Article

In vitro and in vivo anticarcinogenic effects of RNase MC2, a ribonuclease isolated from dietary bitter gourd, toward human liver cancer cells

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong.
The international journal of biochemistry & cell biology (Impact Factor: 4.05). 04/2012; 44(8):1351-60. DOI: 10.1016/j.biocel.2012.04.013
Source: PubMed

ABSTRACT

Hepatocellular carcinoma (HCC) constitutes a predominant part of primary liver cancer which ranks as the fifth most common cancer as well as the third most common cause of cancer mortality. In view of the poor prognosis of unresectable liver cancers, it is of pivotal importance to develop novel chemotherapeutical regimens. RNase MC2 is a 14-kDa ribonuclease isolated from dietary bitter gourd (Momordica charantia) that manifested antitumor potential against breast cancers. In this study, we investigated the potential application of RNase MC2 on Hep G2 cells. We showed that RNase MC2 inhibited cell proliferation and induced cell apoptosis in both in vitro and in vivo studies. RNase MC2 treatment caused cell cycle arrest predominantly at the S-phase and apoptosis, which is associated with the activation of both caspase-8 and caspase-9 regulated caspase pathways. Our further investigation disclosed that RNase MC2 down-regulated the anti-apoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bak. Moreover, the phosphorylation of ERK and JNK was involved in the apoptosis process. Importantly, RNase MC2 significantly suppressed the growth of Hep G2 xenograft-bearing nude mice by inducing apoptosis. This notion is supported by data indicating an increased number of caspase-3- and PARP-positive cells, and TUNEL-positive cells in RNase MC2-treated tumor tissues. In summary, we have revealed the antitumor potential of RNase MC2 toward Hep G2 cells. Considering that bitter gourd is a common dietary component in many countries, this study may help to prompt the clinical application of RNase MC2.

Full-text

Available from: Chris Zhiyi Zhang, Jul 10, 2014
The
International
Journal
of
Biochemistry
&
Cell
Biology
44 (2012) 1351–
1360
Contents
lists
available
at
SciVerse
ScienceDirect
The
International
Journal
of
Biochemistry
&
Cell
Biology
journa
l
h
o
me
page:
www.elsevier.com/locate/biocel
In
vitro
and
in
vivo
anticarcinogenic
effects
of
RNase
MC2,
a
ribonuclease
isolated
from
dietary
bitter
gourd,
toward
human
liver
cancer
cells
Evandro
Fei
Fang
a,1,2
,
Chris
Zhi
Yi
Zhang
b,c,1
,
Lin
Zhang
a
,
Wing
Ping
Fong
d
,
Tzi
Bun
Ng
a,
a
School
of
Biomedical
Sciences,
Faculty
of
Medicine,
The
Chinese
University
of
Hong
Kong,
Shatin,
Hong
Kong
b
State
Key
Laboratory
of
Oncology
in
Southern
China,
Sun
Yat-Sen
University
Cancer
Center,
Guangzhou,
China
c
Department
of
Pathology,
Sun
Yat-Sen
University
Cancer
Center,
Guangzhou,
China
d
School
of
Life
Sciences,
Faculty
of
Science,
The
Chinese
University
of
Hong
Kong,
Shatin,
Hong
Kong
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
17
March
2012
Received
in
revised
form
16
April
2012
Accepted
18
April
2012
Available online xxx
Keywords:
Ribonuclease
Momordica
charantia
Hepatocellular
carcinoma
Nude
mice
Antitumor
a
b
s
t
r
a
c
t
Hepatocellular
carcinoma
(HCC)
constitutes
a
predominant
part
of
primary
liver
cancer
which
ranks
as
the
fifth
most
common
cancer
as
well
as
the
third
most
common
cause
of
cancer
mortality.
In
view
of
the
poor
prognosis
of
unresectable
liver
cancers,
it
is
of
pivotal
importance
to
develop
novel
chemother-
apeutical
regimens.
RNase
MC2
is
a
14-kDa
ribonuclease
isolated
from
dietary
bitter
gourd
(Momordica
charantia)
that
manifested
antitumor
potential
against
breast
cancers.
In
this
study,
we
investigated
the
potential
application
of
RNase
MC2
on
Hep
G2
cells.
We
showed
that
RNase
MC2
inhibited
cell
prolif-
eration
and
induced
cell
apoptosis
in
both
in
vitro
and
in
vivo
studies.
RNase
MC2
treatment
caused
cell
cycle
arrest
predominantly
at
the
S-phase
and
apoptosis,
which
is
associated
with
the
activation
of
both
caspase-8
and
caspase-9
regulated
caspase
pathways.
Our
further
investigation
disclosed
that
RNase
MC2
down-regulated
the
anti-apoptotic
protein
Bcl-2
and
increased
the
expression
of
pro-apoptotic
protein
Bak.
Moreover,
the
phosphorylation
of
ERK
and
JNK
was
involved
in
the
apoptosis
process.
Importantly,
RNase
MC2
significantly
suppressed
the
growth
of
Hep
G2
xenograft-bearing
nude
mice
by
inducing
apo-
ptosis.
This
notion
is
supported
by
data
indicating
an
increased
number
of
caspase-3-
and
PARP-positive
cells,
and
TUNEL-positive
cells
in
RNase
MC2-treated
tumor
tissues.
In
summary,
we
have
revealed
the
antitumor
potential
of
RNase
MC2
toward
Hep
G2
cells.
Considering
that
bitter
gourd
is
a
common
dietary
component
in
many
countries,
this
study
may
help
to
prompt
the
clinical
application
of
RNase
MC2.
© 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Primary
liver
cancer
is
the
fifth
most
common
cancer
and
ranks
third
as
a
cause
of
cancer
death.
It
constitutes
the
bulk
of
hepato-
cellular
carcinoma
(HCC)
(El-Serag
and
Rudolph,
2007).
In
recent
decades,
the
incidence
rate
of
HCC
in
the
United
States
has
been
increasing,
particularly
among
middle-aged
black,
Hispanic,
and
white
men
(Altekruse
et
al.,
2009).
In
Hong
Kong,
HCC
is
a
main
cause
of
cancer
incidence
(10.1%
of
all
cancers
in
males
and
3.6%
of
all
cancers
in
females,
year
2009)
and
mortality
(14.0%
of
all
can-
cers
in
males
and
8.1%
of
all
cancers
in
females,
year
2009)
(2009).
HCC
is
highly
aggressive,
and
only
about
10–20%
of
patients
are
applicable
for
curative
surgery,
while
the
others
are
unresectable
Corresponding
author.
Tel.:
+852
26098031;
fax:
+852
26035123.
E-mail
addresses:
fangfei1030@yahoo.com.cn
(E.F.
Fang),
b021770@mailserv.cuhk.edu.hk
(T.B.
Ng).
1
These
authors
contributed
equally
to
this
work.
2
Current
address:
Laboratory
of
Molecular
Gerontology,
National
Institute
on
Aging,
National
Institutes
of
Health,
Baltimore,
MD
USA.
Emails:
fange@mail.nih.gov,
fangfei1030@yahoo.com.cn
tumors
with
best
supportive
care
as
well
as
systemic
chemother-
apy
as
optimal
palliative
treatment
(Llovet
et
al.,
2002;
Yeo
et
al.,
2005).
In
view
of
the
poor
prognosis
of
HCC,
with
a
median
survival
time
of
4
months
(Yeo
et
al.,
2005),
it
is
of
immense
importance
to
develop
new/novel
therapeutic
agents
against
HCC.
Ribonucleases/RNases
of
different
origins
exhibit
a
wide
spec-
trum
of
antitumor
potentials
(Fang
and
Ng,
2011a,b).
RNases
are
RNA-degrading
enzymes
which
cleave
RNA
and
produce
oligonu-
cleotides
or
mononucleotides
with
2
,
3
-cyclic
phosphate
at
the
3
-side,
and
finally
yielding
3
-mononucleotides
or
oligonucleotides
with
a
3
-phosphate
(Deshpande
and
Shankar,
2002).
The
remark-
able
antitumor
activity
of
RNases
is
linked
to
their
ability
to
destroy
RNA
and
not
to
genotoxicity,
and
therefore,
they
are
a
second
line
of
cancer
chemotherapeutics
(Ribó
et
al.,
2011).
One
example
is
Onconase
(also
known
as
ranpirnase
or
P30)
which
is
an
amphib-
ian
RNase
present
in
both
early
embryos
and
unfertilized
oocytes
of
leopard
frog
(binomial
name
Rana
pipiens)
(Fang
and
Ng,
2011a,b).
There
are
several
strands
of
investigations
indicating
the
antitumor
advantages
of
Onconase
and
currently
it
is
in
Phase
III
trial
on
unre-
sectable
malignant
mesothelioma
(MMe)
(Fang
and
Ng,
2011a,b).
Besides
Onconase,
there
are
some
antitumor
RNases
of
plant
1357-2725/$
see
front
matter ©
2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biocel.2012.04.013
Page 1
1352 E.F.
Fang
et
al.
/
The
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Journal
of
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&
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Biology
44 (2012) 1351–
1360
origin,
including
ginseng
RNases,
wheat
leaf
RNase,
black
pine
pollen
nuclease,
and
others
(Fang
and
Ng,
2011a,b).
RNase
MC2
is
a
new
RNase
isolated
from
bitter
gourd,
named
after
the
first
one
RNase
MC1
from
the
same
plant
(Numata
et
al.,
2000;
Fang
et
al.,
2011a).
This
14-kDa
small
protein
manifested
potent
RNA-cleavage
activity
toward
baker’s
yeast
tRNA,
tumor
cell
rRNA,
and
a
specificity
for
uridine
(Fang
et
al.,
2011a).
We
previously
found
that
RNase
MC2
exhibited
antitumor
potential
toward
human
breast
cancer
MCF-7
cells
by
inducing
apoptosis.
In
this
study,
we
extended
its
possible
ramifications
in
the
treatment
of
HCC.
We
showed
that
RNase
MC2
suppressed
cell
proliferation
and
tumor
growth
in
both
in
vitro
and
in
vivo
studies.
Different
molecular
mechanisms
were
disclosed.
2.
Materials
and
methods
2.1.
Antibodies
and
reagents
The
antibodies
used
in
this
study
were
as
follows:
primary
anti-
bodies
for
Bid
(sc-11423),
p53
(sc-126),
tubulin
(sc-9104),
PARP
(sc-25780),
cleaved
PARP
(sc-23461-R),
and
Bak
(sc-832)
were
purchased
from
Santa
Cruz
Biotechnology
(Santa
Cruz,
CA).
Anti-
bodies
for
caspase
8
(9746),
caspase
9
(9508),
caspase
3
(9665),
p21
(2947),
Bcl-2
(2870),
p-p38
(9215),
p38
(9212),
p-ERK
(4376),
ERK
(4695),
p-JNK
(4671),
JNK
(9252),
p-Akt
(9275),
and
Akt
(9272)
were
provided
by
Cell
Signaling
(Danvers,
MA).
All
reagents
were
from
Sigma,
USA,
unless
otherwise
indicated.
Reagents
including
PD98059,
SP600125,
SB-203580,
and
N-benzyloxycarbonyl-Val-
Ala-Asp-fluoromethyl
ketone
(Z-VAD-FMK)
were
dissolved
in
dimethylsulfoxide
(DMSO).
RNase
MC2
was
prepared
as
we
described
previously
(Fang
et
al.,
2011a,b).
2.2.
Cell
viability,
proliferation,
anchorage-independent,
and
wound
healing
(cell
motility)
assays
Hep
G2
cells
were
cultured
in
DMEM
supplemented
with
10%
FBS
and
1%
penicillin/streptomycin
at
37
C.
Cell
viability
was
determined
by
MTT
assay
as
described
previously
(Fang
et
al.,
2011a,b).
At
the
same
time,
cell
counting
was
assessed
using
a
hemocytometer
under
trypan
blue
exclusion
(Fang
et
al.,
2011a,b).
The
anchorage-independent
assay
was
performed
in
soft
agar
as
described
elsewhere
(Chen
et
al.,
2005).
Wound
healing
assay
under
mono
layer
conditions
was
carried
out
per
the
protocol
described
before
(Kong
et
al.,
2010).
2.3.
Flow
cytometry
and
Hoechst
33342
staining
Cell
cycle
analysis,
apoptosis,
TUNEL
assay,
and
detection
of
mitochondrial
membrane
depolarization
were
conducted
using
a
FACSort
flow
cytometer
(Becton
Dickinson)
(Fang
et
al.,
2011a,b).
Firstly,
cell
cycle
arrest
was
performed
using
propidium
iodide/PI
staining.
Briefly,
cells
were
treated
with
specific
concentrations
of
RNase
MC2
for
48
h,
followed
by
cell
trypsinization
and
fixation
in
70%
ethanol
for
2
h
on
ice.
Fixed
cells
were
washed
with
PBS,
stained
with
50
M
PI
for
15
min,
and
analyzed
by
flow
cytome-
try.
Secondly,
apoptosis
was
assessed
using
Annexin
V-PI
double
staining.
After
the
same
treatment,
cells
were
trypsinized,
and
stained
with
0.5
mg/ml
Annexin
V
in
binding
buffer
(10
mM
HEPES
free
acid,
0.14
M
NaCl,
and
2.5
mM
CaCl
2
)
for
30
min.
Afterward,
PI
(5
g/ml
final
concentration)
was
added
and
incubated
for
another
15
min.
Cells
were
applied
to
a
flow
cytometer
for
data
collection.
Thirdly,
TUNEL
assay
(Roche)
was
applied
for
detection
of
DNA
fragmentation.
Cells
were
stained
following
the
manufacturer’s
instructions,
and
loaded
on
a
flow
cytometry
for
further
analy-
sis.
Finally,
mitochondrial
membrane
depolarization
was
analyzed
using
JC-1
staining
(2.5
g/ml).
On
the
other
hand,
we
used
Hoechst
33342
dye
to
monitor
nuclear
morphological
changes
(Fang
et
al.,
2011a,b,
2012).
2.4.
Western
blotting
Whole
cell
extracts
prepared
by
lysis
buffer
with
phospho-stop
solution
(Roche)
were
applied
for
Western
blotting
with
proce-
dures
as
described
previously
(Fang
et
al.,
2011a,b).
Briefly,
about
20
g
of
protein
was
loaded
on
SDS-PAGE
for
size
fractionation,
and
transferred
onto
polyvinylidene
difluoride
membranes.
Mem-
branes
were
firstly
blocked
in
5%
fresh
milk
for
1
h,
followed
by
incubation
with
a
specific
primary
antibody
overnight
at
4
C.
Finally,
membranes
were
incubated
with
a
horseradish
peroxidase-
conjugated
anti-mouse
or
anti-rabbit
secondary
antibody
(Cell
Signaling,
Danvers,
MA)
for
30
min,
and
bands
were
visualized
by
using
ECL
detection
system
(Amersham
Life
Science).
2.5.
Xenograft
studies
BALB/c
nude
mice
were
provided
by
the
Laboratory
Animal
Services
Centre
at
The
Chinese
University
of
Hong
Kong,
and
all
experimental
procedures
had
been
approved
and
were
performed
under
the
direction
of
the
University
Animal
Research
Ethics
Com-
mittee.
Hep
G2
xenograft-bearing
mouse
model
was
established
by
subcutaneous
inoculation
with
Hep
G2
cells
(2
× 10
7
in
0.2
ml
DMEM)
into
the
right
flank
of
each
nude
mouse.
Mice
were
ran-
domly
divided
into
two
groups
with
five
in
each
group,
and
there
was
no
statistical
difference
of
tumor
volumes
between
the
groups.
For
mice
in
the
RNase
MC2
group,
they
received
an
intraperitoneal
injection
of
2.0
mg
RNase
MC2/kg
every
other
day.
The
control
mice
were
treated
with
dissolving
buffer
(PBS)
instead.
Tumor
dimensions
were
periodically
measured
using
an
electronic
caliper,
and
tumor
volumes
were
calculated
using
the
formula:
tumor
volume
(cm
3
)
=
0.5
×
tumor
length
(cm)
tumor
width
2
(cm
2
).
On
the
thirteenth
day,
all
mice
were
sacrificed.
Tumors
were
excised,
weighted,
followed
by
fixation
in
10%
formalin,
and
embedding
in
paraffin
wax
for
further
immunohistochemical
analysis.
2.6.
Immunohistochemistry
Immunohistochemistry/IHC
was
performed
as
described
else-
where
(Fang
et
al.,
2011a,b).
On
the
one
hand,
deparaffinized
4
m
sections
of
tumor
tissues
were
stained
with
Hematoxylin&Eosin
(H&E)
for
the
evaluation
of
cell
morphology.
On
the
other
hand,
apoptosis
was
examined
by
anti-cleaved
caspase-3
(Cell
Signaling,
MA),
cleaved
PARP
(sc-23461-R,
Santa
Cruz
Biotechnology,
CA),
and
TUNEL
detection
kit
(Roche,
Germany)
by
IHC.
Sections
were
evalu-
ated
by
an
experienced
pathologist
blinded
to
treatment
group.
The
activated
caspase-3,
cleaved
PARP,
and
TUNEL-positive
cells
were
quantitated
as
percentage
of
cells
positive
in
5
microscopic
fields
(40×
magnification)/section
from
5
mice
per
treatment
group.
2.7.
Statistical
analysis
Results
of
all
in
vitro
studies
were
performed
in
two/three
independent
experiments
in
triplicate.
Data
were
expressed
as
mean
±
standard
deviation
(SD)
using
SPSS
11.0
software.
The
two-
tailed
Student’s
t
test
was
chosen
for
between
group
comparisons,
and
a
p
value
of
<0.05
was
considered
significant.
3.
Results
3.1.
RNase
MC2
exhibited
cell
toxicity
in
Hep
G2
cells
MTT
assay
showed
that
RNase
MC2
manifested
the
ability
to
decrease
cell
viability
in
a
time-
and
dose-dependent
manner
Page 2
E.F.
Fang
et
al.
/
The
International
Journal
of
Biochemistry
&
Cell
Biology
44 (2012) 1351–
1360 1353
Fig.
1.
Cytotoxicity
of
RNase
MC2
toward
Hep
G2
cells.
(A)
RNase
MC2
reduced
viability
of
Hep
G2
cells.
Cells
were
incubated
with
different
concentrations
of
RNase
MC2
for
24
h
and
48
h,
and
then
the
residual
cell
viability
was
measured
by
MTT
assay.
(B)
RNase
MC2
exhibited
cytostatic
effects
on
Hep
G2
cells.
After
treatment
with
7.5
M
RNase
MC2
for
specified
durations,
cells
were
trypsinized
and
counted
using
a
hemocytometer
and
the
trypan
blue
exclusion
assay.
(C)
RNase
MC2
arrested
cell
cycle
progression
at
S
phase.
Cells
were
treated
with
different
concentrations
of
RNase
MC2
for
24
h,
and
stained
with
PI
dye
for
the
following
flow
cytometric
analysis.
(D)
RNase
MC2
reduced
anchorage-independent
proliferation
in
Hep
G2
cells.
Cells
were
seeded
into
soft
agar
containing
PBS
(control)
or
3.75
M
RNase
MC2.
After
incubation
for
30
days,
colonies
were
photographed
and
counted
under
a
light
microscope,
and
clonogenicity
was
determined.
Bar,
1
mm.
(E)
RNase
MC2
inhibited
cell
motility.
A
sterile
pipette
tip
was
used
to
make
a
straight
scratch,
simulating
a
wound.
After
cultured
with
7.5
M
RNase
MC2
for
24
h,
a
NIKON
TE2000
microscope
was
applied
for
photo-capturing.
(Fig.
1A).
The
IC
50
(48
h)
value
for
RNase
MC2
toward
Hp
G2
cells
was
about
29.8
M.
We
further
found
that
RNase
exhibited
cyto-
static
activity
against
Hep
G2
cells
as
supported
by
the
reduced
number
to
total
cells
in
cells
treated
with
7.5
M
of
RNase
MC2
compared
with
control
(Fig.
1B).
To
further
explore
the
poten-
tial
mechanism
involved
in
the
cytostatic
process,
PI
staining
was
applied
for
cell
cycle
analysis.
As
shown
in
Fig.
1C,
there
was
sig-
nificant
accumulation
of
cells
in
S
phase.
The
percentage
of
cells
in
S
phase
increased
from
33.53%
in
control
group
to
61.33%
in
cells
treated
with
7.5
M
RNase
MC2.
Furthermore,
RNase
MC2
(3.75
M,
about
one
eighth
of
the
IC
50
value)
inhibited
Hep
G2
colony
growth
in
soft
agar
as
revealed
by
decreased
clonogenic-
ity
(Fig.
1D).
In
addition,
there
was
a
retardation
of
cell
motility
in
RNase
MC2-treated
cells
(7.5
M).
3.2.
RNase
MC2
induced
apoptosis
in
Hep
G2
cells
To
investigate
whether
apoptosis
was
involved
in
the
cytotoxic-
ity
of
RNase
MC2
toward
Hep
G2
cells,
different
assays
of
apoptosis
were
employed.
First,
Annexin
V-PI
staining
showed
that
RNase
MC2
increased
the
number
of
cells
undergoing
early/late
apoptosis
(1.2%
of
apoptosis
for
control
versus
57.43%
of
apoptosis
following
treatment
with
30
M
RNase
MC2,
Fig.
2A).
We
next
investi-
gated
DNA
fragmentation
using
the
TUNEL
assay.
As
depicted
in
Fig.
2B,
treatment
with
30
M
RNase
MC2
for
48
h
induced
DNA
fragmentation
in
33.16%
of
the
cells,
whereas
the
value
was
2.99%
for
the
control.
In
addition,
we
monitored
the
nuclear
morphological
changes
using
Hoechst
33342
dye.
As
expected,
the
stereotypical
morphological
changes
of
apoptosis,
such
as
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Journal
of
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&
Cell
Biology
44 (2012) 1351–
1360
Fig.
2.
RNase
MC2
induces
apoptosis
in
Hep
G2
cells.
(A)
RNase
MC2
induced
apoptosis
in
Hep
G2
cells.
After
treatment
with
different
concentrations
of
RNase
MC2
for
48
h,
cells
were
stained
with
Annexin
V-PI
double
staining
and
analyzed
on
a
flow
cytometer.
(B)
RNase
MC2
induced
DNA
fragmentation
in
Hep
G2
cells.
After
the
same
treatment
as
mentioned
above,
cells
were
harvested
and
DNA
fragmentation
was
detected
with
a
TUNEL
kit
following
the
manufacturer’s
instructions.
(C)
RNase
MC2
induced
typical
nuclear
morphological
changes
associated
with
apoptosis
in
Hep
G2
cells.
Cells
were
treated
with
30
M
RNase
MC2
for
48
h,
and
subsequently
stained
with
Hoechst
33342
dye.
Nuclear
morphology
was
observed
with
a
fluorescence
microscope.
Bar,
50
m.
(D)
RNase
MC2
induced
mitochondrial
membrane
depolarization
in
Hep
G2
cells.
Cells
were
treated
with
different
concentrations
of
RNase
MC2
for
24
h,
and
used
for
flow
cytometric
analysis
after
staining
with
JC-1
dye.
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The
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Biochemistry
&
Cell
Biology
44 (2012) 1351–
1360 1355
Fig.
3.
RNase
MC2
inhibits
protein
expression
of
mutant
p53
and
p21,
and
activates
caspase
signaling
in
Hep
G2
cells.
(A)
Cells
were
treated
with
15
M
and
30
M
RNase
MC2
for
24
and
48
h,
and
the
expression
of
mutant
p53
and
p21
was
detected
by
Western
blotting.
(B)
RNase
MC2
initiated
processing
of
both
caspase-8
and
caspase-9
signaling,
which
in
turn
proteolytically
activated
effector
caspase
3
and
finally
PARP
was
cleaved.
After
different
treatments,
changes
of
protein
levels
were
detected
by
Western
blotting.
(C)
The
effect
of
RNase
MC2
on
expression
of
different
Bcl-2
proteins.
RNase
MC2
significantly
reduced
the
expression
of
Bcl-2,
and
increased
the
expression
of
Bak,
but
had
no
significant
effects
on
Bid.
(D)
The
pancaspase
inhibitor
Z-VAD-FMK
partially
rescued
RNase
MC-2-induced
apoptosis.
Cells
were
pre-incubated
with
20
M
Z-VAD-FMK
for
1
h,
followed
by
addition
of
RNase
MC2
and
incubation
for
24
h.
Apoptosis
was
measured
using
flow
cytometry
after
Annexin
V-PI
double
staining.
Results
represent
mean
±
SD
(n
=
3).
chromatin
condensation,
DNA
fragmentation,
and
karyorrhexis,
were
discerned
(Fig.
2C).
Moreover,
RNase
MC2
treatment
increased
the
percentage
of
cells
with
mitochondrial
membrane
depolarization
from
7.66%
in
control
to
35.44%
(Fig.
2D).
3.3.
RNase
MC2-induced
apoptosis
was
attributed
to
the
activation
of
caspase
cascades
Since
p53
and
p21
are
two
important
proteins
involved
in
cell
proliferation
and
death,
we
used
Western
blotting
to
investigate
the
changes
of
their
protein
levels
in
RNase
MC2-treated
cells.
Results
showed
that
RNase
MC2
suppressed
the
expression
of
both
p53
and
p21
in
a
dose-
and
time-dependent
manner
(Fig.
3A).
Because
apoptosis
has
proven
to
be
tightly
interwoven
with
both
caspase-dependent
and
-independent
mechanisms
(Cregan
et
al.,
2004;
Pradelli
et
al.,
2010),
we
went
on
to
address
the
ability
of
RNase
MC2
to
activate
caspase
signaling.
We
found
that
RNase
MC2
increased
activation
of
both
caspase-8
and
caspase-9,
followed
by
caspase-3
activation,
and
eventually
progression
thorough
cleav-
age
of
PARP
that
in
turn
triggered
apoptosis
(Fig.
3B).
Furthermore,
some
Bcl-2
protein
members
were
implicated
in
the
apoptosis
process,
including
the
suppression
of
antiapoptotic
Bcl-2,
and
the
induction
of
proapoptotic
Bak.
There
were
no
significant
changes
in
another
proapoptotic
protein,
Bid
(Fig.
3C).
The
activation
of
caspase-9
signaling
pathway
was
in
consistent
with
the
damage
of
mitochondrial
membrane
potential
(Fig.
2D),
because
regulation
of
apoptosis-associated
Bcl-2
members
and
activation
of
caspase-
9
were
involved
in
mitochondria-mediated
apoptotic
pathway,
and
they
were
closely
linked
to
mitochondrial
membrane
depo-
larization
(Samraj
et
al.,
2007;
Pradelli
et
al.,
2010;
Fang
et
al.,
2011a,b).
Furthermore,
addition
of
a
pan-caspase
inhibitor
Z-VAD-
FMK
to
RNase
MC2-treated
cells
significantly
abolished
RNase
MC2-induced
cell
apoptosis,
indicating
that
RNase
MC2-induced
apoptosis
was
at
least
partially
ascribed
to
the
activation
of
caspase
signaling.
3.4.
RNase
MC2
mediated
apoptotic
signaling
is
also
contributed
by
dual
phosphorylation
of
ERK
and
JNK
Because
treatment
with
chemotherapeutic
agents
resulted
in
the
modification
of
phosphorylation
of
Akt
and
mitogen-activated
protein
kinases
(MAPKs)
that
were
associated
with
cell
death
(Widenmaier
et
al.,
2009;
Kim
and
Choi,
2010;
Fang
et
al.,
2011a,b),
attempts
were
made
to
identify
the
phosphorylation
changes
of
these
proteins.
A
time-course
analysis
was
performed
on
the
phos-
phorylation
levels
of
Akt
and
three
MAPK
members,
including
extracellular
signal-regulated
kinase
(ERK),
c-Jun
NH
2
-terminal
kinase
(JNK)
and
p38
MAPK.
It
was
found
that
RNase
MC2
increased
the
phosphorylation
of
Akt
from
0.5
h
to
12
h,
with
the
highest
level
at
8
h.
For
MAPKs,
the
phosphorylation
levels
of
ERK
and
JNK
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/
The
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Journal
of
Biochemistry
&
Cell
Biology
44 (2012) 1351–
1360
Fig.
4.
Phosphorylation
of
Akt,
ERK,
and
JNK
contribute
to
RNase
MC2-induced
apoptosis.
(A)
RNase
MC2
increased
phosphorylation
levels
of
Akt,
ERK,
and
JNK.
Hep
G2
cells
were
incubated
with
15
M
RNase
MC2
for
0–12
h.
Protein
expression
was
determined
by
Western
blotting.
(B)
Phosphorylation
of
ERK
and
JNK
was
intimately
associated
with
MAP30-induced
cell
apoptosis.
Cells
were
separately
pre-incubated
with
the
ERK
inhibitor
PD98059
(10
M),
the
JNK
inhibitor
SP600125
(5
M),
and
the
p38
inhibitor
SB203580
(10
M)
for
1
h.
Subsequently,
cells
were
incubated
in
presence
of
15
M
RNase
MC2
for
24
h.
Apoptosis
was
determined
by
flow
cytometry
using
Annexin
V-PI
staining.
Results
represent
mean
±
SD
(n
=
3).
were
detected,
the
highest
level
of
p-ERK
was
observed
at
1
h
while
the
p-JNK
reached
its
highest
level
at
12
h.
However,
no
significant
phosphorylation
changes
of
p38
MAPK
were
detected
(Fig.
4A).
To
further
validate
the
results,
Hep
G2
cells
were
pre-treated
with
three
MAPK
member
inhibitors,
namely
PD98059
(a
specific
ERK
inhibitor),
SP600125
(a
specific
JNK
inhibitor),
and
SB203580
(a
specific
p38
MAPK
inhibitor).
As
Fig.
4B
indicates,
PD98059
and
SP600125
partially
inhibited
RNase
MC2-induced
cytotoxicity,
as
demonstrated
by
the
increase
of
cell
viability
and
decreased
per-
centage
of
apoptotic
cells,
indicating
that
the
phosphorylation
of
ERK
and
JNK
also
contributed
to
RNase
MC2-induced
apoptosis.
On
the
other
hand,
in
line
with
the
Western
blotting
results,
the
p38
MAPK
inhibitor,
SB203580,
had
no
significant
effect
on
RNase
MC2-induced
cell
fate.
3.5.
RNase
MC2
inhibited
Hep
G2
xenograft
growth
in
nude
mice
To
address
the
pre-clinical
significance
of
RNase
MC2
on
HCC,
we
went
on
to
investigate
its
antitumor
efficacy
on
Hep
G2-
bearing
male
nude
mice.
The
mice
were
treated
intraperitoneally
with
2.0
mg/kg
RNase
MC2
every
other
day
for
a
total
of
7
injec-
tions.
Tumor
volumes
were
monitored
every
other
day.
As
shown
in
Fig.
5A,
tumor
volumes
of
RNase
MC2-treated
group
were
smaller
from
the
9th
day
of
treatment
onward.
A
representa-
tive
comparison
between
an
RNase
MC2-treated
mouse
and
the
corresponding
control
is
shown
in
Fig.
5B.
On
the
13th
day
of
RNase
MC2
treatment,
mice
were
sacrificed
and
tumors
were
excised
(Fig.
5C).
In
accordance
with
the
reduced
tumor
volume
in
the
RNase
MC2-treated
group,
the
average
tumor
weight
of
the
same
group
was
also
smaller
in
comparison
with
control
(p
<
0.05,
Fig.
5D).
3.6.
Apoptosis
induction
was
detected
in
tumor
tissues
of
RNase
MC2-treated
mice
In
order
to
investigate
the
molecular
mechanism
of
RNase
MC2-mediated
tumor
suppression
in
nude
mice,
in
situ
apopto-
sis
in
tumor
tissues
were
assessed.
H&E
staining
showed
that
all
tumors
had
a
morphologic
diagnosis
of
cancer.
Compared
with
control
group,
H&E
showed
apoptotic
cells
with
nuclear
conden-
sation/fragmentation
in
RNase
MC2-treated
group
(Fig.
6A).
The
findings
were
further
concomitant
with
IHC
results.
As
shown
in
Fig.
6B
and
C,
RNase
MC2
increased
activation
of
caspase-3
and
production
of
the
downstream
cleaved
PARP,
and
statistically
sig-
nificant
differences
were
found
(p
<
0.05)
(right
panel).
Moreover,
analysis
of
in
situ
apoptosis
showed
that
compared
to
control
group,
RNase
MC2
administration
induced
significant
increase
of
TUNEL-
positive
cells/field,
as
evidenced
by
an
over
10-fold
increase
of
TUNEL-positive
cells
in
average
(Fig.
6D).
4.
Discussion
Bitter
gourd/BG
(Momordica
charantia
from
family
Cucur-
bitaceae)
is
a
common
fruit
in
many
countries.
It
is
also
vernacularly
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/
The
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Journal
of
Biochemistry
&
Cell
Biology
44 (2012) 1351–
1360 1357
Fig.
5.
Effect
of
RNase
MC2
on
tumor
development
in
nude
mice.
Hep
G2
cells
(2
×
10
7
)
were
injected
into
the
right
flank
of
male
nude
mice.
After
tumors
grew
to
around
0.1
cm
3
,
mice
were
divided
into
two
groups,
one
treated
with
RNase
MC2
(2.0
mg/kg,
every
other
day
for
a
total
of
seven
injections,
n
=
5),
and
another
group
left
untreated
(control,
n
=
5).
(A)
Tumor
volumes
of
both
groups
were
calculated.
(B)
Tumor
growth
in
representative
mouse
from
control
and
treated
groups
is
shown.
(C)
Mice
were
sacrificed
on
the
13th
day
of
RNase
MC2
treatment,
and
tumors
were
excised.
(D)
Consistent
with
reduced
tumor
volume,
the
average
tumor
weight
of
RNase
MC2
group
was
smaller
than
that
of
control.
Results
represent
mean
±
SD
(n
=
5).
named
bitter
melon,
bitter
apple,
balsam
pear,
karela,
and
wild
cucumber
(Krawinkel
and
Keding,
2006).
Compelling
evidence
indicates
that
BG
has
the
ability
to
treat
diabetes
and
its
asso-
ciated
complications
(Leung
et
al.,
2009;
Fang
and
Ng,
2011a,b).
In
recent
years,
the
antitumor
potential
of
BG
has
been
raised
from
several
strands
of
evidence.
First,
crude
BG
extract
mani-
fested
antitumor
activity
in
vitro
and
in
vivo
against
both
breast
cancer
cells
and
prostate
cancer
cells
by
modulating
cell
cycle
reg-
ulatory
genes
and
the
induction
of
apoptosis
(Ray
et
al.,
2010;
Ru
et
al.,
2011).
Second,
some
medicinal
components
have
been
puri-
fied
from
BG
and
their
antitumor
potentials
were
investigated.
For
instance,
both
Kuguacin
J
(a
triterpenoid)
and
MCP30
(a
combi-
nation
of
two
ribosome
inactivating
proteins,
alpha-momorcharin
and
beta-momorcharin)
were
effective
against
human
prostate
cancer
(Xiong
et
al.,
2009;
Pitchakarn
et
al.,
2011).
M.
charantia
lectin/MCL
manifested
antitumor
activity
toward
nasopharyngeal
carcinoma
cells
in
vitro
and
in
vivo
(Fang
et
al.,
2011a,b).
In
the
current
study,
we
extended
the
antitumor
potential
of
RNase
MC2
from
breast
cancer
to
HCC.
Same
as
its
action
on
breast
cancer
MCF-7
cells,
RNase
MC2
exhibited
cytotoxicity
on
Hep
G2
cells
which
was
attributed
to
its
cytostatic
action
and
induction
of
cell
death.
Cell
cycle
arrest
in
the
S
phase
may
have
at
least
partially
contributed
to
its
cyto-
static
activity.
Our
results
are
reminiscent
of
the
antitumor
effect
of
crude
BG
extract
on
prostate
cancer
cells
(Ru
et
al.,
2011).
Ru
et
al.
(2011)
found
that
BG
extract
impaired
prostate
cancer
cell
cycle
progression
in
S
phase
associated
with
the
down-regulation
of
cyclin
D1
and
cyclin
E
and
increase
of
p21.
It
should
be
noted
that
different
BG
components
target
different
phases
of
the
cell
cycle.
For
example,
BG
extract
induced
MCF-7
cell
accumulation
in
G2-M
phase
of
the
cell
cycle
by
inhibition
of
cyclin
B1
and
cyclin
D1,
and
the
up-regulation
of
p53,
p21,
and
pChk1/2
(Ray
et
al.,
2010).
In
contrast,
our
results
indicated
that
RNase
MC2
inhibited
the
protein
levels
of
both
p53
and
p21
(Fig.
3A),
which
were
consistent
with
the
results
on
MCL-treated
NPC
cells
(Fang
et
al.,
2011a,b).
This
is
explainable
since
the
Hep
G2
cell
line
used
here
has
been
reported
to
have
an
N-ras
mutation
at
position
2
of
codon
61
(Hsu
et
al.,
1993).
The
down-regulation
of
mutant
p53
by
RNase
MC2
may
cater
the
need
to
develop
therapeutic
strate-
gies
to
eradicate
the
possible
anti-apoptotic
activity
of
mutant
p53,
because
this
mutation
frequently
occurs
in
HCC
(Lee
et
al.,
2000;
Ohnishi
et
al.,
2002).
Moreover,
p21
has
an
‘antagonistic
duality’
in
that
it
often
inhibits
apoptosis
(procancer)
to
counter-
act
its
anticancer
effects
for
it
has
been
reported
to
be
a
negative
regulator
of
both
p53-dependent
and
p53-independent
apoptosis,
and
it
also
induces
apoptosis
(Gartel
and
Tyner,
2002).
How-
ever,
elimination
of
p21
often
increases
sensitivity
of
tumor
cells
to
apoptosis
induced
by
many
chemotherapeutic
agents
(Gartel
and
Tyner,
2002).
In
this
regard,
the
down-regulation
of
p53
and
p21
by
RNase
MC2
may
assist
to
expedite
the
apoptotic
pro-
cess.
RNase
MC2
inhibited
growth
of
Hep
G2
cells
in
soft
agar
and
retarded
cell
motility.
This
is
inspiring
since
HCC
metastasis
is
a
predominant
cause
of
its
high
relapse
risk
and
low
survival
rate
(Katyal
et
al.,
2000).
In
view
of
the
observation
that
apoptosis
is
involved
in
the
antitumor
activity
of
a
host
of
Page 7
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et
al.
/
The
International
Journal
of
Biochemistry
&
Cell
Biology
44 (2012) 1351–
1360
Fig.
6.
RNase
MC2
induces
apoptosis
in
tumor
tissues.
Mice
were
sacrificed
on
the
13th
day
of
RNase
MC2
treatment,
tumors
were
excised
and
processed
for
IHC
staining.
Tissues
were
examined
for
apoptosis
by
H&E
(A),
activated
caspase-3
(B),
cleaved
PARP
(C),
and
TUNEL
(D).
A
representative
picture
of
each
labeling
is
shown.
For
quantitative
analysis,
cells
in
5
microscopic
fields
(40×
magnification)
were
calculated
and
scored
(B–D,
right
panel).
Data
are
represented
as
mean
±
SD
(n
=
5).
p
<
0.05
compared
with
control.
Bars
in
A–C
are
200
M
while
in
D
are
16
M.
chemotherapeutic
agents
(Fang
et
al.,
2011a,b),
we
further
explored
the
apoptosis-inducing
activity
of
RNase
MC2.
The
results
dis-
closed
the
apoptosis-inducing
activity
of
RNase
MC2
as
evidenced
by
the
increase
of
Annexin-V
positive
cells,
increase
in
the
per-
centage
of
cells
with
DNA
fragmentation,
and
the
detection
of
significant
nuclear
morphological
changes
typical
of
apoptosis
in
RNase
MC2-treated
cells.
Our
following
studies
indicated
that
both
caspase-8
regulated
extrinsic
and
caspase-9
regulated
intrin-
sic
(mitochondrial)
caspase
pathways
culminated
in
induction
of
apoptosis.
This
notion
was
supported
by
data
indicating
that
the
activation
of
the
initiator
caspases
(caspase-8
and
-9),
the
executor
caspase-3,
followed
by
cleavage
of
the
downstream
PARP,
that
in
turn
triggered
apoptosis.
These
were
commensu-
rate
with
findings
in
RNase
MC-2-treated
MCF-7
cells
(Fang
et
al.,
2011a,b).
In
recent
years,
the
importance
of
Bcl-2
family
members
in
mitochondrial-associated
apoptotic
network
in
vertebrates
is
noticed
and
corroborated
in
different
investigations
(Martinou
and
Youle,
2011).
Pitchakarn
et
al.
(2011)
found
that
both
BG
leaf
extract
and
a
purified
triterpenoid
Kuguacin
J
inhibited
growth
of
androgen-dependent
LNCaP
prostate
cancer
cells
by
augmenting
Bax/Bcl-2
and
Bad/Bcl-2.
It
has
also
been
reported
that
both
MAP30,
a
RIP
purified
from
BG
(Lee-Huang
et
al.,
1990)
and
BG
seed
fatty
acid
induced
apoptosis
in
human
intestinal
tumor
cells
by
reduc-
tion
of
the
antiapoptotic
member
Bcl-2
(Yasui
et
al.,
2005;
Fan
et
al.,
2008).
There
were
also
exceptions,
for
there
were
no
changes
of
Bcl-
2,
Bak
and
Bid
in
the
process
of
MCL-induced
apoptosis
in
NPC
cells
(Fang
et
al.,
2011a,b).
In
this
case,
our
results
showed
that
Bcl-2
was
significantly
decreased
and
Bak
was
increased
in
RNase
MC2-
treated
Hep
G2
cells.
However,
no
significant
changes
in
Bid
were
detected.
The
function
of
Bid
may
be
substituted
by
other
BH3-only
proapoptotic
proteins.
In
tumor
cells
exposed
to
chemotherapeutic
agents,
several
sig-
naling
pathways
were
constitutively
regulated,
such
as
the
Akt
and
MAPK
pathways
(Meier
et
al.,
2007;
Fang
et
al.,
2011a,b).
It
has
been
reported
that
the
phosphorylation
levels
of
Akt
could
be
increased
or
inhibited
in
tumor
cells
exposed
to
different
medic-
inal
components
(Ng
et
al.,
2001;
Fang
et
al.,
2011a,b).
In
MAPKs
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E.F.
Fang
et
al.
/
The
International
Journal
of
Biochemistry
&
Cell
Biology
44 (2012) 1351–
1360 1359
signaling
pathways,
the
phosphorylation
of
p38
MAPK
and
JNK
is
proapoptotic
(Mansouri
et
al.,
2003;
Chen
et
al.,
2004;
Fang
et
al.,
2011a,b),
whereas
ERK
phosphorylation
has
been
linked
to
both
antitumor
and
pro-tumor
effects
(Chen
et
al.,
2004;
Wilhelm
et
al.,
2004;
Fang
et
al.,
2011a,b).
In
RNase
MC2-treated
Hep
G2
cells,
we
have
detected
increased
phosphorylation
levels
of
Akt
and
two
MAPK
members,
ERK
and
JNK.
To
further
investigate
the
function
of
early
activation
of
Akt
in
RNase
MC2-induced
cell
apoptosis,
the
PI3K/Akt
pathway
inhibitor
LY294002
(10
M,
2
h
pretreatment
before
adding
the
same
dose
of
RNase
MC2)
was
applied.
Our
results
indicated
that
LY294002
slightly
increased
RNase
MC2-induced
cell
apoptosis,
but
no
statistical
significance
was
found
(data
not
shown).
Our
results
are
reminiscent
of
the
studies
done
by
Clark
et
al.
(2002)
who
first
reported
the
iden-
tification
of
early
induction
of
Akt
activity
by
therapeutic
agents.
They
found
that
many
chemotherapeutical
agents,
such
as
doxoru-
bicin,
trastuzumab,
or
tamoxifen
alone
induced
activation
of
Akt
before
the
onset
of
apoptosis
in
a
list
of
breast
cancer
cells
(Clark
et
al.,
2002).
In
view
of
that
Akt
phosphorylation
promotes
cellu-
lar
survival,
and
the
activation
of
Akt
has
also
been
detected
in
the
early
phase
when
tumor
cells
exposed
to
different
chemother-
apeutic
agents,
we
speculate
that
the
activation
of
Akt
may
be
a
common
phenomenon
of
cellular
stress
response
for
cell
sur-
vival.
We
further
validated
that
ERK
and
JNK
activation
was
neces-
sary
for
the
RNase
MC2-induced
apoptosis
in
Hep
G2
cells
because
a
panel
of
pharmacological
inhibitors
reduced
RNase
MC2-induced
apoptosis.
In
view
of
the
observation
that
Akt
and
MAPKs
are
impor-
tant
targets
for
cancer
therapy
for
a
close
linkage
to
numerous
proliferative
and
survival
pathways,
the
multiple
regulatory
activi-
ties
of
RNase
MC2
toward
Akt
and
MAPKs
consolidate
its
medicinal
applications.
Besides
in
vitro
inhibition
of
tumor
cell
proliferation,
the
in
vivo
and
pre-clinical
antitumor
efficacies
of
some
RNases
have
also
been
discovered
(Fang
and
Ng,
2011a,b).
In
this
regard,
we
went
on
to
determine
the
in
vivo
antitumor
effect
of
RNase
MC2.
The
syngeneic
nude
mice
Hep
G2
solid
tumor
model
was
used
to
further
examine
the
in
vivo
antitumor
potential
of
RNase
MC2.
The
murine
stud-
ies
demonstrated
that
RNase
MC2
manifested
excellent
antitumor
activity
against
Hep
G2
xenograft,
as
witnessed
by
the
reduction
of
tumor
volume
as
well
as
retardation
of
tumor
progression.
Our
following
IHC
studies
revealed
that
RNase
MC2
induced
apopto-
sis
in
tumor
tissues
as
supported
by
the
increase
of
the
activated
caspase-3,
cleaved
PARP,
and
TUNEL-positive
cells.
Furthermore,
the
dose
of
RNase
MC2
applied
had
no
detectable
toxicity
to
mice,
such
as
no
effects
on
total
weights
and
no
detectable
toxicity
to
normal
tissues,
highlighting
the
translational
potential
of
this
new
medicinal
component.
In
conclusion,
in
this
study
we
have
identified
RNase
MC2
as
a
medicinal
agent
that
can
induce
apoptosis
in
Hep
G2
cells
in
vitro
and
in
vivo.
Mechanisms
involved
in
RNase
MC2-induced
anti-
proliferative
and
apoptotic
activities
include
(1)
cell
cycle
arrest
at
S
phase;
(2)
reduction
of
mutant
p53
and
p21;
(3)
initiating
both
caspase-8
and
caspase-9-dependent
signaling
pathways
of
apopto-
sis;
(4)
multiple
regulation
of
the
phosphorylation
levels
of
Akt,
ERK
and
JNK;
and
most
importantly
(5)
induction
of
tumor
cell
apoptosis
in
Hep
G2-bearing
mice.
The
manifestation
of
antitumor
proper-
ties
of
RNase
MC2
from
BG
both
in
vitro
and
in
vivo,
in
addition
to
consumption
of
the
knobbly
BG
fruit
as
a
healthy
vegetable
in
many
countries,
suggest
that
RNase
MC2
holds
promise
for
clinical
application
on
HCC.
Conflicts
of
interest
No
potential
conflicts
of
interest
were
disclosed.
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