Influência das vias WNT/β catenina NOTCH e Hedgehog na terapia de neoplasias hematológicas

Abstract

Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless (WNT) and NOTCH, critical for stem cell self-renewal and differentiation in hematopoiesis, have been implicated in the pathogenesis of several hematological malignancies. Acute lymphoblastic leukemia (ALL) is characterized by the abnormal proliferation and accumulation of immature lymphoid cells within the bone marrow and lymphoid tissues, which can develop from the aberrant activation of the WNT/β-catenin, NOTCH and Hedgehog signaling pathways. On account of that, these pathways may constitute new potential candidate targets for ALL therapy. The main goal of this study was to evaluate the therapeutic potential of WNT/B-catenin, NOTCH and Hedgehog inhibitors, respectively IWR-1, Gamma-Secretase inhibitor XXII (GSI) and Vismodegib (GDC 0449), alone and in combination in an ALL cell line. To evaluate the effect of these developmental signaling pathways inhibitors on cell viability, we use an ALL cell line, the CEM cells, submitted to different concentrations of the inhibitors. The IC50 (half maximal inhibitory concentration), was determining using the blue trypan assay. Cell death was assessed by optical microscopy (after May-Grunwald staining) and by flow cytometry (using Propidium Iodide/Annexin V staining and measuring the levels of BAX and BCL-2, proteins). We also tested by flow cytometry, some proteins related with cell cycle regulation, as p53 and Cyclin D1, and we measure the mitochondrial membrane potential, using the fluorescent probe JC1. The results observed showed that GSI, IWR-1 and GDC-0449 induced cytostatic and cytotoxic effects in CEM cells. These compounds suppressed cell growth/proliferation and induced a decrease in cell viability in a time- and dose-dependent manner, when they administrated alone or in combination with each other. The half maximal inhibitory concentration (IC50) of GSI, IWR-1 and GDC 0449 in CEM cells was 25-50 μM, 30-40 μM and 75 μM, respectively, after 24h of treatment. These compounds induce cell death mainly by apoptosis, which may be caspase-dependent and mediated eventually through the mitochondrial apoptotic pathway, as we observe an increase in caspase levels and a decrease in mitochondrial membrane potential. We could also observe that p53/cyclin D1 and BAX/BCL-2 levels where diminished in presence of these cell signalling pathways inhibitors, but that they didn´t show a notable influence in cell cycle arrest. In conclusion, our results suggest that GSI, IWR-1 and GDC 0449 are potential new targeted therapies that could be efficient in ALL treatment in the future.