAC1 removes the acetyl-groups on histones, a more advanced chromosome structure, and inhibits basic transcription complex assembly; therefore, inhibiting transcription (25). The present results demonstrated that solanine and camptothecin increased the expression of ASK1 and TBP-2, but reduced the expression of HDAC1 (Fig. 2).

Next, the present study investigated the effect of solanine on the growth and apoptosis of the HepG2 cells. As revealed by Fig. 3A, the HepG2 cells treated with solanine exhibited notable morphological alterations in contrast to the control cells, which exhibited no alterations in morphology. The solanine-treated cells exhibited numerous cytoplasmic vacuoles, and the cells were round, shrunken and detached. The number of cells was significantly decreased in the solanine-treated (n=3; P=0.0324) and camptothecin-treated groups (n=3; P=0.0026) compared with the control group (Fig. 3B).

Consistent with the cell counting results, flow cytometry analysis confirmed that there was an increased percentage of cells undergoing apoptosis in the solanine and camptothecin-treated groups compared with the control group (Fig. 4). This indicates that following treatment with solanine, the reduced cell growth observed may be due to enhanced cellular apoptosis.