Dulbecco's modified Eagle's medium (DMEM) high glucose and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Carlsbad, CA, USA). 5-FU (>99% purity) was purchased from Shanghai Bangcheng Chemical Co., Ltd. (Shanghai, China). MTT, the Lactate Dehydrogenase (LDH) Cytotoxicity Assay kit and the Caspase-3, -8 and -9 Activity Assay kits were purchased from Beyotime Institute of Biotechnology (Nantong, China). The rabbit polyclonal primary antibodies against β-actin (1:4,000; 20536-1-AP), Fas (1:1,000; 13098-1-AP) and caspase-3 (1:500; 19677-1-AP) were purchased from Proteintech Group, Inc. (Chicago, IL, USA) and the goat anti-rabbit horseradish peroxidase (HRP)-labeled secondary antibody (1:3,000; sc-2004) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All chemicals were of analytical grade unless otherwise specified.

Slices of G. lucidum (Leyss. ex. Fr.) Karst. were provided by the State Key Laboratory of Sub-Health Intervention Technology, State Administration of Traditional Chinese Medicine (Changsha, China). Samples were ground to a fine powder and refluxed in three volumes of methanol/chloroform solvent (1:2, v/v; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and 80% ethanol three times (6 h/each time). The residues were dried, suspended in ~15 vol distilled water and were concentrated with vacuum rotary evaporator (EYELA Auto Jack NAJ-100; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) vacuum filtration three times for 6 h each time at 95°C. The combined concentrated solution was ethanol precipitated and then deproteinized using a Sevage assay (21). The extracts were purified using DEAE-cellulose (DE 52; Sigma-Aldrich, St. Louis, MO, USA), dialyzed with 3000 MWCO regenerated cellulose dialysis tubing (Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA), concentrated and freeze-dried. The crude polysaccharides extraction yield was calculated by dividing the final weight of crude extract by the initial weight of the samples. Purified GLPs (10 mg/ml) were ultrafiltered using Biomax-500, Biomax-300, Ultracel PL-100 and Ultracel PL-30 membranes (EMD Millipore, Billerica, MA, USA) with a molecular weight cut-off of 50, 30, 10 and 3 kDa, and the five freeze-dried fractions were weighed for analysis of molecular weight composition. Three fractions of polysaccharides with high molecular weight (>10 kDa) were pooled and further analyzed. Polysaccharide content was calculated by the phenol-sulfuric acid method (22). Protein and nucleic acid were determined by UV spectrum analysis (UV-1800; Shimadzu Corporation, Kyoto, Japan), and uronic acid was determined by the 3,5-dimethylphenol method (23). The infrared spectra were recorded with a Fourier Transform-Infrared Spectrometer (Thermo Fisher Scientific, Madison, WI, USA) in the range of 400–4,000 cm⁻¹. GLPs were hydrolyzed with 1 mol/l H₂SO₄ (Sinopharm Chemical Reagent Co., Ltd.) for 8 h at 100°C, and cooled prior to centrifugation at 2,400 x g for 30 min at room temperature using centrifugal filters (Amicon Ultra-4; EMD Millipore). The GLPs were then dried in the rotary evaporator, then monosaccharide components were analyzed by high-performance anion exchange chromatography on a Dionex ICS-3000 Ion Chromatograph System (Thermo Fisher Scientific).

The human LoVo colon carcinoma cell line was obtained from the Institute of Basic Medical Sciences (Beijing, China). Cells were maintained in DMEM high glucose supplemented with 10% FBS in an incubator at 37°C and 5% CO₂.

LoVo cells at a density of 3×10⁴ cells/well were cultured in 96-well plates. Following culture for 24 h, cells were incubated with fresh medium containing various concentrations of GLPs (0.313, 0.625, 1.25, 2.5, 5 and 10 mg/ml) at the predetermined time intervals (24, 48 and 72 h). A total of 50 µg/ml 5-FU was used as a positive control, while untreated cells served as a negative control. Subsequently, 20 µl MTT solution (5 mg/ml in phosphate-buffered saline, PBS; pH 7.4; Sinopharm Chemical Reagent Co., Ltd.) was added, followed by incubation for 4 h in the dark. Subsequent to the removal of the media, purple formazan crystals were dissolved in 150 µl dimethyl sulfoxide (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The absorbance at 492 nm was measured using a microplate reader (MK3; Thermo Fisher Scientific, Waltham, MA, USA).

Following the formation of a cell mono-layer in the 24-well plate, lines were scratched in the layer using 10 µl pipette tips. Following the removal of non-adherent cells by PBS, adherent cells were incubated with various concentrations of GLPs (0, 0.625, 1.25, 2.5, 5 and 10 mg/ml) for 48 h at 37°C (50 µg/ml 5-FU as a positive control). Three different views at each concentration were imaged using an inverted microscope (IMT-2; Olympus, Tokyo, Japan) and analyzed using ImageJ software, version 1.42 (National Institutes of Health, Bethesda, MD, USA).

The GLP-treated cell suspension was fixed with 2.5% glutaraldehyde (Sigma-Aldrich) for 2 h, followed by fixation with 1% osmium tetroxide (OsO₄; Sigma-Aldrich) for a further 2 h. Cells were rinsed in PBS three times prior to dehydration in an ascending ethanol series (50, 70, 80, 90, 95 and 100%). Following infiltration in 50% Quetol 651 (Nisshin EM, Tokyo, Japan) for 1 h and 100% Quetol 651 for 6 h, cells were polymerized at 60°C for 39 h. Samples were subsequently sectioned into 100 nm-thick sections with a Leica UC6 micro-tome (Leica Microsystems, Inc., Buffalo Grove, IL, USA) and dyed with 4% uranyl acetate (Sigma-Aldrich) for 10 min. Transmission electron microscopy (TEM) was performed using a JEOL JEM-1230 (JEOL, Ltd., Tokyo, Japan) microscope. For scanning electron microscopy (SEM; JSM-6380LV; JEOL, Ltd.), LoVo cells were exposed to different concentrations of GLPs in six-well plates for 48 h. Subsequently, cells were fixed in 1% glutaraldehyde and 1% OsO₄, dehydrated in an ethanol series (50–100%, v/v) and dried by critical-point drying with the JSM-6380LV microscope. Following gold sputtering also with the JSM-6380LV microscope, digital images were obtained using the scanning electron microscope.

Cells were incubated with GLPs (2.5, 5 and 10 mg/ml) or 5-FU (50 µg/ml) for 24 h, followed by resuspension in 70% ethanol for 4 h at −20°C. Following centrifugation at 1,000 x g for 5 min at 4°C, the precipitate was dissolved in 40 µl phosphate-citric acid buffer (pH 7.8; 91.5 parts 0.5 M Na₂HPO₄ and 8.5 parts of 0.23 M NaH₂PO₄) and continuously incubated at room temperature for 45 min with intermittent agitation. The supernatant was mixed with 3 µl 1 mg/ml RNaseA and 3 µl 0.25% NP40 (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 30 min. DNA fragmentation was analyzed by gel electrophoresis on a 1.2% (w/v) agarose gel (Sigma-Aldrich). The gel was visualized by staining with ethidium bromide (Amresco LLC, Solon, OH, USA) and the images were captured using the UVP GDS-8000 Bioimaging System (UVP, Inc. Upland, CA, USA).

Following culturing for 48 h with or without different concentrations of GLPs (0, 1.25, 2.5 and 5 mg/ml), the supernatant was collected and added to a new 96-well plate (200 µl/well). Subsequently, LDH release was calculated using a LDH Cytotoxicity Assay kit according to the manufacturer's instructions. The plates were analyzed using the MK3 micro-plate reader at a wavelength of 450 nm.

The activity of caspases-3, -8 and -9 was quantified using the Caspase-3, -8 and -9 Activity kits according to the manufacturer's instructions. Briefly, cells were treated for 24 h with GLPs (0, 2.5 and 5 mg/ml). Following production of the pellet by centrifugation at 600 x g for 5 min at 4°C, cells were washed with PBS two times and subsequently lysed using lysis reagent (Beyotime Institute of Biotechnology) in an ice bath for 15 min. The supernatants were incubated with 80 µl reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl and 10% glycerol; Beyotime Institute of Biotechnology) and 10 µl substrate at 37°C for 2 h. At the end of the incubation, the absorbance of cleavage (pNA) was measured with the MK3 microplate reader at 405 nm.

GLP-treated cells were collected, washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer (Applygen Technologies, Inc., Beijing, China) for 30 min at 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total protein was subjected to electrophoresis on 12% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred onto polyvinylidene fluoride membranes (EMD Millipore). Subsequently, the membranes were incubated with rabbit anti-Fas (1:1,000), rabbit anti-caspase-3 (1:500) and mouse anti-β-actin (1:4,000) at 4°C overnight. Membranes were then incubated with HRP-labeled secondary antibodies (1:3,000) at room temperature for 1.5 h. The signal was detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific) for western blot analysis. The relative abundance of bands was quantified using ImageJ software.

All data are presented as the mean ± standard deviation. Statistical analyses were performed with Student's t-test using SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). P<0.01 was considered to indicate a statistically significant difference.

The yield of crude polysaccharides was ~3.08%. The molecular weight composition of polysaccharides >10 kDa was: 10–30 kDa, 32.1%; 30–50 kDa, 21.8%; and >50 kDa, 46.1%. Using UV scanning, the absence of nucleic acids and proteins in the GLPs was indicated by minimal absorption at 260 and 280 nm. GLPs were comprised of 89% carbohydrate and 11% uronic acid. The peaks of infrared spectroscopy at 847 and 922 cm⁻¹ indicated that the purified GLPs predominantly consisted of α-polysaccharides (24). The results of the monosaccharide composition analysis were in agreement and indicated that GLPs were composed of arabinose, galactose, glucose and cellose at the molar ratios of 11:3:3:1. GLPs were prepared as a stock of 10 mg/ml in DMEM high glucose containing 10% FBS and stored at −20°C.

As presented in Fig. 1, the inhibitory rates of GLPs (0.313, 0.625, 1.25, 2.5, 5 and 10 mg/ml) at 72 h were 43.6, 53.0, 62.5, 74.9, 78.6 and 99.3%, respectively, compared with the control. The half maximal inhibitory concentration (IC₅₀) values at 24, 48 and 72 h were 6.94, 1.67 and 0.63 mg/ml, respectively. The results demonstrate that GLPs significantly reduced cell viability in a dose- and time-dependent manner (P<0.01), suggesting that GLPs possess a potent cytotoxicity against human colon cancer cells.

At 48 h, the scratch wounds were completely repaired by the migration of untreated cells, whilst the wounds were maintained in GLP-incubated cells as indicated by examination using light microscopy (Fig. 2A). In particular, LoVo cells treated with GLP concentrations of 5 and 10 mg/ml did not migrate towards the wound area compared with the untreated cells. As presented in Fig. 2B, exposure to GLPs (0.625–10 mg/ml) resulted in a reduction in the number of migratory cells from 95.9–56.9% (P<0.01), indicating that GLPs are able to dose-dependently suppress cell migration in LoVo cells.

To investigate the cell death induced by GLPs, morphological characteristics of cells were observed by SEM and TEM. SEM observations demonstrated that untreated cells exhibited a uniform distribution and numerous microvilli on the cell surface (Fig. 3A–a and A–d). In GLP-treated cells, morphological alterations were observed including low cellular density (Fig. 3A–b and A–c), a reduction in the number or disappearance of microvilli and the formation of apoptotic bodies (Fig. 3A–e and A–f). In the TEM study, untreated cells displayed microvilli on the cell surface and a dispersed chromatin pattern (Fig. 3B–a). Following incubation with 5 mg/ml GLPs, the induction of cell death was indicated by cell shrinkage, microvilli loss, cell membrane blebbing (arrow in Fig. 3B–b) and cytoplasm marginalization (Fig. 3B–b). Subsequently, the cells broke up into membrane-bound apoptotic bodies (arrow in Fig. 3B–c). Following exposure to 10 mg/ml GLPs, apoptotic bodies were degraded by lysosomal enzymes (Fig. 3B–d).

DNA fragmentation and the level of LDH released into the culture medium were used as indexes for evaluating apoptosis. The results demonstrated that GLPs induced DNA fragmentation following treatment with various doses of GLPs (1.25–10 mg/ml), presented in Fig. 4A. The LDH release assay demonstrated that GLP-induced LDH release was significantly increased (P<0.01), and this effect was dose-dependent (Fig. 4B), further supporting the role of apoptosis in GLP-mediated cytotoxicity.

The addition of GLPs to cultured cells increased the activity of caspases-3, -8 and -9 in a dose-dependent manner (P<0.01), indicating GLP-mediated apoptosis occurred via the caspase-dependent pathway in LoVo cells (Fig. 5). Using western blotting, the effect of GLPs on the expression of proteins associated with apoptosis, including Fas, caspase-3 and poly(ADP-ribose) polymerase (PARP) was further investigated. As presented in Fig. 6, the level of caspase-3 protein was significantly increased compared with the control group at 12 h and reached its maximum level at 24 h, which is in agreement with the results of caspase activity assay. In addition, the levels of Fas and PARP were increased and reduced, respectively, in a dose-dependent manner (P<0.01). These results suggest a possible association between GLP-induced apoptosis and the activation of the Fas/caspase-dependent pathway.