The HepG2 cell line was obtained from the Cell Bank, Chinese Academy of Sciences (Shanghai, China). This is a common liver cancer cell line. The HepG2 cell line was derived from a 15-year-old white male with well-differentiated liver cancer. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The HepG2 cells were cultured in a humidified incubator containing 5% CO₂ at 37°C.

Cell viability was assessed by Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA). Vismodegib and entinostat were purchased from Shanghai Biochempartner Co., Ltd. (Shanghai, China). According to the instructions, HepG2 cells cultured in 96-well plates were treated with an indicated concentration of vismodegib (0, 1, 2, 4, 8 and 16 µM) or entinostat (0, 0.5, 1, 2, 4 and 8 µM) or a combination of the two, or DMSO for 72 h. The half-maximal inhibitory concentration (IC₅₀) values were obtained from dose-response curves with GraphPad Prism software (version 7.00; GraphPad Software, Inc., La Jolla, CA, USA). The synergistic effect was determined by calculating the combination index (CI) using the Calcusyn software program (version 2.1; Biosoft, Great Shelford, UK). Data from cell viability assays were expressed as the fraction of growth inhibition by the single drugs or the combination. Synergism was expressed as a CI value <1 and antagonism by a CI value >1 at 0.5 fraction affected (FA).

The effects of the inhibitors on cell growth were evaluated as previously described (18). Briefly, the cells were cultured in 6-well plates at 1,500 cells/well densities and then treated with vismodegib (4 µM) or entinostat (2 µM) or combination (vismodegib and entinostat at the respective concentrations). The cells were treated for ~14 days. Then the cells were fixed, stained with crystal violet, and extracted with glacial acetic acid. The optical density (OD) was assessed at 570 nm by the microplate reader (iMark™ Microplate Absorbance Reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The colony formation of the liver cancer cells in soft agar medium was performed as previously described (19). Briefly, the cell suspension (2×10⁴ cells/well) was mixed with 0.4% soft agar (BD Biosciences, Franklin Lakes, NJ, USA) prepared with DMEM containing 10% FBS and layered onto 0.6% soft agar prepared with DMEM containing 10% FBS. In addition, liver cancer cells were treated with media supplemented with the indicated drugs. The medium was changed twice a week. The colonies were photographed and the area of these colonies was assessed by Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) at the end of the experiment.

HepG2 cells were grown in 6-well plates and treated with vismodegib (4 µM), entinostat (2 µM), combination or DMSO control for 48 h. Following treatment, apoptotic cells were assessed by nuclear morphology and apoptotic cell numbers were detected. Cells exposed to different treatments for 48 h were stained with acridine orange (AO) and ethidium bromide (EB) as previously described (20), and examined with a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). Apoptosis in liver cancer cells was analyzed with an Annexin V-FITC Apoptosis Detection Kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The cultured cells were trypsinized, resuspended in Annexin V binding buffer, and incubated with Annexin V and propidium iodide (PI) in the dark. Flow cytometric analysis was performed using a BD Accuri^™ flow cytometer (BD Biosciences, San Jose, CA, USA) to subject the stained cells.

The treated cells were fixed in ice-cold 70% ethanol and stored at 4°C overnight for cell cycle analysis. After the fixed cells were washed with PBS, they were treated with 100 µg/ml RNase A at 37°C for 20 min, and stained with 50 µg/ml PI (Dojindo Molecular Technologies, Inc.). Then, the cells were subjected to fluorescence-activated cell sorting. The cell populations that were found in G0/G1, S and G2/M phases were quantified using the ModFit software (version 4.0; Verity Software House, Inc., Topsham, ME, USA).

Cells exposed to vismodegib (4 µM) and/or entinostat (2 µM) for 48 h were lysed by RIPA buffer (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) containing protease and phosphatase inhibitors. The BCA method was used to quantify the protein of the lysate. The whole cell extracts (30 µg) was separated by 8% SDS-PAGE and the polyvinylidene fluoride (PVDF) membranes were blocking by 5% skim milk for 2 h at 4°C, then were incubated with the following primary antibodies staying overnight at 4°C: vinculin (dilution 1:2,000; cat. no. V4139; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), CDK1 (dilution 1:500; cat. no. 10762-1-AP), cyclin B1 (dilution 1:500; cat. no. 55004-1-AP), Gli1 (dilution 1:500; cat. no. 25733-1-AP), Gli2 (dilution 1:500; cat. no. 18989-1-AP), Gli3 (dilution 1:500; cat. no. 19949-1-AP) (all from ProteinTech Group, Inc., Chicago, IL, USA), cleaved-PARP (dilution 1:1,000; cat. no. 9541), pS6RP^s255/256 (dilution 1:1,000; cat. no. 4858), pAKT^s473 (dilution 1:1,000; cat. no. 4060) and acetyl-histone H3 (dilution 1:1,000; cat. no. 9677) (all from Cell Signaling Technology, Inc., Danvers, MA, USA). Subsequently, they were incubated with horseradish peroxidase-conjugated secondary antibody (dilution 1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology, Shanghai, China), which was followed by Thermo Scientific™ chemiluminescence (cat. no. 32106; Thermo Fisher Scientific Co., Ltd., Shanghai, China) detection.

The case of a 59-year-old patient with diagnosed primary moderately differentiated liver cancer without treatment history was employed. The acquisition of tumor tissue was executed with an Institutional Review Board protocol approved by Binzhou Medical University (Yantai, China) and written informed consent was provided by this patient. The primary liver cancer specimens from this patient who underwent a hepatic segmentectomy were obtained and subjected to ex vivo culture experiments.

For the ex vivo culture, 1 cm² hemostatic gelatin dental sponges were soaked in the medium, in which DMEM base was supplemented with 10% FBS, hydrocortisone (1 mg/100 ml; Sigma-Aldrich; Merck KGaA), antibiotic/antimycotic solution (Gibco; Thermo Fisher Scientific, Inc.), and insulin (1 mg/100 ml) for 1 h. Then, liver cancer tissues were cut into 1 mm³ blocks, and transferred to the top surface of hemostatic gelatin dental sponges. Tissue blocks were treated with SHH pathway inhibitor vismodegib and HDAC inhibitor entinostat as single-agents or in combination for 48 h, and then fixed with paraformaldehyde. The tissue blocks were embedded in paraffin, sectioned with microtome, and stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was implemented using antibodies Bcl-2 (dilution 1:200; cat. no. 12789-1-AP), Bax (dilution 1:200; cat. no. 50599-2-Ig) and Ki-67 (dilution 1:400; cat. no. 27309-1-AP) (all from ProteinTech Group, Inc.). The results of the staining were quantified by Image-Pro Plus software.

All numerical data were presented as averages and the standard deviation (SD) of the mean. The data was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test. Statistical significance was regarded at P<0.05. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA).

Given that the SHH signaling pathway regulated histone acetylation and led to cancinogenesis (20), the liver cancer cell line, HepG2, was chosen for examination. Cell proliferation assay using CCK-8 revealed that the IC₅₀ value of vismodegib for HepG2 cells was 4.012 µM (Fig. 1A), and the IC₅₀ value of entinostat for these cells was 1.992 µM (Fig. 1A). Then, the HepG2 cells were treated with different concentrations of vismodegib and entinostat, each alone and in combination for 72 h. The median-effect analysis was used to assess the effect of the drug combination on proliferation inhibition. Combined treatment with vismodegib and entinostat resulted in a synergistic increase in proliferation inhibition and synergistic CI value of <1 at 0.5 FA in HepG2 cells (Fig. 1B).

We also observed that SHH signaling inhibitor vismodegib as single-agent slightly reduced the viability of HepG2 cells (Fig. 1C). Conversely, the HDAC inhibitor entinostat revealed a moderate inhibitory effect on the viablity of these liver cancer cells (Fig. 1C). However, the combined use of vismodegib and entinostat almost completely suppressed the growth of HepG2 cells (Fig. 1C).

The observation that the combination treatment inhibited cell proliferation in two-dimensional culture conditions led us to further assess the effect of the drug combination in three-dimensional conditions. Inhibition of the SHH pathway by the use of vismodegib alone slightly inhibited colony formation efficiently and inhibition of entinostat exhibited a moderate inhibitory effect. As anticipated, the combined use of vismodegib and entinostat inhibited the formation of colonies in HepG2 cells almost completely (Fig. 1D). These results indicated that dual use of SHH signaling inhibitor vismodegib and HDAC inhibitor entinostat provided a synergistic inhibitory effect on the proliferation of liver cancer cells.

To investigate whether apoptosis was the pathway of cell death after treatment, cell morphological changes were observed and the proportion of apoptotic cells was evaluated by AO/EB staining and flow cytometric assays, respectively. The results indicated that only sporadic apoptotic cells were observed in the vehicle group, however, more apoptotic cells were observed in the vismodegib and entinostat single-agent treated groups. The number of apoptotic cells significantly increased in the combined treatment group of HepG2 cells (Fig. 2A). While vismodegib or entinostat alone produced a mild increase in Annexin V-positive cells, combined treatment resulted in significantly increased Annexin V-positive cells in HepG2 cells (Fig. 2B). Consistent with this result, the combination treatment also enhanced the abundance of cleaved-PARP, a marker for active apoptosis, in HepG2 cells. (Fig. 2C).

An ex vivo culture model of liver cancer was employed to assess the therapeutic effect of vismodegib combined with entinostat. Briefly, we dissected fresh surgical specimens of primary liver cancer into ~1 mm³ blocks. These blocks were exposed to vismodegib and entinostat as single-agents or in combination for 48 h on the absorbable gelatin sponge. The results of histological examination revealed that the tissue structure of vehicle-treated explants was similar to that of the primary tumor tissue (Fig. 3A). The blocks treated with vismodegib and entinostat displayed marked disrupted cellular integrity compared to that in the single-agent treatment groups (Fig. 3A). Consistently, the combination treatment significantly reduced proliferation as determined by Ki-67, and significantly enhanced apoptosis as determined by downregulated Bcl-2 and upregulated Bax (Fig. 3B). Collectively, these data ascertained the potential use of vismodegib and entinostat to treat liver cancer.

To assess whether the effects of combination treatment on liver cancer cells involved changes in the cell cycle, HepG2 cells were treated with vismodegib and entinostat alone or combined treatment for 48 h. The results revealed that combination treatment significantly increased the number of cells at the G2/M phase and significantly decreased the number of cells at the S phase (Fig. 4A). However, an increase of sub-G1 populations was also observed. While vismodegib and entinostat as single-agents led to a slight increase of sub-G1 populations, dual treatment with vismodegib and entinostat resulted in a marked increase of sub-G1 in HepG2 cells (Fig. 4A). The present study determined that combination therapy induced G2/M arrest and triggered apoptosis of HepG2 cells. With regards to the biomarkers of the cell cycle, vismodegib combined with entinostat reduced the expression of cyclin B1 and cyclin dependent kinase 1 (CDK1) (Fig. 4B), which further indicated that the combination treatment resulted in G2/M phase arrest.

Western blot analysis revealed that vismodegib as a single agent or in combination with entinostat resulted in a marked reduction of the expression of Gli1, Gli2 and Gli3 effectors of SHH signaling in HepG2 cells (Fig. 5A). Notably, a slight effect was observed with SHH signaling in liver cancer cells treated with entinostat as a single-agent. In addition, the results revealed that entinostat as a single agent or in combination with vismodegib led to a marked increase in acetylated histone 3 in HepG2 cells (Fig. 5B).

It has been reported that inhibition of SHH signaling regulated pancreatic tumorigenesis via inhibition of the PI3K/AKT signaling pathway (21). Thus, it was investigated whether the dual inhibition of the SHH pathway and HDAC inhibited the growth of liver cancer cells via inhibition of the PI3K/AKT signaling pathway. Liver cancer cells, HepG2, were treated with vismodegib or entinostat as single-agents or in combination. The results revealed that vismodegib as a single-agent or in combination with entinostat caused markedly reduced phosphorylated levels of AKT and S6RP, a downstream effector of PI3K/mTOR signaling, in HepG2 cells (Fig. 5C). Collectively, the results of the present study indicated that combined use of inhibitors vismodegib and entinostat may downregulate the PI3K/mTOR signaling.