Estudo do potencial citotóxico e do mecanismo de morte celular de novos protótipos à base de rutênio frente a diferentes células tumorais

Abstract

Currently ruthenium complexes have demonstrated interesting anticancer properties and may represent new and effective therapeutic agents for use as drugs alternative to platinum drugs. The present study aimed to investigate in vitro the cytotoxic and pro-apoptotic effect of the ruthenium complex cis-(dichloro)tetrammineruthenium(III) chloride against human lung alveolar carcinoma (A549) and complexes of ruthenium (II) coordinated to different amino acids against Sarcoma 180 tumor cells through the techniques of cell viability assay, kinetics of cell cycle phases assay, annexin V/propidium iodide assay, mitochondrial membrane potential assay, gene expression by real time PCR assay and western blotting assay. The cell viability assay by MTT reduction technique, it was found that complexes of ruthenium (II) coordinated to amino acids showed cytotoxic activity against S180 tumor cells with IC50 values ranging from 22.53 to 70.08 µM. For ruthenium complex (III) cis- (dichloro)tetrammineruthenium(III) chloride the IC50 value against A549 tumor cells was greater than 383 µM (IC50> 383 mM). Additionally, it was observed that complexes of ruthenium (II) exhibited low cytotoxicity on normal cells of mouse fibroblast L929, with IC50 values ranging from 27.39 to 106.72 µM. The clonogenic assay was performed in A549 tumor cells, and by the results obtained it was found that at low concentrations of the complex cis-(dichloro)tetrammineruthenium(III) chloride (0.38 and 3.8 µM) there was a decrease in the ability of cells to form colonies and at high concentrations 95 and 383 µM there was no colonies formation In cell cycle analysis of S180 tumor cells treated with complex of ruthenium (II) RuGly the results showed that this complex increased the percentage of cells in G0/G1, which correlated with consequent reduction in the number of cells in S phase. To complex of ruthenium (III) cis-(dichloro)tetrammineruthenium(III) chloride against A549 tumor cells, it was found that this complex increased the percentage of cells in S phase. In the analysis of apoptosis assays, the results showed that the complex ruthenium (II) RuGly induced cell death via apoptosis in S180 tumor cells as evidenced by the increase in annexin V positive cells, depolarization of the mitochondrial membrane potential, activation of caspase 3, 8 and 9 and increased expression levels of Caspase-3 (mRNA) and Bax (mRNA) and Bak (protein). The expression of genes Caspase-8, Caspase-9 and Tp53 remained unchanged during the period of exposure examined. For complex ruthenium (III) induction of apoptosis on A549 cells was verified by the presence of annexin V positive cells and peak in sub-G1 (indicative of apoptosis) and increased levels of mRNA of Caspase 3. In addition cell death via necrosis was observed as verified by increased number of cells labeling with only propidium iodide. From the results it is concluded that both complexes of ruthenium (II) and (III) to induce cytotoxic activity against cell models tested and this activity correlates with alterations in cell cycle phases and induction of cell death via apoptosis and necrosis, being prototypes promising drugs against tumor models investigated.