The human hepatocarcinoma Hep3B and normal liver HL-7702 cell lines were purchased from Chinese Academy of Sciences Institute of Life Sciences Cell Resource Center (Shanghai, China). They were grown in RPMI-1640 medium and Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences; Hyclone, Logan, UT, USA), respectively. The cell cultures contain 10% fetal bovine serum (GE Healthcare Life Sciences; Hyclone) and 100 U/ml penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO₂. Then, cells were harvested in the logarithmic growth phase for subsequent experiments.

Hep3B cells (2×10⁵ cells) and the normal liver HL-7702 cell (2×10⁵ cells) suspended in 150 µl DMEM and RPMI-1640 medium with 10% FBS, respectively. They were seeded into each well of 96-well microtiter plates overnight, and then treated with 25, 50 and 100 µl/ml Delisheng. Cells were cultured at 37°C and 5% CO₂ for 24, 48 and 72 h at each concentration of Delisheng. Subsequently, 20 µl MTT (5 mg/ml) was added to each well for 4 h at 37°C and formazan crystals were dissolved in 200 µl dimethyl sulfoxide for each well. The optical density (OD) value was recorded at a wavelength of 492 nm. The rate of growth inhibition (%) = (1 - OD of treated group)/(OD of control) × 100. This experiment was repeated in triplicate.

The Hep3Bcells treated with Delisheng (2×10⁵ cells/group) were trypsinized, washed twice with cold PBS and harvested by centrifugation at 4,000 × g and 37°C for 5 min, and then incubated in 100 µg/ml propidium iodide (PI) with RNaseA (5 µg/ml) following fixation with 70% ice-cold ethanol at room temperature (RT) for 30 min. Cellular DNA content was subsequently analyzed by flow cytometry analysis on a BD FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA), as previously described (16).

Hep3B and HL-7702 cells treated with 25, 50 and 100 µl/ml Delisheng were collected and washed twice with cold PBS, and centrifuged at 5,000 × g for 5 min, followed by staining with 5 µl Annexin V-fluorescein isothiocyanate and 5 µl PI for 15 min at 37°C in darkness. The apoptotic cells were subsequently analyzed by fluorescence-activated cell sorting (FACSCalibur; BD Biosciences) using Cell Quest Research 3.3 Software (BD Biosciences).

This procedure has been described previously (15). Briefly, following treatment with 25 µl/ml Delisheng, the Hep3B cells were washed with PBS and then stained with the primary antibodies (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Beijing, China) against VEGF (cat.no., ZA-0509), OPN (cat. no., ZM-0174) and ES (cat.no., TA507124) (dilutions, 1:100) overnight at 4°C. The next day, the cells were washed with PBS 3 times, followed by incubation with a secondary antibody (Biotinylated goat anti-rabbit IgG; cat. no., bs-0294P; Xiamen Bosen Biotechnology Co. Ltd., Xiamen, China) diluted to 1:500 for 20 min at 37°C, and then immunostained using an avidin-biotin complex (ABC) protocol (ABC Staining Kits; Xiamen Bosen Biotechnology Co., Ltd. Xiamen, China) for 30 min, according to the protocol of the manufacturer. The color reaction was visualized through a light microscope (Olympus BH-12, Tokyo, Japan) at magnification, ×400 using 3,3′-diaminobenzidinefor 5 min. The nuclei were counterstained with hematoxylin (10 mg/ml) solution (Beyotime Institute of Biotechnology, Shanghai, China) at 37°C for 10 sec.

The immunoreactivity was evaluated according to the cell staining degree and percentage rate of the positive cell. Staining intensity was graded as follows: 0, no staining; 1, weak; 2, moderate; and 3, strong. The percentage of positively stained cells was scored as follows: 0, ≤5%; 1, 5–25%; 2, 26–50%; and 3, >50%. The score of staining intensity and percentage score of positive cells were multiplied. In these groups, tumors with 0–2 scores were noted as negative, while 3–9 scores were designated as positive. The immunocytochemical results were quantitatively analyzed using LeicaQ550cw imaging analysis system (Leica Microsystems GmbH, Wetzlar, Germany).

Following treatment with 25 µl/ml Delisheng, the Hep3B cells were harvested in ice-cold PBS and lysed with radioimmunoprecipitation cell lysis buffer containing protease and phosphatase inhibitors (Xi'an Wolsen Biotechnology Co. Ltd., Xi'an, China). Protein concentrations in the cell extracts were determined using the BCA Protein Assay reagents (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Each protein sample (30 µg) was subject to 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany), blocked with 8% non-fat milk for 2 h at RT. The membranes were labeled with primary antibodies overnight at 4°C. Subsequently, blots were probed with horseradish peroxidase-conjugated secondary anti-rabbit IgG antibodies (1:5,000; cat. no., sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at RT for 1 h. The primary antibodies VEGF (cat. no., ab46154), OPN (cat. no., ab8448), ES (cat. no., ab64569) at dilutions of 1:1,000 and β-actin (cat. no., ab8227; 1:5,000) were purchased from Abcam (Cambridge, UK). For the quantification of protein levels, films were scanned and analyzed using Labworks 4.0 software (World BioHazTec Corporation, NM, USA).

All results were presented as the mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance followed by Tukey's post hoc test. Student's t-test and Spearman's rank test using SPSS v.19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effects of Delisheng were measured by MTT assay. Based on Table I and Fig. 1, exposure of these cells to Delisheng at the indicated concentrations for 24–72 h periods exhibited growth inhibition effects of Hep3B in a time- and dose-dependent manner. Compared to Hep3B cells, HL-7702 cells were less sensitive to the antiproliferative effects of Delisheng (Table I).

To determine the mechanism underlying the antiproliferative activity of Delisheng on Hep3B cells, cell cycles were tested by flow cytometric analysis. As demonstrated in Table II and Fig. 2, in Hep3B cells treated with 25, 50 and 100 µl/ml Delisheng, G₀/G₁ cell cycle arrest was observed. In addition, the results indicated the cells exhibited G₀/G₁ phase arrest in a concentration-dependent manner.

To additionally determine the potential mechanism of the anticancer effects of Delisheng, cell apoptosis was measured by flow cytometry. As indicated by Fig. 3 and Table III, following treatment with 25, 50 and 100 µl/ml Delisheng at 24 h, the apoptotic rates of Hep3B cells were 21.93±0.98, 26.29±4.45 and 33.02±3.09%, respectively (P<0.05 vs. untreated cells). The HL-7702 cells, which exhibited apoptotic rates of 6.26±3.12, 8.52±2.55 and 15.84±2.67%, at 25, 50 and 100 µl/ml Delisheng at 24 h, respectively, and Delisheng-untreated Hep3B cells did not demonstrate evidence of apoptosis.

Immunocytochemistry analysis demonstrated that the levels of VEGF, OPN and ES expression were observed in the cytoplasm of Hep3B cells. Fig. 4 indicates that Delisheng treatment increased VEGF and OPN and decreased ES protein expression levels (P<0.05). As observed in Fig. 5 and Table IV, western blotting analysis additionally confirmed the results. Furthermore, the Spearman's rank correlation analysis indicated that OPN was positively correlated with VEGF expression (r=0.58, P=0.011). However, ES was negatively correlated with VEGF (r=0.57, P=0.009) and OPN expression (r=0.69, P=0.025).