Atividade citotóxica de complexos de rutênio à base de benzonitrila e bipiridina em tumor de Ehrlich
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2016-04-01
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Universidade Federal de Goiás
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New antitumor agents based on metals have been developed in order to reduce toxicity, to
improve the pharmacological profile and therapeutic efficacy. Among the anticancer agents,
the most promising are the ruthenium complexes for demonstrating antimetastatic properties,
low toxicity to normal cells and high selectivity for tumor cells. This study aimed to determine
the potential cytotoxic, genotoxic and elucidate the pathway of cell death signaling of
ruthenium (II) complexes / bipyridine, in vitro, in cell line murine breast carcinoma, Ehrlich
tumor. To assess the cytotoxicity of the complexes was performed MTT assay. IC50 values
were: 14,93μM and 8,52μM for complexo1 and 2, respectively. In normal cells, the IC50 of
complexes 1 was 0,18μM and 2:34 ± 53.73 ± 5,71μM for 2 complex. The complex 2 had a
better cytotoxic activity and relative potential of 6.31, being selected for further study. For
evaluating the genotoxicity of complex 2 was performed comet assay. After 24h, the
compound 2 at concentrations of 4, 8 and 16μM induced DNA damage of 44, 53 and 44%,
respectively. After 48 hours, induced damage of 48, 45 and 42.5% compared to the negative
control 16.5%. To evaluate the effect of compound 2 on the cell cycle kinetics was performed
testing the cell cycle by flow cytometry. After 24 hours of treatment, an increase in the
percentage of cells in G0 / G1 phase and reduced of cells in S phase and G2 / M was observed, considering a cycle arrest. To evaluate the type of death induced by 2 complex, the test for detection of apoptosis / necrosis by fluorescent microscopy was conducted. After 24 hours of treatment at concentrations of 4, 8 and 16μM were observed 33%, 42% and 30% respectively of cells in early apoptosis compared to negative control 11%. After 48h at concentrations of 4, 8 and 16μM were observed 50%, 64% and 60% of cells in early apoptosis respectively compared to the negative control. Annexin V-FITC staining assay was performed to analyze the type of death induced by compound 2. Both 24 as for 48h, a decrease in the number of viable cells and consequently an increase in the number of apoptotic cells was observed. However, the number of cells in early apoptosis was more significant for the time of 48h. When assessed the expression of the Tp53 genes Bax et Caspase 9 by qPCR, we observed a significant increase in the rate of expression of the Tp53 genes Bax and treatment for 6h. Analysis of expression of proteins involved in cell cycle process, DNA repair and cell death was assessed by western blot. We observed an increase in the levels of Bax and caspase 7. The new complex of ruthenium (II), tested is promising because it has low IC50, selectivity for tumor cells, and induce DNA damage, cell cycle arrest and cell death by apoptosis through caspase activation.
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MAGALHÃES, L. F. Atividade citotóxica de complexos de rutênio à base de benzonitrila e bipiridina em tumor de Ehrlich. 2016. 110 f. Dissertação (Mestrado em Ciências Biológicas) - Universidade Federal de Goiás, Goiânia, 2016.