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Doctoral Thesis
DOI
https://doi.org/10.11606/T.42.2020.tde-12022020-162057
Document
Author
Full name
Matthew Thomas Ferreira
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Colquhoun, Alison (President)
Andrade, Luciana Nogueira de Sousa
Panagopoulos, Alexandros Theodoros
Seelaender, Marilia Cerqueira Leite
Title in Portuguese
Metabólitos da 15-lipoxigenase influenciam crescimento, migração e potencial invasivo de células de glioblastoma.
Keywords in Portuguese
Câncer
Glioblastoma
Lipoxigenases
Mediadores lipídicos
MMPs
Abstract in Portuguese
Introdução e Objetivos: Compreender a interação entre cânceres extremamente agressivos, como o glioblastoma (GBM), e o metabolismo dos ácidos graxos poderia contribuir para a melhora de terapias anti-tumorais. A 15-lipoxigenase-1 (15-LOX-1), uma enzima que metaboliza o ácido linoleico (LA) e o ácido araquidônico (AA), induz efeitos pró ou anti-tumorigênicos em diferentes tipos de câncer. Seu papel no GBM ainda não foi claramente descrito. O objetivo deste estudo foi descrever a influência da 15-LOX e seus metabólitos no crescimento, migração e potencial invasivo de células de GBM. Delineamento Experimental: Duas linhagens celulares de GBM foram cultivadas e tratadas in vitro com metabólitos da 15-LOX (Ácido 13(S)-Hidroxioctadecadienoico (13-HODE), 9-HODE e Ácido 15(S)-Hidroxieicosatetraenoico (15-HETE)) e/ou inibidores de 15-LOX (LUT e NDGA). Curvas de viabilidade com MTT (HODEs [0,1-1M]) e curvas de dose-resposta (HODEs [1-10 µ M]; LUT [7,5-15 µM]; NDGA [20-40 µM]) foram realizadas por 24h, 48h, 72h. Em seguida, foram realizados RT-PCR convencional e/ou quantitativo das células (RNAms de 15-LOX e seus correspondentes downstream: PPARs, GPR132, MMPs). Os ensaios de fechamento de ferida foram realizados após 12 horas de tratamento (HODEs [5 µM]; LUT [15 µM]; NDGA [40µM]) para avaliar a migração. Em seguida, foram realizadas zimografias em gelatina (10mg/mL) e western blot (40g de proteína/amostra) para identificar a influência desses metabólitos e inibidores no potencial invasivo do GBM (definida aqui como a presença/atividade das MMPs). Resultados e Discussão: A via de 15-LOX está presente e ativa nas linhas celulares de GBM. 13-HODE [5 µM] e 9-HODE [5-10 µM] aumentaram a contagem de células em U87MG (n = 3). Inibidores de 13-HODE e 15-LOX-1, LUT [15 µM] e NDGA [40µM], diminuíram a migração na linhagem celular T98G, e NDGA reduziu a atividade de MMP2 e aumentou a forma latente de MMP2. Os tratamentos com 13-HODE / 9-HODE aumentam o RNAm de MMP2 em T98G e U87-MG, respectivamente. Todos os dados foram plotados e analisados usando o GraphPad Prism 5.0. A análise entre dois grupos foi realizada com o teste T de Student, e a ANOVA de duas vias com pós-teste de Bonferroni foi usado para comparar mais de dois grupos de dados. As diferenças foram consideradas estatisticamente significativas quando p< 0,05. Conclusões e Perspectivas: 13-HODE e 9-HODE influenciam o crescimento de células de GBM, 13-HODE e 15-LOX inibiram a migração, e 13-HODE/9-HODE aumentaram o potencial invasivo, enquanto a inibição de 15-LOX diminuiu o potencial invasivo. 15-LOX e seus metabólitos derivados de LA exercem uma influência pró-tumorigênica sobre as células de GBM in vitro. Novos estudos esclarecerão se essas relações se correlacionam positivamente com a malignidade.
Title in English
15-Lipoxygenase Metabolites Influence Growth, Migration and Invasive Potential of Glioblastoma cells.
Keywords in English
Cancer
Glioblastoma
Lipid Mediators
Lipoxygenases
MMPs
Abstract in English
Introduction and Objectives: Understanding the interaction between extremely aggressive cancers, like glioblastoma (GBM), and fatty acid metabolism could contribute to improve anti-tumoral therapies. 15-Lipoxygenase-1 (15-LOX-1), a linoleic acid (LA) and arachidonic acid (AA) metabolizing enzyme, induces both pro- and anti-tumorigenic effects in different cancer types. Its role in glioblastoma has not yet been clearly described. The aim of this study was to describe the influence of 15-LOX and its metabolites on GBM cell growth, migration and invasive potential. Experimental Design: Two GBM cell lines were cultivated and treated in vitro with 15-LOX metabolites (13(S)-Hydroxyoctadecadienoic Acid (13-HODE), 9-HODE and 15(S)-Hydroxyeicosatetraenoic Acid (15-HETE)) and/or 15-LOX inhibitors (LUT and NDGA). MTT viability curves (HODEs [0.1-1 µM]) and dose response curves (HODEs [1-10 µM]; LUT [7.5-15 µM]; NDGA [20-40 µM]) were performed over 24h, 48h, 72h. Next, conventional and/or quantitative RT-PCRs (15-LOX and related downstream mRNAs: PPARs, GPR132, MMPs) of the cells were performed. Wound healing assays were performed after 12h of treatment (HODEs [5 µ M]; LUT [15 µM]; NDGA [40 µM]) to examine migration. Then, gelatin zymography (10mg/mL) and western blots (40 µg of protein/sample) were performed to identify the influence of these metabolites and inhibitors on GBM invasive capacity (defined here as the presence/activity of MMPs). Results and Discussion: The 15-LOX pathway is present and active in the GBM cell lines. Both 13-HODE [5M] and 9-HODE [5-10M] increased cell count in U87MG (n=3). 13-HODE and 15-LOX-1 inhibitors, LUT [15 µM] and NDGA [40 µM], decreased migration in cell line T98G, and NDGA reduced matrix metalloprotease (MMP2) activity and increased the latent form of MMP2. 13-HODE/9-HODE treatments increase MMP2 mRNA in T98-G and U87-MG, respectively. All data were plotted and analyzed using GraphPad Prism 5.0. Analysis between two groups was performed with Student's T test, and a two-way ANOVA with Bonferroni post-test was used to compare multiple groups. The differences were considered statistically significant at p<0.05. Conclusions and Perspectives: 13-HODE and 9-HODE influence GBM cell growth, 13-HODE and 15-LOX inhibition decreased migration, and 13-HODE/9-HODE increased invasive potential, while 15-LOX inhibition decreased invasive potential. 15-LOX and its LA-derived metabolites exercise a certain pro-tumorigenic influence on GBM cells in vitro. Further studies will clarify if these relationships positively correlate with malignancy.
 
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Release Date
2022-02-18
Publishing Date
2020-02-20
 
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