Journal of Ethnopharmacology 120 (2008) 394–401

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G2 /M cell
cycle arrest
Shih-Chung Hsu a,b , Chien-Chih Ou c,d , Jhy-Wei Li e,f , Tzu-Chao Chuang g,h,∗∗ , Han-Pon Kuo i ,
Jah-Yao Liu c , Chin-Shiang Chen j , Song-Chow Lin k , Ching-Hua Su l , Ming-Ching Kao m,n,∗
a
Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
b
Kang-Ning Junior College of Medical Care and Management, Taipei, Taiwan
c
Department of Obstetrics & Gynecology, Tri-Service General Hospital, Taipei, Taiwan
d
Department of Pharmacy, Keelung General Hospital, Keelung, Taiwan
e
Graduate Institute of Pathology, National Defense Medical Center, Taipei, Taiwan
f
Department of Pathology, Da-Chien General Hospital, Miaoli, Taiwan
g
Department of Chemistry, Tamkang University, Tamsui, Taipei, Taiwan
h
Graduate Institute of Life Sciences, Tamkang University, Tamsui, Taipei, Taiwan
i
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
j
Department of Horticulture and Biotechnology, Chinese Culture University, Taipei, Taiwan
k
Department & Institute of Pharmacology, Taipei Medical University, Taipei, Taiwan
l
Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan
m
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan
n
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan

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

Article history: Ethnopharmacological relevance: Ganoderma, known as Lingzhi or Reishi, has been traditionally adminis-
Received 21 April 2008 tered throughout Asia for centuries as a cancer treatment and for other medicinal purposes.
Received in revised form 12 July 2008 Aim of the study: To investigate the inhibitory activity and explore the molecular mechanisms of anti-
Accepted 11 September 2008
tumor effect on colorectal cancer cells in vitro and in vivo as well as to test the side effects of Ganoderma
Available online 2 October 2008
tsugae.
Materials and methods: Methanol fraction was obtained from dried fruiting bodies of Ganoderma. TLC
Keywords:
and HPLC were performed to differentiate and confirm the identification of different species as well as
Ganoderma tsugae
Lingzhi (Reishi)
to quantify the bioactive molecules in methanol extracts of Ganoderma species. MTT and Trypan blue
Colo205 exclusion assay as well as tumorigenesis study were used to assess the anti-tumor effect in vitro and in
Colorectal cancer vivo. Using flow cytometry and Western Blots, we examined further the molecular mechanisms of anti-
Cancer therapy tumor effect. Finally, biochemical and hematological profiles and pathological examinations were used to
evaluate the safety.
Results: The Ganoderma tsugae extracts inhibit colorectal cancer cell proliferation caused by accumulating
cells in G2 /M phase, and it may be through downregulation of cyclin A and B1 and upregulation of p21
and p27. Tumorigenesis study in nude mice revealed the extracts caused tumor shrinkage. Additionally,
safety assay showed Ganoderma tsugae extracts caused no significant side effects in an animal model.
Conclusions: This study provides molecular evidence that Ganoderma tsugae extracts exert anti-tumor
effects both in vitro and in vivo on colorectal adenocarcinoma cells by inducing G2 /M cell cycle arrest.
More importantly, no significant physiological changes resulting from treatment with Ganoderma tsugae
extracts were observed in the animal model. Therefore, these data provide new insights into the possible
therapeutic use of Ganoderma tsugae for treating colorectal cancer.
© 2008 Published by Elsevier Ireland Ltd.

Abbreviations: G. tsugae, Ganoderma tsugae; TCM, traditional Chinese medicine; H&E stain, hematoxylin and eosin stain; TLC, thin layer chromatography; HPLC,
high-performance liquid chromatography; IHC, immunohistochemistry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HPFs, high-power fields;
CDKs, cyclin dependent kinases.
∗ Corresponding author at: Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan, ROC.
Tel.: +886 4 22053366x2206; fax: +886 4 22070465.
∗∗ Corresponding author at: Department of Chemistry, and Graduate Institute of Life Sciences, Tamkang University, Tamsui, Taipei, Taiwan, ROC.
Tel.: +886 2 26215656x2316; fax: +886 2 26209924.
E-mail addresses: tcbc@mail.tku.edu.tw (T.-C. Chuang), mckao@mail.cmu.edu.tw (M.-C. Kao).

0378-8741/$ – see front matter © 2008 Published by Elsevier Ireland Ltd.

doi:10.1016/j.jep.2008.09.025
 S.-C. Hsu et al. / Journal of Ethnopharmacology 120 (2008) 394–401
1. Introduction
Ganoderma (also known as Lingzhi or Reishi), a traditional Chi-
nese medicine (TCM), has recently received considerable attention
from the health care and cancer research communities in Tai-
wan. Colorectal cancer is of particular concern, due to the recent
increases in prevalence and death from this disease; while early
diagnosis and therapy improve the probability of colorectal cancer
survival. In 2006, colorectal cancer was the second leading cause
of cancer-related deaths in the United States (Wolpin et al., 2007)
and the third in Taiwan. To investigate the possible application of
Ganoderma in colon cancer therapy, we performed both in vitro and
in vivo studies of Ganoderma activity.
For centuries, Ganoderma has been used for medicinal purposes
in Asian countries to treat many human diseases, including cancer.
Ganoderma lucidum (G. lucidum) and Ganoderma tsugae (G. tsugae)
Fig. 1. Analysis of Ganoderma species by TLC and HPLC profiling. (A) Identification
of Ganoderma species by TLC. Lanes 1 and 2, Ganoderma lucidium; lanes 3 and 4,
Ganoderma formosanum; lanes 5–7, G. tsugae. (B) Quantification of the methanol
extracts from G. tsugae (GT1, GT2 and GT3) and Ganoderma lucidium (GL1 and GL2)
by HPLC.
395
are the most widely cultivated species of the Ganoderma genus in
Taiwan, and both have a long history of use in folk medicine in
Asia. The biological activities of Ganoderma lucidum, especially its
anti-tumor and immunomodulatory properties, have been well-
documented (Chen et al., 2004; Chien et al., 2004). Several reports
have shown that the two major categories of bioactive ingredients
that can be isolated from Ganoderma lucidum are polysaccharides
and triterpenoids, both of which are potent inhibitors of in vitro
and in vivo tumor growth (Miyazaki and Nishijima, 1981; Min et al.,
2000; Kimura et al., 2002; Shiao, 2003; Lin and Zhang, 2004). More-
over, recent studies have demonstrated that Ganoderma lucidum
suppresses cell motility, inhibits cell proliferation, induces apopto-
sis, and suppresses angiogenesis of highly invasive human breast
and prostate cancer cells (Hu et al., 2002; Sliva et al., 2002; Jiang
et al., 2004; Stanley et al., 2005). Although the anti-tumor activity
of Ganoderma tsugae has been characterized (Wang et al., 1993),
only few clinical or pharmacological studies of its efficacy have
been pursued. Triterpenoids from Ganoderma tsugae can induce
apoptosis and cell cycle arrest in human hepatoma cells (Gan et
al., 1998), although the molecular mechanism of the anti-tumor
effects of Ganoderma tsugae on human colorectal cancer cells has
not been investigated. In this study, extracts from the three Gan-
oderma species (G. tsugae, G. lucidum, and G. formosanum) were
examined by TLC and HPLC profiling to assess their quality prior to
further experimentation. Because the Ganoderma tsugae extracts
demonstrated the highest cytotoxicity in cancer cells as assessed
by MTT assay, this study examined the anti-proliferative effects of
Ganoderma tsugae as well as the possible mechanisms by which
Ganoderma tsugae affects Colo205 human colorectal cancer cells.
Furthermore, the safety of orally administered Ganoderma tsugae
was evaluated in mouse studies.
2. Materials and methods
2.1. Cells and materials
Human colorectal adenocarcinoma Colo205 cells were obtained
from American Type Culture Collection and grown in RPMI 1640
medium supplemented with 10% FBS. Paclitaxel (Taxol) was pur-
chased from Bristol-Myers Squibb (Wallingford, CT) and stored at
−20 ◦ C before use. Paclitaxel was diluted in serum-free media at the
required concentration before use. The Ganoderma tsugae extracts
were directly added to cell cultures at the indicated concentra-
tions. All the primary and secondary antibodies were purchased
from Santa Cruz Biotechnology.
2.2. Sample extraction and preparation
Ganoderma tsugae fruiting bodies were donated by the Luo
Gui-Ying Fungi Agriculture Center (Taoyuan, Taiwan). Ganoderma
lucidum and Ganoderma formosanum were purchased, in a dried
form, at a traditional market in Taipei, Taiwan. The genus and
species of the Ganoderma samples used in this study were iden-
tified and confirmed by Dr. C.H. Su (Graduate Institute of Medical
Sciences, Taipei Medical University, Taiwan) as described (Su et al.,
2001). All Ganoderma samples were dried and ground into fine pow-
der, then extracted with methanol (MeOH) at room temperature.
For MeOH extraction, 5 g of the powdered samples were mixed with
200 mL of methanol and then placed on a rotating shaker at 150 rpm
for 24 h. Filtrate was collected twice by filter paper (Whatman No.
1) and MeOH was evaporated to dryness with a reduced pressure
concentrator for approximately 3 h to obtain dry MeOH extracts.
The extraction yield of the methanol extracts of Ganoderma tsugae
is 30.3 ± 0.7%. All samples of Ganoderma extracts were dissolved in
396 S.-C. Hsu et al. / Journal of Ethnopharmacology 120 (2008) 394–401

MeOH as a stock solution (300 mg/ml) for tests in cells. For in vivo
studies of tumorigenesis, dry Ganoderma tsugae extracts were dis-
solved in ethanol (EtOH, 100 mg/ml) and diluted to the indicated
concentrations (10 mg/ml) with suspension solution (74.5% corn
oil, 16% PEG-400, 4% Tween-80, 4% Cremophor EL, and 1.5% EtOH,
v/v).
2.3. TLC and HPLC analysis
TLC and HPLC analytical procedures were done according to the
report of Su et al., 2001.
obtained by plotting the drug concentration against the percentage
of growth inhibition.
2.5. Cell counting and Trypan blue exclusion assay
Cells (2.5 × 106 ) were seeded onto a 100-mm Petri dish to
measure cell proliferation. Cell growth rate and viability were deter-
mined using a hemacytometer to count the number of cells as a
function of time following Trypan blue staining.
2.6. Cell cycle analysis by flow cytometry and Western blotting

2.4. Drug sensitivity analysis by MTT assay To determine the cell cycle phase distribution and the molecular
markers associated with each phase, flow cytometry and Western
The MTT assay was performed as described (Chuang et al., 2003). blot analysis were performed, respectively, as described (Chuang et
The GI50 (drug concentration causing 50% growth inhibition) was al., 2003).

Fig. 2. Effect of Ganoderma extracts on human colorectal cancer Colo205 cells. (A) Inhibition of cell growth was demonstrated in Colo205 cells by MTT assay. Cells were
treated with three methanol extracts of Ganoderma tsugae, GT1 (a), GT2 (b) and GT3 (c); one methanol extracts of Ganoderma formosanum, GF1 (e); and two methanol extracts
of Ganoderma lucidium, GL1 (f) and GL2 (g) as well as one water extracts of GT2 (d) at the indicated concentrations for 3 days. The Colo205 cells treated with Taxol were used
as positive control (h). Data were expressed as the percentage of positive cells compared to the vehicle-treated control group. (B) Colo205 cells were treated with various
doses of Ganoderma tsugae extracts (0.3, 0.75, 1.5, 2.25 and 3.0 mg/ml) for 72 h. Cell viability was measured by MTT assay and the results were presented as the calculated
cell survival rate. Values are presented as mean ± S.E. of three independent experiments. * P < 0.01 and ** P < 0.001 versus the vehicle-treated control group.
 S.-C. Hsu et al. / Journal of Ethnopharmacology 120 (2008) 394–401
2.7. Laboratory animals and tumorigenicity assay
Four to five week-old nude mice were obtained and bred in the
Animal Center of NDMC, Taipei, Taiwan. Care and use of the ani-
mals was in accordance with institutional guidelines (Confirmed
by AAALAC, USA).
Animal experiments were performed as described (Wang et al.,
2007). Briefly, 5 × 106 viable Colo205 cancer cells were subcuta-
neously inoculated into a dorsal site in nude mice. When tumors
grew to approximately 100–200 mm3 in size, the mice were sorted
into experimental groups of eight animals each for further testing.
Ganoderma tsugae extracts were orally administered in 10 mg/ml of
suspension solution to the nude mice. Either the vehicle or extract
was given p.o. once/day for three weeks at a dose of 0.1 ml/10 g of
body weight. Tumor growth was monitored daily for three weeks,
at which point the animals were sacrificed. Biochemical and hema-
tological measurements were evaluated for the safety of the drug
as described by Xing et al. (1997).
2.8. Histochemical and immunohistochemical (IHC) stains
Xenografted Colo205 tumors and the surrounding mouse tis-
sues were excised, fixed in neutral formalin, embedded in paraffin,
and sliced for hematoxylin and eosin (H&E) staining to measure the
extent of necrosis and mitotic figures. Proliferation was also mea-
sured by Ki-67 IHC analysis. The preparation of samples for H&E
staining and IHC was performed as previously described (Li et al.,
2005).
Five high-power fields (5 HPFs, 400×) of H&E-stained slides
were counted using the image selection function of Adobe Pho-
toshop, Version 7.0 (Adobe Systems, CA). The ratio of necrosis was
defined as the area of the pale-stained, necrotic region of the tumor
divided by the total tumor area on the section. The number of
mitotic figures was counted in 5 HPFs of H&E-stained, necrosis-free
areas.
The anti-human Ki-67 antibody (Clone MIB-1, Dakocytomation,
mouse mAb; 1:150 dilution) was used as the primary antibody. Any
nuclear staining, regardless of intensity, was considered positive for
Ki-67. The Ki-67 labeling index (LI) was expressed as the percentage
of tumor cells with an immunoreactive nucleus in a sample size of
one thousand tumor cells.
2.9. Statistical analysis
All data represent mean ± standard error (mean ± S.E.) from
three independent experiments. Statistical analysis was performed
by Student’s t test. Differences between treatment groups were ana-
lyzed for significance by multiple comparisons using the analysis of
variance. A P value of <0.01 was considered statistically significant.
* P < 0.01 and ** P < 0.001 versus the vehicle-treated control group.
3. Results
3.1. Analysis of Ganoderma sp. by TLC and HPLC
Of the numerous species of Ganoderma, this study classified
seven samples of Ganoderma originating from different places in
Taiwan by the morphological characteristics of the fruiting body
and TLC chromatograms of their triterpenoids (Su et al., 2001). As
Fig. 1A shows, the seven samples could be grouped into the follow-
ing three species: Ganoderma lucidum, Ganoderma formosanum and
Ganoderma tsugae. These three species are among the most com-
mon Ganoderma species used in East Asian folk medicine, but the
morphology of Ganoderma tsugae is more easily confused. There-
fore, HPLC analysis of the triterpenoids was performed, not only
397
to differentiate and confirm the identification of different species
(Fig. 1B), but also to quantify the bioactive molecules in methanol
extracts of Ganoderma lucidum and Ganoderma tsugae (Su et al.,
2001). The analytical results showed that the small molecule con-
tent of Ganoderma tsugae methanol extracts is greater than for
Ganoderma lucidum.
3.2. The Ganoderma extracts inhibit colorectal cancer cell
proliferation
Fig. 2 shows the results of experiments in which the anti-tumor
effects of the methanol extracts from three species of Ganoderma
were examined in human colorectal cancer cells by MTT assay.
The methanol extracts from both Ganoderma lucidum and Gano-
derma tsugae significantly inhibited the growth of colorectal cancer
cells as measured by MTT analysis in Colo205 cells at 72 h com-
pared to vehicle-treated controls (Fig. 2A, a–c and f–g). For growth
inhibition, the effective concentration of methanol extracts from
both Ganoderma lucidum and Ganoderma tsugae ranged from 1 to
3 mg/ml with a GI50 of approximately 1.8 mg/ml. In contrast, expo-
sure of cells to water extracts of Ganoderma tsugae (>1000 mg/ml)
(Fig. 2A, d) and methanol extracts of Ganoderma formosanum
(>3 mg/ml) (Fig. 2A, e) had no effect on Colo205 colorectal cancer
cells in culture. Cells were also treated with Taxol for experi-
mental reference (Fig. 2A, h). Furthermore, colorectal cancer cells
were incubated with various doses of Ganoderma tsugae extracts
(GT3), and cell viability was determined. Our results revealed
that cell viability decreased in response to dose-dependent
treatment with 0.3–3 mg/ml of Ganoderma tsugae extracts
(Fig. 2B).
We next sought to evaluate whether the observed inhibition of
cell growth in cancer cells treated with Ganoderma tsugae extracts
was due to cell death or cell cycle arrest. To do this, we performed
a Trypan blue exclusion assay to assess the viability of the Colo205
cells. As Fig. 3 shows, a significant difference in cell number was
noted between vehicle-treated and extract-treated cells. Addition-
ally, the Trypan blue exclusion assay revealed the extract-treated
cells (3.0 mg/ml) maintained a stable number of cells for up to 48 h
after treatment, suggesting that the growth inhibition observed
in Colo205 cells was not due to cytotoxicity. Taking these find-
ings together, we postulated that G. tsugae extracts caused the
observed inhibition of Colo205 cell growth by arresting the cell
cycle.
Fig. 3. Inhibition of Colo205 cell growth by Ganoderma tsugae extracts. Cell numbers
were counted via Trypan blue stain after treatment for 0.5, 2, 4, 6, 8, 16, 24 and 48 h.
Cell growth was inhibited after treatment for 24- and 48-h by 3 mg/ml of Ganoderma
tsugae extracts but not by 0.3 mg/ml.
398 S.-C. Hsu et al. / Journal of Ethnopharmacology 120 (2008) 394–401

3.3. Ganoderma tsugae extracts accumulate Colo205 cells in
G2 /M phase
To determine the effects of Ganoderma tsugae extracts on the cell
cycle, cell cycle phase distributions were examined by flow cytom-
etry. As Fig. 4A shows, the proportion of cells in G2 /M phase was
markedly increased after treatment with increasing concentrations
of Ganoderma tsugae extracts. Similarly, using increasing concentra-
tions of Taxol to treat Colo205 cells also increases the percentage
of cells in G2 /M phase.
Since the progression of the cell cycle is largely controlled by
cyclins, we examined these proteins as molecular markers of the
cell cycle by immunoblotting. Using these markers, our aim was to
validate the effects of Ganoderma tsugae extracts on the cell cycle
in Colo205 cells. Cyclin D and cyclin E are known to drive the pro-
gression of a cell through G1 into S phase. The cyclin A is important
for G2 phase. Lastly, cyclin B1 interacts with CDK1 to form an active
complex necessary for entry into mitosis. The expression of cyclin
B1 starts in late S phase and remains elevated until its degradation
in anaphase of mitosis (Akli and Keyomarsi, 2003; Muraoka-Cook
et al., 2006).
As Fig. 4B illustrates, treatment of Colo205 with Ganoderma
tsugae extracts decreased cyclin A and B1, but not cyclin D and E. We
also examined the involvement of cyclin dependent kinases (CDKs),
which promote cell cycle progression, and the CDK inhibitors, p21
and p27. Increased expression of p21 and p27 was noted in Colo205
cells treated with Ganoderma tsugae extracts for 24 h (Fig. 4B). Taken
together, these results demonstrate that treatment with Ganoderma

Fig. 4. Effect of Ganoderma tsugae extracts on cell cycle transition in Colo205 cells. (A) Different concentrations of Ganoderma tsugae extracts (0.3, 1.5 and 3.0 mg/ml) (upper
panel) or Taxol (1, 10 and 100 ng/ml) (lower panel) were added and incubated for 24 h, and cellular DNA content was measured by FACS analysis. Both treatment of Ganoderma
tsugae extracts and Taxol caused cell growth arrest at G2 /M phase of Colo205 cells. (B) Effect of Ganoderma tsugae extracts on cyclin and CDK inhibitor protein levels. The
value below the figures represents the change in protein expression normalized against actin.
 S.-C. Hsu et al. / Journal of Ethnopharmacology 120 (2008) 394–401 399

Fig. 5. Inhibition of Colo205-induced tumor growth by Ganoderma tsugae extracts
in nude mice. Tumor volumes were estimated from caliper measurements of three
dimensions of the tumor. Estimated tumor weight was calculated as L × W2 × 0.5,
where L is the major axis, and W is tumor width. Data are represented as mean ± S.E.
(n = 8).
tsugae extracts leads to the downregulation of cyclin A and B1 and
upregulation of p21 and p27, causing G2/M arrest in Colo205 cells.
3.4. Ganoderma tsugae extracts cause regression of Colo205
xenografted tumors
To analyze the in vivo anti-tumor effect of Ganoderma tsugae
extracts, xenografts of Colo205 cells were implanted into nude
mice. Mice were first given oral doses of Ganoderma tsugae extracts
for 21 days to determine maximum tolerable dose (data not shown).
After the tumor volume reached approximately 100–200 mm3 , the
mice were treated for three weeks p.o. with either Ganoderma
tsugae extracts (dissolved in EtOH) at 100 mg/kg daily, or vehicle
only. Fig. 5 shows that mice treated with Ganoderma tsugae extracts
exhibited a marked suppression in Colo205 tumor growth relative
to vehicle-treated controls. These results demonstrate that Gano-
derma tsugae extracts can suppress tumor growth in vivo (Fig. 5).
Tumor xenografts were stained with H&E, which revealed
markedly more advanced necrosis (white zones) in extract-treated
tumors than controls (Fig. 6, A1 vs. A2). Additionally, both mitotic
counts and proliferation (as measured by Ki67-LI) were decreased
after Ganoderma tsugae treatment (Fig. 6, B1 vs. B2; C1 vs. C2), indi-
cating that Ganoderma tsugae extracts both induce cell death and
inhibit cell proliferation in human colorectal cancer cells in vivo.
To investigate the safety of Ganoderma tsugae extracts, patho-
logic examinations, clinical chemistry, and hematological analyses
were performed. Several organs were examined by histopathology
for visceral congestion, inflammation, hemorrhage or hyperplasia:
brain, heart, liver, spleen, lung, kidney, stomach, adrenal glands,
and intestine. No significant microscopic aberrations were noted
comparing to vehicle-treated controls (data not shown). Moreover,
peripheral blood samples were assayed by both clinical chemistry
and hematological analyses. Importantly, the nude mice appar-
ently tolerated repeated treatment with Ganoderma tsugae extracts,

Fig. 6. H&E staining and IHC analysis of representative histological sections taken from tumors of mice inoculated with Colo205 cells. Ganoderma tsugae extract-treated mice
had more extensive necrosis (white zones, necrotic ratio: 57% in A2) and a larger tumor mass than controls (necrotic ratio: 3% in A1). Sections from control mice showed
more mitotic figures (B1, 99/5HPFs) than Ganoderma tsugae extract-treated mice (B2, 37/5 HPFs). The proliferation index was higher in control mice (C1, positive brown-red
nuclei count, Ki67-LI: 2.4) than in Ganoderma tsugae extract-treated mice (C2, Ki67-LI: 1.6). White lines and arrows indicate scale bars and mitotic figures, respectively.
400 S.-C. Hsu et al. / Journal of Ethnopharmacology 120 (2008) 394–401

Table 1 also revealed that the Ganoderma tsugae methanol extracts induced
Biochemical and hematological profiles in mice following twenty-one daily doses of
Colo205 cancer cell arrest at G2 /M phase (Fig. 4), which is similar
the Ganoderma tsugae extracts by oral administration.
to the previous report that the Ganoderma lucidum ethanol extracts
Itema Vehicleb Ganoderma tsugaeb caused G2 /M phase arrest in hepatoma cells (Lin et al., 2003).
BUN (mg/dl) 14.5 ± 0.7 17.6 ± 2.0 Cell cycle arrest in the G2 phase can be triggered by stress
Creatinine (mg/dl) 0.2 ± 0.0 0.2 ± 0.0 from several different stimuli. A recent report demonstrated that
sGOT (IU/l) 359.0 ± 42.2 270.8 ± 37.1 microtubule-interacting agents (i.e., Taxol) influence the expres-
sGPT (IU/l) 45.4 ± 7.0 38.9 ± 5.6
sion of several important regulators of the G2 /M transition. For
Sodium (mmol/l) 151.8 ± 1.3 154.9 ± 0.9
Potassium (mmol/l) 9.8 ± 0.5 9.0 ± 0.5 example, p34cdc2 and topoisomerase II-alpha (TOP2A) were both
Chloride (mmol/l) 132.4 ± 1.7 136.6 ± 1.0 found to be downregulated by agents targeting microtubules, while
Magnesium (mg/dl) 2.2 ± 0.1 2.0 ± 0.1 the CDK inhibitor, p21 was upregulated (Chen et al., 2003; Bani et
WBC (mm3 ) 2650.0 ± 174.4 1821.4 ± 271.4
al., 2004; Skladanowski et al., 2005). Our Western blotting results
Neutro-Seg % 65.6 ± 0.5 62.7 ± 0.8
Neutro-Seg (mm3 ) 1744.9 ± 122.3 1158.1 ± 189.4
demonstrate that Ganoderma tsugae extracts upregulate p21 and
Lymph % 31.4 ± 0.6 34.9 ± 0.8 p27 and downregulate cyclin A and cyclin B1 (Fig. 4B). However,
Lymph (mm3 ) 825.9 ± 47.3 619.6 ± 77.8 further investigation is required to better understand the molecu-
Mono % 3.1 ± 0.2 2.4 ± 0.1 lar mechanisms by which Ganoderma tsugae extracts affect these
Mono (mm3 ) 82.9 ± 9.3 43.7 ± 5.8
cell cycle regulators in Colo205 human colorectal adenocarcinoma
RBC (106 /mm3 ) 6.0 ± 0.2 5.5 ± 0.2
Hb (g/dl) 10.2 ± 0.4 9.2 ± 0.3 cells.
Hct (%) 30.3 ± 1.1 28.0 ± 1.0 In summary, this study provides molecular evidence that Gan-
MCV (␮m6 ) 50.8 ± 0.4 50.8 ± 0.4 oderma tsugae extracts exert anti-tumor effects both in vitro and
MCH (pg) 17.0 ± 0.1 16.7 ± 0.1
in vivo on colorectal adenocarcinoma cells by inducing G2 /M cell
MCHC (%) 33.5 ± 0.2 32.9 ± 0.2
Platelet (103 /mm3 ) 260.1 ± 26.2 274.0 ± 41.4
cycle arrest. More importantly, based on the biochemical, hema-
tological (Table 1) and pathological examinations, no significant
a
BUN, blood urea nitrogen; sGOT, serum glutamic-oxaloacetic transaminase;
physiological toxicities resulting from treatment with Ganoderma
sGPT, serum glutamic-pyruvic transaminase; WBC, white blood cell; Neutro-Seg,
segmented neutrophil; Lymph, lymphocyte; Mono, monocyte; RBC, red blood cell; tsugae extracts were observed in the in vivo animal model. There-
Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean fore, Ganoderma tsugae extracts may prove clinically useful as an
corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. anticancer treatment for human colorectal adenocarcinoma.
b
Values represented as mean ± S.E.

showing no adverse effects on renal, hepatic, and hematological
parameters (Table 1).
4. Discussion
Some chemopreventive extracts of herbs or plants, including
Ganoderma (Lingzhi), are known to be anti-tumorigenic. Among
them, Ganoderma lucidum and Ganoderma tsugae are the most
widely used Ganoderma species for folk remedies in Asia. The anti-
tumor effects of Ganoderma are apparently mediated by numerous
biologically active compounds, such as polysaccharides, triterpenes
and immunomodulatory proteins (Sliva et al., 2002). Because of
variations in the composition of extracts, as well as differences in
culturing methods and climate, individual Ganoderma species may
differ in their bioactivity. Use of TLC and HPLC (Su et al., 2001)
in this study enabled the easy identification and classification of
species from the Ganoderma genus (Fig. 1). Furthermore, as Fig. 2A
shows, the antitumor bioactivity of Ganoderma in Colo205 colorec-
tal cancer cells was observed in Ganoderma lucidum and Ganoderma
tsugae, but not in Ganoderma formosanum.
It has been widely reported that Ganoderma lucidum extracts
interfere with the cell cycle to act as anticancer agents. Ganoderma
lucidum alcohol extracts were found to arrest the cell cycle in G1 /S
phase in cervical tumor HeLa cells and breast tumor MCF-7 cells
(Zhu et al., 2000; Hu et al., 2002); similarly, Ganoderma lucidum
hot water extracts and triterpene-enriched, soluble ethanol frac-
tions caused G2 /M phase arrest in hepatoma cells (Lin et al., 2003).
The differential effects may be due to variations in the composition
of the extracts (Tang et al., 2006). However, little is known about
the anti-tumor properties and mechanisms of action of methanol
extracts of Ganoderma tsugae. This study demonstrated that treat-
ing human colorectal Colo205 tumor cells with Ganoderma tsugae
extracts induced necrosis and inhibited tumor cell growth in a
dose-dependent manner, both in vitro (Figs. 2 and 3) and in vivo
in a xenograft model (Figs. 5 and 6). In addition, our experiments
Acknowledgements
This work was supported by the Department of Health (DOH93-
TD-F-113-030 & DOH94-TD-F-113-023), Taiwan, ROC, and the China
Medical University (CMU95-296), Taichung, Taiwan, granted to
M.C.K.
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