Pharmacology & Pharmacy, 2011, 2, 266-270
doi:10.4236/pp.2011.24034 Published Online October 2011 (http://www.SciRP.org/journal/pp)
Cordyceps sinensis Acts as an Adenosine A3
Receptor Agonist on Mouse Melanoma and Lung
Carcinoma Cells, and Human Fibrosarcoma and
Colon Carcinoma Cells
Noriko Yoshikawa1, Arisa Nishiuchi1, Erika Kubo1, Yu Yamaguchi1, Masaru Kunitomo1,
Satomi Kagota1, Kazumasa Shinozuka1, Kazuki Nakamura1,2
1Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya,
Japan; 2Institute for Biosciences, Mukogawa Women’s University, Nishinomiya, Japan.
Received September 1st, 2011; revised September 25th, 2011; accepted October 15th, 2011.
ABSTRACT
Cordyceps sinensis, a parasitic fungus on the larva of Lapidoptera, has been used as a traditional Chinese medicine.
We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells and mouse Lewis lung carcinoma
(LLC) cells was inhibited by cordycepin (3’-deoxyadenosine), an ingredient of Cordyceps sinensis, and its effect was
antagonized by MRS1191, a selective adenosine A3 receptor (A3-R) antagonist although adenosine (up to 100 μM) had
no effect on the growth of B16-BL6 and LLC cells. In this study, we investigated whether water extracts of Cordyceps
sinensis (WECS) inhibit the growth of B16-BL6 cells, LLC cells, HT1080 human fibrosarcoma (HT1080) cells and
CW-2 human colon carcinoma (CW-2) cells via their A3-R. As a result, the growth of all cell lines were potently inhib-
ited by WECS (10 μg/mL) and the inhibitory effect of WECS was significantly antagonized by MRS1191 (1 μM). Fur-
thermore, WECS included 2.34% w/w cordycepin and 0.12% w/w adenosine as components according to the HPLC-
ECD system. In conclusion, WECS inhibited the proliferation of four cancer cell lines by stimulation of A3-R and the
main component in WECS with anticancer action might be cordycepin instead of adenosine.
Keywords: Cordyceps sinensis, Adenosine A3 Receptor, Cordycepin, HPLC-ECD
1. Introduction
Cordyceps sinensis, a fungus parasitized on the larva of
Lapidoptera, has been used as a valued traditional Chi-
nese medication and a tonic food. Natural products of
Cordyceps sinensis are so rare and difficult to obtain in
uniform composition that cultured products have been
developed. In 1987, CordyMax Cs-4, a mycelial fermen-
tation product of Cordyceps sinensis, was approved by
the National New Drug Review and Approval committee
of the Chinese Ministry of Public Health, and has been
used in clinics for fatigue, night sweating, male and fe-
male hyposexuality including impotence, hyperglycemia,
hyperlipidemia, asthenia after severe illness, respiratory
diseases, renal dysfunction and renal failure, arrhythmias
and other heart diseases, and liver diseases [1]. However,
we focused on the cultural fruiting body of Cordyceps
sinensis instead of the mycelium and reported the anti-
cancer effect of water extracts of Cordyceps sinensis
(WECS) on mouse Lewis lung carcinoma (LLC) cells
and B16 mouse melanoma cells [2].
In this study, we tried to elucidate the mechanism of
the anticancer effect of WECS and speculated an active
ingredient in WECS. In particular, we investigated
pharmacologically whether WECS stimulate adenosine
A3 receptor (A3-R) using a selective A3-R antagonist
since an effective component in WECS, cordycepin
(3’-deoxyadenosine), showed anticancer action via the
stimulation of A3-R, according to our previous experi-
mental data [3]. Finally, we quantified the content of
cordycepin in WECS using the HPLC-ECD system.
2. Materials and Methods
2.1. Chemicals
Dried fruiting bodies of cultured Cordyceps sinensis were

Cordyceps sinensis Acts as an Adenosine A Receptor Agonist on Mouse Melanoma and 267
3
Lung Carcinoma Cells, and Human Fibrosarcoma and Colon Carcinoma Cells
produced by Jiang Men Baode Biological Technology
(Guangdong, China). They were extracted with hot water
(70˚C) for 5 min and the extract was then filtered and ly-
ophilized. The lyophilized powder (WECS) was sealed in
bottles and kept in a refrigerator (4˚C) until use. 3-Ethyl-
5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-
dihydropyridine-3,5-di-carboxylate (MRS1191, a selec-
tive A3-R antagonist), cordycepin (3’-deoxyadenosine),
adenosine and fetal bovine serum (FBS) were purchased
from Sigma Chemical Co. (St. Louis, MO). Dulbecco’s
modified Eagle’s medium (DMEM) with L-glutamine,
Earle’s minimum essential medium (EMEM) plus glu-
tamax, RPMI1640 medium and MEM non-essential
amino acids (NEAA) 100 × solution were from Invitro-
gen Co. (Grand Island, NY). Dulbecco’s phosphate-
buffered saline without calcium and magnesium [DPBS
(–)] was from Nissui Pharmaceutical Co., Ltd (Tokyo,
Japan). EDTA trypsin solution (EDTA, 0.02%; trypsin,
0.1%) and penicillin/streptomycin solution (penicillin,
50,000 U/mL; streptomycin, 50 mg/mL) were obtained
from Cosmo Bio Co., Ltd. (Tokyo, Japan).
2.2. Cells
The mouse B16-BL6 epithelial-like melanoma cell line
was kindly provided by Professor Futoshi Okada of Tot-
tori University (Yonago, Japan). The mouse Lewis lung
carcinoma cell line and CW-2 human colon carcinoma
cell line were provided by the RIKEN CELL BANK
(Tsukuba, Japan). The human HT1080 epithelial-like
fibrosarcoma cell line was purchased from DS Pharma
Biomedical Co., Ltd. (Suita, Japan). B16-BL6 cells were
cultured in DMEM containing 10% FBS and a 0.1%
penicillin/streptomycin solution. LLC cells were cultured
in DMEM containing 10% FBS. HT1080 cells were cul-
tured in EMEM containing 10% FBS and a 1% NEAA.
CW-2 cells were cultured in RPMI1640 containing 10%
FBS.
2.3. Growth Curves of B16-BL6 Cells,
LLC Cells, HT1080 Cells and
CW-2 Cells
Sub-confluent cancer cells were harvested with EDTA
trypsin solution and resuspended as 1 × 105 cells/2 mL in
each well of a 12-well culture plate, and then treated with
10 µg/mL WECS. MRS1191 (0.1 and 1 µM) was added
to the wells 30 min before the addition of WECS. To
evaluate the effect of MRS1191 alone on cell growth, 0,
0.1, 1 and 10 μM of MRS1191 were added to each cul-
ture plate at 0 h. After 24, 48 and 72 h incubation at 37˚C,
viable cells were counted with a Coulter counter (Coulter
Z1; Beckman Coulter, Inc., Tokyo, Japan).
2.4. Determination of Cordycepin and Adenosine
Content in WECS
HPLC with electrochemical detection (HPLC-ECD) was
used to quantify cordycepin and adenosine in WECS,
according to a previously described method [4]. Briefly,
20 μL of WECS (1 mg/mL) was injected into the
HPLC-ECD system. The analytical column was a Cos-
mosil 5C18-AR-II (particle diameter, 5 μm; column size,
150 × 4.6 mm I.D.; Nacalai Tesque, Kyoto, Japan) col-
umn equipped with a guard column (10 × 4.6 mm I.D.)
and the mobile phase was 20 mM sodium phosphate
buffer, pH 7.0, including 10% (v/v) methanol. The flow
rate was 1 mL/min and the column temperature was set
at 25˚C. Quantitation of cordycepin and adenosine was
performed by comparison of the peak area with those of
authentic cordycepin and adenosine, respectively.
2.5. Statistical Analyses
The data are expressed as the mean ± S.D. (N = 3). Sta-
tistical analyses were performed by one-way ANOVA
followed by Tukey’s Multiple Comparison Test using
PRISM Version 4 (GraphPad Software, Inc., San Diego,
CA). Differences were considered significant at P < 0.05.
3. Results
In mouse cancer cell lines, WECS (10 µg/mL) inhibited
the growth of B16-BL6 cells and LLC cells potently to
14% and 23% of the non-treated control at 72 h, respec-
tively. When B16-BL6 cells were premixed with 1 µM
MRS1191, a selective A3-R antagonist, 30 min before the
addition of WECS (10 µg/mL), the cell numbers were
significantly antagonized to 69% of the control value.
When LLC cells were premixed with 0.1 and 1 µM
MRS1191, 30 min before the addition of WECS (10
µg/mL), the cell numbers were significantly antagonized
to 27% and 41% of the control value, respectively (Fig-
ure 1).
In human cell lines, WECS (10 µg/mL) inhibited the
growth of HT1080 cells and CW-2 cells potently to 40%
and 38% of the non-treated control at 72 h, respectively.
When HT1080 cells were premixed with 1 µM MRS1191,
30 min before the addition of WECS (10 µg/mL), the cell
numbers were significantly antagonized to 77% of the
control value. When CW-2 cells were premixed with 0.1
and 1 µM MRS1191, 30 min before the addition of
WECS (10 µg/mL), the cell numbers were significantly
antagonized to 46% and 55% of the control values, re-
spectively (Figure 2).
In Figure 3, showing representative data using CW-2
cells, 10 μM MRS1191 slightly reduced the cell number;
however, 0.1 and 1 µM MRS1191 alone did not have any

Cordyceps sinensis Acts as an Adenosine A Receptor Agonist on Mouse Melanoma and 268 3
Lung Carcinoma Cells, and Human Fibrosarcoma and Colon Carcinoma Cells
(a) (b)
Figure 1. Effects of MRS1191, a selective A3-R antagonist, on mouse B16-BL6 (a) and LLC (b) cell growth inhibited by
WECS. At time 0, 1 × 105 sub-confluent B16-BL6 (a) and LLC (b) cells in 2 mL medium per well, obtained as a monodis-
persed suspension by trypsinization, were seeded into a 12-well culture plate in the presence of 10 μg/mL WECS. At 72 h
after plating, cultures were trypsinized and viable cells of samples were counted using a Coulter counter. MRS1191 (0.1 and 1
μM) was added 30 min before the addition of WECS. Each bar represents the mean ± S.D. (N = 3). *P < 0.05 vs WECS 10
μg/mL.
(a) (b)
Figure 2. Effects of MRS1191, a selective A3-R antagonist, on human HT1080 cell (a) and CW-2 cell (b) growth inhibited by
WECS. At time 0, 1 × 105 sub-confluent HT1080 (a) and CW-2 (b) cells in 2 mL medium per well, obtained as a monodis-
persed suspension by trypsinization, were seeded into a 12-well culture plate in the presence of 10 μg/mL WECS. At 72 h
after plating, cultures were trypsinized and viable cells of samples were counted using a Coulter counter. MRS1191 (0.1 and 1
μM) was added 30 min before the addition of WECS. Each bar represents the mean ± S.D. (N = 3). *P < 0.05 vs WECS 10
g/mL. μ

Cordyceps sinensis Acts as an Adenosine A3 Receptor Agonist on Mouse Melanoma and
Lung Carcinoma Cells, and Human Fibrosarcoma and Colon Carcinoma Cells
269
effect on cancer cell growth at 72 h. be utilized as a marker of the quality of WECS; however,
cordycepin is a potent target candidate in quality checks
because cordycepin was isolated as a pure compound in
WECS and showed anticancer effects, and its structure
and molecular weight were clarified (251.24). Accord-
ingly, we considered cordycepin as an active ingredient
showing anticancer action in WECS and tried to investi-
gate whether the anticancer action of WECS was inhib-
ited by A3-R antagonist, just like cordycepin. As a result,
the anticancer effect of WECS was significantly antago-
nized by MRS1191, a selective A3-R antagonist; that is
to say, cordycepin is a potent ingredient in WECS show-
ing anticancer effects and can also be utilized as a marker
for testing the quality of WECS. Actually, we determined
the content of cordycepin as 2.34% w/w in WECS using
the HPLC-ECD system in this experiment and it is much
higher than that of adenosine (0.12% w/w in WECS).
Although it is apparent that cordycepin might be a com-
ponent showing anticancer action in WECS, other com-
ponents also contribute to the anticancer action of WECS.
Because the inhibitory effects of WECS on HT1080,
B16-BL6, CW-2 and LLC cells were incompletely re-
covered to 77%, 69%, 55% and 41% of non-treated con-
trol values by addition of the selective A3-R antagonist,
respectively, it is conceivable that the different responsi-
bilities among the four cell lines probably occurred based
on the variation in level of expression of A3-R on cell
line membrane. We have already shown that B16-BL6
cells express A3-R according to a radioligand binding
assay using [125I]-AB-MECA (a selective A3-R agonist)
[6] and we speculate that HT1080 cells express a higher
level of A3-R on the cell membrane than LLC, CW-2 and
B16-BL6 cells due to their greatest responsibility to the
selective A3-R antagonist.
As shown in Table 1, the content of cordycepin and
adenosine in WECS were 2.34% w/w and 0.12% w/w,
respectively. WECS included cordycepin more abundant
than adenosine.
4. Discussion
To date, we have reported that WECS inhibited the
growth of B16 melanoma cells and LLC cells [2] and
cordycepin (3’-deoxyadenosine), an effective ingredient
in WECS, also showed anticancer effects using B16-BL6
cells and LLC cells by stimulating A3-R [3]. As an effec-
tive ingredient, in addition to cordycepin in WECS, Chen
et al. demonstrated that polysaccharide in cultivated
Cordyceps sinensis significantly inhibited hepatoma H22
tumor cell growth [5]. In fact, polysaccharide is a potent
candidate in WECS showing anticancer action; however,
it is still a crude ingredient and its exact structure and
molecular weight cannot be determined. WECS is also a
crude product and its quality needs to be checked for
commercial use. In other words, polysaccharide cannot
5. Conclusions
We clarified that the growth inhibitory effect of WECS
on B16-BL6, LLC, HT1080 and CW-2 cells was signifi-
cantly antagonized by the selective A3-R antagonist.
Figure 3. Effects of MRS1191, a selective A3-R antagonist
on human CW-2 cell growth. At time 0, 1 × 105 sub-con-
fluent CW-2 cells in 2 mL medium per well, obtained as a
monodispersed suspension by trypsinization, were seeded
into a 12-well culture plate in the presence of 0, 0.1, 1 and
10 μM MRS1191. At 24, 48 and 72 h after plating, cultures
were trypsinized and viable cells of samples were counted
using a Coulter counter. Data represent the mean of tripli-
cate samples.
It is reasonable that the active ingredient in WECS in-
dicating anticancer action might be cordycepin, which in
WECS should be the target marker for testing the quality
of WECS for commercial use.
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