ARTICLE IN PRESS

Phytomedicine 17 (2010) 606–613

Contents lists available at ScienceDirect

Phytomedicine
journal homepage: www.elsevier.de/phymed

Effects of triterpenes from Ganoderma lucidum on protein expression profile

of HeLa cells
Q.-X. Yue a,b,1, X.-Y. Song a,b,1, C. Ma a, L.-X. Feng a, S.-H. Guan a, W.-Y. Wu a, M. Yang a, B.-H. Jiang a,
X. Liu a, Y.-J. Cui b,n, D.-A. Guo a,b,n
a
Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, PR China
b
College of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, PR China

a r t i c l e in f o a b s t r a c t

To elucidate the cytotoxicity mechanism of Garnoderma triterpenes, a chemoproteomic study using five
Keywords: purified ganoderic acids, ganoderic acid F (GAF), ganoderic acid K (GAK), ganoderic B (GAB), ganoderic
Ganoderic acids acid D (GAD) and ganoderic acid AM1 (GAAM1) was conducted. GAF, GAK, GAB, GAD and GAAM1
Cytotoxicity treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with IC50 values of
Chemoproteomic 19.5 7 0.6 mM, 15.1 70.5 mM, 20.3 70.4 mM, 17.3 7 0.3 mM, 19.8 70.7 mM, respectively. The protein
2-DE expression profiles of HeLa cells treated with each ganoderic acid at dose of 15 mM for 48 h were
Mass spectrometry checked using two-dimensional electrophoresis (2-DE). The possible target-related proteins of
ganoderic acids, i.e. proteins with same change tendency in all five ganoderic acids-treated groups
compared with control, were identified using MALDI-TOF MS/MS. Twelve proteins including human
interleukin-17E, eukaryotic translation initiation factor 5A (eIF5A), peroxiredoxin 2, ubiquilin 2, Cu/Zn-
superoxide dismutase, 14-3-3 beta/alpha, TPM4-ALK fusion oncoprotein type 2, PP2A subunit A PR65-
alpha isoform, nucleobindin-1, heterogeneous nuclear ribonucleoprotein K, reticulocalbin 1 and chain A
of DJ-1 protein were identified. Ganoderic acids might exert their cytotoxicity by altering proteins
involved in cell proliferation and/or cell death, carcinogenosis, oxidative stress, calcium signaling and
ER stress.
& 2009 Elsevier GmbH. All rights reserved.

Introduction et al., 2004). The anticancer effects of Ganoderma lucidum include

Ganoderma lucidum, also called as ‘Lingzhi’, is a medicinal
mushroom that has been used as a home remedy of traditional
Chinese medicine in Asian countries for over 2000 years and is
also popularly accepted as a dietary supplement in Western
countries (Sliva, 2004). In traditional Chinese medicine, it was
believed to have potential in preserving the human vitality and
promoting longevity. Though the mechanism of its announced
effect on human vitality and longevity is still not clear, modern
pharmacological studies did show that Ganoderma lucidum might
be useful in the prevention or treatment of a variety of diseases
including cancer, heart disease and infection (Wachtel-Galor
Abbreviations: GAF, Ganoderic acid F; GAK, Ganoderic acid K; GAB, Ganoderic
acid B; GAD, Ganoderic acid D; GAAM1, Ganoderic acid AM1; 2-DE, two-
dimensional electrophoresis; MTT, 3, [4,5-dimethylthiazol-2-yl-] diphenyltetra-
zolium bromide; IL-17E, interleukin-17E; eIF5A, eukaryotic translation initiation
factor 5A; ER, endoplasmic reticulum
n
Corresponding authors. Tel.: + 86 21 50271516; fax: + 86 21 50272223.
E-mail addresses: Janney808@sina.com (Y.-J. Cui), gda5958@163.com
(D.-A. Guo).
1
Both authors contributed equally to the work.
inhibiting tumor growth, anti-angiogenesis, antimetastasis, im-
muno-enhancement and etc (Yuen and Gohel, 2005). Among
these effects, the immuno-regulating effect of Ganoderma poly-
saccharides and the cytotoxic effect of Ganoderma triterpenes
were of particular interest (Yeung et al., 2004). In our previous
studies, we checked the immuno-regulating effects of Ganoderma
polysaccharides on mononuclear cells (Ma et al., 2008) and
thymus atrophy caused by cyclophosphamide (Ma et al., 2009). In
the present study, we focused on studying the cytotoxicity
mechanism of Ganoderma triterpenes.
The cytotoxicity of Ganoderma triterpenes were reported to
include inhibiting growth, inducing apoptosis and causing cell
cycle arrest of cancer cells (Lin et al., 2003; Yang, 2005; Kimura
et al., 2002; Min et al., 2000). While, the target-related proteins of
Ganoderma triterpenes were still not fully clear. For a compre-
hensive analysis of the molecular targets of Ganoderma triter-
penes, we used a small-scaled chemoproteomic approach to
identify their possible target-related proteins in cancer cells. One
of the categories of chemoproteomic approaches in drug research
is focused application that often involves the detailed study of a
single class of compounds with known biological activity, aiming
to identify a mechanism of action or the use of a compound series

0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2009.12.013
 ARTICLE IN PRESS
Q.-X. Yue et al. / Phytomedicine 17 (2010) 606–613
Fig. 1. Chemical structures of GAF, GAK, GAB, GAD and GAAM1.
in target validation (Hall, 2006). Thus, in the present study, five
purified ganoderic acids (Fig. 1), which are the main components
of Ganoderma triterpenes according to our previous analysis result
(Yang et al., 2007), were used to treat human cervical carcinoma
HeLa cells. Then, 2-DE system which could run six 2-DE gels at the
same time was employed to check the protein expression profiles
of HeLa cells underwent treatment of the five ganoderic acids or
solvent control. The detected proteins with significant change in
expression level under treatment of these ganoderic acids might
be considered as the possible target-related proteins of ganoderic
acids. The change of protein expression detected in proteomic
result was further confirmed using Western blotting analysis.
Materials and Methods
Materials and reagents
Ganoderic acids including ganoderic acid F (GAF), ganoderic
acid K (GAK), ganoderic B (GAB), ganoderic acid D (GAD), and
ganoderic acid AM1 (GAAM1) (Fig. 1) were isolated and purified
from fruit bodies of Ganoderma lucidum as reported before (Wang
et al., 2006; Yang et al., 2007). The purity of the ganoderic acids
was more than 98%. All reagents used in 2-DE were bought from
Bio-Rad Laboratories (Hercules, CA, USA).
Cell culture and Cytotoxicity assay
The HeLa human cervical carcinoma cell line (CCL-2) were
obtained from the American Type Culture Collection (ATCC,
Rockville, MD, USA) and cells were maintained in Minimum
essential medium (Life Technologies, Gaithersburg, MD, USA)
complemented with 2 mM L-glutamine, 1.5 g/l sodium bicarbo-
nate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate,
10% fetal bovine serum, 100 mg/ml streptomycin and 100 units/ml
penicillin (Invitrogen, Karlsruhe, Germany). The cultures were
maintained in a humidified atmosphere of 5% CO2 at 371 C. The
cells were passaged at preconfluent densities with a solution
containing 0.5 mM EDTA and 0.05% trypsin(Life Technologies).
The cytotoxity of ganoderic aicds was examined using a
calorimetric tetrazolium (MTT) assay as reported before (Liu
et al., 2005). Briefly, cells were plated in 96-well flat-bottomed
plates (Corning, Acton, MA, USA) at a density of 1  103 cells/well
in complete medium (with 10% fetal bovine serum) and attached
overnight. Then, the media were changed into fresh complete
medium (with 10% fetal bovine serum) containing various
amounts of ganoderic acids for 48 h and control wells were
treated with 0.1% DMSO solvent control. After the incubation,
607
20 ml of the dye (3, [4,5-dimethylthiazol-2-yl-] diphenyltetrazo-
lium bromide, 5 mg/ml), MTT, was added to each well and the
plates were incubated for 3 h at 371 C. Then, 100 ml of lysis buffer
(20% sodium dodecyl sulfate [SDS] in 50% N,N-dimethylforma-
mide, containing 0.4% [v:v] 1N HCL and 0.5% [v:v] 80% acetic acid)
was added to each well and incubated overnight for 16 h. At the
end of culture, cell viability was determined by measuring the
mitochondrial-dependent conversion of the yellow tetrazolium
salt MTT to purple formazan crystals by metabolically active cells.
The optical density (proportional to the number of live cells) was
assessed with a Genios Microplate Reader (Tecan, Research
Triangle Park, NC, USA) at 570 nm. Each experiment was carried
out in triplicate and results of three independent experiments
were used for statistical analysis. IC50 value (half-maximal
inhibitory concentration) was calculated using the Logit method.
2-DE, image analysis and MALDI-TOF MS/MS
2-DE, image analysis and MALDI-TOF MS/MS were carried out
similar to our previous reports (Ma et al., 2008; Yue et al., 2008a).
For sample preparation, cells were incubated for 48 h with each
ganoderic acid at dose of 15 mM (dose similar to the IC50 value of
the ganoderic acids) or solvent control (0.1% DMSO). For 2-DE,
protein sample (150 mg) from control cells or cells treated with
ganoderic acid was applied for IEF on a Protein IEF cell (Bio-Rad)
using the ReadyStrip IPG Strips, 17 cm, pH 4–7 (Bio-Rad). Then, the
strips were loaded onto constant 12% homogeneous SDS-PAGE
gels for electrophoresis using a PROTEIN II xi Multi-Cell system
(Bio-Rad). Grouped (control and five ganoderic acids-treated)
protein samples from 3 independent experiments were used for
2-DE analysis. And, for each group of protein samples, duplicate
electrophoreses were performed to ensure reproducibility. The
2-D gels were silver stained using Bio-Rad Silver Stain Plus kit
reagents (Bio-Rad). The silver-stained gels were scanned using
a Densitometer GS-800 (Bio-Rad) and then analyzed using the
PD-Quest Version 7.2 software (Bio-Rad). Comparisons were made
between protein profiles of each ganoderic acid-treated group and
control group. Quantitative analysis was performed using the
Student’s t-test and po0.05 was taken as statistically significant.
Protein spots with two fold or more increased or decreased
intensity and statistically significant in each ganoderic acid-
treated group compared with control were found. Then, among
these protein spots, those with similar change tendency in all five
ganoderic acid-treated groups compared with control were
accepted as possible target-related proteins and were then excised
from the gels. After digested with trypsin, the excised spots were
used for identification by MALDI-TOF MS/MS using an ABI 4700
Proteomics Analyzer with delayed ion extraction (Applied Biosys-
tems). All data analysis and database Searching were performed
using the MASCOT search engine (Matrix Science) against the NCBI
protein sequence database.Proteins with protein score more than
62 and best ion score (MS/MS) more than 30 were accepted.
Western blotting analysis
As reported before (Ma et al., 2009), Western blotting analysis
was conducted to confirm the change of protein expression found in
2-DE analysis. Briefly, cells were washed three times with cold TBS,
harvested using a cell scraper, and lysed in 10 volume of cold lysis
buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM
EDTA, 10% glycerol, 1 mM PMSF, 5 mg/ml Aprotinin, 5 mg/ml
Leupeptin) on ice. Lysates were centrifuged and then the super-
natant protein was denatured by mixing with equal volume of 2 
sample loading buffer and then boiling at 1001 C for 5 min. An
aliquot (containing 50 mg protein) of the supernatant was loaded
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onto a 12% SDS gel, separated electrophoretically, and transferred to
a PVDF membrane (Bio-Rad). After the PVDF membrane was
incubated with 10 mM TBS with 1.0% Tween 20 and 10% dehydrated
skim milk to block nonspecific protein binding, the membrane was
incubated with primary antibodies overnight at 41 C. The primary
antibodies used were mouse anti-eIF5A monoclonal antibody
(1:1000, BD Biosciences, San Diego, CA, USA), rabbit anti-14-3-3
beta/alpha polyclonal antibody (1:1000, Abgent, San Diego, CA, USA)
and mouse anti-actin monoclonal antibody (1:2000, Sigma). The
secondary antibodies used were HRP-conjugated goat anti-mouse
Table 1
Cytotoxicity of 48 h treatment of ganoderic acids against HeLa cells.
IgG (Sigma) or HRP-conjugated goat anti-rabbit IgG (Sigma). Blots
were then incubated with the secondary antibodies for 1h at room
temperature at a 1:5000 dilution and then visualized using
chemiluminescence (Pierce Biotechnology, Rockford, IL). Three
independent experiments were carried out.
Statistical analysis
The significance of difference between groups was determined
with the non-paired Student’s t-test (GraphPadPrism, GraphPad
Software Inc., San Diego, CA, US). For each variable, three
independent experiments were carried out. Data were given as
the mean 7SD and p o0.05 was considered significant.

No. Name IC50value (lM)

Results
1 ganoderic acid F (GAF) 19.57 0.6
2 ganoderic acid K (GAK) 15.17 0.5 Cytotoxic effects of ganoderic acids
3 ganoderic acid B (GAB) 20.37 0.4
4 ganoderic acid D (GAD) 17.37 0.3
5 ganoderic acid AM1 (GAAM1) 19.87 0.7 The IC50 values of the five ganoderic acids were shown in
Table 1. To be noted, the IC50 value of GAD was cited from our

Fig. 2. (A) The representative 2-DE images of ganoderic acids-treated HeLa cells. Differentially expressed spots were shown by the arrows. (B) The expanded region of
differentially expressed protein spots. The proteins within the circles were the differentially expressed proteins.

Table 2
Number of protein spots differentially expressed in ganoderic acid-treated group compared with control.

No. Name Number of protein spots Number of protein spots Total number of protein spots
with increased expression with decreased expression with changed expression

1 ganoderic acid F (GAF) 6 16 22

2 ganoderic acid K (GAK) 16 21 37
3 ganoderic acid B (GAB) 7 13 20
4 ganoderic acid D (GAD) 9 17 26
5 ganoderic acid AM1 9 23 32
(GAAM1)
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previous report (Yue et al., 2008a). The results indicated that

ganoderic acids exhibited moderate cytotoxicity against HeLa

compared with
Fold difference
cells.

control
2-DE of control and ganoderic acid-treated HeLa cells

3.31
0.23
2.08
0.46
0.32

0.45
0.17
0.45

0.49
0.22
0.39
0.12
Representative group of 2-D gel images for control, GAF, GAK,

3490.0 7 390.4
1939.87 326.0

677.0 7 102.6
GAB, GAD, GAAM1-treated cells are shown in Fig. 2. Each gel

452.0 7 48.6
257.7 7 52.4
435.2 7 52.5

484.0 7 57.4
120.07 22.8

278.5 7 39.4

247.0 7 51.6
252.0 7 56.4
84.8 7 15.2
Spot volume

(Mean7 SD)
resolved up to 700 protein spots. Gel maps of control and each

GAAM1
ganoderic acid-treated cells were compared with PDQUEST

(ppm)
software to identify the protein spot variations. The number of
protein spots differentially expressed in each ganoderic acid-
treated group compared with control was shown in Table 2.

compared with
Fold difference
Among these spots, 12 protein spots showed similar change
tendency in all ganoderic acids-treated groups compared with
control. Table 3 showed the results of PDQUEST analysis about the

control
average intensity values and their standard deviations of the 12

2.15
7.29
0.21
0.16

0.25
0.44

0.34
0.33

0.26
0.36
0.19
0.10
protein spots, the statistical assay results and the fold differences
between control and each ganoderic acid-treated group. The fold

3606.3 7 498.1
995.5 7 154.4
1867.57 313.1
119.7 7 28.9

269.5 7 57.1
473.5 7 63.2
394.7 7 45.4

286.7 7 57.3
232.5 7 51.9
212.07 30.5
71.7 7 15.0

130.07 30.6
The intensity values and change fold difference (P o 0.05) of the 12 possible target-related proteins in ganoderic acid-treated groups compared with control.

Spot volume
difference was represented by the ratio of the intensity value of

(Mean7 SD)
the ganoderic acid-treated group to the value of the control group.

(ppm)
These 12 protein spots were indicated by the arrowed spots in A

GAD
of Fig. 2 and the expanded plots in B of Fig. 2.

compared with
Identification of differentially expressed proteins

Fold difference
Results of MS/MS identification of the 12 differentially

control
expressed protein spots were summarized in Table 4. For each

2.02
2.10
0.45

0.31
0.31
0.49

0.48
0.49

0.42
0.34
0.13
0.50
protein identified, detailed identification data including accession
number, theoretical molecular mass, theoretical pI, protein score,

662.2 7 136.9
3385.0 7 386.0
2085.5 7 306.2

286.5 7 48.9
252.7 7 38.2

333.5 7 67.5
666.2 7 76.4

468.0 7 55.6
223.5 7 42.9
589.0 7 67.0

89.07 18.0
220.07 40.0
best ion score, percent of sequence coverage and number
Spot volume

(Mean7 SD)

of unique peptides were all shown in Table 4. The MALDI-TOF

MS/MS analysis result of spot 9, which was identified as
(ppm)
GAB

nucleobindin-1, was shown in Fig. 3 as an example.

The 12 differentially expressed protein spots could be
considered as the possible target-related proteins of ganodeirc
compared with
Fold difference

acids. Briefly, based on their biological functions, these 12

proteins could be generally classified into one of the following
control

four categories: (1) cell proliferation and/or cell death, (2)

2.33
5.96
0.23

0.43
0.27
0.14
0.32
0.34

0.25
0.37
0.14
0.50

carcinogenosis, (3) oxidative stress and (4) calcium signaling

and endoplasmic reticulum (ER) stress.
3910.3 7425.8

(1) Proteins including eIF5A, ubiquilin 2, 14-3-3 beta/alpha and

1385.8 7170.2

464.3 761.2
367.3 749.6
95.5 714.5

406.0 753.5

275.3 757.3
244.0 757.6
814.0 783.0

92.2 716.1
127.2 726.0

683.8 770.2
Spot volume

(Mean 7SD)

PP2A subunit A PR65-alpha isoform were related to cell

proliferation and/or cell death. eIF5A is an important protein
(ppm)

in translation initiation and is fundamental to cell survival and

GAK

proliferation (Jao and Chen, 2002). Ubiquilin 2 is suggested to

increase the half-life of proteins destined to be degraded by the
proteasome thus modulate proteasome-mediated protein degra-
compared with
Fold difference

dation. 14-3-3 beta/alpha belongs to the 14-3-3 family that is

involved in many different cellular processes, including mitogen-
control

esis, cell cycle control, and apoptosis (Tzivion et al., 2006). PP2A
3.44
2.07
0.24

0.19
0.33
0.15

0.33
0.23
0.16

0.44

0.18
0.20

subunit A PR65-alpha isoform is a subunit of PP2A, a large family

of highly conserved heterotrimeric enzymes essential for cell
3476.57 373.2
975.3 7 133.0

469.0 7 51.2
134.8 7 24.9

201.0 7 48.2
102.5 7 16.5

457.7 7 68.6
192.2 7 26.9

492.8 7 84.1

120.8 7 34.2
447.0 7 52.0

127.2 7 28.0

survival, cell cycle regulation and DNA damage response (Janssens

Spot volume

(Mean7SD)

and Goris, 2001). Recent studies showed that PP2A was closely
related to regulation of cell apoptosis by affecting the degradation
(ppm)
GAF

of BCL-2 (Lin et al., 2006).

(2) Proteins including IL-17E, TPM4-ALK fusion oncoprotein
1679.37 171.2

1083.5 7 217.3
1342.57 147.6

4275.77 478.9

1118.37 234.4
1374.0 7 290.5
1211.87 220.6

type 2 and heterogeneous nuclear ribonucleoprotein K

136.57 24.3

651.57 66.6
680.8 7 72.8
562.87 60.3

700.5 7 75.0
Spot volume

(Mean7SD)

are carcinogenosis-related proteins. These proteins were

Spot Control

differentially expressed in carcinoma cells or tumors compared

(ppm)

with normal cells or tissues. They might also play roles in

cell proliferation and/or cell death though their roles were not
Table 3

clearly clarified. IL-17E belongs to the IL-17 family, which

11
12
10
6
7
1

8
2
3

9
4
5

had been shown to play a potential role in T-cell mediated

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Table 4
The results of protein identifications of differentially expressed proteins using MALDI-TOF MS/MS.

Spot Target protein NCBI accession TheoreticalMr Protein Sequence Unique Best ion
number (kDa)/pI score coverage (%) peptides score

1 interleukin-17E (IL-17E) 3355455 11.3/7.03 65 19 2 36

2 translation initiation factor 5A (eIF5A) 54037409 16.7/5.08 131 42 3 120
3 peroxiredoxin 2 77744389 22.0/5.66 98 25 4 62
4 ubiquilin 2 16753207 65.7/5.15 72 33 3 45
5 Cu/Zn-superoxide dismutase 1237406 15.9/5.86 69 21 2 37
6 14-3-3 beta/alpha 1345590 28.1/4.76 123 65 3 92
7 TPM4-ALK fusion oncoprotein type 2 10441386 27.5/4.77 355 52 3 41
8 PP2A, subunit A, PR65-alpha isoform 21361399 65.2/5.00 90 20 4 46
9 nucleobindin-1 1144316 53.8/5.15 252 52 3 54
10 heterogeneous nuclear ribonucleoprotein K 55958543 34.0/5.54 66 26 2 50
11 reticulocalbin 1 4506455 38.9/4.86 102 15 4 36
12 chain A, DJ-1 protein 42543006 19.8/6.33 75 29 2 40

angiogenesis and promote tumourigenicity of human cervical
cancer (Wagsater et al., 2006). The expression of TPM4-ALK fusion
oncoprotein type 2 was significantly changed in esophageal
squamous tumors compared with adjacent normal esophageal
tissues (Du et al., 2007). Report about heterogeneous nuclear
ribonucleoprotein K showed that it was overexpressed in
oral squamous cell carcinoma (Roychoudhury and Chaudhuri,
2007).
(3) Proteins including peroxiredoxin 2, Cu/Zn-superoxide
dismutase and chain A of DJ-1 protein are antioxidant proteins
and play important roles in oxidative stress. Peroxiredoxin 2
belongs to the peroxiredoxins family, which control the consti-
tutive level of H2O2 in the cell and thus protect cell against ROS-
induced damage (Chevallet et al., 2003). Cu/Zn-superoxide
dismutase is a well-known anti-oxidant protein. DJ-1 is an
atypical peroxiredoxin-like peroxidase and it could function as a
redox-sensitive chaperone and as a sensor for oxidative stress
(Andres-Mateos et al., 2007).
(4) Proteins including nucleobindin-1 and reticulocalbin 1 are
related to calcium signaling and ER stress. Nucleobindin-1 is the
major Golgi calcium-binding protein though it is present both in
the cytosol and Golgi. It plays role in ER stress by regulating the
function of activating transcription factor 6, an ER membrane-
anchored transcription factor (Tsukumo et al., 2007). Reticulo-
calbin 1 is an endoplasmic reticulum resident Ca(2+ )-binding
protein with multiple EF-hand motifs and a carboxyl-terminal
HDEL sequence. It is involved in the regulation of calcium-
dependent activities in the endoplasmic reticulum lumen or post-
ER compartment (Ozawa and Muramastsu, 1993).
Confirmation of differentially expressed proteins by Western
blotting
Consistent with the proteomic results, eIF5A was found to
be down-regulated while 14-3-3 beta/alpha was found to be
up-regulated in all five ganoderic acids-treated HeLa cells
(Fig. 4).
Discussion
Ganoderma lucidum is a well-known medicinal fungus that has
been used as a traditional drug in China, Korea, Japan and other
Asian countries for a long time. Nowadays, it is still widely
prescribed by traditional Chinese medical doctors for the preven-
tion and treatment of various types of illnesses such as cancer,
hepatopathy, chronic bronchitis, arthritis, hypertension, neur-
asthenia, hyperglycemia, insomnia, cardiovascular diseases, de-
bility and weakness, etc (Lin, 2001). Among the active
components of Ganoderma lucidum, Ganoderma triterpenes have
received considerable attention owing to their conspicuous
pharmacological activities. Ganoderma triterpenes were shown
to have cytotoxic activity on cancer cells, anti-human immuno-
deficiency virus type 1 activity, antihistamine activity, antinoci-
ceptive activity, anticholesterol activity, and inhibitive activity on
angiotensin converting enzyme and glucosyltransferase (El-
Mekkawy et al., 1998; Hada et al., 1989; Kin et al., 1998; Kohda
et al., 1985; Lin et al., 1991; Morigiwa et al., 1986; Sliva, 2006;
Toth et al., 1983; Yeung et al., 2004).
During the last two decades, more than 140 triterpenes have
been isolated from the fruiting bodies, spores and cultured
mycelia of Ganoderma lucidum (Hirotani et al., 1993; Lin et al.,
1988; Min et al., 2004, 2005). The five ganoderic acids used in the
present study belong to major constituents of Ganoderma
triterpenens. According to our previous study, the contents of
these ganoderic acids in the dried fruit bodies of Ganoderma
lucidum were 20-1323 mg/g (unpublished data), 19-1191 mg/g,
19-1759 mg/g, 100-1452 mg/g and 55-829 mg/g for GAF, GAK, GAB,
GAD and GAAM1, respectively (Wang et al., 2006). Furthermore,
our previous work checking the pharmacokinetics characteristics
of GAB and GAK in rat indicated that, after oral administration, the
two ganoderic acids were quickly absorbed into the body fluid
from the gastrointestinal tract and distributed widely in the
organs. At 6.26 min and 32.10 min after oral administration of
1.2 g/kg Ganoderma lucidum extract, the plasma concentrations
of GAB and GAK reached the maximum level of 13.15 mg/ml
(25.5 mM) and 2.86 mg/ml (5 mM), respectively (Wang et al.,
2007a). And, results checking the urinary excretion of these
ganoderic acids indicated that, within 72 h, the cumulative
urinary excretion of GAB and GAK was 0.23 and 0.13, respectively
(Wang et al., 2007b). In the present study, ganoderic acids
including GAB and GAK exhibited cytotoxicity on HeLa human
cervical carcinoma cells with IC50 values of 15-20 mM. By using
proteomic method, the protein expression profiles of HeLa cells
after treatment of ganoderic acids at 15 mM were checked and 12
proteins that might be target-related proteins of ganoderic acids
were found. These 12 proteins were reported to play important
roles in cell proliferation and/or cell death, carcinogenosis,
oxidative stress, calcium signaling and ER stress. Furthermore,
result of the present study was consistent with our previous
proteomic study result using only GAD (Yue et al., 2008a) and
result using Ganoderma lucidum extract (Yue et al., 2008b). Three
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Q.-X. Yue et al. / Phytomedicine 17 (2010) 606–613 611

Fig. 3. The result of the MALDI-TOF MS/MS analysis of the protein spot 9 (identified to be human nucleobindin-1) cut from the 2-DE gels. (A), Peptide mass fingerprint of
the typtic digest of spot 9. (B), MS/MS profile of the peptide with a mass of 1272.79 Da. (C), MS/MS profile of the peptide with a mass of 1506.91 Da. (D), MS/MS profile of
the peptide with a mass of 1934.12 Da. y-ions resulting from fragmentation of the peptides and amino acids they represent are indicated.
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Fig. 4. Western blotting of eIF5A and 14-3-3 beta/alpha. Each blot is the
representative result of three independent experiments.
kinds of proteins, i.e. eIF5A, 14-3-3 protein and peroxiredoxin
protein, were found in all these studies. The results suggested
that eIF5A, 14-3-3 protein and peroxiredoxin protein might be the
most important target-related proteins of ganoderic acids. The
regulation of these proteins might contribute to the cytotoxicity
of ganoderic acids. 14-3-3 proteins are important regulators
in many different cellular processes such as mitogenesis, cell cycle
control, and apoptosis (Tzivion et al., 2006). And, regulation
of eIF5A, an important protein in translation initiation, might
contribute to the growth inhibition of HeLa cells induced by
ganoderic acids. Furthermore, decrease of peroxiredoxin protein, a
regulator of intracellular ROS level, suggested that ganoderic aicds
might increase ROS level of HeLa cells. Our previous study did
show that Ganoderma triterpenes extract caused increase in ROS
level of HeLa cells (Yue et al., 2008b).
Though there was no clinical report about using Ganoderma
triterpenes in cancer therapy, a clinical study was reported
checking the effect of using Ganoderma triterpenes in men with
lower urinary tract symptoms (Noguchi et al., 2008). The clinical
study indicates that Ganoderma triterpenes were well tolerated
and could improve International Prostate Symptom Score.
Presently, we are cooperating with another laboratory to check
in an animal study the potential of Ganoderma triterpenes extract
for complementary cancer therapy. Though further study is
necessary, the results of this study shed light on the anti-cancer
mechanism of ganoderic acids as well as Ganoderma triterpenes
from a molecular perspective point of view and provides useful
information for the possible use of Ganoderma triterpenes in clinic
for complementary cancer therapy.
Acknowledgments
This work was supported in part by grants from the
National Natural Science Foundation of China (30701077) and
the Science and Technology Commission of Shanghai Municipality
(2008DFA30350).
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