Estudo da autofagia e dos mecanismos de morte após privação nutricional para quimiosensibilização de modelos de melanoma humano Wild Type e Braf V600e
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Data
2018-06-28
Autores
Antunes, Fernanda [UNIFESP]
Orientadores
Smaili, Soraya Soubhi [UNIFESP]
Tipo
Tese de doutorado
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O melanoma metastático, embora seja o menos prevalente dentre os tipos de câncer de pele, é o mais letal por ser extremamente resistente aos tratamentos atualmente disponíveis, tornando a busca por novas abordagens terapêuticas urgentemente necessárias. Embora a exposição à radiação UV represente a principal causa da melanomagênese, estudos recentes evidenciam também a presença de várias mutações genéticas, como BRAFV600E, presente em aproximadamente 70% dos melanomas, e que promove a ativação constitutiva da via RASRAFMAPK, levando à proliferação celular descontrolada. Esta mutação também está relacionada à inativação de vias de morte celular, desregulação de autofagia, indução de estresse de retículo e favorecimento do metabolismo glicolítico. que coletivamente resultam em resistência a qualquer tratamento. Recentemente foi demonstrado que alterações na dieta podem influenciar tanto o desenvolvimento como o tratamento de diversos tipos de câncer. Um dos protocolos em estudo consiste em ciclos de restrição severa de nutrientes associados a agentes antitumorais. Entretanto, os mecanismos moleculares responsáveis pelos efeitos quimiossensibilizantes ainda não foram completamente elucidados. Baseado nessas premissas, o objetivo geral deste trabalho foi estudar os efeitos da privação nutricional em combinação com terapias antitumorais para o tratamento do melanoma metastático. Para tanto, linhagens de melanoma humano BRAFWT e BRAFV600E e melanoma murino foram submetidas à avaliação qualiquantitativa de morte celular após privação nutricional e/ou tratamento com cisplatina ou sorafenibe. Observouse que, independetemente do status mutacional de BRAF, a privação nutricional sensibilizou as linhagens de melanoma aos tratamentos antitumorais. A morte celular foi predominantemente apoptótica, com determinante participação da mitocôndria no processo de morte celular. Além disso, o bloqueio da glicólise potencializou os efeitos da privação nutricional associada tanto à cisplatina como ao sorafenibe. Adicionalmente, a privação nutricional e os tratamentos antitumorais, isoladamente, induziram autofagia. No entanto, quando associada privação nutricional e sorafenibe, houve bloqueio dos estágios finais do processo autofágico, que assim como o cotratamento com cloroquina, sensibilizou as linhagens de melanoma à morte celular. Já a análise in vivo demonstrou que o uso de intermittent fasting foi seguro e efetivo em reduzir o crescimento tumoral quando associado ao tratamento com sorafenibe. Além disso, foi observada indução de autofagia nos tecidos tumoral e hepático após intermitente fasting e sorafenibe, mas não na sua associação. Coletivamente, nossos resultados indicam que a privação nutricional em associação a agentes antitumorais pode representar uma nova abordagem terapêutica no tratamento do melanoma metastático tanto BRAFWT como BRAFV600E.
Metastatic melanoma remains one of the most aggressive and invasive human malignancies, with high resistance to the treatments nowadays available. New effective therapeutic approaches are therefore urgently needed to treat this malignancy and sustain patient’s survival. Although UV radiation exposure represents the main cause of melanomagenesis it is now evident the association also with several gene mutations. The RASRAFMAPK pathway seems to be the most affected, with BRAFV600E present in approximately 70% of all melanomas. The constitutively active enzyme determines an uncontrolled cell proliferation, inactivation of cell death pathways, ER stress, autophagy deregulation and high glycolytic metabolism resulting in resistance to any treatment. It has been recently demonstrated that dietary modulation can influence both the development and treatment outcome of many cancers. One of these protocols consists in severe nutrients deprivation cycles (fasting) combined with cytotoxic treatments. However, the molecular mechanisms responsible for the chemosensitizing effects remain poorly understood. Therefore, we aimed to evaluate the opportunity of using nutrient deprivation combined with chemo or targeted therapy as a novel valuable clinical approach to treat human metastatic melanoma. To this end, a panel of wt and oncogenic BRAF melanoma cells were subjected to an in vitro fasting protocol (starvation) in presence or absence of cisplatin or sorafenib, and cell death induction was evaluated. We observed a high sensitive of all cell lines to the combined treatment apart the oncogenic BRAF status. The results indicate an apoptotic cell death with determinant mitochondrial participation in cell death process. In addition, glycolysis blockade potentiated the effects of starvation associated with cisplatin as well with sorafenib. Was also detected autophagy induction after starvation, cisplatin and sorafenib treatment alone. However, nutritional deprivation in presence of sorafenib impaired the final stages of autophagic process, in a similar way as chloroquine, sensitizing melanoma cell lines to cell death. The in vivo analysis demonstrated the feasibility and safety of using intermittent fasting together with sorafenib treatment leading to tumor growth reduction. Moreover, was detected autophagy in tumor and liver samples after intermittent fasting or sorafenib but not in its association. Collectively our results indicate that starvation combined with cytotoxic agents as cisplatin and sorafenib may represent a new therapeutic approach to treat both wt and oncogenic BRAF melanomas.
Metastatic melanoma remains one of the most aggressive and invasive human malignancies, with high resistance to the treatments nowadays available. New effective therapeutic approaches are therefore urgently needed to treat this malignancy and sustain patient’s survival. Although UV radiation exposure represents the main cause of melanomagenesis it is now evident the association also with several gene mutations. The RASRAFMAPK pathway seems to be the most affected, with BRAFV600E present in approximately 70% of all melanomas. The constitutively active enzyme determines an uncontrolled cell proliferation, inactivation of cell death pathways, ER stress, autophagy deregulation and high glycolytic metabolism resulting in resistance to any treatment. It has been recently demonstrated that dietary modulation can influence both the development and treatment outcome of many cancers. One of these protocols consists in severe nutrients deprivation cycles (fasting) combined with cytotoxic treatments. However, the molecular mechanisms responsible for the chemosensitizing effects remain poorly understood. Therefore, we aimed to evaluate the opportunity of using nutrient deprivation combined with chemo or targeted therapy as a novel valuable clinical approach to treat human metastatic melanoma. To this end, a panel of wt and oncogenic BRAF melanoma cells were subjected to an in vitro fasting protocol (starvation) in presence or absence of cisplatin or sorafenib, and cell death induction was evaluated. We observed a high sensitive of all cell lines to the combined treatment apart the oncogenic BRAF status. The results indicate an apoptotic cell death with determinant mitochondrial participation in cell death process. In addition, glycolysis blockade potentiated the effects of starvation associated with cisplatin as well with sorafenib. Was also detected autophagy induction after starvation, cisplatin and sorafenib treatment alone. However, nutritional deprivation in presence of sorafenib impaired the final stages of autophagic process, in a similar way as chloroquine, sensitizing melanoma cell lines to cell death. The in vivo analysis demonstrated the feasibility and safety of using intermittent fasting together with sorafenib treatment leading to tumor growth reduction. Moreover, was detected autophagy in tumor and liver samples after intermittent fasting or sorafenib but not in its association. Collectively our results indicate that starvation combined with cytotoxic agents as cisplatin and sorafenib may represent a new therapeutic approach to treat both wt and oncogenic BRAF melanomas.
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Citação
ANTUNES, Fernanda. Estudo da autofagia e dos mecanismos de morte após privação nutricional para quimiosensibilização em modelos de melanoma murino e humano BRAFwt e BRAFv600e. 2018. 213 f. Tese (Doutorado em Farmacologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2018.