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