Isolamento de fungo endofítico a partir de Achyrocline satureioides: caracterização fitoquímica e análise de efeito antiglioma

Abstract

Glioblastoma multiforme (GBM) is the most malignant primary brain cancer characterized by genomic instability and diffuse infiltration, promoting an oxidative environment, cell proliferation and poor prognosis for patients. Endophytic fungi reside inside the healthy tissues of plants and represent an important source of antitumor compounds. Achyrocline satureioides is a medicinal plant popularly known as “marcela”, which has interesting pharmacological properties. Since there are no evidences reporting of the endophytic fungi composition of A. satureioides, the aim of this study was to isolate an endophytic fungus, obtain crude and fractionated extracts, characterize effectiveness fractions and investigate its antiglioma effects and suppression of oxidative stress in the tumor microenvironment. Metabolites produced by the endophyte isolated were extracted with the organic solvents dichloromethane and ethyl acetate yielding eDCM and eEtAc crude extracts which were submitted to a guided fractionation. The cytotoxic profile of the crude (24-72 h) and fractionated (48 h) extracts was determined by MTT and Sulforodamide B (SRB) assays on C6 and U87MG glioma lines. Clonogenic assay, cycle/cell death analysis and oxidative stress parameters were evaluated on C6 line exposed to crude extracts, the most effective fraction of each extract and compound 1 for 48 h. The data were analyzed by ANOVA followed by Tukey post-hoc and considered significant for P˂0.05. In the present study, the eDCM and AcoEt extracts isolated from endophytic fungus showed significant cytotoxicity with IC50 of 1.23-2.66 μg/mL and 29.92-87.06 μg/mL, respectively, after 48 h, with a consequent increase in the antioxidant system, such as sulfhydryl content (SH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes. F1DCM, F2DCM, F3DCM e F4DCM (obtain from eDCM) and F3EtAc e F4EtAc (obtain from eEtAc) exibinhit significant antiproliferative activity with IC50 1.09 and 21.43 μg/mL, respectively. F3EtAc induced accumulation of cells in late apoptosis at the G2/M stage. On the other hand, F3DCM significantly reduced colony formation by 90%, exhibited increased antioxidant defense and decreased production of reactive oxygen species (ROS). No changes were observed in primary culture of astrocytes exposed to crude and fractionated extracts. Thus, considering the selective antiglioma activity, the F3DCM was characterized by ultra- efficient liquid chromatography and nuclear magnetic resonance. Based on these data, we identified Sch-642305, a lactone produced by endophytic fungi, which inhibited 80% of the cell viability of glioma cells and promoted cell cycle arrest in G2/M inducing apoptosis. Sch-642305 increased the SOD and CAT activity, SH content and suppressed ROS production. In addition, it was possible to isolate a compound from the F4DCM fraction, identified as 5-methyl-melein, however studies are needed to elucidate its antiglioma effects. These results indicate that the compounds produced by the endophytic fungus isolated from A. satureioides represent a promising source for the search for new therapeutic agents against GBM.