Avaliação do potencial citotóxico de alcalóides guanidínicos isolados da esponja Monanchora arbuscula (DUCHASSAING & MICHELOTTI, 1964) coletada no Parque Estadual Marinho Pedra da Risca do Meio - Ce

Abstract

Marine organisms have been highlighted in the treatment of various diseases. Guanidine alkaloids are commonly found among the sponges of the Cambreidae family and have shown a wide range of biological activities. The present work evaluated the cytotoxic potential of guanidine alkaloids isolated from marine sponge Monanchora arbuscula collected at Pedra da Risca do Meio Marine State Park, Ceará, and the mechanism of action involved in the observed cytotoxicity of ptilocaulin. Five guanidine alkaloids were isolated from the hidroalcoholic extract: ptilocaulin (PT), 8b-ydroxyptilocaulin (8HPT), mirabilin B and an epimeres misture of 1,8a;8b,3a-didehydro-8β-hydroxyptilocaulin and 1,8a;8b,3a-didehydro-8α-hydroxyptilocaulin. The citotoxicity of the compounds was evaluated by the MTT method after 72 hours of incubation in human tumor lines HL-60 (leukaemia), MDA-MB-435 (breast), HCT-8 (Colon) and SF-295 (Gioblastoma), and in peripheral blood mononuclear cells (PBMC) by the method of the Alamar Blue. The effect of PT on HL-60 cells was evaluated after 24h treatment at 10, 15 and 20µM using microscopic observation of various parameters by the following assays: Trypan blue exclusion (cell viability); incorporation of BrdU (cell proliferation); May-Grunwald-Giemsa and acridine orange/ethidium bromide (AO/EB) staining (cell morphology). Flow cytometry was used to further access cell membrane integrity, DNA fragmentation, cell cycle pregression and depolarization of mitochondria. The cytotoxic potential of alkaloids PT and 8HPT proved to be significant towards human tumor lines, mostly for melanoma (IC50 7.58-11.34µM) and leukemia (5.77-7.89µM) lines, however not toxic to normal cells (PBMC). The presence of hydroxyl in carbon “8b” seams to underpower the bioactivity of compound 8HPT. PT reduced the count of viable cells at all concentrations tested on the Trypan blue assay, however increase in the number of non-viable cells was affected by only at the highest concentration. Moreover, PT induced an increase in apoptotic cells in the BE/AO assay and these results were reinforced by the observation of condensed chromatin and membrane blebbing. The alkaloid further caused a slight increase of cells at G1 phase of the cell cycle and an boosted the number of cells with fragmented DNA, with 22.29% and 22.97% at concentrations 20 and 15μM, respectively. Thus, the compound caused significant depolarization of mitochondria membrane, except in the lowest concentration. The evidences collected herein suggests that PT induces apoptosis in HL-60 cells in a concentration-dependent manner. Furthermore, these data support the coast of Ceará State as a prosperous source of compounds with biomedical relevance.