Estudo da interação com DNA, citoxicidade e da atividade das topoisomerases I e II do derivado de bufadienolídeo 3-Acetato de bufalina

Abstract

Bufadienolides show a wide variety of biological activities, including antineoplastic activities. The present study evaluated the cytotoxic potential and mechanisms of action of two bufadienolides: bufalin (BUF-05) and 3-acetate bufalin (BUF-06). Cytotoxic potential was evaluated using seven human tumor cell lines, three normal murine cell lines and human peripheral blood mononuclear cells (PBMC). Both bufadienolides were shown to be quite cytotoxic to all tumor cell lines (IC50 from 3 to 32 nM), with BUF06 being slightly more potent than BUF05. In PBMCs, bufadienolides showed IC50 values of about 25nM. The compounds showed no cytotoxicity towards normal murine. The PC-3 prostate cell line was chosen to elucidate the mechanism of action of the bufadienolides. Impedance based real-time growth assay analysis showed that both bufadienolides have a similar profile, increasing impedance in the first few hours, then reducing below the control curves, and finally reaching plateau shortly thereafter. Light scanning photomicrographs showed an increase in cell size for both bufadienolides. Cell viability by flow cytometry indicated a reduction in the number of cells and mebrane integrity at 24 and 48 hours. Morphological analysis by flow cytometry revealed that both compounds increased the number of apoptotic cells. Moreover, morphological analysis using differential staining showed the emergence of apoptotic and necrotic cells. In order to confirm the apoptotic profile, phosphatidylserine externalization detection was performed, which showed an increase of initial apoptosis after 24 hours and late apoptosis after 48 hours incubation for both bufadienolides. The compounds also showed mitochondrial depolarization for both incubation times. Western blot analysis revealed a discrete cleavage of PARP and caspase 8. However, effector caspases 3 and 7 were not activated. Low expression of antiapoptotic components Bcl-2 and Bcl-xL, as well as decrease of proapoptotic Bax suggest non-induction of apoptotic cell death. Evaluation of nuclear DNA content showed that both bufadienolides provoked cell arrest in the G2/M phase after 24 and 48 hours incubation. When analyzing cyclin expression involved in the cell cycle, the absence of cyclin A2 and accumulation of cyclin B1 was observed for both compounds, possibly indicating arrest in M phase. DNA damage was accessed by comet assay and phosphorylated H2AX labeling, which showed that the compounds were not able to produce DNA damage. Evaluation of the involvement of DNA repair pathways using DT40 knockout cells suggested that PARP may be involved in the cell death caused by both bufadienolides tested. PARP hyperactivation is a hallmark of parthanatos, a regulated type of necrotic cell death. Furthermore, topoisomerase-mediated DNA cleavage assays showed the inhibition of topoisomerase II by both BUF-06 and BUF-05. In silico molecular docking assays revealed strong affinity of both bufadienolides for the ATPase portion of topoisomerase IIα, indicating possible catalytic inhibition. Overall, these results indicate that 3-acetate bufalin has a cytotoxic profile similar to bufalin.