RPMI-1640 medium, Opti-MEM^® reduced serum media and newborn bovine serum (NBCs) were purchased from Gibco Laboratories (Grand Island, NE, USA). Penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). The primary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

The human hepatoma cell (SMMC-7721) and normal human liver cell (Chang liver) were obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology, China Academy of Sciences (Shanghai, China). Cav-1 knockdown hepatoma SMMC-7721 cells using RNA interference technology to build early in our laboratory. The cells were grown in RPMI-1640 medium containing 15% newborn bovine serum (NBCs), penicillin (100 μg/ml), and streptomycin (100 μg/ml). Cultures were maintained at 37˚C in a humidified 5% CO₂ atmosphere. Ginseng leaves crude polysaccharides (GLCP) was dissolved in the complete medium and adjusted to the indicated final concentrations with culture medium before use.

The effect of GLCP on the growth of human hepatoma cell SMMC-7721 was evaluated using 5 × 10³ cells seeded onto 96-well plates, which were treated with GLCP after 24h at the time of cell plated. To evaluate the effect of GLCP at concentrations 0.5, 1, 1.5, and 2 mg/ml, cells were maintained in media with various concentrations of GLCP for 48 h and cell numbers were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra- zolium bromide (MTT) assay, and absorbance was read at 570 nm.

Cells were plated in 6-well plates and were treated with GLCP for 48 h. After treatment, the cells were harvested and resuspended in ice-cold cell lysis buffer, and the homogenate was centrifuged at 10,000 g for 15 min at 4˚C. The total supernatant protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Kangchen Biotech, Shanghai, China). An equal volume of sodium dodecyl sulfate (SDS) gel loading buffer [100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol (DTT), 4% SDS, 0.1% bromophenol blue and 20% glycerol] was added to samples, which were subsequently boiled for 7 min. The total proteins from each sample were separated by 15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% fat-free dry milk in PBS-T for 1 h at room temperature followed by incubation with primary antibodies to caveolin-1 (1:1000), LC3B (1:1000), Bcl-2 (1:1000), P53 (1:1000) and β-actin (1:2000) overnight at 4˚C. The next day, the membranes were washed and subsequently incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Following three washes with PBS-T, the membranes were developed using an enhanced chemiluminescence (ECL) kit and exposed to X-ray films. Gel-Pro analyzer 4 software was used to quantify the protein expression levels.

Cells were plated in 24-well plates and were treated with GLCP for 48 h. After washed twice with phosphate buffered saline (PBS), the cells were fixed with 95% ethanol for 10 min. Acridine orange was added at a final concentration of 0.1 mg/ml for 25 min at 4˚C, after washed twice with PBS, added 0.1 mol CaCl₂ in the cells for colour separation. Finally, cells were washed with phosphate buffered saline. Pictures were obtained with a fluorescence microscope.

Cell apoptosis was quantified by flow cytometry using PI and Annexin V double staining, and it was performed as previously described using FACScalibur (Becton Dickinson, Franklin Lakes, NJ).

All of the experiments were performed at least three times. The values were expressed as the means ± S.E.M. Statistical analysis was performed between groups by ANOVA using SPSS 16.0 software (SPSS Inc, Chicago, IL, USA). A probability value of p < 0.05 was considered significant.

MTT analysis showed that compared with the control group, the relative viability of SMMC-7721 cell decreased significantly after adding different concentrations (0.5, 1, 1.5, and 2 mg/ml) of GLCP, at the same time, GLCP activity of normal liver cells in some role in promoting (Figure 1(a)). Using flow cytometry detection after adding the 1.5 mg/ml polysaccharide on apoptosis in SMMC-7721 cells, the results showed that, adding polysaccharide treatment significantly increased apoptosis rate (Figure 1(b)). Western blot to detect the apoptosis associated protein expression changes after adding the GLCP in human hepatoma cell SMMC-7721. We can see from the quantitative, GLCP inhibits the expression of BCL-2 proteins in human hepatoma cell SMMC-7721 and promotes the expression of NF-κ b (Figure 1(c)).

To determine if GLCP induced autophagy in SMMC-7721 cells, acridine orange staining for autolysosome was performed. Results showed that the GLCP at the concentration of 1.5 mg/ml drastically increased the formation of autolysosome in SMMC-7721 cells (Figure 2(a)).Western blot showed the marker protein levels of autophagy, compared with control group the LC3-II/LC3-Ⅰlevel was increased significantly after adding 1.5 mg/ml GLCP (Figure 2(b)).

MTT method and flow cytometry were used to detect the anticancer effect of GLCP in caveolin-1 gene silencing SMMC-7721 cells. Compared with the SMMC-7721 cells, the relative viability of caveolin-1 gene silencing SMMC-7721 cells basically unchanged after adding different concentrations (0.5, 1, 1.5, and 2 mg/ml) of GLCP (Figure 3(a)). Using flow cytometry detection after adding the 1.5 mg/ml polysaccharide on apoptosis in SMMC-7721 cells and the caveolin-1 gene silencing SMMC-7721 cells, the results showed that, adding polysaccharide treatment the apoptosis rate of the caveolin-1 gene silencing SMMC-7721 cells was unchanged

(a) (b)

(c)

Figure 1. Compared with the normal human liver cell (Chang liver), GLCP promote cell apoptosis in human hepatoma cell SMMC-7721. (a) The cells were treated with 0.5 - 2 mg/ml GLCP for 48 h. Cell viability was measured by the MTT assay as described in methods. (b) The SMMC-7721 cell was treated with 1.5 mg/ml GLCP for 48 h. The cell apoptosis level was examined by flow cytometry. (c) The SMMC-7721 cell was treated with 1.5 mg/ml GLCP for 48 h. Western blot was used to examine the relative protein expression of Bcl-2 and NF-κB, the protein level was compaerd with the β-actin. The data represent the mean of three independent experiments ± SD. ^*p < 0.05; ^**p < 0.01 versus control.

(Figure 3(b)). Western blot to detect the apoptosis associated protein expression changes after adding the GLCP in human hepatoma cell SMMC-7721 and the caveolin-1 gene silencing SMMC-7721 cells. The results showed that, caveolin-1 gene silencing inhibits the anticancer effect of GLCP.