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Evaluation of the effects of urokinase-activated (uPA) and metalloproteinases (MMPs) toxin on Canine Hemangiosarcoma: in vitro and in vivo studies

Grant number: 16/20479-7
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): June 01, 2018
Effective date (End): May 31, 2020
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Maria Lucia Zaidan Dagli
Grantee:Márcia Kazumi Nagamine
Home Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil


Hemangiosarcoma (HSA) is a malignant neoplasm originating from endothelial cells. In dogs it is a very aggressive neoplasia, where metastases are frequent and existing therapies provide only a minimal benefit. The investigation of new therapeutic alternatives is necessary for the development of more effective treatments against this neoplasia. A recruited and purified Urokinase (uPA) and metaloproteinase (MMP) activated bacillus anthracis toxin, named PA-U2-R200A + PA-L1-I210A + LF was produced by the group of Prof. Shihui Liu and Dr. Stephen Leppla, and Liu et al., 2016, demonstrated that this toxin also has antineoplastic action against vascular endothelial cells. The objective of this study is to evaluate, in vitro and in vivo, the effects of the reengineered toxins of Bacillus anthracis on canine Hemangiosarcoma, comparing with normal endothelial cells. The toxin will be provided by the Laboratory of Parasitic Diseases of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA in partnership with Prof. Dr. Shihui Liu. Canine Hemangiosarcoma lines will be cultured and tested for expression of metalloproteinases, urokinase (uPA), and urokinase (uPAr) receptor. The strains will be treated with the re-applied toxin, and will later be evaluated for decreased cell viability (MTT test or crystal violet), apoptosis (Click-iT TUNEL Alexa Fluor Imaging) and changes in cell cycle progression (flow cytometry). Normal canine endothelial cell lines (CnAOEC) will also be cultured and treated with toxins for comparison purposes with neoplastic cells. For the in vivo experiment in mice, canine Hemangiosarcoma cells, as well as normal endothelial cells, will be inoculated into hollow fibers which in turn will be implanted into the subcutaneous BALB/c nu/nu mice. Then the mice will be treated systemically with the toxins. The dose required to reduce the population of neoplastic cells in hollow fibers will be checked. In a parallel study, canine Hemangiosarcoma samples from archives (about 42 cases) will be used to create a TMA (tissue microarray) block. Slides obtained from these blocks will be subjected to immunohistochemical marking of metalloproteinases, UPA and UPAr, in addition to TIMP. The results of these immunostainings will be analyzed, and it will be possible to know better if spontaneous cases of canine Hemangiosarcoma are potentially susceptible to treatment with toxins. It is hoped, therefore, to obtain information on the efficacy of the engineered toxin against canine Hemangiosarcoma. Provided the results are positive, it is intended to carry out clinical trials in dogs in the near future. (AU)