The human hepatoma cell line, Huh7, was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific, Inc.) and 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Inc.) in a 37°C, 5% CO₂ cell culture incubator. For passage, Huh7 cells were maintained in 10-cm dishes. For cellular tests, the cells were grown in 6- or 12-well plate.

Following seeding at 3×10⁵ cells/cm², Huh7 cells were administered with three respective doses (140, 280 or 560 µM) of SIN (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) dissolved in PBS, or an equal volume of PBS as vehicle at 37°C for 36 h. Following the SIN exposure, 0.1% crystal violet was added for 30 min for visual observation. The cells were also counted using a TC20TM Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at the end of treatment.

Cell apoptosis was evaluated by flow cytometry (FCM), which was performed on a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, Huh7 cells were treated with the three respective doses (140, 280 or 560 µM) of SIN or vehicle for at 37°C for 36 h. Following harvesting, the cells were washed three times with cold saline. Following centrifugation at 450 × g and 4°C for 10 min, the cell pellets were diluted with annexin V binding buffer (BD Biosciences) at 1×10⁶ cells/ml. Then, 5 µl APC Annexin V (BD Biosciences) was added to 100 μl of the cell suspension, which was followed by incubation for 10 min at room temperature. The cells were washed and resuspended in 200 µl of the annexin V binding buffer, then stained with 5 μl propidium iodide (Sigma-Aldrich; Merck KGaA). CellQuest Pro version 5.1 (BD Biosciences) was used to analyze the data.

Cell cycle distribution was also analyzed by FCM. Briefly, following exposure to the SIN doses indicated or vehicle for 24 h, Huh7 cells were harvested and fixed with 70% ethanol at 4°C for 12 h. The cells were then stained with propidium iodide at room temperature for 30 min for cell cycle analysis. To further assess the effect of SIN on each stage of the cell cycle, nocodazole (NOC; Sigma-Aldrich; Merck KGaA) was introduced to induce cellular mitotic arrest (21). In brief, Huh7 cells were synchronized by adding NOC for 24 h and then the cell cycle distribution was determined. The synchronized cells were also analyzed 24 h after removal of NOC.

WB analysis was used to determine the protein levels of cleaved (active) caspase-3, B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), Bcl-extra large (Bcl-xl), p21 and p27. Briefly, Huh7 cells were treated with the SIN doses indicated or vehicle for 36 h. Following digestion with 0.25% trypsin, the cells were collected and lysed with radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). Following centrifugation at 14,000 × g for 15 min, the supernatant was collected and the total protein concentration determined with a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Samples with equal quantity of total protein were mixed with loading buffer and loaded on 10% SDS-PAGE gel (50 µg/lane). Proteins were separated by electrophoresis and transferred onto polyvinylidene fluoride membranes. Following blocking with blocking buffer (5% non-fat milk in PBS) for 1 h at room temperature, the membranes containing the target protein were incubated with rabbit anti-cleaved caspase-3 antibody (ab2302), rabbit anti-Bax antibody (ab32503), rabbit anti-Bak antibody (ab32371), rabbit anti-Bcl-XL antibody (ab32370), rabbit anti-p21 antibody (ab109520), rabbit anti-p27^KIP1 antibody (ab32034) or mouse anti-β-actin antibody (ab6276; all from Abcam, Cambridge, UK), respectively, with each antibody diluted 1:5,000, at 4°C overnight. Following washing with PBS with Tween-20 (0.05% Tween-20), the membranes were incubated with goat anti-rabbit (A0208) or goat anti-mouse (A0216; both from Beyotime Institute of Biotechnology) immunoglobulin G-horseradish peroxidase conjugate, diluted 1:1,000 in blocking buffer without milk, at room temperature for 2 h. The membranes were then exposed to PierceTM Enhanced Chemiluminescent Western Blotting Substrate (Thermo Fisher Scientific, Inc.), which was followed by detection of the protein bands using X-ray film. β-actin detection was introduced as an internal control. Quantification of the protein bands was performed using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Graphs were prepared with Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA, USA). Statistica 10 (StatSoft, Inc., Tulsa, OK, USA) was used to perform the statistical analyses. Data were expressed as the mean ± standard deviation, and differences among multiple groups were analyzed by one-way analysis of variance followed by Bonferroni's post-hoc tests. P<0.05 was considered to indicate statistical significance.

The effect of three different doses of SIN, 140, 280 and 560 µM, on Huh7 cell survival/proliferation was evaluated. As presented in Fig. 1A, crystal violet was used to stain the cells for visual observations. It was observed that the three doses of SIN markedly inhibited Huh7 cell survival/proliferation when compared with the normal control (NC). The cells were also counted and the resulting data demonstrated that SIN at the doses of 140 (P=0.021), 280 (P=0.007) and 560 µM (P=0.007) significantly suppressed Huh7 cell survival/proliferation (Fig. 1B). However, no differences in effect were observed between the three doses (P>0.05).

To investigate the underlying mechanisms of SIN in suppressing Huh7 cell survival/proliferation, it was first tested whether SIN had an effect on cellular apoptotic death. The FCM results demonstrated that 140 (P=0.004), 280 (P=0.001) and 560 µM (P<0.001) SIN induced cellular apoptosis in Huh7 cells, when compared with apoptotic rate in the NC group (Fig. 2A and B). Notably, this effect occurred in a dose-dependent manner (280 vs. 140 µM, P=0.038; 560 vs. 280 µM, P=0.019). Cleaved caspase 3 (22), Bax (23) and Bak (24) serve as pro-apoptotic regulators, while Bcl-xl (25,26) is an anti-apoptotic protein. In the present study, the levels of these apoptosis-related proteins were determined upon administration of SIN. The WB results demonstrated that the three doses of SIN tested significantly upregulated the levels of the proapoptotic proteins, cleaved caspase 3 (140 µM vs. NC, P=0.006; 280 µM vs. NC, P=0.003; 560 µM vs. NC, P=0.009) and Bax (140 µM vs. NC, P=0.009; 280 µM vs. NC, P=0.023; 560 µM vs. NC, P=0.018), while Bak expression remained unchanged (Fig. 2C and D). Furthermore, the anti-apoptotic protein Bcl-xl was downregulated following exposure to each of the three doses of SIN (140 µM vs. NC, P=0.026; 280 µM vs. NC, P=0.032; 560 µM vs. NC, P=0.040; Fig. 2C and D). Unlike the FCM results, the changes in the expression of these proteins did not occur in an SIN dose-dependent manner. Taken together, these results indicated that SIN treatment dose-dependently induced cellular apoptosis in Huh7 cells, which may potentially be attributed to its effect on the apoptosis-related regulators.

Following confirmation of the effect of SIN on the apoptosis of Huh7 cells, it was then tested whether SIN treatment had an effect on the cell cycle. As presented in Fig. 3A, at baseline, treatment of the tumor cells with 140, 280 and 560 µM SIN led to apparent accumulation of cells at G2/M phase, compared with cell distribution in the NC group, which indicated that SIN treatment may potentially induce G2/M cell cycle arrest. To test the effect of SIN on multiphase cell cycle arrest, NOC was applied to induce mitotic arrest (21). Following exposure to NOC for 24 h, a marked population of Huh7 cells (>60%) was accumulated at G2/M phase in the NC group, while in comparison, administration of the three doses of SIN led to an increased fraction of cells accumulating at G1/S phase, indicating that SIN treatment may delay the cellular G1/S transition (Fig. 3A, ‘NOC for 24 h’). Furthermore, a notable decrease of G2/M population was observed at 24 h after removal of NOC in the NC group, while SIN treatment, particularly at 280 and 560 µM, further increased the cell population at G2/M phase when compared with the NC group, indicating SIN treatment may delay the G2/M transition for Huh7 cells (Fig. 3A, ‘24 h after NOC’). p21 (27,28) and p27 (29,30) are two cell cycle-associated proteins, functioning to stop or slow the cell division cycle. The present study also determined the levels of these cell cycle inhibitor proteins upon SIN treatment. WB results demonstrated that the different doses of SIN were able to significantly increase the protein levels of p21 (140 µM vs. NC, P<0.001; 280 µM vs. NC, P=0.008; 560 µM vs. NC, P=0.009) and p27 (140 µM vs. NC, P=0.017; 280 µM vs. NC, P=0.009; 560 µM vs. NC, P=0.023), in an apparent dose-independent manner (Fig. 3B and C). Taken together, these results indicated that SIN treatment was able to induce multiphase cell cycle arrest in Huh7 cells, potentially due to its effect on the cell cycle-associated regulators.