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GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum, displays anti-tumour activity by increasing both humoral and cellular immune response
Life Sciences 87 (2010) 628–637
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum, displays anti-tumour activity by increasing both humoral and cellular immune response Jingsong Zhang a,b,1, Qingjiu Tang a,b,1, Changyan Zhou a,b, Wei Jia a,b, Luis Da Silva c, Long Duc Nguyen a, Werner Reutter a, Hua Fan a,⁎ a b c
Institut für Biochemie und Molekularbiologie, CBF, Charité-Universitätsmedizin Berlin, Germany The Edible Fungi Institute, Shanghai Academy of Agricultural Sciences, PR China Institut für Physiologie, CBF, Charité-Universitätsmedizin Berlin, Germany
a r t i c l e
i n f o
Article history: Received 9 March 2010 Accepted 23 September 2010 Keywords: Ganoderma lucidum (G. lucidum) Traditional Chinese medicine (TCM) B lymphocytes Macrophages Anti-tumour Proteoglycan
a b s t r a c t Aims: Ganoderma lucidum, a traditional Chinese medicine, is well known as a modulator of functions of the immune system as well as an anti-tumour agent. However, its active compounds and their molecular mechanisms of action are not well established. GLIS, a proteoglycan isolated from the fruiting body of G. lucidum, stimulates directly the activation of B lymphocytes. In this work, the immunoactivation capacities of GLIS as well as its anti-tumour effect were investigated in vitro and in vivo. Main methods: Tumour-bearing mice were prepared by inoculation of mouse sarcoma S180 cells into BALB/c mice. Lymphocytes and bone marrow-derived macrophages were isolated from spleen and tibia/femurs, respectively. After stimulation with GLIS different immune responses of these cells were analysed. Antitumour effect of GLIS was determined. Key findings: After treatment with GLIS, spleen-derived B lymphocytes from tumour-bearing mice became activated, proliferated and produced large amounts of immunoglobulins. Bone marrow-derived macrophages from tumour-bearing mice also became activated after exposure to GLIS, and they produced important immunomodulatory substances, such as IL-1β, TNF-α and reactive nitrogen intermediates, like NO. GLIS markedly increased phagocytosis of macrophages, and very importantly, it markedly raised the macrophagemediated tumour cytotoxicity. Treatment of mice with GLIS caused an inhibition of mouse sarcoma S180 tumour growth by 60% in vivo. Significance: These results indicate that GLIS exhibits a capacity to increase remarkably both humoral and cellular immune activities of tumour-bearing mice and inhibits tumour growth significantly. The anti-tumour effect of GLIS results from its capacity to increase the host's immune activity. © 2010 Elsevier Inc. All rights reserved.
Introduction The mushroom Ganoderma lucidum (G. lucidum), a lamellaless basidio-mycetous fungus belonging to the family Polyporaceae (Leyss. ex Fr. Karst), known as ‘Lingzhi’ in China and ‘Reishi’ in Japan, has played an important role in Chinese traditional medicine for more than 4000 years. Following the views of ancient Chinese medical scholars, people in Asian countries have been widely using G. lucidum for the promotion of general health and longevity (Lin, 2005). G. lucidum has also been used to treat various human diseases such as allergy, arthritis, bronchitis, gastric ulcer, hyperglycemia, hypertension, chronic hepatitis, hepatopathy, insomnia, nephritis, neurasthenia, scleroderma, inflam-
⁎ Corresponding author. Institut für Molekularbiologie und Biochemie, CBF, CharitéUniversitätsmedizin Berlin, Arnimallee 22, D-14195 Berlin, Germany. Tel.: + 49 30 84451552; fax: + 49 30 84451541. E-mail address: [email protected] (H. Fan). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.09.026
mation, and cancer (Sliva, 2003). New reports emphasize its potential in the treatment of viral, especially HIV, infections (Boh et al., 2007). Numerous pharmacological investigations have demonstrated that extracts of G. lucidum inhibit tumour growth in vitro and in several tumour mouse models in vivo, but the mechanism of this effect is not well understood (Boh et al., 2007; Sliva, 2003; Wasser, 2002). It has been determined that the most important pharmacologically active constituents of G. lucidum are triterpenoids and polysaccharides. Triterpenoids have been reported to exert hepatoprotective, antihypertensive, hypocholesterolemic and anti-histaminic properties as well as inhibitory effects on platelet aggregation, complement activation and angiogenesis, while the aqueous extracts and polysaccharide fractions of G. lucidum as well as the triterpenoids exert antitumour activities (Boh et al., 2007). Recently, anti-angiogenic effects in human lung cancer cells have been ascribed to polysaccharide peptides from G. lucidum (Cao and Lin, 2006). Ganoderma polysaccharides represent a structurally diverse class of biological macromolecules with a wide range of physicochemical
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properties (Efferth et al., 2007; Sliva, 2004). A large number of studies have shown that polysaccharides of G. lucidum, especially β-Dglucans, can modulate the functions of many components of the immune system such as the antigen-presenting cells, T and B lymphocytes (Boh et al., 2007; Wasser, 2002), NK cells (Chien et al., 2004), neutrophil granulocytes (Hsu et al., 2003) and dendritic cells (Cao and Lin, 2002; Lin et al., 2005). We have previously reported a bioactive fraction (GLIS) isolated from the aqueous extracts of the fruiting body of G. lucidum using successive chromatographic steps. GLIS is a proteoglycan fraction with a carbohydrate:protein ratio of 11:1 and a molecular weight of about 2000 kDa. The carbohydrate portion consists of heteropolysaccharides composed predominantly of D-glucose, D-galactose and D -mannose. GLIS stimulated the activation, proliferation and differentiation of mouse spleen lymphocytes, especially B lymphocytes, which resulted in an increase in the secretion of large amounts of immunoglobulins (Zhang et al., 2002). It has been postulated that immunosuppression of the tumour-bearing host is one of the reasons for the growth of antigenic tumours despite antitumour immune response (Nicolini and Carpi, 2009). In this work we investigated the capacities of GLIS to activate immune responses and inhibit tumour growth in tumour-bearing mice. We found that GLIS increases remarkably both the humoral and cellular immune activity of tumour-bearing mice and that it inhibits significantly tumour growth. Material and methods Animals BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany). The animals were treated according to the German Law on the Protection of Animals. A permission (VB103-G 140/01) for animal experiments was obtained from the state animal welfare committee.
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(8–10 weeks) were selected for isolating bone marrow macrophages. Tumour-bearing mice were obtained as described earlier. After killing normal and tumour-bearing mice by cervical dislocation, the tibias and the femurs were removed by cutting the proximal end of the femur and the distal end of the tibia, leaving the other ends intact. A 23-gauge needle was inserted into the intact ends and bone marrow was collected in ice-cold DMEM culture medium. The bone marrow cells were dispersed by three passes through a 22gauge needle and then centrifuged (1200 g for 5 min at 4 °C). The cell density was adjusted to 106 cell/ml in complete DMEM medium containing 10% L929 (mouse fibrosarcoma cell line, ATCC, Rockville, MD, CCL1) DMEM conditioned medium. After incubation at 37 °C for 3 days, non-adherent cells were collected, and adherent cells were discarded. Cells were seeded in bacteria-culture Petri dishes containing 10% L929 conditioned medium, and incubated at 37 °C for another three days. The non-adherent cells were washed away with PBS. Adherent macrophages were covered with cold PBS and detached from the dish using a spatula. Isolated macrophages were incubated in complete DMED medium. Determination of proliferation of mouse lymphocytes and activation of macrophages by the Alamar Blue Assay Lymphocyte concentration was adjusted to 2 × 106 cells/ml and macrophage concentration was adjusted to 2 × 105 cells/ml. To each well of a 96-well microplate, 180 μl of the cell suspension and 20 μl of various test substances were added. After incubation at 37 °C in a 5% CO2 atmosphere for the indicated times, 20 μl Alamar Blue reagent (Biosource, Nivelles, Belgium) was added to each well. After incubation for 6 h, optical densities (O.D.) at 570 nm and 600 nm were measured using a micro ELISA autoreader (Bio-Rad). Lymphocyte proliferation and activation rates were calculated according to the Biosource protocol (Nivelles, Belgium). Quantification of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin1ß (IL-1β) and tumour necrosis factor-α (TNF-α)
Preparation and quality control of GLIS GLIS was prepared according to the method of Zhang et al. (2002). The purity and quality of the isolated product were controlled by high-performance liquid chromatography (HPLC), whereby obtaining a single, very sharp and symmetric peak was demonstrated. The carbohydrate content of GLIS was determined by the phenol–sulfuric acid reaction, while the peptide content was measured by UV absorption at 280 nm and the bicinchoninic acid (BCA) assay. The molar ratio of different monosaccharides was determined by high-pH anion-exchange chromatography after hydrolysis with trifluoroacetic acid (TFA). Preparation of lymphocytes from normal (MSLs) and sarcoma-bearing mouse spleens (TMSLs) BALB/c mice, 8–10 weeks old (ca. 28 ± 1 g), were used for lymphocyte preparation. For preparation of lymphocytes from tumour-bearing mice, mouse sarcoma S180 tumour cells (1.0 × 106 cells in 100 μl PBS) were injected intramuscularly in the legs of mice. After 15 days, the mice with growing tumours were killed by cervical dislocation and the spleens were subsequently removed. Spleen lymphocytes were prepared according to the procedure by Zhang et al. (2002). Preparation of bone marrow-derived macrophages from normal mice (BMMs) and tumour-bearing mice (TBMMs) Bone marrow-derived macrophages were prepared according to Stanley (1997). BALB/c mice with similar weights (28 ± 1 g) and ages
Production of the cytokines interleukin-2 (IL-2) and interleukin-4 (IL-4) by lymphocytes and production of the cytokines interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) by macrophages were quantified by ELISA according to the manufacturer's instructions (R & D, Minneapolis, USA). Lymphocytes were adjusted to a concentration of 2 × 106 cells/ml. To each well of a 96-well microplate, 180 μl of the cell suspension and 20 μl of different test substances were added. After incubation with and without substances at 37 °C in a 5% CO2 atmosphere for 24, 48 and 72 h, 50 μl of the suspension of each sample was used for the IL-2 and IL-4 ELISA-assay. Macrophages from normal and tumour-bearing mice were prepared as described earlier. Macrophages were adjusted to a concentration of 1 × 105 cells/ml. To each well of a 96-well microplate, 180 μl of the cell suspension and 20 μl of various test substances were added. After incubation at 37 °C in a 5% CO2 atmosphere for 24 h, 50 μl of cell-free supernatant from each well was used for the IL1β and TNFα ELISA-assay. Analysis of the percentage of subpopulations of lymphocytes using flow cytometry Four milliliters of lymphocyte cell suspension (2 × 106 cells/ml) was added into 6-well plates with or without test substances. After incubation for 72 h, lymphocytes were centrifuged, washed twice with PBS and then labelled with different mAbs in PBS for 30 min at room temperature. After washing three times with PBS, cells were resuspended in 0.3 ml PBS. Fluorescence analysis was performed using a FACScan analyzer (Becton Dickinson, Eremdodegem,
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Belgium). CELLQuest software (Becton Dickinson, Eremdodegem, Belgium) was used to analyse the percentage of different lymphocyte subpopulations. Anti-mouse CD3-fluorescein conjugate (marker for T lymohocytes), anti-mouse CD4-fluorescein conjugate (marker for Th lymphocytes), anti-mouse CD8-R-phycoerythrin conjugate (marker for Tc lymphocytes) and anti-CD19-R-phycoerythrin conjugate (marker for B lymphocytes) were obtained from Dianova (Hamburg, Germany). Determination of immunoglobulin (Ig) production
Anti-tumour test in vivo Sarcoma S180 cells (1 × 106 cells/0.1 ml/mouse) were subcutaneously (s.c.) inoculated into the left leg of BALB/c mice. GLIS (100 μg/ 0.5 ml in PBS/mouse) was injected intraperitoneally (i.p.) into mice 5 times at 5 different time points as follows: 3 days before inoculation, on the day of inoculation, 3 days, 6 days and 9 days after inoculation. Control mice received PBS (0.5 ml/mouse) i.p. Tumour weight were measured 15 days after inoculation. The experiment was repeated 3 times with 5 mice for each test group.
The immunoglobulin (Ig) production of lymphocytes was measured by ELISA. The anti-mouse Ig hybridoma screening reagent (coating antibody) was obtained from Roche (Mannheim, Germany), and goat anti-mouse IgG antibodies were acquired from Amersham Pharmacia Biotech (Uppsala, Sweden). The alkaline phosphatasestreptavidin was purchased from DIANOVA (Hamburg, Germany). Lymphocytes were incubated with and without test substances at the indicated concentrations for 1 to 15 days. Ig concentrations were measured in aliquots drawn from each lymphocyte culture supernatant at the times indicated.
Statistical analysis
Measurement of nitric oxide (NO)
Lymphocytes were isolated from spleens of both normal mice (MSLs) and tumour mice (TMSLs) and treated with different concentrations of GLIS. Lipopolysaccharide from Escherichia coli 0111:B4 (LPS, Sigma), a mitogen for B lymphocytes, and phytohemagglutinin (PHA, Sigma), a mitogen for T lymphocytes, were used as positive controls. Mitogen concentrations at which lymphocytes exhibited significant stimulation and signalling were used in this experiment. Fig. 1 shows that GLIS stimulated the proliferation of lymphocytes significantly. At a GLIS concentration of 50 μg/ml the proliferation rate of MSLs increased 3-fold as compared to untreated controls. This effect was comparable to that of 50 μg/ml of LPS and 6 μg/ml of PHA. TMSLs exhibited a stronger response to GLIS than MSLs. At concentrations of 50, 200 and 500 μg/ml GLIS increased the proliferation rate of TMSLs up to 4, 4.7 and 5.3 folds, respectively (Fig. 1).
Production of nitric oxide (NO) was estimated by measuring nitrite levels by the Griess reaction (Alleva et al. 1994). To each well of a 96well microplate, 180 μl of the macrophage suspension (1 × 106 cells/ ml) and 20 μl of various test substances were added. After incubation at 37 °C in a 5% CO2 atmosphere for 48 h, 100 μl of cell-free supernatants was mixed with 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylethyylene-diamine dihydrochloride, 2.5% phosphoric acid) and incubated for 10 min at room temperature. The optical densities (O.D.) of samples were measured at 543 nm. The nitrite concentration was determined with a standard curve of linear sodium nitrite from 0.1 to 100 μM. Phagocytosis assay The phagocytic activity was measured according to Kreutz et al. (1998). Macrophages were harvested with cold PBS and resuspended at a concentration of 2 × 105 cells/ml. An aliquot (100 μl) of the cell suspension was seeded per well of a 96-well microplate. Latex beads were washed three times with PBS, and then resuspended at 1 × 107 per ml in RPMI medium. To each well, 80 μl of latex beads and 20 μl of sample were added and incubated for 24 h at 37 °C in a 5% CO2 atmosphere. The cells were then washed three times with PBS, and the quantification of phagocytic cells and their percentages were counted under the microscope.
Data obtained from three or more separate experiments were expressed as mean ± SD. A paired, two-tailed Student's t-test was used for the statistical analysis of data and calculation of p values. Results Increase in the proliferation of spleen-derived lymphocytes from normal mice (MSLs) and tumour mice (TMSLs) after stimulation by GLIS
Increase in the B lymphocyte subpopulation of MSLs and TMSLs after stimulation by GLIS In order to identify the proliferating subpopulations of lymphocytes, flow cytometry analysis was performed. LPS, a mitogen for B lymphocytes, and PHA, a mitogen for T lymphocytes, were used as
Measurement of macrophage-mediated tumour cytotoxicity Anti-tumour activity of macrophages was measured as described previously (Moon et al., 1998). An aliquot (100 μl, 2 × 105 cells/ml) of the macrophages was seeded per wells of 96-well plates and incubated for 2 h to allow cell attachment. Then macrophages were co-incubated with L929 tumour cells as target cells at a ratio of 10 macrophages to 1 L929 in the presence of different concentrations of GLIS for 48 h at 37 °C in a 5% CO2 atmosphere. The cells were then stained with crystal violet containing 11.1% ethyl alcohol and 10% formaldehyde for 30 min. Anti-tumour, cytolytic activity of macrophages was expressed as percentage of tumour cells as follows: O:D: of ½ðtarget + macrophagesÞ−macrophages Cytolysisð%Þ = 1− × 100%: O:D: of target ðnontreatedÞ
Fig. 1. Proliferation of MSLs and TMSLs after stimulation by GLIS. Lymphocytes (3.6 × 105 cells/well) were incubated with GLIS (at the indicated concentrations), LPS (50 μg/ml) or PHA (6 μg/ml) for 72 h. Control cells were treated with 0.9% NaCl. The proliferation rate was determined by the Alamar Blue Assay. Each value represents the mean ± SD of 5 separate triplicate experiments. Values significantly higher than those of controls at p b 0.001 are indicated by ***. Values of TMSLs significantly higher than those of MSLs at p b 0.001 are indicated by +++.
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Fig. 2. Percentage changes in the lymphocyte subpopulations of MSLs (A) and TMSLs (B) after stimulation with GLIS. Lymphocytes were incubated with GLIS (500 μg/ml), LPS (50 μg/ ml) or PHA (6 μg/ml) for 72 h. After incubation with the different agents, the lymphocyte subpopulations were labelled with specific fluorophore-conjugated antibodies and analysed by FACScan flow cytometry (Becton Dickinson). Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly higher than those of controls at p b 0.001 are indicated by ***.
controls. After stimulation with GLIS (500 μg/ml) for 72 h, the changes in the percentages of lymphocyte subpopulations were similar to those observed after incubation with LPS, but not to those induced by PHA. The percentage of CD19+ cells in MSLs increased from 14% to 42%, while that of CD19+ cells in TMSLs rose from 25% to 70%. In contrast, the percentages of CD3+ and CD4+ cells decreased, whereas that of CD8+ cells did not change significantly (Fig. 2). These results suggest that most of the proliferating cells are B lymphocytes (CD19+). Additionally, GLIS increased the B lymphocyte number in TMSLs more strongly than in MSLs.
Change in the interleukin-2 (IL-2) and interleukin-4 (IL-4) production of MSLs and TMSLs after stimulation by GLIS After a 24 h exposure to GLIS, low levels of interleukin-2 (IL-2) were found in culture media from MSLs. However, the secretion of IL2 augmented significantly after 48 h, and it reached a maximum level after 72 h. Incubation with LPS had a similar effect as GLIS (Fig. 3). In contrast, only little stimulation of IL-2 secretion into culture media was observed after treatment of TMSLs with GLIS. As to interleukin-4 (IL-4) production, GLIS, unlike PHA, enhanced little the secretion of
Fig. 3. IL-2 and IL-4 production of MSLs and TMSLs after stimulation by GLIS. Lymphocytes (3.6 × 105 cells/well) were incubated with GLIS (500 μg/ml), LPS (50 μg/ml) or PHA (6 μg/ ml) for 24, 48 and 72 h. Control cells were treated with 0.9% NaCl. After incubation, the cell-free supernatants were collected for cytokine measurements. Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly higher than those of controls are indicated by * (p b 0.05), ** (p b 0.01), or *** (p b 0.001).
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this cytokine by MSLs, and it had no effect on the secretion of IL-4 by TMSLs (Fig. 3).
Increase in interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and reactive nitrogen intermediate (RNI) products of BMMs and TBMMs stimulated by GLIS
Production of immunoglobulins (Ig) by MSLs and TMSLs after stimulation by GLIS
The activation of macrophages facilitates the production of many immunomodulatory substances including cytokines. Since IL-1β and TNF-α are currently assumed to be the primary mediators involved in the killing of tumour cells, the ability of GLIS to induce IL-1β and TNFα secretion was examined. GLIS induced an increase of IL-1β and TNFα secretion by BMMs and TBMMs in a dose-dependent manner (Fig. 6B and D). After exposure to GLIS the secretion of IL-1β and TNFα by TBMMs was significantly higher than that of BMMs. Reactive nitrogen intermediates (RNI) play an important role in the capacity of macrophages to kill tumour cells. The level of RNI was estimated by determining the amount of NO, a stable metabolite of RNI. We found that NO generation by BMMs and TBMMs was significantly enhanced by GLIS in a dose-dependent manner, after stimulation of macrophages with GLIS for 24 h. The production of NO by TBMMs in the presence of GLIS was markedly higher than that by BMMs (Fig. 6C).
To study the response of B cells to GLIS, the production of total immunoglobulins (Ig) by MSLs and TMSLs was analysed by ELISA. After stimulation with GLIS, MSLs produced significant amounts of Ig. The Ig production increased until the eighth day and subsequently decreased. As shown in Fig. 4, the effect of GLIS at 500 μg/ml is comparable to that of LPS at 50 μg/ml. It is noticeable that after stimulation with GLIS, as well as with LPS under the same conditions, the Ig production by TMSLs is much higher than that by MSLs. After 8 days, the maximal Ig production by TMSLs was 2000 ng/ml, in comparison to 750 ng/ml by MSLs.
Activation of bone marrow-derived macrophages from normal mice (BMMs) and tumour mice (TBMMs) by GLIS Increase in the phagocytosis of BMMs and TBMMs triggered by GLIS Both BMMs and TBMMs were activated by GLIS in a concentrationdependent manner, in the range between 0.1 and 100 μg/ml. TBMMs showed to be more sensitive to GLIS stimulation than BMMs (Figs. 5 and 6A). No toxicity was found in cultures treated with GLIS at concentrations within the range from 0.1 to 1000 μg/ml as determined by microscopic examination and trypan blue exclusion test (data not shown). At a GLIS concentration of 1.0 μg/ml, both BMMs and TBMMs showed the highest activation rate, which did not augment by further increases of the GLIS concentration. In the presence of 1 μg/ml GLIS, morphological changes in cultured BMMs were observed after 24 h. The cells exposed to GLIS were more elongated and outspread than control cells (Fig. 5).
One of the well-known physiological activities of macrophages is the phagocytosis, whereby those cells ingest, process and/or destroy exogenous particles. The number of phagocytic cells cultured in the presence of GLIS and latex beads was counted microscopically and their percentages were calculated. Morphological changes were found in macrophages cultured with latex beads in the presence of GLIS for 24 h, e.g. most of the cells were able to take up latex beads. Yet, only a few of the control cells (without GLIS treatment) exhibited this capacity (Fig. 7A). GLIS could enhance the percentage of BMMs and TBMMs phagocytic cells from 22% to 90% and from 35% to 90%, respectively (Fig. 7B).
Fig. 4. Production of immunoglobulins by MSLs (A) and TMSLs (B) after stimulation by GLIS. MSLs and TMSLs were incubated with GLIS (500 μg/ml). At the indicated times ELISA was used to determine the immunoglobulin concentration of supernatant aliquots. Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly higher than those of controls are indicated by * (p b 0.05), ** (p b 0.01), or *** (p b 0.001).
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Fig. 5. Activation of BMMs and TBMMs by GLIS. (A) Morphological change of BMMs after stimulation by GLIS (1 μg/ml) for 24 h. (B) Activation of BMMs and TBMMs stimulated with different concentrations of GLIS for 72 h. Each value represents the mean ± SD of 5 separate triplicate experiments. Values significantly higher than those of controls are indicated by * (p b 0.05), or *** (p b 0.001). Activation rates of TBMMs significantly higher than that of BMMs are indicated by ++ (p b 0.01), or +++ (p b 0.001).
Increase in macrophage-mediated tumour cytotoxicity by GLIS In order to examine whether GLIS can stimulate macrophages to kill tumour cells, macrophages were co-cultured with L929 tumour
cells in the presence of GLIS. Fig. 8 shows that GLIS itself had no influence on the growth of L929 tumour cells. Macrophages displayed only very low cytotoxicity (ca. 4%) without stimulation with GLIS. However, the cytotoxicity of macrophages against L929 tumour cells
Fig. 6. Activation, secretion of cytokines and production of reactive nitrogen intermediates by BMMs and TBMMs after stimulation with GLIS. BMMs and TBMMs were incubated with different concentrations of GLIS (0.1–1 μg/ml) for different periods. After a 72 h incubation, the activation of macrophages was determined using the Alamar Blue Assay. Each value represents the mean ± SD of 5 separate triplicate experiments (A). After a 24 h incubation, the supernatants were harvested and assayed for IL-1β (B), reactive nitrogen intermediates (NO) (C), and TNF-α (D). Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly different from controls without GLIS are indicated by *** (p b 0.001). Values of TBMMs significantly different from those of BMMs are indicated by + (p b 0.05), ++ (p b 0.01), or +++ (p b 0.001).
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was increased concentration-dependently after exposure to GLIS. After stimulation of macrophages with 100 μg/ml GLIS for 48 h, the number of L929 tumour cells reduced to 55% of controls, which is comparable to the values obtained with LPS (1 μg/ml). Murine sarcoma S180 cells were also used for cytotoxic assays. We found that GLIS has the similar effect on S180 cells and reduces the viability of S180 cells in the presence of macrophages (data not shown). Increase in IgM levels in serum of mice treated with GLIS In order to evaluate the in vivo effect of GLIS on the humoral immune response of mice, serum IgM levels were measured after treatment with GLIS. After 6 days of administration of GLIS, the IgM levels in serum increased pronouncedly from 100 μg/ml up to 1500 μg/ml (Fig. 9). These elevated IgM levels remained stable up to day 25 after GLIS treatment (Fig. 9). These data indicate that GLIS is able to induce a significant increase of IgM concentrations in serum. Inhibition of S180 tumour growth by GLIS in vivo Our experiments indicate that GLIS could increase both the cellular and humoral immune activity. Particularly, GLIS demonstrates a high capacity to enhance the immune activity of tumour-bearing mice. In order to investigate whether GLIS influences tumour growth in vivo, sarcoma tumour cells S180 were transplanted into BALB/c mice that were treated or not with GLIS. Tumour weight was determined 15 days post-inoculation. In comparison to control mice, tumour growth in GLIS-treated mice was inhibited by about 60% (Figs. 8 and 10). Discussion Tumour growth, defective immunorecognition and immunosuppression are three principal factors considered to be responsible for immune evasion (Nicolini and Carpi, 2009). The ability of tumours to
evade the immune system's surveillance is an important requirement that allows for their growth and survival. Thus, targeting the immune system can be a major strategy to develop novel anti-tumour therapies (Gajewski et al., 2009). The search for drugs with antitumour properties from biological sources is also a promising strategy, judged by the number of publications describing bioactive compounds in the last few years (Efferth et al., 2007). Although it has long been shown that G. lucidum is effective in the treatment of many ailments and diseases throughout its long history of empirical use, it is only recently that studies have been designed to identify its active components and determine the mechanisms by which those compounds may act. We have previously reported the isolation of the bioactive proteoglycan fraction GLIS from aqueous extracts of G. lucidum using successive chromatographic steps. GLIS stimulated the activation, proliferation and differentiation of mouse spleen-derived lymphocytes, especially B lymphocytes, which resulted in an increase in the secretion of immunoglobulins (Zhang et al., 2002). In order to prove whether GLIS can increase the immune activity of tumour-bearing host as well as to inhibit tumour growth, we determined in this work the ability of GLIS to stimulate spleenderived lymphocytes and bone marrow-derived macrophages from non-tumour as well as from tumour-bearing mice in vitro, and measured its capacity to inhibit tumour growth in vivo. We found that the proliferation of mouse spleen-derived lymphocytes was significantly increased after stimulation with GLIS. In this case, spleenderived lymphocytes from tumour-bearing mice showed a stronger response to GLIS than spleen-derived lymphocytes from normal mice. Quantitative analysis of lymphocyte subpopulations revealed that the majority of proliferating cells were B lymphocytes. In response to GLIS, B lymphocytes from non-tumour as well as from tumourbearing mice produced larger amounts of immunoglobulins. Recently the effect of B cells on anti-tumour cellular responses against a lung metastatic tumour (MADB106) was determined. Removal of B cells from lung lymphocyte cultures resulted in diminished IFN-g secretion and tumour lysis, providing evidence of
Fig. 7. Phagocytosis activity of BMMs and TBMMs after stimulation by GLIS. (A) Morphologic observation of the phagocytosis of latex beads by BMMs. After BMMs were treated with GLIS (1 μg/ml) in the presence of latex beads (at a ratio of 50 latex beads per macrophage) for 24 h, morphological changes were observed microscopically. (B) Phagocytosis of latex beads by BMMs and TBMMs. BMMs and TBMMs were treated with different concentrations of GLIS (0.1–100 μg/ml) or LPS (1 μg/ml) for 24 h. After incubation, phagocytic cells were counted under a microscope and their percentages were determined. Each value represents means ± SD of 3 separate triplicate experiments. Values significantly different from controls at p b 0.001 are indicated by ***.
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Fig. 8. Anti-tumour activity of BMMs after stimulation by GLIS. L929 tumour cells, used as target tumour cells, were treated with various concentrations of GLIS (1–100 μg/ml) or LPS (1 μg/ml) in the presence or absence of BMMs (at a ratio of 10 macrophages per L929 cell). After a 48 h incubation, the number of intact tumour cells was determined. Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly different from controls are indicated by ** (p b 0.01) or *** (p b 0.001).
the importance of B cell responses in tumour defenses (Jones et al., 2008). Ollert et al. discovered the presence of natural IgM antibodies in the sera of healthy adults. These antibodies elicit effective killing of neuroblastoma cells by both complement activation and apoptosis (Ollert et al., 1997). Yoshimura et al. have found that alpha1–3 galactosyltransferase transfected M2A PaCa-2 and huH-7 tumour cells were effectively lysed by human natural antibodies (Yoshimura et al., 2001). In this study, we demonstrate that GLIS has a pronounced stimulatory effect on immunoglobulin production of B lymphocytes. Animal experiments also showed a significant increase in IgM levels in serum after stimulation with GLIS. These results are important in the context that the humoral immunity of tumour-bearing mice against tumours could be enhanced by GLIS. However, the stimulatory effects of GLS on IL-2 and IL-4 production of T lymphocytes derived from normal mice and from tumour-bearing mice were too low or non-significant, respectively. This indicates a poor response of T lymphocytes from tumour-bearing mice to this stimulus. Thus, GLIS is a specific stimulus for B lymphocytes, but not for T lymphocytes, indicating that its effects on B cell proliferation and Ig production are T lymphocyte-independent. It is well known that macrophages play an important role in many primary defense mechanisms (Bach et al., 2009). Macrophages can exert a dual influence on tumour growth and progression in response to microenvironmental signals (Mantovani and Sica, 2010). Whether
Fig. 9. Serum IgM concentration of mice treated with GLIS in vivo. Mice were injected intraperitoneally with GLIS (100 μg/0.5 ml in PBS/mouse) or PBS (0.5 ml in PBS/mouse) 5 times at days 1, 3, 6, 9 and 12. Sera at days 1, 6 and 25 were taken for determination of the IgM concentration by ELISA. Each value represents the mean ± SD of 3 separate experiments. Five mice were used for each GLIS and control groups. Values significantly different from controls are indicated by ** (p b 0.01), or *** (p b 0.001).
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Fig. 10. Inhibition of sarcoma 180 tumour growth by GLIS. Sarcoma 180 cells (1 × 106 cells/0.1 ml/mouse) were inoculated into the subcutaneous (s.c.) tissue of the left leg of BALB/c mice. Mice were injected intraperitoneally with GLIS (100 μg/0.5 ml in PBS/ mouse) five times according to the following protocol: 3 days before tumour inoculation, on the day of tumour inoculation, 3 days, 6 days and 9 days postinoculation. Control mice were injected intraperitoneally with PBS (0.5 ml/mouse). Tumour weight was determined 15 days post-inoculation. Each value represents the mean ± SD of three separate experiments. Five mice were used for each GLIS and control groups every experiment. Values significantly different from controls at (p b 0.05) are indicated by *.
tumour-associated macrophages show protumourigenesis or antitumour activity depends on the macrophage activation state. This is probably mirrored by shift in this ying yang balance (Smyth et al., 2006). Certain chemotherapeutic agents can likewise activate a protective macrophage response (Mantovani and Sica, 2010). Thus, the development of effective strategies to shift this balance by activating the protective innate immunity may have an enormous potential in suppressing tumour growth and shaping tumour immunogenicity. In this work we determined that GLIS could activate bone marrow-derived macrophages from non-tumour as well as from tumour-bearing mice in a concentration-dependent manner. After stimulation by GLIS, macrophages spread and elongated, secreted TNF-α and IL-1β, produced NO and showed a marked increase in their phagocytosis and anti-tumour cell cytotoxicity. These results suggest that GLIS could also increase effectively the cellular immune activity of tumour-bearing mice. Phagocytosis represents the final and most crucial step of the immunological defense system, especially in the defense against tumour cells (Popov et al., 1999). The ability of GLIS to stimulate the formation of NO could also be important for its anti-tumour effects, since NO is emerging as a potential anti-oncogenic agent to overcome tumour cell resistance to conventional therapeutic agents (Halama et al., 2008). Macrophage-derived cytokines like e.g. IL-1β and TNF-α have been shown to be cytotoxic for a range of tumour cells (Bach et al., 2009; alSarireh and Eremin, 2000). Furthermore, we show in this work that GLIS increased the macrophage cytotoxicity against L929, as well as S180 tumour cells without having itself any influence on the growth of these tumour cells. These data demonstrate that GLIS can increase the cellular immune activity against tumour cells. Although GLIS and LPS exhibit similar modes of action on B cells and macrophages, our study reveals that they have different properties, as indicated by the inability of polymyxin B, an LPS inhibitor, to attenuate the endotoxic activity of LPS but not that of GLIS (Zhang et al., 2002). We confirmed the differences in functional mechanisms between GLIS and LPS using C3H/HeJ mice, which are LPS hyperesponsers. GLIS, but not LPS, could stimulate NO production of macrophages from C3H/HeJ mice (data not shown). Several forms of immunotherapy are being rigorously explored in laboratories and tested in clinical trials, where they are showing to be promising as effective treatments against cancer. Four types of immunotherapy (cytokine, vaccine, antibody and cellular therapy) have been recently designed to treat neuroblastoma (Rosenberg, 2001; Navid et al., 2009). The expanded knowledge of the molecular basis of tumourigenesis and metastasis, together with the inherently vast structural diversity of natural compounds found in biological organisms like e.g. mushrooms, provides unique opportunities for
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discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to cancer (Zaidman et al., 2005). Mushroom polysaccharides prevent oncogenesis, show direct antitumour activity against various allogeneic and syngeneic tumours, and prevent tumour metastasis. They may not attack cancer cells directly, but produce their anti-tumour effects by activating different immune responses in the host (Wasser, 2002). Several mushroom preparations have shown clinically significant efficacy against human cancers and are used as biological response modifiers (Zaidman et al., 2005; Mitamura et al., 2000; Ng, 1998; Liu et al., 1993). All of these preparations are chemically β-D-glucans in nature or β-D-glucans linked to proteins that have β-(1–3) linkages in the main chain of the glucan and additional β-(1–6) branch points, which are important for their anti-tumour action (Cleary et al., 1999). Large numbers of studies have shown that G. lucidum polysaccharides and, in particular, their active β-D-glucans, modulate the functions of many components of the immune system (Wasser, 2002). Recently, dectin-1 has been reported as a β-glucan receptor that mediates the activities of βglucan (Brown et al., 2003). Our study presents GLIS isolated from the fruiting bodies of G. lucidum as a protein-bound polysaccharide fraction containing more than 90% carbohydrates and exhibiting eight different monosaccharides (Zhang et al., 2002). GLIS has different chemical properties and a molecular mass compared to that of the known active components in G. lucidum and other mushrooms. The elucidation of the chemical structure of GLIS as well as its receptor is being currently performed in our laboratory. Data from multiple epidemiological and clinical studies on the immune effects of conventional cancer treatment, as well as the clinical benefits of polysaccharide immune therapy, suggest that the immune function has a critical role in cancer prevention (Standish et al., 2008). In the present work we demonstrate that treatment with GLIS resulted in an increase of both the humoral and the cellular immune activities. We also show that GLIS can stimulate macrophages to kill tumour cells in vitro and inhibits tumour growth in vivo. On the other hand, GLIS did not inhibit the proliferation of different tumour cell lines in culture, nor did it show any cytotoxicity against tumour cells (data not shown). Our results indicate that the anti-tumour effect of GLIS ensues from its ability to increase the host's immune activities, which thus leads to an increase in the host's capacity to kill tumour cells and suppress tumour growth. Conclusion In this work, we demonstrate that GLIS exhibits a high capacity to increase both humoral and cellular immune response of tumour-bearing mice. We also show that GLIS displays a significant activity to increase the macrophage-mediated tumour cytotoxicity in vitro and inhibit tumour growth in vivo. After treatment with GLIS, spleen-derived B lymphocytes from tumour-bearing mice were activated, proliferated, differentiated and produced large amounts of immunoglobulins. After exposure to GLIS, bone marrow-derived macrophages from tumour-bearing mice were also activated, and important immunomodulatory substances, such as IL-1β, TNF-α and NO were produced. Phagocytosis and tumour cytotoxicity of macrophages were markedly increased after stimulation by GLIS. Our results suggest that GLIS exhibits an effective anti-tumour capacity by increasing both humoral and cellular immune activities. Conflict of interest statement There are no competing interests.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft Bonn (SFB366, Graduiertenkolleg 276/2), SonnenfeldStiftung Berlin and Gesellschaft für Biologische Krebsabwehr e.V.
Heidelberg. We are grateful to M. Zimmerman-Kordmann, U. Schöneberg and D. Grunow for scientific discussions and technical assistance.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum, displays anti-tumour activity by increasing both humoral and cellular immune response Jingsong Zhang a,b,1, Qingjiu Tang a,b,1, Changyan Zhou a,b, Wei Jia a,b, Luis Da Silva c, Long Duc Nguyen a, Werner Reutter a, Hua Fan a,⁎ a b c
Institut für Biochemie und Molekularbiologie, CBF, Charité-Universitätsmedizin Berlin, Germany The Edible Fungi Institute, Shanghai Academy of Agricultural Sciences, PR China Institut für Physiologie, CBF, Charité-Universitätsmedizin Berlin, Germany
a r t i c l e
i n f o
Article history: Received 9 March 2010 Accepted 23 September 2010 Keywords: Ganoderma lucidum (G. lucidum) Traditional Chinese medicine (TCM) B lymphocytes Macrophages Anti-tumour Proteoglycan
a b s t r a c t Aims: Ganoderma lucidum, a traditional Chinese medicine, is well known as a modulator of functions of the immune system as well as an anti-tumour agent. However, its active compounds and their molecular mechanisms of action are not well established. GLIS, a proteoglycan isolated from the fruiting body of G. lucidum, stimulates directly the activation of B lymphocytes. In this work, the immunoactivation capacities of GLIS as well as its anti-tumour effect were investigated in vitro and in vivo. Main methods: Tumour-bearing mice were prepared by inoculation of mouse sarcoma S180 cells into BALB/c mice. Lymphocytes and bone marrow-derived macrophages were isolated from spleen and tibia/femurs, respectively. After stimulation with GLIS different immune responses of these cells were analysed. Antitumour effect of GLIS was determined. Key findings: After treatment with GLIS, spleen-derived B lymphocytes from tumour-bearing mice became activated, proliferated and produced large amounts of immunoglobulins. Bone marrow-derived macrophages from tumour-bearing mice also became activated after exposure to GLIS, and they produced important immunomodulatory substances, such as IL-1β, TNF-α and reactive nitrogen intermediates, like NO. GLIS markedly increased phagocytosis of macrophages, and very importantly, it markedly raised the macrophagemediated tumour cytotoxicity. Treatment of mice with GLIS caused an inhibition of mouse sarcoma S180 tumour growth by 60% in vivo. Significance: These results indicate that GLIS exhibits a capacity to increase remarkably both humoral and cellular immune activities of tumour-bearing mice and inhibits tumour growth significantly. The anti-tumour effect of GLIS results from its capacity to increase the host's immune activity. © 2010 Elsevier Inc. All rights reserved.
Introduction The mushroom Ganoderma lucidum (G. lucidum), a lamellaless basidio-mycetous fungus belonging to the family Polyporaceae (Leyss. ex Fr. Karst), known as ‘Lingzhi’ in China and ‘Reishi’ in Japan, has played an important role in Chinese traditional medicine for more than 4000 years. Following the views of ancient Chinese medical scholars, people in Asian countries have been widely using G. lucidum for the promotion of general health and longevity (Lin, 2005). G. lucidum has also been used to treat various human diseases such as allergy, arthritis, bronchitis, gastric ulcer, hyperglycemia, hypertension, chronic hepatitis, hepatopathy, insomnia, nephritis, neurasthenia, scleroderma, inflam-
⁎ Corresponding author. Institut für Molekularbiologie und Biochemie, CBF, CharitéUniversitätsmedizin Berlin, Arnimallee 22, D-14195 Berlin, Germany. Tel.: + 49 30 84451552; fax: + 49 30 84451541. E-mail address: [email protected] (H. Fan). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.09.026
mation, and cancer (Sliva, 2003). New reports emphasize its potential in the treatment of viral, especially HIV, infections (Boh et al., 2007). Numerous pharmacological investigations have demonstrated that extracts of G. lucidum inhibit tumour growth in vitro and in several tumour mouse models in vivo, but the mechanism of this effect is not well understood (Boh et al., 2007; Sliva, 2003; Wasser, 2002). It has been determined that the most important pharmacologically active constituents of G. lucidum are triterpenoids and polysaccharides. Triterpenoids have been reported to exert hepatoprotective, antihypertensive, hypocholesterolemic and anti-histaminic properties as well as inhibitory effects on platelet aggregation, complement activation and angiogenesis, while the aqueous extracts and polysaccharide fractions of G. lucidum as well as the triterpenoids exert antitumour activities (Boh et al., 2007). Recently, anti-angiogenic effects in human lung cancer cells have been ascribed to polysaccharide peptides from G. lucidum (Cao and Lin, 2006). Ganoderma polysaccharides represent a structurally diverse class of biological macromolecules with a wide range of physicochemical
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properties (Efferth et al., 2007; Sliva, 2004). A large number of studies have shown that polysaccharides of G. lucidum, especially β-Dglucans, can modulate the functions of many components of the immune system such as the antigen-presenting cells, T and B lymphocytes (Boh et al., 2007; Wasser, 2002), NK cells (Chien et al., 2004), neutrophil granulocytes (Hsu et al., 2003) and dendritic cells (Cao and Lin, 2002; Lin et al., 2005). We have previously reported a bioactive fraction (GLIS) isolated from the aqueous extracts of the fruiting body of G. lucidum using successive chromatographic steps. GLIS is a proteoglycan fraction with a carbohydrate:protein ratio of 11:1 and a molecular weight of about 2000 kDa. The carbohydrate portion consists of heteropolysaccharides composed predominantly of D-glucose, D-galactose and D -mannose. GLIS stimulated the activation, proliferation and differentiation of mouse spleen lymphocytes, especially B lymphocytes, which resulted in an increase in the secretion of large amounts of immunoglobulins (Zhang et al., 2002). It has been postulated that immunosuppression of the tumour-bearing host is one of the reasons for the growth of antigenic tumours despite antitumour immune response (Nicolini and Carpi, 2009). In this work we investigated the capacities of GLIS to activate immune responses and inhibit tumour growth in tumour-bearing mice. We found that GLIS increases remarkably both the humoral and cellular immune activity of tumour-bearing mice and that it inhibits significantly tumour growth. Material and methods Animals BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany). The animals were treated according to the German Law on the Protection of Animals. A permission (VB103-G 140/01) for animal experiments was obtained from the state animal welfare committee.
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(8–10 weeks) were selected for isolating bone marrow macrophages. Tumour-bearing mice were obtained as described earlier. After killing normal and tumour-bearing mice by cervical dislocation, the tibias and the femurs were removed by cutting the proximal end of the femur and the distal end of the tibia, leaving the other ends intact. A 23-gauge needle was inserted into the intact ends and bone marrow was collected in ice-cold DMEM culture medium. The bone marrow cells were dispersed by three passes through a 22gauge needle and then centrifuged (1200 g for 5 min at 4 °C). The cell density was adjusted to 106 cell/ml in complete DMEM medium containing 10% L929 (mouse fibrosarcoma cell line, ATCC, Rockville, MD, CCL1) DMEM conditioned medium. After incubation at 37 °C for 3 days, non-adherent cells were collected, and adherent cells were discarded. Cells were seeded in bacteria-culture Petri dishes containing 10% L929 conditioned medium, and incubated at 37 °C for another three days. The non-adherent cells were washed away with PBS. Adherent macrophages were covered with cold PBS and detached from the dish using a spatula. Isolated macrophages were incubated in complete DMED medium. Determination of proliferation of mouse lymphocytes and activation of macrophages by the Alamar Blue Assay Lymphocyte concentration was adjusted to 2 × 106 cells/ml and macrophage concentration was adjusted to 2 × 105 cells/ml. To each well of a 96-well microplate, 180 μl of the cell suspension and 20 μl of various test substances were added. After incubation at 37 °C in a 5% CO2 atmosphere for the indicated times, 20 μl Alamar Blue reagent (Biosource, Nivelles, Belgium) was added to each well. After incubation for 6 h, optical densities (O.D.) at 570 nm and 600 nm were measured using a micro ELISA autoreader (Bio-Rad). Lymphocyte proliferation and activation rates were calculated according to the Biosource protocol (Nivelles, Belgium). Quantification of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin1ß (IL-1β) and tumour necrosis factor-α (TNF-α)
Preparation and quality control of GLIS GLIS was prepared according to the method of Zhang et al. (2002). The purity and quality of the isolated product were controlled by high-performance liquid chromatography (HPLC), whereby obtaining a single, very sharp and symmetric peak was demonstrated. The carbohydrate content of GLIS was determined by the phenol–sulfuric acid reaction, while the peptide content was measured by UV absorption at 280 nm and the bicinchoninic acid (BCA) assay. The molar ratio of different monosaccharides was determined by high-pH anion-exchange chromatography after hydrolysis with trifluoroacetic acid (TFA). Preparation of lymphocytes from normal (MSLs) and sarcoma-bearing mouse spleens (TMSLs) BALB/c mice, 8–10 weeks old (ca. 28 ± 1 g), were used for lymphocyte preparation. For preparation of lymphocytes from tumour-bearing mice, mouse sarcoma S180 tumour cells (1.0 × 106 cells in 100 μl PBS) were injected intramuscularly in the legs of mice. After 15 days, the mice with growing tumours were killed by cervical dislocation and the spleens were subsequently removed. Spleen lymphocytes were prepared according to the procedure by Zhang et al. (2002). Preparation of bone marrow-derived macrophages from normal mice (BMMs) and tumour-bearing mice (TBMMs) Bone marrow-derived macrophages were prepared according to Stanley (1997). BALB/c mice with similar weights (28 ± 1 g) and ages
Production of the cytokines interleukin-2 (IL-2) and interleukin-4 (IL-4) by lymphocytes and production of the cytokines interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) by macrophages were quantified by ELISA according to the manufacturer's instructions (R & D, Minneapolis, USA). Lymphocytes were adjusted to a concentration of 2 × 106 cells/ml. To each well of a 96-well microplate, 180 μl of the cell suspension and 20 μl of different test substances were added. After incubation with and without substances at 37 °C in a 5% CO2 atmosphere for 24, 48 and 72 h, 50 μl of the suspension of each sample was used for the IL-2 and IL-4 ELISA-assay. Macrophages from normal and tumour-bearing mice were prepared as described earlier. Macrophages were adjusted to a concentration of 1 × 105 cells/ml. To each well of a 96-well microplate, 180 μl of the cell suspension and 20 μl of various test substances were added. After incubation at 37 °C in a 5% CO2 atmosphere for 24 h, 50 μl of cell-free supernatant from each well was used for the IL1β and TNFα ELISA-assay. Analysis of the percentage of subpopulations of lymphocytes using flow cytometry Four milliliters of lymphocyte cell suspension (2 × 106 cells/ml) was added into 6-well plates with or without test substances. After incubation for 72 h, lymphocytes were centrifuged, washed twice with PBS and then labelled with different mAbs in PBS for 30 min at room temperature. After washing three times with PBS, cells were resuspended in 0.3 ml PBS. Fluorescence analysis was performed using a FACScan analyzer (Becton Dickinson, Eremdodegem,
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Belgium). CELLQuest software (Becton Dickinson, Eremdodegem, Belgium) was used to analyse the percentage of different lymphocyte subpopulations. Anti-mouse CD3-fluorescein conjugate (marker for T lymohocytes), anti-mouse CD4-fluorescein conjugate (marker for Th lymphocytes), anti-mouse CD8-R-phycoerythrin conjugate (marker for Tc lymphocytes) and anti-CD19-R-phycoerythrin conjugate (marker for B lymphocytes) were obtained from Dianova (Hamburg, Germany). Determination of immunoglobulin (Ig) production
Anti-tumour test in vivo Sarcoma S180 cells (1 × 106 cells/0.1 ml/mouse) were subcutaneously (s.c.) inoculated into the left leg of BALB/c mice. GLIS (100 μg/ 0.5 ml in PBS/mouse) was injected intraperitoneally (i.p.) into mice 5 times at 5 different time points as follows: 3 days before inoculation, on the day of inoculation, 3 days, 6 days and 9 days after inoculation. Control mice received PBS (0.5 ml/mouse) i.p. Tumour weight were measured 15 days after inoculation. The experiment was repeated 3 times with 5 mice for each test group.
The immunoglobulin (Ig) production of lymphocytes was measured by ELISA. The anti-mouse Ig hybridoma screening reagent (coating antibody) was obtained from Roche (Mannheim, Germany), and goat anti-mouse IgG antibodies were acquired from Amersham Pharmacia Biotech (Uppsala, Sweden). The alkaline phosphatasestreptavidin was purchased from DIANOVA (Hamburg, Germany). Lymphocytes were incubated with and without test substances at the indicated concentrations for 1 to 15 days. Ig concentrations were measured in aliquots drawn from each lymphocyte culture supernatant at the times indicated.
Statistical analysis
Measurement of nitric oxide (NO)
Lymphocytes were isolated from spleens of both normal mice (MSLs) and tumour mice (TMSLs) and treated with different concentrations of GLIS. Lipopolysaccharide from Escherichia coli 0111:B4 (LPS, Sigma), a mitogen for B lymphocytes, and phytohemagglutinin (PHA, Sigma), a mitogen for T lymphocytes, were used as positive controls. Mitogen concentrations at which lymphocytes exhibited significant stimulation and signalling were used in this experiment. Fig. 1 shows that GLIS stimulated the proliferation of lymphocytes significantly. At a GLIS concentration of 50 μg/ml the proliferation rate of MSLs increased 3-fold as compared to untreated controls. This effect was comparable to that of 50 μg/ml of LPS and 6 μg/ml of PHA. TMSLs exhibited a stronger response to GLIS than MSLs. At concentrations of 50, 200 and 500 μg/ml GLIS increased the proliferation rate of TMSLs up to 4, 4.7 and 5.3 folds, respectively (Fig. 1).
Production of nitric oxide (NO) was estimated by measuring nitrite levels by the Griess reaction (Alleva et al. 1994). To each well of a 96well microplate, 180 μl of the macrophage suspension (1 × 106 cells/ ml) and 20 μl of various test substances were added. After incubation at 37 °C in a 5% CO2 atmosphere for 48 h, 100 μl of cell-free supernatants was mixed with 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylethyylene-diamine dihydrochloride, 2.5% phosphoric acid) and incubated for 10 min at room temperature. The optical densities (O.D.) of samples were measured at 543 nm. The nitrite concentration was determined with a standard curve of linear sodium nitrite from 0.1 to 100 μM. Phagocytosis assay The phagocytic activity was measured according to Kreutz et al. (1998). Macrophages were harvested with cold PBS and resuspended at a concentration of 2 × 105 cells/ml. An aliquot (100 μl) of the cell suspension was seeded per well of a 96-well microplate. Latex beads were washed three times with PBS, and then resuspended at 1 × 107 per ml in RPMI medium. To each well, 80 μl of latex beads and 20 μl of sample were added and incubated for 24 h at 37 °C in a 5% CO2 atmosphere. The cells were then washed three times with PBS, and the quantification of phagocytic cells and their percentages were counted under the microscope.
Data obtained from three or more separate experiments were expressed as mean ± SD. A paired, two-tailed Student's t-test was used for the statistical analysis of data and calculation of p values. Results Increase in the proliferation of spleen-derived lymphocytes from normal mice (MSLs) and tumour mice (TMSLs) after stimulation by GLIS
Increase in the B lymphocyte subpopulation of MSLs and TMSLs after stimulation by GLIS In order to identify the proliferating subpopulations of lymphocytes, flow cytometry analysis was performed. LPS, a mitogen for B lymphocytes, and PHA, a mitogen for T lymphocytes, were used as
Measurement of macrophage-mediated tumour cytotoxicity Anti-tumour activity of macrophages was measured as described previously (Moon et al., 1998). An aliquot (100 μl, 2 × 105 cells/ml) of the macrophages was seeded per wells of 96-well plates and incubated for 2 h to allow cell attachment. Then macrophages were co-incubated with L929 tumour cells as target cells at a ratio of 10 macrophages to 1 L929 in the presence of different concentrations of GLIS for 48 h at 37 °C in a 5% CO2 atmosphere. The cells were then stained with crystal violet containing 11.1% ethyl alcohol and 10% formaldehyde for 30 min. Anti-tumour, cytolytic activity of macrophages was expressed as percentage of tumour cells as follows: O:D: of ½ðtarget + macrophagesÞ−macrophages Cytolysisð%Þ = 1− × 100%: O:D: of target ðnontreatedÞ
Fig. 1. Proliferation of MSLs and TMSLs after stimulation by GLIS. Lymphocytes (3.6 × 105 cells/well) were incubated with GLIS (at the indicated concentrations), LPS (50 μg/ml) or PHA (6 μg/ml) for 72 h. Control cells were treated with 0.9% NaCl. The proliferation rate was determined by the Alamar Blue Assay. Each value represents the mean ± SD of 5 separate triplicate experiments. Values significantly higher than those of controls at p b 0.001 are indicated by ***. Values of TMSLs significantly higher than those of MSLs at p b 0.001 are indicated by +++.
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Fig. 2. Percentage changes in the lymphocyte subpopulations of MSLs (A) and TMSLs (B) after stimulation with GLIS. Lymphocytes were incubated with GLIS (500 μg/ml), LPS (50 μg/ ml) or PHA (6 μg/ml) for 72 h. After incubation with the different agents, the lymphocyte subpopulations were labelled with specific fluorophore-conjugated antibodies and analysed by FACScan flow cytometry (Becton Dickinson). Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly higher than those of controls at p b 0.001 are indicated by ***.
controls. After stimulation with GLIS (500 μg/ml) for 72 h, the changes in the percentages of lymphocyte subpopulations were similar to those observed after incubation with LPS, but not to those induced by PHA. The percentage of CD19+ cells in MSLs increased from 14% to 42%, while that of CD19+ cells in TMSLs rose from 25% to 70%. In contrast, the percentages of CD3+ and CD4+ cells decreased, whereas that of CD8+ cells did not change significantly (Fig. 2). These results suggest that most of the proliferating cells are B lymphocytes (CD19+). Additionally, GLIS increased the B lymphocyte number in TMSLs more strongly than in MSLs.
Change in the interleukin-2 (IL-2) and interleukin-4 (IL-4) production of MSLs and TMSLs after stimulation by GLIS After a 24 h exposure to GLIS, low levels of interleukin-2 (IL-2) were found in culture media from MSLs. However, the secretion of IL2 augmented significantly after 48 h, and it reached a maximum level after 72 h. Incubation with LPS had a similar effect as GLIS (Fig. 3). In contrast, only little stimulation of IL-2 secretion into culture media was observed after treatment of TMSLs with GLIS. As to interleukin-4 (IL-4) production, GLIS, unlike PHA, enhanced little the secretion of
Fig. 3. IL-2 and IL-4 production of MSLs and TMSLs after stimulation by GLIS. Lymphocytes (3.6 × 105 cells/well) were incubated with GLIS (500 μg/ml), LPS (50 μg/ml) or PHA (6 μg/ ml) for 24, 48 and 72 h. Control cells were treated with 0.9% NaCl. After incubation, the cell-free supernatants were collected for cytokine measurements. Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly higher than those of controls are indicated by * (p b 0.05), ** (p b 0.01), or *** (p b 0.001).
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this cytokine by MSLs, and it had no effect on the secretion of IL-4 by TMSLs (Fig. 3).
Increase in interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and reactive nitrogen intermediate (RNI) products of BMMs and TBMMs stimulated by GLIS
Production of immunoglobulins (Ig) by MSLs and TMSLs after stimulation by GLIS
The activation of macrophages facilitates the production of many immunomodulatory substances including cytokines. Since IL-1β and TNF-α are currently assumed to be the primary mediators involved in the killing of tumour cells, the ability of GLIS to induce IL-1β and TNFα secretion was examined. GLIS induced an increase of IL-1β and TNFα secretion by BMMs and TBMMs in a dose-dependent manner (Fig. 6B and D). After exposure to GLIS the secretion of IL-1β and TNFα by TBMMs was significantly higher than that of BMMs. Reactive nitrogen intermediates (RNI) play an important role in the capacity of macrophages to kill tumour cells. The level of RNI was estimated by determining the amount of NO, a stable metabolite of RNI. We found that NO generation by BMMs and TBMMs was significantly enhanced by GLIS in a dose-dependent manner, after stimulation of macrophages with GLIS for 24 h. The production of NO by TBMMs in the presence of GLIS was markedly higher than that by BMMs (Fig. 6C).
To study the response of B cells to GLIS, the production of total immunoglobulins (Ig) by MSLs and TMSLs was analysed by ELISA. After stimulation with GLIS, MSLs produced significant amounts of Ig. The Ig production increased until the eighth day and subsequently decreased. As shown in Fig. 4, the effect of GLIS at 500 μg/ml is comparable to that of LPS at 50 μg/ml. It is noticeable that after stimulation with GLIS, as well as with LPS under the same conditions, the Ig production by TMSLs is much higher than that by MSLs. After 8 days, the maximal Ig production by TMSLs was 2000 ng/ml, in comparison to 750 ng/ml by MSLs.
Activation of bone marrow-derived macrophages from normal mice (BMMs) and tumour mice (TBMMs) by GLIS Increase in the phagocytosis of BMMs and TBMMs triggered by GLIS Both BMMs and TBMMs were activated by GLIS in a concentrationdependent manner, in the range between 0.1 and 100 μg/ml. TBMMs showed to be more sensitive to GLIS stimulation than BMMs (Figs. 5 and 6A). No toxicity was found in cultures treated with GLIS at concentrations within the range from 0.1 to 1000 μg/ml as determined by microscopic examination and trypan blue exclusion test (data not shown). At a GLIS concentration of 1.0 μg/ml, both BMMs and TBMMs showed the highest activation rate, which did not augment by further increases of the GLIS concentration. In the presence of 1 μg/ml GLIS, morphological changes in cultured BMMs were observed after 24 h. The cells exposed to GLIS were more elongated and outspread than control cells (Fig. 5).
One of the well-known physiological activities of macrophages is the phagocytosis, whereby those cells ingest, process and/or destroy exogenous particles. The number of phagocytic cells cultured in the presence of GLIS and latex beads was counted microscopically and their percentages were calculated. Morphological changes were found in macrophages cultured with latex beads in the presence of GLIS for 24 h, e.g. most of the cells were able to take up latex beads. Yet, only a few of the control cells (without GLIS treatment) exhibited this capacity (Fig. 7A). GLIS could enhance the percentage of BMMs and TBMMs phagocytic cells from 22% to 90% and from 35% to 90%, respectively (Fig. 7B).
Fig. 4. Production of immunoglobulins by MSLs (A) and TMSLs (B) after stimulation by GLIS. MSLs and TMSLs were incubated with GLIS (500 μg/ml). At the indicated times ELISA was used to determine the immunoglobulin concentration of supernatant aliquots. Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly higher than those of controls are indicated by * (p b 0.05), ** (p b 0.01), or *** (p b 0.001).
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Fig. 5. Activation of BMMs and TBMMs by GLIS. (A) Morphological change of BMMs after stimulation by GLIS (1 μg/ml) for 24 h. (B) Activation of BMMs and TBMMs stimulated with different concentrations of GLIS for 72 h. Each value represents the mean ± SD of 5 separate triplicate experiments. Values significantly higher than those of controls are indicated by * (p b 0.05), or *** (p b 0.001). Activation rates of TBMMs significantly higher than that of BMMs are indicated by ++ (p b 0.01), or +++ (p b 0.001).
Increase in macrophage-mediated tumour cytotoxicity by GLIS In order to examine whether GLIS can stimulate macrophages to kill tumour cells, macrophages were co-cultured with L929 tumour
cells in the presence of GLIS. Fig. 8 shows that GLIS itself had no influence on the growth of L929 tumour cells. Macrophages displayed only very low cytotoxicity (ca. 4%) without stimulation with GLIS. However, the cytotoxicity of macrophages against L929 tumour cells
Fig. 6. Activation, secretion of cytokines and production of reactive nitrogen intermediates by BMMs and TBMMs after stimulation with GLIS. BMMs and TBMMs were incubated with different concentrations of GLIS (0.1–1 μg/ml) for different periods. After a 72 h incubation, the activation of macrophages was determined using the Alamar Blue Assay. Each value represents the mean ± SD of 5 separate triplicate experiments (A). After a 24 h incubation, the supernatants were harvested and assayed for IL-1β (B), reactive nitrogen intermediates (NO) (C), and TNF-α (D). Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly different from controls without GLIS are indicated by *** (p b 0.001). Values of TBMMs significantly different from those of BMMs are indicated by + (p b 0.05), ++ (p b 0.01), or +++ (p b 0.001).
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was increased concentration-dependently after exposure to GLIS. After stimulation of macrophages with 100 μg/ml GLIS for 48 h, the number of L929 tumour cells reduced to 55% of controls, which is comparable to the values obtained with LPS (1 μg/ml). Murine sarcoma S180 cells were also used for cytotoxic assays. We found that GLIS has the similar effect on S180 cells and reduces the viability of S180 cells in the presence of macrophages (data not shown). Increase in IgM levels in serum of mice treated with GLIS In order to evaluate the in vivo effect of GLIS on the humoral immune response of mice, serum IgM levels were measured after treatment with GLIS. After 6 days of administration of GLIS, the IgM levels in serum increased pronouncedly from 100 μg/ml up to 1500 μg/ml (Fig. 9). These elevated IgM levels remained stable up to day 25 after GLIS treatment (Fig. 9). These data indicate that GLIS is able to induce a significant increase of IgM concentrations in serum. Inhibition of S180 tumour growth by GLIS in vivo Our experiments indicate that GLIS could increase both the cellular and humoral immune activity. Particularly, GLIS demonstrates a high capacity to enhance the immune activity of tumour-bearing mice. In order to investigate whether GLIS influences tumour growth in vivo, sarcoma tumour cells S180 were transplanted into BALB/c mice that were treated or not with GLIS. Tumour weight was determined 15 days post-inoculation. In comparison to control mice, tumour growth in GLIS-treated mice was inhibited by about 60% (Figs. 8 and 10). Discussion Tumour growth, defective immunorecognition and immunosuppression are three principal factors considered to be responsible for immune evasion (Nicolini and Carpi, 2009). The ability of tumours to
evade the immune system's surveillance is an important requirement that allows for their growth and survival. Thus, targeting the immune system can be a major strategy to develop novel anti-tumour therapies (Gajewski et al., 2009). The search for drugs with antitumour properties from biological sources is also a promising strategy, judged by the number of publications describing bioactive compounds in the last few years (Efferth et al., 2007). Although it has long been shown that G. lucidum is effective in the treatment of many ailments and diseases throughout its long history of empirical use, it is only recently that studies have been designed to identify its active components and determine the mechanisms by which those compounds may act. We have previously reported the isolation of the bioactive proteoglycan fraction GLIS from aqueous extracts of G. lucidum using successive chromatographic steps. GLIS stimulated the activation, proliferation and differentiation of mouse spleen-derived lymphocytes, especially B lymphocytes, which resulted in an increase in the secretion of immunoglobulins (Zhang et al., 2002). In order to prove whether GLIS can increase the immune activity of tumour-bearing host as well as to inhibit tumour growth, we determined in this work the ability of GLIS to stimulate spleenderived lymphocytes and bone marrow-derived macrophages from non-tumour as well as from tumour-bearing mice in vitro, and measured its capacity to inhibit tumour growth in vivo. We found that the proliferation of mouse spleen-derived lymphocytes was significantly increased after stimulation with GLIS. In this case, spleenderived lymphocytes from tumour-bearing mice showed a stronger response to GLIS than spleen-derived lymphocytes from normal mice. Quantitative analysis of lymphocyte subpopulations revealed that the majority of proliferating cells were B lymphocytes. In response to GLIS, B lymphocytes from non-tumour as well as from tumourbearing mice produced larger amounts of immunoglobulins. Recently the effect of B cells on anti-tumour cellular responses against a lung metastatic tumour (MADB106) was determined. Removal of B cells from lung lymphocyte cultures resulted in diminished IFN-g secretion and tumour lysis, providing evidence of
Fig. 7. Phagocytosis activity of BMMs and TBMMs after stimulation by GLIS. (A) Morphologic observation of the phagocytosis of latex beads by BMMs. After BMMs were treated with GLIS (1 μg/ml) in the presence of latex beads (at a ratio of 50 latex beads per macrophage) for 24 h, morphological changes were observed microscopically. (B) Phagocytosis of latex beads by BMMs and TBMMs. BMMs and TBMMs were treated with different concentrations of GLIS (0.1–100 μg/ml) or LPS (1 μg/ml) for 24 h. After incubation, phagocytic cells were counted under a microscope and their percentages were determined. Each value represents means ± SD of 3 separate triplicate experiments. Values significantly different from controls at p b 0.001 are indicated by ***.
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Fig. 8. Anti-tumour activity of BMMs after stimulation by GLIS. L929 tumour cells, used as target tumour cells, were treated with various concentrations of GLIS (1–100 μg/ml) or LPS (1 μg/ml) in the presence or absence of BMMs (at a ratio of 10 macrophages per L929 cell). After a 48 h incubation, the number of intact tumour cells was determined. Each value represents the mean ± SD of 3 separate triplicate experiments. Values significantly different from controls are indicated by ** (p b 0.01) or *** (p b 0.001).
the importance of B cell responses in tumour defenses (Jones et al., 2008). Ollert et al. discovered the presence of natural IgM antibodies in the sera of healthy adults. These antibodies elicit effective killing of neuroblastoma cells by both complement activation and apoptosis (Ollert et al., 1997). Yoshimura et al. have found that alpha1–3 galactosyltransferase transfected M2A PaCa-2 and huH-7 tumour cells were effectively lysed by human natural antibodies (Yoshimura et al., 2001). In this study, we demonstrate that GLIS has a pronounced stimulatory effect on immunoglobulin production of B lymphocytes. Animal experiments also showed a significant increase in IgM levels in serum after stimulation with GLIS. These results are important in the context that the humoral immunity of tumour-bearing mice against tumours could be enhanced by GLIS. However, the stimulatory effects of GLS on IL-2 and IL-4 production of T lymphocytes derived from normal mice and from tumour-bearing mice were too low or non-significant, respectively. This indicates a poor response of T lymphocytes from tumour-bearing mice to this stimulus. Thus, GLIS is a specific stimulus for B lymphocytes, but not for T lymphocytes, indicating that its effects on B cell proliferation and Ig production are T lymphocyte-independent. It is well known that macrophages play an important role in many primary defense mechanisms (Bach et al., 2009). Macrophages can exert a dual influence on tumour growth and progression in response to microenvironmental signals (Mantovani and Sica, 2010). Whether
Fig. 9. Serum IgM concentration of mice treated with GLIS in vivo. Mice were injected intraperitoneally with GLIS (100 μg/0.5 ml in PBS/mouse) or PBS (0.5 ml in PBS/mouse) 5 times at days 1, 3, 6, 9 and 12. Sera at days 1, 6 and 25 were taken for determination of the IgM concentration by ELISA. Each value represents the mean ± SD of 3 separate experiments. Five mice were used for each GLIS and control groups. Values significantly different from controls are indicated by ** (p b 0.01), or *** (p b 0.001).
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Fig. 10. Inhibition of sarcoma 180 tumour growth by GLIS. Sarcoma 180 cells (1 × 106 cells/0.1 ml/mouse) were inoculated into the subcutaneous (s.c.) tissue of the left leg of BALB/c mice. Mice were injected intraperitoneally with GLIS (100 μg/0.5 ml in PBS/ mouse) five times according to the following protocol: 3 days before tumour inoculation, on the day of tumour inoculation, 3 days, 6 days and 9 days postinoculation. Control mice were injected intraperitoneally with PBS (0.5 ml/mouse). Tumour weight was determined 15 days post-inoculation. Each value represents the mean ± SD of three separate experiments. Five mice were used for each GLIS and control groups every experiment. Values significantly different from controls at (p b 0.05) are indicated by *.
tumour-associated macrophages show protumourigenesis or antitumour activity depends on the macrophage activation state. This is probably mirrored by shift in this ying yang balance (Smyth et al., 2006). Certain chemotherapeutic agents can likewise activate a protective macrophage response (Mantovani and Sica, 2010). Thus, the development of effective strategies to shift this balance by activating the protective innate immunity may have an enormous potential in suppressing tumour growth and shaping tumour immunogenicity. In this work we determined that GLIS could activate bone marrow-derived macrophages from non-tumour as well as from tumour-bearing mice in a concentration-dependent manner. After stimulation by GLIS, macrophages spread and elongated, secreted TNF-α and IL-1β, produced NO and showed a marked increase in their phagocytosis and anti-tumour cell cytotoxicity. These results suggest that GLIS could also increase effectively the cellular immune activity of tumour-bearing mice. Phagocytosis represents the final and most crucial step of the immunological defense system, especially in the defense against tumour cells (Popov et al., 1999). The ability of GLIS to stimulate the formation of NO could also be important for its anti-tumour effects, since NO is emerging as a potential anti-oncogenic agent to overcome tumour cell resistance to conventional therapeutic agents (Halama et al., 2008). Macrophage-derived cytokines like e.g. IL-1β and TNF-α have been shown to be cytotoxic for a range of tumour cells (Bach et al., 2009; alSarireh and Eremin, 2000). Furthermore, we show in this work that GLIS increased the macrophage cytotoxicity against L929, as well as S180 tumour cells without having itself any influence on the growth of these tumour cells. These data demonstrate that GLIS can increase the cellular immune activity against tumour cells. Although GLIS and LPS exhibit similar modes of action on B cells and macrophages, our study reveals that they have different properties, as indicated by the inability of polymyxin B, an LPS inhibitor, to attenuate the endotoxic activity of LPS but not that of GLIS (Zhang et al., 2002). We confirmed the differences in functional mechanisms between GLIS and LPS using C3H/HeJ mice, which are LPS hyperesponsers. GLIS, but not LPS, could stimulate NO production of macrophages from C3H/HeJ mice (data not shown). Several forms of immunotherapy are being rigorously explored in laboratories and tested in clinical trials, where they are showing to be promising as effective treatments against cancer. Four types of immunotherapy (cytokine, vaccine, antibody and cellular therapy) have been recently designed to treat neuroblastoma (Rosenberg, 2001; Navid et al., 2009). The expanded knowledge of the molecular basis of tumourigenesis and metastasis, together with the inherently vast structural diversity of natural compounds found in biological organisms like e.g. mushrooms, provides unique opportunities for
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discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to cancer (Zaidman et al., 2005). Mushroom polysaccharides prevent oncogenesis, show direct antitumour activity against various allogeneic and syngeneic tumours, and prevent tumour metastasis. They may not attack cancer cells directly, but produce their anti-tumour effects by activating different immune responses in the host (Wasser, 2002). Several mushroom preparations have shown clinically significant efficacy against human cancers and are used as biological response modifiers (Zaidman et al., 2005; Mitamura et al., 2000; Ng, 1998; Liu et al., 1993). All of these preparations are chemically β-D-glucans in nature or β-D-glucans linked to proteins that have β-(1–3) linkages in the main chain of the glucan and additional β-(1–6) branch points, which are important for their anti-tumour action (Cleary et al., 1999). Large numbers of studies have shown that G. lucidum polysaccharides and, in particular, their active β-D-glucans, modulate the functions of many components of the immune system (Wasser, 2002). Recently, dectin-1 has been reported as a β-glucan receptor that mediates the activities of βglucan (Brown et al., 2003). Our study presents GLIS isolated from the fruiting bodies of G. lucidum as a protein-bound polysaccharide fraction containing more than 90% carbohydrates and exhibiting eight different monosaccharides (Zhang et al., 2002). GLIS has different chemical properties and a molecular mass compared to that of the known active components in G. lucidum and other mushrooms. The elucidation of the chemical structure of GLIS as well as its receptor is being currently performed in our laboratory. Data from multiple epidemiological and clinical studies on the immune effects of conventional cancer treatment, as well as the clinical benefits of polysaccharide immune therapy, suggest that the immune function has a critical role in cancer prevention (Standish et al., 2008). In the present work we demonstrate that treatment with GLIS resulted in an increase of both the humoral and the cellular immune activities. We also show that GLIS can stimulate macrophages to kill tumour cells in vitro and inhibits tumour growth in vivo. On the other hand, GLIS did not inhibit the proliferation of different tumour cell lines in culture, nor did it show any cytotoxicity against tumour cells (data not shown). Our results indicate that the anti-tumour effect of GLIS ensues from its ability to increase the host's immune activities, which thus leads to an increase in the host's capacity to kill tumour cells and suppress tumour growth. Conclusion In this work, we demonstrate that GLIS exhibits a high capacity to increase both humoral and cellular immune response of tumour-bearing mice. We also show that GLIS displays a significant activity to increase the macrophage-mediated tumour cytotoxicity in vitro and inhibit tumour growth in vivo. After treatment with GLIS, spleen-derived B lymphocytes from tumour-bearing mice were activated, proliferated, differentiated and produced large amounts of immunoglobulins. After exposure to GLIS, bone marrow-derived macrophages from tumour-bearing mice were also activated, and important immunomodulatory substances, such as IL-1β, TNF-α and NO were produced. Phagocytosis and tumour cytotoxicity of macrophages were markedly increased after stimulation by GLIS. Our results suggest that GLIS exhibits an effective anti-tumour capacity by increasing both humoral and cellular immune activities. Conflict of interest statement There are no competing interests.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft Bonn (SFB366, Graduiertenkolleg 276/2), SonnenfeldStiftung Berlin and Gesellschaft für Biologische Krebsabwehr e.V.
Heidelberg. We are grateful to M. Zimmerman-Kordmann, U. Schöneberg and D. Grunow for scientific discussions and technical assistance.
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