molecules
Article
Anticancer Activity of γ-Bisabolene in Human
Neuroblastoma Cells via Induction of p53-Mediated
Mitochondrial Apoptosis
Yu-Jen Jou 1,† , Chun-Hung Hua 2,† , Chen-Sheng Lin 3,† , Ching-Ying Wang 1 , Lei Wan 4 ,
Ying-Ju Lin 4 , Su-Hua Huang 5, * and Cheng-Wen Lin 1,5, *
1
2
3
4
5
*
†
Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91,
Hsueh-Shih Road, Taichung 404, Taiwan; [email protected] (Y.-J.J.); [email protected] (C.-Y.W.)
Department of Otolaryngology, China Medical University Hospital, Taichung 404, Taiwan;
[email protected]
Division of Gastroenterology, Kuang Tien General Hospital, Taichung 433, Taiwan; [email protected]
Department of Medical Genetics and Medical Research, China Medical University Hospital,
Taichung 404, Taiwan; [email protected] (L.W.); [email protected] (Y.-J.L.)
Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung 413, Taiwan
Correspondence: [email protected] (S.-H.H.); [email protected] (C.-W.L.); Tel.: +886-4-23323456
(ext. 6353) (S.-H.H.); +886-4-22053366 (ext. 7210) (C.-W.L.); Fax: +886-4-22057414 (C.-W.L.)
These authors contributed equally to this work.
Academic Editor: Derek J. McPhee
Received: 23 March 2016; Accepted: 3 May 2016; Published: 7 May 2016
Abstract: γ-Bisabolene has demonstrated antiproliferative activities against several human cancer
cell lines. This study first discloses the antiproliferative and apoptosis induction activities of
γ-bisabolene to human neuroblastoma TE671 cells. A CC50 value of γ-bisabolene was 8.2 µM to TE671
cells. Cell cycle analysis with PI staining showed γ-bisabolene elevating the sub-G1 fractions in a
time-dependent manner. In addition, annexin V-FITC/PI staining showed γ-bisabolene significantly
triggering early (annexin-V positive/PI negative) and late (annexin-V positive/PI positive) apoptosis
in dose-dependent manners. γ-Bisabolene induced caspase 3/8/9 activation, intracellular ROS
increase, and mitochondrial membrane potential decrease in apoptosis of human neuro-blastoma cells.
Moreover, γ-bisabolene increased p53 phosphorylation and up-regulated p53-mediated apoptotic
genes Bim and PUMA, as well as decreased the mRNA and protein levels of CK2α. Notably, the
results indicated the involvement of CK2α-p53 pathways in mitochondria-mediated apoptosis of
human neuroblastoma cells treated with γ-bisabolene. This study elucidated the apoptosis induction
pathways of γ-bisabolene-treated neuroblastoma cells, in which could be useful for developing
anti-neuroblastoma drugs.
Keywords: γ-bisabolene; neuroblastoma; anticancer; apoptosis; p53; CK2α
1. Introduction
Neuroblastoma is a tumor of neural progenitor cells, which widely metastasizes to skin, bone
marrow, liver, and non-contiguous lymph nodes, and then becomes the most common extracranial
solid tumor in children [1]. Neuroblastoma is found in a greater than 7% of malignancies and near
15% of oncology deaths in children [2]. The infant patients with metastasis only to skin, bone marrow,
and liver have the good prognosis post treatment with the current anticancer drugs. The 5-year
event-free survival rate of children with disseminated neuroblastoma (Stage 4) is still less than 40%
post-treatment [3,4]. In addition, neuroblastoma has resistance to chemotherapeutic drugs, and even
Molecules 2016, 21, 601; doi:10.3390/molecules21050601
www.mdpi.com/journal/molecules
Molecules 2016, 21, 601
2 of 11
recurrence
the development of novel anti-neuroblastoma agents is still recommended
to
Molecules[2].
2016,Therefore,
21, 601
2 of 11
improve the patient outcomes.
recurrence
Therefore,
the development
of novel
anti-neuroblastoma
is still recommended
Apoptotic[2].
inducers
have
been widely
regarded
as potential agents
anti-cancer
therapeutics and
to improve the patient outcomes.
evaluated in pre-clinical or clinical trials [5]. Apoptosis results from initiating Fas receptor-mediated
Apoptotic inducers have been widely regarded as potential anti-cancer therapeutics and
or mitochondria-dependent pathways, subsequently converting DNA fragmentation, cytoskeletal
evaluated in pre-clinical or clinical trials [5]. Apoptosis results from initiating Fas receptor-mediated
protein
degradation, and apoptotic body formation [6]. Anti- and pro-apoptotic molecules included
or mitochondria-dependent pathways, subsequently converting DNA fragmentation, cytoskeletal
Bcl-2,
BclXL,
Bax, Bak,and
BAD,
BIM,body
andformation
BclXS, regulate
in mitochondria-dependent
apoptotic
protein degradation,
apoptotic
[6]. Anti- and
pro-apoptotic molecules included
pathways
[6]. Tumor
suppressor
protein
withinproapoptotic
activity is apoptotic
the key pathways
transcription
Bcl-2, BclXL,
Bax, Bak,
BAD, BIM, and
BclXS,p53
regulate
mitochondria-dependent
factor
up-regulation
of protein
pro-apoptotic
genes
like PUMA
and Noxa.
[6].forTumor
suppressor
p53 with
proapoptotic
activity
is the key transcription factor for
up-regulation
pro-apoptotic
genesinlike
and Noxa. and apoptotic activities against human
γ-Bisaboleneofshows
in vitro and
vivoPUMA
antiproliferative
γ-Bisabolene
shows
in
vitro
and
in
vivo
antiproliferative
and apoptotic
activities
against
oral squamous cell carcinoma [7]. γ-Bisabolene induces the apoptosis
of oral
squamous
cellhuman
carcinoma
oral
squamous
cell
carcinoma
[7].
γ-Bisabolene
induces
the
apoptosis
of
oral
squamous
cell
carcinoma and
via p53-medaited signaling pathways. This study further investigates the antiproliferative
via p53-medaited
pathways.
This human
study further
investigates the antiproliferative and
apoptotic
mechanismssignaling
of γ-bisabolene
against
neuroblastoma.
apoptotic mechanisms of γ-bisabolene against human neuroblastoma.
2. Results
2. Results
2.1. Growth Inhibition and Apoptosis Induction of γ-Bisabolene to Human Neuroblastoma
2.1. Growth Inhibition and Apoptosis Induction of γ-Bisabolene to Human Neuroblastoma
To examine the growth inhibitory ability of γ-bisabolene, the survival rates of human
To examine the growth inhibitory ability of γ-bisabolene, the survival rates of human neuroblastoma
neuroblastoma TE671 cells were examined using MTT assays 2 days post-treatment (Figure 1B).
TE671 cells were examined using MTT assays 2 days post-treatment (Figure 1B). γ-Bisabolene
γ-Bisabolene
inhibited
growth
ofinTE671
cells in a concentration-dependent
manner,
exhibiting
a
inhibited the
growththe
of TE671
cells
a concentration-dependent
manner, exhibiting
a CC50 value
of
CC508.2
value
of
8.2
µM
(Figure
1B).
μM (Figure 1B).
Figure 1. Survival rates and cell cycle analysis of human neuroblastom cells in response to γ-bisabolene.
Figure 1. Survival rates and cell cycle analysis of human neuroblastom cells in response to γ-bisabolene.
The structure of γ-bisabolene ((Z)-1-Methyl-4-(6-methylhept-5-en-2-ylidene) cyclohex-1-ene) was
The shown
structure
((Z)-1-Methyl-4-(6-methylhept-5-en-2-ylidene)
(A). of
Forγ-bisabolene
antiproliferative
assay (B), TE671 cells were grown in the indicatedcyclohex-1-ene)
concentrations of was
shown
(A).
For
antiproliferative
assay
(B),
TE671
cells
were
grown
in
the
indicated
concentrations
γ-bisabolene for 48 h. Survival rate was calculated based on MTT assays; For cell cycle analysis
(C),
of γ-bisabolene
for
48
h.
Survival
rate
was
calculated
based
on
MTT
assays;
For
cell
cycle then
analysis
treated cells with 20 μM γ-bisabolene were fixed by 70% ethanol, incubated with PI solution,
(C), examined
treated cells
with
µM γ-bisabolene
wereoffixed
by 70% ethanol,
incubated(D).
with
solution,
using
flow20
cytometry;
The percentage
cell distribution
was represented
***, PI
p value
then< examined
using
flow
cytometry;
0.001 compared
with
untreated
cells. The percentage of cell distribution was represented (D).
***, p value < 0.001 compared with untreated cells.
Molecules 2016, 21, 601
Molecules 2016, 21, 601
3 of 11
3 of 11
Meanwhile,
Meanwhile, cell
cell cycle
cycle analysis
analysisof
of flow
flow cytometry
cytometry with
with PI
PI staining
staining showed
showed the
the increase
increase in
in the
the
sub-G1
sub-G1fractions
fractionsand
andthe
thedecrease
decreasein
inthe
theG1
G1fractions
fractionsof
ofγ-bisabolene-treated
γ-bisabolene-treatedcells
cellscompared
comparedto
tomock
mock
controls
manner (Figure
(Figure1C,D).
1C,D).The
Theresults
resultsindicated
indicatedanti-proliferative
anti-proliferative
activity
controls in
in aa time-dependent
time-dependent manner
activity
of
of
γ-bisabolene
to
human
neuroblastoma
cells.
γ-bisabolene to human neuroblastoma cells.
2.2.
2.2.Apoptosis
ApoptosisofofNeuroblastoma
NeuroblastomaCells
Cells Induced
Induced by
by γ-Bisabolene
γ-Bisabolene
To
To test
test whether
whether γ-bisabolene
γ-bisabolene induces
induces apoptosis
apoptosisof
of human
human neuroblastoma
neuroblastomacells,
cells,the
the fractions
fractions of
of
early
early (annexin-V
(annexin-V positive/PI
positive/PInegative)
negative)and
andlate
late (annexin-V
(annexin-V positive/PI
positive/PIpositive)
positive)apoptosis
apoptosisin
in treated
treated
were
were determined
determined by
by flow
flow cytometer
cytometer with
with annexin
annexinV-FITC
V-FITC and
and PI
PI staining
staining (Figure
(Figure 2A–C).
2A–C). γ-Bisabolene
γ-Bisabolene
triggered
triggeredthe
thesignificant
significantincrease
increaseof
ofearly
earlyand
andlate
lateapoptosis
apoptosison
onTE671
TE671cells
cellsinindose-dependent
dose-dependentmanners.
manners.
To
To further
further examine
examine the
the mRNA
mRNA and
and protein
protein levels
levels of
of caspases
caspases3,3, 88 and
and 99 in
in treated
treated cells,
cells, TE671
TE671 cells
cells
treated
were
harvested
48
h
post
treatment
for
total
RNA
extraction
and
the
lysate
preparation.
treated were harvested 48 h post treatment for total RNA extraction and the lysate preparation.
Quantitative
Quantitative RT-PCR
RT-PCR revealed
revealed that
that γ-bisabolene
γ-bisabolene significantly
significantly induced
induced the
the mRNA
mRNA expression
expression of
of
caspases
(Figure
3A).
Caspases
3, 83,and
9 were
activated
to
caspases3,3,8,8,and
and99inindose-dependent
dose-dependentmanners
manners
(Figure
3A).
Caspases
8 and
9 were
activated
greater
than
5 folds
in in
response
toto
1010μM
to greater
than
5 folds
response
µMγ-bisabolene.
γ-bisabolene.Subsequently,
Subsequently,western
westernblots
blotsindicated
indicatedthe
the
dose-dependent
dose-dependentincrease
increase in
in propro- and
and active
active forms
forms of
of caspases
caspases3,
3,8,
8,and
and99in
inγ-bisabolene-treated
γ-bisabolene-treatedcells
cells
48
post treatment
treatment(Figure
(Figure3B–E).
3B–E).
Active
forms
of caspases
8, 9and
9 exhibited
3.5-,
1.6-,
and
48 h post
Active
forms
of caspases
3, 8, 3,
and
exhibited
3.5-, 1.6-,
and
2.3-fold
2.3-fold
post-treatment
with
10 μM γ-bisabolene,
respectively.
The results demonstrated
increasesincreases
post-treatment
with 10 µM
γ-bisabolene,
respectively.
The results demonstrated
γ-bisabolene
γ-bisabolene
induces
intrinsic
apoptosis
of human neuroblastoma.
induces extrinsic
andextrinsic
intrinsic and
apoptosis
of human
neuroblastoma.
A.
γ-Bisabolene
1
5
10
20
μM
C.
60
% Annexin V+/PI+ cells
(late apoptosis)
***
40
***
60
50
50
40
30
20
10
0
0
1
5
10
γ-Bisabolene (μM)
***
10
***
20
20
***
30
***
% Annexin V+/PI- cells
(early apoptosis)
B.
0
0
0
1
5
10
γ-Bisabolene (μM)
20
Figure 2.
2. Apoptosis
Apoptosisanalysis
analysisof
of human
human neuroblastom
neuroblastomcells
cells in
in responses
responses to
to γ-bisabolene.
γ-bisabolene. Cells
Cellswere
were
Figure
harvested 48
48 hh post
post treatment,
treatment, stained
stained by
by Annexin
Annexin V-FITC/PI
V-FITC/PIdye,
dye,and
andthen
thenanalyzed
analyzedusing
usingflow
flow
harvested
cytometry
(A);
Annexin
V
positive/PI
negative
indicated
early
phase
of
apoptosis
(B);
Annexin
cytometry (A); Annexin V positive/PI negative indicated early phase of apoptosis (B); Annexin VV
positive/PIpositive
positivepresented
presentedas
aslate
lateapoptosis
apoptosis(C).
(C).***,
***,ppvalue
value<<0.001
0.001compared
comparedwith
withuntreated
untreatedcells.
cells.
positive/PI
*
***
1
5
γ-Bisabolene (μM)
4
3
γ-Bisabolene
(μM)
***
2
Caspase 3
1
Active0Caspase 3
10
***
0
1
**
5
17 kDa
02.5
E.
2
**
**
**
***
Caspase 8
Active caspase 8
0
1
D.0.5 3
10
32 kDa
*
D.
5
γ-Bisabolene (μM)
10
**
***
**
***
3
2.5
3
2
2.5
1.5
21
0.5
1.5
0
1
5
10
γ-Bisabolene (μM)
Caspase 8
Active
caspase 18
0
5
10
γ-Bisabolene (μM)
**
***
***
Caspase 3
0Active caspase
1 3
*****
***
caspase 3
caspase 8
caspase 9
***
B.
6
11
5
10
4
9
3
8
2
7
1
6
0
5
4 of 11
4 of 11
**
A.
Caspase 3
Active caspase 3
***
**
***
8 2016, 21, 601
Molecules
7
Caspase/β actin
Relative expression levels
Relative expression levels
***
4
3.5
3
2.5
2
1.5
1
C.0.5 4
0
3.5
Caspase/βCaspase/β
actin
actin
caspase 3
caspase 8
Molecules
601 9
9 2016, 21,caspase
10
***
C.
11
Caspase/βCaspase/β
actin
actin
A.
4 of 11
***
Molecules 2016, 21, 601
**
**
***
**
**
***
**
**
Caspase/β actin
***
**
10
55 kDa
**
5
γ-Bisabolene (μM)
***
4
1.5
Caspase 9
3.5
Active caspase 9
1
3
Caspase 8
B.
2.5
0.5
0
1
5
10 18 kDa
γ-Bisabolene
(μM)8
2
Active Caspase
1.50
4632kDa
kDa
0
1
5
10
Caspase
3
Caspase 9
1
γ-Bisabolene
(μM)
17kDa
kDa
E.0.5 4
Active Caspase
Caspase 93
34
Active
Caspase 9
0
3.5
Active
caspase19
0
5
10
55
kDa
43
kDa
β-actin
3
Caspase 8
γ-Bisabolene (μM)
2.5
18 kDa
2
Active Caspase 8
Figure 3. Relative mRNA and protein levels of caspases-3, 8, and
1.5 9 in γ-bisabolene-treated cells. Cells
46 kDa
9 for total RNA extraction and western blotting 48
1 h post treatment. The relative gene
were Caspase
harvested
0.5
34 kDa
Active
Caspase
9 normalized to GAPDH in real-time
expression
was
PCR assays 0(A); Active forms of caspases 3, 8 and
5
10
9 in TE671
were characterized using western blotting
(B); Relative band0 intensity1 of indicated
caspase
43 kDa
β-actin
1
γ-Bisabolene (μM)
or active caspase was normalized by β actin, compared to the mock cells, and quantified using imageJ
based
on3.triplicate
of each
experiment
(C–E). **, p value
***, p value < 0.001 compared
Figure
Relativereplicates
mRNA and
protein
levels of caspases-3,
8, and<90.01;
in γ-bisabolene-treated
cells. Cells
Figure 3. Relative mRNA and protein levels of caspases-3, 8, and 9 in γ-bisabolene-treated cells. Cells
with
untreated
cells.
were
harvested
for
total
RNA
extraction
and
western
blotting
48
h
post
treatment.
The
relative
gene
were harvested for total RNA extraction and western blotting 48 h post treatment. The relative gene
expressionwas
wasnormalized
normalized
GAPDH
real-time
PCR
assays
(A);
Active
forms
of caspases
8 and
expression
toto
GAPDH
inin
real-time
PCR
assays
(A);
Active
forms
of caspases
3, 83,and
9
2.3. ROS
Production
Increase
and Mitochondrial
Membrane
Potential
Decrease
in Treated
Cells caspase
9
in
TE671
were
characterized
using
western
blotting
(B);
Relative
band
intensity
of
indicated
in TE671 were characterized using western blotting (B); Relative band intensity of indicated caspase or
or active
caspase
normalized
byactin,
β actin,
to the
mock
cells,
and
quantified
using
imageJ
active
caspase
waswas
normalized
by β
compared
to the
mock
cells,
and
quantified
image
J the
To
examine
the
apoptotic
pathways
of compared
γ-bisabolene-induced
apoptosis,
theusing
changes
in
based
on
triplicate
replicates
of
each
experiment
(C–E).
**,
p
value
<
0.01;
***,
p
value
<
0.001
compared
based
on
triplicate
replicates
of
each
experiment
(C–E).
**,
p
value
<
0.01;
***,
p
value
<
0.001
compared
intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) in
withuntreated
untreatedcells.
cells.
with
γ-bisabolene-treated
cancer cells was subsequently surveyed using flow cytometry analysis with
DCFH-DA and DiOC6(3) staining, respectively (Figures 4 and 5). γ-Bisabolene treatment induced
2.3. ROS Production Increase and Mitochondrial Membrane Potential Decrease in Treated Cells
2.3. ROS
Production
Increaseneuroblastom
and Mitochondrial
Potential Decrease
in (Figure
Treated Cells
ROS
production
in human
cellsMembrane
in a dose-dependent
manner
4).
To examineanalysis
the apoptotic
pathways
of γ-bisabolene-induced
apoptosis, the
changes
in the
Quantitative
of
MMP
changes
using
DiOC6(3)
staining
demonstrated
that
γ-bisabolene
To examine the apoptotic pathways of γ-bisabolene-induced apoptosis, the changes
in the
intracellular
reactive
oxygen
species
(ROS)
levels
and
mitochondrial
membrane
potential
(MMP)
significantly
the MMP
of human
neuroblastom
cells (Figure 5A,B);
a 97% decrease
MMPin
intracellular decreased
reactive oxygen
species
(ROS)
levels and mitochondrial
membrane
potentialin(MMP)
γ-bisabolene-treated
cancer
cells was
subsequently
surveyed using
flow
cytometry
analysis
with
was
found in TE671 cells
treated
5 μM of γ-bisabolene
5B).cytometry
Therefore,
the results
in γ-bisabolene-treated
cancer
cells with
was subsequently
surveyed (Figure
using flow
analysis
with
DCFH-DA
and DiOC6(3)
staining,
respectivelyand
(Figures
4the
and
5). γ-Bisabolene
treatment
induced
indicated
γ-bisabolene
induces
ROS
production
injures
mitochondria
in
apoptosis
of
human
DCFH-DA and DiOC6(3) staining, respectively (Figures 4 and 5). γ-Bisabolene treatment induced ROS
ROS production
in human neuroblastom cells in a dose-dependent manner (Figure 4).
neuroblastom
cells.
production in human
neuroblastom cells in a dose-dependent manner (Figure 4).
Quantitative analysis of MMP changes using DiOC6(3) staining demonstrated that γ-bisabolene
significantly decreased the MMP of human neuroblastom cells (Figure 5A,B); a 97% decrease in MMP
was found in TE671 cells treated with 5 μM of γ-bisabolene (Figure 5B). Therefore, the results
indicated γ-bisabolene induces ROS production and injures the mitochondria in apoptosis of human
neuroblastom cells.
Figure
Figure 4.
4. Cont.
Cont.
Figure 4. Cont.
Molecules
2016,
Molecules
2016,
21,21,
601601
Molecules 2016, 21, 601
5 of
5 of
1111
5 of 11
Figure
ROS production
productionininγ-bisabolene-treated
γ-bisabolene-treated
cells.
Cells
were
treated
Figure4.4.Increase
Increaseof
of intracellular
intracellular ROS
cells.
Cells
were
treated
with
Figure
4.
Increase
of
intracellular
ROS
production
in
γ-bisabolene-treated
cells.
Cells
were
treated
with
γ-bisabolene
for
48
h,
harvested
stained
using
DCF-DA,
and
then
analyzed
by
flow
cytometry
γ-bisabolene for 48 h, harvested stained using DCF-DA, and then analyzed by flow cytometry with
with
γ-bisabolene
48spectra
h, harvested
stained
using
and(A);
then
analyzed
by flow
cytometry
with
excitation
and for
emission
spectra
495
and
529
nm respectively
(A); fluorescent
Relative
fluorescent
excitation
and emission
of 495of
nm
andnm
529
nmDCF-DA,
respectively
Relative
intensity
of
with
excitation
and
emission
spectra
of
495
nm
and
529
nm
respectively
(A);
Relative
fluorescent
intensity
of
DCF
was
further
calculated
(B).
***,
p
value
<
0.001
compared
with
untreated
cells.
DCF was further calculated (B). ***, p value < 0.001 compared with untreated cells.
intensity of DCF was further calculated (B). ***, p value < 0.001 compared with untreated cells.
A.
A.
High MMP
High MMP
Low MMP
Low MMP
5
1
0
5
1
B.
B. 120
20
20
***
10
10
20
20
(%)
Low
MMP
(%)
Low
MMP
***
***
120
100
100
80
80
60
60
40
40
20
20
0
00
10
10
***
0
γ-Bisabolene (μM)
γ-Bisabolene (μM)
1
0
1
5
γ-Bisabolene
5 (μM)
γ-Bisabolene (μM)
Figure 5. Loss of mitochondrial membrane potential (ΔΨM) in human neuroblastom cells treated with
Figure 5. Loss of mitochondrial membrane potential (∆ΨM) in human neuroblastom cells treated with
Figure 5. Loss
of mitochondrial
membrane
potential
(ΔΨM)
human
neuroblastom
treated(A);
with
γ-bisabolene.
TE671
cells were stained
using
DiOC 6(3),
andinthen
measured
by flowcells
cytometry
γ-bisabolene. TE671 cells were stained using DiOC6(3), and then measured by flow cytometry (A);
γ-bisabolene.
TE671
cells
were
usingwith
DiOC
6(3), and then
measured
by ***,
flowp cytometry
(A);
Relative
changes
in low
MMP
ofstained
cells treated
γ-bisabolene
were
shown (B).
value < 0.001
Relative changes in low MMP of cells treated with γ-bisabolene were shown (B). ***, p value < 0.001
Relative changes
in low MMP
compared
with untreated
cells. of cells treated with γ-bisabolene were shown (B). ***, p value < 0.001
compared with untreated cells.
compared with untreated cells.
Molecules 2016, 21, 601
6 of 11
Quantitative analysis of MMP changes using DiOC6(3) staining demonstrated that γ-bisabolene
significantly decreased the MMP of human neuroblastom cells (Figure 5A,B); a 97% decrease in
MMP was found in TE671 cells treated with 5 µM of γ-bisabolene (Figure 5B). Therefore, the results
indicated γ-bisabolene induces ROS production and injures the mitochondria in apoptosis of human
neuroblastom cells.
Molecules 2016, 21, 601
6 of 11
2.4. The Increase of p53-Mediated Transcription in γ-Bisabolene-Treated Cells
2.4. The Increase of p53-Mediated Transcription in γ-Bisabolene-Treated Cells
10
Phospho-p53
53 kDa
Phospho-p21
21 kDa
β-actin
43 kDa
phospho-p21
2
**
5
***
1
**
0
phospho-p53
2.5
**
γ-Bisabolene (μM)
3
**
B.
phospho-protein/β actin
A.
***
To examine the γ-bisabolene-induced apoptotic pathways of human neuroblastom cells,
To examine
the γ-bisabolene-induced
apoptoticinduction
pathwayswere
of human
neuroblastom
cells,(Figure
p53- 6).
p53-mediated
transcriptional
activities for apoptosis
subsequently
analyzed
mediated
transcriptional
activities
for
apoptosis
induction
were
subsequently
analyzed
(Figure
Western blotting showed γ-bisabolene elevating the phosphorylation of p53 and p21 in 6).
TE671
elevating the phosphorylation of p53 and p21 in TE671 cells
cellsWestern
(Figureblotting
6A,B),showed
linkedγ-bisabolene
with activation
of PUMA and Bim expression, but not Noxa, in
(Figure 6A,B), linked with activation of PUMA and Bim expression, but not Noxa, in dose-dependent
dose-dependent manners quantified using real-time RT-PCR (Figure 6C). Since down-regulation
manners quantified using real-time RT-PCR (Figure 6C). Since down-regulation of casein kinase 2α
of casein
kinase 2α (CK2α) was associated with activation of p53 transcriptional activities in apoptosis
(CK2α) was associated with activation of p53 transcriptional activities in apoptosis of γ-bisaboleneof γ-bisabolene-treated
oral[7],
cancer
cells and
[7], the
CK2α
and CK2β
expression in γ-bisabolene-treated
treated oral cancer cells
the CK2α
CK2β
expression
in γ-bisabolene-treated
neuroblastom
neuroblastom
cells was
further characterized
7A–C).
Real-time
RT-PCR
anddemonstrated
western blotting
cells was further
characterized
(Figure 7A–C).(Figure
Real-time
RT-PCR
and western
blotting
demonstrated
that γ-bisabolene
the CK2β
CK2α expression
and CK2β in
expression
in treated
that γ-bisabolene
significantlysignificantly
reduced the reduced
CK2α and
treated human
neuroblastom
cells. Therefore,
CK2 down-regulation
was suggested
to be
associated to
with
human
neuroblastom
cells. Therefore,
CK2 down-regulation
was
suggested
bep53-mediated
associated with
apoptosis of
γ-bisabolene-treated
neuroblastom neuroblastom
cells.
p53-mediated
apoptosis
of γ-bisabolene-treated
cells.
1.5
1
0.5
0
0
1
5
10
γ-Bisabolene (μM)
***
***
3
**
**
2
Noxa
Bim
1
PUMA
**
Relative expression levels
C.
0
1
5
10
γ-Bisabolene (μM)
Figure
6. Activation
p53
mediatedtranscriptional
transcriptional activity
Western
blotblot
analysis
Figure
6. Activation
of of
p53
mediated
activitybybyγ-bisabolene.
γ-bisabolene.
Western
analysis
of lysates from treated and mock cells was performed; the blots were probed with primary and
of lysates from treated and mock cells was performed; the blots were probed with primary and
secondary antibodies (A); Relative band intensity of phospho-p53 or phosphor-p21 was normalized
secondary antibodies (A); Relative band intensity of phospho-p53 or phosphor-p21 was normalized
by β actin, compared to the mock cells, and quantified using imageJ based on triplicate replicates of
by β actin, compared to the mock cells, and quantified using imageJ based on triplicate replicates
each experiment (B); Relative fold levels in treated and mock cells appear as ratio of indicated
of each
experimentmRNA
(B); Relative
fold levels
treated
and (C).
mock
appear
as ratio
of< indicated
after performing
real in
time
PCR assays
**, pcells
value
< 0.01; ***,
p value
0.001
mRNA/GAPDH
mRNA/GAPDH
mRNA
after
performing
real
time
PCR
assays
(C).
**,
p
value
<
0.01;
***,
p value <
compared with untreated cells.
0.001 compared with untreated cells.
Molecules 2016, 21, 601
Molecules 2016, 21, 601
B.
CK2β
γ-Bisabolene
CK2α/β actin
**
C.
**
**
0.5
**
1
0
1
5
γ-Bisabolene (μM)
10
0
1
5
10
(μM)
CK2α
46 kDa
β-actin
43 kDa
1.4
1.2
1
0.8
0.6
0.4
0.2
0
**
CK2α
1.5
*
2
**
Relative expression levels
A.
7 of 11
7 of 11
0
1
5
10
γ-Bisabolene (μM)
D.
γ- Bisabolene
Up-regulation
Down-regulation
CK2α
CK2β
ROS
MMP
PUMA, Bim
active Caspase 9
p53
active Caspase 3
PUMA, Bim mRNA
p53-response element
Apoptosis
Figure
Figure 7.
7. Decrease
Decrease of
of CK2α/β
CK2α/βexpression
expressioninin γ-bisabolene-treated
γ-bisabolene-treated cells.
cells. Cells
Cellswere
were treated
treated with
with
γ-bisabolene
for
48
h,
and
then
harvested
for
real
time
PCR
(A)
and
western
blot
assays
(B,C);
γ-bisabolene for 48 h, and then harvested for real time PCR (A) and western blot assays (B,C);Relative
Relative
mRNA
mRNAlevels
levelsof
ofCK2α
CK2αand
andCK-2β
CK-2βwere
werenormalized
normalizedby
byGAPDH,
GAPDH,and
andthen
thencompared
comparedtotoun-treated
un-treatedcells,
cells,
respectively.
respectively. Relative
Relative band
bandintensity
intensityof
ofCK2α
CK2αwas
wasnormalized
normalizedby
byββactin,
actin,compared
comparedtotothe
themock
mockcells,
cells,
and
andquantified
quantifiedusing
usingimageJ
image Jbased
basedon
ontriplicate
triplicatereplicates
replicatesofofeach
eachexperiment
experiment(C);
(C);GeneGO
GeneGOMeta-Core
Meta-Core
pathway
analysis
of
the
results
predicts
p53-mediated
apoptosis
pathway
in
(D).
pathway analysis of the results predicts p53-mediated apoptosis pathway in treated treated
cells (D).cells
*, p value
*,< p0.05;
value
value
< 0.01 compared
with untreated
cells.
**, <p 0.05;
value**,< p0.01
compared
with untreated
cells.
3.3.Discussion
Discussion
Essential
oils containing
containingγ-bisabolene
γ-bisabolene
have
demonstrated
anti-proliferative
activities
to
Essential oils
have
demonstrated
anti-proliferative
activities
to human
human
oral
cancer,
prostate
cancer,
glioblastoma,
lung
carcinoma,
breast
carcinoma,
and
colon
oral cancer, prostate cancer, glioblastoma, lung carcinoma, breast carcinoma, and colon adenocarcinoma
adenocarcinoma
lines
[7–10].
studythe
firstpotent
reports
the potent antiproliferative
and
apoptotic
cell lines [7–10].cell
The
study
firstThe
reports
antiproliferative
and apoptotic
activities
of
activities
of
γ-bisabolene
on
human
neuroblastom
cells
(Figures
1–3).
γ-Bisabolene
induced
apoptosis
γ-bisabolene on human neuroblastom cells (Figures 1–3). γ-Bisabolene induced apoptosis of human
of
human neuroblastom
cells
through intracellular
increasing intracellular
ROS,
declining
MMP, and
activating
neuroblastom
cells through
increasing
ROS, declining
MMP,
and activating
p53-mediated
p53-mediated
activities
4–7).indicated
MTT assays
indicatedatγ-bisabolene
at high
transcriptionaltranscriptional
activities Figures
(4–7). (Figures
MTT assays
γ-bisabolene
high concentrations
concentrations
(15
to
200
μM)
was
less
cytotoxic
than
low
concentrations
(0.1
to
10
μM)
(Figure
1B),
(15 to 200 µM) was less cytotoxic than low concentrations (0.1 to 10 µM) (Figure 1B), but cell cycle
but
cell cycle
analysis demonstrated
sub-G1
fraction
90%cells
in treated
cells
with
μM treatment
48 h post
analysis
demonstrated
sub-G1 fraction
as near
90%asinnear
treated
with 20
µM
48 h20post
treatment
(Figure
1C,D).
Since
the
MTT
assay
relies
on
mitochondrial
dehydrogenase
activity,
(Figure 1C,D). Since the MTT assay relies on mitochondrial dehydrogenase activity, γ-bisabolene
at
γ-bisabolene
at
low
concentrations
(0.1
to
10
μM)
was
effective
at
triggering
the
mitochondrialow concentrations (0.1 to 10 µM) was effective at triggering the mitochondria-dependent apoptosis of
dependent
apoptosis ofcells,
human
neuroblastom
as consistent
with2–7.
the findings in Figures 2–7.
human neuroblastom
as consistent
with cells,
the findings
in Figures
Tumor
suppressor
p53
has
demonstrated
its
involvement
in
the mitochondrial
Tumor suppressor p53 has demonstrated its involvement in the mitochondrial
apoptoticapoptotic
pathway,
pathway,
exerting
transcriptional
activities
on
regulating
the
expression
of
pro-apoptotic
genes:
exerting transcriptional activities on regulating the expression of pro-apoptotic genes: e.g., PUMA,
e.g.,
Bim,[6,11,12].
and NOXA
[6,11,12].concentration-dependently
γ-Bisabolene concentration-dependently
increased p53
Bim,PUMA,
and NOXA
γ-Bisabolene
increased p53 phosphorylation
phosphorylation
that
linked
with
up-regulation
of
p53
transcriptional
activities
on
apoptotic
genes
that linked with up-regulation of p53 transcriptional activities on apoptotic genes PUMA and
Bim
PUMA
andneuroblastom
Bim in humancells
neuroblastom
cells (Figure
Since
andknown
Bim have
been
known for
as
in human
(Figure 6). Since
PUMA6).
and
BimPUMA
have been
as the
activator
the activator for Bax involved in mitochondria-mediated apoptosis [13], γ-Bisabolene-induced p53mediated PUMA and Bim up-regulation correlated with the MMP decreases in treated neuroblastom
Molecules 2016, 21, 601
8 of 11
Bax involved in mitochondria-mediated apoptosis [13], γ-Bisabolene-induced p53-mediated PUMA
and Bim up-regulation correlated with the MMP decreases in treated neuroblastom cells (Figure 5).
In addition, p53 could induce the Fas up-regulation that activates the extrinsic apoptotic pathway [14].
Thus, p53 activation induced by γ-bisabolene might be responsible for increasing the expression and
activity of caspase 8 (Figure 3). Results demonstrated γ-bisabolene triggering p53-mediated apoptosis
of human neuroblastom cells.
Protein kinase CK2α participates in heart and neural tube development, maintains cell viability,
and regulates cell cycle stages [15–18]. CK2α has been identified to overexpress in human colorectal
cancer [19]; gene silence of CK2α small interfering RNA was associated with elevating the expression of
p53/p21 and decreasing the expression of C-myc [20]. Therefore, suppression of CK2α was suggested
to a novel therapeutic approach for human colorectal cancer. This study demonstrated γ-bisabolene
down-regulating the CK2α and CK2β expression in human neuroblastom cells (Figure 7A–C). Thus,
down-regulation of CK2 was proposed to as one of promising mechanisms for activating p53-mediated
apoptosis of human neuroblastom cells treated with γ-bisabolene (Figure 7D).
4. Materials and Methods
4.1. Cell Cultures
Human neuroblastoma TE671 cells were cultured in MEM medium (HyClone Laboratories,
South Logan, UT, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL
streptomycin, 2 mM glutamine and 1 mM sodium pyruvate at 37 ˝ C in a humidified atmosphere of
5% CO2 .
4.2. In Vitro Cytotoxicity Test
Cytotoxic activity of γ-bisabolene (4-(1,5-dimethyl-4-hexenylidene)-1-methylcyclohexene)
(Figure 1A) was assessed by MTT assay. Cells (5 ˆ 104 cells/mL) plated in 96-well plates overnight
and then treated with indicated concentrations (0.1, 1, 5, 10, 15, 20, 50, 100, and 200 µM) for 48 h at
37 ˝ C in humidified atmosphere of 5% CO2 . Cell were incubated with 10 µL of a MTT solution for
4 h, reacted with 100 µL of stop solution for dissolving formazan crystals, and then measured with
micro-ELISA reader by optical density (OD) at 570–630 nm. 50% cytotoxic concentration (CC50 ) of
γ-bisabolene on TE671 cells was determined using the ID50 software developed by Dr. John Spouge
with the survival rate of treated cells to mock cells.
4.3. Apoptosis and Cell Cycle Assay by Flow Cytometry
Cells were treated with γ-bisabolene at 10 µM for 24 and 48 h, and harvested for cell cycle analysis
by propidium iodide (PI) staining, and apoptosis assays with γ-bisabolene at 1, 5, 10, and 20 µM for
48 h by Annexin V-FITC apoptosis Detection Kit with PI (BioVision, Milpitas, CA, USA). Cell cycle and
apoptosis assays were performed as described in our prior report [7].
4.4. Mitochondrial Membrane Potential (MMP) Detection Assays
Cells were treated with γ-bisabolene at 1, 5, 10, and 20 µM for 48 h; MMP changes were quantitated
using flow cytometry with DiOC6(3) staining (Calbiochem, San Diego, CA, USA). Cells were stained
with the DiOC6(3) solution at 37 ˝ C in the dark for 1 h, and then measured using a flow cytometer
with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
4.5. Detecting Intracellular Reactive Oxygen Species (ROS) by Flow Cytometry
Cells were treated with γ-bisabolene at 1, 5, 10, and 20 µM for 48 h, harvested, and then stained
with 10 µM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma, Saint Louis, MO, USA) at
37 ˝ C for 30 min in darkroom. DCF fluorescence converted from DCFH-DA by ROS was detected
Molecules 2016, 21, 601
9 of 11
by flow cytometry with excitation wavelength of 485 nm and emission wavelength of 530 nm, as
described in our prior report [21].
4.6. Western Blot
TE671 cells were treated with γ-bisabolene at 1, 5 and 10 µM for 48 h, collected, and lysed in
the RIPA lysis buffer (Roche Diagnostics, New York, NY, USA). Lysates in sample buffer were boiled
for 8 min, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
and then followed by western blotting assays, as described in our prior report [7,22]. The blots were
probed with specific primary antibodies against caspase 3 (Calbiocem), capspase 8, caspase 9 (upstate),
phospho-p53(S15), p21 (Millipore, Billerica, MA, USA), phospho-p21(T145) (ABGENT, San Diego,
CA, USA), casein kinase 2α (CK2α), and β-actin (Cell Signalling, Danvers, MA, USA), and then
reacted with HRP-conjugated anti-mouse or anti-rabbit IgG antibodies (Invitrogen, Carlsbad, CA,
USA). Immunoreactive bands were reacted with chemiluminescent western blotting detection reagents,
and then pictured by autoradiography.
4.7. Real-Time RT-PCR
Total RNA was extracted from mock control and treated cells with γ-bisabolene using the RNA
purification kit (Invitrogen), and then converted into cDNA with oligo dT primer and SuperScript III
reverse transcriptase (Thermo Fisher, San Jose, CA, USA). Two-step RT-PCR was performed using
SYBR Green I, as described in our prior report [23]. Oligonucleotide primer pairs in our study
were listed as the following: (1) forward primer 51 -CAGTGG AG GCCGACTTCTTG-31 and reverse
primer 51 -TGGCA CAAAGCGACTGGAT-31 for human caspase 3; (2) forward primer 51 -TGTCCTA
CTCTACTTTCCCAGGTTTT-31 and reverse primer 51 -GTGAGCCCACTGCTCAAAGAT-31 for
human caspase 9; (3) forward primer 51 -AGTGGGTATTTCTCTTTTGACACAG-31 and reverse
primer 51 -GTCTCCAATACGCCGCAACT-31 for human Bim; (4) forward primer 51 -GACGA
CCTCAACGCACAGTA-31 and reverse primer 51 -CACCTAATTGGGCTCCATCT-31 for human PUMA;
(5) forward primer 51 -GAGATGCCTGGGAAGAAGG-31 and reverse primer 51 -TTCTGCCGGAAGTT
CAGTTT-31 for human Noxa; (6) forward primer 51 -GGATGGCCACTGTGAATAACTG-31
and reverse primer 51 -TCGAGGACATCGCTCTCTCA-31 for caspase 8; (7) forward primer
51 -GAACGCTTTGTCCACAGTGA-31 and reverse primer 51 -TATCGCAGCAGTTTGTCCAG-31
for human protein kinase CK2α; (8) forward primer 51 -CCCATTGGCCTTTCAGACAT-31
and reverse primer 51 -CCGTGTGATGGTGTCTTGATG-31 for CK2β; and (9) forward primer
51 -CCACCCATGGCAAATTCC-31 and reverse primer 51 -TGGGATTTCCATTGATGACAAG-31 for
human GAPDH. Real-time PCR reaction mixture contained 2 µL of cDNA (reverse transcription
mixture), 200 nM of each primer pair in SYBR Green I master mix (LightCycler TaqMAn Master,
Roche Diagnostics). Relative mRNA levels of indicated genes were gene normalized by housekeeping
gene GAPDH.
4.8. Statistical Analysis
All data from three independent experiments were used for measuring mean ˘ standard
error (mean ˘ S.E.) that was compared using Student’s t-test. p < 0.05 was considered as
statistically significant.
5. Conclusions
This study demonstrated that γ-bisabolene has potential to inhibit the growth and inducing
apoptosis of human neuroblastom cells. γ-Bisabolene reduced the CK2α expression and activated
p53-mediated mitochondrial apoptosis pathway in human neuroblastom cell. The results suggested
γ-bisabolene as a potential agent of anti-proliferative and apoptosis-inducing activities for treating
human neuroblastom cells.
Molecules 2016, 21, 601
Acknowledgments:
This work was supported by the National Science Council of
(NSC102-2320-B-039-044-MY3) and China Medical University (CMU102-ASIA-15, CMU103-ASIA-07).
10 of 11
Taiwan
Author Contributions: C.-W.L., S.-H.H. and C.-H.H. conceived and designed the experiments; Y.-J.J., C.-S.L., and
C.-Y.W. performed the experiments; C.-W.L., S.-H.H. and C.-H.H. analyzed the data; L.W. and Y.-J.L. contributed
reagents/materials/analysis tools; C.-W.L., S.-H.H. and C.-S.L. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Park, J.R.; Eggert, A.; Caron, H. Neuroblastoma: Biology, prognosis, and treatment. Pediatr. Clin. N. Am.
2008, 55, 97–120. [CrossRef] [PubMed]
Maris, J.M.; Hogarty, M.D.; Bagatell, R.; Cohn, S.L. Neuroblastoma. Lancet 2007, 369, 2106–2120. [CrossRef]
De Bernardi, B.; Nicolas, B.; Boni, L.; Indolfi, P.; Carli, M.; Cordero, D.; Montezemolo, L.; Donfrancesco, A.;
Pession, A.; Provenzi, M.; et al. Disseminated neuroblastoma in children older than one year at diagnosis:
Comparable results with three consecutive high-dose protocols adopted by the Italian Co-Operative Group
for Neuroblastoma. J. Clin. Oncol. 2003, 21, 1592–1601. [CrossRef] [PubMed]
Cohn, S.L.; Pearson, A.D.; London, W.B.; Monclair, T.; Ambros, P.F.; Brodeur, G.M.; Faldum, A.; Hero, B.;
Iehara, T.; Machin, D.; et al. The International Neuroblastoma Risk Group (INRG) classification system:
An INRG Task Force report. J. Clin. Oncol. 2009, 27, 289–297. [CrossRef] [PubMed]
Liu, Q.; Wang, H.G. Anti-cancer drug discovery and development: Bcl-2 family small molecule inhibitors.
Commun. Integr. Biol. 2012, 5, 557–565. [CrossRef] [PubMed]
Elmore, S. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 2007, 35, 495–516. [CrossRef]
[PubMed]
Jou, Y.J.; Chen, C.J.; Liu, Y.C.; Way, T.D.; Lai, C.H.; Hua, C.H.; Wang, C.Y.; Huang, S.H.; Kao, J.Y.; Lin, C.W.
Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human
oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation. Proteomics 2015, 15, 3296–3309.
[CrossRef] [PubMed]
Sylvestre, M.; Pichette, A.; Longtin, A.; Nagau, F.; Legault, J. Essential oil analysis and anticancer activity
of leaf essential oil of Croton flavens L. from Guadeloupe. J. Ethnopharmacol. 2006, 103, 99–102. [CrossRef]
[PubMed]
Bayala, B.; Bassole, I.H.; Gnoula, C.; Nebie, R.; Yonli, A.; Morel, L.; Figueredo, G.; Nikiema, J.B.;
Lobaccaro, J.M.; Simpore, J. Chemical composition, antioxidant, anti-inflammatory and anti-proliferative
activities of essential oils of plants from burkina faso. PLoS ONE 2014, 9, e92122. [CrossRef] [PubMed]
Farag, M.A.; Al-Mahdy, D.A. Comparative study of the chemical composition and biological activities of
Magnolia grandiflora and Magnolia virginiana flower essential oils. Nat. Prod. Res. 2013, 27, 1091–1097.
[CrossRef] [PubMed]
Mancini, F.; Moretti, F. Mitochondrial MDM4 (MDMX): An unpredicted role in the p53-mediated intrinsic
apoptotic pathway. Cell Cycle 2009, 8, 3854–3859. [CrossRef] [PubMed]
Fujita, T.; Ishikawa, Y. Apoptosis in heart failure. The role of the β-adrenergic receptor-mediated signaling
pathway and p53-mediated signaling pathway in the apoptosis of cardiomyocytes. Circ. J. 2011, 75,
1811–1818. [PubMed]
Shukla, S.; Sharma, A.; Pandey, V.K.; Raisuddin, S.; Kakkar, P. Concurrent acetylation of FoxO1/3a and
p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in
berberine-treated HepG2 cells. Toxicol. Appl. Pharmacol. 2016, 291, 70–83. [CrossRef] [PubMed]
Haupt, S.; Berger, M.; Goldberg, Z.; Haupt, Y. Apoptosis - the p53 network. J. Cell Sci. 2003, 116, 4077–4085.
[CrossRef] [PubMed]
Seldin, D.C.; Lou, D.Y.; Toselli, P.; Landesman-Bollag, E.; Dominguez, I. Gene targeting of CK2 catalytic
subunits. Mol. Cell. Biochem. 2008, 316, 141–147. [CrossRef] [PubMed]
Padmanabha, R.; Chen-Wu, J.L.; Hanna, D.E.; Glover, C.V. Isolation, sequencing, and disruption of the yeast
CKA2 gene: Casein kinase II is essential for viability in Saccharomyces cerevisiae. Mol. Cell. Biol. 1990, 10,
4089–4099. [CrossRef] [PubMed]
Molecules 2016, 21, 601
17.
18.
19.
20.
21.
22.
23.
11 of 11
Buchou, T.; Vernet, M.; Blond, O.; Jensen, H.H.; Pointu, H.; Olsen, B.B.; Cochet, C.; Issinger, O.G.; Boldyreff, B.
Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect
and early embryonic lethality. Mol. Cell. Biol. 2003, 23, 908–915. [CrossRef] [PubMed]
Kulartz, M.; Hiller, E.; Kappes, F.; Pinna, L.A.; Knippers, R. Protein kinase CK2 phosphorylates the cell cycle
regulatory protein Geminin. Biochem. Biophys. Res. Commun. 2004, 315, 1011–1017. [CrossRef] [PubMed]
Lin, K.Y.; Tai, C.; Hsu, J.C.; Li, C.F.; Fang, C.L.; Lai, H.C.; Hseu, Y.C.; Lin, Y.F.; Uen, Y.H. Overexpression of
nuclear protein kinase CK2 α catalytic subunit (CK2α) as a poor prognosticator in human colorectal cancer.
PLoS ONE 2011, 6, e17193. [CrossRef] [PubMed]
Zou, J.; Luo, H.; Zeng, Q.; Dong, Z.; Wu, D.; Liu, L. Protein kinase CK2α is overexpressed in colorectal cancer
and modulates cell proliferation and invasion via regulating EMT-related genes. J. Transl. Med. 2011, 9, 97.
[CrossRef] [PubMed]
Yang, T.C.; Lai, C.C.; Shiu, S.L.; Chuang, P.H.; Tzou, B.C.; Lin, Y.Y.; Tsai, F.J.; Lin, C.W. Japanese encephalitis
virus down-regulates thioredoxin and induces ROS-mediated ASK1-ERK/p38 MAPK activation in human
promonocyte cells. Microbes Infect. 2010, 12, 643–651. [CrossRef] [PubMed]
Yang, T.C.; Li, S.W.; Lai, C.C.; Lu, K.Z.; Chiu, M.T.; Hsieh, T.H.; Wan, L.; Lin, C.W. Proteomic analysis for
Type I interferon antagonism of Japanese encephalitis virus NS5 protein. Proteomics 2013, 13, 3442–3456.
[CrossRef] [PubMed]
Wang, C.Y.; Huang, S.C.; Zhang, Y.; Lai, Z.R.; Kung, S.H.; Chang, Y.S.; Lin, C.W. Antiviral Ability of
Kalanchoe gracilis Leaf Extract against Enterovirus 71 and Coxsackievirus A16. Evid. Based Complement.
Altern. Med. 2012, 2012, 503165. [CrossRef]
Sample Availability: Not Available.
© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).