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Introduction
Among the numerous antitumor cellular components in
mammals, natural killer (NK) cells play important roles in
inhibiting tumor development, growth, and metastasis, as well
as having a significant role in tumor
treatment[1_3]. Tumor cells are able to evade the adaptive immune system by
downregulating the expression of major histocompatibility
complex (MHC) molecules; however, NK cells are cytotoxic
against MHC class I-deficient tumor
cells[4,5]. Besides killing cancer cells directly through cell-mediated cytolysis, NK cells
produce cytokines, such as interferon-gamma (IFN-gamma),
which stimulate adaptive immunity and restrict tumor
angiogenesis[6_8]. Therefore, maintaining or enhancing NK
cell-based immune responses is very important for cancer
treatment[9]. Accordingly, many cancer immunotherapies that
enhance the activities of NK cells have been proven to be
effective in both experimental animal models and cancer
patients[10,11].
Natural resource-derived compounds such as ginseng
extract or echinacea root extract have been investigated as
potential immunomodulators for the treatment of cancer
patients[12,13]. Many of these studies have focused on the
activation of NK cells and demonstrated positive
results[14,15]. Furthermore, polysaccharides found in various mushrooms
have recently attracted attention due to their NK cell-related
immune-activating properties[16_19].
Hericium erinaceum is a widely cultivated edible
mushroom and has been used in several Asian countries to treat
various human diseases. Recently, H erinaceum
was report-ed to have cytotoxic effects on cancer cell lines, as well as
nematicidal and antimicrobial
activities[20,21]. We previously reported that a water extract of
H erinaceum (WEHE) induced the production of NO and interleukin (IL)-1-beta in rat
macrophages and a macrophage-like cell line, RAW
264.7[22,23].
In the present study, we examined the immunomodulatory
effects of WEHE on the cytolytic function of NK cells against
an MHC class I-deficient cell line, Yac-1. Our results
indicated that WEHE indirectly facilitated the cytolytic ability of
mouse splenocytes by amplifying the NK cell activity
induced by IL-12.
Materials and methods
Reagents and chemicals 51Cr-source was obtained from
PerkinElmer (Boston, MA, USA), and lipopolysaccharide
(LPS, Salmonella typhosa) was obtained from Sigma_Aldrich
(St Louis, MO, USA). All RT-PCR reagents were purchased
from Bio-Rad (Hercules, CA, USA). Mouse IL-12 and the
anti-mouse IL-12 rat antibody were purchased from eBioscience (San Diego, CA, USA).
β-D-(1,3)-(1,6)-glucan was obtained from VP GmbH (Hergestellt, Germany).
Extraction, composition analysis, and fingerprinting of
WEHE Dried H erinaceum was obtained from the Korean
Mushroom Corporation (Pochon, Korea). The mushrooms
(100 g) were washed 3 times with pyrogen-free water, then
soaked in 1.5 L pyrogen-free water for 2 h and boiled for 2 h.
Solid particles and aggregates were removed by
centrifugation at 3000×g for 30 min, and the supernatant was lyophilized.
A total of 26.35 g lyophilized water extract was obtained for
use in this experiment. According to the Association of
Analytical Communities[24], the general chemical composition of
WEHE is crude protein (44.82%), carbohydrate (27.63%),
crude ash (16.84%), moisture (9.05%), crude fiber (0.94%),
and crude fat (0.72%). For additional quality control of the
tested samples, high-performance-thin layer
chromatography (HPTLC)-based fingerprinting was performed using the
CAMAG Application System (Muttenz, Switzerland) as
follows. WEHE and β-D-(1,3)-(1,6)-glucan were dissolved in
90% HPLC-grade methanol and applied to a pre-washed silica
gel 60 F254 HPTLC plate (10×10 cm, 0.2 mm thick silica gel,
Merck, Darmstadt, Germany) with an automated applicator
(Linomat IV, CAMAG, Muttenz, Switzerland). The samples
were then separated (migration distance: 60 mm) using
HPLC-grade n-butanol/methanol/water (50:25:20). Thereafter,
glucose-specific staining with
aniline-diphenylamine-phosphoric acid or protein-specific staining with ninhydrin reagent
was separately performed. Because the β-D-(1,3)-(1,6)-glucan
preparation used in our experiment was likely to have
proteins, bands with the same Rf value in
β-D-(1,3)-(1,6)-glucan were stained with both reagents. The developed plate
was visualized at 254 nm using a Reprostar 3 Digital Camera
System (CAMAG; Figure 1A_1D).
Preparation of splenocytes and purified NK cells, and
fluorescence-activated cell sorter analysis The spleens were
removed aseptically from BALB/c mice and homogenized
with an iron mesh and syringe plunge. The red blood cells
were lysed by adding lysis buffer (0.15 mol/L ammomium
chloride) to the cell pellet and washed with phosphate
buffered saline (PBS). The single-cell suspension was used for
NK cell isolation or cultured in RPMI-1640 medium (JBI,
Daegu, Korea) containing 10% fetal bovine serum (FBS, JBI,
Korea) without antibiotics for use in other assays. NK cell
isolation was performed by magnetic cell separation (MACS)
following the manufacturer's instructions (Miltenyi Biotec,
Bergisch Gladbach, Germany). Briefly, the splenocytes were
labeled with CD49B (DX5) microbeads and then loaded onto
a MACS column to capture the NK cells. The
magnetically-retained NK cells were eluted as a positively-selected cell
fraction. The NK cells and NK cell-depleted splenocytes
were then used for 51Cr release assays. To evaluate the
efficacy of the NK cell selection, fluorescence-activated cell
sorter (FACS) analysis was performed with anti-CD3-PE,
B220-PE, and anti-DX5 FITC antibody labeling (BD Pharmingen, San Diego, CA, USA).
Culture of Yac-1 cells and radio-labeling
Yac-1 cells were cultured in RPMI-1640 medium supplemented with 10%
FBS without antibiotics at 37 oC in a humidified incubator
with 5% CO2. To test for NK cell-derived cytolytic activity,
4×106 Yac-1 cells were labeled with 100 µCi
51Cr by incubation for 2 h at 37
oC in a humidified incubator set with 5%
CO2. After washing twice with RPMI-1640 medium
containing 10% FBS, the cells were resuspended in 10 mL RPMI-
1640 medium supplemented with 10% FBS for use as target
cells.
51Cr release assay with splenocytes or NK cells
The 51Cr release assay was performed as previously described
with slight modifications[25]. Briefly, the total splenocytes,
isolated NK cells, or NK cell-depleted splenocytes were
prepared in RPMI-1640 medium as effector cells. Aliquots (100
µL) of each cell suspension were plated onto round-bottom
96-well plates (3 wells per group), with 50 µL WEHE at
various concentrations (0, 1, 10, or 100 mg/L) or LPS (0.1 mg/L),
with or without IL-2 (300 U/mL), and incubated for 20 h at
37 oC in a humidified incubator with 5%
CO2. Thereafter, 50 µL target cells
(1×104 cells) were mixed with the effector cells
(5×105 or 1×106 spleen cells or NK cell-depleted splenocytes,
and 5×104 or 1×105
isolated NK cells) and incubated for an additional 4 h. The maximum-release groups were induced
by adding 50 μL 2% NP-40, and the spontaneous-release
groups were induced by adding 150 μL complete medium.
Gamma irradiation from each well was then assessed using a
scintillation counter (Packard Instruments, Meriden, CT,
USA). Cytotoxic activity was defined as the percentage of
specific 51Cr released using the following equation:
Specific lysis (%)=(RAexperimental-RAspontaneous
)/(RAmaximal-RAspontaneous)×100.
RA =radioactivity
In addition to the above procedure, 2000 pg/mL anti-IL-12
(p40) neutralizing antibody was added to the splenocytes to
confirm the effect of WEHE on IL-12-mediated NK cell
activation.
RT-PCR for the analysis of IL-12 The total RNA was
extracted from splenocytes treated for 12 h with various
concentrations of WEHE (0, 1, 10, or 100 mg/L) or LPS (0.1 mg/L)
using Trizol (Invitrogen, Carlsbad, CA, USA) and RNeasy
columns (Qiagen, Valencia, CA, USA). cDNA was
synthesized using 10 pmol oligo dT and 10 pmol random hexamer
(Bioneer, Daejeon, Korea). After the cDNA synthesis,
RT-PCR was performed using the following primers (forward and
reverse, respectively). β-actin:
5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGATTTC-3';
IL-12 (p40): 5'-TCA GGG GAA CTG CTA CTG CT3' and
5'-TGA CAC GCC TGA AGA AGATG-3'. The reactions were
performed with 0.2 μL Go Taq DNA polymerase (5 U/mL;
Promera, Madison, Wisconsin, USA), 1 μL 10 pmol primer
pairs, 3 μL 10 mmol dNTP, 6 μL 5x reaction buffer, 19.8
μL distilled water, and 1 μL cDNA. PCR was performed for 27
and 40 amplification cycles for β-actin and IL-12 (p40),
respectively, under the following conditions: initial
denaturation at 95 oC for 5 min, denaturation for each cycle at 95
oC for 1 min, annealing at 58
oC for 40 s, and elongation at 72
oC for 40 s.
ELISA analysis for IL-12 and IFN-gamma The
splenocytes (3×107 cells) were plated on 24-well plates and
pre-cultured for 4 h before being incubated with various
concentrations of WEHE (0, 1, 10, or 100 mg/L) or LPS (0.1 mg/L)
for 12 or 24 h in RPMI-1640 medium containing 10% FBS.
The cell-free supernatant was collected and used for
measuring the concentration of released IL-12 (p40) and
IFN-gamma using an ELISA assay kit according to the manufacturer's
instructions (BD Biosciences, San Jose, CA, USA). Briefly,
a 96-well microplate was coated with a capture antibody by
incubating overnight at room temperature, then washed,
blocked, and rewashed. The samples and standards were
added to the plate and incubated for 2 h at room temperature.
After adding streptavidin-horseradish peroxidase and
mixing the substrate solution, the plate was read at 450 nm and
560 nm (ie, the reference wavelengths) using a microplate
reader (Molecular Devices, Union City, CA, USA).
Results
WEHE activates NK cells among splenocytes to lyse
Yac-1 cells Yac-1, a NK cell-sensitive target cell
line[26], was labeled with
51Cr to examine the effect of WEHE on NK cell
cytotoxicity using BALB/c splenocytes. The splenocytes
treated for 20 h with 1, 10, or 100 mg/L WEHE or 0.1 mg/L LPS
as a positive control were co-cultured with
51Cr-labeled Yac-1 cells for 4 h. Upon exposure of the cells to WEHE, the
cytolytic activity of the splenocytes was significantly
increased in a dose-dependent manner (P<0.01, Figure 2A).
A synergic effect was observed following co-treatment with
WEHE and IL-2 as expected (P<0.05 or 0.01, Figure 2B). We
then investigated whether the WEHE-driven cytolytic effect
was caused by NK cells and not by other cellular
components within the spleen. Thus, we repeated the assay with
NK cell-depleted splenocytes, and found no enhancement
of cytotoxicity following treatment with WEHE (Figure 2C).
Those results showed that WEHE-activated NK cells among
the splenocytes were responsible for lysis of the Yac-1 cells.
WEHE indirectly activates NK cells to kill Yac-1 cells
Since WEHE stimulated splenocyte-derived NK cells to lyse
Yac-1 cells, we next examined whether WEHE could directly
activate NK cells using NK cells purified from splenocytes
by MACS (Figure 3A). Interestingly, regardless of whether
the NK cells were treated with WEHE alone or in
combination with IL-2, no enhancement of NK cell cytolytic activity
was observed (Figure 3B, 3C). This result indicated that
WEHE activated NK cells indirectly via the induction of other
immuno-mediators or cellular components.
Treatment with anti-IL-12 antibody reduces WEHE-
induced cytolytic activity towards Yac-1 cells
The above results led us to hypothesize that WEHE generates the
cytolytic activity of NK cells by inducing IL-12 production,
which is the main activator of NK
cells[27]. We found that
IL-12-treated splenocytes were strongly activated to
lyse Yac-1 cells, but this activity was abolished by co-treatment
with a neutralizing antibody against IL-12
(P<0.01, Figure 4A). We also observed a significant decrease of
WEHE-derived cytolytic activity in splenocytes by treatment with
an anti-IL-12 antibody as expected (P<0.05, Figure 4B). These
results strongly suggest the involvement of IL-12 in the
WEHE-derived augmentation of NK cell activity.
WEHE induces the transcription and translation of
IL-12 and IFN-gamma in splenocytes Based on the above
results indicating that IL-12 mediates the activation of NK
cells by WEHE, we examined the changes in the expression
of IL-12 (p40) and protein production in splenocytes treated
with WEHE for 12 h. In addition, we measured the
production of IFN-gamma in WEHE-treated splenocytes. WEHE
treatment increased the expression of the IL-12 (p40) in
splenocytes in a dose-dependent manner (Figure 5A). In
addition, the production of the IL-12 (p40) and IFN-gamma
proteins increased in a dose-dependent manner at 12 and
24 h (P<0.05 and 0.01, respectively, Figure 5B, 5C). No
increase in IL-12 p70 production was observed under the same
experimental condition (data not shown).
Discussion
NK cells are an important part of the innate immune
system and efficiently eliminate cancer cells in the body. Thus,
the development of NK cell-based immunomodulating agents
with minimal side effects could be very important for treating
cancer patients[28_30]. Here, we report that WEHE enhances
NK cell activity in mouse splenocytes. In addition, WEHE
induces the expression of IL-12, which efficiently activates
the cytolytic ability of NK cells.
The increase in NK cell cytolytic activity appears to be
an indirect effect of WEHE. Indeed, isolated NK cells treated
with WEHE do not exhibit increased cytolytic activities
towards Yac-1 cells, although a significant increase in NK
cell activity by WEHE was observed in splenocytes
containing mixed immune cells. It is possible that mediators
positively influence NK cells. For example, the cytokine
IL-12 may mediate the WEHE-derived activation of NK cells,
since IL-12 remarkably increased the cytotoxic activities of
NK cells. In addition, when WEHE plus an anti-IL-12
neutralizing antibody were co-administered to splenocytes,
Yac-1 cell lysis was significantly attenuated. Moreover, IFN-gamma,
whose expression is strongly induced by IL-12, was highly
expressed in splenocytes cultured with WEHE. These
results support previous data showing that IL-12 and
IFN-gamma are important anticancer cytokines with
anti-angiogenic and antimetastatic
effects[31_34].
Although we found that IL-12 was involved in the
activation of NK cells, we did not identify which cell types among
the splenocytes preferentially respond to WEHE.
Spleno-cytes include various cell types, including B cells, T cells,
macrophages, NK cells, and dendritic cells. Previous
studies have shown that IL-12 is largely produced by activated
antigen presenting cells, such as dendritic cells, B cells, and
macrophages[35]. Thus, future studies are needed to identify
which cells respond to WEHE, leading to the production of
cytokines, including IL-12, that subsequently enhance NK
cell activity. It remains to be clarified how WEHE controls
the expression of IL-12. Even though IL-12 is comprised
with two distinct subunits, p35 and p40, we here verified the
induction of IL-12 as only p40 at the gene and protein levels.
So, further study should be performed for the expression of
p35 and p70).
Despite these unresolved matters, the enhanced NK cell
cytotoxicity and production of IL-12 and IFN-gamma
suggest that WEHE may be a valuable immunomodulating agent
for cancer patients. We previously reported that
approximately 28% (w/w) of WEHE was composed of
polysac-charides, mainly β-glucan, that participate in the induction
of NO and IL-1-beta expression via increased NF-κB binding
activity in rat macrophages and RAW 264.7
cells[22,23]. Similarly, several studies have shown that
mushroom-derived fractions or polysaccharides activate NK cell function
and cytokine production[37,37]. One group reported an
increase in the activity and number of NK cells, including a
high IFN-gamma plasma concentration following a 12-week
treatment with Gano-derma lucidum polysaccharide extract
in advanced cancer patients[38].
Taken together, the current data indicate that WEHE has
an inductive effect on splenocyte-derived NK cell activation
leading to cytolysis of Yac-1 cells. We also verified that the
underlying mechanism of the immunomodulatory effect of
WEHE on splenocytes involves the stimulation of IL-12
production. This study enhances our understanding of the
cancer-related biological properties of H erinaceum
and supports its clinical applications as an immunomodulator.
References
1 Hayakawa Y, Smyth MJ. Innate immune recognition and
suppression of tumors. Adv Cancer Res 2006; 95: 293_322.
2 Dewan MZ, Terunuma H, Ahmed S, Ohba K, Takada M, Tanaka
Y, et al. Natural killer cells in breast cancer cell growth and
metastasis in SCID mice. Biomed Pharmacother 2005; 59:
S375_9.
3 Waller EK. Cellular immunotherapy and cancer. Semin Oncol
2004; 31: 87_90.
4 Smyth MJ, Hayakawa Y, Takeda K, Yagita H. New aspects of
natural-killer-cell surveillance and therapy of cancer. Nat Rev
Cancer 2002; 2: 850_61.
5 Takaki R, Hayakawa Y, Nelson A, Sivakumar PV, Hughes S,
Smyth MJ, et al. IL-21 enhances tumor rejection through a
NKG2D-dependent mechanism. J Immunol 2005; 175:
2167_73.
6 Liebau C, Merk H, Schmidt S, Roesel C, Karreman C, Prisack JB,
et al. Interleukin-12 and interleukin-18 change ICAM-I
expres-sion, and enhance natural killer cell mediated cytolysis of human
osteosarcoma cells. Cytokines Cell Mol Ther 2002; 7: 135_42.
7 Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari
MC, et al. Activating receptors and coreceptors involved in
human natural killer cell-mediated cytolysis. Annu Rev Immunol
2001; 19: 197_223.
8 Hayakawa Y, Takeda K, Yagita H, Smyth MJ, Van Kaer L, Okumura
K, et al. IFN-gamma-mediated inhibition of tumor angiogenesis
by natural killer T-cell ligand, alpha-galactosylceramide. Blood
2002; 100: 1728_33.
9 Boyiadzis M, Foon KA. Natural killer cells: from bench to
cancer therapy. Expert Opin Biol Ther 2006; 6: 967_70.
10 Sentman CL, Barber MA, Barber A, Zhang T. NK cell receptors
as tools in cancer immunotherapy. Adv Cancer Res 2006; 95:
249_52.
11 Guven H, Gilljam M, Chambers BJ, Ljunggren HG, Christensson
B, Kimby E, et al. Expansion of natural killer (NK) and natural
killer-like T (NKT)-cell populations derived from patients with
B-chronic lymphocytic leukemia (B-CLL): a potential source
for cellular immunotherapy. Leukemia 2003; 17: 1973_80.
12 Liou CJ, Huang WC, Tseng J. Short-term oral administration of
ginseng extract induces type-1 cytokine production.
Immuno-pharmacol Immunotoxicol 2006; 28: 227_40.
13 Brousseau M, Miller SC. Enhancement of natural killer cells and
increased survival of aging mice fed daily
Echinacea root extract from youth. Biogerontology 2005; 6: 157_63.
14 Li Q, Nakadai A, Matsushima H, Miyazaki Y, Krensky AM,
Kawada T, et al. Phytoncides (wood essential oils) induce human
natural killer cell activity. Immunopharmacol Immunotoxicol
2006; 28: 319_33.
15 Ishikawa H, Saeki T, Otani T, Suzuki T, Shimozuma K, Nishino
H, et al. Aged garlic extract prevents a decline of NK cell number
and activity in patients with advanced cancer. J Nutr 2006;136:
S816_20.
16 Kodama N, Asakawa A, Inui A, Masuda Y, Nanba H.
Enhancement of cytotoxicity of NK cells by D-Fraction, a
polysaccharide from Grifola frondosa. Oncol Rep 2005; 13: 497_502.
17 Sarangi I, Ghosh D, Bhutia SK, Mallick SK, Maiti TK.
Anti-tumor and immunomodulating effects of Pleurotus ostreatus
mycelia-derived proteoglycans. Int Immunopharmacol 2006; 6:
1287_97.
18 Kim GY, Lee JY, Lee JO, Ryu CH, Choi BT, Jeong YK,
et al. Partial characterization and immunostimulatory effect of a novel
polysaccharide-protein complex extracted from Phellinus
linteus. Biosci Biotechnol Biochem 2006; 70: 1218_26.
19 Jia LM, Liu L, Dong Q, Fang JN. Structural investigation of a
novel rhamnoglucogalactan isolated from the fruiting bodies of
the fungus Hericium erinaceus. Carbohydr Res 2004; 339:
2667_71.
20 Mizuno T, Wasa T, Ito H, Suzuki C, Ukai N.
Antitumor-active polysaccharides isolated from the fruiting body of
Hericium erinaceum, an edible and medicinal mushroom called yamabushitake
or houtou. Biosci Biotechnol Biochem 1992; 56: 347_8.
21 Stadler M, Mayer A, Anke H, Sterner O. Fatty acids and
other compounds with nematicidal activity from cultures of
Basidiomycetes. Planta Med 1994; 60: 128_32.
22 Son CG, Shin JW, Cho JH, Cho CK, Yun CH, Chung W,
et al. Macrophage activation and nitric oxide production by water soluble
components of Hericium erinaceum. Int Immunopharmacol 2006;
6: 1363_9.
23 Son CG, Shin JW, Cho JH, Cho CK, Yun CH, Han SH. Induction
of murine interleukin-1 beta expression by water-soluble
components from Hericium erinaceum. Acta Pharmacol Sin 2006; 27:
1058_64.
24 Association of Analytical Communities
(AOAC). Official methods of analysis of the Association of Official Analytical Chemists.
15th ed. Washington DC: Association of Official Analytical
Chemists; 1990.
25 Langhans B, Ahrendt M, Nattermann J, Sauerbruch T, Spengler
U. Comparative study of NK cell-mediated cytotoxicity using
radioactive and flow cytometric cytotoxicity assays. J Immunol
Methods 2005; 306: 161_8.
26 Piontek GE, Taniguchi K, Ljunggren HG, Gronberg A, Kiessling
R, Klein G, et al. YAC-1 MHC class I variants reveal an
association between decreased NK sensitivity and increased H-2
expression after interferon treatment or in vivo
passage. J Immunol 1985; 135: 4281_8.
27 Miller G, Lahrs S, Dematteo RP. Overexpression of
interleukin-12 enables dendritic cells to activate NK cells and confer
systemic antitumor immunity. FASEB J 2003; 17: 728_30.
28 Ahn WS, Kim DJ, Chae GT, Lee JM, Bae SM, Sin JI,
et al. Natural killer cell activity and quality of life were improved by
consumption of a mushroom extract, Agaricus blazei
Murill Kyowa, in gynecological cancer patients undergoing
chemo-therapy. Int J Gynecol Cancer 2004; 14: 589_94.
29 Jensen GS, Hart AN. Immunomodulation by SanPharma fungal
metabolic products. J Altern Complement Med 2006; 12:
409_16.
30 Farag SS, VanDeusen JB, Fehniger TA, Caligiuri MA. Biology and
clinical impact of human natural killer cells. Int J Hematol
2003; 78: 7_17.
31 Siddiqui F, Ehrhart EJ, Charles B, Chubb L, Li CY, Zhang X,
et al. Anti-angiogenic effects of interleukin-12 delivered by a novel
hyperthermia induced gene construct. Int J Hyperthermia 2006;
22: 587_606.
32 Shao X, Liu C. Influence of IFN-alpha and IFN-gamma on
lymphangiogenesis. J Interferon Cytokine Res 2006; 26:
568_74.
33 Pulaski BA, Smyth MJ, Ostrand-Rosenberg S.
Interferon-gamma-dependent phagocytic cells are a critical component of innate
immunity against metastatic mammary carcinoma. Cancer Res
2002; 62: 4406_12.
34 Rodrigues EG, Garofalo AS, Travassos LR. Endogenous
accumulation of IFN-gamma in IFN-gamma-R(-/-) mice increases
resistance to B16F10-Nex2 murine melanoma: a model for direct
IFN-gamma anti-tumor cytotoxicity in vitro and
in vivo. Cytokines Cell Mol Ther 2002; 7: 107_16.
35 Carra G, Gerosa F, Trinchieri G. Biosynthesis and
posttranslational regulation of human IL-12. J Immunol 2000; 164:
4752_61.
36 Zhong M, Tai A, Yamamoto I. In vitro
augmentation of natural killer activity and interferon-gamma production in murine spleen
cells with Agaricus blazei fruiting body fractions. Biosci
Biotechnol Biochem 2005; 69: 2466_9.
37 Kodama N, Harada N, Nanba H. A polysaccharide, extract
from Grifola frondosa, induces Th-1 dominant responses in
carcinoma-bearing BALB/c mice. Jpn J Pharmacol 2002; 90:
357_60.
38 Gao Y, Zhou S, Jiang W, Huang M, Dai X. Effects of ganopoly
(a Ganoderma lucidum polysaccharide extract) on the immune
functions in advanced-stage cancer patients. Immunol Inves
2003; 32: 201_15. |
