Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI)
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFMS |
Texto Completo: | https://repositorio.ufms.br/handle/123456789/4787 |
Resumo: | Kunitz family peptidase inhibitors have several anticancer mechanisms of action. Inga laurina Trypsin inhibitor (ILTI) is known for its insecticidal, fungicidal and antibiofilm activities. Recently, ILTI was produced by heterologous expression and exhibited potential in vitro anticancer activity. However, the anticancer mechanism of action has not been elucidated. In order to increase the available knowledge about the anticancer action of plant peptidase inhibitors from the Kunitz family, we propose to investigate the anticancer properties of ILTI in a leukemia model. The screening test was performed with the tetrazolium salt MTT using the leukemic strains KG-1, Kasumi and K-562 exposed to ILTI at 0.5 and 5 μM for 24 h. The test with resazurin was performed on the Raji, CCRF-CEM, KG-1, Kasumi-1 and K-562 strains, exposed to ILTI between 0.1 and 0.5 μM, in 24 h, to obtain the curves of cytotoxicity. The anticancer mechanism of action of ILTI was verified in the model of chronic myeloid leukemia K-562 by flow cytometry. ILTI was used at 0.07 μM, concentration corresponding to the IC50 obtained in the cytotoxicity assay with propidium iodide (PI), in 24 h. The type of cell death was verified using annexin-V-FITC/PI double labeling after 24 h of ILTI exposure. The caspase-3 activation assay was also performed with PE Rabbit Anti-Active Caspase-3 antibody after 12 h and 24 h. The cell cycle was analyzed using PI labeling after 24 h. ILTI was cytotoxic to Raji, CCRF-CEM, KG-1, Kasumi-1 and K-562 strains. K-562 leukemic cells treated with ILTI underwent apoptosis process mediated by caspase-3 activation. Apparently, apoptosis was the main pathway of activated death, since there was insignificant increase in death by necrosis. It is possible that ILTI-induced apoptosis is involved with cell cycle arrest in the G2/M phase observed in K-562 cells. Future experiments should include activation of caspase-9, release of cytochrome c and loss of mitochondrial potential, to corroborate current results and bring a better understanding of the anticancer activity of ILTI. Keywords: leukemias, chronic myeloid leukemia, inhibitors, apoptosis. |
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2022-05-12T23:04:45Z2022-05-12T23:04:45Z2022https://repositorio.ufms.br/handle/123456789/4787Kunitz family peptidase inhibitors have several anticancer mechanisms of action. Inga laurina Trypsin inhibitor (ILTI) is known for its insecticidal, fungicidal and antibiofilm activities. Recently, ILTI was produced by heterologous expression and exhibited potential in vitro anticancer activity. However, the anticancer mechanism of action has not been elucidated. In order to increase the available knowledge about the anticancer action of plant peptidase inhibitors from the Kunitz family, we propose to investigate the anticancer properties of ILTI in a leukemia model. The screening test was performed with the tetrazolium salt MTT using the leukemic strains KG-1, Kasumi and K-562 exposed to ILTI at 0.5 and 5 μM for 24 h. The test with resazurin was performed on the Raji, CCRF-CEM, KG-1, Kasumi-1 and K-562 strains, exposed to ILTI between 0.1 and 0.5 μM, in 24 h, to obtain the curves of cytotoxicity. The anticancer mechanism of action of ILTI was verified in the model of chronic myeloid leukemia K-562 by flow cytometry. ILTI was used at 0.07 μM, concentration corresponding to the IC50 obtained in the cytotoxicity assay with propidium iodide (PI), in 24 h. The type of cell death was verified using annexin-V-FITC/PI double labeling after 24 h of ILTI exposure. The caspase-3 activation assay was also performed with PE Rabbit Anti-Active Caspase-3 antibody after 12 h and 24 h. The cell cycle was analyzed using PI labeling after 24 h. ILTI was cytotoxic to Raji, CCRF-CEM, KG-1, Kasumi-1 and K-562 strains. K-562 leukemic cells treated with ILTI underwent apoptosis process mediated by caspase-3 activation. Apparently, apoptosis was the main pathway of activated death, since there was insignificant increase in death by necrosis. It is possible that ILTI-induced apoptosis is involved with cell cycle arrest in the G2/M phase observed in K-562 cells. Future experiments should include activation of caspase-9, release of cytochrome c and loss of mitochondrial potential, to corroborate current results and bring a better understanding of the anticancer activity of ILTI. Keywords: leukemias, chronic myeloid leukemia, inhibitors, apoptosis.Inibidores de peptidase da família Kunitz possuem diversos mecanismos de ação anticâncer. O Inga laurina Trypsin inhibitor (ILTI) possui atividade inseticida, fungicida e antibiofilme e foi recentemente produzido por expressão heteróloga com potencial atividade anticâncer in vitro. Entretanto, seu mecanismo de ação anticâncer não foi elucidado. A fim de incrementar os conhecimentos disponíveis sobre a ação anticâncer de inibidores de peptidase vegetais, propomos investigar as propriedades anticâncer do ILTI em modelo de leucemia. Para a obtenção das curvas de citotoxicidade do ILTI, realizou-se o teste de triagem com o sal tetrazólio MTT nas linhagens leucêmicas KG-1, Kasumi e K-562 expostas às concentrações de 0,5 e 5 μM de ILTI por 24 h. Seguiu-se com a avaliação da citotoxicidade com resazurina nas linhagens Raji, CCRF-CEM, KG-1, Kasumi-1 e K-562, expostas as concentrações de 0,1 e 0,5 μM de ILTI, por 24 h. O mecanismo de ação anticâncer do ILTI foi verificado no modelo de leucemia mielóide crônica K-562 por citometria de fluxo. Utilizou-se o ILTI a 0,07 μM, concentração correspondente ao IC50 obtido no ensaio de citotoxicidade com o iodeto de propidio (PI), em 24 h. O tipo de morte celular foi verificado utilizando-se dupla marcação com anexina-V-FITC/PI após 24 h de exposição ao ILTI. Realizou-se também o ensaio de ativação de caspase-3 com o anticorpo PE Rabbit Anti-Active Caspase-3 após 12 e 24 h. O ciclo celular foi analisado utilizando-se marcação com PI, após 24 h. O ILTI foi citotóxico para as linhagens Raji, CCRF-CEM, KG-1, Kasumi-1 e K-562. Células leucêmicas K-562 tratadas com o ILTI sofreram processo de apoptose mediado pela ativação de caspase-3. Aparentemente, apoptose foi a principal via de morte ativada, uma vez que houve um aumento insignificante de morte por necrose. É possível que a apoptose provocada por ILTI possua envolvimento com a parada do ciclo celular na fase G2/M observado em células K-562. Experimentos futuros incluem a ativação de caspase-9, liberação de citocromo C e perda de potencial mitocondrial, para trazer um melhor entendimento da atividade anticâncer do ILTI. Palavras-chave: leucemias, leucemia mielóide crônica, inibidores, apoptose.Fundação Universidade Federal de Mato Grosso do SulUFMSBrasilAvaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI)Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI)info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisMaria Ligia Rodrigues MacedoQueisielle Magalhães Carvalho de Souzainfo:eu-repo/semantics/openAccessporreponame:Repositório Institucional da UFMSinstname:Universidade Federal de Mato Grosso do Sul (UFMS)instacron:UFMSORIGINALDissertação Queisielle.pdfDissertação Queisielle.pdfapplication/pdf3851387https://repositorio.ufms.br/bitstream/123456789/4787/-1/Disserta%c3%a7%c3%a3o%20Queisielle.pdf70c27370050b8da344f5eb4dd8ef84e6MD5-1123456789/47872022-05-12 19:04:46.834oai:repositorio.ufms.br:123456789/4787Repositório InstitucionalPUBhttps://repositorio.ufms.br/oai/requestri.prograd@ufms.bropendoar:21242022-05-12T23:04:46Repositório Institucional da UFMS - Universidade Federal de Mato Grosso do Sul (UFMS)false |
dc.title.pt_BR.fl_str_mv |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
title |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
spellingShingle |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) Queisielle Magalhães Carvalho de Souza Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
title_short |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
title_full |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
title_fullStr |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
title_full_unstemmed |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
title_sort |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
author |
Queisielle Magalhães Carvalho de Souza |
author_facet |
Queisielle Magalhães Carvalho de Souza |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Maria Ligia Rodrigues Macedo |
dc.contributor.author.fl_str_mv |
Queisielle Magalhães Carvalho de Souza |
contributor_str_mv |
Maria Ligia Rodrigues Macedo |
dc.subject.por.fl_str_mv |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
topic |
Avaliação da Atividade Anticâncer do Inibidor de Peptidase Inga laurina Trypsin Inhibitor (ILTI) |
description |
Kunitz family peptidase inhibitors have several anticancer mechanisms of action. Inga laurina Trypsin inhibitor (ILTI) is known for its insecticidal, fungicidal and antibiofilm activities. Recently, ILTI was produced by heterologous expression and exhibited potential in vitro anticancer activity. However, the anticancer mechanism of action has not been elucidated. In order to increase the available knowledge about the anticancer action of plant peptidase inhibitors from the Kunitz family, we propose to investigate the anticancer properties of ILTI in a leukemia model. The screening test was performed with the tetrazolium salt MTT using the leukemic strains KG-1, Kasumi and K-562 exposed to ILTI at 0.5 and 5 μM for 24 h. The test with resazurin was performed on the Raji, CCRF-CEM, KG-1, Kasumi-1 and K-562 strains, exposed to ILTI between 0.1 and 0.5 μM, in 24 h, to obtain the curves of cytotoxicity. The anticancer mechanism of action of ILTI was verified in the model of chronic myeloid leukemia K-562 by flow cytometry. ILTI was used at 0.07 μM, concentration corresponding to the IC50 obtained in the cytotoxicity assay with propidium iodide (PI), in 24 h. The type of cell death was verified using annexin-V-FITC/PI double labeling after 24 h of ILTI exposure. The caspase-3 activation assay was also performed with PE Rabbit Anti-Active Caspase-3 antibody after 12 h and 24 h. The cell cycle was analyzed using PI labeling after 24 h. ILTI was cytotoxic to Raji, CCRF-CEM, KG-1, Kasumi-1 and K-562 strains. K-562 leukemic cells treated with ILTI underwent apoptosis process mediated by caspase-3 activation. Apparently, apoptosis was the main pathway of activated death, since there was insignificant increase in death by necrosis. It is possible that ILTI-induced apoptosis is involved with cell cycle arrest in the G2/M phase observed in K-562 cells. Future experiments should include activation of caspase-9, release of cytochrome c and loss of mitochondrial potential, to corroborate current results and bring a better understanding of the anticancer activity of ILTI. Keywords: leukemias, chronic myeloid leukemia, inhibitors, apoptosis. |
publishDate |
2022 |
dc.date.accessioned.fl_str_mv |
2022-05-12T23:04:45Z |
dc.date.available.fl_str_mv |
2022-05-12T23:04:45Z |
dc.date.issued.fl_str_mv |
2022 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
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masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufms.br/handle/123456789/4787 |
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https://repositorio.ufms.br/handle/123456789/4787 |
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por |
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por |
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info:eu-repo/semantics/openAccess |
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openAccess |
dc.publisher.none.fl_str_mv |
Fundação Universidade Federal de Mato Grosso do Sul |
dc.publisher.initials.fl_str_mv |
UFMS |
dc.publisher.country.fl_str_mv |
Brasil |
publisher.none.fl_str_mv |
Fundação Universidade Federal de Mato Grosso do Sul |
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reponame:Repositório Institucional da UFMS instname:Universidade Federal de Mato Grosso do Sul (UFMS) instacron:UFMS |
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UFMS |
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UFMS |
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Repositório Institucional da UFMS |
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https://repositorio.ufms.br/bitstream/123456789/4787/-1/Disserta%c3%a7%c3%a3o%20Queisielle.pdf |
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Repositório Institucional da UFMS - Universidade Federal de Mato Grosso do Sul (UFMS) |
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