RESEARCH PAPER
Cell Adhesion & Migration 9:6, 422--431; November/December 2015; Published with license by Taylor & Francis Group, LLC
Hydrophilic extract from Posidonia oceanica
inhibits activity and expression of gelatinases
and prevents HT1080 human fibrosarcoma
cell line invasion
Emanuela Barletta1,y, Matteo Ramazzotti1,y, Florinda Fratianni2, Daniela Pessani3,4, and Donatella Degl’Innocenti1,4,*
1
Dipartimento Scienze Biomediche Sperimentali e Cliniche; Universita degli Studi di Firenze; Firenze, Italy; 2Istituto di Scienze dell’Alimentazione; Consiglio Nazionale delle
Ricerche (ISA-CNR); Avellino, Italy; 3Laboratorio di Zoologia e Biologia Marina; Dipartimento di Biologia Animale e dell’Uomo; Universita degli Studi di Torino; Torino, Italy;
4
Centro Interuniversitario di Biologia Marina ed Ecologia Applicata (CIBM); Livorno, Italy
y
These authors equally contributed to this work.
Keywords: cell migration and invasion, gelatinase, hydrophilic extract, MMP-2, MMP-9, Polyphenols, Posidonia oceanica
Posidonia oceanica (L.) Delile is an endemic Mediterranean sea-grass distributed in the infralittoral zones, where it
forms meadows playing a recognized ecological role in the coastal marine habitat. Although its use as a traditional
herbal remedy is poorly documented, recent literature reports interesting pharmacological activities as antidiabetic,
antioxidant and vasoprotective. Differently from previous literature, this study presents a hydrophilic extraction method
that recovers metabolites that may be tested in biological buffers. We showed for the first time in the highly invasive
HT1080 human fibrosarcoma cell line that our hydrophilic extract from P. oceanica was able to strongly decrease gene
and protein expression of gelatinases MMP-2 and MMP-9 and to directly inhibit in a dose-dependent manner
gelatinolytic activity in vitro. Moreover, we have revealed that our extract strongly inhibited HT1080 cell migration and
invasion. Biochemical analysis of the hydrophilic extract showed that catechins were the major constituents with minor
contribution of gallic acid, ferulic acid and chlorogenic plus a fraction of uncharacterized phenols. However, if each
individual compound was tested independently, none by itself was able to induce a direct inhibition of gelatinases as
strong as that observed in total extract, opening up new routes to the identification of novel compounds. These results
indicate that our hydrophilic extract from P. oceanica might be a source of new pharmacological natural products for
treatment or prevention of several diseases related to an altered MMP-2 and MMP-9 expression.
Introduction
Degradation of the extracellular matrix by specific proteases
is involved in numerous physiological processes such as cell
proliferation, cell adhesion and migration, angiogenesis, bone
development, wound healing.1,2 Among the different proteases
responsible for the degradation of the extracellular matrix in vivo,
an important role is played by the matrix metalloproteinases
(MMPs), a family of zinc-dependent endopeptidases.3,4 In particular, MMP-2, also known as gelatinase A, and MMP-9, also
known as gelatinase B, degrade type IV collagen, one of the extracellular matrix component of basement membrane, or denatured
collagen (gelatin), and therefore these MMPs play a pivotal role
in cell invasion and migration.
An increased expression and activity of MMPs has been
reported in various pathological processes such as inflammation
and vascular diseases.5,6 Moreover, MMP activity represents one
of the main mechanisms responsible for the process of invasion
and metastasis of tumor cells.7,8
In the last few years, several studies have shown that polyphenolic compounds of dietary origin are able to play an inhibitory
role on the process of activation of MMPs.9,10 Polyphenols are
one of the most common classes of secondary metabolites in terrestrial and marine plants. Since these compounds have low toxicity, in recent years growing interest has been focused on
evaluating their role as chemopreventive agents in cancer. Indeed,
polyphenols are able to inhibit gene and protein expression as
well as activity of gelatinases in various malignancies.9-14
The angiosperm Posidonia oceanica (PO) is a sea-grass belonging to the Posidoniaceae family. Despite its name, it is endemic
in the Mediterranean sea and its dense underwater meadows
cover tens of thousands square kilometres. It is considered as
© Emanuala Barletta, Matteo Ramazzotti, Florinda Fratianni, Daniela Pessani, Donatella Degl’Innocenti
*Correspondence to: Donatella Degl’Innocenti; Email: donatella.deglinnocenti@unifi.it
Submitted: 09/22/2014; Revised: 11/07/2014; Accepted: 01/12/2015
http://dx.doi.org/10.1080/19336918.2015.1008330
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The
moral rights of the named author(s) have been asserted.
422
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Volume 9 Issue 6
fundamental for the Mediterranean environment and an important ecological indicator.15-17 Studies addressing PO as a bioremediator agent have been drawn since long time.18
Although PO is widely distributed, its role as a traditional
herbal remedy is poorly documented, except for the benefit of
the decoction of the leaves of PO against diabetes mellitus and
hypertension used by the villagers who live near the west coast of
Anatolia.19 The folk medicine reports that Egyptians attributed
to the seagrass curative properties, especially for sore throats and
skin problems, and PO has also been reported in the popular
pharmacopoeia by an old botanical handbook of Cazzuola.20,21
PO has emerged as an important reservoir of bioactive compounds with important antidiabetic, antioxidant, vasoprotective,
antibacterial and antifungine actions.19,22,23 PO has been shown
to contain partially known phenolic mixtures composed of flavonoids and condensed tannins such as proanthocyanidins as well
as new compounds (e.g. a novel sesquiterpene).24-28 A comparison of results from different extraction methods reported in literature is hardly feasible because of the different strategies used and
because PO leaves were not collected from the same seasonal
periods and ecological conditions (e.g., fresh and polluted
waters). The main phenols found in PO are 4-hydroxybenzoic
acid, 4-coumaric acid, cinnamic acid, caffeic acid, ferulic acid,
myricetin, quercetin, isorhamnetin, kaempferol, gentisic acid,
chicoric acid and vanillin.25,26,29
Several polyphenols found in PO might be able to inhibit
activity and expression of specific MMPs involved in cell invasion
and migration.30-33 Nevertheless, to the best of our knowledge, a
possible activity of Posidonia oceanica compounds on cells with
such aggressive phenotype has never been reported. Therefore, in
the present study we aimed at investigating whether PO extracts
might play a role in cell migration and cell invasiveness through
inhibition of the MMP-2 and MMP-9. We optimized a waterethanol extraction system that recovers hydrophilic compounds
and we analyzed the effect of this hydrophilic extract on cell
migration/invasion and on expression of MMP-2 and MMP-9 in
the highly invasive HT1080 human fibrosarcoma cell line which
constitutively expresses MMP-2 and MMP-9.
Results
Hydrophilic extraction recovers water-soluble compounds
Since we aimed at collecting substantially hydrophilic compounds from PO leaves in order to perform experiments in
biological buffers, we optimized an extraction method based on
70% ethanol followed by hexan-based removal of hydrophobic
compounds. This procedure allowed us a consistent extraction
(0.13 g dry extract from 1 gr of dry leaves, a yield of 7,7%, see
Table 1), and an easy and complete solubilization of the dried
material in 20% ethanol.
Biochemical characterization and antioxidant activity
As shown in Table 1, Posidonia oceanica extract (POE) was
found to contain carbohydrates (33.4 § 10.8 mg glucose/mL)
and polyphenols (7.40 § 0.40 mg gallic acid/mL). Since phenols
and polyphenols are known to act as scavenger for free radicals
and as antioxidant in general, we tested these POE activities with
DPPH and FRAP assays, as reported in Table 1. POE showed a
radical scavenging and antioxidant activity of 6.01 § 0.55 mg/
mL and to 8.03 § 0.34 mg/mL ascorbic acid equivalents,
respectively.
Natural products are known to have a limited stability during
time. We tested our POE at 1 week after solubilisation and storage at 4 C. We verified that its composition and activities were
very similar to that of fresh batches, demonstrating its stability
(Table 1). However, in order to ensure the reproducibility of the
experiments, all experiments were done with fresh POE.
Phenolic composition
We investigated the phenolic fraction of POE using UPLC
and a battery of reference compounds (Fig. 1). We were able to
identify and quantify about 88% of POE phenols. The principal
compound (over 83% of the total of identified phenols) was (C)
catechin and the remaining 5% fraction was a mixture of gallic
acid, ferulic acid, (¡) catechin, epicatechin and chlorogenic acid.
Using the area under the chromatographic peaks we estimated
the concentration of each known compound, in particular we
measured (C) catechin at a concentration of 420 mg/mL.
Table 2 reports the relative abundance and the estimated concentration of the known phenolic constituents in POE. The remaining 12% fraction was composed by several minor peaks,
indicating the presence of additional compounds that, although
detectable as phenols, results as unknown/uncharacterized.
Effect of POE on cell viability
Cell viability of HT1080 cells exposed to POE was tested
using the MTT assay. As shown in Fig. 2, during exposure of
HT1080 cells to 1:2000 dilution (corresponding to »13 mg dry
extract/mL and 0.21 mg/mL or »4.5 mM catechin) and to
Table 1. Biochemical characterization of POE. Values represent the mean and the standard deviation of at least 4 independent extractions. Abbreviations:
TC, total carbohydrate; TP, total polyphenols; RS, radical scavenging activity
Dry extract
TC
TP
Antioxidant
RS
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Method
Fresh POE mg/mL
1 week POE mg/mL
Reference compound
Weighting
Phenol-sulfuric acid
Folin-Ciocalteau
FerroZineTM
DPPH
43.0 § 0.5
33.4 § 10.8
7.4 § 0.4
8.03 § 0.34
6.01 § 0.55
43.0 § 0.5
31.0 § 9.7
7.3 § 0.5
8.20 § 0.41
5.80 § 0.62
—
D-glucose
Gallic acid
Ascorbic acid
Ascorbic acid
Cell Adhesion & Migration
423
1:1000 dilution of POE (corresponding to
»26 mg dry extract/mL and 0.42 mg/mL
or »9 mM catechin) cell viability was the
same as untreated control cells. In addition, even higher concentrations of POE
had a very slight effect on cell viability: at
1:62.5 dilution, corresponding to 416.87
mg/mL of dry extract, we observed 87.2
§5.1% of cell viability with respect to
control untreated cells.
POE reduces cell migration and
invasiveness
When cell migration was evaluated by
the scratch wound healing assay (Fig. 3),
POE exposure determined a dramatic
Figure 1. UPLC chromatogram of POE. The names and retention times of phenolic standards are
indicated. In the box, the structures of the main constituents of POE are depicted.
reduction of cellular motility, that in turn
determined a very limited closure of the
wound area (see a representative time lapse movie in supplemen- medium evidenced that zymographic bands comprise MMP-9
tary materials). In particular, control untreated cultures HT1080 (MMP-9, 94 KDa), pro-active MMP-2 (pro-MMP-2, 72 KDa)
cells completely closed the wound as early as 6 h from the initial and active MMP-2 (64 KDa) respectively. Treatment of cells
scratching (20.7 § 9.4% of wound area) and a complete closure with POE showed a 3 to 5 fold reduction of the amount of
(9,6 § 1,9% of wound area) after 12 h, while in the presence of MMPs released in the medium (Fig. 5A), also confirmed by
POE (1:1000 dilution, previously shown to be non-toxic to cells Western blot of MMP-9 and MMP-2 (Fig. 5B). Analysis of
by MTT assay) about 70% of wound area was still open (72.3 § MMP-9 and MMP-2 gene expression by real time PCR revealed
that inhibition of MMP secretion by exposure to POE was likely
2,3%) at 12 h.
Figure 4 shows the degree of invasiveness of HT1080 cells induced by a reduction of the MMP gene expression (Fig. 5C).
trough the reconstituted basement membrane MatrigelÒ . During The expression of TIMP-1, TIMP-2 and MMP-14, the physiocontrol culture conditions, 182.5 § 6.4 cells/filter were able to logical inhibitors and activator of gelatinases, respectively, assayed
invade MatrigelÒ . When cells were treated with POE (1:1000 by RT-PCR, was not affected by POE treatment (P-value > 0.5,
dilution) a marked and significant reduction of invasiveness was 2-tails t-test, data not shown).
In a second set of zymographic experiments we showed that
observed (53.75 § 2.9 cells/filter, about 70% reduction, P <
POE has a direct effect on MMP-2 and MMP-9 activities. In
0.05, t-test) with respect to untreated cells.
fact, incubation for 16 h of zymographic gel-lanes (prepared
from untreated HT1080 control cells) in reaction buffer suppleModulation of gelatinase activity and expression
Gelatine zymography of conditioned medium from control mented with POE, showed a dose-dependent inhibition of gelatiHT1080 cell line showed the presence of gelatinolytic activity of nases (Fig. 5D and E), that was not abolished by the addition in
an apparent molecular weight of 92 kDa, 72 kDa and 64 kDa large excess of the divalent cations Ca2C and Zn2C (data not
(Fig. 5A). Western-blotting (Fig. 5B) of the same conditioned shown).
Table 2. Characterization of the polyphenolic component of POE by UPLC.
Phenol name*
Gallic acid
NA
NA
NA
Chlorogenic acid
(C) Catechin
Epicatechin
NA
Ferulic acid
NA
NA
Retention Time (min)
Area (%)
Concentration (mg/mL)**
0.882
1.133
2.078
2.815
3.059
3.361
4.022
4.384
5.483
5.815
6.434
0.374
4.303
0.410
1.352
0.639
84.762
1.383
3.634
1.729
0.043
1.371
1.94 § 0.78
NA
NA
NA
8.46 § 0.03
418.44 § 9.00
33.31 § 0.60
NA
10.68 § 1.19
NA
NA
*NA indicates peaks with unknown attributions
**Concentrations are reported for known polyphenols only
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Volume 9 Issue 6
Figure 2. Effect of POE on HT1080 cell viability. Values are expressed as
percent with respect to control untreated cells. The labels of bars report
the used POE dilution and the corresponding concentration in term of
mg dry extract / mL. Error bars represent standard deviation of 3 different
experiments. POE at 1:1000 dilution was chosen as safe concentration for
further experiments.
Catechin alone can not explain POE direct inhibition
of MMP activity
In order to dissect the relative contribution of each identified
phenolic compounds of POE, we incubated the zymographic gel
slabs with pure (C) catechin, (¡) catechin, epicatechin, ferulic
acid, gallic acid and chlorogenic acid (together constituting the
88% of POE polyphenols) at concentrations equivalents to that
achieved with 1:10 extract, i.e. the dilution that determined a
nearly complete (>90%) direct in vitro MMP inhibition
(Fig. 5D, last strip). As shown in Fig. 5F1-3 (and quantified in
Fig. 5G), apart from (C) catechin, that showed a reduction in
Figure 4. POE reduces MatrigelÒ invasion by HT1080 cells. Top panels
show microscopic images of MatrigelÒ with cells stained with hematoxylin and eosin. Bottom panels report the corresponding number of cells
per filter.
the overall intensity of both MMP bands of about 30%, all other
compounds evidenced a very small reduction of MMP activity
(in most cases lower than 10%). These results let us suppose that
the fraction of phenols we were not able to identify (comprising
about 12% of the total phenolic composition) should confer
most of the in vitro inhibitory activity of POE.
Discussion
Figure 3. POE delays wound healing by HT1080 cells (scratch test). Time
course analysis of the percent of wounded area in untreated (circles) and
treated (squares) cells. Points are expressed in terms of mean § standard
deviation of 3 consecutive frames (spanning 1 minute).
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Cell invasion and migration play a pivotal role in several physiological and pathological conditions, particularly these processes
that are essential for malignant cancer cells to invade the tissue
micro-environment by degrading extracellular matrix components and to disseminate far from their primitive site, a necessary
step for the metastatic process. Matrix metalloproteases MMP-2
and MMP-9, by degrading type IV collagen and other extracellular matrix adhesive proteins, have been shown to play a crucial
role in tumor cell migration and invasion. In this study we demonstrated for the first time that POE is able to inhibit MMP-2
and MMP-9 expression and to drastically reduce migration and
invasion of the highly invasive human fibrosarcoma HT1080 cell
line, indicating that PO might be a suitable source of phytochemicals against tumor cell dissemination.
We introduced an extraction method that combines a water/
ethanol extraction and a degumming step with n-hexane. Most
literature on PO describes methods mainly aimed at recovering
the hydrophobic polyphenolic components in the organic phase
(e.g. ethyl-ether), therefore a direct comparison of our results
with these studies is hardly feasible. First, our method allowed
recovering water-soluble secondary metabolites and compounds
compatible with biological buffers for in vitro and in vivo studies.
Moreover, our POE retains antioxidant activities as those
Cell Adhesion & Migration
425
Figure 5. Gelatinase inhibition by POE. (A) Gelatin zymography: incubation of HT1080 with POE (C) reduces the amount of MMP-9 and MMP-2 released
in the medium with respect to control (¡). (B) Western blotting: identification of MMP-9 (left) or MMP-2 (right) in conditioned medium showed in A by
specific antibodies. (C) Real time PCR: incubation of HT1080 with POE (C) reduces the expression levels of MMP-9 and MMP-2 with respect to control
(¡). (D) Gelatin zymography: in vitro dose dependent inhibition of MMP-2 and MMP-9 by POE. (E) Densitometric quantification of signals form bands in
panel D. (F) in vitro effect of single, known phenols determined to be present in POE by UPLC at concentrations equivalent to those at 1:10 POE. (G) Densitometric quantification of signals form bands in panel F. In E and G the values are expressed in terms of inhibition percent with respect to controls and
error bars represent the standard deviation of at least 3 independent experiments.
observed in hydrophilic and hydrophobic extracts isolated from
PO by previous studies, suggesting that our extraction method
makes no depletion of the main bioactive properties of PO. It is
widely accepted that antioxidant and radical scavenger compounds might play a role in regulation of MMPs since reactive
oxygen species induce activation and release of MMPs, especially
MMP-2 and MMP-934 and they also modulate the redox state of
the cell and influence the NF-kB pathway, that in turn modulates
MMP transcription. Therefore, in this study POE might affect
cell migration and invasion through its antioxidant properties.
Finally, we revealed that our hydrophilic POE mostly contains
(C) catechin (>80%) and minor amounts of ferulic, gallic and
chlorogenic acids. All these biochemical characteristics were not
previously reported by any literature on phenolic composition of
PO leaves.35
Catechins, are not only well known antioxidants, but they are
also able to inhibit MMPs, as documented in a wealth of literature related to epigallocatechin gallate and its relatives.36,37
Through inhibition of MMP-2 and MMP-9, catechins and its
epimer epicatechin as well as their gallic acid conjugates are
strongly implicated in cell migration and invasion.38
Although ferulic acid, which was in minor concentrations in
our POE, seems to possess diverse pharmacological functions
some of which still remaining unknown, most of its beneficial
effects were ascribed to its antioxidant activities which might
modulate MMP expression/activity. In fact, ferulic acid is able to
modulate protein expression of MMP-2 and MMP-9 in liver
alcoholic fibrosis and in end-stage cirrhosis.39
Gallic acid, the other minor compound in POE, inhibits gene
expression of gelatinases MMP-2 and MMP-9 in human leukemia cells by reducing the binding of c-Jun/ATF-2 and c-Jun/cFos with promoter region of MMP-2 and MMP-9 genes and it
also inhibits cell migration and invasion of a human squamous
426
cell carcinoma cell line by suppressing MMP-2 and MMP-9
expression through inhibition of NF-kB.40,41
Similarly, chlorogenic acid, another minor compound of
POE, exhibits inhibitory effects on cell migration and MMP2 secretion in a glioblastoma cell line and it is also able to
inhibit other matrix metalloproteinases such as MMP-1,
MMP-3, and MMP-13 which play a role in cell migration
and invasion.42,43
Although anti-invasive properties of the above mentioned polyphenols were revealed in pure substances, their concomitant
presence in the same extract (POE in this case) is reasonably leading to even more powerful effect (a principle of synergy well
known to the pharmacognosy discipline44). In fact, POE showed
a powerful inhibition of MMP-2 and MMP-9 both at protein
and gene levels.
Further, our data clearly depict an additional role of POE in
cell migration and invasion that is possibly correlated to a direct
effect of POE on MMP-2 and MMP-9 activities. In fact, when
zymography of conditioned medium of HT1080 was incubated
in the presence of POE, we observed a dose dependent inhibition
of gelatinolysis that was not related to chelating properties of polyphenols, since a large excess of Zn2C or Ca2C was not able to
recover the inhibition by POE, in agreement with previous
results.45,46 It is important to underline that none of the identified POE phenols was by itself able to induce a direct inhibition
of MMP-2 and MMP-9 gelatinolytic activity as strong as that
observed in POE and their cumulative effect was insufficient.
This lead us to speculate that POE could contain some not yet
identified component with strong inhibitory activity on MMPs.
Since this bioactive component or mixture is not supposed to
have either a lipid, a complex carbohydrate or a protein origin,
due to our extraction method, it is reasonable to suppose its phenolic origin. However, it is beyond the scope of this study to
Cell Adhesion & Migration
Volume 9 Issue 6
identify this compound and to elucidate its mechanism of inhibition on both MMPs and cell migration/invasion.
In conclusion, in this study we characterized POE at biochemical and compositional levels and proved its important
role in modulating cell migration and invasion. We showed
that gelatinases, which promote tumor invasion and metastasis through degradation type IV collagen, are affected by
POE at a dual level: on the one hand, POE was very effective
in inhibiting gene and protein expression of MMP-2 and
MMP-9; on the other hand, POE directly inhibited the activity of synthesized and secreted gelatinases, thereby reducing
the capability of degrading extracellular matrix components.
Therefore, Posidonia oceanica can be considered as an interesting reservoir for new natural compounds useful for treatment of pathologies related to altered expression of MMP-2
and MMP-9.
Materials and Methods
Materials
a,a-Diphenyl-b-picrylhydrazyl (DPPH), 3-(2-Pyridyl)-5,6diphenyl-1,2,4-triazine-40 ,400 isulfonic acid sodium salt
(FerrozineÒ ), Folin–Ciocalteu’s phenol reagent, gallic acid,
ascorbic acid, D-glucose, gelatin and Coomassie brilliant blue R250, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan, Cell
Growth Determination Kit MTT based (Cat. CGD1-1KT),
Dulbecco’s Modified Eagle’s Medium (DMEM), heat inactivated Fetal Bovine Serum (FBS) and UPLC phenol standards
were purchased from Sigma-Aldrich. Cell culture plastics were
from Nunc (Thermo Fisher Scientific). Transwell filter units
were from Costar. 30% Acrylamide/Bis 37.5:1 solution, ammonium persulfate (APS), 1,2-Bis (dimethylamino) ethane
(TEMED), and Tris/Glycine buffers were purchased from BioRad. UPLC solvents and equipment were from Waters.
MatrigelÒ was purchased from Collaborative Research. Monoclonal antibodies against MMP-2 (clone 42-5D11) and MMP-9
(clone 56-2A4), were purchased from Calbiochem-Novabiochem
International. Blotting membranes, Immobilon polyvinylidene
difluoride (PVDF), were from Millipore. Goat anti-mouse
IRDye 680LT antibody (Cat# 926-68020) was purchased
from Li-Cor Biosciences. The RT-PCR primers for matrix metalloproteases (MMP-2: Hs00234422_m1 and MMP-9:
Hs00234579_m1), tissue inhibitors of MMPs (TIMP-1:
Hs00171558_m1 and TIMP-2: Hs00234278_m1), membranetype 1 MMP (MMP-14/MT1-MMP: Hs00237119_m1) and for
glyceraldehyde-3-phosphate dehydrogenase (Hs00266705_g1),
as well as TaqMan universal PCR master mix, were purchased
from Applied Biosystems. RT-PCR ABI 7500 PCR instrument
was from Applied Biosystems. Photometric measurements in
multiwell plates were recorded on an iMARK microplate reader
(Bio-Rad). Western blotting signals were acquired using an infrared imaging system (Odyssey; Li-Cor Biosciences).When not
otherwise specified, all chemicals and solvents, such as ethanol,
methanol and n-hexane were of the highest analytical grade and
were purchased from Sigma-Aldrich.
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Sample collection
Leaves of Posidonia oceanica (L.) Delile (see algaeBASE species
id 18577 and references therein) were collected from scuba divers
in July 2013 in Le Ghiaie meadow (Elba Island, Tyrrhenian Sea,
Italy, 42 490 100 N, 10 180 1500 E) at -10 m. Even though the sampling zone is a Biological Protection Reserve, at the depth of
10 m the plant was lusher and more protected. Soon after their
collection, the leaves were removed from the rhizomes and kept
at low temperature (10 C) and dark during the transport to the
laboratory.
In the laboratory only adult leaves47 were selected and washed
repeatedly in fresh water. From the leaves, fragments (1–5 cm in
length) free from visible epiphytes were cut off with non metallic
tools. The fragments were washed with distilled water and dried
at 60 C in darkness (to preserve polyphenol composition,
according to24) for about 24 h and stored in plastic food bags
until processing.
Extraction method
Extraction of phenolic compounds involved the central part of
the leaves. Dried leaves were minced and suspended in 10 mL
70% ethanol per gram of leave. Extraction was performed overnight at room temperature, under continuous agitation and at
65 C for further 3 h. The PO ethanol extract was separated from
debris by filtration and mixed with n-hexane (ratio 1:1), then vigorously mixed in a separatory funnel. The organic phase containing hydrophobic compounds was discarded and 1 mL batches of
the cleaned PO extract, mainly containing hydrophilic compounds, were dried by UnivapoTM vacuum-spin concentration.
Dry extracts were then stored at room temperature in the dark.
For assays, batches of dry extracts were suspended until complete
solubilisation in 0.5 mL 20% ethanol in sterile water and directly
used. Hydrophilic Extract from PO were hereinafter called POE.
Ultra performance liquid chromatography
The lyophilized extract dissolved in ethanol and the standards
(previously dissolved in methanol) were filtered with 0.45 mm
microfilters before analysis. The analysis of polyphenols present
in the extract was carried out by using an ACQUITYTM Ultra
Performance Liquid Chromatography (UPLC) system linked to
a PDA 2996 photodiode array detector. Empower software
(Waters) was used to control the instruments and for data acquisition and processing. The analyses were performed at 30 C
using a reversed phase column (BEH C18, 1.7 mm, 2.1 £
100 mm, Waters).48 The mobile phase consisted of 7.5 mM acetic acid (solvent A) and acetonitrile (solvent B) at a flow rate of
250 mL/min. Gradient elution was employed, starting with 5%
B for 0.8 min, then 5% – 20% B over 5.2 min, isocratic 20% B
for 0.5 min, 20% – 30% B for 1 min, isocratic 30% B for
0.2 min, 30% – 50% B over 2.3 min, 50% – 100% B over
1 min, isocratic 100% B for 1 min, and finally 100% – 5% B
over 1.0 min. At the end of this sequence, the column was equilibrated under the initial conditions for 2 min. The pressure
ranged from 6000 to 8000 psi during the chromatographic run.
The effluent was introduced into an LC detector (scanning range:
Cell Adhesion & Migration
427
210 – 400 nm, resolution: 1.2 nm). The injection volume was
5 mL.
Carbohydrate content
The total carbohydrate content (TCC) of POE was determined according to the phenol-sulfuric acid method optimized
from with minor modifications.49 Briefly, scalar aliquots of POE
were added to 96-well microplate and diluted with water (final
volume 70 ml), then 150 mL of concentrated sulfuric acid was
added to each well. After 5 min of incubation at RT under continuous shaking, 30 mL of 5% phenol solution was added to
each well and heated for 5 min at 90 C. After cooling to room
temperature for 20 min, the absorbance at 490 nm was recorded
with a microplate reader. Carbohydrate content was determined
by linear regression using D-glucose as a reference in the range
0–50 mg.
Total polyphenol content
The total polyphenol content (TPC) of POE was determined
according to the colorimetric Folin-Ciocalteau method.50 Scalar
volumes of POE (final volume 20 mL) were added to 100 mL of
Folin–Ciocalteu (Folin–Ciocalteu’s phenol reagent diluted 1:10
in H2O). After incubation for 5 min at RT, 80 mL of 7,5%
sodium carbonate solution was added and incubated for further
2 h. The absorbance at 595 nm was recorded with a microplate
reader. Polyphenol content was determined by linear regression
with gallic acid as a reference in the range 0–10 mg.
Radical scavenging activity
The radical-scavenging activity (RSA) of POE was determined
adapting the method from Fukumoto and Mazza.51 In a 96-well
microplate, scalar aliquots of POE were diluted with 95% methanol (final volume 100 mL) and mixed with 100 mL of freshly
prepared DPPH solution (0.15 mg/mL methanol). After 30 min
incubation in the dark at room temperature, the absorbance was
read at 490 nm with a microplate reader. Radical scavenging
activity was determined by linear regression with ascorbic acid
reference in the range 0–4 mg.
Antioxidant activity
The antioxidant activity (TAA) of POE was estimated using
the FRAP (ferric-reducing/antioxidant power) method adapted
from Pulido et al.52 Scalar aliquots of POE were diluted with
water (final volume 50 mL) and 200 mL of FerrozineTM reagent
(10 mM FerrozineTM in 40 mM HCl : 20 mM ferric chloride :
0.03 M acetate buffer pH 3.6 ratio 1:1:10) were added to each
aliquot. After 4 min incubation at 37 C, the absorbance was
measured at 595 nm at room temperature with a microplate
reader. Antioxidant activity was determined by linear regression
with ascorbic acid reference in the range 0–4 mg.
Cell lines and culture conditions
The HT1080 human fibrosarcoma cell line (ATCC CCL121) was grown in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% of heat inactivated fetal
bovine serum (FBS). Cell cultures were incubated at 37 C in a
428
5% CO2 -humidified atmosphere and sub-confluent cultures
were propagated by trypsinization (trypsin 0.025% – EDTA
0.5 mM).
Cell viability
Cell viability was assessed by the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolic activity assay according to the cell growth determination kit
from Sigma-Aldrich. Briefly, HT1080 cells were seeded in 96well culture dishes in the presence of complete culture medium
at a density of 1£104 cells/well. After 24 h of incubation at
37 C in a 5% CO2-humidified atmosphere, cell monolayers
were washed with serum-free medium supplemented with
25 mg/mL heat-treated bovine serum albumin (BSA) and
exposed to dried Posidonia extract resuspended in serum-free
medium at 6 1:2 serial dilutions from 1:62.25 to 1:2000. Cells
incubated in serum-free medium served as control untreated
group. After 24 h of incubation at 37 C in a 5% CO2-humidified atmosphere, culture medium was removed, cell monolayers
were washed with phosphate buffered saline (PBS) and then
tested for cell viability. All experiments were performed in triplicate and absorbance values at 595 nm were averaged. Cell viability was expressed as percent relative to untreated control.
Invasion assay
The invasiveness of HT1080 cells through reconstituted basement membrane represented by MatrigelÒ was evaluated in
Transwell filter units with pores of 8 mm in diameter. Aliquots
(100 ml) of a Matrigel solution in serum-free culture medium
(2 mg protein per mL) were layered on the top of each filters and
dried overnight. Then, cells detached from stock cultures with
EGTA (0.5 mM in PBS) were suspended at a density of 3 £ 105
cells/mL in serum-free medium supplemented with 25 mg/mL
heat-treated BSA and POE 1:1000 dilution. 0.5 mL of cell suspension was layered on the upper surface of filters that were then
immersed in wells filled with 1 mL serum-free medium supplemented with BSA and POE 1:1000 dilution. Control untreated
cells were treated as above except for the absence of POE. Transwell filter units were then incubated at 37 C in a 5% CO2
-humidified atmosphere for 18 h. Non-invading cells were
removed from the upper surface of the filter by wiping with a cotton swab, and the filters were then fixed in a methanol:acetic acid
mixture (70:30 v/v) for 1 h at room temperature. The filters
were stained with hematoxylin and eosin, and the invading cells
were quantified by examining the whole lower surface of the filter
under a light microscope (200-fold magnification). Each assay
was performed in duplicate.
Cell migration assay
The scratch wound healing assay of tissue-culture cell monolayers was used in order to analyze the effects of POE on cell
migration. Briefly, HT1080 cells were seeded in 9 cm2 slide
flasks at high density (5£105 cells/flask) and allowed to form
monolayers overnight in the presence of medium supplemented
with serum. Cell monolayers were then wounded with a sterile
rounded glass tip and washed several times with PBS to discard
Cell Adhesion & Migration
Volume 9 Issue 6
detached cells and cell debris. In order to maintain proper pH
without requirement of CO2, cell monolayers were then exposed
to serum-free medium supplemented with BSA and 20 mM
HEPES and POE 1:1000 dilution. Cells treated with serum-free
adhesion medium supplemented with BSA and 20 mM HEPES
without POE served as control untreated cells. The wounded
cell-free area was then observed under phase contrast microscopy
for 48–72 h at 37 C. Three frames from the same optical field
were captured every minute by time-lapse recording and wound
size was then analyzed with the TScratch software (ETH CSElab,
Zurich, Swiss).
Gelatin zymography
Conditioned medium from HT1080 cell cultures was prepared by incubating cells for 12 h in serum-free medium or in
serum-free medium in absence or supplemented with POE
1:1000 dilution. In brief, cells detached from stock cultures with
EGTA were seeded at a density of 2.5 £ 104 cells/cm2 in 6-well
culture plates in the presence of complete culture medium supplemented with 10% FBS. After 18 h of incubation at 37 C in a
5% CO2 -humidified atmosphere, culture medium was removed,
cell monolayers were washed twice with PBS and incubated in
serum-free medium supplemented with 25 mg/mL heat-treated
BSA or in serum-free medium supplemented with BSA and POE
1:1000 dilution. After 24 h of incubation, conditioned medium
was collected and centrifuged at 12000 rpm for 1 min at 4 C in
order to pellet non adherent cells. Then, same volumes of conditioned media from control or treated HT1080 cells were assayed
for gelatinase activity by gelatin zymography. Briefly, 25 ml aliquots of conditioned medium were electrophoresed under nonreducing conditions in 8% polyacrylamide gels containing 1 mg/
mL gelatin. After the electrophoretic separation, gels were washed
twice in 2.5% Triton X-100 for 1 h to remove SDS, rinsed
briefly, and incubated at 37 C for 24 h in reaction buffer
(50 mM Tris-HCl pH 7.4, 0.2 M NaCl, 5 mM CaCl2, 1 1 mM
mu;M ZnCl2).
When the effect of POE on gelatinase activity was assayed in
vitro, the medium of untreated cells was electrophoresed as
above, then strips of gels corresponding to lanes were cut, treated
with Triton X-100 and incubated separately in reaction buffer
containing scalar dilutions of POE or pure phenols at appropriate
concentrations.
In order to exclude inhibition by a chelating effects, the reaction buffer was optionally supplemented with concentration of
Ca2C and Zn2C ranging form 5 mM to 100 and from 1 mM to
20 M respectively.
Gels or gel strips were stained with 0.05% Coomassie Brillant
Blue R-250 dissolved in 40% methanol and 10% acetic acid and
destained with the same Coomassie-free solution. Zones of enzymatic activity appeared as clear bands against a blue background.
Images were acquired with a digital scanner.
Western blotting
In order to identify individual protease activities, conditioned
medium form treated or untreated cells was analyzed for
immuno-reactive MMP-2 and MMP-9. In brief, 25 ml aliquots
www.tandfonline.com
form the same conditioned medium used for zymography (see
above for details) were electrophoresed under non reducing conditions on 8% SDS-polyacrylamide gel and transferred by electroblotting onto Immobilon PVDF membranes. Red Ponceau
staining was used to check for equal loading and protein transfer.
After blocking with 2% fat-free dry milk in 20 mM Tris-HCl
(pH 7.4), 0.9% NaCl, 0.1% Tween-20 (TBS-Tween), the membrane was incubated with 2 mg/mL anti human matrix metalloproteinase MMP-2 monoclonal antibody (clone 42–5D11) or
with anti human matrix metalloproteinase MMP-9 monoclonal
antibody (clone 56–2A4). After washing with TBS, the membranes were incubated with goat anti-mouse and revealed by an
Odissey infrared imaging system.
Real time PCR
Total cytoplasmic RNA was isolated as described elsewhere53
from sub-confluent HT1080 cells cultured for 24 h in control
serum free-medium or in serum-free medium supplemented with
1:1000 dilution of POE. RNA concentration after extraction was
measured using a NanoDrop (Thermo Scientific) and first-strand
cDNA was synthesized from 1 mg of total RNA. Relative expression of MMP-2, MMP-9, TIMP1, TIMP2, MMP14/MT1MMP and GAPDH gene, which represents the housekeeping
gene, were quantified using TaqMan real-time polymerase chain
reaction using the primer sets described above. Each reaction was
performed in triplicate using the following protocol: 95 C for
10 min for one cycle, 50 cycles of 95 C for 15 s and 58 C for
1 min. Negative controls for amplification consisted in non-template control and no-RT enzyme control. The relative quantification (RQ) of gene expression was obtained by the comparative
CT method, 2-DDCt, as follows: first, the mean of the triplicate
threshold cycle (CT) values for the target gene was normalized to
the mean of the triplicate CT values for the internal control
GAPDH gene in the same samples (DCT D mean CTTarget –
mean CTGAPDH); second it was normalized with the control –
i.e., cells unexposed to POE – (DDCT D DCT – DCTControl)
and the fold change in gene expression was obtained by calculating 2-DDCT.54 Reported data are representative of 3 independent experiments with similar results.
Statistical analysis
All the results were expressed in terms of mean § standard
deviation (SD) on the basis of at least 3 independent extractions
for the main biochemical activities. Replicates for other experiments are detailed in appropriate sections. Linear regression analysis of reference compounds were performed with Microsoft
Office Excel (Microsoft Corporation).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The Authors wish to thank the researchers of the Cooperative
Society Pelagosphera for having collected the P. oceanica plants.
Cell Adhesion & Migration
429
Funding
This work was supported by the following grants: 1. Contract
grant sponsor: Ente Cassa di Risparmio di Firenze; Contract
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