The human liver cancer HepG2 cell line was obtained from Guangzhou Jini Ou Biological Technology Co., Ltd. (Guangzhou, China). High glucose Dulbecco's modified Eagle's medium (H-DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin, trypsin (non-EDTA) and phosphate-buffered saline (PBS) were acquired from Gibco Chemical Co. (Rockville, MD, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Gigantol was purchased from Chinese Materials Research Center (Daxing, Beijing, China). The chemical structure of gigantol is shown in Fig. 1. The Annexin V-FITC apoptosis assay and Hoechst 33258 assay kits were provided by KeyGen Biotech Co., Ltd. (Jiangsu, China). The PI3K/AKT inhibitor LY294002 was purchased from Selleck Chemicals (Houston, TX, USA). All other chemicals used were of analytical grade.

HepG2 cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% penicillin-streptomycin and incubated at 37°C in a humidified atmosphere containing 5% CO₂. The control group included HepG2 cells in the absence of gigantol in serum-free medium. To study the effects from the intervention of the inhibitor, the cells were pretreated with PI3K/AKT inhibitor LY294002 (25 µmol/l) for 1 h, followed by treatment with gigantol.

The cytotoxicity was measured using MTT assays. Briefly, HepG2 cells were seeded in a 96-well plate (5×10⁴ cells/well) and serum-starved for 24 h using H-DMEM (without FBS). Then the culture medium was replaced with H-DMEM containing a series of concentrations of gigantol (1, 10, 40, 80 and 150 µM) for different time intervals (12, 24 and 48 h). After treatment, 20 µl of MTT (5 mg/ml) was added to each well and continuously cultured for an additional 4 h at 37°C. The cell supernatant was removed by aspiration and the formazan was dissolved with 150 µl DMSO. The absorbance was read at 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The cell viability was calculated as follows: cell viability = (A₄₉₀ experimental/A₄₉₀ control) × 100%.

The effect of gigantol on the morphological changes of HepG2 cells was assessed using the chromatin dye Hoechst 33258. The HepG2 cells were cultured in 24-well plates (5×10⁴ cells/well) overnight and then exposed to gigantol with concentrations of 0, 1, 40 and 150 µM for 48 h. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 min at 4°C and then dyed with Hoechst 33258 for 5 min in the dark after being washed by PBS twice. Morphological changes were observed by phase-contrast microscopy and the images of the apoptotic cells were visualized using a fluorescence microscope (Leica, Microsystems GmbH, Wetzlar, Germany).

HepG2 cells were seeded in 6-well plates (5×10⁵ cells/well). After the cells had attached to the culture plate, gigantol was added at concentrations of 0, 1, 40 and 150 µM for 48 h. According to the manufacturer's instructions, HepG2 cells which were digested with trypsin (non-EDTA) were washed with PBS and harvested and then suspended in 500 µl binding buffer. Following staining with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) for 10 min at room temperature in the dark, the samples were immediately analyzed using BD FACSCanto II flow cytometer.

HepG2 cells were seeded in 6-well plates (1×10⁶ cells/well) overnight. After treatment with or without gigantol or in combination with the inhibitor LY294002, the cells were collected, washed with cold PBS, and resuspended in cold RIPA buffer with a protein inhibitor. Then the cellular protein was collected and the protein concentration was determined using the Bradford assay. Equal amounts of protein (40 µg/lane) were separated on 15% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes via electrophoresis. After blocking with Tris-buffered saline (TBS) containing 5% skimmed milk and 0.05% Tween-20 for 1 h, the membranes were incubated with primary antibodies Akt, p-Akt, PARP, p53 and β-actin at 4°C overnight. Following three washes with TBS for 5 min, the membranes were incubated with horseradish peroxidase-conjugated with goat anti-rabbit immunoglobulin G for 1 h at room temperature. Finally, the protein bands were washed with TBST three times and visualized using an ECL western blot analysis kit according to the manufacturer's instructions.

Data shown in this study are presented as the mean ± SEM and the results from each group were obtained using triplicate samples independently. A one-way ANOVA was used to determine the differences among the groups. A probability value of *P<0.05 was considered to be statistically significant, while **P<0.01 was considered to be highly statistically significant and are indicated in the figures. All statistical analyses were carried out using Prism5 software package (GraphPad Software Inc., La Jolla, CA, USA).

The effect of gigantol on the viability of the HepG2 cells was measured by MTT assay. As indicated in Fig. 2, gigantol inhibited the growth of HepG2 cells in a dose- and time-dependent manner, particularly at a concentration beyond 10 µM at 48 h. Gigantol at concentrations of 1, 40 and 150 µM markedly decreased the cell viability by 11.7, 30.0 and 56.4% at 24 h and 21.1, 66.8 and 85.5% at 48 h, respectively. The IC₅₀ (48 h) value was 9.30 µM. Thus, the concentrations of 1, 40 and 150 µM and the incubation period of 48 h were the optimal parameters to carry out further experiments.

The morphological changes and apoptotic morphology of HepG2 cells were investigated by fluorescence microscopy, as shown in Fig. 3. Following treatment with gigantol (1, 40 and 150 µM) for 48 h, the cells exhibited membrane blebbing, cytoplasm condensation, and nuclear fragmentation with condensed chromatin typical characteristics of apoptosis. This suggests that gigantol inhibits the growth of HepG2 cells by inducing the apoptotis of tumor cells.

We evaluated cell apoptosis using the Annexin V and PI double-staining assay using flow cytometry. As depicted in Fig. 4, after incubation with gigantol (1, 40 and 150 µM) for 48 h, the apoptosis rates were 9.9, 14.9 and 20.7%, respectively. All of the values were higher than that in the control group, which demonstrated a significant increase in the cell apoptosis rate.

As previously reported, the PI3K/Akt pathway plays an important role in cell survival under stimulation (23). Therefore, to investigate whether gigantol inhibits the phosphorylation of Akt, HepG2 cells were treated with gigantol at concentrations of 1, 40 and 150 µM for 48 h (Fig. 5). Western blot analysis showed that the p-Akt protein expression level was decreased in a dose-dependent manner. In addition, there was a significant decrease in the ratio of p-Akt/Akt following treatment with gigantol, which was further suppressed by LY294002.

Anticancer effect of gigantol involves the induction of PARP signaling in human liver cancer cells. Western blot analysis was performed to detect PARP protein expression in HepG2 cells which were treated with gigantol. As shown in Fig. 6, significant upregulation of PARP protein expression was observed in the HepG2 cells treated with gigantol (1, 40 and 150 µM) and LY294002 further increased the expression of PARP.

We investigated whether the anticancer effect of gigantol involves p53 signaling in human liver cancer cells by western blot analysis. As shown in Fig. 7, following gigantol treatment, p53 protein expression was increased in the HepG2 cells and LY294002 further increased the expression of p53.

To determine whether gigantol induces the apoptosis of HepG2 cells, caspase-3 activity was assessed. Fig. 8 demonstrates that gigantol significantly increased caspase-3 activity in the HepG2 cells.