A secretion of the mollusc Cryptomphalus aspersa promotes proliferation, migration and survival of keratinocytes and dermal fibroblasts in vitro

Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, C/ Darwin, 2. 28049 Madrid, Spain.
International journal of cosmetic science (Impact Factor: 1.38). 12/2011; 34(2):183-9. DOI: 10.1111/j.1468-2494.2011.00699.x
Source: PubMed
ABSTRACT
Regenerative properties of skin decrease with age, and thus, the search for substances that minimize cutaneous ageing has increased in the last few years. The secretion of the mollusc Cryptomphalus Aspersa (SCA) is a natural product that bears regenerative properties when applied topically. The purpose of this work is to study the in vitro effects of SCA on cell proliferation and migration, as well as on cell-cell (E-cadherin and β-catenin) and cell-substrate (vinculin and β1-integrin) adhesion proteins expression, using a human keratinocyte cell line (HaCaT cells) and primary dermal fibroblasts (HF). We tested the effects of SCA on cell proliferation using a colorimetric assay. In addition, SCA-induced changes on cell migration were studied by wound-healing assays. Besides, Western blot and immunofluorescence microscopy were carried out to test the expression of different cell adhesion proteins. We found that SCA promotes proliferation and migration of HaCaT cells in a time- and dose-dependent manner. Moreover, treatment with SCA increases the migratory behaviour and the expression of adhesion molecules in both HaCaT and HF. Finally, SCA also improves cell survival and promotes phosphorylation of FAK and nuclear localization of β-catenin. These results shed light on the molecular mechanisms underlying the regenerative properties of SCA, based on its promoting effect on skin cell migration, proliferation and survival. Moreover, these results support future clinical uses of SCA in the regeneration of wounded tissues.

Full-text

Available from: M. Carmen Iglesias-de la Cruz, Sep 24, 2015
A secretion of the mollusc Cryptomphalus aspersa promotes
proliferation, migration and survival of keratinocytes and dermal
fibroblasts in vitro
M. C. Iglesias-de la Cruz*, F. Sanz-Rodrı
´
guez*, A. Zamarro
´
n*, E. Reyes, E. Carrasco*, S. Gonza
´
lezà and A. Juarranz*
*Departamento de Biologı´a, Facultad de Ciencias, Universidad Auto
´
noma de Madrid, C/ Darwin, 2. 28049 Madrid, Spain, Industrial Farmace
´
utica
Cantabria, C/ Arequipa, 1, 28043 Madrid, Spain, àDermatology Service, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,
NY 10065, U.S.A. and §Dermatology Group, O’Donnell 41, 28009 Madrid, Spain
Received 20 May 2011, Accepted 12 November 2011
Keywords: cell adhesion components, Cryptomphalus aspersa, cytoskeleton, fibroblasts, keratinocytes, proliferation
Synopsis
Regenerative properties of skin decrease with age, and thus, the
search for substances that minimize cutaneous ageing has
increased in the last few years. The secretion of the mollusc Cryp-
tomphalus Aspersa (SCA) is a natural product that bears regenera-
tive properties when applied topically. The purpose of this work is
to study the in vitro effects of SCA on cell proliferation and migra-
tion, as well as on cell–cell (E-cadherin and b-catenin) and cell–
substrate (vinculin and b1-integrin) adhesion proteins expression,
using a human keratinocyte cell line (HaCaT cells) and primary
dermal fibroblasts (HF). We tested the effects of SCA on cell prolif-
eration using a colorimetric assay. In addition, SCA-induced
changes on cell migration were studied by wound-healing assays.
Besides, Western blot and immunofluorescence microscopy were
carried out to test the expression of different cell adhesion proteins.
We found that SCA promotes proliferation and migration of HaCaT
cells in a time- and dose-dependent manner. Moreover, treatment
with SCA increases the migratory behaviour and the expression of
adhesion molecules in both HaCaT and HF. Finally, SCA also
improves cell survival and promotes phosphorylation of FAK and
nuclear localization of b-catenin. These results shed light on the
molecular mechanisms underlying the regenerative properties of
SCA, based on its promoting effect on skin cell migration, prolifera-
tion and survival. Moreover, these results support future clinical
uses of SCA in the regeneration of wounded tissues.
Introduction
Skin degeneration is a complex process induced by ageing and
environmental damage, for example UV light. When considering
the skin, UV light is the most important damaging factor, leading
to skin photoageing and cancer. The main mechanism of UV-
induced photoageing is a marked increase in the production of
reactive oxygen species (ROS), which causes photo-oxidative dam-
age and decreases cell migration and proliferation [1, 2]. Photoage-
ing is characterized by the appearance of wrinkles, loss of
structural integrity and impaired wound healing [3]. These effects
are closely related with alterations in the remodelling process of
the extracellular matrix (ECM), a task carried out by dermal fibro-
blasts [4, 5].
When the skin is injured by any external aggression, the machin-
ery to repair the damage is rapidly activated, including fibroblast
proliferation and migration to the wound site to secrete and assem-
ble new ECM components (i.e. collagen, elastin and fibronectin) that
renew elasticity and consistency of the skin. In healthy skin, the
basal keratinocyte layer attaches to a carpet of specialized matrix,
the basal lamina. Therefore, physiological regenerative events
include proliferation of keratinocytes and the establishment of new
cell–cell and cell–substrate (basal lamina) adhesions. One of the
most important molecules involved in intercellular attachment is E-
cadherin (ECD), a transmembrane protein that mediates epithelial
cell–cell adhesion through other intracellular anchor proteins such
as b-catenin in adhesion junctions [6]. Keratinocytes also establish
hemidesmosomes, which anchors to the laminin in the basal lamina
through specific integrins belonging to the b1 subfamily [7]. In
these cells, hemidesmosomes connect the ECM to the intermediate
filament cytoskeleton (cytokeratins) inside the cell. These interac-
tions are crucial not only for the integrity of the skin but also for
signal transduction leading to cell migration and proliferation, for
example phosphorylation of the Tyr kinase focal adhesion kinase
(FAK) [7, 8]. Finally, survival signals are also needed to maintain
the integrity of the new repaired tissue. In this sense, elevated levels
of nuclear b-catenin also have been associated with improved cell
survival [9]. Among the plethora of signals involved in cellular pro-
liferation, b-catenin translocation to the nucleus plays a pivotal
role. In the nucleus, b-catenin is incorporated to the transcriptional
machinery, stimulating the expression of proliferation-inducing
gene products [10].
Research of natural substances that can improve and stimulate
skin regeneration has increased in the last few years. In this
regard, the secretion of some snails has proven beneficial in the
regeneration of burnt skin [11] and in the management of
open wounds [12]. In particular, a secretion from the mollusc
Cryptomphalus aspersa (SCA) has been proven to have antioxidant
activity [13] and to induce skin regeneration after wound-healing
impairment from acute radiodermatitis [14]. Moreover, a recent
study demonstrated that SCA stimulates fibroblast proliferation,
Correspondence: Salvador Gonza
´
lez, Dermatology Group, O’Donnell 41,
Madrid 28009, Spain. Tel.: +34610270045; fax: +34914317805;
e-mail: gonzals6@mskcc.org
International Journal of Cosmetic Science, 2012, 34, 183–189 doi: 10.1111/j.1468-2494.2011.00699.x
ª 2011 The Authors
ICS ª 2011 Society of Cosmetic Scientists and the Socie
´
te
´
Franc¸aise de Cosme
´
tologie
183
Page 1
rearrangement of the actin cytoskeleton and stimulates ECM assem-
bly [15]. However, the molecular mechanisms underlying the
regenerative properties of SCA are not completely understood. In
the present study, we have investigated the regenerative properties
of SCA using different in vitro approaches. We found that SCA
increased fibroblast and keratinocyte migration and increased the
expression of cell–cell and cell–substrate adhesion molecules in
both cell types. Finally, we demonstrated a role for SCA in cell sur-
vival signalling pathways, because it induces the expression of mol-
ecules related with this process, such as active b-catenin, FAK and
phosphorylated FAK. Taken together, our results support the use of
SCA as a putative cutaneous regenerative product.
Material and methods
SCA preparation
SCA was prepared according to US patent US 5538740. Briefly, the
gastropod was physically stimulated by centrifugation to increase
the secretion naturally produced by the mucinous, albuminous and
salivary glands. Then, the secreted fluids were separated and col-
lected from the live gastropod, clarified by centrifugation and fur-
ther clarified by filtration through 0.22-lm filters. Further dilutions
were performed in aqueous solution (pH 7.4). SCA toxicity was
assayed by trypan blue exclusion method. The highest effect/toxic-
ity ratio was achieved at 100 lgmL
)1
SCA; thus, we employed
this concentration for most of the assays described.
Cell cultures
The cells included in the study are HaCaT cells, an immortalized
human keratinocytes cell line and human dermal adult fibroblasts
(NHDF Cambrex, Charles City, IA, U.S.A.). Cells were grown in
F-25 flasks, or 35-mm culture dishes (Costar, Corning, NY, U.S.A.)
or on 22-mm square glass coverslips placed into dishes, using Dul-
becco’s modified Eagle’s medium containing 10% (v/v) foetal calf
serum, 50 U mL
)1
penicillin, 50 mg mL
)1
streptomycin and 1%
(v/v) 0.2 M l-glutamine (all from Gibco, Paisley, Scotland, U.K.).
Cells were incubated at 37C in an atmosphere containing 5%
CO
2
.
MTT viability assay
Cell viability was documented by the MTT assay [16]. HaCaT
cells cultured on 6-well Petri dishes were treated with different
concentrations of SCA (0, 25, 50 and 100 lgmL
)1
) during sev-
eral time points (2, 4, 6 and 8 days). Following these incuba-
tions, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide
(MTT) solution was added to each well at a concentration of
0.5 ng mL
)1
, and plates were incubated at 37C for 2–3 h. The
resulting formazan crystals were dissolved by the addition of
Dimethyl sulfoxide (DMSO), and absorbance was measured at
560 nm. The viability was also established by microscopy with a
trypan blue exclusion test using a Neubauer chamber counter.
In both cases, similar results were obtained.
Migration (wound-healing) assays
In vitro wound-healing assays were performed as previously
described [15]. Briefly, HaCaT and NHDF cells were seeded in T6-
well culture dishes at a density of 3 · 10
5
cells per well. Confluent
cell monolayers were then gently scratched with a pipette tip
across the entire diameter of the dish. Next, cells were rinsed with
medium to remove all cellular debris, and treatment with SCA was
applied to three wells, whereas the rest of the wells served as con-
trol. Cultures were observed immediately after wounding and again
12 and 24 h later, and phase-contrast pictures were taken of the
wounded area using a high-resolution microscopic camera Axio-
cam MRm vers.3 FireWire (D) (Zeiss, Le Pecq, France) connected to
the microscope. Migrated cells were quantified by measuring the
healed area. Data are given as means ± SD from three independent
experiments. Statistical significance was evaluated using the Stu-
dent’s t-test, and differences were considered to be significant at a
value of P < 0.05 (*).
Immunostaining
For general immunodetection, cells grown on coverslips were fixed
in 3% paraformaldehyde for 15 min. Samples were rehydrated in
PBS, permeabilized with 1% Triton X-100 in PBS for 10 min and
incubated for 1 h at 37C with the primary mouse monoclonal
antibodies against ECD, b-catenin, vinculin, FAK and phospho-FAK
(BD Transduction Laboratories, Palo Alto, CA, U.S.A.) and active
b-catenin (Millipore, Billerica, MA, U.S.A.) at 1 : 50 dilution in
0.1% BSA (Santa Cruz Biotechnologies, Santa Cruz, CA, U.S.A.).
Afterwards, coverslips were washed with PBS, incubated with rab-
bit anti-IgG of FITC-labelled secondary antibody (1 : 500 dilution
in 0.1% BSA; Santa Cruz Biotechnologies) and finally washed with
PBS. The preparations were mounted with Prolong (Invitrogen,
Carlsbad, CA, U.S.A.). Microscopic observations and photographs
were performed in an Olympus photomicroscope BX61, equipped
with a HBO 100 W mercury lamp and the corresponding filter sets
for fluorescence microscopy: blue (450–490, exciting filter BP 490)
and green (545 nm, exciting filter BP 545). The fluorescence inten-
sity of nuclear active b-catenin was measured using Imagej 1.37v
software (http://rsb.info.nih.gov/ij/); at least 100 nuclei were
counted for untreated (controls) and treated SCA cells, and the val-
ues are given as means ± SD.
Western Blot analysis
Semiconfluent monolayers were washed and then lysed with
RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.05% deoxycho-
late, 0.1% SDS, 1% Non idet-40, 50 mM Tris, pH 8) containing
phosphatase cocktail 2 and protease inhibitor cocktail (Sigma, St.
Louis, MI, U.S.A.). The samples were adjusted to the same protein
concentration (BCA Protein Assay Reagent; Pierce, Rockford, IL,
U.S.A.) and denatured by boiling in Laemmli sample buffer with
5% b-mercaptoethanol. 20 lg of each sample was subjected to
electrophoresis separation in SDS–PAGE. Gels were then trans-
ferred to a PDVF Immobilon-P membrane (Millipore, Bedford, MA,
U.S.A.) for 2 h. Membranes were stained with Ponceau S (Sigma)
to control loading, and after destained, they were blocked with
5% skimmed milk in Tris-buffered saline (Tris–HCl 10 mM pH
7.6, NaCl 0.9%, 0.05% Tween 20). The membranes were incu-
bated with the following specific antibodies: anti-ECD, vinculin,
FAK, P-FAK (BD Transduction Laboratories), active b-catenin
(Millipore) and G-actin (Sigma), diluted 1 : 500 in blocking buffer
at 4 C overnight. HRP secondary monoclonal antibody anti-IgG of
mouse was used, diluted 1 : 1000 and incubated 2 h at room
temperature. Detection of bands was performed using ECL Plus
Western blotting detection system (GE Healthcare, Hertfordshire,
U.K.). To quantify the bands obtained by Western blot, we applied
Imagej software-based analysis.
ª 2011 The Authors
ICS ª 2011 Society of C osmetic Scie ntists and the Socie
´
te
´
Franc¸aisedeCosme
´
tologie
International Journal of Cosmetic Science, 34,183189184
Skin effects of secretion of Cryptomphalus aspersa M. C. Iglesias-de la Cruz et al.
Page 2
Results
SCA treatment induces proliferation of HaCaT cells
To test whether SCA affected the proliferation of HaCaT cells, we
incubated the cells with different concentrations of SCA for different
times (Fig. 1). We found a non-significant increase in cell prolifera-
tion compared with control cells at 25 lgmL
)1
, whereas higher
concentrations of SCA (50 or 100 lgmL
)1
) induced a gradual and
significant increase with the time of treatment (Fig. 1).
SCA treatment stimulates migration of HaCaT cells and human
fibroblasts
We evaluated whether exogenous treatment with SCA had an
effect on the migration of keratinocytes and fibroblasts, using a
classical wound-healing assay. Confluent cultures of HaCaT cells
and human fibroblasts were gently scratched with a pipette tip and
treated with 100 lgmL
)1
SCA for 24 h. Photographs were taken
before (t = 0), and 12 and 24 h after wounding (Fig. 2). As shown
in Fig. 2(A,C), SCA promotes rapid occupation of the space
between both limits of the wound compared with control,
untreated HaCaT cells. At 24 h after SCA addition, the wound was
completely occupied with cells, whereas there was still an open
space in control cells. Similar results were found with human der-
mal fibroblasts; however, closure occurred at a slower rate
(Fig. 2B,C).
SCA treatment increases the expression of cell–cell and cell–
substrate adhesion molecules
We then focused on the expression of molecules that mediates
intercellular adhesion and attachment to the substrate. Immunoflu-
orescence and Western blot experiments revealed that treatment of
HaCaT cells with 100 l gmL
)1
SCA for 48 h stimulated the expres-
sion of both ECD and b-catenin (Fig. 3A). Densitrometry analysis of
Western blot bands revealed a 3.2-fold increase of ECD expression
and, more remarkably, a 28-fold increase for b-catenin (Fig. 3B)
with SCA treatment. Moreover, SCA not only increased the expres-
sion of ECD in keratinocytes, but it also seems to contribute to the
organization of ECD in cell–cell junctions (Fig. 3A, lower panel).
To study the effect of SCA treatment in cell–matrix adhesion, we
analysed the expression and distribution of the focal adhesion mar-
ker vinculin in both cultured HaCaT cells and in human fibroblasts
with SCA for 48 h (100 lgmL
)1
). As seen in Fig. 4(A), immuno-
fluorescence studies revealed that SCA promotes the recruitment of
vinculin to focal adhesions in HaCaT cells, which correlated with
increased vinculin expression as determined by Western blot analy-
sis (Fig. 4B). Moreover, we also studied by Western blot the effect
of SCA in b1-integrin production, a very important molecule
involved in cell–substrate adhesion. Results from these experiments
are shown in the lower panel of Fig. 4B, and it can be seen that
SCA treatment induced the expression of vinculin and b1-integrin
in HaCaT cells by 3.5- and 9.5-fold increase, respectively,
compared with control, untreated cells. With respect to human
fibroblast, we also found an important increase in the expression of
b1-integrin and vinculin mediated by exogenous treatment with
SCA, shown by both, immunofluorescence (Fig. 4A) and Western
blot analysis (4- and 5.3-fold increase vs. untreated cells, Fig. 4B).
SCA treatment promotes the expression of survival molecules
in keratinocytes and human fibroblasts
To assess the effect of SCA treatment on cell survival, we studied
the expression of molecules involved in this process, both by immu-
nofluorescence and Western blot. As seen in Fig. 5A (left panels),
we found an increase in b -catenin expression in HaCaT cells, when
treated with 100 lgmL
)1
SCA for 48 h, compared with untreated
cells. Furthermore, distribution of this molecule was predominantly
at the nuclear area in SCA-treated cells, whereas in control cells,
b-catenin was mainly located at the plasma membrane (Fig. 5A,
left panels). The fluorescence intensity because of nuclear b-catenin
was notably higher in SCA-treated cells compared with that of con-
trols (14.06 ± 3.28 vs. 1.3 ± 0.42; Fig 5B). Western blot studies
also revealed a 13.6-fold increase in protein production of active
nuclear b-catenin in SCA-treated vs. control HaCaT cells (Fig. 5C).
We next investigated the expression and activation by phosphor-
ylation of FAK, which is another molecule involved in cell survival,
in human fibroblasts treated with SCA 100 lgmL
)1
for 48 h.
Immunofluorescence studies show a clear increase in both expres-
sion and phosphorylation of FAK when SCA is present, compared
with control cells (Fig. 5A, right panels). Comparable results were
obtained by Western blot in human fibroblasts (data not shown)
and in HaCaT cells (Fig. 5C). In these cells, we found that expres-
sion of FAK is increased 5-fold, whereas its phosphorylation
increased 24-fold compared with control, untreated cells.
Discussion
Skin ageing is a natural biological process. However, environmental
aggression and genetic predisposition can accelerate this process.
For example, exposure to UV wavelengths promotes oxidative dam-
age, in a process referred to as photoageing. Photoageing affects the
sun-exposed areas and is characterized clinically by wrinkles,
roughness, dryness, laxity, telangiectasia, loss of tensile strength
and pigmentary changes in the skin. There is also an increase in
the development of benign and malignant neoplasms on photoaged
skin. Several pathological mechanisms are involved in the process
of skin ageing, such as an increase in ROS production [17],
impaired remodelling capacity of the skin, reduced cell migration,
adhesion and survival [1]. The search for products that can
150
100 50 25 Control
50
100
150
No. cells
×
10
6
0
50
02468
Days
200
250
Figure 1 SCA treatment increases the proliferation of HaCaT cells. Increase
in the number of HaCaT cells as a function of the days of treatment with dif-
ferent concentrations of SCA (25, 50 and 100 lgmL
)1
). Each point corre-
sponds to the mean value ± SD from three different experiments. SCA,
secretion from the mollusc Cryptomphalus aspersa.
ª 2011 The Aut hors
ICS ª 2011 Society o f Cosmetic Scientists and the So cie
´
te
´
Franc¸aise de Cosme
´
tologie
International Journal of Cosmetic Science, 34,183189 185
Skin effects of secretion of Cryptomphalus aspersa M. C. Iglesias-de la Cruz et al.
Page 3
diminish the effects of either chronological ageing or UV-induced
photoageing is increasing dramatically in the last few years.
Among these natural products are fern leaves [18], green tea
[19], or retinoids [20]. SCA is a natural secretion from the mollusc
C. aspersa endowed with regenerative properties in experimental
acute radiodermatitis produced in a rat model [21]. In a recent
study, Brieva et al. [15] demonstrated that SCA also exhibits anti-
oxidant properties and promotes fibroblasts’ survival and prolifera-
tion and ECM assembly. However, the effects of SCA on
keratinocyte biology or other aspects of fibroblast behaviour
remained unexplored.
In this report, we show that SCA induces both fibroblast and
keratinocyte migration in vitro. Throughout the whole repair pro-
cess, proliferation and migration of different cell types provide forma-
tion of the scar. Migration of fibroblasts, which occurs from
surrounding unwounded skin towards the provisional matrix of the
haemostatic plug, is a crucial step in the wound-healing process
[22] that is impaired with ageing [23]. The molecular mechanisms
underlying cell migration involve attachment to the ECM,
rearrangement of cytoskeleton and subsequent detachment of the
cell from the matrix. SCA has already been shown to induce actin
reorganization bundling and microfilament alignment in human
0 h 24 h12 h
(A)
(B)
(C)
– SCA+ SCA– SCA
+ SCA
0.3
0.4
0.5
0.6
0.7
0.8
0 h
12 h
*
0
0.1
0.2
– SCA
+ SCA – SCA + SCA
24 h
Wound (mm)
HaCaT HF
*
*
Figure 2 SCA treat ment increases the migra-
tory behaviour of HaCaT cells (panels A, C) and
human fibroblasts (panels B, C). The motility/
migratory behaviour of non-treated (control) or
SCA-treated (100 lgmL
)1
, SCA) cells was
analysed in an in vitro wound model. Confluent
cultures were gently scratched with a pipette
tip to produce a wound. Photographs of the
cultures were taken immediately after the inci-
sion (0 h), after 12 h and after 24 h and
wounds measured in wide. Pictures are repre-
sentative of at least 6 experiments. Data of
wounds in panel C are given as the mean ± SD
from at least three independen t experiments.
*P < 0.05. SCA, secretion from the mollusc
Cryptomphalus aspersa.
ª 2011 The Authors
ICS ª 2011 Society of C osmetic Scie ntists and the Socie
´
te
´
Franc¸aisedeCosme
´
tologie
International Journal of Cosmetic Science, 34,183189186
Skin effects of secretion of Cryptomphalus aspersa M. C. Iglesias-de la Cruz et al.
Page 4
dermal fibroblasts [15]. Our results show that SCA is able to increase
the migration rate of human fibroblasts as well as keratinocytes at
24 h of study, confirming its positive role in the first steps of the
wound-healing process. It is of particular interest that SCA also pro-
motes the expression of motility-related molecules, such as b1 inte-
grins (epidermal and mucosal keratinocytes express a2b1, a3b1 and
a6b4 [24], vinculin and FAK), and promotes phosphorylation of
FAK, which is a mandatory step in the process of cell migration by
participating in the release and reformation of adhesive contacts
with the ECM [25]. Our results clearly show that SCA improves the
protein production of both molecules in keratinocytes and fibro-
blasts, thus stimulating the attachment of these cell types to the sub-
strate. Taken together, these findings suggest an important role for
SCA in some of the most important events during wound healing
that are weakened with age, such as cell–cell and cell–substrate
adhesion.
Additionally, SCA promotes nuclear appearance of b catenin.
Differentiated keratinocytes are characterized by strong intercellular
attachment by both desmosomes and adhesion junctions. Adherens
junctions are characterized by the presence of ECD, a- and b-cate-
nins, and c-catenin (plakoglobin) at the membrane [26]. b-Catenin
is a multifunctional protein that plays an important role during
embryonic development and neoplasia as a mediator of the Wnt
signalling pathway [27]. When the Wnt pathway is quiescent,
b-catenin participates in adherens junctions [28]. When b-catenin
becomes cytoplasmic, it gets phosphorylated and targeted for ubiq-
uitination and degradation. Activation of the Wnt pathway inhibits
this phosphorylation, leading to cytosolic stabilization of b-catenin,
which consequently translocates to the nucleus where it binds
Tcf–Lef transcription factors and regulates transcription [29]. Once
in the nucleus, b-catenin interacts with members of the Lef/Tcf
family of transcription factors to activate the expression of target
genes related to the cell cycle, proliferation and survival. b-catenin
translocation to nucleus is associated with several epithelial can-
cers [30], because it inhibits apoptosis signals and promotes migra-
tion and proliferation of epithelial cells [10]. Moreover, in a recent
report, it was demonstrated that UVB radiation causes apoptosis,
and this process was directly related with cleavage of ECD and its
downstream components, including b-catenin [31]. Our results
(B)
(A)
Figure 3 SCA treatment increases the expression of cell–cell adhesion mole-
cules in HaCaT cells. Panel A: Immunofluorescence (IF) staining for E-cadh-
erin (ECD) or b-catenin (b-Cat) in control cells and SCA-treated
(100 lgmL
)1
) for 48 h. Preparations were observed under blue excitation
light. Note the enhanced ECD and b-catenin expression in SCA-treated cells
compared with control cells. Panel B: The SCA-induced increase in cell–cell
adhesion molecules expression was also confirmed with Western blot analy-
sis; Densitometric analysis of the bands revealed a 3.2- or 28-fold increase
for ECD or b-Cat expression, respectively (lower panel). Scale bar = 20 lm.
SCA, secretion from the mollusc Cryptomphalus aspersa.
(B)
(A)
a
Figure 4 SCA treatment increases the expression of cell–substrate adhesion
molecules in HaCaT cells and human fibroblasts. Panel A: Immunofluores-
cence (IF) staining for vinculin in control cells and SCA-treated
(100 lgmL
)1
) for 48 h using HaCaT cells (left panels) or human fibroblasts
(right panels). Preparations were observed under blue excitation light. Panel
B: The SCA-induced increase in vinculin in HaCaT cells was also confirmed
with WB analysis. Expression of b1-integrin, assayed by Western blot in
both cell types, is also increased with SCA treatment (lower panels). Scale
bar = 20 lm. SCA, secretion from the mollusc Cryptomphalus aspersa.
ª 2011 The Aut hors
ICS ª 2011 Society o f Cosmetic Scientists and the So cie
´
te
´
Franc¸aise de Cosme
´
tologie
International Journal of Cosmetic Science, 34,183189 187
Skin effects of secretion of Cryptomphalus aspersa M. C. Iglesias-de la Cruz et al.
Page 5
indicate a clear increase in the expression of nuclear b-catenin in
cultured keratinocytes after treatment with SCA. This observation
clearly points to the ability of SCA in inhibiting keratinocyte apop-
tosis, although additional studies should be performed.
In addition to its role in regulating cell motility, phosphorylation
of FAK also mediates cell survival [32]. At focal adhesions, inte-
grins can activate various protein tyrosine kinases, including FAK.
The cytoplasmic tail of integrin b subunits activate FAK, and then,
it autophosphorylates Tyr
397
creating a binding site for several pro-
teins. These proteins eventually promote signalling pathways that
modify the cytoskeleton and activate the extracellular signal-regu-
lated kinase (ERK) family of mitogen-activated protein kinases,
leading to cell proliferation [32]. According to Manohar et al., [33]
b1 integrin promotes keratinocyte survival through activation of
the ERK cascade, also involving phosphorylation of FAK. Here, we
have shown that SCA induces an increase in the expression of FAK
and P-FAK in cultured keratinocytes. Thus, through increased
expression of b1 integrin and phosphorylated FAK, SCA plays a
dual role in promoting cytoskeletal reorganization and promoting
cell survival.
In summary, our results provide new insights into molecular
mechanisms underlying the regenerative properties of SCA.
Furthermore, they may suggest dermatologic or cosmetic uses of
SCA-based products as to diminish ageing-induced cutaneous man-
ifestations.
Acknowledgements
The authors declare that the main subject of this research (SCA) is
subject to US patent US 5538740. The study was partially sup-
ported by a research grant from Industrial Farmace
´
utica Cantabria,
S.A., Madrid, Spain. S.G. is a consultant for I.F.C.
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Figure 5 SCA treatment increases the expres-
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for 48 h) in both cases. Note the enhanced
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Skin effects of secretion of Cryptomphalus aspersa M. C. Iglesias-de la Cruz et al.
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    • "Significant correlations are in bold panormitanum and C. aspersus. Both of these species are favoured by human land management because they are related to cultivated areas or gardens and may be considered pests for cultivation, especially D. panormitanum (Cordoba et al. 2011; Fabian et al. 2012; Iglesias-de la Cruz et al. 2012). The only species strongly related to cemented areas was C. itala, a typical rock-dwelling species that feeds on lichens (Kerney and Cameron 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: One of the classical and traditional wall typology built in agricultural or pas- toral landscapes are dry stone walls (walls built only of stones without concrete). These vertical surfaces are expected to increase habitat heterogeneity and to play an important role for biodiversity. This study focused on two groups of organisms: amphibians, repre- sented by the rock-dwelling salamander Hydromantes strinatii , that are expected to use walls mainly as shelters, and molluscs, which use of walls may be affected mainly by the trophic resources available. A mountain area of the northern Appennines (NW-Italy) was surveyed to assess the differences between dry stone walls and the wall typologies in terms of morphology, surrounding landscape and salamander and mollusc occurrence; the rela- tionships between wall typology features and salamander and mollusc distribution were assessed. Dry stone walls were more heterogeneous than concrete walls and hosted more lichens than natural rocky walls. They were more used by H. strinatii juveniles than the other walls and played an important role for their distribution. They were positively related to the occurrence of several molluscan species, including species with high ecological plasticity and rock-dwelling species. Among wall features, the most important for molluscs species distribution was vegetation cover, followed by lichen cover and heterogeneity, confirming the importance of trophic content for mollusc exploitation, while vegetated without concrete walls hosted higher number of species. The results suggest that dry stone walls can be important for fauna biodiversity and should be maintained and preserved as a part of landscape management.
    No preview · Article · Apr 2014 · Biodiversity and Conservation
  • [Show abstract] [Hide abstract] ABSTRACT: Growth factors (GFs) are chemical messengers that regulate specific cellular activities such as cell proliferation and formation of the extracellular matrix. GFs may be derived from a variety of sources, including animals. Evaluate the safety and efficacy of a topical antiphotoaging product containing secretions of the snail Cryptomphalus aspersa (SCA) for the improvement of facial rhytides. MATERIALS and This was a 2-center, double-blind, randomized, 14-week study in which 25 patients with moderate to severe facial photodamage were treated with an emulsion (with 8% SCA) and liquid serum (with 40% SCA) on one side of the face and placebo on the contralateral side for 12 weeks. Silicone skin impressions of periocular rhytides were performed at baseline and after 12 weeks of treatment. Patient and physician assessments were also performed at 8, 12, and 14 weeks. Periocular rhytides on the active ingredient side showed significant improvement after 12 weeks (P=.03) and improved texture to a greater degree than placebo at 8 and 12 weeks, as well as 2 weeks after discontinuing the product (14 weeks). Daily application of topical products containing SCA proved effective and well tolerated for improvement in coarse periocular rhytides and fine facial rhytides. Subjects noted a significant degree of improvement in fines lines at the 8-week time point on the SCA-treated side (P≤.05) but did not report a significant difference in the quality of their skin. J Drugs Dermatol. 2013;12(4):453-457.
    No preview · Article · Apr 2013 · Journal of drugs in dermatology: JDD
  • [Show abstract] [Hide abstract] ABSTRACT: Synopsis Background The search of substances that minimize cutaneous ageing has increased in the last few years. Previous studies have described the regenerative properties of the secretion of the mollusc Cryptomphalus aspersa (C.aspersa) when applied topically. Objective We evaluate the in vitro effects of a new product derived from the eggs of C.aspersa, IFC-CAF, on cell proliferation, migration, distribution of cytoskeletal proteins, production of extracellular components as well as its ability to prevent cutaneous ageing because of intrinsic or extrinsic factors (exposure to UVB) by determination of ageing markers. Methods We have used the human keratinocyte cell line (HaCaT cells), primary dermal fibroblasts (HDF) and senescent dermal fibroblasts (SHDF). The effects of the compound on cell proliferation and on the cell cycle were determined by the MTT colorimetric assay, estimation of total protein and/or trypan blue test and by flow cytometry, respectively. We also studied cell migration using the wound-healing migration assay, whereas ELISA assays, Western Blot and immunofluorescence microscopy were carried out to test the expression of proteins related to cytoskeleton, extracellular matrix and with ageing. ResultsWe have found that IFC-CAF does not promote proliferation but induces migration of HaCaT, HDF and SHDF in a time- and dose-dependent manner; a better organization of cytoskeletal proteins (F-actin and vimentin) and promotes the production of extracellular components (fibronectin, collagen 1 and MMPs) and the adhesion to cell-substrate vinculin protein. IFC-CAF also prevents cutaneous ageing. The treatment decreases the expression of the ageing-related markers b-Gal, p53 and p16INK4 in SDDF cells, and improves cell survival after UVB irradiation and nuclear repair in HaCaT cells. ConclusionIFC-CAF has regenerative properties and protects against ageing factors being, therefore, a potential therapeutic agent for treating or preventing skin ageing.
    No preview · Article · Sep 2014 · International journal of cosmetic science