Thirty patients were treated at the Department of Pediatric Surgery, The First Hospital of Jilin University (Changchun, China) between August 2011 and August 2014. The present study was approved by the Research Ethics Committee of Jilin University (Changchun, China). Written informed consent was obtained from all of the patients. Fresh hepatoblastoma and corresponding normal liver tissues 5 cm away from the edge of the hepatoblastoma tissue were taken from the patients with hepatoblastoma who had not received any chemotherapy or radiotherapy prior to surgery. The samples were then snap-frozen in liquid nitrogen immediately after resection and kept at −80°C.

Human HepG2 and HuH6 hepatoblastoma cell lines, purchased from the Cell bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell biology (Shanghai, China) were cultured in DMEM and RPMI-1640 medium, respectively, with 10% fetal bovine serum (FBS; Gibco-Life Technologies, Carlsbad, CA, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Primary human fibroblasts were isolated from the skin biopsies of healthy adult donors, and were cultured as adherent monolayers in DMEM containing 15% FBS, 1% glutamine and 1% penicillin/streptomycin. The cells were subcultured every 2 or 3 days.

YM155 was obtained from Selleck Chemicals (Houston, TX, USA). Cisplatin was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The antibodies used for the western blot analysis were: mouse anti-human survivin, and mouse anti-human GAPDH, mouse anti-human cleaved caspase-3, mouse anti-human cleaved caspase-8 and mouse anti-human RARP monoclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Total RNA of liver tissue and cultured cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and quantified with the NanoDrop 2000 (Thermo Fisher Scientific, Tokyo, Japan). Reverse transcription of 1 µg of total RNA was performed using oligo(dT) primers (Invitrogen) and reverse transcriptase (Toyobo, Co., Osaka, Japan) to obtain complementary deoxyribonucleic acid (cDNA). qPCR assays were carried out using SYBR-Green Real-Time PCR Master Mix (Toyobo) and amplification equipment ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA). GADPH was used as the endogenous control for quantifying mRNA levels. The survivin and GAPDH primer sequences were designed as described by Zhang et al (26). The 2−^ΔΔCT method was used to calculate the relative abundance of target gene expression generated by Rotor-Gene Real-Time Analysis software 6.1.81 (Qiagen, Hilden, Germany).

To determine the inhibitory effect of YM155 alone or in combination on cell proliferation, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. Cells (1×10⁴/well) were plated in 96-well plates and then cultured in medium with or without various concentrations of YM155 and/or CDDP alone or in combination. Control cultures received 0.1% DMSO. Three days after the treatment, the percentage of viable cells in each well was examined by MTT assay (Sigma Chemical), using the SpectraMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 570 nm by a spectrophotometer. The percentage of viable cells for each treatment group was calculated by adjusting the untreated control group to 100%.

Cells were seeded in 6-well plates at a density of 1×10³ cells and were exposed to their respective half maximal inhibitory concentration (IC₅₀) values of YM155, CDDP or both for 48 h. The cells were then washed with drugs-free medium and allowed to grow for 14 days in drugs-free conditions. The percentage colony formation was calculated by adjusting untreated cells to 100%. All the experiments were performed in triplicate.

Apoptosis was performed using an Annexin V binding assay (ApoAlert Annexin V-FITC apoptosis kit; Clontech Laboratories, Inc., Mountain view, CA, USA). Briefly, the cells were treated with their respective IC₅₀ values of YM155, CDDP or both for 48 h. Then cells were washed with PBS twice and resuspended in 100 µl binding buffer (Sigma). Subsequently, 5 µl Annexin V FITC was added and incubated for 15 min in the dark at room temperature. The cells were analyzed with a Becton-Dickinson FACScan flow cytometer using the CellsQuest program 2.6 (Becton-Dickinson).

In addition, the expression of cleaved caspase-3, cleaved caspase-8 and cleaved PARP was determined by western blot analysis 24 h after treatment with their respective IC₅₀ values of YM155, CDDP or both.

Protein was extracted from tissues or cells using lysis buffer (RIPA Buffer; Sigma Chemical). Protein concentrations of lysates were determined using a BCA protein assay reagent kit (Tiangen Biotech, Beijing, China). Equal amounts of total protein (20 µg) were resolved by 10–12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking at room temperature with with 5% dry milk in TBS-T, each membrane was incubated overnight at 4°C with the primary antibodies. After washing with TBS-T twice, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Proteins of interest were visualized by enhanced chemiluminescence using ECL Prime (GE Healthcare, Buckinghamshire, UK). Blots were stripped and reprobed with anti-GAPDH to control for loading variations. Quantity One software (Bio-Rad, Hercules, CA, USA) was used for quantification of the protein bands.

To assess the effect of CDDP and/or YM155 on tumor growth in vivo, we used a nude mouse tumor xenograft model. Six-week-old male nude mice (BALB/c nu/nu) were purchased from the Experiments Animal Center of Changchun Biological Institute (Changchun, China). HepG2 cells (2×10⁶) were injected into the flanks of the mice and allowed to reach a tumor volume of >100 mm³ in tumor volume (length × width2)/2. The mice were randomly divided into four groups (n=10 mice/group). The control group received 1% polysorbate resuspended in deionized water. The remaining three groups were treated with YM155 (10 mg/kg body weight), CDDP (3 mg/kg body weight), or YM155 plus CDDP (3 and 1 mg/kg body weight, respectively) intraperitoneally for three days over 4 weeks. The tumor volume was measured on day 7, 14, 21 and 28 of the treatment. On day 28, the animals were euthanized using chloroform, tumor tissues were resected and volume and weight were measured. The survivin expression levels of tumor tissues were analyzed by western blot analysis. The study was approved by the Jilin University Animal Care and Experimentation Committee (Changchun, China).

Data were presented as mean ± SD (standard deviation). Comparisons between two groups were analyzed by the Student’s paired t-test. A comparison of ≥2 groups was performed using one-way ANOVA followed by a Tukey’s post hoc test. Statistical analyses were performed using GraphPad Prism, version 5 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA). P<0.05 was considered statistically significant.

To identify the potential roles of survivin in the occurrence and development of hepatoblastoma, we detected survivin expression levels in tumor tissues and adjacent normal tissues from 30 patients with hepatoblastoma by reverse-transcriptase quantitative PCR (RT-qPCR) and western blot analysis. Results of RT-qPCR showed that the mRNA expression level of surivivn in hepatoblastoma tumor tissues was significantly increased compared to matched adjacent normal tissues (Fig. 1A; P<0.05). In addition, elevated levels of survivin protein were found in hepatoblastoma tumor tissues compared with the paired adjacent normal tissues by western blot analysis (Fig. 1B).

The expression levels of survivin in human HepG2 and HuH6 hepatoblastoma cell lines, and normal fibroblasts were detected by RT-qPCR and western blot analysis. It was found that the expression of survivin on the mRNA (Fig. 1C) and protein (Fig. 1D) levels were clearly upregulated in HepG2 and HuH6 cells compared to the normal fibroblasts (P<0.05).

YM155 (Fig. 2A), a novel small molecule inhibitor of survivin, has been shown to inhibit survivin expression in various types of cancer (17–22). We examined whether YM155 was effective in inhibiting survivin expression on protein level in hepatoblastoma cell lines by western blot analysis 24 h following treatment with different doses of YM155 (0, 0.001, 0.01 and 1 µM). It was found that YM155 significantly downregulated survivin protein expression in HepG2 and HuH6 cells in a dose-dependent manner (Fig. 2B and C).

To assess the effect of YM155 and CDDP alone or in combination on the cell viability of hepatoblastoma cells in vitro, HepG2 and HuH6 cells were treated with different drug concentrations of YM155 (0–1 µM), CDDP (0–10 µM), or both (0.001 µM YM155 plus 0–10 µM CDDP) for 72 h, respectively, and an MTT assay were performed. It was found that YM155 inhibited the cell proliferation of the HepG2 and HuH6 in a dose-dependent manner. The IC₅₀ values of YM155 were 64 and 76 nM for HepG2 and HuH6 cell line, respectively (Fig. 3A). CDDP alone also reduced cell proliferation in hepatoblastoma cell lines in a dose-dependent manner with an IC₅₀ of 5.4 µM in HepG2 cells and an IC₅₀ of 6.1 µM in HuH6 cells (Fig. 3b). Combination treatment (0–10 µM CDDP in the presence of 0.001 µM YM155) reduced cell proliferation in hepatoblastoma cell lines in a dose-dependent manner with an IC₅₀ of 2.1 µM in HepG2 cells and an IC₅₀ of 2.4 µM in HuH6 cells (Fig. 3C), indicating that YM155 enhanced the sensitivity of CDDP to hepatoblastoma cell lines. Based on the results, we selected respective IC₅₀ values of drugs for subsequent treatments throughout the study.

The effects of YM155 and CDDP alone or in combination on the cell colony formation of hepatoblastoma cell lines were also determined. As shown Fig. 3D, the colony formation number of the HepG2 and HuH6 hepatoblastoma cells was significantly reduced in the YM155 and CDDP alone or combination groups. Combination treatment with YM155 and CDDP resulted in an even greater percentage of reduction.

We examined the in vitro effect of YM155 and CDDP alone or in combination on cell apoptosis in hepatoblastoma cell lines. The results showed that exposure to YM155 increased the apoptotic cells by 21.7% in HepG2 cells and 25.6% in HuH6 cells (Fig. 4), and exposure to CDDP increased the apoptotic cells by 22.4% in HepG2 cells and 27.8% in HuH6 cells. By contrast, the combination of YM155 and CDDP showed that the apoptotic cells were increased by 47.3 and 58.8% in HepG2 and HuH6 cells, respectively, which was greater than either agent alone (Fig. 4).

To assess the ability of YM155 and CDDP alone or in combination to activate caspase-3 and -8 activity, we determined cleaved caspase-3 and -8 expression by western blot analysis following treatment with YM155 and CDDP alone or in combination. We found that cleaved caspase-3 and -8 expression was significantly upregualted in the YM155 and CDDP alone or combination treatment groups as compared to the controls group (Fig. 5). Combination treatment significantly increased the cleaved caspase-3 and -8 expression levels as compared to YM155 or CDDP treatment alone (Fig. 5). Furthermore, we determined the cleaved PARP expression in the hepatoblastoma cell lines following treatment with YM155 and CDDP alone or in combination. Western blot analysis revealed that the expression of cleaved PARP was upregulated in cells treated with YM155 and CDDP alone or in combination as compared to the control (Fig. 5). Combination treatment led to maximal addition for cleaved PARP expression.

The in vitro data suggested that YM155 and CDDP alone or in combination significantly inhibited cancer cell growth. To validate our in vitro results further, we carried out YM155 and CDDP combination treatment experiment in a HepG2 xenograft mouse model. Tumors grown in nude mice were respectively treated with YM155, CDDP or both for four weeks. The tumors were then extracted and it was found that they were significantly smaller in the YM155 and CDDP alone or in combination groups than that of the control group. The combination treatment group led to marked inhibition of tumor growth as compared to the monotherapy groups (Fig. 6A). Additionally, tumor volume (Fig. 6b) and tumor weight (Fig. 6C) following treatment with YM155 and CDDP alone or in combination were significantly reduced as compared to the control group (P<0.05; Fig. 6B and C). Treatment with the combination of YM155 and CDDP resulted in marked inhibitoin of tumor growth as compared to monotherapy (P<0.05; Fig. 6B and C). We also determined survivin expression in xenograft tumors by western blot analysis. The results revealed that survivin expression was upregulated in the CDDP treatment group as compared to the remaining groups, while survivin expression was downregulated in the YM155 treatment group as compared to the remaining groups (Fig. 6D). Of note, survivin expression had no significant change in the combination group as compared to the control group (Fig. 6D). These results showed that YM155 in combination with CDDP treatment markedly suppressed hepatoblastoma tumorigenicity in nude mice.