Influência dos glicosaminoglicanos na citotoxidade de peptídeos catiônicos da família dos mastoparanos
Data
2021-08-30
Autores
Muniz Seif, Elias Jorge [UNIFESP]
Orientadores
Arcísio Miranda, Manoel
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
OBJETIVO: Mensurar a influência dos glicosaminoglicanos no potencial citotóxico dos peptídeos MPX, HR1 e MP1. METODOLOGIA: As células de CHO-K1 (ATCC CCL-61TM), tipo selvagem, e CHO-745 (ATCC CRL-2242TM) com produção reduzida de glicosaminoglicano foram cultivadas em meio F12 suplementado com 5% de soro fetal bovino e 1% de antibióticos (penicilina e estreptomicina).O brometo de 3-(4,5-dimetil-2- tiazolil)-2, 5-difenil-2H-tetrazólio (MTT) foi utilizado para determinar as concentrações de citotoxicidade média de cada peptídeo para o tratamento durante 24 horas. Na sequência, foi realizada citometria de fluxo para determinação da via de morte celular. A interação dos peptídeos com a membrana plasmática foi monitorada através sonda FPE (N-(Fluorescein-5-Thiocarbamoyl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanol-amine, riethyl-ammonium Salt). RESULTADO: Os experimentos de MTT e citometria de fluxo mostraram que os peptídeos foram potencialmente citotóxicos para as duas linhagens estudadas, sendo mais eficaz contra as células de linhagem CHO-K1. Para o experimento de MTT observou-se diferença de 13,77%, 24,73% e 11,79% para a viabilidade celular após tratamento com MPX, HR1 e MP1, respectivamente. Já a citometria de fluxo mostrou diferença de viabilidade celular de 28,52%, 20,34% e 18,40% após o tratamento com MPX, HR1 e MP1, respectivamente. Além disso, a necrose foi observada como a principal via de morte celular para todos os peptídeos nas duas linhagens celulares utilizadas nesse estudo. Os peptídeos foram capazes de se ligar na membrana de ambas as linhagens. Todavia, essa ligação foi maior na linhagem CHO-K1, sendo 58%, 48% e 11% maior para MPX, HR1 e MP1, respetivamente. CONCLUSÃO: Os peptídeos mastoparanos são potencialmente mais citotóxicos à linhagem CHO-K1 quando comparados a linhagem CHO-745, resultando em uma maior ligação de peptídeos em sua membrana. Essa pesquisa sugere que a ausência de glicosaminoglicanos é um fator protetivo contra atividade membranolítica desses peptídeos.
OBJECTIVE: To measure the influence of glycosaminoglycans on the cytotoxic potential of MPX, HR1 and MP1 peptides. METHODOLOGY: We employed used the cell lines CHO-K1 (ATCC CCL-61TM) wild type and CHO-745 (ATCC CRL-2242TM) mutant type to redussed glycosaminoglycan production. Both cell ware cultivated in F12 medium suppplemented with 5% fetal bovine serum and 1% antibiotics (penicillin and streptomycin). It was used 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) to determine the average cytotoxicity concentrations of each peptide the 24 hours treatment. Flow cytometry assays were performed to determine the cell death pathway. Finally, the peptide binding experiment was performed through the kinetics of the probe N- (Fluorescein-5-Tiocarbamoil) -1,2-Dihexadecanoil-sn-Glicero-3-Phosphoethanol-amine, Triethyl-ammonium salt) FPE as a function of time and concentration of the peptides used. RESULT: The MTT and flow cytometry experiments showed that the peptides were potentially cytotoxic to the two cell lines, however with more efficiently against CHO-k1 cells. It was observed a difference of 13.77%, 24.73% and 11.79% for cell viability after treatment with MPX, HR1 and MP1, respectively for MTT and 28.52%, 20.34% and 18.40% between the viability after treatment with MPX, HR1 and MP1 respectively for flow cytometry. In addition, necrosis was observed as the main cell death pathway after treatment with all peptides, without alteractions between the cell lines studied. The peptides were bound to the membrane of both cell lines, occurring with increased intensity in CHO-k1, with 58%, 48% and 11% higher intensity for MPX, HR1 and MP1 respectively. CONCLUSION: Mastoparan peptides are potentially more cytotoxic to the CHO-k1 cell line when compared to the CHO-745 cell line resulting in an increase in peptide binding in this membrane. This research suggests that the absence of glycosaminoglycans in membrane is a protective factor against membranolytic activity of these peptides.
OBJECTIVE: To measure the influence of glycosaminoglycans on the cytotoxic potential of MPX, HR1 and MP1 peptides. METHODOLOGY: We employed used the cell lines CHO-K1 (ATCC CCL-61TM) wild type and CHO-745 (ATCC CRL-2242TM) mutant type to redussed glycosaminoglycan production. Both cell ware cultivated in F12 medium suppplemented with 5% fetal bovine serum and 1% antibiotics (penicillin and streptomycin). It was used 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) to determine the average cytotoxicity concentrations of each peptide the 24 hours treatment. Flow cytometry assays were performed to determine the cell death pathway. Finally, the peptide binding experiment was performed through the kinetics of the probe N- (Fluorescein-5-Tiocarbamoil) -1,2-Dihexadecanoil-sn-Glicero-3-Phosphoethanol-amine, Triethyl-ammonium salt) FPE as a function of time and concentration of the peptides used. RESULT: The MTT and flow cytometry experiments showed that the peptides were potentially cytotoxic to the two cell lines, however with more efficiently against CHO-k1 cells. It was observed a difference of 13.77%, 24.73% and 11.79% for cell viability after treatment with MPX, HR1 and MP1, respectively for MTT and 28.52%, 20.34% and 18.40% between the viability after treatment with MPX, HR1 and MP1 respectively for flow cytometry. In addition, necrosis was observed as the main cell death pathway after treatment with all peptides, without alteractions between the cell lines studied. The peptides were bound to the membrane of both cell lines, occurring with increased intensity in CHO-k1, with 58%, 48% and 11% higher intensity for MPX, HR1 and MP1 respectively. CONCLUSION: Mastoparan peptides are potentially more cytotoxic to the CHO-k1 cell line when compared to the CHO-745 cell line resulting in an increase in peptide binding in this membrane. This research suggests that the absence of glycosaminoglycans in membrane is a protective factor against membranolytic activity of these peptides.
Descrição
Citação
SEIF, EJM. Influência dos glicosaminoglicanos na citotoxidade de peptídeos catiônicos da família dos mastoparanos. São Paulo, 2021. 47p. Dissertação (Mestrado em Ciências Biológicas) - Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2021.