MECCANISMI DELLA CANCEROGENESI

Pancreatic

Skin

Breast

SOLID CANCERS

Lung

Prostate

Brain

LIQUID CANCERS
Leukemia Hodgkins Non-Hodgkins

Multistadi / field cancerization

CRESCITA DELLA MASSA NEOPLASTICA Divisioni cellulari No. Di cellule Peso (g)

PROCESSO DI CANCEROGENESI
TOSSICOCINETICA E METABOLISMO DANNO E RIPARO DEL DNA INIZIAZIONE (giorni - settimane)

Dose espositiva Dose farmacologica Dose cellulare Dose bersaglio Dose molecolare

STRATEGIE DI INTERVENTO

0 5 10 15 20 25 30 35 40

10 0 10 1 10 2 10 3 10
4

10-9 10-8 10-7 10-6 10

-5

Ambiente e stile di vita

PREVENZIONE PRIMARIA

PROMOZIONE (anni - decenni)

10 5 10 6 10 7 10 10
8 9

10-4 10-3 10-2 10 10 10 10 10

-1 0 1 TUMORE BENIGNO

Organismo ospite (chemio prevenzione)

PROGRESSIONE (~ 1 anno)
CANCRO

PREVENZIONE SECONDARIA

C H E M I O P R E V E N Z I O N E

1010 1011 10 12

INVASIONE
2 3

METASTASI

TERAPIA, RIABILITAZIONE e PREVENZIONE TERZIARIA

MASSA NEOPLASTICA

S. De Flora et al., Mutat. Res. 480481, 922, 2001

CELLULE EPITELIALI MAMMARIE UMANE DI ORIGINE STAMINALE

Cellule M13SV1 Cellule M13SV1R2 Cellule M13SV1R2N1

Espressione di Cx43

Controlli / DMBA

Cellule apoptotiche (%)

Controlli NAC DMBA DMBA + NAC 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10

Addotti al DNA/ 108 nucleotidi

Controlli NAC DMBA DMBA + NAC 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

8-oxo-dG/ 105 nucleotidi

Controlli NAC DMBA DMBA + NAC .0 .1 .2 .3 .4 .5 .0 .1 .2 .3 .4 .5 .0 .1 .2 .3 .4 .5

Differenze significative DMBA vs. controlli S. De Flora et al., Int. J. Oncol., 29, 521529, 2006

CELLULE EPITELIALI MAMMARIE UMANE DI ORIGINE STAMINALE

Cellule M13SV1 Cellule M13SV1R2 Cellule M13SV1R2N1

Espressione di Cx43

Controlli / DMBA

Cellule apoptotiche (%)

Controlli NAC DMBA DMBA + NAC 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10

Addotti al DNA/ 108 nucleotidi

Controlli NAC DMBA DMBA + NAC 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

8-oxo-dG/ 105 nucleotidi

Controlli NAC DMBA DMBA + NAC .0 .1 .2 .3 .4 .5 .0 .1 .2 .3 .4 .5 .0 .1 .2 .3 .4 .5

Differenze significative DMBA vs. controlli S. De Flora et al., Int. J. Oncol., 29, 521529, 2006

INIZIAZIONE DEL CANCRO

Tossicocinetica e metabolismo Danno genotossico Riparazione del DNA

METABOLISMO DEGLI XENOBIOTICI

ATTIVITA METABOLICHE DI FASE I E DI FASE II E LORO POLIMORFISMI GENETICI

Phase I
Epoxide hydroxylase DPD oxidases others NQO1 CYP1A1/2 CYP1B1 CYP2A6 CYP2B6 CYP2C8 others CYP2C9 ALDH ADH CYP2C19 UGTS STS

Phase II
NAT2 NAT1 GST-M GST-T GST-P GST-A

CYP2O6 CYP3A4/5/7

HMT CYP2E1 TPWT COWT

Drug-metabolizing enzymes known to exhibit common polymorphisms at the genomic level that have been associated with changes in drug effects; however, almost every gene involved in the drug metabolism is subject to common genetic polymorphisms that may contribute to interindividual variability in drug response and drug toxicity.

Attivazione metabolica del benzo[a]pirene

12 1 2 3 4 7 6 5

Bay region
10 9 8

11

Phenols and quinones

Benzo[a]pyrene

K region

Mono- oxygenase
O

Simple epoxides
O 9,10Oxide 7,8Oxide 4,5Oxide O

Epoxide hydratase
OH HO HO 9,10Dihydrodiol OH 7,8Dihydrodiol OH 4,5Dihydrodiol OH

Dihydrodiols

Mono-oxygenase
OH HO HO O 9,10Diol7,8oxide OH 7,8Diol9,10oxide O

Diol- epoxides

CICLO DEL GLUTATIONE

GLUCOSO GLUCOSE

NADPH

GSSG
GSSG

2H2 O 2

G6P

G6PD

6PGD 6PG

REDUTTASI REDUCTASE

GSH PEROSSIDASI PEROXIDASE

NADP + H+

2 GSH

O2 H2 2 2

GSH = Glutatione ridotto GST = Glutatione S-trasferasi GSTM1 GSTT1

GSTM1 GENOTYPE STATUS AND DNA ADDUCT LEVELS

IN SMC OF HUMAN ATHEROSCLEROTIC LESIONS
32P

postlabelled DNA adducts Spot 1 Spot 2 Spot 3 Spot 4 Spot 5 Spot 6 Spot 7 Spot 8 Spot 9 DRZ Total

GSTM1+ (n = 36) 0.46 0.11 0.23 0.10 0.13 0.26 0.36 0.25 0.64 10.80 13.80 0.63 0.25 0.49 0.18 0.35 0.27 0.48 0.35 1.07 6.16 9.77

GSTM1 (n = 39) 0.58 0.74 0.20 0.28 0.51 0.74* 0.28 0.43 0.28 0.57** 0.46 0.52 0.86 1.23* 0.77 1.40** 2.74 5.97** 16.41 10.63** 22.67 18.63**

Ratio (/+) 1.3 1.8 2.2 2.8 2.2 1.8 2.4 3.1 4.3 1.5 1.6

*P < 0.05, **P < 0.01

A. Izzotti et al., FASEB J. 15, 752-757, 2001

METABOLIC DEACTIVATION OF MUTAGENS

S. De Flora, Nature 271, 455-456, 1978

METABOLIC DEACTIVATION OF MUTAGENS

S. De Flora, Nature 271, 455-456, 1978

CHROMIUM(VI) REDUCING CAPACITY OF ORGANS, CELL POPULATIONS AND FLUIDS IN THE HUMAN DIGESTIVE SYSTEM
SALIVA [0.7 2.1 mg Cr(VI)/day]

BRONCHIAL TREE CELLS

PERIPHERAL LUNG PARENCHYMA CELLS [260 mg Cr(VI)]

ELF [0.9 1.8 mg Cr(VI)] PAM [136 mg Cr(VI)] BLOOD [187 234 mg Cr(VI)] LIVER CELLS [3,300 mg Cr(VI)]

PORTAL SYSTEM BLOOD [187 - 234 mg Cr(VI)]

GASTRIC JUICE [> 84 88 mg Cr(VI)/day]

INTESTINAL BACTERIA [11 24 mg Cr(VI) eliminated daily with bacteria in feces]

De Flora et al., Carcinogenesis 18, 531537, 1997

THRESHOLDS IN CARCINOGENESIS?
CARCINOGENESIS PROCESS
TOXICOKINETIKS AND METABOLISM DNA DAMAGE AND REPAIR INITIATION (days - weeks)

Exposure dose Pharmacologic dose Cellular dose Target dose Molecular dose

DNA repair

Apoptosis

PROMOTION (years - decades)

BENIGN TUMOR

PROGRESSION (~ 1 year)
CANCER

INVASION

METASTASIS

NEOPLASTIC MASS

INIZIAZIONE DEL CANCRO

Tossicocinetica e metabolismo Danno genotossico Riparazione del DNA

CARIOTIPO UMANO

CORRELAZIONE FRA
C

MUTAGENICITA E CANCEROGENICITA
Ames reversion test NG
Accuracy = 74.7% (68/91)

Sensitivity 76.8% (53/69)

23.2% (16/69) G 31.8% (7/22) 78.2% (15/22) Specificity NC

Sensitivity 79.3% (46/58)

20.7% (12/58)

DNA-repair test
Accuracy = 73.7% (59/80)

NG 40.9% (9/22)

59.1% (13/22) Specificity NC

EFFETTI PATOLOGICI DELLE MUTAZIONI NELLUOMO MUTAZIONI IN CELLULE SOMATICHE (in et adulta o in epoca embrionale)

Morte cellulare (apoptosi)

Tumori e altre malattie cronico-degenerative

Invecchiamento e patologia associata

EFFETTI PATOLOGICI DELLE MUTAZIONI NELLUOMO MUTAZIONI IN CELLULE GERMINALI

Sterilit, aborto, morte perinatale (mutazioni dominanti letali) Malattie genetiche nei neonati (mutazioni dominanti non letali) Malattie genetiche in generazioni successive (mutazioni recessive)

AGEING
mtDNA

ADDOTTI AL DNA POLMONARE IN RATTI ESPOSTI A FUMO DI SIGARETTA mtDNA NAC

nDNA

NAC +

R. Balansky et al., Cancer Res. 56, 1642-1647, 1996

INIZIAZIONE DEL CANCRO

Tossicocinetica e metabolismo Danno genotossico Riparazione del DNA

RIPARAZIONE DEL DNA

XP AT AF Suscettibilit individuale

UV Light
R hH B 23

DNA Repair Mechanisms

SS
OH

TF IIH
C S B

X PX CP
A
C S A

NER

Br X- eak b Ra ys y P

G1
ellll C Ce c yc C Cy e lle

PN K

S
RNA Pol UV & Bulky Adducts
TF IIH

Trans Trans cripti cripti on on Coupl Coupl ededNER NER

X P D

XA B2
X P B

X P G

DNA-Damage Checkpoint

Ra d9 Ra d17 Mec 1 Rad 24 p 2 1 NB S1

G2

Estro Estro gen gen

R P A

ERC C1

NER

X P F

B DS

Ku70 /80

BRC A1
IG F1

Ra d51 Ra d51 BRC A1 FA NCs

Ra d52

UV Light Ionizing Radiation, Bulky Adducts & ROS

B DS

D DN NA A-D Da am m a ag ge e C Ch he ec k kp c po oiin n tt

BAR D1

BRC A2 P
P

om H

XRC tion C4 ina Li mb gI Reco V us go olo

DN NHE AJ PKc s Ra d50

M
p 5 3 Chk 2 Chk 1

70 Ku 0 /8

MR E11

AT M AT R Ra Ra d9 d1 XRC C1 PA RP Li Po gII I l- Depurination & Deamination

S Sm m o ok kii n ng g

C P BR A1

sos Cr king Lin

X-Rays & Anti-Tumor Agents

JNK/SA PK
D45 GAD

Apoptosis

ir pa Re

PC NA

ads NA Lo -P n to D RF o NA PC

Lig ase

PC NA

xo O

Abasic Site

B B E E R R

Po l- SN M1

/
DN Po A l

Pol-

s reak SS-B Rays X by

Hu s1

U b U U b bU b

MD M2 p 5 3

Recom Recom binatio binatio nal nal Repair Repair

a ge IR Dam

PC Proo f NA Readin g

FE N1

Proteas ome

BER

Cell Proliferation
M S H 6

MS H2

Endogenous Agents (Water & ROS) MMR

of lation methy n & De o ti la e ty Deace Guanin

Repli catio n E (DNA rrors Pol & IDLs )

hOG G1
TRE X1
E TR X2

AP E1

p53 p53 Degrada Degrada tion tion

MM R

MS H2

MS H3

M M u u tt S S

M M u u tt S S

MG MT Oxygen Radicals Alkylating Agents Spontaneous Reactions

G
R BE
hN TH

Me

5
U BER

3
MB D4

UN G2 SMU G1

Nucleus
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PROMOZIONE DEI TUMORI

PROMOZIONE DEL CANCRO

Promotori fisici / meccanici Promotori biologici Promotori chimici

PROMOZIONE DEL CANCRO

Promotori fisici / meccanici Promotori biologici Promotori chimici

SPECIE REATTIVE DELLOSSIGENO O2

e OSSIGENO MOLECOLARE

O2
e

ANIONE SUPEROSSIDO

H2O2
e

IDROGENO PEROSSIDO

OH
e

RADICALE IDROSSILE

H2O

SPECIE REATTIVE DELLOSSIGENO

O2

OSSIGENO MOLECOLARE

Luce coloranti

O2

OSSIGENO SINGOLETTO

PROMOZIONE DEL CANCRO

Promotori fisici / meccanici Promotori biologici Promotori chimici

ORGANOSPECIFICITA DEI PROMOTORI TPA NaCl Saccarina Componenti della bile Estrogeni Fenobarbital Componenti del fumo Cute Stomaco Vescica Colon Mammella Fegato Polmone

CRESCITA DELLA MASSA NEOPLASTICA Divisioni cellulari No. Di cellule Peso (g)

PROCESSO DI CANCEROGENESI
TOSSICOCINETICA E METABOLISMO DANNO E RIPARO DEL DNA INIZIAZIONE (giorni - settimane)

Dose espositiva Dose farmacologica Dose cellulare Dose bersaglio Dose molecolare

STRATEGIE DI INTERVENTO

0 5 10 15 20 25 30 35

10

10

-9

Ambiente e stile di vita

10 1 10 2 10 3 10
4

10-8 10-7 10-6 10

-5

PREVENZIONE PRIMARIA

PROMOZIONE (anni - decenni)

10 5 10 6 10 7 10 10
8 9

10-4 10-3 10-2 10 10 10 10 10

-1 0 1 TUMORE BENIGNO

Organismo ospite (chemio prevenzione)

PROGRESSIONE (~ 1 anno)
CANCRO

PREVENZIONE SECONDARIA

1010 10 40
11

INVASIONE
2 3

METASTASI

TERAPIA e PREVENZIONE TERZIARIA

10 12

MASSA NEOPLASTICA

ANGIOGENESI E ANTIANGIOGENESI

ANGIOGENESI E LINFANGIOGENESI

GENI IMPLICATI IN LINFANGIOGENESI (K. Alitalo et al., Nature, 2006)

VEGF Pathway
PIP2
ell lC lia e h dot En

VEGFA, VEGFC, VEGFD VEGFR2

P P

PI3K PIP3 Src

PLC VRAP Sck

PIP2

IP3 DAG GRB2 MKK3/6 Akt/ PKB p38

P P

SHC FAK

SOS PKC Ca2+

Paxillin
P

Ras Focal Adhesion Turnover ProstaglandinProd uction Raf1

eNOS BAD
P

HSP90 Caspase9

MAPKAPK2/3 MAPKAPK2/3 Cell Migration HSP27 Nitric Oxide Production cPLA ERK1/2 Actin Actin Reorganization Reorganization Gene Expression & Cell Proliferation ANGIOGENESIS Vascular Cell Permeability

MEK1/2

Cell Survival

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REGOLAZIONE GENETICA DELLA PROLIFERAZIONE CELLULARE

IL CICLO CELLULARE

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Replicative Replicative Senescence Senescence

TGF

Cyclins and Cell Cycle Regulation

C Co on nt ta ac IIn ct t nh hiib biit tiio on n

r tto or c c a ll a F a w tth h Fra wa w a ow hd ro dr G Gr Wiitth W

p19(INK4D)
GS K3

p16(INK4A) p15(INK4B) p18(INK4C)

CDC25A

P
p ( K 27 IP1 ) C CIIP P //K F KI Fa am P miil IP ly y

ATR

D DN NA AD Da am ma ag ge e

ATM p53 p21 Replicative Replicative Scenescece Scenescece

CDK4/6 CyclinD/D1

K1 CD

Ubiquitination Ubiquitination
P

Cyclin-E CDK2 P P P Rb
1 W ee

G1
Ubiquitination Ubiquitination

Cyc

lin-A

K1 CD

P Rb E2F

HDACs
DP1

P Off Off On On
DP1

P P Cyclin-H

PP2A

Nucleus
Cell Cell Cycle Cycle Progression Progression Cyclin-H
A

MYT1
Wee1

CDC

25A

E 2F

Cyc

B lin-

E2F P
1 We e

Rb P Rb P
lin-A

S-Phase S-Phase Genes Genes [Cyclin-A, [Cyclin-A, E,E2F, E,E2F, CDC2] CDC2]
E2F

P p21

K1 CD

linCyc

CDK7 P P P P

Rb

Rb
Wee

2 CDK

Cyc

p21
lin-A Cyc
p CDK2 (K 27 IP1 P )

CDC25A

K1 CD P P

P Raf1

A C25 CD

p27(KIP1) p27(KIP1) Degradation Degradation

p2 P (KIP 7 1)

G2

MYT1

SCF Ub SCF

Ub

Ub

PROTOONCOGENI

ONCOGENI

TRASLOCAZIONE CROMOSOMICA

MUTAZIONI PUNTIFORMI

AMPLIFICAZIONE GENICA

GENI ONCOSOPPRESSORI

Sindrome di Li-Fraumeni Sindrome di von Hippel-Lindau

Hypoxia Hypoxia

UV UV

Chemotherapy Chemotherapy

Ionizing Ionizing Radiation Radiation

p53 Signaling
F a s
D R 5

p53 p53 Degradation Degradation

JNK

HIPK2

CSNK1

p38

Proteasome Proteasome Hypoxia Induced Hypoxia Induced HIF1 HIF1 Expression Expression PTEN p53 HIF1 P p53 P ATR

DNA DNA Damage Damage Apoptosis Apoptosis ATM P P Chk2 DNA-PK BRCA1 Cell Cell Survival Survival PCAF CAK Ac p53 Ac p53 P P PTEN GSK3 Akt Caspase Bax

Chk1 PIAS1 PML

Ub Ub p53 MDM2

Ub

c-Abl Ac HDAC p53 p300 Sirt p21CIP MDM2 CyclinD1 CDK2 E2F Rb CyclinE CDK4 Cell Cell Cycle Cycle Progression Progression
C

BCL2 Gene Gene Expression Expression DNA DNA Repair Repair TSP1 BAI1 c-Fos GADD45 P Rb Angiogenesis Angiogenesis Inhibition Inhibition

14-3-3

E2F

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Cellular Apoptosis Pathway

C

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Apo2L / Apo3L

TNF TNFR1

G Grro ow wth th F Fa ac cto torr

Growth Factor Receptor

FasL
Fas
D FA e sD aspa c o r P 8

Apo2/ Apo3
T AD R
D FA Procaspase8 D

Withdrawl of Growth Factors

RIP TRAF2

DD Procaspase8

FA

TR A D D

PKC p90RS K

PI3K Akt1 BAD

BID FLIP CIAP1 SMAC Caspase9 Arts APAF 1 HTRA 2 Cytoc Apoptosis EndoG POD POD 4 PML PAR ZIPK Daxx CAD AT M NF-B Chk2 Endo G tBI BCLXLD MMP IKKs IB NF-B
BAX

Caspase8 Caspase3 Caspase7 Apoptosis

BAK

ICAD CAD

Apoptosis

Noxa AIF

PUMA

PARP

DNA Fragmentation Apoptosis Apoptosis

DN AIF Fragm A entati on FLIP,CIAP2,BFL1, FLIP,CIAP2,BFL1, BCL2 BAK,BAK,BID, BCL2 Ras,Noxa,PUMA, p53 APAF1,Survivin, BCL2 MDM2 p53

Mit oc h dr i on a

V D A PMR ANT C CypD

BCL2
BIM

1 AI P 3 p5
Depolarization Oncogenic Signal p14(ARF)

LOSS OF FHIT EXPRESSION IN SPRAGUE-DAWLEY RATS EXPOSED TO CIGARETTE SMOKE FOR 28 DAYS
1.4 0.74% 4.9 1.96%
P < 0.001 compared to SHAM

3.1 1.7%
P < 0.05 compared to ECS

Bronchial epithelium

1.1 0.64%

1.4 0.52%

1.3 0.71%

Kidney tubular epithelium

SHAM

ECS

ECS + NAC

F. DAgostini et al., Cancer Res. 66, 393641, 2006

TUMORI DELLA MAMMELLA O DELLOVAIO

Colorectal carcinogenesis: adenomacarcinoma sequence. (Adapted from Kelloff GJ, Hawk ET, Crowell J, et al: Oncology 10(10):147184, 1996 and Ilyas M, Straub J, Tomlinson IP, et al: Eur J Cancer 35:33551, 1999 and reprinted with permission (131, 132).