Resumo

Brain tumors have been studied for a long time. Nevertheless, the impact of technological advances on clinical outcome has not been satisfactory. The drug resistance is an important cause of this problem. Then, new therapeutic approaches, treatments and drugs are necessary. The purpose of this study was to evaluate the antiproliferative and apoptosis-inducing activities of the furanocoumarin-derivative 8-methoxypsoralen (8-MOP) against glioma cell lines, and its GST (glutathione S-transferase) inhibitory activity. GST is an enzyme with detoxification function that is often super-expressed in tumors and related to resistance for promoting conjugation of drugs with glutathione (GSH). For methodology, Rat glioma C6 cells and human glioblastoma GL-15 cells were used in tests with 8-MOP at increasing concentrations, as well as rat astrocytes for comparison. Cell viability was measured by the MTT assay, changes on cellularity was evaluated by phase contrast microscopy and quantified by total proteins content measurement. Analysis of cell proliferation was performed by direct counting by using the trypan blue dye exclusion assay. Flow cytometry was used for elucidation of the kind of cell death. In order to investigate if 8-MOP inhibits GST (isoform pi), in silico and in vitro tests were done. A docking calculation for 8-MOP was carried out (using recognized inhibitors of this enzyme as controls) and GST activity test was also performed. The mechanism was accessed by classic enzymatic kinetic analysis using GSH and chloro-dinitrobenzene (CDNB) as substrates. Monochlorobimane (MCB) assay was used for evaluation of in situ GSH depletion. As results, 8-MOP reduced cell viability and cellularity, time- and dose-dependently, more efficiently in tumor than in normal cells. The compound also showed considerable antiproliferative effect against tumor cells and induced cell death by apoptosis. Necrosis was not observed. In addition, phase contrast microscopy analysis suggested an anti migratory effect of the drug. In the in silico assays, best 8-MOP binding mode presented an energy of -6.4 kcal/mol, indicating better predictive value of binding affinity than any other inhibitor tested. Some relevant interactions were identified. In vitro tests confirmed the inhibitory activity (IC50: 82.25 µM). This inhibitor changed both Vmax and Km, leading parallel lines on the Lineweaver-Burk plot, which indicates an incompetitive inhibition as mechanism, what is interesting, since computational data suggest that 8-MOP binds in the active site. The MCB assay clearly demonstrated that 8-MOP do not deplete GSH, showing that this drug inhibits the enzyme despite it is not a substrate for conjugation with GSH. It is know that many cellular proteins have theirs activities modulated by direct interaction with GST, and inhibitors of this enzyme can, by different pathways, lead to apoptosis, what confirms our findings. In conclusion, 8-methoxypsoralen presented antiproliferative and apoptosis-inducing activities in glioma cells. Additionally, it did not affected normal astrocytes to the extent that affected tumor cells. The pro-apoptotic action could be explained by GST inhibition that is also important to improve therapy. Then, 8-methoxypsoralen is a good prototype of new anti-glioma drug as well as a possible chemosensitizer for treatment of cancer.