GL-PP was isolated from the boiled extract of G. lucidum cultivated with JUNCAO grasses (National Engineering Research Center of Juncao Technology, Fujian, China). An ethanol precipitation was then performed, followed by dialysis and de-proteination according to the Sevag method, as previously described (35).

The homogeneity and molecular weight of GL-PP was identified using high-performance gel permeation chromatography (HPGPC) with a Waters 2695 HPLC apparatus (Waters, Milford, MA, USA), equipped with a Waters 2515 HPLC pump (Waters), a gel permeation column TSK4000PW (21.5×300 mm, 10 µm; Tosoh, Tokyo, Japan) and a Waters 2414 refractive index detector (Waters). Water (HPLC grade) was used as the mobile phase with a gradient elution (flow rate of 0.8 ml/min) at 35°C. Dextran standards were obtained from the National Institutes for Food and Drug Control and molecular weights ranging from 2,500 to 84,400 Da were used to generate a calibration curve (36). The molecular weight of GL-PP was estimated using Waters Empower software (version 5.0; Waters).

Monosaccharides were determined using hydrophilic interaction liquid interface chromatography and an evaporative light scattering detector (HILIC-ELSD) via a Waters Alliance 2695 HPLC system and 2424 ELSD (both from Waters). Chromatographic separation was performed on a Sugar-D column (4.6×250 mm; 5 µm; Nacalai Tesque, Inc., Kyoto, Japan). The column was run at 35°C and the mobile phase consisted of 75% acetonitrile and 25% water, at a flow rate of 0.8 ml/min. The drift tube temperature and air carrier gas pressure of the ELSD was set to 55°C and 45 psi, respectively. The injection volume was 10 µl. The identity of sample monosaccharides were determined by comparing the retention time of peaks with those of known standards including, rhamnose, fructose, xylose, arabinose, glucose, galactose and mannose.

The amino acid composition of GL-PP was determined using the Hitachi-L8800 amino acid analyzer (Hitachi High-Technologies Co., Tokyo, Japan) according to the Chinese National Standard (37).

GL-PP was dissolved in serum-free Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Chicago, IL, USA), then filtered through a 0.22-µm filter and stored at 4°C. This medium was further diluted to the indicated concentration prior to each assay.

The presence of an endotoxin in GL-PP was detected using Tachypleus Amebocyte Lysate (TAL; Fuzhou Xin Bei Biochemical Industry Co., Ltd., Fuzhou, China) according to the manufacturer's instructions. A total of 100 µl TAL reagent was added to 100 µl of 800 µg/ml GL-PP, 100 µl endotoxin standard (Fuzhou Xin Bei Biochemical Industry Co., Ltd.) and 100 µl endotoxin-free water. The mixture was gently agitated, covered with foil and incubated at 37°C for 1 h. Since endotoxin contamination results in the formation of a hard gel substance, samples were observed for the formation of this gel by performing a gentle 180° tube inversion following the incubation period.

The human glioma U251 cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) where it was routinely maintained in DMEM containing 10% inactivated fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences) and 100 U/ml penicillin/streptomycin. Cells were grown at 37°C in an atmosphere of 5% CO₂.

Cell proliferation and viability were analyzed using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). U251 cells were seeded onto 96-well culture plates at a density of 2,000 cells/well and cultured for 24 h prior to treatment. GL-PP was added to the culture at a final concentration of 50, 100, 200, 400 or 800 µg/ml and cells were incubated for a further 24, 48 or 72 h. Wells that were not treated with GL-PP were used as negative controls. The CCK-8 reagent was then added to the cultures according to manufacturer's instructions and absorbance was measured at 450 nm using an ELISA MK3 microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). All measurements were performed in triplicate.

U251 cells (2.5×10⁴ cells/ml) were plated onto a 6-well plate and harvested at 48 h following treatment. Cells were fixed in 70% ice-cold ethanol, washed with ice cold PBS and stained with 50 mg/ml propidium iodide in the presence of 50 mg/ml RNase A for 30 min. Staining procedures were performed under low light at room temperature. Analyses were performed using a flow cytometer (Cytomics FC 500; Beckman Coulter Inc., Brea, CA, USA). Cell proportions in sub-G₁, G₀/G₁, S and G₂/M phases were analyzed using ModFit LT software (version 2.0; Verity Software House, Topsham, ME, USA). Tests were performed in triplicate for each sample.

A 6-well cell culture plate was prepared as aforementioned. Total protein was purified from cells using a cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM phenylmethylsulfonyl fluoride. Protein concentrations were subsequently measured using a BCA kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Samples (150 µg protein/lane) were then separated on 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. β-actin (Cell Signaling Technology Inc., Danvers, MA, USA) was used as a loading control. The membrane was blocked with 5% bovine serum albumin (Sangon Biotech, Shanghai, China) at room temperature for 2 h and incubated overnight at 4°C with a rabbit polyclonal anti-active caspase-3 antibody (1:1,000, cat. no. ab2302; Abcam, Cambridge, UK). The membrane was then incubated with a horseradish peroxidase conjugated goat anti-rabbit immunoglobulin G (1:2,000, cat. no. sc-2004; Santa Cruz Biotechnology Inc., Dallas, TX, USA) at room temperature for 2 h. Protein expression was detected using a Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA) and exposed on X-ray film. The optical density of each band was determined using Quantity One software (version 4.6.1; Bio-Rad Laboratories Inc., Hercules, CA, USA). The expression of cleaved caspase-3 was expressed as a ratio to β-actin used as an internal control. Tests were performed in triplicate for each sample.

The activity of caspase-3 was determined using a Caspase 3 Activity Assay kit (Beyotime Institute of Biotechnology). To evaluate the activity of caspase-3, cell lysates were prepared following cell treatment with GL-PP for 48 h. Each 10 µl cell lysate was incubated with 80 µl reaction buffer [1% NP-40, 20 mmol/l Tris-HCl (pH 7.5), 137 mmol/l NaCl, 10% glycerol] containing 10 µl caspase-3 substrate [2 mmol/l acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA)] at 37°C for 2 h. Caspase-3 can catalyze the substrate Ac-DEVD-pNA to produce pNA. Thereafter, absorbance of pNA at 405 nm was measured using an ELISA plate reader (MK3; Thermo Fisher Scientific Inc.). The analysis procedure followed the manufacturer's instructions. All experiments were performed in triplicate.

The data were expressed as mean ± standard deviation. Multigroup comparisons of the means were carried out by one-way analysis of variance test with post hoc Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

GL-PP was determined to be a polysaccharide peptide with a mean molecular weight (Mw) of 42,635 Da (Fig. 1), a purity of 96.89% and a polysaccharide to peptide ratio of 88.70:11.30%. HILIC-ELSD data indicated that polysaccharides were primarily composed of l-arabinose, d-mannose and d-glucose at a molar ratio of 1.329:0.372:2.953 (Fig. 2). The amino acid automatic analyzer indicated that the peptide contained 17 amino acids, including Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Pro, with a respective mass ratio of: 1.45:1.08:1.12:1.45:0.84:0.87:0.19:0.64:0.01:0.38:0.56:0.23:0.47:0.58:0.30:0.53:0.60 (Table I and Fig. 3). No detectable level of endotoxin (≤0.10 EU/ml) was identified in the GL-PP samples.

Cells were incubated with varying concentrations of GL-PP (50, 100, 200, 400 and 800 µg/ml) for 24, 48 or 72 h. GL-PP markedly inhibited the proliferation of U251 cells in a dose-dependent manner compared with untreated controls. The maximum inhibition observed at 48 h among all treatment groups except 400 µg/ml group (Fig. 4). The half-maximal inhibitory concentration of GL-PP at 48 h was 274.1 µg/ml.

To examine how GL-PP inhibited cell proliferation and viability, flow cytometry was utilized to determine the effect of GL-PP on cell cycle progression. Results revealed that the presence of G0/G1 phase cells was increased from 62.9 to 74.94% at 48 h with increasing concentrations of GL-PP among all groups. This increase was accompanied by a significant decrease in the percentage of S phase cells, whereas the fraction of G₂/M phase cells was primarily unchanged. The sub-G₁ phase cell population, which indicate the number of late-stage apoptotic cells, also increased significantly in GL-PP treated cells in a dose-dependent manner (Fig. 5). This result demonstrated that GL-PP induced G₀/G₁ phase arrest and apoptosis induction.

To examine the induction of apoptosis by GL-PP, the activation of caspase-3, a primary enzyme in cell apoptosis, was assessed. The expression of active caspase-3 in GL-PP-treated U251 cells was determined using western blotting. The active caspase-3 level was increased in a dose-dependent manner following treatment with 50, 100, 200, 400 or 800 µg/ml GL-PP for 48 h (Fig. 6). In addition, the activation of caspase-3 was analyzed by measuring the catalytic capability to the substrate Ac-DEVD-pNA. The result showed the activation of caspase-3 significantly increased in GL-PP treated groups in a dose-dependent manner (Fig. 7). This agrees with the result obtained by western blot analysis (Fig. 6).