Human HCC cell lines HepG2 and Huh7 were purchased from Shanghai Laboratory Animal Centre (SLRC; Shanghai, China). YM155 was obtained from Selleck Chemicals (Houston, TX, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit (cat. no. 556547) was purchased from BD Pharmingen (San Diego, CA, USA). Bicinchoninic Acid (BCA) Protein Assay and Protein Lysis kits, phosphate-buffered saline (PBS), streptomycin and penicillin were purchased from the Beyotime Institute of Biotechnology (Nanjing, China). The primary rabbit monoclonal anti-survivin (cat. no. 76424), anti-procaspase 3 (cat. no. 32499), anti-β-actin (cat. no. 8227) and anti-phosphorylated retinoblastoma tumor suppressor protein (p-Rb; Ser807/811; cat. no. D20B12) antibodies, as well as secondary goat anti-rabbit horseradish peroxidase-conjugated antibody (cat. no. 97080) were purchased from Abcam (Cambridge, MA, USA).

To explore the protein expression profiles of survivin and p-Rb, eight paraffin-embedded HCC specimens (T group) and matched adjacent normal tissues (N group) were retrieved from the Department of Pathology (Taizhou People's Hospital, Taizhou, China) for immunohistochemical assessment. All patient samples recruited to the present study were approved by the ethical review committee (Institutional Ethical Board of Taizhou People's Hospital).

Consecutive sections (4 µm) of paraffin-embedded normal and tumor specimens were prepared as described previously (12). Survivin and p-Rb protein expression in these sections was detected by incubation with respective antibodies against survivin (1:150) and p-Rb (1:150) at 37°C for 1 h. Two individual pathologists, blinded to patient characteristics, scored the sections using an Olympus CX32 microscope (Olympus Corp., Tokyo, Japan). Protein expression was classified according to the staining intensity as follows: 0, absence of staining; 1, mild expression; 2, moderate expression and 3, high expression. A mean percentage of positive tumor cell was determined in at least five areas at x400 magnification. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each case. Theoretically, the total scores ranged from 0 (0% of cells stained) to 3 (100×3/100).

Human HCC cell lines HepG2 and Huh7 were cultured in DMEM supplemented with 10% FBS and 1% streptomycin and penicillin, in an atmosphere of 5% CO₂ at 37°C. Cells were allowed to adhere overnight. Subsequently, HCC cells incubated with and without YM155 were designated as the YM155 and control groups, respectively.

For cell viability assessment, HCC cells were seeded in 96-well plates (Nunc, Roskilde, Denmark) at 5000 cells/well and subsequently exposed to YM155 at the IC₅₀ concentration of 100 nM according to previous literature (13) for up to 72 h. At various time-points (0, 24, 48 and 72 h), 20 µl MTT (5 mg/ml) was added to each well. The HCC cells were then incubated at 37°C for a further 4 h. The culture medium was removed and 150 µl 0.1% dimethylsulfoxide (DMSO) was added to each well to resolve the MTT. The absorbance of each well was measured at 450 nm on a Multiskan FC 51119000 photometer (BioTek Instruments, Inc., Winooski, VT, USA). For apoptosis measurements and protein sample preparation, cells were cultured in 6-well plates (Nunc) with YM155 or without YM155 (control group) and harvested 48 h later.

The apoptotic degree of YM155-treated HCC cells was quantified by flow cytometry using the FITC Annexin V Apoptosis Detection kit I. Briefly, cell samples were sequentially incubated for 15 min at 28°C with 5 mg/ml Annexin V-FITC (AV-FITC) and 10 mg/ml propidium iodide (PI) diluted in Annexin V binding buffer. The cells were then resuspended in PBS and analyzed with a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA), using a 530/30 nm signal detector for AV-FITC and a 582/42 nm signal detector for PI. The data were subsequently analyzed by Flow J software (version 7.6.5; Tree Star, Inc., San Carlos, CA, USA). The upper left and lower left quadrants represented late and early apoptosis, respectively. The total apoptosis ratio was calculated by adding the late and early apoptosis proportions.

Cell samples grown in 6-well plates were incubated with ice-cold lysis buffer [0.1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 1.5 mM MgCl₂, 1 mM dithiothreitol, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and 2 mg/ml leupeptin and aprotinin]. The total protein sols were centrifuged at 12,000 × g at 4°C for 10 min and the supernatants were bolted at 100°C by iron heating for a further 10 min. A BCA Protein Assay kit was used to determine the protein concentration. Equivalent samples (30 mg protein) were then subjected to 12% SDS-PAGE (Beyotime Institute of Biotechnology). The proteins were transferred to nitrocellulose membranes (Beyotime Institute of Biotechnology) and probed with specific primary antibodies. The antibodies were used at the following concentrations: survivin (1:1,500), procaspase-3 (1:2,000), p-Rb (1:2,000), β-actin (1:2,000), followed by secondary antibody (1:2,500). The molecular sizes of the target proteins were determined by comparison with prestained Bio-Rad protein markers (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The protein bands were then scanned using the Western Lightning Chemiluminescent Reagent Plus (Perkin Elmer, Boston, MA, USA) detection system. Protein immunoreactivity was quantified by densitometry analysis using the Gel DocXR System 170–8170 device and (Bio-Rad Laboratories, Inc., Nazareth, Belgium) and Quantity One software (version 4.4; Bio-Rad Laboratories, Inc.). β-actin was used as a loading control.

All parametric data are expressed as the mean ± standard error of the mean, and analyzed by Student's t-test, whereas non-parametric data analysis was performed by Mann-Whitney U test. For all tests, analyses were performed using SPSS 19.0 statistical software (IBM SPSS, Armonk, NY, USA) and two-sided P<0.05 was considered to indicate a statistically significant difference.

To investigate the variance in protein expression of survivin between human HCC and normal liver tissues, immunohistochemistry assays were performed. As shown in Fig. 1, HCC specimens presented with markedly greater survivin expression than that of noncancerous tissues, and the distribution was characterized by expression primarily in the nucleus, but also the cytoplasm.

It is well established that Rb protein functions as a cell-cycle suppressor, and its functional loss through protein phosphorylation has been implicated to the tumorigenesis of various types of neoplasm (14,15). Therefore, the phosphorylation level of Rb protein in HCC was investigated. Immunohistochemical analysis revealed that HCC samples displayed elevated p-Rb expression, as compared with that of normal liver tissues, in a nucleus-exclusive distribution model (Fig. 1).

Cell apoptosis status was determined by flow cytometry. Following 48 h of exposure to YM155, the cell apoptosis ratios in HepG2 and Huh7 cells were 47.3±5.5 and 37.3±7.8%, respectively, markedly higher than those of the corresponding control groups, which were 3.7±1.2 and 4.2±0.4%, respectively (P<0.05; Fig. 2A and B). These results indicated a significant pro-apoptotic effect of YM155 on HCC cell lines.

To verify the antitumor activity of YM155, HCC cell viabilities were investigated by MTT assay. Following YM155 exposure, HepG2 cell viabilities at 24, 48 and 72 h were 0.81±0.02, 0.55±0.09 and 0.25±0.05 respectively, which were markedly lower than those at the corresponding times in the control group (1.00±0.04, 1.74±0.10 and 1.97±0.19) (P<0.05; Fig. 2C). Concurrently, the Huh7 cell survival at 24, 48 and 72 h in the YM155 group was 0.93±0.06, 0.78±0.06 and 0.38±0.06, respectively. These results were markedly lower than those at the corresponding time-points in the control group: 1.30±0.06, 1.56±0.12 and 1.92±0.10 (P<0.05; Fig. 2C). These data demonstrated a significant anti-proliferative effect of YM155 in HCC cells.

To detect the variations in the protein expression of survivin, p-Rb and procaspase-3 in HCC cells following YM155 pretreatment, immunoblotting assays were performed (Fig. 2D and E). The relative survivin protein expression levels of YM155-treated HepG2 and Huh7 cells were 0.40±0.07 and 0.50±0.09, respectively; these were markedly lower than those of the corresponding control groups (0.89±0.15 and 1.02±0.12, respectively). Relative p-Rb protein levels in HepG2 and Huh7 cells following YM155 exposure (0.72±0.08 and 0.57±0.12, respectively) were also lower than those in the associated control group (1.01±0.09 and 1.11±0.13, respectively). The relative protein expression levels of procaspase-3 in control HepG2 and Huh7 cells were 0.69±0.06 and 0.34±0.08, respectively, which were significantly lower than those of the corresponding YM155-treated groups (0.86±0.07 and 0.53±0.08, respectively). These results revealed that YM155-treatment was not only able to directly suppress survivin protein expression, but also indirectly inhibited p-Rb and enhanced procaspase-3 expression.