Estudo do mecanismo de citotóxicidade da oncocalixona-A em leucemia promiolocítica humana – linhagem HL-60

Abstract

Auxemma oncocalyx Taub. belongs to the Boraginaceae family. It is known as “pau branco” and is frequently found in the State of Ceará. The skin of that tree is an astringent and is commonly used as a medicine for wounds. Oncocalyxone A (Onco-A) is a quinone isolated from the ethanolic extract of A. oncocalyx, it has exhibited a series of pharmacological properties, such as analgesic, anti-inflammatory, antioxidant, cytotoxic and antitumor properties. The present study tried to provide a basic set of data on the cytotoxic effects of Onco-A against human promyelocytic leukemia cell line HL-60. The cytotoxic potential of Onco-A was evaluated by the MTT assay, colorimetric analysis, after 24 hours exposure in HL-60 cells with increasing concentrations (8, 16,5 e 33 µM). After the cells were exposure, there were performed experiments to identify the mecanisms of action in vitro (flow cytometry) by the cellular viability, phosphatidyl externalization, internucleosomal DNA fragmentation, identifying apoptotic pathway; the experiments to morphological analysis were made with tripan blue, May-Grunwald-Giemsa, and acridine orange/ethidium bromide; DNA integrity’s test by cometa assay; and the avaliation of interection of enzyme topoisomerase with Onco-A. Results: according to MTT test results, Onco-A showed a significant cytotoxic activity against HL-60 cells (IC50 11 µM). In addition, morphological analysis of cells treated with lower concentration of Onco – A demonstrated that there were reduction in cell’s volume, formation of apoptotic bodies, chromatin condensation, phosphatidylserine externalization, also reduction of cell viability and DNA fragmentation. The Onco-A caused mitochondrial depolarization, activated caspase starters -9 and -8, and the effectors -3 and -7, cleaved Poly ADP-Ribose polymerase – intrinsic and extrinsic pathways. Although our results had shown that none of the doses was able to generate Reactive Oxigen Species, after pre-treated for 1 hour with N-acetylcysteine, the cytotoxicity of Onco-A was changed, an increase in membrane integrity, decreased DNA fragmentation, drag on the G2/M cell cycle phase and low levels of DNA damage. Besides the Onco-A does not interact with the enzyme topoisomerase, the data suggest that their target as Michael acceptor, can be the DNA. These results suggest that apoptosis is one of the major forms of cell’s death caused by Onco-A, and reinforces the compound may be a prototype for cancer therapy.