Modelos para avaliação
experimental da atividade
anticâncer
I Simpósio Ibero-americano de Investigação em Câncer
24-25 de setembro de 2013
CYTED - RIBECANCER
CPQBA cancer screening
• 1998 – CYTED – NCI – Panamá
▫ Mahabir Gupta, Gordon Cragg
• 1999 - Frederick Cancer Research
and Development Center
In vivo screening - NCI
• 1955 - 1975
▫ Murine Leukemias: L1210 and P388
• 1975 - 1985
▫ 1st stage: P388
(5 to 9 days)
▫ 2nd stage: 4 to 8 in vivo models
(murine and xenotransplant)
▫ formulation / toxicology
▫ clinical studies
• 1955 - 1985
▫ 600.000 tested compounds
In vivo screening
• Advantages
▫ practical, reproducible, low cost (?),
metabolic activation, sensibility
• Disadvantages
▫ time, drug consuming, facilities,
no. of compounds tested, no of animals
In vivo screening - Results
• Discovery of specific drugs for the treatment of
rapid growing tumors (leukemias, lymphomas)
 selective tendency
•  drugs for the treatment of solid tumors
• Results predictability: animals  men
NCI tumor cell lines
Lung
(9) (3)
Colon
(7) (4)
Melanoma
(8) (8)
Prostate
(2) (1)
Breast
(8) (2)
Ovarium
(6) (3)
Kidney
(8) (3)
Brain
(6) (1)
Hematopoietic
(6) (2)
( ) CPQBA
In vitro screening
• Disadvantages
▫ false-positives
▫ drugs solubility
▫ metabolism activation
▫ immunomodulation, angiogenesis,
metastasis
▫ culture cells vs. tumor
In vitro screening
• Advantages
▫ oriented disease screening / specificity (?)
▫ drug amount
▫ costs
▫ space facilities
▫ time / no. of compounds tested
Anticancer activity: human cancer cells
•
•
•
•
•
•
•
•
•
•
Leukaemia (K 562)
Lung (NCI 460)
Breast (MCF 7)
Breast expressing phenotype multiple drugs
resistance (NCI ADR)
Melanoma (UACC 62)
Renal (786 0)
Colon (HT 29)
Ovarian (OVCAR)
Prostate (PCO 3)
U251 (CNS)
Anticancer test
Cells Inoculation
96 wells plates
RPMI +
bovine calf serum
Incubation
24 hours
Drugs addition
Acid fixation
control plate
time zero
Incubation
48 hours
Acid fixation
trichloroacetic
acid
Dye addition
SRB
Dye
solubilization
OD Reading
(540 nm)
Quimioterápicos
Doxorrubicina
Etoposídeo
100
100
75
50
Proliferação celular (%)
Crescimento Celular (%)
75
25
0
U251
UACC-62
MCF-7
NCI-ADR/RES
786-0
NCI-H460
PC-3
OVCAR-03
HT29
K562
VERO
-25
-50
-75
50
25
0
U251
UACC-62
MCF-7
786-0
NCI-H460
PC-3
OVCAR-03
HT29
K562
Vero
-25
-50
-75
-100
-100
-3
10
-2
10
-1
0,025
10
0
0,25
10
Concentração (g/mL)
-3
1
2,5
10
25
10
-2
10
0,025
-1
10
0,25
0
10
Concentração (g/mL)
2,5
1
10
25
Quimioterápicos
Paclitaxel
Vincristina
100
100
75
50
Crescimento Celular (%)
Crescimento Celular (%)
75
25
0
U251
UACC-62
MCF-7
NCI-ADR/RES
786-0
NCI-H460
PC-3
OVCAR-03
HT29
K562
VERO
-25
-50
-75
50
25
0
U251
UACC-62
MCF-7
NCI-ADR/RES
786-0
NCI-H460
PC-3
OVCAR-03
HT29
K562
VERO
-25
-50
-75
-100
-100
-3
10
-2
10
-1
0,025
10
0
0,25
10
Concentração (g/mL)
-3
1
2,5
10
25
10
-2
10
-1
0,025
10
0
0,25
10
Concentração (g/mL)
1
2,5
10
25
Quimioterápicos
5-Fluoruracila
100
100
75
75
50
50
Crescimento Celular (%)
Crescimento Celular (%)
Cisplatina
25
0
U251
UACC-62
MCF-7
NCI-ADR/RES
786-0
NCI-H460
PC-3
OVCAR-03
HT29
K562
VERO
-25
-50
-75
25
0
U251
UACC-62
MCF-7
NCI-ADR/RES
786-0
NCI-H460
PC-3
OVCAR-03
HT29
K562
VERO
-25
-50
-75
-100
-100
-3
10
-2
10
-1
0,025
10
0
0,25
10
Concentração (g/mL)
-3
1
2,5
10
25
10
0,025
10
1
0
-1
-2
10
0,25
10
Concentração (g/mL)
2,5
10
25
CPQBA – cancer research
• Collection and identification of plants
• Extractions with different solvents
• Anti-cancer screening of crude extracts
• Anti-cancer screening of synthetic compounds
• Anti-cancer studies with animal models (in vivo)
• Isolation and identification of active principles,
monitored by biological assays
• Flow cytometry
Plant acquisition and identification
Grinded plant material
dichloromethane
Organic extract
Plant residue
ethanol 70%
Anti-cancer tests
Isolation and identification
of active principles
Ethanolic
extract
Active products
07/72 dcm
07/81 dcm
100
100
75
Porcentagem de Crescimento
Porcentagem de Crescimento
75
50
25
0
-25
UACC62
MCF7
NCIADR
786O
NCI460
PCO3
OVCAR03
HT29
K562
-50
-75
-100
50
25
0
-25
UACC62
MCF7
NCIADR
786O
NCI460
PCO3
OVCAR03
HT29
K562
-50
-75
-100
-3
10
-2
10
-1
0
10
1
10
0
2,5
0,25
2
10
10
25
3
250
10
-3
-2
10
-1
10
75
75
Porcentagem de Crescimento
Porcentagem de Crescimento
100
50
25
0
UACC.62
MCF.7
NCI.460
K.562
OVCAR
PC0.3
HT.29
786
NCI.ADR
-3
0
-2
10
10
-1
0
10
10
2,5
10
25
0
UACC.62
MCF.7
NCI.460
K.562
OVCAR
PC0.3
HT.29
786
NCI.ADR
-25
-50
-75
1
10
2
3
-100
10
-3
0,25
Concentração (g/mL)
3
250
50
-100
10
2
10
25
Ensaio 060828- cod37dicloro
100
-75
1
10
2,5
Concentração (g/mL)
Ensaio 060918- cod 53 CH2Cl2
-50
10
0,25
Concentração (g/mL)
-25
0
10
0
25
250
10
0
-2
10
10
-1
0,25
0
10
10
2,5
Concentração (g/mL)
1
10
25
2
3
10
250
Inoculation 0,5 mL 104 cells of Ehrlich
Tumor
Intraperitoneal injection
Ehrlich ascitic
tumor in mice
4th, 11th and 18th day: treatments: negative
control: saline; positive control:
doxorrubicin 5 mg/ kg and EBD in doses
100, 200 and 400 mg/ kg
Post-inoculation
n=10
Number of deaths and day of
death: ANOVA and KaplanMeyer Survival Curve
Ehrlich ascitic tumor in mice: survival curve
Ehrlich solid tumor in mice: paw tumor
• Ehrlich cancer cells are injected in the hind paw
• The evaluation is done by paw volume determination
• Relative weight of the tumor
▫ tumor weight / body weight
• Histological analysis
• Weight gain of the animals
Pletismometer: paw tumor
Ehrlich solid tumor in mice: paw tumor
Veiculo
5FU 10 mg/kg
rac-GTN 30 mg/kg
rac-GTN 100 mg/kg
rac-GTN 300 mg/kg
0,12
Volume tumoral (mL)
0,10
0,08
0,06
**
*
***
***
***
*
*
*
*
0,04
0,02
**
**
***
***
***
***
***
0,00
0
4
7
Dias
10
13
15
Melanoma B16: lung metastasis
concentração de melanina/massa pulmão
9
veículo
rac-GTN 100mg/kg
8
7
6
5
4
3
2
1
0
*
Psidium guajava L. Ehrlich paw tumor
Saline
Doxorrubicin (5mg/Kg)
P. guajava (10mg/Kg)
P. guajava (30mg/Kg)
P. guajava (50mg/Kg)
0.35
0.30
Tumor volume (ml)
0.25
*
0.20
0.15
0.10
#
#
*
*
15
18
*
0.05
*
0.00
3
6
9
12
21
Treatment (days)
*
uterus relative weight
control
0.012
0.011
0.010
Doxo
Uterus weight (%)
0.009
*
Saline
Doxorrubicin (5mg/kg)
P. guajava (10mg/kg)
*
P. guajava (30mg/kg)
*
P. guajava (50mg/kg)
0.008
0.007
0.006
0.005
*
0.004
0.003
0.002
P. guajava
50 mg/kg
0.001
0.000
Salina
Doxo
PG 10
Groups
PG 30
PG 50
Hollow Fiber
Cancer cells in hollow fibers: abdominal
and dorsal implants
Hollow-Fiber model – (Fr7)
MCF7
NCIADR
OVCAR
1,2
1,0
Absorbance
0,8
0,6
**
**
**
0,4
**
0,2
0,0
Salina
Doxorrubicina
Fr7
Hollow-fiber: abdominal implant
Hollow-Fiber model – (Fr7)
1,4
MCF7
NCIADR
OVCAR
1,2
Absorbance
1,0
**
0,8
**
0,6
0,4
0,2
0,0
Salina
Doxorrubicina
Fr7
Hollow-fiber : dorsal subcutaneous implant
Hanahan and Weinberg. Hallmarks of cancer: the next generation. Cell:144 (5), 646-74, 2011
Targeted screening
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growth factors
signaling molecules
cell-cycle proteins
modulators of apoptosis
molecules that promoted angiogenesis
anti-inflammatory drugs
Malignant growth and normal
growth are so genetically
intertwined that unbraiding the two
might be one of the most significant
scientific challenges faced by our
species.
Crescimento maligno e crescimento normal são tão entrelaçados geneticamente
que separá-los pode ser o desafio científico mais importante que nossa espécie
tem diante de si.
The Emperor of All Maladies: A Biography of Cancer
Siddhartha Mukherjee
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Adriana Della Torre
Ana Lúcia T. G. Ruiz
Ana Possenti
Débora B. Vendramini Costa
Fabiana R. Nonato
Gabriela Marchetti
Giovanna B. Longato
Giovanna Fiorito
Humberto M. Spindola
Jéssica Giroto
Karin Maia Monteiro
Larissa Shiozawa
Lucas Lopes
Mariana Cecchetto
Mary Ann Foglio
Michelle Pedroza Jorge
Paula A. Monteiro
Paula P. de Paiva
Rafael Rosolen T. Zafred
Sirlene Valério Tinti
Vanessa Helena S. Souza
Gracias
Obrigado
Thank you
João Ernesto de Carvalho
carvalho@cpqba.unicamp.br