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Doctoral Thesis
DOI
Document
Author
Full name
Leandro Figueiredo dos Santos
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Leopoldino, Andréia Machado (President)
Antunes, Lusania Maria Greggi
Coletta, Ricardo Della
Freitas, Luiz Carlos Conti de
Oliveira, Paulo Tambasco de
Russo, Elisa Maria de Sousa
Title in Portuguese
Análise dos mecanismos antitumorais de FTY720 em linhagens humanas de carcinoma espinocelular de cabeça e pescoço
Keywords in Portuguese
Antitumoral
Autofagia
Câncer de cabeça e pescoço
Fingolimod
Abstract in Portuguese
O FTY720 foi considerado um agente antitumoral contra vários tipos de câncer na promoção da morte celular, diminuição da proliferação, angiogênese e metástase; no carcinoma espinocelular de cabeça e pescoço (CECP), ainda não foi descrito sua ação em todas as vias ativadas ou inibidas por este composto. O objetivo deste estudo foi avaliar a ação do FTY720 em modelo in vitro (cultura celular) e in vivo (camundongos) de CECP. Analisamos a atividade da PP2A, uma fosfatase antitumoral inibida em CECP, em um painel de linhagens de células de CECP, além de avaliar o tratamento com FTY720 nestas células e eleger duas linhagens para os posteriores experimentos. As células HN12 e SCC9 foram selecionadas para os estudos de mecanismo baseados nos menores níveis de atividade da PP2A sendo posteriormente incubadas com FTY720 e tendo as principais proteínas alvo analisadas por Westernblotting. Determinamos a viabilidade celular por citometria de fluxo, e estudos funcionais de sinalização de morte celular foram realizados utilizando-se inibidores de caspases/apoptose (Q-VD-OPh), necroptose (Necrostatina-1) e autofagia (3-metiladenina). A degradação de proteínas foi analisada com o inibidor de proteassoma MG132. A formação de tumor de xenoenxerto em camundongos Balb/c Nude foi utilizada, com a linhagem HN12, para avaliar o efeito de FTY720 in vivo. Como esperado, FTY720 aumentou as atividades de fosfatases em células CECP, incluindo PP2A, diminuiu os níveis de proteína SphK1 e ativou caspase-8 e -3 em ambas as células, bem como aumentou a lactato desidrogenase, que é marcador de necrose quando presente no meio extracelular. O bloqueio da apoptose recuperou a viabilidade celular enquanto a inibição da necroptose não afetou a morte celular promovida por FTY720. Por outro lado, a inibição da autofagia aumentou drasticamente a morte celular promovida pelo FTY720 in vitro, sugerindo autofagia como uma resposta protetora. In vivo, foi confirmado que a combinação de FTY720 com inibidor de autofagia (3-MA) reduz a formação do tumor xenoenxerto (HN12) em camundongos combinado ao aumento de ceramidas no sangue dos animais. Nós evidenciamos o FTY720, pelo seus efeitos antitumorais sinérgicos com inibidor de autofagia, como potencial estratégia antitumoral em CECP e talvez outros tipos de câncer, assim como os níveis de ceramidas no sangue como potencial marcador de resposta terapêutica
Title in English
Analysis of the antitumor mechanisms of FTY720 in human cell lines of head and neck squamous carcinoma
Keywords in English
Antitumoral
Autophagy
Fingolimod
Head and neck cancer
Abstract in English
FTY720 has been considered an antitumor agent against several cancer types decreased proliferation, angiogenesis and metastasis; in head and neck squamous cell carcinoma (HNSCC), it has not been addressed yet in all pathways activated or inhibited by this compound. The objective of this study was to evaluate the action of FTY720 in vitro (cell culture) and in vivo (mice) HNSCC models. PP2A, an inhibited antitumor phosphatase in HNSCC, activity was assessed in a panel of HNSCC cell lines. HN12 and SCC9 cells were selected for the studies of mechanisms based on the lower levels of PP2A activity were subsequently incubated with FTY720 and having the main target proteins analyzed by Western blot. Cell viability was determined by flow cytometry, and functional studies of cell death signaling were performed by using inhibitors for apoptosis (Q-VD-OPh), necroptosis (Necrostatin-1) and autophagy (3-Methyladenine). Protein degradation was analyzed with the proteasome inhibitor MG132. Xenograft tumor formation in Balb/c Nude, with HN12, mice was used to evaluate the FTY720 effect in vivo. As expected, FTY720 increased phosphatases activities in HNSCC cells, including PP2A, decreased SphK1 protein levels, and activated caspase-8 and -3 in both cells, as well as increased the release of lactate dehydrogenase activity, which is a marker of necrosis when present in the medium extracellular. Apoptosis blockage recovered cell viability while necroptosis inhibition did not affect FTY720-promoted cell death. On the other hand, autophagy inhibition drastically increased cell death promoted by FTY720 in vitro, suggesting autophagy as a protective response. In vivo, it was confirmed that the combination of FTY720 with autophagy inhibitor (3-MA) reduces the formation of xenograft tumor (HN12) in mice combined with increased blood ceramides in animals. We have shown FTY720, for its synergistic antitumor effects with autophagy inhibitor, as a potential antitumor strategy in HNSCC and perhaps other cancers types, as well as blood ceramides levels as a potential marker of therapeutic response
 
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Release Date
2021-05-22
Publishing Date
2019-11-20
 
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