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Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata are mediated via downregulation of the expressions of four tumor-related genes in human non-small cell lung carcinoma A549 cell
Lan-Hsiang Chien (簡嵐翔), Yu-Yi Chan (詹于宜), Chun-Sheng Chang (張春生), Ting-Feng Wu* (吳定峰)
Department of Biotechnology and Biotechnology Research Center, Southern-Taiwan University, Tainan, Taiwan
Abstract
Antrodia camphorata (niu-chang-chih) is a fungus
native to Taiwan which is believed to be effective in
preventing diseases. Recent reports demonstrate that A.
camphorata products induce the apoptosis of various
kinds of tumor cells. In this study we determined the
inhibitory effects of alcohol extract and individual fractions
of alcohol extract on the proliferation of human non-small
cell lung carcinoma A549 cell and clarified the
mechanism underlying the anti-cancer activities.
Three HPLC fractions, fractions 5 to 7, had robust
inhibition of human A549 cells and among them fraction 6
(Fr-6) possessed the most potent effectiveness. Apoptotic
assay showed that Fr-6 induced human A549 cell
apoptosis by triggering the mitochondrial pathway and
endothelium reticulum (ER) stress. Immunoblotting
results demonstrated that Fr-6 possibly activated the
mitochondrial pathway and ER stress by lowering the
expression level of calpain 1/2 small subunit and Fr-6mediated decrease in cell proliferation might attribute to
the suppressive effect on the Erk 1/2 pathway, which
arose from Fr-6-derived low galectin-1 expression.
Furthermore Fr-6 could diminish Rho GDP dissociation
inhibitor  (RhoGDI-) expression and subsequently
activated c-Jun NH2-terminal kinase (JNK) pathway,
which is linked to cell apoptosis. Fr-6 also could decrease
the production level of eukaryotic translation initiation
factor 5A, which is a potential cancer intervention target.
Results and discussion
A. camphorata mycelia
Hexane
ppt.
Sup.
EtoAc
Assay
ppt.
Sup.
EtoH
ppt.
Assay
Sup.
H2O
ppt.
Assay
Sup.
Assay
mAU
1
2
3
4
5
6
7
8
2000
2000
1000
1000
0
mAU
B
0
0
25
50
75
100
125
Minutes
Figure 1. Preparation for Antrodia camphorata extracts
( A ) The extraction flow chart ( B ) The ethanol extracts of A.
camphorata were separated using HPLC column and the
elute was collected every fifteen minutes. Totally eight
B
Afractions were obtained.
A
B
0hr 15min 30min 1hr
60
40
20
80
3
4
5
6
7
Separation of the ethanol extract of A. camphorata
by high performance liquid chromatography (HPLC)
The ethanol extracts were separated using HPLC
column (250 x 4.6 ㎜, Hypersil, 5μm ) with a linear
solvent gradient elution system composed of solvents
A and B ( A：H2O；B：100 % acetonitrite ) The elute
was collected every 15 minutes and totally eight
fractions were obtained. Each fraction was dissolved
in dimethyl sulfoxide ( DMSO ) and control cultures
received the carrier solvent ( 0.1 % DMSO ).
Cell viability
Cell viability was measured by MTT assay.
10μg/ml
25μg/ml
2hr
4hr
0.00
8hr
0hr
50μg/ml
100μg/ml
150μg/ml
C
200μg/ml
2hr
4hr
48h
2hr
4hr
8hr
eIF-5a
Actin
Actin
100
1.60
2.10
50
5μg/ml
10μg/ml
25μg/ml
50μg/ml
100μg/ml
150μg/ml
200μg/ml
Concentration
Treatment Times
1.40
1.90
1.80
1.70
1.60
15min
30min
1hr
0.60
0.40
4hr
0hr
8hr
15min
30min
1hr
2hr
4hr
8hr
B
0hr 15min 30min
24h
2hr
A
50μg/ml
control
0.80
Figure 5. Effect of fraction 6 on galectin-1, calpain1/2, RhoGDI-α and eIF-5a expression To
investigate if Fr-6 had the same effect on the
expression of these four tumor-related genes, western
blotting with the antibody against each of these four
proteins was carried out. (A), (B), (C), (D) Fraction 6
(50μg / ml) caused a time-dependent decrease in
galectine-1, calpain-1 / 2, RhoGDI-α and eIF-5a
expression after treatment. These results suggest
ed that fraction 6 could reduce the expression of the
proteins as indicated above on A549 cell.
C
50μg/ml
1.00
0.00
0hr
Figure 2. The inhibitory effects of HPLC fractions on
human A549 cells. (A) Treatment with 200μg/ml for 72 h,
the maximum inhibitory effect on proliferation was fractions
5-7. ( B ) Treatment with various concentration of fraction
5-7 for 72 h, 25 μg / ml fraction 6 inhibited cell proliferation
at 72 % , indicating
that human A549 cell was more
sensitive to fraction 6 than to fraction 5 and 7.
( C )
Treatment with 50μg / ml of fraction 6 for 0~72 h, which
inhibited cell proliferation at 80% and the result was similar
with Fig. 2B. (D) Treatment with various concentration of
fraction 6 on MRC-5 cell, the inhibitory effect was observed
with 100μg/ml. These results suggested that normal ( MRC5 ) and cancer (A549) cell lines exhibit differential sensitivity
to fraction 6.
1.20
0.20
1.50
control
2hr
150
72hr
A
1hr
0hr 15min 30min 1hr
8hr
eIF-5a / actin
24h
30min
D
RhoGDI-α
200
0
0
12hr
15min
250
1hr
2hr
4hr
8hr
0hr 15min 30min 1hr
ERK-1
p-ERK-1
ERK-2
p-ERK-2
Actin
Actin
0.90
ERK-1
0.80
ERK-2
4hr
8hr
p-ERK-2
0.9
0.60
0.50
p-ERK / actin
ERK / actin
48h
2hr
p-ERK-1
0.70
0.40
0.30
0.6
0.3
0.20
0.10
0.00
0hr
72h
15min
30min
1hr
2hr
4hr
0.0
8hr
0hr
C
15min
30min
1hr
2hr
4hr
8hr
D
0hr
4hr
8hr
12hr
0hr
24hr
4hr
8hr
12hr
24hr
p-JNK1
D
Fraction-6
70
60
50
40
30
20
10
0
24hr
48hr
100.00
90.00
Early apoptosis
80.00
60.00
50.00
0.70
40.00
0.60
20.00
0.50
10.00
0.00
Control24hr
Fraction
6-24hr
Control48hr
Fraction
6-48hr
Control72hr
Fraction
6-72hr
Figure 3. Apoptosis assay
( A ) , ( B ) The
remarkable accumulation of sub-G1 was considered
apoptosis.
Fraction- 6 (50μg/ml) elevated the
percentage of human A549 cells in the sub-G1 phase
from 24% to 34%, 64% (24h~72h) when compared
with the control group. (C),(D) Compared with
vehicle-treated cells, 50μg / ml fraction 6 induced
17%, 29%, 13% of early apoptosis cells and 6%,
11%, 75% of late apoptosis cells at 24h, 48h and 72
h. These results suggest edthat fraction 6 could
induce apoptosis on A549 cell. However, cytometric
analysis could not determine whether Fr-6 leaded to
the apoptosis by death receptor signaling,
mitochondrial damage or ER stress. To explore the
mechanism by which Fr-6 invoked the apoptosis,
western blotting with anti-cappase-3, -12 and bax/bcl2 were carried out in Fig. 4.
A
0.7
48hr
72hr
Pro-caspase3
Actin
0.40
0.30
0.10
0.10
0.00
0.00
0hr
0hr
4hr
8hr
12hr
4hr
8hr
12hr
P-JNK1
P-JNK2/3
24hr
48hr
12hr
72hr
24hr
3.00
Bax
2.00
100
80
60
40
0
0hr
0hr
2hr
4hr
8hr
8hr
1.50
24hr
48hr
72hr
Treatment Time
1.00
0.50
Actin
50μg/ml fraction-6
fraction-6+15uM SP600125
20
Fraction-6+SP600125
B
2.50
24hr
140
120
0.3
0.2
12hr
C
JNK
0.4
8hr
Figure 6. Effect of fraction 6 on ERK and JNK activation.
Recent findings showed that the recruiting of H-Ras to the
cell membrane is dependent on galectin-1 and galectin-1
binds H-Ras to stimulate the transformation (Belanis et al.,
2008; Paz et al., 2001). Since Ras take part in Erk pathway,
western blotting with the antibodies against Erk and
phospho-Erk (p-Erk) to examine if Fr-6 could interfere with
Erk pathway. (A), (B) The down-regulation of ERK and
phospho-ERK expression were detected at time dependent after treatment. Recent investigations showed
that he inhibition of RhoGDI- production invokes the
apoptosis of insulin-secreting cells by the activation of cJun NH2-terminal kinase (JNK) pathway. (C) The downregulation of JNK expression was detected at timedependent after treatment. (D) The up-regulation of
phospho-JNK expression was detected at timedependent after treatment. These results suggest that
fraction 6 could prevent the activation of ERK, but promote
the activation of JNK.
Actin
8hr
4hr
24hr
0.5
0hr
4hr
0.30
0.20
0hr
0.1
0hr
0.40
0.20
0.6
0
B
p-JNK-2/3
0.50
A
0.8
p-JNK-1
0.60
30.00
72hr
24hr
Actin
Actin
70.00
Treatment Time
0hr
p-JNK2/3
Late apoptosis
p-JNK / actin
Control
80
JNK
Apoptotic cells (% of tatal cells)
90
JNK / actin
B
RhoGDI
0.00
0hr
4hr
8hr
12hr
24hr
Actin
C
1
Actin
4hr
8hr
12hr
0.9
24hr
0.8
caspase12 / actin
0hr
Pro-caspase12
0.7
Fraction-6+SP600125
0.6
F-6
0.5
0.4
0.3
0.2
0.1
0
0hr
After treatment as indicated above , cells
were collected and lysed in the lysis buffer. Total cell
lysates were separated by 12.5 % SDS- PAGE and
transferred onto PVDF membranes. The membrane was
blotted with primary antibody overnight , followed by
incubation
with
HRP-conjugated
secondary
antibody and visualized using chemiluminescence.
1hr
2.00
Apoptosis analysis
Western blotting
30min
0hr 15min 30min 1hr
Survival ratio (% of control)
Survival ratio (% of control)
20
Bcl-2
SubG1 analysis and Annexin V/propidium iodide (PI)
assay were used to observe human A549 cell apoptosis
15min
Survival ratio (% of control)
A.
camphorata
mycelia
were
provided
by
Biotechnology Center, Southern Taiwan University. A.
camphorata mycelia (about 555 g) were sequentially
extracted using the solvent with increasing polarity,
including n-hexane, ethylacetate, ethanol and wtaer
(Figure 1A). 20.1 g, 16 g and 127.6 g of the products
were obtained respectively in the EtOAc, EtOH and
water extraction experiments.
40
8hr
0.20
20
caspase3 / actin
Preparation for Antrodia camphorata extracts
60
4hr
0.30
0.10
0hr
Bax / Bcl-2
Methods
80
0hr
0.40
Concentration
100
sub-G1(% of total cells)
However, the wild fruiting bodies of A. camphorata
are rarity in nature, very expensive and the failure of
artificial cultivation . Therefore , using a submerged
cultured method to obtain useful cellular materials or to
produce effective substances from cultured mycelia ,
might be a possible way to overcome the disadvantage
of the fruiting bodies ( Sone et al., Agricultural and
Biological Chemistry , 1985 ). The mycelia of Antrodia
camphorata in submerged culture induced apoptosis
in human hepatoma cells ( Tuzz et al., Journal of
Ethnopharmacology , 2005 ). A. camphorata inhibited
proliferation and induced apoptosis of human breast
cancer cells in vitro and in vivo ( Hseu et al., Food and
Chemical Toxicology , 2008 ) . Our previous study
demonstrates that ethanol extracts of A. camphorata
cultivated by solid -state fermentation could effectively
impede the proliferation of human lung cancer A549
cells and inhibit the expression of galectin-1, eIF-5a,
RhoGDI-α and calpain-1/2 ( Hung et al., Proteomics,
2006). However, no information about their mechanism
of action and significant compounds. To explore their
significant compounds , the ethanol extracts of A.
camphorata (AC-EtOH) were separated by HPLC and
collect to one fraction every fifteen minutes. This study
evaluates the whether the eight fractions also have the
inhibitory effect on A549 cell line and explore their
mechanism of action.
0.40
0.00
Fraction
120
8hr
Actin
0.20
5μg/ml
D
4hr
(small subunit)
0.60
40
8
C
8hr
Calpain-1/2
0.80
60
0
2
4hr
0.50
Fraction-7
0
1
2hr
0.60
RhoGDI / actin
Survival ratio
(% of control)
80
0hr 15min 30min 1hr
1.00
Fraction-6
100
8hr
1.20
Fraction-5
120
4hr
Actin
galectin-1 / actin
Survival ratio (% of control)
Introduction
100
2hr
Galectin-1
140
120
Antrodia camphorata is the traditional Taiwan crude
drug for treating diarrhea, hypertension , inflammation
and cancer. Previous studies have shown that the
Antrodia camphorata crude extract has anti-proliferation
and anti-migration on the transitional cell carcinomas
(Chiung, et al., 2007) and ethanol extract from wild
fruiting bodies could induce leukemia HL-60 cells
apoptosis ( Mei et al., 2008 ). Recent studies has
identified that lanostane and ergostane - type
triterpenes isolated from the fruiting bodies of A.
camphorata could inhibit proliferation of human colon,
live, breast, lung cancer cells ( Chi et al., 2009).
Figure 4. Effects of fraction 6 on caspase-3,
caspase-12 activation and Bax, Bcl-2 expression
( A ) A549 cells treated with fraction 6 caused a timedependent decrease in pro-caspase3. (B) Bax could
induce apoptosis, but Bcl-2 could inhibit apoptosis on
the mitochondria pathway. The up-regulation of Bax
/ Bcl-2 expression was detected at time-dependent
after treatment.
(C) ER-induced apoptosis is
associated with the activation of caspase12. Fraction 6
also caused a time-dependent decrease in procaspase12. These results suggested that fraction 6
could induce apoptosis on
A549
cells through
mitochondria pathway and ER-induced pathway. Our
previous examinations suggested that the ethanol
extracts of A. camphorata (SACE) can evoke the
apoptosis of human A549 cells possibly by the downregulation of human galectin-1, human eIF5A, human
Rho GDI-, human calpain small (regulatory) subunit
cells (Wu et al., 2006).
calpain-1/2 / actin
A
4hr
8hr
12hr
24hr
Figure 7. Effect of fraction 6 on activated
JNK expression in relation to RhoGDI-α Cells were
treated with 50μg / ml fraction 6 alone or 15μM JNK
inhibitor ( SP600125 ) for the time periods indicated. (A),
(B) The SP600125 was administered to cells 24 h prior to
fraction- 6 treatment. Pre-treatment with JNK inhibitor
could prevent the activation of JNK and the reduction
in RhoGDI-α expression caused by fraction 6. ( C )
Viability of cells treated with both fraction 6 and the JNK
inhibitor was 42 % greater than that of cells treated with
fraction 6 alone at 72 h. These results suggested that
fraction 6-induced cell death might occur through an