Avaliação do potencial antitumoral de flavonóides polihidroxilados sobre células de glioblastoma humano

Abstract

In this work, we investigated the effect of polyhydroxilic flavonoids 5,7-dihydroxiflavone (crisin), 3’,4’-dihydroxyflavone, 4’,5,7-trihidroxiflavone (apigenin), 3,4',5,7-tetrahidroxiflavone (kaempferol), 3,3′,4′,5,6-pentahydroxyflavone (quercetin), and 3,3′,4′,5,7-pentahydroxyflavone-3-rutinoside (rutin) on cell viability, phenotypic change, in migratory and invasive capacity in cultured of human glioblastoma multiform cells, using as a model the highly proliferative human cell line GL-15. In a first study we investigated the effects of the flavonoid rutin (3,3',4',5,7-pentahydroxyflavone-3-rutinoside) on growth, viability and morphology of glioblastoma cells. We observed that rutin (50-100 μM) reduced proliferation and viability of GL-15 cells, leading to decreased levels of ERK1/2 phosphorylation (P-ERK1/2) and accumulation of cells in the G2 phase of the cell cycle. On the other hand, 87.4% of GL-15 cells exposed to 100 μM rutin entered apoptosis, as revealed by flow cytometry after AnnexinV/PI staining. Nuclear condensation and DNA fragmentation were also observed, further confirming that apoptosis had occurred. Moreover, the remaining cells that were treated with 50 μM rutin presented a morphological pattern of astroglial differentiation in culture, characterised by a condensed cell body and thin processes with over expression of GFAP. In second study, we investigated the effects of rutin and the others polyhydroxylated flavonoids on viability, morphology, and structure of GL-15 gliobastoma cells, and also the effects on regulation of elements involved in cellular invasion, as cell migration, and expression of MEC, MMPs, and CX43. We observed that, measuring mitochondrial metabolism through MTT-teste, that flavonoids chrysin, apigenin and 3',4'-dihydroxyflavone induced a significative reduction in viability GL-15 cells, besides ultrastructural features of apoptosis. Flavonoids tested also induced morphological changes in GL-15 cells, which acquired a phenotype pattern of astrocytic differentiation, characterized by the presence of a condensed cell body and thin and long cellular processes, expressing GFAP. Scanning electron microscopy showed that flavonoids induced a reduction in filopodia-like structures on the cell surface of GL-15 cells. We found that flavonoids induced a delay in the migration capacity of glioblastoma cells, after the first 12 h after treatment, and cells exhibited increase in intra and extracellular expression fibronectin, and mainly intracellular expression of laminin. Moreover, we also observed that flavonoids induced reduction on MMP-2 expression and MMP activity, and a reduction on Cx43 expression after treatment with rutin. Taken together, these finds show that rutin and polyhydroxylated flavonoids can inhibit the growth, induce morphological changes or apoptosis, and regulate the expression of components involved in tumor cell migration and invasion, these molecules may be considered as potential adjuvants for therapy of CNS malignant tumor as glioblastomas.