Inbred male 6–8 weeks old (body weight 18–22 g) BALB/c (H-2 d) mice (Grade II, Certificate No scxk11-00-0004) were purchased from the Department of Experimental Animals, Health Science Center, Peking University, Beijing, China. All procedures were in accordance to the Institute Ethical Committee for Experimental Use of Animals.

Murine Sarcoma 180 cell lines (S180) were obtained from Beijing Tumor Institute. Human lung carcinoma cell lines (PG) and YAC-1 cell lines were provided by Department of Pathology and Immunology, Peking University Health Center. The cells were maintained in RMPI-1640 supplemented with 10% fetal calf serum (FCS) in a humidified environment at 37°C and 5% CO₂, and cultures were passaged every 2 or 3 days.

Gl-BSP was kindly provided by Fuzhou Institute of Green Valley Bio-Pharm Technology. It was a sandy beige water soluble powder and was isolated from boiling water extract of the broken-spore of G. lucidum (Leyss. et Fr.) Karst. (Gl), followed by ethanol precipitation, dialysis, and protein depletion using Sevag method. The purity of the Gl-BSP was 97%, which was analyzed by high-performance liquid chromatography (HPLC). The component sugars and molecular-weight distributions of the glycopeptides were determined by gel permeation chromatography (GPC) and HPLC. The structures of the glycopeptides were detected by IR, ¹H NMR, and ¹³C NMR. Gl-BSP had a molecular weight of 512,500 with a ratio of polysaccharides to peptides of 94.8:5.2%. The polysaccharides consisted of d-rhamnose, d-xylose, d-fructose, d-galactose, and d-glucose with a molar ratio of 0.549:3.614:3.167:0.556:6.89 linked together by β-glycosidic linkages. The peptides contained the following 16 amino acids: Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Phe, Lys, His, Arg, and Pro. Gl-BSP was dissolved in physiological saline for in vivo experiments or in serum-free RPMI-1640 (Gibco Laboratories, Grand Island, NY, USA) for in vitro experiments, filtered through a 0.22 μm filter and stored at 4°C for future use. Endotoxins concentration in Gl-BSP samples were assayed under endotoxin-free experimental conditions by using a limulus amebocytes lysate chromogenic assay kit (Beijing BXGK Technology Development Co., Ltd., Beijing, China) according to the manufacture’s instruction. The quantity of endotoxin in Gl-BSP was less than 0.01 EU mg⁻¹, indicating that endotoxin contamination in Gl-BSP was negligible.

Murine Sarcoma 180 cell lines were injected into peritoneal cavity of mouse, proliferated there to produce ascites, and were maintained by weekly transplantation of the tumor cells from ascites into another peritoneal cavity of mouse. S180 were taken out from the ascites, adjusted concentration as 1 × 10⁷ cells ml⁻¹ with physiological saline, and then injected in 0.2 ml (2 × 10⁶ cells) into the axillary fossa of a mice right foreleg to prepare tumor-bearing mice. The 50 mice were divided into five groups randomly (10 mice in each group) 24 h after the tumor inoculation and treated for 14 consecutive days with (1) low-dose Gl-BSP (50 mg kg⁻¹) once daily by gavage, (2) intermediate-dose Gl-BSP (100 mg kg⁻¹) once daily by gavage, (3) high-dose Gl-BSP (200 mg kg⁻¹) once daily by gavage, (4) cyclophosphamide (CY, an antitumor agent, 30 mg kg⁻¹) once every other day by intraperitoneal injection as positive controls, and (5) sterile physiological saline (10 ml kg⁻¹) once daily by gavage as negative controls (model group), and the sixth group of normal mice administrated sterile physiological saline intragastrically once daily (10 ml kg⁻¹) served as normal control.

After completion of treatment on the 14th day, the mice were killed and the tumors were removed and weighted. At the same time, blood samples were collected from the orbital vein and the serum was sterilized by filtration and preserved at −70°C.

PG or S180 cells were maintained in RMPI-1640 supplemented with 10% FCS. A cell suspension (2 × 10⁷ cell per liter) was planted onto 96-well plates (0.1 ml per well), and incubated at 37°C, 5% CO₂ for 24 h. The media was replaced with 0.1 ml of RPMI-1640 supplemented with 5% FCS and Gl-BSP 0.1, 1, 10, 100, or 400 mg l⁻¹. In another series, RPMI-1640 was supplemented with 5% Gl-BSP treated serum 50, 100, or 200 mg kg⁻¹. After 48 h, cells proliferation was estimated based on the cellular reduction of tetrazolium salt MTT by a microplate reader (BIO-RAD, Model 550), using a test wavelength of 540 nm.

Mice in all treated groups were killed and the spleens were aseptically removed, chopped with two slides and filtered over a fine nylon mesh to obtain single-cell suspensions. The cells were washed and lymphocytes were separated from red blood cells by Tris-HCl-buffered NH₄Cl solution [mix 9 volumes of 0.83% NH₄Cl with 1 volume of Tris-HCl (2.06%, pH 7.65), adjust pH 7.2]. Cells were finally suspended in 10% FCS RPMI-1640 supplemented with benzylpenicillin 100 kU l⁻¹, streptomycin 100 mg l⁻¹ for further experiments.

Lymphocytes were planted into 96-well plates (2 × 10⁶ cells per well) with or without ConA 1 mg l⁻¹ or LPS 5 mg l⁻¹, and incubated at 37°C, 5% CO₂ for 72 h. Cell proliferation was estimated based on the method of MTT.

The NK cell activity of spleen cells was determined by a 4 h ⁵¹Cr-release assay. Viable trypan blue-excluded lymphocytes were counted. Cell survival rate was greater than 95%. Target cells of YAC-1 were incubated for 1 h at 37°C with Na⁵¹CrO₄ and were coated at a concentration of 1 × 10⁴ cells per well in 96-well U-bottomed culture plates after washing. Effectors and targets were mixed at a ratio of 40:1 and incubated for 4 h at 37°C. Spontaneous release was determined from wells that contained labeled target cells alone, and maximum ⁵¹Cr-release was determined by addition of 1% Triton X-100 (Sigma). The radioactivity of the supernatants was counted in γ-counter (Beckman LS 6500). Specific cytotoxicity was calculated as: percentage specific release = 100 × (cpm experimental release − cpm spontaneous release)/(cpm maximum release − cpm spontaneous release).

Macrophages were obtained from mice peritoneal exudates cells (PECs). PECs were washed twice and resuspended in a RPMI-1640 medium containing 10% FCS. Peritoneal macrophages were further isolated from the PECs by incubating the PECs (2 × 10⁵ cells per well) in a 96-well plate at 37°C, 5% CO₂ for 4 h in a humidified atmosphere to allow peritoneal macrophages to adhere. The supernatants were discarded, and 0.075% Neutral red (NR) were added and incubated for another 1 h. Cells were then washed with PBS for three times to remove excess dye and incubated with cell lysis buffer (1 M acetic acid: ethanol = 1:1) overnight. The absorbance (OD) was measured at 540 nm in a microplate reader (BIO-RAD, Model 550) and translated into phagocytosis ratio for comparison: phagocytosis ratio = test_OD/normal control_OD × 100%.

The cell concentration of lymphocytes was adjusted to 1 × 0⁷ ml⁻¹. A volume of 20 μl of FITC-labeled anti-mouse CD4 and 20 μl of PE-labeled anti-mouse CD8 was added to 100 μl of the cell suspension. After incubation for 30 min at 4°C, the lymphocytes were rinsed three times with 1 ml of PBS, pH 7.4, containing 0.5% BSA and 0.1% NaN₃. CD4 and CD8 T-cell subpopulations were analyzed by flow cytometry using a FACS Calibur (Becton Dickinson).

Serum collected from tested mice were assayed for the level of IL-2, TNF-α, and IFN-γ using commercially available kits from R&D systems, with a solid-phase enzyme-linked immunoabsorbent assay (ELISA) as described by the manufacturers.

The amount of NO2− present in fresh serum collected from tested mice were assayed by Griess reagent, using NaNO₂ as a standard. Briefly, 50 μl of serum was reacted with 50 μl of Griess reagent (1% sulfanilamide in 5% H₃PO₄ −0.1% naphthalene ethylendiamine dihydrochloride) for 10 min. The absorbance was then read at 540 nm using a microplate reader.

Statistical significance was determined by one-way ANOVA employing the computer SPSS statistic package. P < 0.05 was considered significant.

In in vivo experiment, mice were implanted with Sarcoma 180 and different dosages of Gl-BPS or physiological saline (model group) were administrated intragastrically for 14 days. Compared with the model group, Gl-BSP 100 and 200 mg kg⁻¹ significantly inhibited growth of Sarcoma 180 by 49.1 and 59.9%, respectively, with no effect on body weight (Table 1).

Gl-BSP did not inhibit Sarcoma 180 and PG cell proliferation in vitro when added directly to the cultured medium (Table 2); but Gl-BSP (50, 100, and 200 mg kg⁻¹)-treated serum of S180-bearing mice markedly inhibited S180 or PG cell proliferation (Figure 1).

Compared with normal mice, proliferation of spleen lymphocyte induced by Con A and LPS declined significantly in S180-bearing mice administrated with physiological saline (model group), but Gl-BSP administrated intragastrically promoted spleen lymphocytes proliferation induced by Con A and LPS in S180-bearing mice (Table 3).

Compared with normal mice, splenic NK cytotoxic activity in model group was significantly lower, reduced from 35.6% down to 8.3%, while Gl-BSP (50, 100, and 200 mg kg⁻¹) administrated intragastrically increased the splenic NK cytotoxic activity of S180-bearing mice (Table 4).

Gl-BSP (50, 100, or 200 mg kg⁻¹) could augment the percentage of phagocytosis of NR by peritoneal macrophages in S180-bearing mice (Table 5).

Compared with normal mice, the percentage of the CD4⁺ or CD8⁺ subset decreased markedly, while the CD4⁺/CD8⁺ ratio increased significantly in the model group. The results indicated that the number of CD8⁺ subset decreased more than that of CD4⁺ subset. Compared with the model group, the percentage of the CD4⁺ and CD8⁺ subset in S180-bearing mice treated with Gl-BSP (50, 100, or 200 mg kg⁻¹) was greatly augmented, and the ratio of CD4⁺/CD8⁺ reduced nearly to that in normal mice (Table 6).

On the basis of the effect of Gl-BSP on splenic lymphocytes, NK cells, and peritoneal macrophages, the production of serum cytokines was detected. We found that IL-2, IFN-γ, and TNF-α in serum were undetectable in normal mice. Only IFN-γ in serum was detectable in S180-bearing mice. The serum levels of IL-2, IFN-γ, and TNF-α were markedly increased in S180-bearing mice administrated with Gl-BSP (200 mg kg⁻¹), compared with that in S180-bearing control mice administrated with physiological saline (Table 7).

To determine whether the growth inhibition in Sarcoma 180 cells or PG cells induced by Gl-BSP 200 mg kg⁻¹-treated serum was related to the cytokines released from immune system activated by Gl-BSP in S180-bearing tumor mice. Serums collected from mice in all groups were preincubated with one or two cytokine-neutralizing antibodies before addition to cell cultures. Neutralization with anti-TNF-α apparently diminished S180 or PG cell lines growth inhibition induced by Gl-BSP 200 mg kg⁻¹-treated serum, and a similar result was obtained by use of anti-IFN-γ. Blocking effect was noted in the combination of anti-TNF-α and anti-IFN-γ, which reduced the inhibition rate from 55.6 or 45.1% (before neutralization in S180 or PG cell lines) down to 11 or 15.8%, respectively. However, the cytokine antibodies used above did not completely block growth inhibition induced by the Gl-BSP 200 mg kg⁻¹-treated serum of S180-bearing mice (Table 8).

We found that the Gl-BSP-treated serum of S180-bearing mice had the different effects on Sarcoma 180 cell proliferation in vitro. After Gl-BSP 200 mg kg⁻¹-treated serum underwent heat-inactive at 56°C for 30 min, the inhibition rate reduced from 57.1% down to 10.9%; while Gl-BSP 50 mg kg⁻¹ and 100 mg kg⁻¹-treated serum with pretreatment at 56°C for 30 min did not show significantly effect on the proliferation of Sarcoma 180 cells in vitro (Figure 2).