Avaliação da inibição da FAK e o efeito na interação entre FAK e αB-cristalina em células de glioblastoma

Abstract

Glioblastoma Multiforme (GBM) is one of the most aggressive human cancers in existence and has one of the lowest survival rates. The limitations in the treatment make it necessary to discover new alternatives that allow patients a longer survival time and better quality of life. FAK and αB-crystalline are proteins that are highly expressed in several types of cancers, including glioblastoma, and are associated with tumor cell survival and proliferation. Previous studies have demonstrated the importance of the FAK/αB-crystalline interaction in the viability of U87MG glioblastoma cells. In the present study, we aimed to evaluate the effects of inhibition of FAK activation on the survival and migration of glioblastoma cells, and to verify the effect of this inhibition on the interaction between FAK/αB- crystalline proteins. The FAK kinase inhibitor PF 562.271 (PF) and glioblastoma cell lines U87MG and T98G were used to perform the assays. The cells were cultured and treated with PF at different concentrations and treatment times, after which cell viability assays were performed by MTT, Western Blotting, quantitative PCR (qPCR), co-immunoprecipitation and slotting assay. MTT assays demonstrated that GBM cells (U87MG and T98G) treated at different times and PF concentrations significantly reduced glioblastoma cell viability, indicating that FAK phosphorylation in tyrosine residue 397 is important for GBM cell survival. Western blotting assays demonstrated that there was inhibition of FAK activation after PF treatment and there was no change in αB-crystalline protein expression. On the other hand, qPCR assays showed no significant alteration in FAK expression, which suggests that PF treatment only inhibits FAK activation and does not alter its expression; there was also no significant expression of Caspase-3, indicating that cell death caused by PF occurs by alternative pathways to apoptosis. αB-crystalline gene expression, in turn, increased significantly after PF treatment; the data obtained indicated that there was an increase in gene expression, however, there was no increase in protein expression. The interaction between FAK/αB-crystalline proteins occurred even after inhibition of FAK activation, which indicates that the interaction between proteins does not depend on FAK activation. Finally, it was found that there was no change in the migration of glioblastoma cells after treatment with PF. Taken together, the results obtained in the present study demonstrated the effect of inhibition of FAK activation on the interaction with αB-crystalline and its importance in the survival and migration of GBM cells, indicating it as a potential therapeutic target for the treatment of the disease.