The oleanolic acid derivative was provided by Dr Lei of Beijing University of Chinese Medicine (Beijing, China) and dissolved in DMSO. The chemical structure of the oleanolic acid derivative is presented in Fig. 1A. RPMI-1640, fetal bovine serum, penicillin, streptomycin and trypsin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). SDS, ponceaus, dithiothreitol, phenylmethylsulfonylfluoride, bovine serum albumin, MTT, the Annexin V-fluorescein isthocyanate (FITC)/propidium iodide (PI) Apoptosis Detection kit and JC-1 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-caspase-9 (catalog no., 9502), anti-caspase-3 (catalog no., 9662) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Bcl-2 (catalog no., BS1031) and Bax (catalog no., BS1030) were obtained from Biogot Technology Co., Ltd. (Nanjing, China). Horseradish peroxidase-conjugated goat anti-rabbit antibodies (catalog no., BA1054) were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). Polyvinylidene difluoride (PVDF) microporous membranes and the enhanced chemiluminescence detection system were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). RIPA cell lysis buffer for western blot analysis was purchased from Beyotime Institute of Biotechnology (Haimen, China). The kit for detection of adenosine triphosphate (ATP) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Human cytochrome c (Cyt-C) ELISA kit was purchased from Shanghai Ximei Chemical (Shanghai, China). The Cytoplasmic and Mitochondrial Protein Extraction kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

SMMC-7721 human HCC cells were obtained from the American Type Culture Collection (Manassas, VA, USA). HL-7702 normal human liver cells were obtained from Obio Technology (Shanghai, China). The cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO₂ incubator. Cells were treated with vehicle (0.15% DMSO) alone or with 3, 7, 9, 11, 13 or 15 µM oleanolic acid derivative for 24, 48 and 72 h.

Cell viability was determined using the MTT assay. SMMC-7721 and HL-7702 cells were seeded at a density of 1×10⁵ cells/well in 96-well plates. Following incubation for ~24 h at 37°C in a 5% CO₂ incubator, the cells were treated with various concentrations of oleanolic acid derivative (0, 3, 5, 7, 9, 11, 13 and 15 µM). The MTT assay was performed after 24, 48 and 72 h of treatment. The culture medium was discarded, then 30 µl 0.5% (w/v) MTT dissolved in 1X phosphate-buffered saline was added to each well, and the plate was incubated for 3 h at 37°C. Following incubation for 3 h, the culture medium was discarded and 120 µl DMSO was added into each well. Following incubation for 30 min and gentle agitation for 15 min at 37°C, the absorbance at 490 nm was evaluated using a microplate reader. The experiment was repeated in triplicate. Cell viability was expressed as a percentage of proliferation against the controls (untreated cells), which was set at 100%.

The cell membrane integrity of SMMC-7721 cells was assessed using annexin V-FITC and PI double staining. Briefly, ~1×10⁵ SMMC-7721 cells were seeded/well in 6-well plates. Following incubation for 24 h, the cells were treated with various concentrations of oleanolic acid derivative (7, 9 or 11 µM) or vehicle (0.11% DMSO) alone for 12, 24 or 36 h. Subsequently, the drug-containing medium was removed at each time point, and the SMMC-7721 cells were harvested, washed and stained with annexin V-FITC/PI, according to the manufacturer's protocol. Samples were then diluted with binding buffer and were analyzed using a fluorescence microscope (×200) within 1 h.

Following treatment with the oleanolic acid derivative (7, 9 or 11 µM) for 48 h at room temperature, the SMMC-7721 cells were harvested. Total protein was extracted using RIPA cell lysis buffer on ice. The protein concentration was determined using a BCA protein assay. Proteins were then mixed with loading buffer (Beijing ComWin Biotech Co., Ltd., Beijing, China) and boiled at 95°C for 10 min. Equal amounts of proteins (60 µg) were separated by SDS-PAGE (12% gradient gel), transferred to PVDF membranes and detected using the specific antibodies. The PVDF membranes were then incubated overnight at 4°C with anti-caspase-9 and anti-caspase-3 (dilution, 1:1,000). The immunoreactive proteins following incubation at 37°C with the horseradish peroxidase-conjugated goat anti-rabbit antibodies (dilution, 1:10,000) were detected using an enhanced chemiluminescence detection kit. The results were normalized to those of β-actin (dilution, 1:1,000).

Cells (~1×10⁵) were cultured in 96-well plates and incubated with 0, 7, 9 or 11 µM oleanolic acid derivative or vehicle (0.11% DMSO) alone for 36 h. Intracellular ATP levels were determined using an ATP determination kit, according to the manufacturer's protocol. The entire cell population, including any floating cells, were assayed. Luminescence was determined using a multimode microplate reader. The experiment was repeated in triplicate.

The lipophilic cationic fluorescent probe, JC-1, a mitochondrial dye that stains mitochondria in living cells in a membrane potential-dependent fashion, was used to quantify the ΔΨm. The reduction in the red/green fluorescence intensity ratio of JC-1 is directly proportional to the loss of ΔΨm. In brief, cells were treated with or without the oleanolic acid derivative (7, 9 or 11 µM) in 6-well plates for 12, 24 and 36 h. The medium was removed and replaced with 1 ml fresh culture medium (RPMI-1640 medium supplemented with 2% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin), followed by the addition of 1 ml JC-1 staining solution (final concentration of 2 mM) and incubation at 37°C for 20 min. The cells were then harvested and washed twice with JC-1 Dyeing buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Finally, 2 ml cell culture medium was added to each well, and JC-1 fluorescence was determined using a fluorescence microscope (×100).

SMMC-7721 cells were harvested following treatment with various concentrations of the oleanolic acid derivative (7, 9 and 11 µM) for 12 h. Cytoplasmic and mitochondrial proteins were isolated using a Cytoplasmic and Mitochondrial Protein Extraction kit, according to the manufacturer's protocol. The concentrations of cytoplasmic and mitochondrial proteins were detected using the BCA assay. The concentrations of Cyt-C in the cytoplasm and mitochondria were detected according to the manufacturer's instructions using a Human Cyt-C ELISA kit, In order to ensure that the Cyt-C concentration was accurate, the cytoplasmic and mitochondrial protein were adjusted to the same level and then the sample was added to 50 µl of diluent to each well at the end. The Cyt-C content of the sample was determined by the regression equation of the standard curve of the reference standard.

All experiments were performed at least three times. All data were analyzed using SPSS version 19.0 statistical software (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation. Statistical analysis of the data for multiple groups was performed using one-way analysis of variance and Dunnett's test. GraphPad Prism version 5.02 (GraphPad Software, Inc., La Jolla, CA, USA) and Excel (2010) were used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

To determine the cytotoxicity of the oleanolic acid derivative, the present study treated SMMC-7721 cells and HL-7702 normal liver cells with various concentrations of the oleanolic acid derivative (0, 3, 5, 7, 9, 11, 13 and 15 µM) for 24, 48 and 72 h, and analyzed the cell viability by performing an MTT assay. In comparison with the DMSO control, the oleanolic acid derivative inhibited the proliferation of SMMC-7721 and HL-7702 cells in a dose- and time-dependent manner (Fig. 1B). The maximum inhibition rates for the oleanolic acid derivative on SMMC-7721 cells were 30.30, 63.00 and 82.46%, and on HL-7702 cells were 37.20, 64.45 and 84.68% at 24, 48 and 72 h, respectively. The inhibition rates demonstrated that the oleanolic acid derivative had the same inhibitory effect on SMMC-7721 cells and HL-7702 normal liver cells (Fig. 1B). These results indicated that the oleanolic acid derivative dose- and time-dependently inhibited the proliferation of SMMC-7721 and HL-7702 cells.

To determine whether the oleanolic acid derivative induces cell apoptosis, the present study treated SMMC-7721 cells with 0, 7, 9 or 11 µM oleanolic acid derivative for 36 h and observed cell morphology under a microscope. Compared with the DMSO-treated cells, the SMMC-7721 cells underwent the typical morphological changes of apoptosis, including cell shrinkage, membrane blebbing and accumulation of small apoptotic bodies (as indicated by the arrows in Fig. 2A). The induction of apoptosis was more evident when the oleanolic acid derivative concentration was 9 or 11 µM. These results demonstrated that the oleanolic acid derivative induced apoptosis of SMMC-7721 cells in a dose-dependent manner.

Annexin V is a protein that interacts strongly and specifically with phosphatidylserine residues on the cell membrane and is widely used for the detection of apoptosis (8). PI, a nucleic acid dye, cannot cross the intact cell membrane; however, in the late-apoptotic cells and dead cells, PI is able to penetrate the cell membrane and interact with DNA, revealing red staining under a microscope. Thus, PI has been widely applied to assess cell viability. To further support the evidence that the oleanolic acid derivative induced apoptosis of SMMC-7721 cells, the present study treated SMMC-7721 cells with 0, 7, 9 or 11 µM oleanolic acid derivative for 12, 24 and 36 h and performed annexin V/PI double staining. As presented in Fig. 2B, there were markedly few red (PI-stained) and green (annexin V-stained) fluorescent cells in the control and DMSO groups, whereas the red fluorescence was increased with the increase of oleanolic acid derivative concentration and the extension of treatment time. The red fluorescence was highest at 11 µM oleanolic acid derivative treatment for 36 h. These results revealed that the oleanolic acid derivative induced membrane disintegrity of SMMC-7721 cells in a dose- and time-dependent manner.

Oleanolic acid derivative induces activation of the intrinsic apoptosis pathway in SMMC-7721 cells. To explore the mechanism underlying apoptosis induction by the oleanolic acid derivative, the present study determined the change of apoptosis-associated protein expression levels in SMMC-7721 cells following treatment with various concentrations of the oleanolic acid derivative (5, 9 and 11 µM) for 48 h by western blotting. The results revealed that anti-apoptotic protein, Bcl-2, was downregulated following 11 µM oleanolic acid derivative treatment for 48 h. Conversely, the proapoptotic protein, Bax, was upregulated following treatment of 5, 9 or 11 µM oleanolic acid derivative for 48 h (Fig. 3A). It is well known that caspase-3 is the ultimate executioner caspase of apoptosis and that activated caspase-9 cleaves downstream caspases, including caspase-3, to initiate the caspase cascade (9). Indeed, treatment of SMMC-7721 cells with the oleanolic acid derivative for 48 h induced obvious cleavage of caspase-9 and caspase-3 in a concentration-dependent manner (Fig. 3B). These results indicated that the oleanolic acid derivative induced the intrinsic pathway of apoptosis in SMMC-7721 cells.

Mitochondria serve a key role in activation of the intrinsic cell apoptosis pathway. The activation of caspase-9 following oleanolic acid derivative treatment suggested that the oleanolic acid derivative may disrupt mitochondrial function. To investigate this hypothesis, the present study first assessed the intracellular ATP expression level, a decrease of which indicates an impairment of mitochondrial function. As presented Fig. 4, the ATP expression levels were significantly decreased in a dose-dependent manner following oleanolic acid derivative treatment. These results demonstrated that the oleanolic acid derivative impaired mitochondrial function.

Impairment of mitochondrial function is closely associated with the loss of ΔΨm. Subsequently, the present study treated SMMC-7721 cells with 0, 7, 9 or 11 µM oleanolic acid derivative for 12, 24 and 36 h, and determined the ΔΨm using the lipophilic cationic fluorescent probe JC-1 assay. Compared with the blank and DMSO controls, the oleanolic acid derivative significantly decreased the red/green fluorescence ratio in SMMC-7721 cells in a dose- and time-dependent manner (Fig. 5). Furthermore, the cell adherence rate declined markedly when the cells were exposed to 11 µM oleanolic acid derivative. Numerous floating cells were removed during the experiments (data not shown). These results indicated that the oleanolic acid derivative induced a decrease of ΔΨm in SMMC-7721 cells.

It is well known that Cyt-C release from the mitochondria during apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization, indicating that a reduction in ΔΨm and caspase activation occurs considerably later than Cyt-C release (10). In the present study, depolarization of ΔΨm was revealed in SMMC-7721 cells exposed to the oleanolic acid derivative for 12 h (Fig. 5). Therefore, the present study detected the mitochondrial and cytoplasmic content of Cyt-C in SMMC-7721 cells treated with various concentrations of the oleanolic acid derivative for 12 h. The standard curve regression equation was y=0.0114×-0.0391 (R²=0.9989; Fig. 6A). The content of the sample was determined by the regression equation of the standard curve of the reference standard. The results demonstrated that when SMMC-7721 cells were treated with various concentrations of the oleanolic acid derivative for 12 h, the mitochondrial content of Cyt-C was decreased significantly in a dose-dependent manner, with a 65.48% decrease by 11 µM oleanolic acid derivative compared with the controls. In the cytoplasm, the content of Cyt-C was increased when the cells were treated with 7 µM oleanolic acid derivative and then gradually decreased with 9 and 11 µM oleanolic acid derivative (Fig. 6B). These results demonstrated that the oleanolic acid derivative induced Cyt-C release from the mitochondria in SMMC-7721 cells.