Fotossensibilização de células derivadas dos carcinomas de orofaringe e mamário pela Eritrosina B: de efeitos moleculares em sistema modelos aos mecanismos de morte celular em ensaios in vitro
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Data
2022-02-07
Autores
Bistaffa, Maria Julia
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Universidade Estadual Paulista (Unesp)
Resumo
A fim de desenvolver terapias mais eficientes e menos invasivas no tratamento de tumores, a terapia fotodinâmica (TFD) tem ganhado destaque. Além da elucidação dos mecanismos de ação, os mecanismos subjacentes à oxidação lipídica em sistemas complexos, como as membranas plasmáticas, são importantes para eficiência da TFD. Contudo, ambos os mecanismos permanecem elusivos. Nesta dissertação de mestrado, investigamos através de filmes de Langmuir as interações moleculares que permitem a adsorção do FS eritrosina em monocamadas lipídicas e os resultados da fotooxidação subsequentes. Além disso, a eficiência fotodinâmica e os mecanismos de morte celular induzido pelo FS fotoativado em células derivadas do carcinoma de orofaringe (HEp-2) e mamário (MCF7) foram avaliados com citometria de fluxo e marcação de fluorescência. Os filmes de Langmuir foram inicialmente construídos com os fosfolipídeos 2-dioleoil-sn-glicero-3-fosfocolina (DOPC), 1,2-dioleoil-sn-glicero-3-fosfo-L-serina (DOPS) e a mistura DOPC/DOPS, mimetizando membranas plasmáticas tumorais. Em um segundo momento, as monocamadas foram construídas com o extrato lipídico extraído das células MCF7. Os efeitos fotodinâmicos nas células MCF7 foram mais pronunciados do que nas células HEp-2. Em ambos os casos, a incubação da eritrosina não irradiada, não gera efeitos citotóxicos significativos, desencadeando apoptose no cultivo in vitro a partir de 24 h incubação. Por outro lado, a viabilidade celular é significativamente reduzida sob irradiação, desencadeando o mecanismo de morte celular por necrose. Modelos inspirados na membrana tumoral baseados em monocamadas de Langmuir mistas de DOPC e DOPS revelaram que interações eletrostáticas com a cabeça dos lipídeos são as principais forças de adsorção da eritrosina. Nas monocamadas construídas com extrato lipídico da MCF7, a adsorção da eritrosina é dirigida por interações secundárias que afetam a orientação dos grupos carbonil e a organização das cadeias lipídicas. A irradiação do FS nas monocamadas induz a hidroperoxidação lipídica, que pode ainda sofrer decomposição, resultando na clivagem da cadeia de fosfolipídeos e permeabilização da membrana. De fato, a clivagem lipídica, e os subprodutos gerados, foram detectados por espectroscopia de massa, sustentando o caminho necrótico de morte celular observadas nos ensaios in vitro.
In order to develop more efficient and less invasive therapies in the treatment of tumors, photodynamic therapy (PDT) has gained prominence. In addition to elucidate the mechanism of action, the subjacent mechanism of lipid oxidation in complex systems, such as plasma membranes, are important for PDT efficiency. However, both mechanisms remain elusive. In this work, the molecular interactions allowing the adsorption of the PS erythrosine and the subsequent photooxidation reactions were investigated using Langmuir films. Besides, the photodynamic efficiency and cell death mechanism induced by the photoactivated PS in cells derived from oropharyngeal (HEp-2) and mammary (MCF7) carcinomas were evaluated by flow cytometry and fluorescence labeling. Langmuir films were first built with 2-dioleoyl-sn- glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) phospholipids and the DOPC/DOPS mixture, simulating the literature regarding tumor plasma membranes. In a second step, the monolayers were built with the lipid extract from the MCF7 cells. Photodynamic effects in MCF7 cells were more pronounced than in HEp-2 cells. In both cases, the incubation of non-irradiated erythrosine does not generate significant cytotoxic effects, triggering apoptosis on in vitro culture after 24 h of incubation. On the other hand, cell viability is significantly reduced under irradiation, triggering the mechanism of cell death by necrosis. Tumor membrane models based on mixed Langmuir monolayers of DOPC and DOPS revealed that electrostatic interactions with the lipid head is the main driving forces allowing erythrosine adsorption. In monolayers built with MCF7 lipid extract, erythrosine adsorption is driven by secondary interactions that affect the orientation of carbonyl groups and the organization of lipid chains. Irradiation of FS on monolayers induces lipid hydroperoxidation, which can further undergo decomposition, resulting in phospholipid chain cleavage and membrane permeabilization. Indeed, lipid cleavage, and the by-products generated, were detected by mass spectroscopy, supporting the necrotic pathway of cell death observed in in vitro assays.
In order to develop more efficient and less invasive therapies in the treatment of tumors, photodynamic therapy (PDT) has gained prominence. In addition to elucidate the mechanism of action, the subjacent mechanism of lipid oxidation in complex systems, such as plasma membranes, are important for PDT efficiency. However, both mechanisms remain elusive. In this work, the molecular interactions allowing the adsorption of the PS erythrosine and the subsequent photooxidation reactions were investigated using Langmuir films. Besides, the photodynamic efficiency and cell death mechanism induced by the photoactivated PS in cells derived from oropharyngeal (HEp-2) and mammary (MCF7) carcinomas were evaluated by flow cytometry and fluorescence labeling. Langmuir films were first built with 2-dioleoyl-sn- glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) phospholipids and the DOPC/DOPS mixture, simulating the literature regarding tumor plasma membranes. In a second step, the monolayers were built with the lipid extract from the MCF7 cells. Photodynamic effects in MCF7 cells were more pronounced than in HEp-2 cells. In both cases, the incubation of non-irradiated erythrosine does not generate significant cytotoxic effects, triggering apoptosis on in vitro culture after 24 h of incubation. On the other hand, cell viability is significantly reduced under irradiation, triggering the mechanism of cell death by necrosis. Tumor membrane models based on mixed Langmuir monolayers of DOPC and DOPS revealed that electrostatic interactions with the lipid head is the main driving forces allowing erythrosine adsorption. In monolayers built with MCF7 lipid extract, erythrosine adsorption is driven by secondary interactions that affect the orientation of carbonyl groups and the organization of lipid chains. Irradiation of FS on monolayers induces lipid hydroperoxidation, which can further undergo decomposition, resulting in phospholipid chain cleavage and membrane permeabilization. Indeed, lipid cleavage, and the by-products generated, were detected by mass spectroscopy, supporting the necrotic pathway of cell death observed in in vitro assays.
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Palavras-chave
Eritrosina B, Filmes de Langmuir, Cultivo in vitro, Terapia fotodinâmica, Photodynamic therapy, Erythrosine B, Langmuir films, In vitro culture