Araújo Júnior, Raimundo Fernandes dePessoa, Jonas Bispo2017-06-122017-06-122016-12-06PESSOA, Jonas Bispo. Avaliação da ação antitumoral isolada e combinada do anti-hipertensivo carvedilol e nanopartícula de ouro em células de carcinoma hepatocelular humano. 2016. 91f. Dissertação (Mestrado em Biologia Estrutural e Funcional) - Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 2016.https://repositorio.ufrn.br/jspui/handle/123456789/23485Gold nanoparticles (NPO) have emerged as an important modality of treatment for cancer, due to their physicochemical characteristics. In addition to this, the antihypertensive carvedilol has been shown in the recent literature to be antitumor potential. Therefore, the objective of this study was to evaluate the antitumor action, isolated and combined, of the gold nanoparticle and antihypertensive carvedilol on tumor cells (HepG2) and non-tumor cells (HEK-293) at 24 hours and 48 hours. The viability test was carried out by exclusion of tripan blue at doses of NPO (1 μg / ml, 3 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml and 50 μg / Ml) and carvedilol (1.5 μM, 3 μM, 6.25 μM, 12.5 μM, 50 μM, 100 μM, 200 μM and 300 μM) were used to select those that would cause low inhibition of growth of HepG2 cells . In the combined treatment, cells were treated with NPO and 24 hours later treated with carvedilol at different doses (3 μg / ml NPO + 1.5 μM carvedilol and 6.25 μg / ml NPO + 3 ΜM carvedilol). Selected doses of NPO viability test (3 μg / ml and 6.25 μg / ml) and carvedilol (1.5 μM and 3 μM) were used, alone and in combination, for cell death analysis by flow cytometry By the labeling of Anexins V-FITC and propidium iodide (PI). Oxidative stress analysis was performed by the determination of GSH and the levels of Malondialdehyde (MDA). Subsequently, tumor cells were analyzed for expression of caspase-3, caspase-8, Bcl-2 and MAPK / ERK proteins by immunofluorescence microscopy. Then, mRNA levels of FADD, Apaf-1, survivin, MDR-1, EGFR, Akt and mTOR were analyzed by relating them to resistance and cell death. Protein levels of Akt, mTOR and MAPK, for the combined treatment, were measured by western blotting. The evaluation of the intracellular targets of NPO, isolated and in combination, was performed by transmission electron microscopy (MET). The best doses of the cell viability test with NPO (3 μg / ml and 6.25 μg / ml) and carvedilol (1.5 μM and 3 μM) showed, by flow cytometry, pro-apoptotic activity on cells (HepG2) with statically significant results for the combination (P <0.001). In relation to the non-tumor cells (HEK-293), there was a reduction of total apoptosis (P <0.05), for the highest dose of the combination, and reduction of oxidative stress in which GSH levels were elevated P = 0.0003) and those of reduced MDA (P <0.01), showing protective capacity. When the immunofluorescence was observed, there were strong markers for caspase-3 (P <0.01) and caspase-8 (P <0.001) in the groups treated with NPO (6.25 μg / ml) and carvedilol (3 μM) Isolated and in combination, absence of MAPK / ERK labeling (P <0.001) and maintenance of Bcl-2 expression (P> 0.05) for the same groups. In addition, the expression of mRNA of anti-apoptotic proteins (EGFR Akt, mTOR), with P <0.001, and resistance (MDR-1), with P <0.01, was shown to be underregulated, whereas expression (PADF), with P <0.01, was overregulated for 48 hours for the combination of 6.25 μg / ml NPO and 3 μM carvedilol. In addition, the protein levels of Akt, mTOR and MAPK were reduced. The MET demonstrated the internalization of NPO alone, in the vicinity of the plasma membrane and, in combination, in the vicinity of the nucleus. Thus, it can be concluded that the combined action of NPO and carvedilol shows proapoptotic effects on tumor cells more effective than isolated doses And protection of non-tumor cells.Acesso AbertoCâncer de fígadoApoptoseNanopartícula de ouroCarvedilolmasterThesisCNPQ::CIENCIAS BIOLOGICAS: BIOLOGIA ESTRUTURAL E FUNCIONAL