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Previous issue date: 2016-12-06 / As nanopart?culas de ouro (NPO) t?m despontado como uma importante modalidade de tratamento para o c?ncer, devido ?s suas caracter?sticas f?sico-qu?micas. Somado a isso, o anti-hipertensivo carvedilol tem demonstrado, na literatura recente, potencial antitumoral. Diante disto, o objetivo do trabalho foi avaliar a a??o antitumoral, isolada e combinada, da nanopart?cula de ouro e do anti-hipertensivo carvedilol sobre c?lulas tumorais (HepG2) e n?o tumorais (HEK-293) nos tempos de 24 horas e 48 horas. Para tanto, foi realizado o teste de viabilidade pela exclus?o do azul tripan para as doses de NPO (1 ?g/ml, 3 ?g/ml, 6,25 ?g/ml, 12,5 ?g/ml, 25 ?g/ml e 50?g/ml) e carvedilol (1,5 ?M, 3 ?M, 6,25 ?M, 12,5 ?M, 50 ?M, 100 ?M, 200 ?M e 300 ?M) para selecionar as que provocassem baixa inibi??o do crescimento das c?lulas da linhagem HepG2. No tratamento combinado, as c?lulas foram submetidas ao tratamento com a NPO e, 24 horas depois, tratadas com carvedilol em diferentes doses (3 ?g/ml de NPO + 1,5 ?M de carvedilol e 6,25 ?g/ml de NPO + 3 ?M de carvedilol). As doses selecionadas do teste de viabilidade da NPO (3 ?g/ml e 6,25 ?g/ml) e carvedilol (1,5 ?M e 3 ?M) foram utilizadas, isoladas e em combina??o, para an?lise de morte celular por citometria de fluxo pela marca??o da Anexins V-FITC e iodeto de prop?dio (PI). A an?lise de estresse oxidativo foi realizada atrav?s da dosagem de GSH e dos n?veis de Malondialde?do (MDA). Posteriormente, as c?lulas tumorais foram analisadas quanto a express?o das prote?nas caspase-3, caspase-8, Bcl-2 e MAPK/ERK atrav?s da microscopia de imunofluoresc?ncia. Em seguida, os n?veis de mRNA de FADD, Apaf-1, survivin, MDR-1, EGFR, Akt e mTOR foram analisados relacionando-os a resist?ncia e morte celular. Os n?veis proteicos de Akt, mTOR e MAPK, para o tratamento combinado, foram mensurados atrav?s do western blotting. A avalia??o dos alvos intracelulares da NPO, isolada e em combina??o, foi realizada atrav?s da microscopia eletr?nica de transmiss?o (MET). As melhores doses do teste de viabilidade celular com a NPO (3 ?g/ml e 6,25 ?g/ml) e carvedilol (1,5 ?M e 3 ?M) apresentaram, atrav?s da citometria de fluxo, atividade pr?-apopt?tica sobre as c?lulas tumorais humanas (HepG2) com resultados estaticamente significativos para a combina??o (P<0,001). Em rela??o ?s c?lulas n?o tumorais (HEK-293), houve redu??o da apoptose total (P<0,05), para a maior dose da combina??o, e redu??o do estresse oxidativo na qual, os n?veis de GSH, mostrou-se elevado (P=0,0003) e os de MDA reduzidos (P<0,01), demonstrando capacidade protetora. Ao se observar a imunofluoresc?ncia, houve forte marca??o para caspase-3 (P<0,01) e caspase-8 (P<0,001), nos grupos tratados com a NPO (6,25 ?g/ml) e carvedilol (3?M), isoladamente e em combina??o, aus?ncia de marca??o de MAPK/ERK (P<0,001) e manuten??o da express?o de Bcl-2 (P>0,05) para os mesmos grupos. Al?m disso, a express?o de mRNA de prote?nas anti-apopt?ticas (EGFR Akt, mTOR), com P<0,001, e de resist?ncia (MDR-1), com P<0,01, mostrou-se subregulado, enquanto que, a express?o g?nica de prote?nas proapopt?ticas (FADD), com P<0,01, apresentou-se sobre-regulado para o tempo de 48 horas para combina??o de 6,25 ?g/ml de NPO e 3 ?M de carvedilol. Al?m disso, os n?veis proteico de Akt, mTOR e MAPK se encontraram reduzidos. A MET demonstrou a internaliza??o da NPO isolada, nas proximidades da membrana plasm?tica e, em combina??o, nas proximidades do n?cleo Com isso, pode-se concluir que a a??o combinada da NPO e carvedilol mostra efeitos proapopt?ticos sobre c?lulas tumorais mais efetivos que as doses isoladas e prote??o de c?lulas n?o tumorais. / Gold 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 ?g / ml, 3 ?g / ml, 6.25 ?g / ml, 12.5 ?g / ml, 25 ?g / ml and 50 ?g / Ml) and carvedilol (1.5 ?M, 3 ?M, 6.25 ?M, 12.5 ?M, 50 ?M, 100 ?M, 200 ?M and 300 ?M) 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 ?g / ml NPO + 1.5 ?M carvedilol and 6.25 ?g / ml NPO + 3 ?M carvedilol). Selected doses of NPO viability test (3 ?g / ml and 6.25 ?g / ml) and carvedilol (1.5 ?M and 3 ?M) 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 ?g / ml and 6.25 ?g / ml) and carvedilol (1.5 ?M and 3 ?M) 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 ?g / ml) and carvedilol (3 ?M) 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 ?g / ml NPO and 3 ?M 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.
Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/23485 |
Date | 06 December 2016 |
Creators | Pessoa, Jonas Bispo |
Contributors | 01847881459, http://lattes.cnpq.br/1903940945895093, Ara?jo, Aurigena Antunes de, 83806059420, http://lattes.cnpq.br/3531154240424211, Vieira, Jeymesson Raphael Cardoso, 02930177438, http://lattes.cnpq.br/6727125662817949, Ara?jo J?nior, Raimundo Fernandes de |
Publisher | PROGRAMA DE P?S-GRADUA??O EM BIOLOGIA ESTRUTURAL E FUNCIONAL, UFRN, Brasil |
Source Sets | IBICT Brazilian ETDs |
Language | Portuguese |
Detected Language | English |
Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
Rights | info:eu-repo/semantics/openAccess |
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