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Phytomedicine
23
(2016)
550–557
Contents
lists
available
at
ScienceDirect
Phytomedicine
journal
homepage:
www.elsevier.com/locate/phymed
Isololiolide,
a
carotenoid
metabolite
isolated
from
the
brown
alga
Cystoseira
tamariscifolia
,
is
cytotoxic
and
able
to
induce
apoptosis
in
hepatocarcinoma
cells
through
caspase-3
activation,
decreased
Bcl-2
levels,
increased
p53
expression
and
PARP
cleavage
Catarina
Vizetto-Duarte
a
,
Luísa
Custódio
a
,
KatkamN.
Gangadhar
a
,
JoãoHenriqueG.
Lago
b
,
Catarina
Dias
c
,
Ana Marta
Matos
c
,
Nuno
Neng
c
,
José Manuel Florêncio
Nogueira
c
,
Luísa
Barreira
a
,
Fernando
Albericio
d
,
e
,
f
,
Amelia
P.
Rauter
c
,
João
Varela
a
,
∗
a
Centre
of
Marine
Sciences,
University
of
Algarve,
Faculty
of
Sciences
and
Technology,
Ed.
7,
Campus
of
Gambelas,
Faro,
Portugal
b
Institute
of
Environmental,
Chemical
and
Pharmaceutical
Sciences,
Federal
University
of
Sao
Paulo,
09972-270,
Sao
Paulo,
Brazil
c
Center
of
Chemistry
and
Biochemistry,
Department
of
Chemistry
and
Biochemistry,
Faculty
of
Sciences
University
of
Lisbon,
Campo
Grande,
Ed.
C8,
Piso
5,
1749-016
Lisbon,
Portugal
d
Institute
for
Research
in
Biomedicine,
Barcelona
Science
Park,
Baldiri
Reixac
10,
08028,
Barcelona,
Spain
e
CIBER-BBN,
Networking
Centre
on
Bioengineering,
Biomaterials
and
Nanomedicine,
Barcelona
Science
Park,
Baldiri
Reixac
10,
08028
Barcelona,
Spain
f
University
of
Barcelona,
Department
of
Organic
Chemistry,
Martí i
Franqués
1-11,
08028
Barcelona,
Spain
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
30
January
2016
Accepted
9
February
2016
Keywords:
Marine
natural
product
Cystoseira
Isololiolide
Carotenoid
metabolite
Cell
cycle
Apoptosis
a
b
s
t
r
a
c
t
Background:
Brown
macroalgae
have
attracted
attention
because
they
display
a
wide
range
of
biological
activities,
including
antitumoral
properties.
Inthis
study
we
isolated
isololiolide
from
Cystoseira
tamarisci-
folia
for
the
first
time.
Purpose:
To
examine
the
therapeutical
potential
of
isololiolide
against
tumor
cell
lines.
Methods/Study
design:
The
structure
of
the
compound
was
established
and
confirmed
by
1D
and
2D
NMR
as
well
as
HRMS
spectral
analysis.
The
in
vitro
cytotoxicity
was
analyzed
by
colorimetric
3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
assay
in
tumoral
as
well
as
in
non-tumoral
cell
lines.
Cell
cycle
arrest
and
induction
of
apoptosis
were
assessed
by
flow
cytometry.
Alteration
of
expres-
sion
levels
in
proteins
important
in
the
apoptotic
cascade
was
analyzed
by
western
blotting.
Results:
Isololiolidewas
isolated
for
the
first
time
from
the
brown
macroalga
C.tamariscifolia
.
Isololiolide
exhibited
significant
cytotoxic
activity
against
three
human
tumoral
cell
lines,
namely
hepatocarcinoma
HepG2
cells,
whereas
no
cytotoxicity
was
found
in
non-malignant
MRC-5
and
HFF-1
human
fibroblasts.
Isololiolide
completely
disrupted
the
HepG2
normal
cell
cycle
and
induced
significant
apoptosis.
More-
over,
western
blot
analysis
showed
that
isololiolide
altered
the
expression
of
proteins
that
are
important
in
the
apoptotic
cascade,
increasing
PARP
cleavage
and
p53
expression
while
decreasing
procaspase-3
and
Bcl-2
levels.
Conclusion:
Isololiolide
isolated
from
C.
tamariscifolia
is
able
to
exert
a
selective
cytotoxic
activity
on
hepatocarcinoma
HepG2
cells
as
well
as
induce
apoptosis
through
the
modulation
of
apoptosis-related
proteins.
© 2016 Elsevier GmbH. All rights reserved.
Abbreviations:
1D
and
2D
NMR,
one-dimensional
and
two-dimensional
nuclear
magnetic
resonance;
1
H
and
13
C
NMR,
proton
and
carbon-13
nuclear
magnetic
res-
onance;
ANOVA,
analysis
of
variance;
Bak,
Bcl-2
homologous
antagonist
killer;
Bax,
Bcl-2-associated
X
protein;
Bcl-2,
B-cell
lymphoma
2;
CDCl
3
,
deuterated
chloro-
form;
CNT,
compound-induced
cytotoxicity
on
non-tumoral
cells;
CT,
compound-
induced
cytotoxicity
on
tumoral
cells;
DEPT,
distortionless
enhancement
by
polarization
transfer;
DMEM,
Dulbecco’s
Modified
Eagle’s
medium;
DMSO,
dimethyl
sulfoxide;
ECL,
enhanced
chemiluminescence;
EDTA,
ethylenediaminetetraacetic
acid;
EtOAc,
ethyl
acetate;
FBS,
fetal
bovine
serum;
FITC,
fluorescein
isothiocyanate;
HCC,
hepatocellular
carcinoma;
HPLC,
high
performance
liquid
chromatography;
HRESIMS,
high-resolution
electrospray
ionization
mass
spectrometry;
HRMS,
high
resolution
mass
spectrometry;
HRP,
horseradish
peroxidase;
HSD,
honest
significant
difference;
IC
50
,
half
maximal
inhibitory
concentration;
m/z
,
mass-to-charge
ratio;
MeOH,
methanol;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide;
NP-40,
nonidet
P-40;
p53,
protein
53;
PARP,
poly
(ADP-ribose)
polymerase;
PI,
propidium
iodide;
RPMI,
Roswell
Park
Memorial
Institute
medium;
SARs,
structure-
activity
relationships;
SDS,
sodium
dodecyl
sulfate;
SEM,
standard
error
of
mean;
SiO
2
,
silica;
TLC,
thin
layer
chromatography;
TMS,
tetramethylsilane;
T-TBS,
tween-
tris
buffered
saline;
WHO,
World
Health
Organization.
∗
Corresponding
author.
Tel.:
+351
289
80
0
051;
fax:
+351
28980
0
051.
E-mail
address:
jvarela@ualg.pt
,
joaocvarela@gmail.com
(J. Varela).
http://dx.doi.org/10.1016/j.phymed.2016.02.008
0944-7113/© 2016 Elsevier GmbH. All rights reserved.
C.
Vizetto-Duarte
et
al.
/
Phytomedicine
23
(2016)
550–557
551
Introduction
Cancer
is
a
major
public
health
problem
with
an
estimated
prevalence
of
about
3%
in
Europe,
increasing
to
15%
at
old
age.
Moreover,
cancer
related
deaths
are
estimated
to
increase
to
over
11
million
in
2030
(WHO,
2010).
Hepatocellular
carcinoma
(HCC)
is
the
third
leading
cause
of
cancer-related
death
worldwide,
af-
ter
lung
and
stomach
cancer
(
Ferenci
et
al.
2010
).
The
current
therapeutics
used
for
HCC
treatment
involves
surgical
resection,
transplantation
and/or
systemic
chemotherapy;
however,
surgery
and
transplantation
may
not
be
appropriate
for
many
patients
and
chemotherapy
often
fails
(
Liu
et
al.
2014
).
Chemotherapy
is
also
constrained
by
its
toxicity,
significant
resistance
to
available
chemotherapeutic
agents
and
side
effects,
including
neutropenia
and
myelosuppression
(
Chau
et
al.
2006
).
Current
studies
involved
in
developing
effective
cancer
prevention
approaches
have
focused
on
the
use
of
bioactive
natural
agents
that
may
have
less
adverse
effects
and
can
exert
selective
cytoxicity
against
cancer
cells
(
Ghate
et
al.
2014
).
The
chemical
and
biological
diversity
of
the
marine
environ-
ment
is
immeasurable
and
therefore
is
an
extraordinary
resource
for
the
discovery
of
novel
anticancer
drugs.
Brown
algae
are
a
rich
source
of
secondary
metabolites
displaying
a
wide
variety
of
bioactivities
with
important
features
for
pharmaceutical
pur-
poses.
Cystoseira
tamariscifolia
has
demonstrated
interesting
bi-
ological
activities
such
as
antibacterial,
antifungal,
antiprotozoal,
cell
division
inhibition,
anti-inflammatory,
antioxidant
and
cyto-
toxic
properties
(
Bennamara
et
al.
1999
;
Spavieri
et
al.
2010
;
Lopes
et
al.
2012
;
Andrade
et
al.
2013
).
These
properties
have
been
ascribed
to
the
presence
of
different
classes
of
molecules
that
were
identified
in
C.
tamariscifolia
,
such
as
phlorotannins
(fu-
cophloroethol,
fucodiphloroethol,
fucotriphloroethol,
7-phloroeckol,
phlorofucofuroeckol
and
bieckol/dieckol),
phloroglucinol,
proline,
β
-sitosterol,
fucosterol,
and
diverse
fatty
acids
(
Ferreres
et
al.
2012
;
Andrade
et
al.
2013
;
Vizetto-Duarte
et
al.
2015
).
As
C.
tamarisci-
folia
extracts
have
previously
demonstrated
cytotoxic
potential,
in
this
study
we
describe
the
identification
of
isololiolide,
a
known
carotenoid
metabolite,
as
a
selective
cytotoxic
compound
that
was
isolated
from
the
brown
macroalga
C.tamariscifolia
for
the
first
time
.
Here
we
show
evidence
that
exposure
of
hepatocarcinoma
HepG2
cells
to
isololiolide
is
associated
with
changes
in
the
ex-
pression
of
p53,
PARP,
Bcl-2
and
procaspase-3.
These
results
might
explain
the
dramatic
suppression
of
the
S
phase
as
well
as
the
in-
duction
of
apoptosis
caused
by
this
monoterpene.
Material
and
methods
Chemicals
and
reagents
Hexane
and
ethyl
acetate
were
purchased
from
Prolabo
(VWR
International,
Leuven,
Belgium).
Merck
(Darmstadt,
Germany)
sup-
plied
dimethyl
sulfoxide
(DMSO).
Roswell
Park
Memorial
Institute
medium
(RPMI),
Dulbecco’s
Modified
Eagle’s
medium
(DMEM),
fe-
tal
bovine
serum
(FBS),
L-glutamine
and
penicillin/streptomycin
were
obtained
from
Lonza
Ibérica
(Barcelona,
Spain).
3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
was
obtained
from
Calbiochem.
Primary
antibodies
for
poly
(ADP-
ribose)
polymerase
(PARP),
p53,
Bcl-2,
procaspase-3,
actin
and
re-
spective
secondary
antibodies
were
from
Santa
Cruz
Biotechnol-
ogy
Inc.,
Heidelberg,
Germany.
FITC-conjugated
annexin
V/
pro-
pidium
iodide
(PI)
assay
kit
was
acquired
from
Cayman
Chemi-
cal
Company,
USA.
Silica
gel
(Merck,
40–63
μ
m
mesh)
was
used
for
column
chromatographic
separation,
while
silica
gel
60
PF
254
(Merck)
was
used
for
analytical
(0.25
mm)
TLC.
CDCl
3
(Aldrich)
was
used
as
solvent
for
1
H
and
13
C
NMR
spectra
acquisition
and
TMS
(Aldrich)
was
used
as
internal
standard.
1D
and
2D
NMR
spec-
tra
were
recorded
at
Bruker
Digital
Avance
800
MHz
spectrometer.
Additional
reagents
and
necessary
solvents
were
purchased
from
VWR
International
(Leuven,
Belgium).
Sampling
Cystoseira
tamariscifolia
was
collected
in
the
middle/lower
inter-
tidal
areas,
during
the
low
tide,
between
May
and
September
2012
on
the
Portuguese
coast.
Biomass
was
rinsed
with
distilled
water
and
macroscopic
epiphytes
and
extraneous
matter
were
carefully
removed.
Identification
of
specimens
was
made
by
Dr
Aschwin
En-
gelen
(Centre
of
Marine
Sciences,
University
of
Algarve,
Portugal)
and
Dr
Javier
Cremades
Ugarte
(Facultade
de
Ciencias,
University
of
A
Coruña)
and
a
voucher
specimen
of
C.
tamariscifolia
(code
num-
ber
MB016)
was
deposited
at
the
Centre
of
Marine
Sciences,
Uni-
versity
of
Algarve.
Samples
were
freeze-dried
and
stored
at
–20
°
C
until
the
extraction
procedure.
Extraction
Biomass
was
mixed
with
hexane
(1:10,
w/v)
and
homogenized
for
2
min
using
a
disperser
IKA
T10B
Ultra-Turrax
at
room
tem-
perature
(RT).
The
tubes
were
then
vortexed
for
1
min,
centrifuged
(50
0
0
g
,
10
min,
RT)
and
the
supernatants
recovered.
The
extraction
procedure
was
repeated
3
times
and
the
supernatants
combined
and
filtered.
The
extract
was
dried
at
40
º
C
under
vacuum
and
dis-
solved
in
DMSO
for
biological
activities
screening
or
in
the
ade-
quate
solvent
for
chemical
characterization,
aliquoted
and
stored
(–20
°
C).
Isolation
and
elucidation
of
isololiolide
C.
tamariscifolia
hexane
extract
(9
g)
was
fractionated
by
col-
umn
chromatography
(2.5
cm
×
18
cm)
over
silica
gel
(SiO
2
)
using
increasing
amounts
of
EtOAc
in
hexane
(9:1;
85:15;
4:1;
75:25;
7:3;
3:2;
1:1)
and
increasing
amounts
of
MeOH
in
EtOAc
(9:1;
8:1;
5:1;
2:1;
1:1),
MeOH
(100%)
and
H
2
O
(100%)
as
eluents.
This
procedure
afforded
57
fractions,
which
were
analyzed
by
TLC
and
pooled
together
in
21
groups
(A
–U).
Fraction
14
(70
mg)
was
re-
fractionated
over
SiO
2
eluted
with
hexane
(100%);
hexane/EtOAc
(9:1,
8:2,
7.5:2.5,
7:3,
6.5:3.5,
6:4,
5.5:4.5,
1:1,
4:6),
EtOAc
(100%)
and
MeOH
(100%)
to
afford
151
fractions
which
were
pooled
to-
gether
in
9
groups
after
TLC
analysis.
Group
6–8,
obtained
from
the
hexane/
EtOAc
elution
(6:4
through
1:1),
was
purified
by
re-
verse
phase
preparative
HPLC
to
afford
3
mg
of
isololiolide.
Isololiolide:
Pale
yellow
oil;
1
H
NMR
(800
MHz,
CDCl
3
,
TMS,
ppm)
δ
5.71
(1H,
s,
H-7),
4.21
(1H,
m,
H-3),
2.55
(2H,
br
d,
J
=
2.4
Hz,
H-4),
2.03
(1H,
br
d,
J
=
2.4
Hz,
H-2),
1.59
(3H,
s,
H-
11),
1.23
(3H,
s,
H-10),
1.21
(3H,
s,
H-9).
13
C
NMR
δ
(200
MHz,
CDCl
3
,
TMS,
ppm):
181.2
(C-6),
171.5
(C-8),
113.3
(C-7),
86.4
(C-5),
65.1
(C-3),
49.8
(C-2),
47.9
(C-4),
35.0
(C-1),
29.9
(C-9),
25.6
(C-11),
25.1
(C-10);
HRESIMS
m/z
219.0993
[M
+
Na]
+
(calc
to
C
11
H
16
O
3
Na
219.0997).
Cell
culture
HepG2
cells
(human
hepatocellular
carcinoma)
were
main-
tained
in
RPMI-1640
culture
media
supplemented
with
glucose
(10
0
0
mg/ml),
10%
FBS,
L-glutamine
(2
mM),
penicillin
(50
U/ml)
and
streptomycin
(50
μ
g/ml).
MRC-5
and
HFF-1
human
fibrob-
lasts,
AGS
human
gastric
cancer,
HCT-15
human
colon
cancer
cells
were
grown
in
DMEM
culture
media
supplemented
with
glucose
(10
0
0
mg/ml),
10%
FBS,
L-glutamine
(2
mM),
penicillin
(50
U/ml)
and
streptomycin
(50
μ
g/ml).
Cell
lines
were
grown
in
an
incuba-
tor
at
37
º
C
and
5.0%
CO
2
in
humidified
atmosphere.
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