Mechanisms Involved in the Anticarcinogenic Effects of Olive...

Mechanisms Involved in the AnticarcinogenicEffects of Olive Oil Phenols

Roberto FabianiGuido Morozzi

DIPARTIMENTO DI SPECIALITA’ MEDICO-CHIRURGICHE E SANITA’ PUBBLICAUniversity of PERUGIA - ITALY

Countries with a relatively high consumption of olive oil

Lower cancer incidence and mortality

100100181890906688262637373388United StateUnited State

303010107070668822223131661616UnitedUnitedKingdomKingdom

484813136464668818182222771414ScandinavianScandinavian171710104343446614141818881919MediterraneanMediterranean

FemalesFemalesMalesMalesFemalesFemalesMalesMalesFemalesFemalesMalesMales

ProstateProstateEndometriumEndometriumBreastBreastPancreasPancreasLarge bowelLarge bowelStomachStomach

Age-adjusted incidence rate for selected cancer sites per 100,000 person-year, 1990Age-adjusted incidence rate for selected cancer sites per 100,000 person-year, 1990

A. Trichopoulou et. al., Cancer Epid. Biom. Prev. 2000

DESCRIPTIVE STUDIES

EPIDEMIOLOGICAL EVIDENCES SUGGESTING THEPROTECTIVE EFFECT OF OLIVE OIL ON CANCER

Inverse correlation between olive oil intake and rates of cancer

Es. Colorectal cancer

28 Countries

(Stoneham et al. 2000)

-0.24-0.24CerealsCereals-0.40-0.40VegetablesVegetables-0.55-0.55Vegetables oilVegetables oil-0.33-0.33FishFish-0.57-0.57FruitFruit-0.36-0.36MilkMilk-0.62-0.62Total fatTotal fat-0.32-0.32Animal fatAnimal fat-0.47-0.47MeatMeat

Partial correlation coefficient, controlling forPartial correlation coefficient, controlling for

--0.290.29Correlation coefficient, unadjustedCorrelation coefficient, unadjusted

ECOLOGICAL-CORRELATION STUDIES


CASE-CONTROL STUDIES-N1

0.50.578786565BreastBreastSpainSpain19981998Morales et al.Morales et al.

0.810.810.880.88

4,1544,1541,2251,225728728

ColonColonRectumRectum

ItalyItaly19981998BragaBraga et al. et al.0.60.61,5021,502362362PancreasPancreasItalyItaly19971997La La VecchiaVecchia et al. et al.

GreeceGreece

ItalyItaly

GreeceGreece

SpainSpain

ItalyItaly

CountryCountry

0.740.74298298145145EndometriumEndometrium19961996TzonouTzonou et al. et al.0.870.872,5882,5882,5642,564BreastBreast19951995La La VecchiaVecchia et al. et al.0.750.751,5481,548820820BreastBreast19951995TrichopoulouTrichopoulou et al. et al.0.60.6988988762762BreastBreast19941994Martin-Moreno et al.Martin-Moreno et al.<1<11,1591,1591,0161,016StomachStomach19891989BuiattiBuiatti et al. et al.

ORORControlControlCaseCaseCancer-SiteCancer-SiteYearYearAuthorAuthor


0.40.41,2971,297527527LarynxLarynxItalyItalySwitzerlandSwitzerland

20022002BosettiBosetti et al. et al.0.850.85106106106106Oral cavityOral cavityGreeceGreece20022002PetridouPetridou et al. et al.0.50.5480480317317ProstateProstateNew ZealandNew Zealand20002000NorrishNorrish et al. et al.

ItalyItaly

ItalyItaly

ItalyItaly

GreeceGreece

ItalyItaly

CountryCountry

0.40.4743743304304EsophagusEsophagus20002000BosettiBosetti et al. et al.

≅≅ 114,4544,4541,9531,953Colon -Colon -RectumRectum

19991999FranceschiFranceschi et al. et al.0.50.51.4911.491598598Oral cavityOral cavity19991999FranceschiFranceschi et al. et al.≅≅ 11246246320320ProstateProstate19991999TzonouTzonou et al. et al.0.580.581,5521,552362362PancreasPancreas19981998SolerSoler et al. et al.




0.270.27464464291291BreastBreastGranGranCanariaCanaria

20062006Garcia-SegoviaGarcia-Segovia

FranceFrance

AustraliaAustralia

ItalyItaly

ItalyItalySwitzerlandSwitzerland

ItalyItaly

CountryCountry

0.40.4426426154154Colon -Colon -SmallSmallAdenomaAdenoma

20052005RoullierRoullier et al. et al.0.80.8905905858858ProstateProstate20042004Hodge et alHodge et al0.670.67292292342342LungLung20032003Fortes et al.Fortes et al.

0.30.33403406868LarynxLarynx20032003Gallus et al.Gallus et al.0.680.682,4112,4111,0311,031OvaryOvary20022002BosettiBosetti et al. et al.




MOLECULAR EPIDEMIOLOGY

213 healthy subjects

Biomarker: DNA-adducts in peripheral leukocytes

0.030.03-41.0-41.05.875.877.797.799.959.95Oleic acidOleic acid0.050.05-18.4-18.46.766.768.888.888.288.28Olive oilOlive oil

PP value valuefor trendfor trend

%%changechange

IIIIIIIIIIIITertileTertile of consumption/intake of consumption/intake

DNA-adducts per 109 nucleotides

Palli et al. 2000


INTERVENTION STUDIES

•Salvini et al. 2006

10 postmenopausal women

high-phenol EVOO (592 mg total phenols/kg)

low-phenol EVOO (147 mg/kg)

30% Reduction of oxidative DNA damage inlymphocytes


ANIMAL STUDIES SUGGESTING THEPROTECTIVE EFFECT OF OLIVE OIL ON

CANCER

Olive oil protects animals fromchemically induced carcinogenesis

DMBA-induced mammarytumors (Lasekan et al. 1990)

AZM-induced carcinoma of thecolon (Bartoli et al. 2000)

UV-induced skin cancer (Ichihashi et al . 2000)

Olive oil reduce the incidence of the spontaneous liver tumours in mice(Thuy et al. 2001)

WHY PHENOLS?

•EXAMPLES OF WINE AND TEA

•PubMed Search - 14.09.07

2020HydroxytyrosolHydroxytyrosol Cancer Cancer693693EpigallocatechinEpigallocatechin Cancer Cancer632632ResveratrolResveratrol Cancer Cancer

N° ofN° ofpublicationspublications

Key wordsKey wordsResveratrol

Epigallocatechin

Hydroxytyrosol

WHY PHENOLS?

SOME OF THE PROPERTIES BY WHICH PHENOLSMAY PREVENT CARDIOVASCULAR DISESASESCOULD ALSO BE INVOLVED IN THE PROTECTIONOF CANCER

•ANTI-OXIDANT

•ANTI-INFLAMMATORY

WHY PHENOLS?

•1998 (Simonsen et al.) MULTICENTRE STUDY (Germany,Northern Ireland, Spain, Netherlands, Switzerland)

•relation of adipose tissue fatty acid content to breast cancer

•Oleic acid showed a strong inverse association with breastcancer only in the Spanish centre. OR= 0.40

•OR= 1.27 in all other centres pooled

•The protective effect of olive oil may be due to minorcomponents instead of the direct effect of oleic acid.

CARCINOGENESIS PROCESS

Normal

DNA damagingagents

Anti-INITIATION•Anti carcinogen metabolism•Enhance carcinogen detoxification•Scavenge electrophiles/ROS•Enhance DNA repair

Anti-PROMOTION/PROGRESSION•Scavenge ROS•Alter gene expression•Decrease inflammation•Suppress proliferation•Induced differentiation•Encourage apoptosis

Initiated

Preneoplastic

Neoplastic

Increased proliferation

Additional geneticalterations

Effect of hydroxytyrosol (3,4-DHPEA) onH2O2-induced DNA damage in PBMC

0

100

200

300

Control 0 1 3 5 10

DN

A d

am

ag

e, A

.U.

aa

b

c

d

3,4-DHPEA, !M

H2O2, 20 !M

The cells were co-incubated with H2O2 and 3,4-DHPEA for 30 min. at 37C°

0

1

2

3

0 24 48 72 96 120 144 168

Incubation time, h

N° o

f v

iab

le cell

s (1

06/m

l)

0 !M

10 !M

25 !M

50 !M

100 !M

250 !M

Effect of 3,4-DHPEA on proliferationof HL60 cells

0

25

50

75

100

0 10 25 50 100 250

Concentration of 3,4-DHPEA, !M

N°

of

ce

ll,

%

Necrotic

Late apoptosis

Early apoptosis

Viable

Effect of 3,4-DHPEA on apoptosisof HL60 cells

3,4-DHPEA, µM

0 10 25 50 100 250 M

500 bp

800 bp

3000 bp

2000 bp

1500 bp

1030 bp

Are the effects of 3,4-DHPEA due to a pro-oxidantactivity? The effect of N-acetyl-cysteine (NAC) on

apoptosis

0

20

40

60

0 50 100

3,4-DHPEA concentration, !M

% a

po

pto

tic

ce

lls

NAC 0mM

NAC 5mM

NAC 10mM

** ***

*

Are the effects of 3,4-DHPEA due to a pro-oxidantactivity? The effect of N-acetyl-cysteine (NAC) on

proliferation

0.0

0.5

1.0

1.5

2.0

0 24 48 72 96 120 144 168

Incubation time, h

N°

of

ce

lls

, 1

06/m

l

Control

3,4-DHPEA

NAC

3,4-DHPEA + NAC

Effect of 3,4-DHPEA on the differentiation ofHL60 cells

0,0

0,5

1,0

1,5

2,0

0 50 75 100 DMSO

(1.3%)

ATRA

(1!M)

3,4-DHPEA, !M

NB

T r

ed

uc

tio

n,

O.D

./1

06 v

iab

le c

ell

s

*

**

***

***

***

Control 3,4-DHPEA, 75 µM DMSO 1.3 %3,4-DHPEA, 100µM

Effect of 3,4-DHPEA on cycle phasedistribution of HL60 cells

6.0 ± 1.76.0 ± 1.7bb14.9 ± 2.214.9 ± 2.2bb79.1 ± 3.979.1 ± 3.9bb100100

8.4 ± 1.58.4 ± 1.5bb14.3 ± 1.614.3 ± 1.6bb77.23 ± 0.277.23 ± 0.2bb5050

11.2 ± 4.211.2 ± 4.2bb18.7 ± 1.318.7 ± 1.3bb70.1 ± 2.970.1 ± 2.9bb2525

18.0 ± 2.218.0 ± 2.2aa24.2 ± 2.024.2 ± 2.0aa57.8 ± 4.357.8 ± 4.3aa1010

19.9 ± 1.019.9 ± 1.0aa29.8 ± 2.529.8 ± 2.5aa50.3 ± 3.550.3 ± 3.5aa00

GG22/M/MSSGG00/G/G11

% Cell population% Cell population3,4-DHPEA3,4-DHPEAµMµM

0

50

100

150

200

0 100 200 300 400 500

DNA content (FL2)C

ell

nu

mb

er

Control

3,4-DHPEA 50 !MG0/G1G0/G1

SSG2/MG2/M

Phenol composition of the olive oil extract

0 5 10 15 20 25 30 35 40 45

mAU

350

300

250

200

150

100

50

0

Retention time, min.

1 2 3

4

5

6

7 9

8

0.890.892020ppHPEAHPEA-EDA-EDA

0.0690.0690.70.7ppHPEAHPEA

12.512.5311311Total 3,4-DHPEATotal 3,4-DHPEAcontaining compoundscontaining compounds

3.943.941101103,4-DHPEA-EA3,4-DHPEA-EA

8.4658.4652002003,4-DHPEA-EDA3,4-DHPEA-EDA

0.0950.095113,4-DHPEA3,4-DHPEA

µM, cultureµM, culturemediummedium

µg/mgµg/mgextractextract

[1] 3,4-DHPEA; [2] p-HPEA; [3] vanillic acid; [4] 3,4-DHPEA-EDA; [5] p-HPEA-EDA; [6] (+)-1-acetossipinoresinolo; [7] 3,4-DHPEA-EA; [8] ligstroside aglicone; [9] apigenina aglicone.

The Phenol Extract (PE) inhibits theproliferation and induces apoptosis on HL60

cells

0.0

0.1

0.2

0.3

0.4

0.5

0 3.125 6.25 12.5 25

PE concentration, !M

MT

T r

ed

uc

tio

n (

56

5n

m)

aa

b

c

d

0

25

50

75

100

0 3.125 6.25 12.5 25

PE concentration, !mol/LA

po

pto

tic c

ells, %

Fluorescence microscopy

Flow cytometry

d

c,dc,d

c,d

b,c

c,d

b

b

a

a

PROLIFERATION APOPTOSIS

The Phenol Extract (PE) blocks the cell cycleand induces differentiation on HL60 cells

0

25

50

75

100

G0/G1 S G2/M

N°

of

cells, %

0 !mol/L

3.125 !mol/L

6.25 !mol/L

12.5 !mol/L

25 !mol/L

a,c

a,c

a,cb,c

b

d,ed

d,ee

d,e d,ed,e d,e

d,e

d,e

0.0

0.5

1.0

1.5

2.0

control 6.25 9 12.5 DMSO

1.25%

PE concentration, !mol/L

Dif

fere

nti

ati

on

, O

.D.

715n

m/1

06 c

ell

s

24h

48h

72h

c

b,c

c

b

c

b,c

b

c

b,d

a

c,dc,d

c,d

cc

CELL CYCLE DIFFERENTIATION

Regulation of the cell cycle

I. Cyclins (D, E, A, B)

II. Cyclin-Dependent protein Kinases (CDKs)

III. CDK inhibitors (p15, p21, p27)

Regulation of transition from G1 to S phase

Rb: Retinoblastoma protein

Effect of 3,4-DHPEA on the expression ofCDK6 and cyclins

0

50

100

150

200

CyD3

3,4-DHPEA, µM

0 25 50 75 100 0 25 50 75 100

3,4-DHPEA, µM

0

50

100

150

200

CyE

0

50

100

150

CyA

0

50

100

150

CyB1

0

50

100

150

CDK6CDK6

Cyclin D

Cyclin E

Cyclin A

Cyclin B

Effect of 3,4-DHPEA on the expression of cellcycle inhibitors

3,4-DHPEA, µM 0 25 50 75 100

0

100

200

p27

p21

p27

β-actin

0 25 50 75 100

3,4-DHPEA, µM

0

100

200

p21

0

50

100

150

p15

p21

p27

p15

p21

p27

p15

mRNA expression

Effect of 3,4-DHPEA on the phosphorylationstate of Retinoblastoma Protein

3,4-DHPEA, µM

0 50 100

Total Rb

Phosphorylation at serine 780

Phosphorylation at serine 795

Phosphorylation at serine 807 and 811

=

=

CONCLUSIONS

Phenols may be responsible of the anti-Phenols may be responsible of the anti-carcinogenic effects of olive oilcarcinogenic effects of olive oil

Phenols are able to interfere on both initiation andPhenols are able to interfere on both initiation andpromotion/progression stepspromotion/progression steps

Phenols are able to alter the expression of somePhenols are able to alter the expression of somegenes involved in the regulation of cell cyclegenes involved in the regulation of cell cycle

The effects could be mediated by the anti-/pro-The effects could be mediated by the anti-/pro-oxidant properties of these compoundsoxidant properties of these compounds

Mechanisms Involved in the Anticarcinogenic Effects of Olive...

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