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Milan, June 11, 2015
ROLE OF FOOD – DERIVED ANTIOXIDANTS IN DERMATOLOGY AND COSMETOLOGY
Gabriella Calviello - Leonardo Celleno Institutes of General Pathology and Clinical Dermatology, and Research Center for Biotechnologies Applied to Cosmetology School of Medicine, Università Cattolica S. Cuore, Rome, Italy
Inhibit the activity of other compounds
Instable compounds highly reactive very short half-life
• Free radicals
Oxidants
• Non free radicals
ROS RNS
Skin
ANTIOXIDANTS
ANTIOXIDANTS Late ’80 Early ‘90
Public attention
Involved in the modulation of the pathogenesis of several chronic diseases (atherosclerosis, cancer, visual disorders, etc)
Low dietary intake of fruit and vegetables rich in antioxidants
risk of these chronic diseases
Preclinical Studies
Human Observational
Studies
Increasing clinical experimental evidences
to demonstrate the usefulness of these antioxidant molecules
have contributed
ANTIOXIDANT DEFENSE
Normal redox state
Exogenous sources
UVR Environmental toxins
Chemicals
Ionizing radiations
The most vulnerable organ to radiations and environmental chemicals
ROS Endogenous sources
Melanogenesis
Mitochondria
Cytochrome P450 NADPH oxidase
Lipoxygenases
Peroxisomes
Inflammation Skin
ANTIOXIDANT DEFENSE
Exogenous sources
Normal redox state
Enzymatic: Catalase SOD, Superoxide dismutase GPX, Glutathione peroxidase
HO-1, Heme oxygenase 1 GST, Glutathione S-transferase NQO1, NAD(P)H dehydrogenase quinone 1 PRX, Peroxiredoxin
Non enzymatic: Eumelanin Ferritin Glutathione Vitamin A Vitamin C Vitamin E
ROS Endogenous
sources
ANTIOXIDANT DEFENSE OXIDATIVE STRESS
Cellular substructures
Photoaging Immunosuppression Photocarcinogenesis
CLINICAL EFFECTS
UVR
ROS
Lipid peroxidation
Protein oxidation
DNA mutation
Counteracting
In the prevention and treatment
A main challenge
ROS
Skin photoaging
Skin photocarcinogenesis
at skin level
ROS
largely derived from plants and naturally contained in foods
Request for more targeted antioxidants
These substances are now widely used in many fields of medical therapy and also in dermatology
administered topically or systemically
for inflammatory dermatosis and photodermatology
Naturally-derived compounds with antioxidant properties
Dermatology field In the search for new preventive or therapeutic strategies for chronic skin pathologies
Natural extracts as safe source of antioxidants
Cosmetic field
To test the potential antioxidant efficacy of natural compounds alone or in combination
Our AIM
an experimental procedure which involves the use of different in vitro and in vivo models
The antioxidant ability of a substance is evaluated
First step
Human and mouse-derived cultured cells
a) The intracellular production of ROS
b) Cellular lipid peroxidation
Cytofluorimetric analysis using the fluorescent probe dichlorofluorescein diacetate (DCFA)
Spectrophotometrical analysis using the TBARS (thiobarbituric acid reactive substances) assay.
c) Cellular total antioxidant capacity (TAC)
Colorimetric analysis (ELISA KIT)
By measuring its ability to modulate:
in
On the basis of the results obtained
Evaluating the ability of the substance to regulate the cellular level/expression of various endogenous antioxidants
Tot GSH CuZnSOD
HO-1 GSH
reductase
Second step
Designing of experimental clinical trials on a significant number of people monitored during the treatment
Third step
If the outcomes are satisfactory
To verify the effectiveness of the compound in vivo
UVR
UV as a stimulus capable of producing an excess of free radicals
ROS
• Omega-3 Normal and neoplastic human skin cell lines, animal models, humans
• Glycerophosphoinositol Human normal skin cell lines
THE MOST IMPORTANT RESULTS
• Combination of antioxidants
Evolved as a part of a complex organization where each single component
Neither of them can do the work of the entire team
ANTIOXIDANTS
play a different role
Human normal skin cell lines
Green tea • Combination
Resveratrol
γ-Oryzanol
Soy Isoflavones
Serini S. et al. Esperienze Dermatologiche, 2012
Human normal skin cell lines
Coenzyme Q10
Krill oil
• Combination
Lipoic acid Resveratrol
Grape seed oil α-Tocopherol
Na-Selenite
Fasano et al. BioMed Res International, 2014
• Combination Sulforaphan
Fernblock® XP
Human normal and neoplastic skin cell lines
Serini S. et al. (manuscript in preparation)
ROS PRODUCTION (+/- H2O2)
LIPID PEROXIDATION (+/- PROOXIDANTS)
IL-6
MCP-1
CYTOKINE PRODUCTION (+/- TNF-α)
Coenzyme Q10
Krill oil
Combination
Lipoic acid
Resveratrol
Grape seed oil
α-Tocopherol
Na-Selenite
NF-κB ACTIVATION (+/- TNF-α)
IkBα PHOSPHORILATION
NF-κB(p65) EXPRESSION
Fasano et al. BioMed Res International, 2014
Sulforaphan (SFN)
[1-Isothiocyanato-4-(methylsulfinyl) butane]
Found at high levels in vegetables of Brassicaceae (o Cruciferae) family,
especially in broccoli and cauliflowers
• Combination Sulforaphan
Fernblock® XP
Human normal and neoplastic skin cell lines
Serini S. et al. (manuscript in preparation)
Fernblock® XP: Extract of Polypodium leucotomos (PL)
A tropical fern rich in phenolic compounds
Previous studies on not-skin human and murine cell lines
SFN PL
• Antitumoral • Antioxidant • Anti-inflammatory
in vitro treatment
• Photoprotective • Antioxidant • Inti-inflammatory
Effects: Effects:
• Cell growth • Cell migration • MMP Production
Human melanoma cells
Primary (WM115)
Metastatic (WM266-4)
Human Immortalized Keratinocytes
HaCaT
NCTC 2544
Treatment: • SFN 2.5 - 20 µM • PL 1 - 2 mg/ml
• ROS Production
Both SFN and PL inhibit the of melanoma cell growth in dose- and time-dependent manners
WM115
Cel
ls (
x1
03)
0 24 48 72 (h)
Ctrl
SFN 5 μM
SFN 10 μM FB 1 mg/ml FB 2 mg/ml
SFN 20 μM
0
50000
100000
150000
200000
250000
300000
350000
0
50
100
150
200
250
300
350
Cel
ls (
x1
03) WM266-4
0 24 48 72 (h)
Ctrl
SFN 5 μM FB 1 mg/ml FB 2 mg/ml
SFN 20 μM
SFN 10 μM
0
50
100
150
200
250
300
350
Cell growth
The combined treatment inhibits melanoma cell growth more efficiently than each single treatment
Cel
l g
row
th in
hib
itio
n
(% v
s co
ntr
ol)
Cell growth
Anti-invasive effect Wound Healing assay
0
20
40
60
80
100
120
SFN 10 μM
ctrl
0 24 48 72 h 0
20
40
60
80
100
120
0 24 48 72 h
WM266-4
0
5
10
SFN(μM) WM115 c c c
c c
c
b, c
c
c
b
a
a
d d d
b
b, c
c, d
a
a
a
a
b
b
Cel
l fr
ee a
rea
(%
)
SFN powerfully inhibits human melanoma cell invasiveness in dose- and time-dependent manners
PL powerfully inhibits cell invasive potential, more than SFN
0
20
40
60
80
100
120
ctrl
PL 2 mg/ml
0 24 48 72 h
WM115
0
20
40
60
80
100
120
0 24 48 72 h
WM266-4
0
1
2
PL (mg/ml) d d d
c c
c, d c, d c, d c, d c, d
b
a
c c c c c
c
b
a a
a
b, c
b, c
Cel
l fr
ee a
rea
(%
) Anti-invasive effect Wound Healing assay
The combination powerfully inhibits cell invasiveness more than SFN e PL individually administered
Cel
l fr
ee a
rea (
%)
C
ell
free
are
a (
%)
Anti-invasive effect Wound Healing assay
MMP evaluation: ELISA assay
The combined treatment inhibits MMP-2, MMP-3 and MMP-9 production
more than each single compound
MMP-3
MMP-2 MMP-9
ROS evaluation: Cytofuorimetry
The combined teatment inhibits ROS production more than each single compound
RO
S p
rod
uct
ion
(% v
s co
ntr
ol)
RO
S p
rod
uct
ion
(% v
s co
ntr
ol)
RO
S p
rod
uct
ion
(% v
s co
ntr
ol)
RO
S p
rod
uct
ion
(% v
s co
ntr
ol)
• SFN and PL
The use of a combination of SFN and PL may represent an important preventive strategy against the development of invasive melanoma
and the oxidative damage of keratinocytes
• SFN and PL
• Combination SFN + PL
Conclusions
cell growth
cell invasiveness
human melanoma cell lines
inhibit
inhibit intracellular ROS production
in human keratinocytes
than SFN or PL given separately
markedly more effective
Experimental clinical trial on a significant number of patients monitored during the treatment
To verify the effectiveness of the combinations
Further step
SFN/PL
in vivo
Thank you for your attention!
Acknowledgments
Prof. Simona Serini Dr. Roberta Guarino Dr. Nadia Mondella Dr. Elena Fasano Prof. Leonardo Celleno