A 50 mmol/l stock solution of SGI-1027 (Selleck Chemicals, Houston, TX, USA) was prepared in dimethylsulfoxide (DMSO), which was used to prepare dosing solutions of 5, 10, 15, 20, 25, 30 and 35 µmol/l in culture medium, and 0.1% DMSO in the culture medium used as a control. The stock solution was kept at −20°C for long term storage.

The human HCC cell line-Huh7 was purchased from the Cell bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Huh7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum (FBS; Life Technologies; Thermo Fisher Scientific, Inc.), and incubated in a humidified atmosphere containing 5% CO₂ at 37°C.

Cell viability was measured using a CellTiter 96 Aqueous One Solution cell proliferation assay kit (Promega Corporation, Madison, WI, USA). Huh7 cells were seeded onto 96-well plates at a concentration of 1×10⁴ cells/well and incubated in a humidified atmosphere containing 5% CO₂ at 37°C for 24 h in DMEM supplemented with 10% FBS. The Huh7 cells were subsequently treated with SGI-1027 (SGI-1027 was dissolved in DMSO) at different concentrations (5, 10, 15, 20, 25, 30 and 35 µmol/l, with 0.1% DMSO as a control). Cell viability was measured according to the manufacturer's protocol after 24 h. An ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA) was used to measure the absorbance value at 490 nm. The viability ratio was calculated according to the following formula: [(The absorbance of experimental group-the absorbance of blank group)/(the absorbance of untreated group-the absorbance of blank group)] ×100%.

The cell cycle was measured using a Cell Cycle Detection kit (KeyGen Biotech Co., Ltd., Nanjing, China). A total of 2 ×10⁵ Huh7 cells/well were seeded into a 6-well plate and starved in serum-free medium on the second day of culture. After 12 h starvation, the cells were treated with different concentrations of SGI-1027 (10, 20 and 30 µmol/l, and 0.1%DMSO as control) for 24 h. The cells were trypsinized and washed with cold PBS, then fixed overnight with 70% ethanol at 4°C. The next day, the fixed cells were centrifuged at 1,200 × g at 4°C for 5 min and washed once with cold PBS. Subsequently, the cells were suspended in propidium iodide (PI)/RNase staining buffer at room temperature for 30 min in the dark according to the manufacturer's protocol. A FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the DNA contents of the cells. Each measurement collected at least 1×10⁴ cells.

Cell apoptosis was assessed using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGen Biotech Co., Ltd.). A total of 2×10⁵ Huh7 cells/well were seeded into a 6-well plate. After 24 h exposures to different concentrations of SGI-1027 (10, 20 and 30 µmol/l, with 0.1% DMSO as control), the cells were treated with 0.25% trypsin without EDTA. According to the manufacturer's protocols, Annexin V-FITC and PI were used for staining. The double-stained cells were measured using a FACScan flow cytometer (BD Biosciences). At least 1×10⁴ cells were measured.

The TUNEL method was used to label apoptotic cells using a TUNEL apoptosis detection kit (KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Following treatment with SGI-1027 at a number of concentrations (10, 20 and 30 µmol/l, with 0.1% DMSO as control) for up to 24 h in a humidified atmosphere containing 5% CO₂ at 37°C in a 6-well plate, the cells were washed twice with PBS and fixed in 1 ml of 4% paraformaldehyde for 10 min at 4°C, and permeabilized with 0.1% Triton X100 at 25°C for 5 min. The cells washed twice with PBS, stained by the TUNEL mixture for 1 h at 37°C in darkness, and then stained by the Streptavidin-Tetramethylrhodamine for 30 min at 37°C in darkness, followed by staining with DAPI at 37°C for 10 min. The cells were washed with PBS, then mounted and examined using fluorescence microscopy (magnification, ×200) (Olympus IX71; Olympus Corporation, Tokyo, Japan). Apoptotic cells were identified by the condensation and fragmentation of their nucleus. The apoptotic ratio was calculated using the following formula: Apoptotic cell number/seeded cell number ×100%.

Huh7 cells were treated with SGI-1027 at different concentrations (10, 20 and 30 µmol/l, with 0.1% DMSO as control) for 24 h, and then washed twice with ice-cold PBS and lysed in ice-cold protein lysis buffer containing 1% protease inhibitor cocktail and phenylmethylsulfonyl fluoride. The lysates were centrifuged at 10,000 × g for 10 min at 4°C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Total proteins (25 µg/lane) were resolved using 12% SDS-PAGE, then transferred to polyvinylidene difluoride (PVDF) membranes using a wet transfer system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 70V at 4 °C. The PVDF membranes were blocked with Tris-buffered saline plus Tween-20 (TBST; pH 8.0; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing 5% nonfat milk at room temperature for 1 h, and then incubated with primary antibodies overnight at 4°C. Subsequent to washing with TBST at room temperature (15 min/time, 3 times), membranes were incubated with horseradish peroxidase-conjugated Immunoglobulin G (IgG) at room temperature for 2 h. The probed proteins were detected using an Enhanced Chemiluminescence kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The primary antibodies used in this study were rabbit anti-B cell lymphoma (Bcl)-2 polyclonal antibody (1:1,000; cat. no. 12789-1-AP; Proteintech Group, Inc. Chicago, IL, USA), mouse anti-Bcl associated X-protein (BAX) monoclonal antibody (1:1,000; cat. no. 60267-1-Ig; Proteintech Group, Inc.) and mouse anti-β-actin monoclonal antibody (1:1,000; cat. no. CW0096; CwBiotech, Beijing, China). The secondary antibodies included goat anti-rabbit IgG serum (1:20,000; cat, no. TA130015; OriGene Technologies, Inc., Beijing, China) and goat anti-mouse IgG serum (1:20,000; cat, no. TA130001; OriGene Technologies, Inc.). The images were analyzed using the MicroChemi Bio-Imaging system (DNR Bio-Imaging Systems Ltd., Neve Yamin, Israel) and quantified using Quantity One software (version 4.5.0; Bio-Rad Laboratories, Inc.).

All experiments were performed three times. The data were expressed as the mean ± standard deviation. GraphPad Prism software (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA) was used for data analysis. The differences were analyzed using a one-way analysis of variance test followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

In order to evaluate the effect of SGI-1027 on Huh7 cells, the cell viability was investigated using an MTS assay once Huh7 cells were dose-dependently treated with SGI-1027 for 24 h. The results revealed that SGI-1027 significantly suppressed the cell viability of Huh7 cells in a dose-dependent manner compared with the DMSO control group, with an IC₅₀ value of 27.30 µmol/l (Fig. 1).

To investigate the effect of SGI-1027 on the cell cycle, the cell cycle profiles of Huh7 cells were analyzed using flow cytometry. However, no significant alterations of the cell cycle phases were observed following treatment with distinct concentrations of SGI-1027 for 24 h (P>0.05). The percentage of cells accumulated in the G1 phase was 73.81±1.357, 69.17±2.021, 69.517±0.904 and 72.547±2.724% in the DMSO control, 10, 20 and 30 µmol/l SGI-1027 groups, respectively. The percentage of cells accumulated in the S phase was 17.017±1.443, 25.623±0.869, 26.377±3.107 and 14.140±1.067% in the DMSO control, 10, 20 and 30 µmol/l groups, respectively. The percentage of cells accumulated in the G2 phase was 8.377±1.542, 3.577±1.452, 4.633±0.503 and 6.11±4.116% in the DMSO control, 10, 20 and 30 µmol/l SGI-1027 groups, respectively (Fig. 2).

To confirm whether the SGI-1027 induced inhibition of proliferation was due to a direct effect on apoptosis in Huh7 cells, flow cytometry analysis was performed using Annexin V-FITC and PI double staining. It was identified that following dose-dependent treatment with SGI-1027 for 24 h, in the control group there were normal cells and rarely apoptotic cells, while in SGI-1027 groups, the rates of apoptotic cells significantly increased in a dose-dependent manner. The rates of apoptosis in response to different concentrations of SGI-1027 were 3.242±0.204 (DMSO group), 3.672±0.123 (10 µmol/l), 33.49±1.317 (20 µmol/l) and 46.57±2.512% (30 µmol/l) (Fig. 3).

In the present study, the apoptotic effects of SGI-1027 were ascertained in Huh7 cells by assessing morphological changes. Subsequent to treatment with different doses of SGI-1027 for 24 h, Huh7 cells were stained with TUNEL. The results revealed that in the chromatin of apoptotic cells the fluorescence dye stains were observed more brightly compared with that of control cells. The number of Huh7 cells adhering to the culture plates in SGI-1027 treated groups was decreased compared with the control group. The morphological changes of cell apoptosis included blebbing, cell shrinkage, nuclear or chromatin condensation, DNA fragmentation and apoptotic body formation. In contrast, the morphology of normal cells was round and homogenous. In the present results, the apoptotic morphological changes were observed in the SGI-1027 treated groups, while few apoptotic cells were observed in the control group. The percentage of apoptotic cells was 0.987±0.34, 3.883±0.667, 42.44±3.244 and 58.24±8.427% in the DMSO control, 10, 20 and 30 µmol/l SGI-1027 groups, respectively (Fig. 4).

In order to investigate whether Bcl-2 and BAX expression were involved in the apoptosis of Huh7 cells, the expression levels of Bcl-2 and BAX were assessed using western blotting. The results indicated that following treatment with different concentrations of SGI-1027 (0.1% DMSO as control, 10, 20 and 30 µmol/l SGI-1027), Bcl-2 protein expression was significantly downregulated, whereas BAX expression was significantly upregulated, in a dose-dependent manner, in Huh7 cells (Fig. 5).