Dr. Guacyara MottaAssociate Professor
Department of Biochemistry, Escola Paulista de Medicina (EPM), Federal University of São Paulo (EPM/UNIFESP), Brazil
Speech Title: Plasma Kallikrein-Kinin System Proteins Interaction with Breast Cancer Cells
Abstract: Introduction: The plasma kallikrein-kinin system (KKS) is related to contact system of coagulation, fibrinolysis and inflammation. Plasma kallikrein (PKa) activates factor XII, pro-urokinase (pro-uPA) and releases bradykinin (BK) from high molecular weight kininogen (HK) generating HKa. In tumor cells (CHO) we have shown the role of heparan sulfate proteoglycans (HSPG) in assembly and endocytosis of KKS proteins on cell surface (Motta and Tersariol, 2017). The aim of the present work is to study in human breast epithelial cells the interaction of HK, the presence of PKa and BK release. Methods: The cell lineages studied were MCF-10A (non-metastatic), MCF-7 (less-metastatic) and MDA-MB-231 (highly-metastatic). The techniques employed were confocal microscopy, radioimmunoassay (RIA) and RT-PCR. The confocal microscopy analysis showed HK-Dylight-650 bound on cell surface in MCF-10A (1,947.0±0.003 pixels/cell), MCF-7 (648.0±0.002 pixels/cell) and MDA-MB-231 (1,251.00±0.002 pixels/cell). In endocytic vesicles HK-Dylight-650 and LT-green colocalized in MCF-10A (974.0±0.002 pixels/cell), MCF-7 (61.0±0.001 pixels/cell) and MDA-MB-231 (385.0±0.001 pixels/cell). In comparison, the HK binding and endocytosis was MCF10A > MDAMB-231 > MCF-7. By confocal microscopy the BK colocalized with LT-red in MCF-10A (19.7%), in MCF-7 (40.9%), in MDA-MB-231 (53%) and BK in endocytic vesicles of MDA-MB-231 allows its proliferative effect in metastatic cells. The kininogenase activity was analyzed by RIA, after incubation of cells with HK, and the amount of BK released in supernatant in presence of kininases inhibitors was MCF-10A (1.13±0.01 ng/106cell), MCF-7 (0.29±0.01 ng/106cell), MDA-MB-231 (0.78±0.00 ng/106cell); in presence of both serine proteases and kininases inhibitors the BK released was MCF-10A (0.03±0.01 ng /106cell), MCF-7 (0.19±0.00 ng/106cell) and MDA-MB-231 (0.21±0.00 ng/106cell). The higher BK release in MCF-10A cells is in agreement with its proangiogenic effect. In MDA-MB-231 plasma prekallikrein (PK) mRNA was detected, and by confocal microscopy, in both non-permeabilized and permeabilized cells, PK/PKa was shown colocalized with pro-uPA/uPA. Conclusion: Our data suggest a role for plasma KKS in breast cancer through interaction of HK with cell surface that may result in endocytosis, BK release through endogenous and pericellular proteolysis by PKa and interaction with pro-uPA that may activate uPA/uPAR system.
Keywords: breast cancer, kininogen, plasma kallikrein, endocytosis, proteolysis
Biography: Dr. Motta graduated in Biological Sciences at Escola Paulista de Medicina (EPM), Federal University of São Paulo (EPM/UNIFESP), São Paulo, Brazil. She got her Master degree and Ph.D. in Molecular Biology at EPM/UNIFESP, and during her graduate program, she held an internship at the Division of Clinical Chemistry and Biochemistry - University of Munich (Ludwig - Maximilians) - Germany. Her postdoctoral training was at the Department of Internal Medicine, Division of Hematology/Oncology at University of Michigan (Ann Arbor) - USA.
Dr. Motta's position is currently as Associate Professor at the Department of Biochemistry at EPM/UNIFESP. Her expertise in Biochemistry field is on Protein Chemistry, Intermediate Metabolism and Cell Biology. Her research area expertise is focused on the relationship between the plasma kallikrein-kinin system (plasma prekallikrein/kallikrein, kininogens, bradykinin) and fibrinolysis (urokinase and urokinase receptor) considering the structure, function and interaction of these proteins with biology of cancer.