FOCAL ADHESION KINASE INHIBITION DECREASES CELL VIABILITY AND INDUCES APOPTOSIS IN SET-2 CELLS

Abstract

Myeloproliferative Neoplasms (MPN) are diseases characterized by accumulation of myeloid cells. Polycythemia vera (PV), Essential Thrombocythemia (TE) and Primary Myelofibrosis (MF) are MPN BCR-ABL negative and mutations in JAK2, MPL and CALR genes are involved in their pathophysiology. For some PV and PMF patients, Ruxolitinib, a JAK inhibitor, has been used, leading to an improvement of constitutional symptoms. However, the allele burden is not completely reduced, and bone marrow transplantation is still the only curative option. Therefore, more studies are necessary to elucidate the mechanisms involved in these diseases. The Focal Adhesion Kinase (FAK) is a tyrosine kinase which participates in adhesion and migration and also has been described as participating in proliferation, cell survival and apoptosis process mainly by activating SRC kinase. FAK has been described as overexpressed in many types of cancer, including acute myeloid leukemia. Moreover, FAK seems to participate in inflammatory process. The aims of this study was to verify the participation of FAK protein in cell viability and apoptosis of SET-2 cells. With this purpose, SET-2 cells, a JAK2 V617F positive cell line, were treated with the FAK inhibitor PF-562,271 at 5 μM and 10 μM for 72h and cell viability and apoptosis were evaluated. The concentration that inhibits 50% of cell viability (IC50) was also determined. DMSO was used as negative control and Doxorrubicin 10 μM (DOXO) as positive control. SET-2 cells were also treated with Ruxolitinib at 300 nM for 72h. The cell viability and IC50 value for PF-562,271 were evaluated using MTT assay and cleaved PARP and cleaved Caspase 3 expression were detected by Western Blotting to verify apoptosis. P-SRC and SRC detection was performed to verify whether FAK inhibition inhibited SRC phosphorylation. The IC50 determined was 3,215μM. PF-562,271 reduced SET-2 cells viability by 70,2% and 85,4% at 5μM and 10μM, respectively. Ruxolitinib reduced cell viability by 88,4% at 300nM, compared to DMSO. It was possible to observe that cleaved PARP and cleaved CASP3 expression was increased in SET-2 cells treated with PF-562,271 at 5μM and 10μM compared to DMSO and also compared to Ruxolitinib 300nM. Therefore, it was possible to verify that FAK participate in cell viability and apoptosis of SET-2 cells. Considering that many studies have demonstrated that in MPN occurs modifications in bone marrow microenvironment, involving inflammation, resistance to apoptosis and excess of proliferation, FAK may participate in the deregulation of this process, justifying more studies regarding signaling pathways involving FAK in MPN. FAPEMIG (APQ02185/16); CT_INFRA2013/FINEP; ACMMV receives master's scholarship from FAPEMIG.