The roots of V. souliei were identified by Professor Min Chen, the corresponding author of the present study, of the College of Pharmaceutical Sciences, Southwest University (Chongqing, China). The plants were collected at Luding County (Sichuan, China) in October 2015, and a voucher specimen (no. 2015-14) was deposited at the College of Pharmaceutical Sciences, Southwest University.

Powder derived from the air-dried roots (11.0 kg) of V. souliei was extracted three times with 95% ethanol overnight at room temperature. The ethanol extract was evaporated in vacuo to yield a semisolid (1.12 kg), which was then suspended in water and partitioned successively with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate solution was concentrated to yield 296 g of residue, which was subjected to silica gel column chromatography elution with petroleum ether/ethyl acetate, using mixtures of increasing polarity (99:1 to 10:1) to obtain a total of 16 fractions. Cos (4.23 g) was purified from fraction 7 (purity, >98%) by crystallization and recrystallization, and its structure was confirmed by spectroscopic methods, including 1H-nuclear magnetic resonance and mass spectrometric analyses, by comparing with data in the literature (21).

Human liver cancer HepG2 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Dimethyl sulfoxide (DMSO), the Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), streptomycin and penicillin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The MTT assay kit, cell cycle analysis kit, rabbit monoclonal primary antibodies against β-actin (cat. no. AF0003), Bax (cat. no. AF1270), Bcl-2 (cat. no. AF1915), caspase-3 (cat. no. AF1213), caspase-8 (cat. no. AF1243) and caspase-9 (cat. no. AF1264), and horseradish peroxidase-conjugated secondary antibodies (cat. no. A0208) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Cos (purified via recrystallization to a purity >98%, as described above) was dissolved in DMSO and stored at 4°C.

HepG2 cells were cultured in DMEM supplemented with 10% FBS, 100 µg/ml streptomycin and 100 µg/ml penicillin at 37°C in a humidified atmosphere containing 5% CO2. Cells in the control group were treated with DMSO, whereas HepG2 cells in the experimental groups were exposed to 2.5, 5, 10, 20 and 40 µmol/l cos for 3, 6, 12, 24, 36 and 48 h. In the cos treatment groups, the final concentration of DMSO added to the cells was <0.1%.

Morphological changes associated with apoptosis were assessed using light microscopy. Following the corresponding treatments, the morphology of HepG2 cells was observed under an inverted light microscope (Olympus CKX53; Olympus Corporation, Tokyo, Japan), and images were captured at a magnification of ×100.

MTT assay was used to measure the viability of the HepG2 cells. Briefly, cells were plated in 96-well culture plates (1×104 cells/well) and incubated at 37°C with cos at various concentrations (2.5, 5, 10, 20, and 40 µmol/l) for 48 h. In addition, cells were treated with 10 µmol/l cos for 3, 6, 12, 24, 36 and 48 h. Subsequently, MTT solution (5 mg/ml) was added to each well. After 3 h of incubation, the formazan precipitate was dissolved in 100 ml DMSO, and the absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The half-maximal inhibitory concentration (IC50) values were calculated by nonlinear regression analysis using Origin version 8.0 (OriginLab Software, Inc., Northampton, MA, USA) and GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) software packages. Each sample was analyzed in triplicate.

Cell cycle distribution was analyzed using flow cytometric analysis. Briefly, cells were plated in 10-cm plates, and treated with DMSO or with the various concentrations (2.5, 5, 10, 20, and 40 µmol/l) of cos for 48 h. Subsequently, the treated cells were collected, washed twice with cold phosphate-buffered saline (PBS), fixed with 70% ethanol and stained with propidium iodide (PI). The cells were then analyzed using a flow cytometer (Accuri C6; Bio-Rad Laboratories, Inc.), and the data obtained were analyzed with FlowJo software (version X; FlowJo LLC, Ashland, OR, USA).

Cells were treated with DMSO or various concentrations (2.5, 5, 10, 20, and 40 µmol/l) of cos. After 48 h of incubation, the cells were harvested and washed twice in cold PBS for cell apoptotic analysis. The cells were subsequently treated with 5 µl Annexin V/FITC and 5 µl PI solution, and then incubated at room temperature for 15 min in the dark. Finally, the cells were analyzed for apoptosis using an Accuri C6 flow cytometer. According to the protocol provided with the apoptosis kit, the number of cells in each cell cycle phase was calculated using Cell ModFit software (BD Biosciences, Franklin Lakes, NJ, USA). The experiments were performed in triplicate. The apoptotic rate was the sum of the proportion of cells in the early (Annexin V/FITC+ PI-) and late (Annexin V/FITC+ PI+) stages of apoptosis.

Approximately 5×105 HepG2 cells plated in 6-well culture plates were used for western blotting. Total protein fractions were isolated, the medium was removed, and cells were washed twice with ice-cold PBS prior to lysis using cell lysis buffer (containing 20 mM Tris, 150 mM NaCl and 1% Triton X-100). The lysates were collected by scraping from the plates and subsequently centrifuged at 12,000 × g at 4°C for 5 min. Protein concentrations were determined using the BCA protein assay method (Beyotime Institute of Biotechnology). Samples (50 µg) were separated by SDS-PAGE (10% gels) and subsequently transferred onto nitrocellulose membranes for 1.5 h. Following blocking with 10% dried fat-free milk in Tris-buffered saline buffer with 0.05% Tween-20 for 1 h at 37°C, the membranes were incubated with the primary antibodies overnight at 4°C. The primary antibodies used were as follows: Anti-Bax (1:1,000 dilution), anti-Bcl-2 (1:1,000), anti-caspases-3, −8 and −9 (1:1,000), and anti-β-actin (1:1,500). Subsequently, the membranes were incubated with secondary goat anti-rabbit antibodies (1:1,000) at 37°C for 1 h. Proteins were detected using an EasyBlot ECL kit (cat. no. C506668-0100; Sangon Biotech Co., Ltd., Shanghai, China). Using ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA) for grayscale analysis, the relative expression levels of the target proteins were expressed as the ratio of the gray value of Bax, caspase-3, caspase-8, caspase-9 and Bcl-2 protein to the gray value of β-actin.

Data are expressed as the mean ± standard deviation. SPSS software version 18.0 (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analyses. One-way analysis of variance followed by least significant difference post hoc test was used to compare data containing multiple groups. Student's t-test was performed to determine statistically significant differences between two groups. P<0.05 was considered to indicate statistically significant values.

The chemical structure of cos, which is a sesquiterpene lactone with an α-methylene-γ-lactone ring, is shown in Fig. 1A. HepG2 cells in the DMSO group appeared to have a regular phenotype and to adhere to the wall of the plate, as observed in images captured under an inverted microscope (Fig. 1B). By contrast, the majority of the cells pretreated with 10 µmol/l cos for 48 h appeared to have a smaller volume, irregular shape and smaller size, while they were separated from adjacent cells and lost their adhesive property, revealing a clear reduction in their proliferative capacity. At the end of the treatment period, the adhesion of these cells gradually declined, and they eventually separated and floated freely (Fig. 1B). Furthermore, the results revealed that cos effectively inhibited the proliferation of HepG2 cells in a dose-dependent manner, as compared with the DMSO-treated cell group (Fig. 1C). After 48 h of treatment with cos, the IC50 was determined to be 18.09±1.74 µM. The inhibitory effect of cos was also induced in a time-dependent manner, with the proliferation of HepG2 cells progressively decreasing over the time course of the experiment (3, 6, 12, 24, 36 and 48 h), as shown in Fig. 1D. Taken together, these results suggested that cos effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner.

HepG2 cells in the control group were treated with DMSO, whereas cells in the experimental groups were exposed to 2.5, 5, 10, 20 and 40 µmol/l cos for 48 h. PI staining and flow cytometric analysis were used to detect the effect of cos on the cell cycle distribution of HepG2 cells, and the results are shown in Fig. 2. Compared with the DMSO-treated group, the percentage of cells in G1/G0 phase following treatment with the various concentrations of cos did not change significantly, whereas the percentage of cells in the G2/M phase was significantly increased and that in S phase was significantly decreased. Following treatment with 0, 2.5, 5, 10, 20 and 40 µmol/l cos for 48 h, the percentage of HepG2 cells in the G2/M phase was 18.52±2.58, 20.90±4.21, 26.54±3.67, 29.08±6.32, 32.17±3.14 and 33.42±8.97%, respectively. These results suggested that cos treatment led to cell cycle arrest at G2/M phase in a dose-dependent manner.

Translocation of phosphatidylserine (PS) to the outer leaflet of the cellular membrane is the key step in the early stages of apoptosis. Annexin V selectively binds to PS, and this process enables the identification of cells undergoing apoptosis. Different populations of cells may be observed when cells are double-stained with Annexin V and PI (22). HepG2 cells in the control group were treated with DMSO, whereas the treatment groups were exposed to 2.5, 5, 10, 20 and 40 µmol/l cos for 48 h. As revealed in Fig. 3, the apoptotic rate of the HepG2 cells increased significantly with an increasing concentration of cos. The apoptotic rates of HepG2 cells upon treatment with 2.5, 5, 10, 20 and 40 µmol/l cos were 6.04±1.24, 8.04±1.57, 17.03±2.31, 37.19±3.19 and 55.33±2.12%, respectively.

Bax and Bcl-2 proteins exert pivotal roles in caspase activation and the regulation of apoptosis (23). In addition, caspase family proteins, the central components of the apoptotic response, are a conserved family of enzymes that induce irreversible cell death (24). Caspases-3, −8 and −9 stand at the nexus of critical regulatory networks controlling cell apoptosis, and are components of the pathway that ultimately mediates the activation and execution of apoptosis. Therefore, the levels of caspases-3, −8 and −9, as well as Bax and Bcl-2 proteins, were investigated in the present study. Cos treatment was observed to induce the apoptosis of HepG2 cells by upregulating the protein expression levels of Bax, and caspases-3, −8 and −9, and by downregulating the expression of Bcl-2 protein in a dose-dependent manner after 48 h of treatment (Fig. 4). Taken together, these results suggested that cos exerted an inhibitory effect on the growth of HepG2 cells.