Glicogênio sintase quinase 3 e autofagia como alvos moleculares do carbonato de lítio e ácido ursólico em leucemias: possível aumento da atividade da Ara-c

• Ano de defesa: 2016
• Autor(a) principal: Santos, Caroline Palmeira dos [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• dARK ID: ark:/48912/001300002kj86
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Leukemia; Cell death; Ara-c; Gsk3-beta; Ursolic acid; Lithiun; Leucemia; Morte celular; Acido ursólico; Litio
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3773648; http://repositorio.unifesp.br/handle/11600/47180

Objectives: To evaluate the molecular mechanisms involved in the increased cytotoxic effects of Ara-C for lithium carbonate (LIT) in leukemic cells HL-60, and examine possible antileukemic properties of ursolic acid (URS), firstly alone, aiming studies in combination therapy with Ara-C. Methods: The leukemic lines HL-60 and NB4 were used as study models in the following tests: MTT reduction; incorporation of trypan blue dye; evaluation of cell death by labeling fragmented DNA; analysis of the cell cycle phases with propidium iodide; evaluation of modalities of cell death by simultaneous labeling with propidium iodide (PI) and annexin-V-FITC; semi- quantitation of the p62, LC3-II PRAM-1, RAR? and GSK3-? proteins by Western Blotting; assessing the activation of caspase-3 protein; clonal culture assay; immunophenotyping to CD11b and CD15; production and infections of lentivirus and assessment of morphological changes by Leishman stain. Results: Initially, referring to the possible increase of the cytotoxic effects of Ara-C by LIT in NB4 cells, it was found that LIT was not cytotoxic when used alone and did not increase the cytotoxic effects of Ara-C in this lineage. In relation to the increase of Ara-C activity in HL-60 cells, verified in previous studies, using the LIT as inhibitor of glycogen synthase kinase 3 (GSK3), it was verified na partial involvement of caspase-3 protein in AC+LIT association. Although the cells treated with Ara-C have shown significant decrease in protein expression levels of RAR?, increase in CD11b marker and decrease in CD15, these parameters were not altered when these associated LIT. It was also observed that silencing GSK3-? inhibited the increase of the cytotoxic effects of Ara-C by LIT, denoting the importance of this signaling pathway in the increase of the cytotoxic activity of Ara-C by LIT. The studies using the URS demonstrated significant cytotoxic effects in a concentration and time dependent manner, triggering many different cell death types, such as necrosis, apoptosis and late apoptosis after 4 hours of exposure. The URS also decreased the number of colonies, modulates autophagy and led the formation of vacuoles. The URS did not affect the cell differentiation markers CD11b and CD15, nor levels of PRAM-1 protein in NB4 cell line. The effects of URS for both cell lines were not cycle-dependent, once changes in cell cycle phases were not observed. URS did not alter the levels of GSK3-? protein, and did not increase the cytotoxic effects caused by Ara-C. Conclusions: It is suggested that the increase in cytotoxic effects of Ara-C depend on LIT inhibiting GSK3-?, thus stimulating further studies aimed at elucidating this action for future therapeutic targets in leucemias.Regarding the antileukemic effects of URS, this was significantly cytotoxic to the cell lines, possibly due to its modulation of the autophagic process and formation of vacuoles, effects that occurred independently of actions on the cell cycle and in GSK3-?, suggesting thus lower cytotoxic activity for normal tissues, that stimulates preclinical studies for possible application in antileukemic therapy.