CARCINOGENESIS PROCESS

INITIATOR
CHEMICAL
CARCINOGEN ANIMAL’S MODEL
PHYSICAL (UV, X-RAY)
VIRAL GENETIC
SPESIFIC ANIMAL LABORATORY
SEX / GENDER
AGE
PROMOTOR ORGAN SPESIFIC
SEASON
DIET (HIGH FAT DIET)
IMMUNE SUPPRESSION
OXYDATIVE STRESS
HORMONE
CANCER CAUSED FACTOR
CHECK POINT REGULATION
CHEMICAL CARCINOGENS
Model of chemical carcinogenesis
CHEMICAL CARCINOGENS
INITIATION AND PROMOTION PHASE OF
CARCINOGENESIS IN MICE
 BREAST CANCER MODEL
DEPEND ON

CHEMICAL CARCINOGEN
PAH (DIMETHYLBENZ(A)ANTRASENE)
N-METHYL-N-NITROSOUREA (NMU).

SPECIFIC ORGAN
MAMMARY GLAND

SPECIFIC ENZYME
CYP1A1 AND GSTµ
(Lewis et al., 1998).
(Lewis et al., 1998)
 Flavonoid

Menghambat
CYP1A1

Induksi GST

Russo, et al., 2005

KEAP1 : Kelch-like ECH-associated protein 1

Nrf2 : NF-E2 p45-related factor

TUMOR SUPPRESSION GEN REGULATOR
Phytochemicals
Target Surh, 2003
 RAT STRAIN
Mammary cancer in the rat best models
human breast cancer. The induced rat carcinoma
recapitulates the same histopathologic progression
stages to malignant breast cancer seen in women
(Laurie A. Shepel, 1998).

The period after postnatal day 40-50, is the

factor significantly increasing incidence and
frequency of mammary carcinogenesis in the
Sprague-Dawley strain (Kubatka, 2002).
AGE / SEX
High susceptibility of female rats can be
observed in carcinogen application between postnatal
days 40-60 in early puberty with highly proliferating
terminal end buds (TEBs) in the mammary gland
(Huggins et al. 1999).

SEASON
A seasonal variability in the incidence of DMBA-
induced mammary carcinogenesis in female Sprague-
Dawley rats: autumn values (34 %) differed from those
in the spring (62 %). Nelson and Blom (1994) supposed
that both people and animals increase the adjustment.
of the immune system during the short day, thus
helping in the protection against. winter. stress factors.
 DOSE and APLICATION

1ST TRIAL
-
Inisiator : DMBA
30 mg/kgBB, ig Promotor
+ corn oil Estradiol 0,018 mg/kgBB Po
2 x week interval for 30 days

4 MONTHS
RESULT
NO TUMORS
NO MAMMARY
- Macroscopic /palpasion Analysis:
EPITHELIAL Toumor Incidency and Multiplicity
PROLIFERATION Microscopic Analysis :
AND HYPERPLASIA
Histopatology Mammary Gland
 DOSE and APLICATION

2ND TRIAL

Inisiator : DMBA
10 mg/kgBB, ig Promotor
+ corn oil Estradiol 0,018 mg/kgBB Po
3 x week interval for 30 days

4 MONTHS
RESULT
NO TUMORS
NO MAMMARY
- Macroscopic /palpasion Analysis:
EPITHELIAL Toumor Incidency and Multiplicity
PROLIFERATION Microscopic Analysis :
AND HYPERPLASIA
Histopatology Mammary Gland
 DOSE and APLICATION

3TH TRIAL

Inisiator : DMBA
20 mg/kgBB, ig
+ corn oil 4 MONTHS
10x, 2x / weeks

RESULT Macroscopic /palpasion Analysis:

TUMORS Toumor Incidency and Multiplicity
MAMMARY
- Microscopic Analysis :
EPITHELIAL
PROLIFERATION AND
Histopatology Mammary Gland
HYPERPLASIA
Mammary Gland Morphology
 C
A

B D

A & B: Negative Control, No Growth Tumor Nodule

C & D: Positive Control, Growth Tumor Nodule (Arrow Sign)
NORMAL EPITHEL HYPERPLASIA EPITHEL

NORMAL OVER ACTIVITY

PROLIFERATION PROLIFERATION
 LUNG CANCER MODEL
 Asbestos : exposure from asbestos industry Mesothelioma
 Cygarette Smoke : Smoking exposure Carcinoma

 Coke oven workers have among the highest exposure to coal-tar-

pitch volatiles rich in PAHs and particularly Benzo[a]-pyrene
(B[a]P). SCC, SCLC and NSCLC

Benzo[a]pyrene (BaP), a prototypical environmental PAH, present

in cigarette smoke, charbroiled meat and industrial waste by-
products, is mainly metabolized by human cytochrome (CYP)
1A1/1B1 and epoxide hydrolase to carcinogenic BaP 7,8-diol-9,10-
epoxides, which covalently binds to cellular DNA to start the
carcinogenic process.

In contrast, some detoxification enzymes, such as glutathione S-

transferases (GSTs) and UDP-glucuronosyltransferases (UGTs).
After AhR translocation, including binding to the aryl
hydrocarbon nuclear translocator (ARNT), the CYP1A1
gene is transcriptionally activated.
7,8-dihydrodiol-9,10 epoxide
B(a)P 7,8-dihydrodiol

epoxide
hydrolase monooxygenase N-2 dan O6 guanin,
N-6 adenin
carbonium ion
Carsinogen Proximate Ultimate DNA adduct
carcinogen Carsinogen

Repair Mutation

Detoxycation
GST, GSH Normal Death Cancer
 DOSE and APLICATION

Inisiator : Benzo(a)pyrene
New Born Mice
0,2 ; 0,4 ; 0,4 µmol
Intraperitoneal
+ DMSO Inj. Volume 20 µl
(1st, 8th, 15th days)
4th Month

RESULT Macroscopic /palpasion Analysis:

TUMORS NODULE Toumor Incidency and Multiplicity
EPITHELIAL
- Microscopic Analysis :
PROLIFERATION AND
HYPERPLASIA
Histopatology Mammary Gland

Kapitulnik, et al., 1977, 1978

 Control group DMSO, No growth lung nodule tumor

Control B[a]P group, growth lung nodule tumor

(see arrow)
 b
c

Terlihat bahwa pada kelompok Sel epitel bronkus yang normal

kontrol B[a]P (perbesaran 40X ditunjukkan dengan selapis sel
objektif) terdapat fokus tumor (c) yang tidak ditemukan
pada daerah septa alveoli (a). Sel adanya aktivitas mitotik yang
epitel bronkiolus terdiri dari berlebihan seperti pada
berlapis sel yang mengarah pada kelompok kontrol DMSO
lumen (huruf b, ditunjukkan (perbesaran 40X objektif).
tanda panah)
 A B

Gambar di atas menunjukkan bagian bronkiolus sel paru hewan uji

kelompok kontrol B[a]P (A) dan kontrol DMSO (E) dengan
perbesaran 100x objektif.
ANGIOGENESIS
  
1. Diseased or injured tissues produce and release angiogenic
growth factors.

2. The angiogenic growth factors bind to specific receptors

located on the endothelial cells (EC) of nearby preexisting
blood vessels.

3. Once growth factors bind to their receptors, the endothelial

cells become activated. Signals are sent from the cell's surface
to the nucleus. The endothelial cell's machinery begins to
produce new molecules including enzymes.

4. Enzymes dissolve tiny holes in the basement membrane

surrounding all existing blood vessels.

5. The endothelial cells begin to divide, and they migrate out

through the dissolved holes of the existing vessel
6. Specialized molecules called adhesion molecules, or integrins
(avb3, avb5) serve as grappling hooks to help pull the new
blood vessel sprout forward.

7. Additional enzymes (matrix metalloproteinases, or MMP) are

produced to dissolve the tissue in front of the sprouting vessel
tip in order to accommodate it. As the vessel extends, the
tissue is remolded around the vessel.

8. Sprouting endothelial cells roll up to form a blood vessel tube.

9. Individual blood vessel tubes connect to form blood vessel

loops that can circulate blood.

10. Finally, newly formed blood vessel tubes are stabilized by

specialized muscle cells (smooth muscle cells, pericytes) that
provide structural support.
 Chemical stimulation of angiogenesis is performed
by various angiogenic proteins, including several growth factors.
Stimulator Mechanism
Promotes proliferation & differentiation of
FGF endothelial cells, smooth muscle cells, and
fibroblasts
VEGF Affects permeability
VEGFR and NRP-1 Integrate survival signals
Ang1 and Tie2 Stabilize vessels
PDGF (BB-homodimer) and PDGFR recruit smooth muscle cells
TGF-β, endoglin and TGF-β receptors ↑extracellular matrix production
MCP-1
Integrins αVβ3, αVβ5 (?[9]) and α5β1 Bind matrix macromolecules and proteinases

VE-cadherin and CD31 endothelial junctional molecules

ephrin Determine formation of arteries or veins
remodels extracellular matrix, releases and
plasminogen activators
activates growth factors
plasminogen activator inhibitor-1 stabilizes nearby vessels
NOS and COX-2
AC133 regulates angioblast differentiation
Id1/Id3 Regulates endothalial transdifferentiation
The fibroblast growth factor (FGF) family with its prototype
members FGF-1 (acidic FGF) and FGF-2 (basic FGF)
consists to date of at least 22 known members. Most are 16-18
kDa single chain peptides.

aFGF (FGF-1) and bFGF (FGF-2) are important players

in wound healing. They stimulate the proliferation of
fibroblasts and endothelial cells that give rise to
angiogenesis and developing granulation tissue, both
increase blood supply and fill up a wound space/cavity
early in the wound healing process.

FGF-2 is a more potent angiogenic factor than VEGF or

PDGF (platelet-derived growth factor), however, less potent
than FGF-1.
 ANGIOGENESIS
MODEL
The principal methods now in use:
The in vivo Matrigel plug and corneal neovascularization assays
The in vivo/in vitro chick chorioallantoic membrane (CAM) assay
The in vitro cellular (proliferation, migration, tube formation)
organotypic (aortic ring) assays.

FGF-2 (bFGF)-induced
angiogenesis in the mouse
cornea (top), and example of the
sponge-Matrigel system (bottom).

Panel A shows the total explant

area. Panel B shows more details
of the outgrowth area, in which
endothelial cells are seen to form
A B tube-like structures.
 ANGIOGENESIS
Membran MODEL
Korio Alantois (CAM)
dapat digunakan sebagai salah satu metode untuk
mempelajari respon angiogenesis, karena
merupakan membran yang sangat kaya dengan
pembuluh darah.
The three most widely used assays are CAM, the
rabbit corneal micropocket and the subcutaneous
implants. The main advantages of the CAM assay
are its low cost, simplicity, reliability, lends itself to
large-scale screening, which are important
determinants of the choise of a method. (Ribatti, at
al., 1997)
 METHOD

bFGF 25 ug
Telur ayam berembrio Pengumpulan & identifikasi
diencerkan dengan
umur 8-9 hari bahan tanaman
Tris HCl 10 mM, pH 7,5

Simplisia kering daun

Dicandling Gynura procumbens diserbuk
bFGF kadar 1 ng/ul
Diekstrak dengan etanol 80%,
Dipindah rongga udaranya
dikeringkan

Dibuat ‘jendela’ pada Dibuat seri konsentrasi

cangkang telur 50, 100, 200 dan 400 ug/ml

Uji induksi angiogenesis Uji penghambatan

dengan bFGF angiogenesis
Induction Angiogenesis Test Inhibition Angiogenesis Test

Paper bFGF bFGF+

bFGF bFGF bFGF bFGF
Disc 30 ng Dilution
10ug 20ug 40ug 80ug
Control Control Control
1st Dose 2nd Dose 3rd Dose 2th Dose
Group Group Group

Inoculated to Embryonic Chicken Egg

Incubated, T 39C, for 3 days

Pada hari ke-3, telur dibuka untuk dilakukan pengamatan

makroskopik dan mikroskopik
 Respon Angiogenesis CAM

N I
O N
R D
M U
A C
L T
E
C D
A
M b
F
G
F
 D C B A

Cheiradone inhibits in vivo angiogenesis in the CAM assay. (A) control,

methylcellulose alone showed no evidence of angiogenesis or inflammation (B)
cheiradone (10 μg) failed to induce blood vessel formation (C) VEGF, a network
of new blood vessels was established (see arrow) at the point of application. (D)
cheiradone (10 μg) + VEGF inhibited vessel formation. There was no evidence of
an inflammatory response in any preparation. Representative
photomicrographs are shown.