CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O lapachol, uma naftoquinona natural, e seus derivados sintÃticos tÃm demonstrado, nos Ãltimos anos, importantes aÃÃes citotÃxicas contra varias linhagens de cÃlulas tumorais, assim como significante atividade antitumoral contra alguns tumores. Assim, o objetivo desse trabalho foi avaliar o mecanismos de aÃÃo citotoxica em cÃlulas HL-60 de duas naftoquinonas sintÃticas anÃlogas do lapachol (compostos 1 e 2). Inicialmente foi investigado a atividade antiproliferativa dessas naftoquinonas apÃs um perÃodo de incubaÃÃo de 72h em cÃlulas leucÃmicas (HL-60) e cÃlulas mononucleadas do sangue perifÃrico (CMSP) onde foi observado que essas naftoquinonas mostraram-se ativas para estas linhagens com CI50 de 12 ÂM, 2,3 ÂM e 4,3 ÂM para o lapachol, composto 1 e composto 2, respectivamente em cÃlulas HL-60 e 13,7 ÂM e 34,0 ÂM para o composto 1 e 2 em cÃlulas CMSP, respectivamente. A atividade antiproliferativa em cÃlulas HL-60 apÃs 24 horas de incubaÃÃo foi avaliada com e sem co-tratamento com o antioxidante n-acetilcisteÃna (NAC). Assim, a CI50 sem NAC apÃs 24 horas de exposiÃÃo ao lapachol, composto 1 e composto 2 foi de 42,9 μM, 2,7 ÂM e 4,3 ÂM , respectivamente. JÃ CI50 com NAC (5 ÂM) apÃs 24 horas de exposiÃÃo ao lapachol, composto 1 e composto 2 foi de 180,0 μM, 46,0 ÂM e 18,0 ÂM , respectivamente. Estudos feitos em cÃlulas HL-60 indicaram que o lapachol e seus dois anÃlogos induzem morte celular por apoptose e necrose, como mostrado pelas mudanÃas morfolÃgicas avaliadas atravÃs do uso de coloraÃÃo May-GrÃnwald-Giemsa. Nos ensaios realisados por citometria de fluxo foi revelado que estes compostos promovem a geraÃÃo de espÃcies reativas de oxigÃnio (EROs) 18,86%, 13,31% e 39,11% respectivamente para o lapachol (82 ÂM) e compostos 1 e 2 (3,5 ÂM) e 40,94% e 60,49% para os compostos 1 e 2 (7,0 ÂM), respectivamente. O lapachol (82 ÂM) e os compostos 1 e 2 (3,5 ÂM) diminuÃram o nÃmero de cÃlulas com membrana Ãntegra 26,51%, 34,78% e 29,58% respectivamente e os compostos 1 e 2 (7,0 ÂM) diminuÃram 75,3% e 71,1%, respectivamente. A fragmentaÃÃo do DNA promovida por esses compostos foi observada a partir de 3 horas de exposiÃÃo sendo mais intensa apÃs 24 horas de exposiÃÃo aos compostos testados. O lapachol e os compostos 1 e 2 tambÃm promoveram a ativaÃÃo de caspases relacionadas com a via intrÃnseca de morte celular. AlÃm disso, mostraram induzir a quebra de fitas de DNA. Todos os efeitos citotÃxicos foram abolidos quando os compostos 1 e 2 foram co-incubados com o NAC, mostrando, dessa forma, a participaÃÃo de EROs na citotÃxicidade destas naftoquinonas. / The lapachol, one naphthoquinone natural, and its derivatives synthetic have demonstrated, in recent years, important actions cytotoxic against several lineages of tumor cells, well as signifier antitumoral activity against some tumors. Thus, the objective this work was to evaluate the mechanisms of action cytotoxic in cells HL-60 of two naphthoquinones synthetic analogous of lapachol (compounds 1 and 2). Initially was investigated at antiproliferative activity these naphthoquinones after a incubation period of 72 hours in leukemic cells (HL-60) and peripheral blood mononucleated cells (PBMC) where was observed that these naphthoquinones were active for these lines with IC50 of 12 ÂM , 2.3 ÂM and 4.3 ÂM for the lapachol, compound 1 and compound 2, respectively in cells HL-60 and 13.7 ÂM and 34.0 ÂM for compound 1 and 2 in cells PBMC, respectively. The antiproliferative activity in cells HL-60 after 24 hours incubation was evaluated with and without co-treatment with the antioxidant n-acetylcysteine (NAC). Thus, IC50 without NAC after 24 hours of exposure to lapachol, compound 1 and compound 2 was 42.9 ÂM, 2.7 ÂM and 4.3 ÂM, respectively. Have IC50 with NAC (5 ÂM) after 24 hours of exposure to lapachol, compound 1 and compound 2 was 180.0 ÂM, 46.0 ÂM and 18.0 ÂM, respectively. Studies done in HL-60 cells indicated that the lapachol and its two analogues induce cell death by apoptosis and necrosis, as shown by morphological changes evaluated through the use of staining May-GrÃnwald-Giemsa. In trials realisados by flow cytometry was revealed that these compounds promote the generation of reactive oxygen species (ROS) 18.86%, 13.31% and 39.11% respectively for the lapachol (82 ÂM) and compounds 1 and 2 (3,5 ÂM) and 40.94% and 60.49% for the compounds 1 and 2 (7.0 ÂM), respectively. The lapachol (82 ÂM) and the compounds 1 and 2 (3.5 ÂM) decreased the number of cells with intact membrane 26.51%, 34.78% and 29.58% respectively and the compounds 1 and 2 (7, 0 ÂM) decreased 75.3% and 71.1%, respectively. The DNA fragmentation promoted by such compounds was observed starting from 3 hours of exposure being more intense after 24 hours of exposure to tested compounds. The lapachol and the compounds 1 and 2 also promoted the activation of caspases related to intrinsic pathway of cell death. Furthermore, showed induce the synthesis of DNA strands. All cytotoxic effects were abolished when the compounds 1 and 2 were co-incubated with the NAC, showing thus the participation ROS in cytotoxicity these naphthoquinones.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:7521 |
| Date | 24 August 2012 |
| Creators | Arinice de Menezes Costa |
| Contributors | LetÃcia Veras Costa-Lotufo, Pedro Jorge Caldas MagalhÃes, Paula Christine Jimenez |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em Farmacologia, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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