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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

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Modelos para avaliação experimental da atividade anticâncer

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  1. Modelos para avaliação experimental da atividade anticâncer I Simpósio Ibero-americano de Investigação em Câncer 24-25 de setembro de 2013 CYTED - RIBECANCER

  2. CPQBA cancer screening • 1998 – CYTED – NCI – Panamá • Mahabir Gupta, Gordon Cragg • 1999 - Frederick Cancer Research and Development Center

  3. In vivo screening - NCI • 1955 - 1975 • Murine Leukemias: L1210 and P388 • 1975 - 1985 • 1ststage: P388(5 to 9 days) • 2ndstage: 4 to 8 in vivo models(murine and xenotransplant) • formulation / toxicology • clinical studies • 1955 - 1985 • 600.000 tested compounds

  4. In vivo screening • Advantages • practical, reproducible, low cost (?), metabolic activation, sensibility • Disadvantages • time, drug consuming, facilities,no. of compounds tested, no of animals

  5. 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

  6. NCI tumor cell lines Colon (7) (4) Lung (9) (3) Prostate (2) (1) Melanoma (8) (8) Ovarium (6) (3) Breast (8) (2) Brain (6) (1) Kidney (8) (3) Hematopoietic (6) (2) ( )CPQBA

  7. In vitro screening • Disadvantages • false-positives • drugs solubility • metabolism activation • immunomodulation, angiogenesis, metastasis • culture cells vs. tumor

  8. In vitro screening • Advantages • oriented disease screening / specificity (?) • drug amount • costs • space facilities • time / no. of compounds tested

  9. 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)

  10. Acid fixation trichloroacetic acid Dye addition SRB Dye solubilization OD Reading (540 nm) Anticancer test Cells Inoculation 96 wells plates RPMI + bovine calf serum Incubation 24 hours Acid fixation control plate time zero Drugs addition Incubation 48 hours

  11. Quimioterápicos

  12. Quimioterápicos

  13. Quimioterápicos

  14. 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

  15. Plant acquisition and identification Grinded plant material dichloromethane Organic extract Plant residue ethanol 70% Anti-cancer tests Ethanolicextract Isolation and identificationof active principles

  16. Active products

  17. Ehrlich ascitic tumor in mice Inoculation 0,5 mL 104 cells of Ehrlich Tumor 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 Intraperitoneal injection Post-inoculation n=10 Number of deaths and day of death: ANOVA and Kaplan-Meyer Survival Curve

  18. Ehrlich ascitic tumor in mice: survival curve

  19. 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

  20. Pletismometer:paw tumor

  21. Ehrlich solid tumor in mice: paw tumor

  22. Melanoma B16: lung metastasis

  23. PsidiumguajavaL.Ehrlich paw tumor

  24. uterus relative weight control Doxo P. guajava 50 mg/kg

  25. Hollow Fiber Cancer cells in hollow fibers: abdominal and dorsal implants

  26. Hollow-Fiber model – (Fr7) Hollow-fiber: abdominal implant

  27. Hollow-Fiber model – (Fr7) Hollow-fiber : dorsal subcutaneous implant

  28. Hanahan and Weinberg. Hallmarks of cancer: the next generation. Cell:144 (5), 646-74, 2011

  29. Targeted screening • growth factors • signaling molecules • cell-cycle proteins • modulators of apoptosis • molecules that promoted angiogenesis • anti-inflammatory drugs

  30. 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

  31. 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

  32. Gracias Obrigado Thank you João Ernesto de Carvalho carvalho@cpqba.unicamp.br

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