Huachansu injection (5 ml/dose) was purchased from Anhui Jinchan Biochemical Co., Ltd. (batch no. 120811-3; Anhui, China). 20(R)Ginseng saponin Rg3 standard (20 mg/bottle) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (batch no. 20120506; Shanghai, China). Lentinan standard (1 g/bag) was obtained from Nanjing Zelang Medical Technology Co., Ltd., Nanjing, China). Xuesaitong injection (lyophilized; 400 mg/ampule) was purchased from Kunming Shenghuo Pharmaceutical Group Ltd. (batch no. 12GA12; Kunming, China), and DDP for injection (10 mg/branch) was obtained from Qilu Pharmaceutical Co., Ltd. (batch no. 012027CF; Jinan, China). The QHF preparation contained huachansu, Rg3, notoginseng total saponin and lentinan at a ratio of 57:1:0.4:7.

Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin was obtained from Shanghai Source Leaves Biological Technology, Ltd. (Shanghai, China), and MTT was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Matrigel™ glue was obtained from Wuhan Kori Biological Technology, Ltd. (Wuhan, China). A Multiskan™ Spectrum full-wavelength enzyme standard instrument was obtained from Thermo Fisher Scientific (Waltham, MA, USA). An XS-213 optical microscope and a CKX41 inverted phase contrast microscope were purchased from Olympus Corp. (Tokyo, Japan).

The human HCC cell line HepG2 was obtained from the China Center for Type Culture Collection of Wuhan University (Wuhan, China). The cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

An MTT assay was used to detect cell viability following exposure of the HCC cells to QHF. The HepG2 cells were grown on 96-well plates, at a concentration of 2×10³ cells/well, incubated at 37°C in 5% CO₂ for 24 h and then treated with different concentrations of QHF (QHF1-5: 20, 40, 80, 160 and 320 µg/ml). Incubation was subsequently continued for 24, 48 and 72-h periods, respectively (5 wells/treatment group). A total of 20 µl MTT was added 4 h prior to the end of the final incubation time, and the cells were then incubated for a further 4 h. Following incubation, the density value for each well plate was determined using a microplate reader at a detection wavelength of 570 nm.

Using a marker pen and a straight edge guide, lines were drawn evenly across each well on the reverse side of six-well tissue culture plates, with 6 rows per well at 0.5-cm intervals. Next, 1.5×10⁶ cells were aliquoted into each well, and a microscope was used the following day to confirm that each well was coated with cells. A needle was used to scratch and remove the cells from a discrete area of the confluent monolayer. Lines were marked using 10-µl spearheads along the ruler, perpendicular to the horizontal line scratches. The plates were washed with phosphate-buffered saline three times to remove the displaced scratched cells, and serum-free DMEM was added. Cells exposed to different concentrations of QHF (QHF1-5: 20, 40, 80, 160 and 320 µg/ml) and the appropriate control groups were cultivated simultaneously in an incubator at 37°C in 5% CO₂. At 0 and 24 h, images of the samples were captured, and the procedure was repeated three times.

Cell suspensions (100-µl) were sampled from the upper wells and exposed to different QHF concentrations (QHF1-3: 20, 40 and 80 µg/ml). The total volume of fluid in each chamber was 200 µl. A total of 600 µl DMEM containing 10% FBS was placed in each lower chamber and incubated at 37°C in 5% CO₂ for 24 h. Subsequently, the small indoor culture medium was discarded and the cells on the inner surface of the membrane were gently cleaned using cotton. The cells were exposed to 4% paraformaldehyde for 1 h for fixation and stained using crystal violet for 30 min. The cells were then washed with sterile water three times, and the membrane filter was gently placed on a microscope slide using forceps. The outside membrane cells were observed at ×200 magnification using an optical microscope, and five fields were randomly selected to calculate an average cell count. This procedure was repeated three times.

Transwell^® chambers with a fiber membrane pore size of 8 µm were used. The lower compartment was filled with 600 µl serum media, containing 10% bovine serum albumin (BSA). The upper compartment was filled with 600 µl serum-free media, containing 10% BSA, and 1×10⁵/ml cell suspension was added in a 30-µl volume. The cells were incubated for 24 h at 37°C in a humidified 5% CO₂ atmosphere. The membrane was subsequently removed, fixed using methanol and stained using Giemsa. Five randomly selected fields were counted, and each sample was assayed in duplicate to give an average quantitative measure of the degree of invasiveness of each tumor cell.

Cells were collected and washed twice with ice-cold phosphate-buffered saline, prior to lysis with 100 µl radioimmunoprecipitation assay buffer (Takara Biotechnology Co., Ltd., Dalian, China) for 30 min. For nuclear extraction the cells were lysed with Nuclear-Cytosol Extraction kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), according to the manufacturer's instructions. Lysates were centrifuged at 1613 × g for 10 min at 4°C. Total protein content was determined using bicinchoninic acid protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). A total of 50 µg of proteins were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (Shanghai Source Leaves Biological Technology Ltd.). The membranes were blocked in Tris-buffered saline + Tween-20 (TBST; Beyotime Institute of Biotechnology, Haimen, China) containing 5% non-fat dried milk for 1 hour at room temperature, and then incubated overnight at 4°C with primary antibodies (1:800 dilution; ERK (cat. no. ab50011); p38 (cat. no. ab31828), JNK (cat. no. ab179461)) against extracellular signal-regulated kinase (ERK) and phosphorylated-(p-)ERK (both Santa Cruz Biotechnology, Inc.), and p38, c-Jun N-terminal kinase (JNK), p-p38 and p-JNK (all Cell Signaling Technology, Inc., Beverly, MA, USA). The membranes were then washed with TBST three times and incubated with the corresponding secondary antibody (Goat anti-mouse IgG Fc, 1:3000 dilution; cat. no. ab97261; Sigma-Aldrich) for 1 h. The membranes were then washed again and the proteins were visualized using an enhanced chemiluminescence assay kit (Eastman Kodak, Rochester, NY, USA). Images were captured as a permanent record of the data.

The experimental groups were as follows: Blank control, QHF4, QHF4 + JNK inhibitor (SP600125; JNK inhibitor concentration, 10 mM; Beyotime Institute of Biotechnology), QHF4 + p38 inhibitor (SB203580; p38 inhibitor concentration, 10 mM; Beyotime Institute of Biotechnology), QHF4 + ERK inhibitor (PD98059; ERK inhibitor concentration, 10 mM; Selleck Biological Technology, Nanjing, China). The method described for the cell invasion assay was followed.

Data are expressed as the mean ± standard deviation and a Student's t-test was performed for the statistical analysis of single comparisons. P<0.05 was considered to indicate a statistically significant difference.

The results of the MTT assay demonstrated that 24, 48 and 72 h after the exposure of HepG2 cells to different concentrations of QHF, cell proliferation was inhibited in a concentration-dependent manner. As the QHF concentration increased, a more marked inhibitory effect was exerted against HepG2 cell proliferation (P<0.05). A significant difference was observed between the degrees of inhibition produced in each of the groups, although the inhibitory effect was not time-dependent. The inhibition rate in each of the concentration groups after 24 h was not reduced in the groups at 48 h; however, the largest inhibition rate was observed in the groups at 72 h (Table I).

The effect of QHF on the proliferation of cells within 24 h of exposure to QHF1-QHF3 was limited, with an inhibition rate of 14–22%; therefore, the following three concentrations were selected for subsequent experiments into cell invasion and metastasis.

An inverted microscope was used to observe the difference in the scratch cell areas of HepG2 cells within 24 h of QHF treatment. The results showed that the scratch damage zones of the QHF intervention groups were significantly reduced compared with those of the control group. In the QHF groups, it was difficult to detect cells migrating to the scratch area; by contrast, the scratch damage area of the control group was infiltrated with migrated liver cancer cells. These data suggest that QHF is able to inhibit HepG2 cell migration (Fig. 1).

The cell migration assay results showed that the number of HepG2 cells passing through the membrane filter in the QHF intervention group was significantly reduced compared with that in the control group (P<0.05 or P<0.01), and the migration inhibition rate increased with concentration. It was therefore concluded that QHF exerted a marked concentration-dependent inhibitory effect on the ability of HepG2 cells to migrate. Compared with the control group, the number of cells migrating was significantly different in the groups exposed to various concentrations of QHF (P<0.05) (Fig. 2).

The cell invasion assay results were consistent with the migration assay results. Compared with the control group, QHF significantly reduced the number of HepG2 cells migrating through the membrane filter (P<0.05 or P<0.01), and the invasive inhibition rate increased with the increase in QHF concentration. Thus, QHF exhibited marked inhibitory activity against the invasive ability of HepG2 cells in a concentration-dependent manner. A statistically significant difference was detected in cell number between the various QHF concentrations (QHF1-3: 20, 40 and 80 µg/ml) (P<0.05) (Fig. 3).

Using western blot analysis, it was demonstrated that, at 24 h after intervention, the expression levels of p-ERK were decreased significantly in the HepG2 cells treated with QHF compared with those in the control group cells, in a concentration-dependent manner (P<0.05); however, differences in the expression of ERK protein between the different experimental groups were not always detected. Overall the results suggest that QHF inhibits the ERK signaling pathway (Fig. 4).

The results of western blot analysis showed that the expression levels of p-p38 and p-JNK were significantly increased in the HepG2 cells exposed to QHF for 24 h compared with those in the control group cells, in a concentration-dependent manner (P<0.05); however, the total expression levels of p38 and JNK protein in each group were not obviously altered. The results indicate that QHF is able to activate the p38 and JNK signaling pathways (Figs. 5 and 6).

PD98059 is an ERK-specific inhibitor that blocks the ERK signaling pathway. SB203580 is a specific p38 inhibitor that is able to inhibit p38 activity by blocking p38 signaling pathways. SP600125 specifically inhibits the actions of JNK by blocking the JNK signaling pathway. The results of the cell invasion experiments indicated that, following the blockage of the ERK pathway, the activity of the ERK pathway was decreased significantly compared with that in the cells treated solely with QHF (15±3.54 vs. 25±4.12; P<0.05). ERK inhibitors thus appear to enhance the ability of QHF to inhibit liver cancer cell invasion. After blocking p38, and therefore the JNK signaling pathway, the number of cells passing through the membrane filters was increased significantly compared with the cells treated with QHF alone (53±7.84 (QHF4 + SB203580) vs. 25±4.12 (QHF4), 45±8.92 (QHF4 + PD98059) vs. 25±4.12). This difference was statistically significant (P<0.01) indicating that the inhibition of liver cancer cell invasion by QHF is partially reversed by the p38 and JNK inhibitors (Fig. 7).