Effects of lycopene and apigenin on human umbilical vein endothelial cells in vitro under angiogenic stimulation

Article (PDF Available)inActa histochemica 114(2):94-100 · April 2011with35 Reads
DOI: 10.1016/j.acthis.2011.03.004 · Source: PubMed
Abstract
Angiogenesis is the formation process of new blood vessels from preexisting vessels. Solid tumors need angiogenesis for growth and metastasis. The suppression of tumor growth by inhibition of neoangiogenic processes represents a potential approach to cancer treatment. Lycopene has powerful antioxidant capacities and anticarcinogenic properties. The aim of this study was to investigate the effects of lycopene on angiogenesis in vitro. For this reason, we measured in vitro angiogenesis in human umbilical vein endothelial cells including parameters of cell proliferation, tube formation, cell migration. Lycopene and apigenin were observed to block the endothelial cell proliferation in a dose-dependent manner. In addition, they significantly decreased the capillary-like tube lengths, tube formation and endothelial cell migration. This study provides indications that apigenin and lycopene, which are considered as chemopreventive agents, to be effective in vitro on endothelial cells and angiogenesis.
Acta
Histochemica
114 (2012) 94–
100
Contents
lists
available
at
ScienceDirect
Acta
Histochemica
j
o
ur
nal
homepage:
www.elsevier.de/acthis
Effects
of
lycopene
and
apigenin
on
human
umbilical
vein
endothelial
cells
in
vitro
under
angiogenic
stimulation
Mehmet
S¸
ahin
a
,
Emel
S¸
ahin
b
,
Saadet
Gümüs¸
c,
a
Health
Sciences
Research
Centre,
Faculty
of
Medicine,
Akdeniz
University,
07070
Antalya,
Turkey
b
Central
Laboratory,
Clinical
Biochemistry
Unit,
Faculty
of
Medicine,
Akdeniz
University,
07070
Antalya,
Turkey
c
Department
of
Biochemistry,
Faculty
of
Medicine,
Akdeniz
University,
07070
Antalya,
Turkey
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
28
December
2010
Received
in
revised
form
4
March
2011
Accepted
6
March
2011
Keywords:
Angiogenesis
Lycopene
Apigenin
Cancer
HUVEC
a
b
s
t
r
a
c
t
Angiogenesis
is
the
formation
process
of
new
blood
vessels
from
preexisting
vessels.
Solid
tumors
need
angiogenesis
for
growth
and
metastasis.
The
suppression
of
tumor
growth
by
inhibition
of
neoangiogenic
processes
represents
a
potential
approach
to
cancer
treatment.
Lycopene
has
powerful
antioxidant
capac-
ities
and
anticarcinogenic
properties.
The
aim
of
this
study
was
to
investigate
the
effects
of
lycopene
on
angiogenesis
in
vitro.
For
this
reason,
we
measured
in
vitro
angiogenesis
in
human
umbilical
vein
endothe-
lial
cells
including
parameters
of
cell
proliferation,
tube
formation,
cell
migration.
Lycopene
and
apigenin
were
observed
to
block
the
endothelial
cell
proliferation
in
a
dose-dependent
manner.
In
addition,
they
significantly
decreased
the
capillary-like
tube
lengths,
tube
formation
and
endothelial
cell
migration.
This
study
provides
indications
that
apigenin
and
lycopene,
which
are
considered
as
chemopreventive
agents,
to
be
effective
in
vitro
on
endothelial
cells
and
angiogenesis.
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Angiogenesis,
the
formation
of
new
blood
vessels,
plays
several
roles
in
various
human
pathologies
including:
rheumatoid
arthritis,
diabetic
retinopathy,
atherosclerosis,
psoriasis,
and
chronic
airway
inflammation.
One
of
the
most
important
roles
of
angiogenesis
is
to
support
tumor
growth,
which
is
dependent
on
nutritional
support
derived
from
the
local
blood
supply
(Sahin
et
al.,
2009).
Targeting
inhibition
of
angiogenesis
represents
a
potential
approach
in
the
treatment
of
solid
tumors
and
such
antiangio-
Abbreviations:
BHT,
butylated
hydroxytoluene;
DMSO,
dimethyl
sulfoxide;
EBM-2,
endothelial
basal
medium;
EGM-2,
endothelial
growth
medium;
EGF,
epi-
dermal
growth
factor;
FBS,
fetal
bovine
serum;
FGF,
fibroblast
growth
factor;
Hep3B,
a
human
hepatoma
cell
line;
HUVEC,
human
umbilical
vein
endothelial
cells;
IGF-
1,
insulin-like
growth
factor;
KB-1,
a
cell
line
derived
from
a
human
oral
cavity
tumor;
LNCaP,
an
androgen-sensitive
human
prostate
adenocarcinoma
cell
line;
MCF-7,
a
breast
cancer
cell
line;
MDA-MB-231,
an
estrogen-independent
human
breast
cancer
cell
line;
MMP,
matrix
metalloproteinase;
MTT,
(3
(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide);
PC3,
a
metastatic
cell
line
derived
from
advanced
androgen
independent
prostate
cancer;
RPMI,
Roswell
Park
Memorial
Institute
cell
culture
medium;
SK-Hep1,
an
immortal
and
metastatic
cell
line
derived
from
a
liver
adenocarcinoma;
THF,
tetrahydrofuran;
VEGF,
vascular
endothelial
growth
factor.
Corresponding
author.
Tel.:
+90
2422496896;
fax:
+90
2422274495.
E-mail
addresses:
msahin@akdeniz.edu.tr
(M.
S¸
ahin),
esahin@akdeniz.edu.tr
(E.
S¸
ahin),
sgumuslu@akdeniz.edu.tr
(S.
Gümüs¸
lü).
genic
strategies
inhibiting
the
growth
of
endothelial
cells
may
be
more
advantageous
than
targeting
cancer
cells.
The
inhibition
of
tumor
growth
requires
a
chronic
inhibition
of
vascular
orga-
nization,
defined
as
‘angiostasis’
(Hanahan
and
Folkman,
1996).
Long-term
treatment
is
necessary
to
achieve
angiostasis
and
it
is
necessary
to
identify
and
characterize
angiostatic
molecules
show-
ing
low
or
no
toxicity.
Current
dietary
guidelines
to
combat
chronic
diseases
including
cancer,
recommend
increased
intake
of
plant
foods
that
are
rich
in
antioxidants.
The
role
of
dietary
antioxidants,
including
carotenoids
and
flavonoids
in
disease
prevention
has
received
much
attention
(Kim
et
al.,
1998;
Agarwal
and
Rao,
2000).
Apigenin
is
a
flavonoid
with
anti-inflammatory,
anticarcinogenic
and
free
radical
scavenger
properties
(Kim
et
al.,
1998).
Lycopene,
one
of
more
than
600
carotenoids,
is
a
natural
pigment
synthesized
by
plants
and
photosynthetic
microorganisms.
Several
epidemio-
logical
studies
have
strongly
implied
that
consumption
of
foods
containing
high
concentrations
of
lycopene
may
reduce
the
risk
of
certain
types
of
cancer
(Gann
et
al.,
1999;
Giovannucci
et
al.,
2002).
The
mechanism
of
lycopene
action
is
still
under
investigation.
Lycopene
was
shown
to
induce
apoptosis
in
a
dose-dependent
manner
in
prostate
cancer
cells
(Hantz
et
al.,
2005).
Similarly,
treat-
ment
of
human
colon
carcinoma
cells
with
lycopene
at
2.0
or
4.0
M
was
found
to
be
able
to
induce
apoptosis
(Salman
et
al.,
2007).
There
have
been
several
reports
that
lycopene
can
induce
cell
cycle
arrest.
It
has
been
reported
that
the
growth
of
Hep3B
human
hepatoma
cells
was
inhibited
20–50%
by
lycopene
(Park
et
al.,
2005).
A
simi-
0065-1281/$
see
front
matter ©
2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.acthis.2011.03.004
M.
S¸
ahin
et
al.
/
Acta
Histochemica
114 (2012) 94–
100 95
Fig.
1.
Lycopene
inhibited
tube
formation
produced
by
human
umbilical
vein
endothelial
cells.
Representative
micrographs
of
HUVECs
cultured
for
24
h
on
Matrigel
in
the
presence
of
the
indicated
compound
are
illustrated.
Serum-starved
cells
were
seeded
to
each
well
of
24-well
plate
as
2.5
×
10
4
cells
in
EGM-2
complete
medium.
HUVEC
cells
were
incubated
in
(A)
THF
(Control),
(B)
Lyc
1
M,
(C)
Lyc
5
M
and
(D)
Lyc
10
M.
Scale
bar
=
250
m.
lar
study
with
the
human
prostate
cancer
cell
lines,
LNCaP
and
PC3,
also
found
that
lycopene
induced
mitotic
arrest
at
the
G0/G1
phase
(Ivanov
et
al.,
2007).
With
respect
to
anti-invasive
and
anti-metastatic
activities,
lycopene
(5–10
M)
has
been
shown
to
decrease
the
activities
of
the
gelatinolytic
matrix
metalloproteinases,
MMP-2
and
MMP-9,
and
to
inhibit
adhesion,
invasion
and
migration
of
SK-Hep1
cells,
which
is
a
highly
invasive
human
hepatoma
cell
line
(Hwang
and
Lee,
2006).
Lycopene
at
similar
concentrations
was
also
found
to
induce
the
metastasis
suppressor
gene
nm23-H1
(Huang
et
al.,
2005).
Although
there
have
been
several
studies
based
on
the
antiox-
idant
and
anticarcinogenic
activities
of
lycopene,
its
role
on
endothelial
cells
and
angiogenesis
has
remained
unclear.
The
aim
of
this
study
was
to
investigate
whether
lycopene
is
effective
on
endothelial
cells
and
angiogenesis
inhibition
at
different
concen-
trations,
and
to
compare
the
effects
of
lycopene
with
apigenin
with
regard
to
antiangiogenic
activities.
Materials
and
methods
Reagents
Human
umbilical
vein
endothelial
cells
(HUVECs)
and
Matrigel
TM
were
purchased
from
BD
Biosciences
(BD-354234;
Bedford,
MA,
USA).
EGM-2
®
BulletKit
®
(CC-3162,
EBM-
2
+
supplements)
for
HUVEC
culture
was
purchased
from
Lonza
(Walkersville,
MD,
USA).
Lycopene
and
apigenin
were
purchased
from
Sigma–Aldrich
(St.
Louis,
MO,
USA).
Cell
culture
Human
umbilical
vein
endothelial
cells
(HUVECs)
were
cul-
tured
in
EGM-2
complete
medium
consisting
of
EBM-2
basal
medium
supplemented
with
hydrocortisone,
ascorbic
acid,
hep-
arin,
GA-1000
and
2%
FBS
plus
growth
factors
(epidermal
growth
factor
(EGF),
vascular
endothelial
growth
factor
(VEGF),
fibroblast
growth
factor
(FGF),
insulin-like
growth
factor
(IGF-1))
except
for
serum
starvation
in
which
FBS
concentration
was
reduced
to
0.1%
and
no
growth
factors
were
added.
Cell
culture
flasks
or
well
plates,
pre-coated
with
gelatin,
were
used
for
culture
of
HUVECs
(Sigma–Aldrich,
Irvine,
Ayrshire,
UK).
All
cells
were
grown
in
a
humidified
atmosphere,
95%
air
and
5%
CO
2
at
37
C
and
passaged
every
4–6
days.
Cells
from
the
fourth
to
the
sixth
passage
were
used
for
experiments.
Lycopene
was
dissolved
in
tetrahydrofuran
(THF;
stabilized
with
0.025%
BHT)
and
applied
to
the
cells
at
0
M,
1
M,
5
M
and
10
M
concentrations.
Lycopene
was
prepared
according
to
methods
reported
by
Martin
et
al.
(2000)
and
Lin
et
al.
(2007)
to
increase
its
stability
and
to
facilitate
its
uptake
by
cells.
Apigenin
was
dissolved
in
dimethyl
sulfoxide
(DMSO)
and
added
at
5
mg/l
(18.5
M)
and
10
mg/l
(37
M)
concentrations.
The
final
concen-
tration
of
DMSO
and
THF
did
not
exceed
respectively
0.2%
(v/v)
and
0.1%
(v/v)
in
any
case.
In
vitro
angiogenesis
assay
Matrigel
TM
(BD-354234,
Bedford,
MA,
USA)
was
thawed
at
4
C,
and
250
l
were
quickly
added
to
each
well
of
a
24-well
plate
and
allowed
to
solidify
for
1
h
at
37
C.
Once
solid,
HUVECs,
serum-
96 M.
S¸
ahin
et
al.
/
Acta
Histochemica
114 (2012) 94–
100
starved
for
24
h,
were
added
to
each
well
(2.5
×
10
4
cells/well)
in
EGM-2
complete
medium.
After
cells
had
adhered
to
the
Matrigel,
DMSO,
THF,
apigenin
or
lycopene
was
added,
and
cells
were
incu-
bated
at
37
C
for
24
h.
The
tube
formation
of
HUVECs
was
recorded
with
an
Olympus
IX81
Photomicroscope
(Olympus,
Tokyo,
Japan)
with
a
×4
objective.
Tube
lengths
of
three
different
fields
of
images
were
assayed
with
the
UTHSCSA
ImageTool
Version
3.0
software
programme
(University
of
Texas
Health
Science
Center,
San
Anto-
nio,
TX,
USA).
Experiments
were
repeated
five
times
and
the
results
were
expressed
as
mean
±
standard
deviation
(SD).
Cell
proliferation
assay
The
effects
of
lycopene
and
apigenin
on
the
viability
of
cells
were
determined
by
MTT
(3
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium
bromide)
assay
as
described
previously
with
some
modifications
(Mosmann,
1983).
MTT
was
purchased
from
AppliChem
(Darmstadt,
Germany).
MTT
stock
solution
(5
mg/ml)
in
RPMI-1640
without
phenol
red
(Sigma–Aldrich,
St.
Louis,
MO,
USA)
was
diluted
(1:10)
for
experiments.
The
cells
were
plated
at
2.5
×
10
4
cells
per
well.
70–80%
confluent
HUVECs
in
24-
well
plates
were
serum-starved
for
24
h.
After
24
h,
HUVECs
were
treated
with
DMSO,
THF,
apigenin,
or
lycopene
and
incubated
at
37
C
for
24
h.
After
medium
was
exchanged
with
500
l
MTT
solution,
cells
were
incubated
at
37
C
for
3
h.
At
the
end
of
the
incu-
bation
period,
the
converted
dye
was
solubilized
with
500
l
acidic
isopropanol
(0.04
M
HCl
in
absolute
isopropanol).
Absorbance
of
the
converted
dye
was
measured
at
570
nm
with
background
sub-
traction
at
650
nm
by
colorimetric
plate
reader
(Model
No.:
1500;
Thermo
Labsystem,
Finland).
Experiments
were
repeated
five
times
and
the
results
were
expressed
as
mean
±
SD.
Cell
migration
assay
We
used
a
Millipore
QCM
TM
Endothelial
Migration
Assay
Kit
containing
fibronectin
coated
24-well
Boyden
Chamber
System
with
3
m
pore
width
(Millipore
ECM201,
Temecula,
CA,
USA).
Experiments
were
carried
out
according
to
manufacturer’s
kit
assay
procedures.
Briefly,
migrated
cells
at
the
bottom
plate
were
dyed
with
CyQUANT
®
GR
fluorescent
dye
(Millipore,
Temecula,
CA,
USA).
This
green-fluorescent
dye
was
assayed
with
a
fluorescence
plate
reader
using
480/520
nm
filter
set
(Type:
374;
Thermo
Fluoroskan
Ascent
FL,
Finland).
Experiments
were
repeated
five
times
and
results
were
expressed
as
mean
±
SD
of
five
different
experiments.
Wound
healing
assay
For
another
cell
migration
assay,
a
wound
healing
assay
was
applied
as
horizontal
migration.
The
experiment
was
applied
using
methods
of
Schleef
and
Birdwell
(1982)
with
some
mod-
ifications.
After
full-confluent
HUVECs
were
starved
of
serum
and
growth
factors
for
24
h,
cells
in
12-well
plate
were
scratched
with
a
sterile
200
l
plastic
pipette
tip
across
mono-
layer
of
cells.
Cells
were
treated
with
DMSO,
THF,
apigenin
or
lycopene.
When
HUVECs
in
control
groups
had
almost
com-
pletely
migrated
(approximately
12–14
h),
cells
were
dyed
with
1,1
-dioctadecyl-3,3,3
,3
-tetramethylene
indocarbocynanine
per-
chlorate
(Dil)
(Aldrich
468495)
and
photographed
by
integrated
Olympus
DP70
digital
camera
(Olympus,
Japan)
on
an
inverted
microscope
(Olympus
IX81S1F-2,
Japan)
with
x40
magnification.
Dil
were
prepared
as
stock
solution
(1
mg/ml)
in
DMSO
and
diluted
as
1:200
in
growth
medium
for
experiments.
Migration
distances
of
ten
different
fields
at
photographs
were
assayed
with
UTHSCSA
ImageTool
Version
3.0
software
programme
(University
of
Texas
Health
Science
Center,
San
Antonio,
TX,
USA).
Experiments
were
repeated
five
times.
Fig.
2.
Inhibition
of
the
in
vitro
angiogenesis
of
human
umbilical
vein
endothe-
lial
cells
by
apigenin.
Representative
micrographs
of
HUVECs
cultured
for
24
h
on
Matrigel
as
mentioned
in
material
and
methods
section
in
the
presence
of
the
indi-
cated
compound
are
illustrated.
HUVECs
were
incubated
with
(A)
DMSO
(Control),
(B)
Apigenin
(5
g/ml)
and
(C)
Apigenin
(10
g/ml).
Scale
bar
=
250
m.
Statistical
analysis
Data
were
expressed
as
mean
±
standard
deviation
(SD).
Com-
parison
of
groups
according
to
the
parameters
was
performed
using
the
Student’s
t-test
and
test
of
one-way
ANOVA
(Tukey’s
Multiple
Comparison)
in
Prism
Program
of
GraphPad
Software
(San
Diego,
CA).
P
values
less
than
0.05
were
considered
statistically
significant.
Results
Lycopene
and
apigenin
inhibit
capillary-like
tube
formation
In
this
study,
we
found
that
1
M,
5
M
and
10
M
concen-
trations
of
lycopene
have
significantly
suppressed
the
endothelial
tube
formations
(7102
±
497,
4448
±
426,
2718
±
287,
respectively)
on
Matrigel
matrix
as
compared
with
controls
(14574
±
575)
in
which
the
cells
were
treated
with
THF
(p
<
0.001)
(Fig.
1,
Fig.
3A).
M.
S¸
ahin
et
al.
/
Acta
Histochemica
114 (2012) 94–
100 97
Fig.
3.
Tube
lengths
of
capillary-like
structures
are
affected
by
the
doses
of
lycopene
in
(A)
and
apigenin
in
(B).
***
Shows
p
<
0.001.
We
found
that
the
inhibition
of
tube
lengths
and
organization
was
dose-dependent.
In
apigenin-treated
HUVECs,
capillary-like
tube
formation
was
also
dramatically
inhibited
at
5
g/ml
(18.5
M)
or
10
g/ml
(37
M)
concentrations
(8098
±
479,
3842
±
461,
respectively)
as
compared
with
control
(16431
±
502,
p
<
0.001)
(Figs.
2
and
3
Figs.
2
and
3B).
Lycopene
and
apigenin
suppress
cell
proliferation
Treatment
of
cells
with
three
different
concentrations
of
lycopene
(Fig.
4A)
and
two
different
concentrations
of
apigenin
(Fig.
4B)
decreased
the
proliferation
of
endothelial
cells
in
a
dose-
dependent
manner.
As
compared
with
controls,
percentage
of
the
proliferating
cells
in
groups
treated
with
1
M,
5
M
and
10
M
concentrations
of
lycopene
(63.34
±
2.34,
41.53
±
2.17,
30.24
±
1.96
expressed
as
the
percentage
of
control
cells,
respectively)
was
found
to
be
significantly
decreased
(p
<
0.001).
Percentage
of
prolif-
eration
was
seen
to
be
dose-dependent.
Difference
between
doses
was
found
significant
(p
<
0.001)
(Fig.
4A).
Apigenin
was
also
shown
to
be
effective
against
endothelial
proliferation
at
5
g/ml
and
10
g/ml
concentrations
(54.24
±
2.33
and
38.15
±
2.38,
respec-
tively)
compared
with
control
groups
(expressed
as
100)
(p
<
0.001).
In
addition,
there
was
a
significant
difference
among
the
doses,
as
well
(p
<
0.001)
(Fig.
4B).
Lycopene
and
apigenin
block
endothelial
cell
migration
Endothelial
cells
migrate
towards
the
chemo-attractants
dur-
ing
angiogenesis.
In
this
system
endothelial
cells
migrate
through
the
pores
and
move
to
the
bottom
of
the
plate
containing
EGM-2
medium
and
serum.
Lycopene
at
1
M,
5
M
and
10
M
con-
centrations
suppressed
endothelial
cell
migration
through
pores
compared
with
controls
(56.02
±
3.11,
52.10
±
2.44,
42.06
±
3.24,
respectively)
(Fig.
5A).
Values
of
fluorescence
intensity
in
control
groups
were
expressed
as
100%.
We
did
not
find
any
significant
difference
between
1
and
5
M
lycopene.
However,
differences
among
other
doses
were
found
as
significant
(p
<
0.001).
It
was
shown
5
g/ml
and
10
g/ml
apigenin
concentrations
decreased
fluorescence
intensity
of
cells
(57.98
±
3.22,
49.72
±
2.84,
respec-
tively)
as
compared
with
control
groups
(expressed
as
100%)
(p
<
0.001).
10
g/ml
apigenin
was
more
effective
than
5
g/ml
(p
<
0.01)
(Fig.
5B).
Lycopene
and
apigenin
decrease
directional
migration
Additionally,
we
have
also
used
another
migration
assay
method
named
“wound
healing
assay”
showing
directional
migration.
We
only
assayed
10
M
lycopene
and
10
g/ml
apigenin
for
this
assay.
Similarly,
migration
of
cells
towards
each
other
was
found
to
be
suppressed
(or
migration
length
increase)
both
group
treated
with
lycopene
(610.17
±
36.17)
and
apigenin
(670.62
±
39.39)
as
com-
pared
to
controls
THF
(68.41
±
15.36)
and
DMSO
(99.46
±
19.39)
(Fig.
6A–D)
(p
<
0.001).
Discussion
The
critical
role
of
tumor
angiogenesis
in
cancer
progression
was
postulated
about
40
years
ago
in
pioneering
studies
by
Folkman
et
al.
(1971).
However,
only
in
recent
years
has
the
knowledge
of
endothelial
cell
physiology
and
tumor
angiogenesis
provided
the
necessary
background
to
develop
effective
antiangiogenic
strate-
gies.
Developing
antiangiogenic
strategies
against
the
growth
of
endothelial
cells
may
be
more
advantageous
than
directly
target-
ing
cancer
cells
in
shrinkage
of
tumors.
The
endothelial
cell,
which
is
a
cell
type
common
to
all
solid
tumors,
represents
a
preferential
target
for
therapy.
Even
though
every
cancer
is
virtually
a
unique
disease,
the
tumor
endothelium
is
a
relatively
uniform,
normal
cell
type
(Tosetti
et
al.,
2002).
Another
importance
of
this
approach
is
the
apparent
inability
by
the
endothelial
cells
to
counteract
ther-
apy
through
development
of
multi-drug
resistance
mechanisms,
due
to
the
low
mutagenesis
rate
of
this
normal
cell
type
(Boehm
et
al.,
1997).
Chronic
inhibition
of
vascular
recruitment
may
be
required
to
block
tumor
growth;
thus,
long-term
treatment
may
be
necessary
(Hanahan
and
Folkman,
1996).
Therefore,
we
need
to
identify
and
characterize
angiostatic
molecules
endowed
with
low
or
no
toxi-
city.
Substantial
efforts
have
been
dedicated
to
identifying
natural
and
synthetic
compounds
that
can
be
used
to
either
prevent
insur-
gence
of
primary
tumors
in
subjects
at
high
risk
to
develop
cancer
or
prevent
tumor
relapse
after
surgical
removal.
Cancer
chemo-
prevention
involves
the
use
of
agents
to
slow
the
progression
of
carcinogenesis,
reverse,
or
inhibit
it,
with
the
aim
of
lowering
the
risk
of
developing
invasive
or
clinically
significant
disease.
Chemo-
preventive
drugs
must
be
devoid
of
toxicity
and
well
tolerated
since
they
must
be
used
over
extended
periods
(Hanahan
and
Folkman,
1996).
In
this
study,
we
investigated
the
effects
of
lycopene
on
endothe-
lial
cells.
Because
of
its
chemical
structure,
lycopene
is
a
molecule
that
has
very
powerful
antioxidant
activity.
As
described
earlier,
growth
of
endothelial
cells
occur
under
tight
regulation.
However,
conditions
around
the
cancerous
tissue
change
the
regulation
to
constitute
new
vessels.
Under
these
conditions,
endothelial
cells
increasingly
proliferate,
migrate
towards
chemoattractants
pro-
duced
by
cancer
cells,
and
finally
generate
a
new
capillary
network.
In
this
study,
we
have
investigated
whether
these
steps
are
affected
98 M.
S¸
ahin
et
al.
/
Acta
Histochemica
114 (2012) 94–
100
Fig.
4.
Percentage
proliferation
of
endothelial
cells.
HUVECs
were
plated
at
2.5
×
10
4
cells
per
well.
70–80%
confluent
HUVECs
in
24-well
plate
were
serum-starved
for
24
h.
After
this
period,
cells
in
growth
medium
were
treated
with
DMSO,
THF,
apigenin
and
lycopene.
After
incubated
at
37
C
for
24
h,
cells
were
treated
with
MTT
solution
and
the
converted
dye
was
measured
at
570
nm.
(A)
Percentage
of
proliferating
cells
exposed
to
lycopene.
Values
represent
mean
±
SD
of
five
different
experiments:
(a)
C
vs.
Lyc
(1
M),
(b)
C
vs.
Lyc
(5
M),
(c)
Lyc
(1
M)
vs.
Lyc
(5
M),
(d)
C
vs.
Lyc
(10
M),
(e)
Lyc
(1
M)
vs.
Lyc
(10
M)
and
(f)
Lyc
(5
M)
vs.
Lyc
(10
M).
(B)
Percentage
of
living
cells
exposed
to
apigenin:
(a)
C
vs.
Api
(5
g/ml),
(b)
C
vs.
Api
(10
g/ml)
and
(c)
Api
(5
g/ml)
vs.
Api
(10
g/ml).
(C)
Control;
Lyc:
lycopene;
Api:
apigenin.
by
lycopene.
Matrigel
TM
matrix
used
in
our
study
is
an
extracellu-
lar
matrix
provided
from
a
cancer
type
and
containing
an
agent
essential
for
angiogenesis.
In
addition,
we
added
EGM
containing
growth
factors
such
as
VEGF
and
FGF
important
for
the
angiogen-
esis
process
of
cells.
Under
these
environmental
conditions,
while
we
observed
widespread
tube
formation
in
cells
treated
with
DMSO
or
THF
alone,
capillary-like
tubular
formation
appeared
to
be
sup-
pressed
in
cells
treated
with
lycopene.
In
our
experiment,
lycopene
was
applied
at
concentrations
of
0,
1,
5
and
10
M.
We
observed
that
lycopene
significantly
inhibited
in
vitro
angiogenesis
in
a
dose-
dependent
manner.
Apigenin
which
is
used
for
positive
control
is
known
to
display
antiangiogenic
effects
in
a
variety
of
studies
(Trochon
et
al.,
2000;
Kim,
2003;
Erdogan
et
al.,
2007).
We
also
demonstrated
that
api-
genin
inhibited
tube
formation
in
a
dose-dependent
manner.
The
proliferation
rate
of
endothelial
cell
increases
during
angiogene-
sis.
Therefore,
we
investigated
the
effects
of
lycopene
via
the
MTT
method
on
cells
in
medium
containing
serum
and
growth
factors
and
observed
lycopene
significantly
suppressed
cell
proliferation
for
all
doses
used.
While
lycopene
at
the
most
effective
dose
10
M
decreased
proliferation
by
69.76%,
5
M
and
1
M
lycopene
inhib-
ited
proliferation
by
58.47%
and
36.66%,
respectively.
Differences
among
the
concentrations
are
statistically
significant.
Ivanov
et
al.
(2007)
suggested
treatment
of
LNCaP
prostate
carcinoma
cells
with
lycopene
decreased
the
cell
proliferation.
In
another
study,
it
was
shown
that
lycopene
suppressed
the
proliferative
capacity
of
sev-
eral
malignant
cell
lines
(Salman
et
al.,
2007).
Moreover,
in
cell
culture
system,
lycopene
was
demonstrated
to
strongly
inhibit
IGF-
1-mediated
proliferation
of
human
endometrial,
breast
and
lung
cancer
cells
(Karas
et
al.,
2000).
Lycopene
is
also
known
to
inhibit
proliferation
of
human
oral
cavity
tumor
(KB-1)
(Livny
et
al.,
2002)
and
human
breast
cancer
cells
(MCF-7
and
MDA-MB-231)
(Prakash
et
al.,
2001).
Even
if
the
types
of
the
cells
are
different,
the
general
suppressive
effects
of
lycopene
on
proliferation
are
consistent
with
our
study.
We
revealed
that
apigenin
at
both
5
g/ml
and
10
g/ml
also
significantly
inhibited
cell
proliferation
45.76%
and
61.85%,
respectively.
As
regards
the
migration
assay,
another
important
parameter
for
in
vitro
angiogenesis,
we
used
two
different
methods
to
assay
endothelial
cell
migration.
One
of
the
methods
is
based
on
cell
movements
towards
the
chemoattractant.
In
this
experiment,
we
assayed
migration
of
cells
from
the
upper
micro-pore
(3
m)
plate
coated
with
fibronectin
to
the
bottom
plate
containing
serum
and
growth
factors.
Control
of
the
experiment
involves
using
albumin
coated
plates
instead
of
fibronectin.
Consequently,
we
observed
all
concentrations
of
lycopene,
as
compared
with
THF,
inhibited
cell
migration.
Similarly,
apigenin
also
significantly
suppressed
the
migration.
Another
method,
named
wound
healing
assay,
is
based
Fig.
5.
Lycopene
(A)
and
apigenin
(B)
treatments
suppress
the
migration
of
endothelial
cells
from
fibronectin-coated
plates
with
pores
(3
m)
to
bottom
plate
containing
serum
and
growth
factors.
Migrated
cells
were
dyed
and
assayed
with
fluorescence
plate
reader.
Data
were
given
as
relative
percent
fluorescence
intensity
according
to
controls
(DMSO
or
THF).
No
significant
difference
between
1
and
5
M
lycopene.
***p
<
0.001,
**p
<
0.01.
M.
S¸
ahin
et
al.
/
Acta
Histochemica
114 (2012) 94–
100 99
Fig.
6.
The
fluorescence
microphotographs
of
wound
healing
assayed
cells
treated
with
(A)
THF
(Control),
(B)
lycopene
(10
M),
(C)
DMSO
(Control)
and
(D)
Apigenin
(10
g/ml).
After
full-confluent
HUVECs
were
starved
with
serum
and
growth
factors
for
24
h,
cells
in
12-well
plate
were
scratched
with
sterile
200
l
plastic
pipette
tip.
Cells
were
treated
with
the
indicated
compound.
When
HUVECs
in
control
groups
almost
completely
migrated,
cells
were
stained
with
Dil
fluorescence
dye
as
mentioned
in
material
and
methods
section.
Microphotographs
were
taken
under
fluorescence
microscope
at
×40
magnification.
Reciprocal
distance
(pixel)
between
cells
at
ten
different
fields
of
each
of
five
independent
experiments
was
calculated
as
mean
±
SD.
Scale
bar
=
100
m.
***p
<
0.001.
on
mimicking
the
cell
migration
during
wound
healing
in
vivo.
Apigenin
and
lycopene
in
this
assay
significantly
decrease
the
movement
of
cells.
Hwang
and
Lee
(2006)
suggested
that
lycopene
at
5
and
10
M
inhibited
the
adhesion,
invasion
and
migration
of
a
highly
inva-
sive
human
hepatoma
cell
line,
SK-Hep1.
Moreover,
it
was
found
that
the
metastasis
suppressor
gene
nm23-H1
was
induced
in
these
cells
(Huang
et
al.,
2005).
The
studies
performed
with
SK-Hep1
are
similar
to
our
study
in
terms
of
anti-migration
properties
of
lycopene.
Our
study
is
consistent
with
other
reported
studies
defend-
ing
the
protective
effects
of
flavonoids
and
carotenoids
against
chronic
diseases
(Kim
et
al.,
1998;
Agarwal
and
Rao,
2000).
In
our
study,
it
was
revealed
apigenin
and
lycopene
showed
similar
effects
under
the
same
experimental
conditions.
It
may
be
concluded
that
lycopene
is
more
effective
than
apigenin
when
comparing
their
concentrations.
Various
epidemiological
and
experimental
studies
have
revealed
that
lycopene
is
an
important
agent
to
prevent
from
cancer,
however,
effects
of
lycopene
on
endothelial
cells
or
angio-
genesis
could
not
be
found
in
the
literature.
In
conclusion,
the
present
results
suggest
that
lycopene
has
some
antiangiogenic
properties,
though
additional
studies
are
needed
to
demonstrate
the
effects
of
lycopene
on
molecular
mechanisms
in
endothelial
cells.
Conflict
of
interest
The
authors
have
no
conflict
of
interest.
Acknowledgements
Funding
for
this
study
was
provided
by
grants
from
the
Akd-
eniz
University
Scientific
Research
Project
Unit
(Project
Number:
2006.03.0122.005).
Also
this
study
was
supported
by
Akdeniz
Uni-
versity
Health
Sciences
Institute.
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    • "Such inhibitory effect has been previously observed on human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480), [24] . These inhibitory effects were reported to be mediated by the suppression of DNA synthesis [31]. Further, to investigate the anti-proliferative mechanism of apigenin- 7-glucoside, genkwanin and naringenin, we analyzed changes in B16F10 cell cycle progression induced by the aforementioned molecules , using flow cytometry. "
    [Show abstract] [Hide abstract] ABSTRACT: Aims: In this study, we have investigated the effects of apigenin-7-glucoside, genkwanin and naringenin, on mouse melanoma B16F10 cell proliferation. Influence of these natural products on percentage cell distribution in cycle phases and melanogenesis was also studied. Main methods: Cell viability was determined at various periods using the MTT assay, whereas effects of tested compounds on progression through the cell cycle were analyzed by flow cytometry. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475nm. Besides, the mechanism involved on the death route induced by the tested molecules was evaluated using the bis-benzimide trihydrochloride coloration method (Hoechst 33258). Key findings: Apigenin-7-glucoside, genkwanin and naringenin exhibited significant anti-proliferative activity against B16F10 melanoma cells after 24 and 48h of incubation. Furthermore, apigenin-7-glucoside, genkwanin and naringenin provoked an increase of subG0/G1, S and G2/M phase cell proportion with a significant decrease of cell proportion in G0/G1 phases. The results evaluated using Hoechst 33,258, confirm that the percentage of B16F10 cells observed in the sub G0/G1 phase were undergoing apoptosis. Moreover, apigenin-7-glucoside and naringenin revealed an ability to enhance melanogenesis synthesis and tyrosinase activity of B16F10 melanoma cells. Whereas genkwanin induces a decrease of melanin synthesis by inhibiting tyrosinase activity. Significance: Our results promote the introduction of genkwanin in cosmetic preparations, as skin whitening agent, whereas apigenin-7-glucoside and naringenin should be introduced into cosmetic products as natural tanning agents.
    Full-text · Article · Nov 2015
    • "Recent studies have shown that lycopene inhibits human umbilical vascular endothelial cell (HUVEC) migration and tube formation [53]. Moreover, high doses of lycopene can inhibit tumour growth in nude mice xenotransplanted with the PC-3 prostate carcinoma and Sk-Hep-1 hepatocellular carcinoma cell lines. "
    [Show abstract] [Hide abstract] ABSTRACT: Epidemiological studies suggest that including fruits, vegetables, and whole grains in regular dietary intake might prevent and reverse cellular carcinogenesis, reducing the incidence of primary tumours. Bioactive components present in food can simultaneously modulate more than one carcinogenic process, including cancer metabolism, hormonal balance, transcriptional activity, cell-cycle control, apoptosis, inflammation, angiogenesis and metastasis. Some studies have shown an inverse correlation between a diet rich in fruits, vegetables, and carotenoids and a low incidence of different types of cancer. Lycopene, the predominant carotenoid found in tomatoes, exhibits a high antioxidant capacity and has been shown to prevent cancer, as evidenced by clinical trials and studies in cell culture and animal models. In vitro studies have shown that lycopene treatment can selectively arrest cell growth and induce apoptosis in cancer cells without affecting normal cells. In vivo studies have revealed that lycopene treatment inhibits tumour growth in the liver, lung, prostate, breast, and colon. Clinical studies have shown that lycopene protects against prostate cancer. One of the main challenges in cancer prevention is the integration of new molecular findings into clinical practice. Thus, the identification of molecular biomarkers associated with lycopene levels is essential for improving our understanding of the mechanisms underlying its antineoplastic activity.
    Full-text · Article · Jul 2013
    • "In contrast, it was shown that prolonged tomato juice consumption (containing 47.1 mg/d lycopene) increases plasma lycopene concentrations without significantly affecting cell-mediated immunity in well-nourished elderly subjects. Lycopene has been shown to significantly decrease endothelial cell proliferation, migration and tube formation; the latter result suggests that lycopene may inhibit angiogenesis [34]. We previously showed that oral supplementation with lycopene decreases the number of lung tumors and the tumor cross-sectional area in athymic nude mice after tail vein injection with SK-Hep-1 cells, and this effect involves angiogenic factors, including up-regulation of IL-12 secretion [35]. "
    [Show abstract] [Hide abstract] ABSTRACT: The carotenoid lycopene has been reported to possess anti-metastatic activity which may be associated with immunomodulation. However, the anti-angiogenic effects and mechanisms of action of lycopene have not been reported. In this study, we investigated the immunomodulatory effect on in vitro and ex vivo angiogenesis of lycopene. We found that the proliferation, migration and the matrigel tube formation of human umbilical vein endothelial cells (HUVECs) was remarkably inhibited by conditioned medium (CM) of human peripheral blood mononuclear cells (MNC-CM) stimulated with various dose (1-10 μmol/L) of lycopene (LP-MNC-CM). LP-MNC-CM treatment inhibited ex vivo angiogenesis, as revealed by chicken egg chorioallantoic membrane assay. We further examined the effects of lycopene stimulation on cytokine levels in MNC and showed that, as compared to the control, lycopene (10 μmol/L) significantly (P<.001) up-regulated interleukin (IL)-12 by 163% and interferon (IFN)-γ by 531%. Furthermore, pre-treatment of HUVECs with dexamethasone, an IL-12 inhibitor, blocked the anti-angiogenic effects of LP-MNC-CM in parallel with inhibition of IL-12 and IFN-γ induction in MNC. These results demonstrate that lycopene has a potent anti-angiogenic effect and that these effect may be associated with its up-regulation of IL-12 and IFN-γ.
    Full-text · Article · Jun 2012
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