EFEITOS DE UM INIBIDOR DO TIPO
KUNITZ PURIFICADO DE SEMENTES DE JUQUIRI (Mimosa regnellii Benth) SOBRE EVENTOS CELULARES DA LINHAGEM TUMORAL B16-f10
Mimosa regnellii Benth; Trypsin inhibitor; Melanoma; Apoptosis; Angiogenesis; Cell migration; Mitochondrial membrane potential.
Cancer is a term used to represent a set of more than 200 diseases, including malignant tumors of different localizations. There are several mechanisms that contribute to carcinogenesis: sustained proliferative signal, deregulation of cellular energy, evasion of apoptosis, angiogenesis induction, unlimited replication, among others. Among the main types of cancer, skin cancer stands out: arises in melanocytes and is the most common in Brazil, accounting for 30% of all malignant tumors registered in the country. Melanomas, at an early stage, can be treated with surgery, but the most advanced cancers require other treatments. In this work a Kunitz-type trypsin inhibitor was purified from Mimosa regnellii Benth (ITJ) legume seeds, partially characterized and evaluated for its toxicity against tumor cell lines, specifically acting in cell line B16-F10, with an IC50 of 0.65 uM, showing no toxicity compared to non-transformed cell lines. Its ability to induce cell death by apoptotic pathway in mouse B16-F10 melanoma cells was evaluated by flow cytometry with Annexin V-FITC / PI markers. In addition, the inhibitor was also evaluated for its ability to: change the mitochondrial membrane potential, visualized by flow cytometry experiments using Rhodamine123 probe and confocal microscopy with Mitotracker Red marker; Reactive oxygen and nitrogen species release through specific probes visualized by microscopy techniques; Liberation of cytosolic calcium, an event that influences apoptosis activation; Inhibit angiogenic activity on rabbit endothelial cells through experiments of inhibition of new vessel formation in Matrigel; Analysis of VEGF expression by western blotting techniques and reduction of IL-6 expression analyzed by confocal microscopy; Inhibition of cell migration process by induction of wound assay, analyzed by microscopy and, finally, cell morphologic changes of B16-F10, analyzed by incubation with specific antibodies of extracellular matrix components and intermediate filaments of melanoma cells, conducted in fluorescence microscopy. All these combined results favor the construction of a possible ITJ action mechanism in the induction of cell death by apoptosis in B16-F10 cells, where the inhibitor would act primarily by altering the mitochondrial function, wherein the trigger initiation of the apoptotic process is the loss of mitochondrial membrane potential, release of cytosolic calcium and ROS, activating a death pathway dependent on caspases. These results suggest that ITJ has the potential to be used as a drug adjuvant treatment for melanomas, due to their specificity and low-dose when compared to other bioactive molecules.