The HepG2 and Huh7 human hepatocellular carcinoma cell lines and the RKO human colorectal cancer cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in McCoy’s 5A medium (Applichem, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA). Cells were incubated in an atmosphere of 5% CO₂ at 37°C. Cells were treated in serum-free medium with the indicated concentrations of oleuropein (Sigma-Aldrich, St. Louis, MO, USA; 12247), dimethyl sulfoxide (Sigma-Aldrich; D2650) and glutathione (GSH; Sigma-Aldrich; V900456).

The effects of oleuropein on cell viability were determined using a Dojindo Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan). HepG2, Huh7 and RKO cells were cultured in 96-well plates at approximately 10⁴ cells per well for 24 h at 37°C. Cells were treated with (0, 20, 40, 60, 80 or 100 μM) oleuropein for 24 h. Subsequently, 10 μl of CCK-8 solution was added to each well and incubated for 1 h at 37°C. The absorbance at 450 nm was measured using a spectrophotometer (Shimadzu UV-1800; Shimadzu Scientific Instruments, Columbia, MD, USA). Independent experiments were performed three times in triplicate.

HepG2 cells (2000 cells/well) were seeded into six-well plates (in triplicate). Cells were permitted to adhere for 24 h at 37°C, following which the media was changed for media mixed with (0, 20, 40, 60, 80 or 100 μM) oleuropein. After 48 h, cells were cultured in normal media for 10 d. In order to visualize colonies, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min and stained with crystal violet staining solution (BD Biosciences, San Jose, CA, USA) for 30 min. Colonies were counted and presented as the mean number of colonies formed from three independent experiments.

HepG2 cells were harvested, washed with phosphate-buffered saline (PBS) and resuspended in 40 μl lysis buffer, which contained 100 mM Tris (pH 8.0; Amresco LLC, Solon, OH, USA), 20 mM EDTA (Sinopharm, Shanghai, China), 0.8% sodium dodecyl sulfate (SDS; W/V; Sinopharm) and 0.5 mg/ml proteinase K (Merck Millipore, Darmstadt, Germany), at 65°C for 3 h. RNase A (0.5 mg/ml; Sigma-Aldrich) was added to the cell lysates and incubated for a further 3 h at 55°C. Genomic DNA was extracted using phenol-chloroformisoamyl alcohol (25:24:1), separated by 1% agarose gel electrophoresis and scanned with an Image Analyzer (Tanon-2500; Tanon Science and Technology Co., Ltd., Shanghai, China).

Detection of apoptosis induced by oleuropein was performed using Annexin V and Propidium Iodide (PI) staining, according to the manufacturer’s instructions (BD, Pharmingen San Diego, CA, USA). Cells (1×10⁵) were suspended in 100 μl binding buffer and stained with Annexin V-PE and PI (1 μg/ml). Following incubation at room temperature, a further 400 μl binding buffer was added and the apoptotic cells were quantification using flow cytometry (Becton Dickinson FACsCalibur™, BD Biosciences, Franklin Lakes, NJ, USA).

HepG2 cells in a 24-well plate were harvested following treatment for 24 h. Cells were washed twice with PBS, labeled with 10 μM 2,7-dichlorodihydrofluorescein diacetate (Sigma-Aldrich) at 37°C for 30 min in darkness, and then subjected to flow cytometry for the measurement of reactive oxygen species (ROS).

Signaling pathway arrays were conducted using a luciferase assay. Reporters, NF-κB-luc, PI3K/AKT-luc and Notch-luc, were purchased from Qiagen (Qiagen-SABiosciences, Valencia, CA, USA). HepG2 cells were cotransfected with reporter plasmids and a control reporter in 24-well plates. After 48 h, the luciferase activity of firefly and Renilla cells was measured using a dual Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA).

Total RNA from HepG2 cells, with or without oleuropein treatment, was isolated using RNAiso Plus (Takara Bio, Inc., Otsu, Japan) according to the manufacturer’s instructions. cDNA was synthesized using a PrimeScript™ RT-PCR kit (Takara Bio, Inc.). qPCR was performed using LightCycler (Roche, Basel, Switzerland) according to the manufacturer’s recommendations. The following primer sequences (Sagon Biotech, Shanghai, China) were used: Forward: 5′-GAGGATGATTGCCGCCGTGGACA-3′ and reverse: 5′-GGTGGGGGAGGAGGCTTGAGG-3′ for BAX, forward: 5′-ATGTGTGTGGAGAGCGTCAACC-3′ and reverse: 5′-TGAGCAGAGTCTTCAGAGACAGCC-3′ for Bcl-2 and forward: 5′-TGCACCACCAACTGCTTAGC-3′ and reverse: 5′-GCATGGACTGTGGTCATGAG-3′ for GAPDH. All samples were read in triplicate, and values were normalized to the expression of GAPDH.

HepG2 cells, with or without oleuropein treatment, were lysed in ice-cold radioimmunoprecipitation assay buffer containing protease inhibitors [20 mmol/l Tris, 150 mmol/l NaCl (Sinopharm), 1% NP40 (Sigma-Aldrich), 0.5% sodium deoxycholate (Sigma-Aldrich), 1 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride (Sigma-Aldrich), 0.15 units/ml aprotinin and 10 mmol/l leupeptin (Roche Diagnostics GmBH, Mannheim, Germany)]. Cells were lysed for 30 min on ice and lysates were then centrifuged at 11,340 × g for 10 min at 4°C. The protein concentration was determined by a bicinchoninic acid protein assay kit (Pierce, 23227; Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer’s instructions. Lysates were dissolved in 4X loading buffer and boiled for 8 min. Samples were separated by 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Bedford, MA, USA). Samples were blocked with 5% non-fat milk in 50 mM Tris-buffered saline (pH 7.4) with 0.1% Tween-20). Membranes were probed with primary antibodies for 3 h, followed by a horseradish peroxidase-conjugated IgG (Thermo Fisher Scientific, Rockford, IL, USA). Bands were revealed using an enhanced chemiluminescence detection system (Immobilon Western Chemiluminescent HRP Substrate; EMD Millipore). The following primary antibodies were used: Rabbit monoclonal anti-poly ADP ribose polymerase (PARP) antibody (1:1,000; #9532; Cell Signaling Technology, Inc., Danvers, MA, USA); rabbit polyclonal anti-caspase-9 antibody (1:1,000; #9504; Cell Signaling Technology, Inc.); rabbit polyclonal anti-caspase-8 antibody (1:1,000; #4927; Cell Signaling Technology, Inc.); mouse polyclonal anti-caspase-3 antibody (1:1,000; #9665; Cell Signaling Technology, Inc.); rabbit polyclonal anti-BAX antibody (1:1,000; #2772; Cell Signaling Technology, Inc.); rabbit polyclonal anti-Bcl-2 antibody (1:1,000; #2872; Cell Signaling Technology, Inc.); mouse monoclonal anti-flag antibody (1:2,000; #3165; Sigma-Aldrich) and rabbit monoclonal anti-GAPDH antibody (1:5,000; #3683; Cell Signaling Technology, Inc.).

Analysis of variance using SPSS 19.0 (IBM SPSS, Armonk, NY, USA) or Student’s t-test were employed to analyze the data, P<0.05 were considered to indicate a statistically significant difference.

Oleuropein, whose structure is exhibited in Fig. 1A, was the bioactive polyphenol in olive oil. In order to determine whether oleuropein affects the tumorigenic properties of human HCC cells, HepG2 and Huh7 human hepatoma cells were treated with various concentrations of oleuropein. The human RKO colorectal cancer cell line was also treated with oleuropein under the same conditions. Using a cell viability assay, a significant dose-dependent inhibition of cell growth was observed when the concentration of oleuropein was in the 20–80 μM range (Fig. 1B). In addition, alterations in the morphology of HepG2 tumor cells, including cell shrinkage and increased cell death, were observed following treatment with oleuropein (Fig. 1C). Furthermore, the colony formation assay demonstrated a marked reduction in the number of colonies formed in the oleuropein-treated cells, when compared with the control cells (Fig. 1D). These findings suggest that oleuropein is involved in the growth of liver cancer cells.

In order to examine the mechanisms involved in growth inhibition following treatment with oleuropein, the effect of oleuropein on the cascade of caspases was investigated. Caspases are essential initiators or effectors in cell apoptosis. The results of the present study showed that oleuropein induces cleavage of caspase-8, caspase-9, caspase-3 and PARP, which is a nuclear enzyme with a wide range of functions, including the regulation of DNA repair, cell differentiation and gene expression (Fig. 2A). To further confirm the involvement of apoptotic pathways in cell death due to oleuropein, a DNA fragmentation assay was conducted. A higher level of DNA fragmentation was observed in oleuropein-treated cells compared with control cells (Fig. 2B). Furthermore, cell apoptosis was assessed by Annexin V-FITC/PI staining in HepG2 cells treated with 30 μM oleuropein. Following 48 h of treatment, a significant percentage of HepG2 cells were observed to be undergoing apoptosis (Fig. 2C). T he Bcl-2 family of mitochondria proteins are involved in the regulation of apoptosis (23). In order to determine whether Bcl-2 family members are involved in oleuropein-induced apoptosis, the expression of BAX and Bcl-2 was measured. The results indicated that oleuropein treatment leads to the upregulation of the expression of BAX mRNA and protein and the downregulation of that of Bcl-2 (Fig. 2D and E). This suggests that oleuropein induces HCC cell apoptosis and that this progress is mediated via the activation of caspases, in addition to the regulation of the expression of mitochondrial proteins.

In order to explore the primary regulators involved in oleuropein-induced apoptosis, a pathway array was performed using a luciferase assay. A significant reduction in the activity of (PI3K/AKT; Fig. 3A) was observed. AKT is involved in cell proliferation, apoptosis and cell survival. The activation of AKT by phosphorylation is associated with the protection of cells from apoptosis (24). The level of the phosphorylated AKT (S473) and total AKT proteins were analyzed. In accordance with the results from the luciferase assay, a high concentration of oleuropein (60 μM) reduced the level of phosphorylated active AKT, while the level of total AKT remained unchanged with the various concentrations used (Fig. 3B).

To examine the role of AKT in oleuropein-induced apoptosis, a rescue experiment was conducted, in which HepG2 cells were transfected with pcDNA5-flag-AKT or pcDNA5-control constructs prior to treatment with oleuropein. The results indicated that AKT overexpression (Fig. 3C) causes less cleavage of PARP and caspase-3 compared with intrinsic AKT (Fig. 3D). LY294002, a specific PI3K inhibitor (25), led to increased cleavage of PARP and caspase-3 when used together with oleuropein (Fig. 3E). These results suggest that the PI3K/AKT signaling pathway contributes to oleuropein-induced apoptosis in HCC cells.

It has been reported that AKT activation induces the production of ROS. Elevated levels of intracellular ROS contribute to tumorigenesis by activation of signaling pathways and by increasing mutation rates (26,27). In order to determine whether the dose-dependent inhibition of AKT activation decreases the accumulation of intracellular ROS, HepG2 cells were treated with 50 μM oleuropein for 48 h and the level of ROS was measured by FACS analysis. ROS accumulation was observed following 24 h of oleuropein treatment in HepG2 cells, as shown in Fig. 4A. We hypothesized that ROS generation resulted in PI3K/AKT inactivation. ROS blockers were then used to investigate this hypothesis. HepG2 cells were pretreated with 20 mM GSH for 2 h, followed by 50 μM oleuropein treatment for an additional 24 h. A cell viability assay demonstrated that GSH pretreatment promoted cell survival (Fig. 4B). The results of the western blot analysis showed that the expression of phosphorylated AKT was increased following the removal of ROS by pretreatment with GSH (Fig. 4C). Pretreatment with GSH consistently inhibited the oleuropein-induced cleavage of PARP and caspase-3 (Fig. 4D). These results indicate that oleuropein-induced ROS generation leads to PI3K/AKT inactivation and is thus has an important function in the apoptotic pathway.