Enzyme and Microbial Technology 40 (2006) 177185

Ganoderma lucidum inhibits tumour cell proliferation

and induces tumour cell death
Yi-Zhen Xie a,1 , Sen-Zhu Li a,1 , Albert Yee c , David P. La Pierre c,d , Zhaoqun Deng c ,
Daniel Y. Lee c,d , Qing-Ping Wu a , Qi Chen a , Chong Li a , Zhi Zhang a ,
Jun Guo a , Zide Jiang b , Burton B. Yang c,d,
a Guangdong Institute of Microbiology, Guangdong Academy of Sciences, 100 Central Xian-Lie Road, Guangzhou, China
b Department of Plant Pathology, South China Agricultural University, 483 Wu-Shan Road, Guangzhou, China
c Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Toronto, Canada M4N 3M5
d Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ont., Canada

Received 18 March 2005; received in revised form 8 October 2005; accepted 11 October 2005

Abstract
Ganoderma lucidum, a traditional Chinese medicinal fungus, has been a favourite remedy in oriental medicine for centuries. The objective
of this study is to analyze whether G. lucidum affects cancer cell proliferation and cell death. Malignant human breast carcinoma cells were
used in our studies. Different preparations of G. lucidum spores were added to the cancer cells at a final concentration of 1 mg/ml followed by
incubation of the cultures for two days. Treatment with G. lucidum resulted in tumour cells detachment from the tissue culture plates and death.
The proliferation of the adherent cells was also inhibited. The experiments indicated that the inhibitory effects of G. lucidum on cancer cell growth
were sporoderm-broken spores (broken by enzymatic method) > sporoderm-broken spores (broken by physical method) > intact spores > buffer
control. The polysaccharides isolated from G. lucidum cultivated with wood-logs exhibited the greatest inhibitory effect on cell proliferation,
which was concentration-dependent. These results were confirmed by trypan blue staining and MTT assay. The inhibitory effect of G. lucidum on
cell proliferation appeared to occur through the Erk pathway: The expression of Erk was reduced in the presence of G. lucidum polysaccharide.
2006 Elsevier Inc. All rights reserved.

Keywords: Polysaccharide; Spore; Proliferation; Cancer; Erk activation

1. Introduction are G. lucidum polysaccharides, ganoderic acid (triterpene), and

Ganoderma lucidum is a favourite remedy in oriental
medicine for centuries. Its fruiting body is called Lingzhi in
China and Reishi in Japan. For hundreds of years, this mush-
room has been regarded as a traditional Chinese medicine, or a
folk medicine, used for the prevention and treatment of many
human diseases. The major bioactive components in G. lucidum
Abbreviations: DMEM, Dulbeccos modified Eagles medium; FBS, fetal
bovine serum; HBSS, Hanks balanced salt solution; PAGE, polyacrylamide gel
electrophoresis; MTT assay, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenly
tetrazolium bromide] assay; Erk, extracellular regulated kinase
Corresponding author at: Research Building, Sunnybrook Sciences Centre,
2075 Bayview Avenue, Toronto, Canada M4N 3M5. Tel.: +1 416 480 5874; fax:
+1 416 480 5737.
E-mail address: burton.yang@sri.utoronto.ca (B.B. Yang).
1 The first two authors contributed equally to this study.
adenosine. While the polysaccharides are the major source of its
biological activity and therapeutic use [15], ganoderic acid pos-
sesses interesting anti-tumour and anti-HIV-1 activities [6,7]. To
date, more than 100 species of oxygenated triterpenes have been
isolated from this fungus, many of which have been identified
only in this species [8]. There have been a few reports on bio-
logical activities including inhibition of histamine release [9],
immunomodulatory activity [10], cytokine production [11], and
differentiation-inducing activity [12].
Traditionally, the fruiting body has been the only part of G.
lucidum used for medical purposes. However, it has recently
been recognized that the spores of G. lucidum are more potent
[13]. As a result of their containing some unique compo-
nents, the spores have been shown to be much more effective
in disease treatment. Although the sporoderm-broken spores
are more effective than the intact spores, there are far fewer
publications on the roles of sporoderm-broken spores, and

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.10.051
178 Y.-Z. Xie et al. / Enzyme and Microbial Technology 40 (2006) 177185

the polysaccharides in the spores have been less studied due
to the difficulty in breaking the sporoderm [14]. With the
improvement of cultivation techniques, it has become possible
to harvest spores. We have recently developed an enzymatic
method to digest the sporoderm and obtain a large quantity of
sporoderm-broken spores of G. lucidum, and we have isolated
the polysaccharides from different types of fruiting bodies of
G. lucidum.
This study was designed to investigate the roles of the
sporoderm-broken spores on the growth of human tumour cells.
We demonstrated that different preparations of G. lucidum
spores were able to inhibit cancer cell proliferation and induced
cell death. Nevertheless, the sporoderm-broken spores produced
greater effects than the intact spores. Furthermore, sporoderm-
broken spores generated by enzymatic methods produced the
best results. Interestingly, the polysaccharides isolated from
G. lucidum cultivated from wood-logs exhibited the greatest
inhibitory effect on cell proliferation and cell death as com-
pared with polysaccharides isolated from other sources. The
inhibitory effect of G. lucidum on cell proliferation appeared
to occur through the Erk pathway.
2.3. Trypan blue staining
Cells were seeded on 12-well tissue culture plates at a density of
1 105 cells/well in DMEM containing 10% serum. G. lucidum products were
added to the cultures, which were maintained in a tissue culture incubator at
37 C containing 5% CO2 for two days. Cells were harvested and mixed with
trypan blue (invitrogen) in a 1:1 ratio for exclusion staining [17,18]. Dead cells
were stained as blue, while living cells were not stained by the dye due to the
presence of the intact cell membrane. The number of living cells was determined
with a Coulter counter. Each experiment was repeated three times.
2.4. MTT assay
Cell growth was determined either by direct cell counting or by MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenly tetrazolium bromide] assay. Cells were
seeded on 96 well plates at a density of 1 104 cells/well in DMEM containing
10% serum. G. lucidum products were added at different concentrations to the
cultures, which were maintained in a tissue culture incubator at 37 C containing
5% CO2 . The number of metabolically active cells was measured using an MTT
cell proliferation kit I (Roche Diagnostics, Mannheim, Germany) according to
the manufacturers instructions. Briefly, MTT labeling solution was added to the
culture medium at the end of each experiment and the cells were further incubated
for 3 h. Mitochondrial dehydrogenase activity reduced the yellow MTT dye to
a purple foramazan, which was then solubilized by overnight incubation with

2. Materials and methods

2.1. Materials

Dulbeccos modified Eagles medium (DMEM), fetal bovine serum (FBS),

Hanks balanced salt solution (HBSS), and trypsin/EDTA, were purchased from
invitrogen (Burlington, ON, Canada). The ECL Western blot detection kit was
from Amersham. Antibodies against Erk and -actin were from Santa Cruz
Biotechnology. Horseradish peroxidase-conjugated goat anti-mouse IgG sec-
ondary antibody was from Sigma (St. Louis, MO). Tissue culture plates were
from Nunc Inc. All chemicals were from Sigma. Jurkat cells were obtained from
the American Type Culture Collection (Rockville, MD).
High quality and purity of G. lucidum spores and the fruiting body of G.
lucidum were identified and selected for use by the experts from the Center for
Research and Development of Edible Fungi, Guangdong Institute of Microbi-
ology, Guangdong Academy of Sciences. The sporoderm of G. lucidum spores
were broken using physical methods by grinding the spores with an ultra smash-
ing machine or enzymatic methods. The enzymatic method was conducted by
inoculating the heat-treated G. lucidum spores with G. lucidum mycelia, which
were obtained from germinating spores. The mycelia released different types of
enzymes which digested the sporoderm of the spores. In this way, the bioactive
components were slowly released.
Fig. 1. The effect of different G. lucidum products on cell growth. Human malig-
The polysaccharide of G. lucidum was extracted from the fruiting body using
nant breast carcinoma cells (MT-1), maintained as monolayer cultures on tissue
hot water, concentrated and powdered. These procedures were performed at the
culture plates in 1 ml medium containing 10% FBS, were used in our studies. A
Center for Research and Development of Edible Fungi, Guangdong Institute of
large number of G. lucidum products were commercially obtained in Toronto.
Microbiology.
The G. lucidum products were suspended in PBS (20 mg/ml), followed by incu-
Commercial products of G. lucidum were either purchased from the Greater
bation at 100 C for 60 min. We examined the potential of different G. lucidum
Toronto Area or provided by Guangdong Yue-Wei Edible Fungi Technology Co.
products to inhibit tumour cell growth. G. lucidum products of equal concen-
Ltd.
tration (1 mg/ml) were added to the breast cancer cells and the cultures were
maintained in the same conditions. Each bar represents one commercial prod-
2.2. Cell counting uct. Control (ctrl) was buffer vehicle alone: (1) Polysaccharides isolated from
fruiting body cultivated on logs of wood; (2) Lucid Ganoderma from M&A Phar-
Cells were maintained in DMEM containing penicillin, streptomycin, and maceutical Factory Co Ltd., Japan; (3) Wild-Lingzhi from Well Herbs Nutrition
10% fetal bovine serum (FBS). The cells were seeded on 12-well tissue culture Ltd., Fendalton Christchurch, New Zealand; (4) Arashi Kuni Dual-Breakage
plates at a density of 1 105 cells/well in DMEM containing 10% serum. The Ganoderma Spores from Natural Corporation Ltd., Hong Kong; (5) The Origi-
isolated components, total extract of sporoderm-broken spores of G. lucidum nal Premium Lingzhi from PuraPharm International Ltd., Hong Kong; (6) Vita
were added to the cultures at different concentrations (02 mg/ml). The buffer Green Lingzhi from Vita Green Pharmaceutical Ltd., Hong Kong; (7) Mikei
(PBS) used to dissolve the products of G. lucidum served as a control. The Reishi from Nikkei Co, Sawa-Gun, Gumma-Ken, Japan; (8) Japan Ganoderma
cultures were maintained in a tissue culture incubator at 37 C containing 5% Lucidum Powder from Tokyo Herb Co Ltd., Tokyo, Japan; (9) Lingzhi Mas-
CO2 for two days. Cells were harvested and the cell number was determined ter from Care & Health Ltd., Hong Kong; (10) Extra Strength Lingzhi Craked
with a Coulter counter as described [15,16]. Spores from Eu Yan Sang Ltd., Hong Kong.
 Y.-Z. Xie et al. / Enzyme and Microbial Technology 40 (2006) 177185 179

MTT solubilizing reagent (10% SDS in 0.01 HCl) and absorbance was read at
595 nm on an enzyme linked immunosorbent assay (ELISA) plate reader (Bio-
Tek Instruments Inc.).
2.5. Protein extraction and Western blot analysis
After being treated with polysaccharide, the cells were washed twice with
ice-cold phosphate buffer saline (PBS) and lysed with RIPA buffer (50 mM
TrisHCl, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate and 0.1% SDS)
containing 1 mM DTT, 1 mM Na3 VO4 , 5 mM NaF, 100 mM EDTA, 10 mg/ml
aprotinin, and 100 mM PMSF. Protein samples were subjected to SDS-PAGE
on separating gels containing 10% polyacrylamide in reducing loading dye
(1x) containing 50 mM TrisCl, pH 7.2, 2% SDS, 10% glycerol, and 0.02%
bromophenol blue. The buffer system was 1x-Tris/glycine buffer (Amresco)
containing 1% SDS. Separated proteins were transblotted onto a nitrocellulose
membrane (Bio-Rad) in 1x TG buffer (Amresco) containing 20% methanol.
The membrane was blocked in TBST (10 mM TrisCl, pH 8.0, 150 mM NaCl,
0.05% Tween 20) containing 10% non-fat dry milk powder (TBSTM) for 1 h at
room temperature, and then incubated at 4 C overnight with primary antibody,
prepared in TBSTM. The membranes were washed with TBST (3 30 min) and
then incubated for 1 h in TBSTM with goat anti-mouse secondary antibody con-
jugated to horseradish peroxidase. After washing as above, the bound antibody
was visualized with an ECL kit according to the manufacturers instructions.
The Western blot analysis technique is routinely used in our laboratory [1922].
2.6. Statistical analysis
Differences among treatment means were determined by Students t-test.
P < 0.05 was considered significant.
3. Results and discussion
3.1. G. lucidum products inhibit cancer cell growth
Products of the micro-organism G. lucidum are known to
help in improving conditions of cancer patients [23]. G. lucidum
is widely taken by patients with different types of cancers in
advanced stages, when the cancers become too advanced for
surgery, chemotherapy, or radiotherapy, especially in oriental
countries. For those who have gone through one of these ther-
apeutic treatments, G. lucidum is used as a dietary supplement
for an alternative therapy. The outcomes have been encourag-
ing. In clinical studies at the Sloan-Kettering Cancer Center, it
was reported that applications of G. lucidum should be studied
and considered for (1) chemoprophylaxis of cancer in individ-
uals at high risk for developing cancer; (2) adjuvent use in the
prevention of metastasis or recurrence of cancer; (3) palliation
of cancer-related cachexia and pain; and (4) adjunctive use with
concurrent chemotherapy to reduce side-effects, maintain leuko-
cyte counts, and allow a more optimal dosing of chemo- or
radiotherapeutics. We hypothesize that G. lucidum improves the
health of cancer patients by modulating the activities of cancer
cells. The objective of this study is to analyze whether various
preparations of G. lucidum affect cancer cell proliferation and
cancer cell death.
We have thus developed a model of cancer cell culture to
study the effect of G. lucidum on mediating cell activities. Using
human breast malignant carcinoma cells MT-1 [24], maintained
as monolayer cultures on tissue culture plates, we examined a
large number of G. lucidum products obtained commercially.
These products were imported from China (Mainland and Hong
Kong), Japan, the United States, and New Zealand or produced
in Canada. The G. lucidum products were suspended in PBS
(20 mg/ml), followed by incubation at 100 C for 60 min. The
preparations were added to the cell cultures at the amount of
50 l/well (1 mg/ml), followed by incubation at 37 C for two
days. The addition of PBS alone served as a control. Treated

Fig. 2. The effect of sporoderm-broken spores on cancer cell growth. (A) Human malignant breast carcinoma cells (MT-1), maintained as monolayer cultures on
tissue culture plates in 1 ml medium containing 10% FBS, were used in our studies. Intact G. lucidum spores and sporoderm-broken spores were suspended in PBS
(20 mg/ml), followed by incubation at 100 C for 60 min. The supernatant and mix (the mixture of supernatant and pellet) of the preparations were added to the cell
cultures at the amount of 100 ml/well, followed by incubation at 37 C for two days. Addition of PBS alone served as a control. Treated with G. lucidum products,
some tumour cells detached from the tissue culture plates and died. The experiments indicated that the sporoderm-broken spores of G. lucidum produced greater
inhibitory effects on cancer cell growth than intact G. lucidum spores. The supernatant exhibited a lower inhibitory effect on tumour cell growth than the mixture.
(B) Statistical analysis of the growth inhibition indicated that the sporoderm-broken spores of G. lucidum produced very significant inhibitory effects on cell growth
(n = 3, ** p < 0.01).
180 Y.-Z. Xie et al. / Enzyme and Microbial Technology 40 (2006) 177185

with G. lucidum products, some tumour cells detached from the
tissue culture plates and died (Fig. 1). The experiments indicated
that the G. lucidum products exerted a direct effect on the can-
cer cells. This cellular model thus allows us to test the effect
of G. lucidum products on cancer cell activities. Our exper-
iments also indicated that all G. lucidum products had some
inhibitory effect on cancer cell growth. This provided direct
evidence of how G. lucidum products might improve the condi-
tions of cancer patients. However, different G. lucidum products
exerted very different effects on cancer cell growth. This indi-
cated that it is crucial to improve the quality of G. lucidum
products to produce a better outcome for patients taking the
products.
3.2. Effect of G. lucidum spores on cancer cell growth
latter allows slow digestion of the spores, resulting in the
release of bioactive components. Our experiments indicated
the inhibitory effects on cancer cell growth were sporoderm-
broken spores (enzymatic method) > sporoderm-broken spores
(physical method) > intact spores > buffer control (Fig. 3A),
which was statistically significant (Fig. 3B). Our study sug-
gests that breaking the sporoderm of G. lucidum spores is
essential to improve the quality of the G. lucidum products.
Furthermore, the methods used to break the sporoderm are
also important. Enzymatic treatment allows the sporoderm
to be digested and the bioactive components to be released.
However, it should be pointed out that extensive digestion
should be avoided, as it would cleave the bioactive compo-
nents.

Recent studies have indicated that the spores of G. lucidum

have better outcome in disease treatments than the fruiting body
[13]. This may be due to the presence of the glucan complex in
the spores [25]. The expended chains of sulfated glucan possess
higher anti-tumour activity [26]. As such, the sporoderm-broken
spores of Ganoderma have much higher bioactivities than the
whole spores in anti-tumour growth in an animal model [27],
since the bioactive substances in the spores are not well utilized
in vivo when the sporoderm is not broken. The whole spores
have been found in the dejecta of animals and humans who take
the whole spores during treatment [27]. When the sporoderm is
not broken, the spores have little effect on HeLa cells in vitro
[28].
Using the cellular model described above, we examined the
effect of intact spores and the sporoderm-broken spores of
G. lucidum on cancer cell growth. Human malignant breast
carcinoma cells were maintained as monolayer cultures on
tissue culture plates as above. Intact G. lucidum spores and
sporoderm-broken spores were suspended in PBS (20 mg/ml),
then incubated at 100 C for 60 min. The supernatant and mix-
ture, containing supernatant and pellet, of the preparations
were added to the cell cultures at the amount of 100 l/well,
then incubated at 37 C for two days. The addition of PBS
alone served as a control. The experiments indicated that the
sporoderm-broken spores of G. lucidum inhibited the growth
of cancer cells more than intact G. lucidum spores (Fig. 2A).
The supernatant exhibited lower inhibitory effect on tumour
cell growth than the mixture. Perhaps, the spore surface-binding
molecules exerted some inhibitory effect on tumour cell growth.
Statistical analysis of the growth inhibition indicated that the
sporoderm-broken spores of G. lucidum produced very sig-
nificant inhibitory effects on cell growth (Fig. 2B). It is not
clear whether enzymatic treatment produced some active com- Fig. 3. The effect of sporoderm-broken spores generated by different methods
on cancer cell growth. (A) Human malignant breast carcinoma cells (MT-1),
ponents inhibiting cancer cell growth. We have also observed maintained as monolayer cultures on tissue culture plates, were used in our
that G. lucidum oil fraction prepared from the enzymatic prepa- studies. Different preparations of G. lucidum spores were added to MT-1 cells at
ration of the sporoderm-broken spores exhibited greater effects a final concentration of 1 mg/ml followed by incubation at 37 C for two days.
on cell death as compared with oil prepared from the physi- Treated with sufficient concentrations of G. lucidum products, some tumour cells
cal preparation of the sporoderm-broken spores (unpublished detached from the tissue culture plates and died. (B) The growth inhibition was
analyzed statistically. The experiments indicated that the inhibitory effects of
data). G. lucidum on cancer cell growth were: sporoderm-broken spores (enzymatic
We further tested the effect of the sporoderm-broken method) > sporoderm-broken spores (physical method) > intact spores > buffer
spores generated by physical and enzymatic methods. The control. (n = 3, * p < 0.05, ** p < 0.01).
 Y.-Z. Xie et al. / Enzyme and Microbial Technology 40 (2006) 177185 181

3.3. Effect of G. lucidum polysaccharides on cancer cell
growth
It is known that the major bioactive components in G. lucidum
are polysaccharides, ganoderic acid (triterpene), and adenosine.
The polysaccharides of G. lucidum significantly improved the
immune parameters of patients with advanced cancers [23]. The
polysaccharide fraction of Ganoderma can suppress the activity
of colon cancer cells and may act as a potent chemopreventive
agent for colon carcinogenesis [29,30]. The anti-tumour activi-
ties of the polysaccharides appear to be due to the promotion of
the expression of TNF and IFN [31].
We tested if the polysaccharides could function directly in
the cancer cells. Polysaccharides were prepared from differ-
ent sources, including the fruiting body grown in wood-logs,
mycelia, and the wild type fruiting body obtained from the
mountains in South China. Human malignant breast carcinoma
cells were cultured as monolayer, to which polysaccharides of
G. lucidum were added at a final concentration of 0.5 mg/ml,
followed by incubation at 37 C for two days. The growth
inhibition was examined under a light microscope and cell
numbers were counted. The experiments indicated that the
inhibitory effects of polysaccharides of G. lucidum from dif-
ferent sources were as follows: wood log > mycelium = wild
type > buffer control (Fig. 4A) and the difference was statisti-
cally significant (Fig. 4B). The inhibitory effect of polysaccha-
rides was concentration-dependent (Fig. 4C).
It has been reported that the polysaccharides are composed
of heteroglycans and glycans, with or without a protein core
[32,33]. A similar structure of polysaccharide was also iso-
lated from mycelium of G. lucidum [34]. After repeated iso-
lation and purification through DEAE-Sephadex and DEAE-
Cellulose, two active polysaccharides, named GLMB0 and
GLMB1, were isolated from the mycelium of G. lucidum. The
structures of polysaccharides isolated from spores appear to
be similar [25,35]. Recently, a polysaccharide, named Lzps-1,

Fig. 4. The effect of polysaccharides of G. lucidum from different sources on tumour cell growth. Human breast carcinoma cells were maintained as monolayer
cultures on tissue culture plates, to which polysaccharides of G. lucidum obtained from different sources were added at a final concentration of 0.5 mg/ml, followed
by incubation at 37 C for two days. The growth inhibition was examined under a light microscope (A) and cell numbers were counted (B). The experiments indicated
that the inhibitory effects of polysaccharides of G. lucidum from different sources were as follows: wood-log (1) > mycelium (2) = wild type (3) > buffer control
(n = 3, ** p < 0.01). The inhibitory effect of polysaccharides (from wood log) was concentration-dependent (C).
182 Y.-Z. Xie et al. / Enzyme and Microbial Technology 40 (2006) 177185

Fig. 5. The inhibitory effect of polysaccharides on the growth of different cancer cells. Human breast carcinoma cells (MT-1) and human leukemia cells (Jurkat) were
maintained as monolayer cultures on tissue culture plates, to which the G. lucidum products were added at different concentrations (mg/ml), followed by incubation at
37 C for two days. These products were produced by the Center for Research and Development of Edible Fungi, Guangdong Institute of Microbiology, Guangdong
Academy of Sciences. Treated with sufficient concentrations of G. lucidum product, some tumour cells detached from the tissue culture plates and died.

Fig. 6. The polysaccharide of G. lucidum inhibits cancer cell proliferation. Human breast carcinoma cells (A) and human leukemia cells (B) were maintained as
monolayer cultures on tissue culture plates, to which the G. lucidum products were added at different concentrations (mg/ml), followed by incubation at 37 C
containing 5% CO2 for two days. The number of metabolically active cells was measured using MTT assay. Data represent means S.E.M. (n = 8 wells). The
experiment has been repeated three times and similar results were obtained (n = 3, ** p < 0.01).
 Y.-Z. Xie et al. / Enzyme and Microbial Technology 40 (2006) 177185 183

Fig. 7. The polysaccharide of G. lucidum reduces cancer cell survivability. Human breast carcinoma cells (A) and human leukemia cells (B) were maintained as
monolayer cultures on tissue culture plates, to which the G. lucidum products were added at different concentrations (mg/ml), followed by incubation at 37 C
containing 5% CO2 . Cells were harvested and mixed with trypan blue. The number of living cells was determined with a Coulter counter (n = 3, ** p < 0.01).

was obtained from G. lucidum spores through water extract.
Its molecular weight was estimated by HPGPC to be 8000. Its
structure was confirmed to be glucan. The total polysaccha-
rides, Lzps, exhibit anti-tumour activity against sarcoma 180
and Lewis lung cancer in mice and enhanced the activity of
natural killer cells. Lzps-1 is the first reported polysaccharide
obtained from G. lucidum spore Lzps that exerts anti-tumour
activity [36].
We also tested the effect of the polysaccharides on human
malignant breast carcinoma cells and human leukimia cells.
Treated with sufficient concentrations of G. lucidum polysaccha-
rides, the tumour cells detached from the tissue culture plates

Fig. 8. The polysaccharide of G. lucidum reduces Erk expression. Human breast carcinoma cells were incubated with the polysaccharide of G. lucidum suspended
in PBS (0.5 mg/ml) at 37 C for 24 h. Culture medium was centrifuged to collect cells detached from the plates. Cell lysate was prepared from the cell pellet and
cells of the monolayer using lysate buffer. Equal amounts of cell lysate were separated on SDS-PAGE and subjected to Western blot analysis probed with anti-Erk
and anti-actin (for loading control) antibodies. Cells treated with the polysaccharide expressed a lower level of Erk protein compared to the untreated cells. Little
difference was detected in actin expression between the treated and untreated cells (A). The intensity of the protein bands was scanned with a densitometer (B).
184 Y.-Z. Xie et al. / Enzyme and Microbial Technology 40 (2006) 177185
and died (Fig. 5). These results indicate that G. lucidum can
function in different types of tumours.
3.4. G. lucidum reduces cancer cell proliferation and
survival
We further tested how G. lucidum products inhibited can-
cer cell growth and induced cancer cell death, using an MTT
assay. Human malignant breast carcinoma cells and human
leukemia cells were treated with different concentrations of the
polysaccharides derived from G. lucidum fruiting body culti-
vated with wood-logs, followed by incubation at 37 C con-
taining 5% CO2 . The number of metabolically active cells was
measured using the MTT assay. The experiment indicated that
the number of metabolically active cells decreased when treated
with the polysaccharides as compared with the untreated cells
(Fig. 6). These results indicate that the polysaccharides reduced
the growth rate of cancer cells by inhibiting cell growth. We
further validated these results with trypan blue staining. Our
experiments indicated that the cancer cells exhibited a lower
rate of survivability when treated with the polysaccharides than
the untreated cells (Fig. 7). These results were consistent with
the reduced rate of growth and proliferation and increased cell
death when treated with polysaccharides.
3.5. G. lucidum modulates Erk signal pathway
Epidermal growth factor receptor (EGFR) has been docu-
mented as a prototypic receptor tyrosine kinase (RTK) which
modulates cell proliferation. The classic signalling cascade
downstream of EGFR is the extracellular regulated kinase
(Erk) pathway [37]. To examine whether G. lucidum was
able to regulate the EGFR downstream signalling pathway,
human malignant breast carcinoma cells were incubated with
the polysaccharide of G. lucidum at different concentrations
for 24 h. Cell lysate was prepared and subjected to Western
blot analysis probed with anti-Erk and anti-actin (for loading
control) antibodies. We detected strong signals of Erk expres-
sion in the control cells. Cells treated with the polysaccha-
ride expressed a lower level of Erk protein, and a maximum
reduction was observed when the cells were treated with the
polysaccharides for 24 h (Fig. 8). Little difference was detected
in actin expression between the cells treated with polysac-
charides and those not treated. These results correlated with
those of proliferation assay, suggesting that the polysaccharides
were able to regulate Erk expression and modulate this signal
pathway.
4. Conclusion
In this study, we examined the role of G. lucidum in anti-
cancer cell growth and induced cancer cell death. We demon-
strated that G. lucidum products directly modulate on tumour
cell growth and cell death. The sporoderm-broken spores of
G. lucidum produced greater inhibitory effects on cancer cell
growth than intact G. lucidum spores, and spores broken by
enzymatic methods produced the best results in the inhibition of
cancer cell growth. The polysaccharides appear to exert a major
effect on tumour cell growth, and those obtained from wood
log-cultured G. lucidum appeared to be the best. The inhibitory
effect of G. lucidum on cell proliferation is mediated, at least in
part, through the Erk signal pathway.
Acknowledgements
This work was funded by an operating grant from The Depart-
ment of Sciences and Technologies of Guangdong Province,
China, and by Grant NHP-78380 from the Natural Health Prod-
uct Directorate (Health Canada/Canadian Institutes of Health
Research Partnership), and Grant MOP-74469 from the Cana-
dian Institutes of Health Research to B.B.Y.
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