Modelos para avaliação experimental da atividade anticâncer
34
Modelos para avaliação experimental da atividade anticâncer I Simpósio Ibero-americano de Investigação em Câncer 24-25 de setembro de 2013 CYTED - RIBECANCER
description
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 - PowerPoint PPT Presentation
Transcript of Modelos para avaliação experimental da atividade anticâncer
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
LungLung (9)(9) (3)(3) ColonColon (7)(7) (4)(4)
MelanomaMelanoma (8)(8) (8)(8) ProstateProstate (2)(2) (1)(1)
BreastBreast (8)(8) (2)(2) OvariumOvarium (6)(6) (3)(3)
KidneyKidney (8)(8) (3)(3) BrainBrain (6)(6) (1)(1)
HematopoieticHematopoietic (6)(6) (2)(2)
( )( ) CPQBACPQBA
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 InoculationCells Inoculation96 wells plates96 wells plates
Cells InoculationCells Inoculation96 wells plates96 wells plates
Drugs additionDrugs additionDrugs additionDrugs additionAcid fixationAcid fixationcontrol platecontrol plate
time zerotime zero
Acid fixationAcid fixationcontrol platecontrol plate
time zerotime zero
Acid fixationAcid fixationtrichloroacetictrichloroacetic
acidacid
Acid fixationAcid fixationtrichloroacetictrichloroacetic
acidacid
Dye additionDye addition
SRBSRB
Dye additionDye addition
SRBSRB
DyeDye solubilizationsolubilization
DyeDye solubilizationsolubilization
OD Reading(540 nm)
OD Reading(540 nm)
IncubationIncubation24 hours24 hours
IncubationIncubation24 hours24 hours
IncubationIncubation48 hours48 hours
IncubationIncubation48 hours48 hours
RPMI +bovine calf serumRPMI +bovine calf serum
Quimioterápicos
Quimioterápicos
Quimioterápicos
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 identificationPlant acquisition and identificationPlant acquisition and identificationPlant acquisition and identification
Grinded plant materialGrinded plant materialGrinded plant materialGrinded plant material
Organic extractOrganic extractOrganic extractOrganic extract Plant residuePlant residuePlant residuePlant residue
EthanolicEthanolicextractextractEthanolicEthanolicextractextract
dichloromethanedichloromethane
ethanol 70%ethanol 70%
Anti-cancer testsAnti-cancer testsAnti-cancer testsAnti-cancer tests
Isolation and identificationIsolation and identificationof active principlesof active principlesIsolation and identificationIsolation and identificationof active principlesof active principles
Active products
10-3 10-2 10-1 100 101 102 103
-100
-75
-50
-25
0
25
50
75
100
Por
cent
agem
de
Cre
scim
ento
Concentração (g/mL)
UACC62 MCF7 NCIADR 786O NCI460 PCO3 OVCAR03 HT29 K562
07/72 dcm
0 0,25 2,5 25 250 10-3 10-2 10-1 100 101 102 103
-100
-75
-50
-25
0
25
50
75
100
Por
cent
agem
de
Cre
scim
ento
Concentração (g/mL)
UACC62 MCF7 NCIADR 786O NCI460 PCO3 OVCAR03 HT29 K562
07/81 dcm
0 0,25 2,5 25 250
10-3 10-2 10-1 100 101 102 103
-100
-75
-50
-25
0
25
50
75
100
Ensaio 060918- cod 53 CH2Cl
2
250252,50,250
UACC.62 MCF.7 NCI.460 K.562 OVCAR PC0.3 HT.29 786 NCI.ADR
Por
cent
agem
de
Cre
scim
ento
Concentração (g/mL)10-3 10-2 10-1 100 101 102 103
-100
-75
-50
-25
0
25
50
75
100
Ensaio 060828- cod37dicloro
250252,50,250
UACC.62 MCF.7 NCI.460 K.562 OVCAR PC0.3 HT.29 786 NCI.ADR
Por
cent
agem
de
Cre
scim
ento
Concentração (g/mL)
Ehrlich ascitic tumor in miceInoculation 0,5 mL 104 cells of Ehrlich
TumorInoculation 0,5 mL 104 cells of Ehrlich
Tumor
Intraperitoneal Intraperitoneal injectioninjection
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
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
Number of deaths and day of death: ANOVA and
Kaplan-Meyer Survival Curve
Number of deaths and day of death: ANOVA and
Kaplan-Meyer Survival Curve
n=10n=10
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
0,00
0,02
0,04
0,06
0,08
0,10
0,12
151310740
*
*
* ******
**
**
***
******
*****
*******
*
Vol
ume
tum
oral
(m
L)
Dias
Veiculo 5FU 10 mg/kg rac-GTN 30 mg/kg rac-GTN 100 mg/kg rac-GTN 300 mg/kg
Melanoma B16: lung metastasis
0
1
2
3
4
5
6
7
8
9
conc
entra
ção
de m
elan
ina/
mas
sa p
ulm
ão
veículo rac-GTN 100mg/kg
*
Psidium guajava L. Ehrlich paw tumor
3 6 9 12 15 18 21
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
#
*
*
Saline Doxorrubicin (5mg/Kg) P. guajava (10mg/Kg) P. guajava (30mg/Kg) P. guajava (50mg/Kg)
Tum
or v
olum
e (m
l)
Treatment (days)
#
*
*
*
*
control
Doxo
P. guajava 50 mg/kg
uterus relative weight
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.010
0.011
0.012
*
PG 30 PG 50PG 10Doxo
Ute
rus
wei
ght (
%)
Groups
Saline
Doxorrubicin (5mg/kg)
P. guajava (10mg/kg)
P. guajava (30mg/kg)
P. guajava (50mg/kg)
Salina
*
*
*
Hollow Fiber
Cancer cells in hollow fibers: abdominal and dorsal implants
Hollow-Fiber model – (Fr7)
Salina Doxorrubicina Fr7
0,0
0,2
0,4
0,6
0,8
1,0
1,2
**
**
****
Abs
orba
nce
MCF7 NCIADR OVCAR
Hollow-fiber: abdominal implant Hollow-fiber: abdominal implant
Salina Doxorrubicina Fr70,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
****
Ab
so
rba
nce
MCF7 NCIADR OVCAR
Hollow-fiber : dorsal subcutaneous implantHollow-fiber : dorsal subcutaneous implant
Hollow-Fiber model – (Fr7)
Hanahan and Weinberg. Hallmarks of cancer: the next generation. Cell:144 (5), 646-74, 2011
Targeted screening
•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 CancerSiddhartha Mukherjee
• 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
