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Inhibition of Growth and Induction of Apoptosis in Human Cancer Cell Lines by an Ethyl Acetate Fraction from Shiitake Mushrooms

Authors:

Abstract

Shiitake (Lentinus edodes) mushrooms have been reported to have cancer-preventing properties. However, little research has been conducted verifying the antitumor activities of "mycochemicals" in shiitake mushrooms. In this study, potential roles of an ethyl acetate fraction from shiitake mushrooms were investigated by in vitro bioassays. The activities of an ethyl acetate fraction were evaluated by [3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide] (MTT), apoptosis bioassay, cell cycle analysis, and Western blot analysis using two human breast carcinoma cell lines (MDA-MB-453 and MCF-7), one human nonmalignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI-8226 and IM-9). Concentration-dependent antiproliferative effects of the fraction were observed in all cell lines using the MTT assay. Approximately 50 mg/L concentration of the fraction induced apoptosis in 50% of the population of four human tumor cell lines and the fraction-induced apoptosis may have been mediated through the pro-apoptotic bax protein which was up-regulated. Cell cycle analysis revealed that the fraction induced cell cycle arrest by significant decrease of S phase, which was associated with the induction of cdk inhibitors p21 and the suppression of cdk4 and cyclin D1 activity. Compared to malignant tumor cells, nonmalignant cells were less sensitive to the fraction for the suppression of cell growth and regulation of bax, p21, cyclin D1, and cdk4 expression. A 51% antiproliferative effect occurred at the highest concentration of the fraction (800 mg/L). These data suggest that inhibition of growth in tumor cells by "mycochemicals" in shiitake mushrooms may result from induction of apoptosis.
THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINE
Volume 12, Number 2, 2006, pp. 125–132
© Mary Ann Liebert, Inc.
Inhibition of Growth and Induction of Apoptosis in Human
Cancer Cell Lines by an Ethyl Acetate Fraction from
Shiitake Mushrooms
NIANBAI FANG, Ph.D.,
1.2
QINGLIN LI, Ph.D.,
1,2
SHANGGONG YU, B.Sc.,
2
JIANXIANG ZHANG, Ph.D.,
1,2
LING HE, M.D.,
1,3
MARTIN J.J. RONIS, Ph.D.,
1,4
AND THOMAS M. BADGER, Ph.D.
1,3
ABSTRACT
Objective: Shiitake (Lentinus edodes) mushrooms have been reported to have cancer-preventing properties.
However, little research has been conducted verifying the antitumor activities of “mycochemicals” in shiitake
mushrooms. In this study, potential roles of an ethyl acetate fraction from shiitake mushrooms were investi-
gated by in vitro bioassays.
Design: The activities of an ethyl acetate fraction were evaluated by [3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-
tetrazolium bromide] (MTT), apoptosis bioassay, cell cycle analysis, and Western blot analysis using two hu-
man breast carcinoma cell lines (MDA-MB-453 and MCF-7), one human nonmalignant breast epithelial cell
line (MCF-10F), and two myeloma cell lines (RPMI-8226 and IM-9).
Results: Concentration-dependent antiproliferative effects of the fraction were observed in all cell lines us-
ing the MTT assay. Approximately 50 mg/L concentration of the fraction induced apoptosis in 50% of the pop-
ulation of four human tumor cell lines and the fraction-induced apoptosis may have been mediated through the
pro-apoptotic bax protein which was up-regulated. Cell cycle analysis revealed that the fraction induced cell
cycle arrest by significant decrease of S phase, which was associated with the induction of cdk inhibitors (p21)
and the suppression of cdk4 and cyclin D1 activity. Compared to malignant tumor cells, nonmalignant cells
were less sensitive to the fraction for the suppression of cell growth and regulation of bax, p21, cyclin D1, and
cdk4 expression. A 51% antiproliferative effect occurred at the highest concentration of the fraction (800 mg/L).
Conclusions: These data suggest that inhibition of growth in tumor cells by “mycochemicals” in shiitake
mushrooms may result from induction of apoptosis.
125
INTRODUCTION
S
hiitake mushrooms (Lentinus edodes) have been de-
scribed as having antiviral, antibiotic, anti-inflammatory,
antihypertensive, and anticancer properties,
1–3
and have
been popular as a health food for thousands of years in the
East and more recently in the West.
Orally administered shiitake mushroom fruiting bodies have
been reported to have anticarcinogenic effects against some
tumor types and that these effects appear to be strain specific.
4,5
Diets containing shiitake mushrooms significantly reduced the
growth of Sarcoma-180 cells in female ICR mice and MM-46
carcinoma in C3H mice, but had little effect on growth of other
tumor types such as B-16 melanoma, Lewis lung carcinoma,
1
Arkansas Children’s Nutrition Center, University of Arkansas for Medical Sciences, Little Rock, AR.
2
Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR.
3
Department of Physiology/Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR.
4
Department of Pharmacology/Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR.
or meth-A fibrosarcoma.
4,5
Moreover, the anti-cancer effects
of the shiitake diets were found to be accompanied by activa-
tion of macrophages and increased cytotoxic effects of both
NK and killer T cells,
4,5
suggesting that one possible mecha-
nism of this tumor-inhibiting activity may involve boosting
immune surveillance.
With regard to identification of anticancer constituents in
shiitake mushrooms, an antitumor polysaccharide was iso-
lated from fruiting bodies and named lentinan.
6,7
Its struc-
ture was confirmed as -(1 3)-glucans (C
6
H
10
O
5
)
n
, with
the mean molecular weight of 5 10
5
kDa.
8
Lentinan has
been established to be a major antitumor-active component
in shiitake mushrooms. However, mammals lack the en-
zyme, -1 3-glucanase, required to digest lentinan and
purified lentinan has been reported to possess no antitumor
activity when administered orally.
3,9
One study has been
conducted on fractionation of chemical constituents in shi-
itake mushrooms and orally administered the fractions.
4
When tumor-bearing mice received the ethyl ether-ethanol
fraction in the diet, the growth of tumors was inhibited by
24.7%. It appears that in addition to anticancer polysaccha-
rides, shiitake mushroom also contains as yet uncharacter-
ized ethyl ether-ethanol soluble anticancer mycochemicals.
To date, very little research has been conducted on health
effects of the ethyl ether-ethanol soluble portion from shi-
itake mushrooms.
In the present study, the anticancer effects of an ethyl ac-
etate fraction from shiitake mushrooms were assessed against
human breast carcinoma cell lines (MDA-MB-453 and
MCF-7), the human nonmalignant breast epithelial cell line
(MCF-10F), and myeloma cell lines (RPMI-8226 and IM-9).
The effects on the suppression of cell growth were measured
by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] (MTT) assay. Apoptosis assay and cell cycle analysis
were used to elucidate the basis of the antiproliferative prop-
erties of the fraction. In addition, the authors evaluated the ef-
fects of the fraction on the levels of Bax, p21, cyclin D1, and
cdk4 in the treated cells by Western blot analysis.
MATERIALS AND METHODS
Preparation of an ethyl acetate fraction from
shiitake mushrooms
Log-grown shiitake mushrooms were obtained from
Shirley Community Development Corporation (Shirley,
AR). Frozen shiitake mushrooms (2000 g) were extracted
with 80% aqueous methanol (3000 mL) and 50% aqueous
methanol (3000 mL) at 5°C. The extracts were combined
and concentrated on a rotary evaporator under reduced pres-
sure at room temperature until the methanol was removed.
The aqueous extract (200 mL) was partitioned with ethyl ac-
etate two times (300 mL 2). The combined ethyl acetate
fraction was evaporated under reduced pressure at room tem-
perature followed by drying in a freeze-dryer. The ethyl ac-
etate fraction represented 2.12% of frozen weight of the shi-
itake mushrooms.
Cell cultures and treatment
Two human breast carcinoma cell lines (ER-negative
MDA-MB-453 and ER-positive MCF-7), one human non-
malignant breast epithelial cell line (ER-negative MCF-
10F), and two myeloma cell lines (EBV-negative RPMI-
8226 and EBV-positive IM-9) were obtained from the
American Type Culture Collection (Rockville, MD). The
cell lines were cultured as described in the instructions from
the American Type Culture Collection. The fraction from
shiitake mushrooms was dissolved in dimethylsulfoxide
(DMSO) in different concentrations and mixed with medium
before treatment. The final concentration of DMSO in
treated cell medium was 0.1% (0.1% DMSO alone as con-
trol).
Cell proliferation assay
The effects of the fraction from shiitake mushrooms on
cell proliferation were measured by MTT assay. The MTT
assay of myeloma cell lines was conducted according to pub-
lished procedure
10
and the MTT assay of human breast car-
cinoma cell lines was carried out using the methods de-
scribed previously.
11
The cells were treated with 0, 0 (with
0.1% DMSO), 12.5, 50, 200, and 800 mg/L fraction and in-
cubated for 24, 48, or 72 h before treatment with MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(Sigma, St. Louis, MO).
Apoptosis assays
The apoptotic effect of the fraction from shiitake mush-
rooms was determined by staining cells with Annexin V-
fluorescein isothiocyanate (FITC) (BD Bioscences, Palo
Alto, CA) and propidium iodide (PI) (Sigma).
12
The cells
were cultured in medium containing various concentrations
of the fraction for 24, 48, or 72 h before apoptosis assays.
Data plots were generated from analysis of ungated data and
representative dot plots indicate apoptotic tumor cells de-
termined by PI and Annexin V–flourescein isothiocyanate
(FITC) staining. The total apoptotic cells are the sum of
PI
/Annexin V
(newly apoptotic) and PI
/Annexin V
(already apoptotic) cell populations.
12
Cell cycle analysis
To determine the effects of the fraction from shiitake
mushrooms on cell cycle progression, 5-bromo-2-de-
oxyuridine [BrdU] (Sigma) incorporation was used.
13
The
cells were cultured in medium containing various concen-
trations of the fraction for 24 h before cell cycle analysis.
Cell cycle distribution was analyzed by flow cytometry.
13
FANG ET AL.
126
Protein extraction and Western blot analysis
Two human breast carcinoma cell lines (MDA-MB-453
and MCF-7) and one human nonmalignant breast epithe-
lial cell line (MCF-10F) were used in Western blot analy-
sis. The cells were incubated in 75-cm
2
flasks and treated
with the fraction from shiitake mushrooms at 100, 400, or
800 mg/L concentrations for 24 h. Protein from treated
cells were fractionated by electrophoresis on 15% SDS-
PAGE and electrotransferred to immuneblot PVDF mem-
branes (Bio-Rad laboratories, Hercules, CA). PVDF mem-
branes incubated with appropriate dilutions of primary
antibodies in blocking buffer: anti-cdk4 (1:600), anti-
p21
WAF1
(1:500), anti-Bax (1:600), and anti-cyclin D1
(1:2000). Subsequently, the membranes were incubated
with the peroxidase-conjugated anti-rabbit secondary anti-
body diluted in blocking buffer: 1:6000 for anti-cdk4,
1:5000 for anti-p21
WAF1
, and anti-Bax; or the peroxidase-
conjugated anti-mouse secondary antibody 1:3000 for anti-
cyclin D1. Antibody-bound proteins were visualized by
ECL plus Western blotting analysis system (Amersham
Pharmacia Biotech, UK). The primary antibodies against
cdk4, p21
WAF1
, Bax, and two secondary antibodies were
purchased from Santa-Cruz Biotechnology (Santa Cruz,
CA), and anti-cyclin D1 antibody was from Cell Signaling
Technology (Beverly, MA).
LC-MS analysis
The fraction from shiitake mushrooms was directly ana-
lyzed by LC-MS. LC-MS was performed using a Bruker Es-
quire-LC multiple ion trap mass spectrometer equipped with
an Agilent 1100 series liquid chromatograph. An HP Chem-
Station was used for data collection and manipulation. A
150 4.6 mm i.d. Eclipse XDB-C8 column (Agilent Tech-
nologies, Wilmington, DE) was used with LC solvent at a
flow rate of 0.8 mL/min. The LC gradient was 0.1% formic
acid/acetonitrile (solvent B) in 0.1% formic acid/H
2
O (sol-
vent A) as follows: 10% to 15% in 15 min; 15% to 18%
from 15 to 16 min; 18% to 22% from 16 to 33 min; 22% to
40% from 33 to 40 min; held at 40% from 40 to 45 min;
40% to 42% from 45 to 49 min; 42% to 45% from 49 to 50
min; 45% to 50% from 50 to 65 min; 50% to 65% from 65
to 75 min; 65% to 85% from 75 to 76 min; 85% to 100%
from 76 to 80 min; held at 100% from 80 to 85 min and fi-
nally back to 10% in 90 min. Mycochemicals in the eluant
were analyzed by MS with positive ion modes. LC-MS
analysis was carried out using the methods described previ-
ously.
14
Statistical analysis
Results are means SD for at least three replicate de-
terminations for each assay. Significant differences between
two groups were determined by student’s t-test. Differences
with p-values 0.05 were considered significant.
RESULTS
Antiproliferative properties of the ethyl
acetate fraction
The authors assessed antiproliferative effects of the frac-
tion from shiitake mushrooms against five cell lines using
the MTT assay. Following incubation with the fraction at 0
without DMSO, 0 with 0.1% DMSO, 12.5, 50, 200, and 800
mg/L concentrations for 24 h, the fraction led to a concen-
tration-dependent reduction (p 0.05) of relative cell via-
bility (Fig. 1). IC
50
s for the suppression of cell growth for
two human breast carcinoma cell lines (MDA-MB-453 and
MCF-7) and two myeloma cell lines (RPMI-8226 and IM-
9) were 289 108, 179 34, 444 40, and 477 88
mg/L, respectively. However, at the highest concentration
of the fraction (800 mg/L), only 51% antiproliferative effect
occurred in the nonmalignant breast epithelial cell line MCF-
10F.
Fraction-induced apoptosis
To evaluate the fraction-induced apoptosis, two human
breast carcinoma cells and two multiple myeloma cells were
treated with the ethyl acetate fraction from shiitake mush-
rooms at 0 without DMSO, and 0 with 0.1% DMSO, 12.5,
CANCER-PREVENTING PROPERTIES OF SHIITAKE
127
FIG. 1. Antiproliferative effects on 5 cell lines by four concentra-
tions of an ethyl acetate extract from shiitake (Lentinus edodes) mush-
rooms for 24 hours of treatment as determined by [3-(4,5-dimethyl-
thiazol-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. 1
Medium as control; 2 0.1% dimethylsulfoxide; 3 12.5 mg/L;
4 50 mg/L; 5 200 mg/L; 6 800 mg/L. Each value repre-
sents the mean standard deviation of more than three replicates.
*P values 0.05, which were considered significant versus control.
50, 200, and 800 mg/L concentrations. The cells were se-
quentially stained with annexin V-FITC and PI following
24, 48, and 72 h of fraction treatments. The pattern of the
population stained with annexin V-FITC and/or PI of the
treated cells and their controls were quite different, which
indicated that the ethyl acetate fraction led to significant in-
duction of apoptosis in human breast carcinoma cells and
meyloma cells. The apoptotic effect of the fraction was dose-
dependent. The degrees of apoptosis were not significantly
different between human breast carcinoma cells and mey-
loma cells. Figure 2 shows approximately 50% of cells bind
annexin V-FITC and/or PI at 50 mg/L concentration of the
ethyl acetate fraction from shiitake mushrooms.
Fraction arrests the cell cycle of tumor cells
The cell cycle distribution of two human breast carcinoma
cell lines (MDA-MB-453 and MCF-7) and two myeloma
FANG ET AL.
128
10
0
10
0
10
1
10
2
Propidium Iodide Propidium Iodide
Propidium Iodide Propidium Iodide
10
3
10
4
10
1
10
2
Ammexom v-FITC
C
B
D
A
B
C
D
A
B
C
D
Ammexom v-FITC
10
3
10
4
0 DMSO 12.5 50
Mushroom fraction (mg/L)
200 800
0 50
% of Apoptotic cells
100
10
0
10
1
MDA
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
24 h
48 h
72 h
10
0
10
0
10
1
10
2
10
3
10
4
10
1
10
2
Ammexom v-FITC Ammexom v-FITC
10
3
10
4
0 DMSO 12.5 50
Mushroom fraction (mg/L)
200 800
*
*
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0 50
% of Apoptotic cells
100
10
0
10
1
MCF-7
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
24 h
48 h
72 h
10
0
10
0
10
1
10
2
10
3
10
4
10
1
10
2
Ammexom v-FITC Ammexom v-FITC
10
3
10
4
10
0
10
1
RPMI-8226
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
0
10
1
10
2
10
3
10
4
10
1
10
2
Ammexom v-FITC Ammexom v-FITC
10
3
10
4
10
0
10
1
Im-9
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
0 DMSO 12.5 50
Mushroom fraction (mg/L)
200 800
0 50
% of Apoptotic cells
100
24 h
48 h
72 h
0 DMSO 12.5 50
Mushroom fraction (mg/L)
200 800
0 50
% of Apoptotic cells
100
24 h
48 h
72 h
AA
CD
B
Propidium IodidePropidium Iodide
Propidium IodidePropidium Iodide
FIG. 2. Ethyl acetate extract from shiitake
(Lentinus edodes) mushroom–induced apop-
tosis in two human breast carcinoma cell
lines and two myeloma cell lines. Repre-
sentative dot plots showing apoptotic tumor
cells determined by PI and Annexin V–flu-
orescein isothiocyanate (FITC) staining.
Live cells (PI
/Annexin V
), newly apop-
totic cells (PI
/Annexin V
) and already
apoptotic cells (PI
/Annexin V
) are lo-
cated at LL, LR, and UR, respectively. In
each part of this figure where letters appear:
A control; B 50 mg/L for 48 hours;
C 200 mg/L for 48 hours; and D 800
mg/L for 48 hours. The percentages of
apoptotic cells are the sum of PI
/Annexin
V
(newly apoptotic cells) and PI
/An-
nexin V
(already apoptotic) cells popula-
tion by Flow cytometry analysis. Each value
represents the mean standard deviation of
more than three replicates. *P values 0.05,
which were considered significant versus
control. DMSO, dimethylsulfoxide.
cell lines (RPMI-8226 and IM-9) were examined 24 h after
treatment of the ethyl acetate fraction from shiitake mush-
rooms at 0 (0.1% DMSO), 50, 200, and 800 mg/L. The frac-
tion-treated cells exhibited prominent increases in the per-
centage of cells arresting in G0/G1 of all four cells (Fig. 3).
The effect of the fraction was dose-dependent.
Expression of bax, cdk4, cyclin D1, and p21
WAF1
To elucidate the mechanisms of the effects of the ethyl
acetate fraction from shiitake mushroom on apoptosis and
cell cycle distribution of the tumor cells, MDA-MB-453,
MCF-7, and MCF-10F cells were treated with the frac-
tion at 0 (0.1% DMSO), 100, 400, and 800 mg/L con-
centrations and the expression of related proteins in the
treated cells were examined by Western blot analysis. As
shown in Figure 4, the levels of bax and p21 protein ex-
pression were increased and, in contrast, the levels of cy-
clin D1 and cdk4 expression were decreased 24 h after
fraction treatment. As expected, nonmalignant breast ep-
ithelial cells MCF-10F were least sensitive to mushroom
extract.
LC-MS/MS fingerprint of the ethyl acetate
fraction from mushrooms
The ethyl acetate fraction was analyzed by LC-MS and
total ion chromatogram (TIC) from positive MS was estab-
lished as the fingerprint of the fraction. Although the iden-
tification of the chemical structures of bioactive compounds
has not been accomplished as yet, the fingerprint indicated
numerous components (peaks overlapped in TIC chro-
matogram) in the fraction (Fig. 5). The base peaks in the
mass spectra of TIC peaks a through j were shown in Fig-
ure 5 for the characterization of the fingerprint. Preparation
of fraction was repeated several times and the variability of
LC-MS profiles of different fractions from same prepara-
tion procedure did not have significant difference on the ba-
sis of the comparison of the number and areas of peaks in
LC-MS profiles.
CANCER-PREVENTING PROPERTIES OF SHIITAKE
129
Control 50
Mushroom fraction (mg/L)
200 800
0
20
40
60
80
100
G0/G1
Linear (G0/G1)
S
Linear (S)
G2/M
Linear (G2/M)
MDA-MB-453
Control 50
Mushroom fraction (mg/L)
200 800
0
20
40
60
80
100
MCF-7
Control 50
*
*
*
*
*
*
*
*
*
Mushroom fraction (mg/L)
200 800
0
20
40
60
80
100
RPMI-8226
Control 50
Mushroom fraction (mg/L)
200 800
0
20
40
60
80
100
IM-9
FIG. 3. Cell cycle analysis of two human breast carcinoma cell lines and two myeloma cell lines after exposure to an ethyl acetate
extract from shiitake (Lentinus edodes) mushrooms for 24 hours. Cell cycle distribution was analyzed by flow cytometry. *P values
0.05 were considered significant versus control.
DISCUSSION
Shiitake mushrooms are a good source of protein, fiber,
and certain vitamins (thiamine, riboflavin, niacin, D
2
, B
2
,
and B
12
). Improved growing techniques have resulted in
increased marketing of low-priced, high-quality shiitake
mushrooms and the consumption by Americans is at an all-
time high.
Shiitake mushrooms have been reported to have cancer-
preventing properties.
4,5,15
Most cancer-related research on
shiitake mushrooms has been conducted on the -(1 3)-
glucans polysaccharides (named lentinan), which are pro-
moted as major anticancer components in the shiitake mush-
room and are being used in some parts of the world as agents
to improve the outcome of cancer therapy. However, mam-
mals lack the necessary enzyme, -1 3-glucanase, and
lentinan has been reported to possess no antitumor activity
when administered orally.
3,9
These data suggest that myco-
chemicals may be responsible for the anticancer effects of
the shiitake mushroom. This report describes the antitumor
activities of mycochemicals in the ethyl acetate fraction
(nonpolysaccharide) from shiitake mushrooms by in vitro
bioassay in two human breast carcinoma cell lines (MDA-
MB-453 and MCF-7), one nonmalignant breast epithelial
cell line (MCF-10F), and two myeloma cell lines (RPMI-
8226 and IM-9).
Antiproliferative properties of the ethyl acetate fraction
from shiitake mushrooms were observed in all cell lines us-
ing the MTT assay. Antiproliferative effects of the fraction
were concentration-dependent and the degree of antiprolif-
erative activity in human breast carcinoma cell lines was
higher than in nonmalignant breast epithelial cell line (Fig.
1). Similarly, there was a dose-dependent increase of apop-
tosis in the treated cells (Fig. 2), consistent with the cell
growth suppression detected in the MTT assay. Fraction–in-
duced apoptosis occurred at lower concentrations than the
FANG ET AL.
130
Bax p21
*
*
Cdk4Cyclin D1
Protein levels (ADU)
0.0
2.0
4.0
6.0
8.0
Cdk4
Cyclin D1
P21
Bax
DMSO
DMSO 100 400 800 DMSO 100 400 800 DMSO 100 400
MDA-MB-453 MCF-7 MCF-10F
800
*
*
*
*
*
*
*
*
*
Bax p21
*
*
Cdk4Cyclin D1
0.0
2.0
4.0
6.0
8.0
*
*
*
*
*
*
*
*
Bax p21
*
*
*
*
*
Cdk4Cyclin D1
0.0
0.5
1.0
1.5
2.0
*
100 mg/L 400 mg/L 800 mg/L
FIG. 4. Expression of bax, p21, cyclinD1, and cdk4 in MDA-MB-453, MCF-7, and MCF-10F cells with Western blot analysis. The
cells were treated with the ethyl acetate fraction from shiitake (Lentinus edodes) mushrooms in various doses for 24 hour. Each value
represents the mean standard deviation of three replicates. *P values 0.05, which was considered significant versus control.
FIG. 5. LC-MS fingerprint of the ethyl acetate fraction from shi-
itake (Lentinus edodes) mushrooms that have anticancer activities.
The base peaks in the mass spectra of peaks a through j were at
m/z 277, 279, 498, 498, 377, 377, 413, 379, 737, and 313, respec-
tively, and used for the characterization of the fingerprint. Inten,
intensity.
concentration for the antiproliferative effect determined by
MTT because MTT was used to determine the dead cells
and the apoptosis bioassay measured the damaged cells and
dead cells together. Western blot analysis showed that treat-
ment with the fraction from shiitake mushrooms enhanced
expression of the proapoptotic bax protein levels in human
breast carcinoma cell lines (Fig. 4), suggesting that fraction-
induced apoptosis of the cells might be mediated by in-
creasing bax expression.
Cell cycle analysis showed an increased proportion of
cells in G0/G1 phase, indicating an arrest of cell cycle pro-
gression from the G1 to the S phase (Fig. 3). Cell cycle pro-
gression is accelerated by cyclins and cdks, and decelerated
by cdk inhibitors, such as p21.
16
The D-type cyclins (cyclin
D1, D2, and D3) are involved in regulation of transition from
G1 to S during cell cycle. In this study, MDA-MB-453 and
MCF-7 cells treated with the fraction showed upregulation
of p21, and downregulation of cyclin D1 and cdk4. These
results suggest that the fraction from shiitake mushrooms
induces G1 arrest in cells via the induction of cdk inhibitors
(p21), with the suppression of cyclin D1 and cdk4 activi-
ties. Compared to malignant cell lines, nonmalignant MCF-
10F cells were less sensitive to effects of the fraction on the
suppression of cell growth and the regulation of expression
of cdk4, cyclin D1, and p21 (Fig. 4).
Concentrations of an ethyl acetate fraction from shiitake
mushrooms that elicited bioactivity in this study were high
(50 mg/L). However, LC-MS fingerprint revealed that this
bioactive fraction consists of more than 100 mycochemicals
(peaks) without major components (Fig. 5). It is reasonable
to assume that the bioactivity in the fraction results from
one or more specific mycochemicals, but not all myco-
chemicals in the fraction. The potency of the bioactive my-
cochemicals, alone or together, should be much higher than
the potency of the whole fraction.
CONCLUSIONS
Shiitake mushrooms have been reported to have cancer-
preventing properties and lentinan (-1 3-glucans poly-
saccharides) isolated from shiitake mushrooms are being
used in some parts of the world as an adjuvant in cancer
chemotherapy. However, mammals lack the necessary en-
zyme, -1 3-glucanase, and lentinan has been reported to
possess no antitumor activity when administered orally.
3,9
The inhibitory activities of the ethyl acetate fraction from
shiitake mushrooms against several human cancer cells lines
were detected by in vitro bioassay in this study, which sug-
gests that mycochemicals may be responsible for the anti-
cancer effects of the shiitake mushroom in humans,
1–3
and
in in vivo study.
4,5
LC-MS analysis of the fraction reveals
more than 100 chemicals in the fraction without quantita-
tively dominant mycochemical constituent. Clearly, further
work is needed to identify the antitumor mycochemicals in
the ethyl acetate fraction from shiitake mushrooms and de-
lineate their potency and mechanisms of action.
ACKNOWLEDGMENTS
The authors thank Drs. Frank Simmen and R. Terry Pivik
for helpful comments. Funds for this research were provided
in part by NIH/NCI (#MBS-95016-13) and the USDA
Arkansas Children’s Nutrition Center (#6251-51000-003-
06S).
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Address reprint requests to:
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Arkansas Children’s Nutrition Center
1120 South Marshall Street
Little Rock, AR 72202
E-mail: fangnianbai@uams.edu
FANG ET AL.
132
... β-glucan, a bioactive polysaccharide derived from Maitake mushroom, exhibited antitumor activity by promoting apoptosis in human prostate cancer cells [117]. Ethyl acetate fraction of shiitake (Lentinus edodes) mushrooms have shown to possess anti-cancer properties by inducing growth inhibition and apoptosis in human cancer cell lines [118][119][120]. In vitro study reported that Grifolin, isolated from Albatrellus confluens, exhibited anti-cancer effect by inducing apoptosis [121]. ...
... One of the advantages of using mushroom-derived metabolites in cancer treatment is that these metabolites can act as therapeutic agents that can stimulate the immune system against cancer cells. Most of them stimulate the innate immune response of the host and hence can be used as supportive treatments in conjunction with conventional therapies for the prevention of cancer [119,120]. Therefore, many of the mushrooms derived products were considered as either immunomodulators or biological response modifiers (BRMs) [47]. Thus, it can be conclusively said that medicinal mushrooms can act both as strong immunostimulators and a source of good anti-cancer agents and are capable of disrupting cellular signal pathways linked to cancer complications [121]. ...
... Grifola frondosa inhibits the cyclooxygenase activity towards cancer inhibition [184]. Polyporenic acid C isolated from Piptoropus betulinus and Daedalea dickinsii found an effective inhibitor of Murine double minute 2 (Mdm2), which is a proto-oncogene and encodes a negative regulator of the p53 tumor suppressor and thus, promotes cancer [120,185]. 5,6-epoxy-24-methylcholesta-7,22-dien-3β-ol (EMCD) is another potent Mdm2 inhibitor, isolated from the mushroom species, Cordyceps sinensis, and is shown to inhibit the proliferation of K562, Jurkat, WM-1341, HL-60, and RPMI-8226 tumor cell lines [186]. ...
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