Efeito do peptídeo ligante de heparam sulfato na progressão tumoral
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Data
2018-02-22
Autores
Melo, Carina Mucciolo [UNIFESP]
Orientadores
Pinhal, Maria Aparecida Da Silva [UNIFESP]
Tipo
Tese de doutorado
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Resumo
O heparam sulfato (HS) é amplamente distribuído e conservado evolutivamente, um fato que remonta à importância vital desta molécula no desenvolvimento celular e funções como proliferação, migração, adesão e diferenciação celular. Os proteoglicanos de heparam sulfato podem atuar como co-receptores, interagindo com numerosos fatores de crescimento, fatores angiogênicos e citocinas e modulam a patogênese de muitas doenças, incluindo a carcinogênese e inflamação. O objetivo deste estudo foi avaliar o peptídeo ligante de HS e verificar o efeitosobre a progressão tumoral. O peptídeo ligante de HS foi selecionado usando a técnica de Phage display. A ligação do peptídeo ao HS foi avaliado in vitro, utilizando a emissão de triptofano e a análise de FRET (transferência de energia ressonante por fluorescência), enquanto, o ensaio in vivo foi determinado por análise em microscopia confocal time-lapse. e imunomarcação dos embriões de Danio rerio. Os resultados mostraram que o peptídeo ligante de HS interage especificamente com HS/heparina. Os ensaios In vivo (modelo zebrafish), ex vivo (ensaio de aorta de camundongos) e in vitro, utilizando células endoteliais de veia umbilical humana (HUVEC), evidenciaram que o peptídeo ligante de HS é capaz de inibir a angiogênese. O teste de proliferação celular mostrou que FGF-2, mas não o VEGF-A, está envolvido com a resposta do peptídeo ligante de HS no processo angiogênico. A migração de células humanas tumorais de mama triplo negativas, MDA-MB-231, diminuiu na presença do peptídeo ligante de HS. No entanto, a linhagem MDA-MB-231-BR, que apresentam alta capacidade de desenvolver metástase cerebral, quando injetadas em embriões de galinha em presença do peptídeo ligante de HS, mostrou que tal peptídeo não foi capaz de inibir extravasão e colonização metastática. As células de câncer de mama triplo negativas, derivadas da ressecção cirúrgica de paciente com câncer de mama (pacient derived xenotransplant, PDX) foram plaqueadas em cultura 3D. A proliferação e a viabilidade de tais células de câncer de mama PDX não foram afetadas pelo peptídeo ligante de HS. As células PDX estavelmente transduzidas com proteína fluorescente vermelha (RFP), foram injetadas no saco de vitelínico de embriões de zebrafish e tratados com peptídeo ligante de HS. O peptídeo diminuiu a progressão do tumor in vivo e aumentou significativamente a sobrevida dos animais. Os resultados combinados permitem concluir que o peptídeo se liga ao HS e inibe a angiogênese, possivelmente por sinalização mediada pelo FGF-2. Ainda, a inibição da angiogênese parece ser suficiente para diminuir o desenvolvimento do tumor nos ensaios in vivo. O peptídeo ligante de HS pode ser útil como um potencial composto anti-neoplásico.
Heparan sulfate (HS) is widely distributed and evolutionarily conserved, a fact that dates back to the vital importance of this molecule in cell development and functions such as cell proliferation, migration, adhesion, and differentiation. Heparan sulfate proteoglycans are known to function as co-receptors to interact with numerous growth factors, angiogenic factors and cytokines modulating the pathogenesis of many diseases, including carcinogenesis and inflammation. The study aimed to evaluate HS-binding peptide and check the effect on tumor progression. The HSbinding peptide was selected using Phage display technique. Peptide targeting was assessed in vitro, using tryptophan emission and FRET (Fluorescence resonance energy transfer) analysis, while in vivo assay was determined by confocal time-lapse, followed by zebrafish embryo HS labeling. The results showed that anti-HS-peptide interacts specifically with HS/heparin. In vivo (zebrafish model), ex vivo (mice aorta assay) and in vitro using umbilical vein endothelial cells showed that the peptide was able to inhibit angiogenesis. Proliferation assay showed that FGF-2 signaling, but not VEGF-A, was affected by the peptide. MDA-MB-231 cell line migration was decreased by in the presence of anti-HS-peptide. However, MDA-MB-231-BR, lineage metastatic to the brain, was injected into chicken embryos. It was observed that extravasation and metastatic colonization was not inhibited in the presence of anti-HS-peptide. Triple-negative breast cancer cells derived from patient resection patient-derived xenograft cells (PDX) was plated in 3D-culture. Proliferation and viability of such PDX breast cancer cells were not affected by anti-HS-peptide. Finally, Lentivirus transduction assay was performed using to obtain PDX-red fluorescence protein cells. The PDX-RFP cells were injected into the zebrafish yolk sac of zebrafish embryos and treated with anti-HS-peptide. The peptide decreased tumor progression in vivo and raised survival significantly. The combined results let us conclude that anti-HSpeptide binds to HS and inhibits angiogenesis, possibly affected by FGF-2 signaling. Since inhibition of angiogenesis seems to be sufficient to decrease cancer development, anti-HSpeptide might be useful as a potential anti-neoplastic compound as perspective target therapy.
Heparan sulfate (HS) is widely distributed and evolutionarily conserved, a fact that dates back to the vital importance of this molecule in cell development and functions such as cell proliferation, migration, adhesion, and differentiation. Heparan sulfate proteoglycans are known to function as co-receptors to interact with numerous growth factors, angiogenic factors and cytokines modulating the pathogenesis of many diseases, including carcinogenesis and inflammation. The study aimed to evaluate HS-binding peptide and check the effect on tumor progression. The HSbinding peptide was selected using Phage display technique. Peptide targeting was assessed in vitro, using tryptophan emission and FRET (Fluorescence resonance energy transfer) analysis, while in vivo assay was determined by confocal time-lapse, followed by zebrafish embryo HS labeling. The results showed that anti-HS-peptide interacts specifically with HS/heparin. In vivo (zebrafish model), ex vivo (mice aorta assay) and in vitro using umbilical vein endothelial cells showed that the peptide was able to inhibit angiogenesis. Proliferation assay showed that FGF-2 signaling, but not VEGF-A, was affected by the peptide. MDA-MB-231 cell line migration was decreased by in the presence of anti-HS-peptide. However, MDA-MB-231-BR, lineage metastatic to the brain, was injected into chicken embryos. It was observed that extravasation and metastatic colonization was not inhibited in the presence of anti-HS-peptide. Triple-negative breast cancer cells derived from patient resection patient-derived xenograft cells (PDX) was plated in 3D-culture. Proliferation and viability of such PDX breast cancer cells were not affected by anti-HS-peptide. Finally, Lentivirus transduction assay was performed using to obtain PDX-red fluorescence protein cells. The PDX-RFP cells were injected into the zebrafish yolk sac of zebrafish embryos and treated with anti-HS-peptide. The peptide decreased tumor progression in vivo and raised survival significantly. The combined results let us conclude that anti-HSpeptide binds to HS and inhibits angiogenesis, possibly affected by FGF-2 signaling. Since inhibition of angiogenesis seems to be sufficient to decrease cancer development, anti-HSpeptide might be useful as a potential anti-neoplastic compound as perspective target therapy.
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Citação
MELO, Carina Mucciolo. Efeito do peptídeo ligante de heparam sulfato na progressão tumoral Carina Mucciolo Melo. 2018. 123 f. Tese (Doutorado em Biologia Moecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2018.