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Master's Dissertation
DOI
https://doi.org/10.11606/D.5.2021.tde-17082021-101606
Document
Author
Full name
Laertty Garcia de Sousa Cabral
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2021
Supervisor
Committee
Maria, Durvanei Augusto (President)
Bydlowski, Sergio Paulo
Oliveira Junior, Vani Xavier de
Oliveira, Caio Fernando Ramalho de
Title in Portuguese
Potencial antiproliferativo do monofosfoester 2-aminoetil dihidrogeno fosfato associado ao peptídeo penetrante de tumor BR2 em células tumorais de mama triplo-negativo
Keywords in Portuguese
Monofosfoester
Nanocarreadores
Neoplasias da mama
Peptídeos
Abstract in Portuguese
O câncer de mama é o tipo de câncer mais comum entre as mulheres no mundo. A maioria das mortes relacionadas ao câncer de mama é devido ao rápido crescimento de tumores e formação de metastase. As terapias atuais são limitadas e a resistência à quimioterapia constitui um importante obstáculo para o sucesso do tratamento. O 2- aminoetil dihidrogeno fosfato (2-AEH2F) é um monofosfoester que está envolvido no turnover de fosfolipídios, apresentando efeitos antiproliferativos em uma ampla variedade de linhagens de células tumorais. O peptídeo BR2 é um derivado não específico de penetração celular derivado do buforin IIb, tendo mostrado maior especificidade às células tumorais. Foi avaliado os efeitos da toxicidade e determinado a concentração inibitória (IC50%) pelo teste colorimétrico MTT. Foram avaliadas as alterações morfológicas e estruturais envolvidas no processo de apoptose pela marcação do citoesqueleto com faloidina e mitocôndria com a sonda MitoRED. A expressão de marcadores envolvidos nas vias de morte celular, proliferação, bem como o potencial elétrico mitocondrial foi avaliado por citometria de fluxo. Os resultados obtidos mostraram que o peptídeo BR2 e a associação 2-AEH2F+BR2 promoveu maior toxicidade nas linhagens tumorais, em comparação ao monofosfoester 2-AEH2F, além disto, o peptídeo BR2 promoveu parada na fase S para célula tumoral MDA MB-231 e G0/G1 para a célula tumoral 4T1, já a associação 2-AEH2F+BR2 ocasionou parada nas fases G0/G1, nas células tumorais 4T1 e MDA MB-231. As células tumorais MDA MB-231 foram sensíveis a todos os tratamentos. Os tratamentos levaram a mudanças na morfologia das células tumorais, principalmente nas mitocôndrias, que perdem sua integridade e se reorganizaram na região perinuclear, aumentando a porcentagem de células com mitocôndrias inativas. Os tratamentos com o peptídeo BR2 e a associação 2-AEH2F+BR2 foram efetivos em induzir morte celular, modulando a via intrínseca do apoptose e promovendo: redução da expressão de marcadores CD44, CD34, CD24, ciclina D1 e Bcl-2, aumento de p53, p21, Bax e caspases 8 e 3 ativas e liberação do citocromo c nas células tumorais MDA MB-231. Houve diminuição considerável da resposta proliferativa das células tumorais após os tratamentos, corroborando a redução da expressão do marcador PCNA. Redução da expressão do Fator de crescimento endotelial vascular (VEGF) e Fator de Crescimento Epidérmico (EGF) foram observados após os tratamentos para células tumorais MDA MB-231. A associação 2- AEH2F+BR2 se mostrou mais efetiva, provendo modulação das proteínas envolvidas na morte celular regulada e senescência, bem como citoxicidade seletiva para as células tumorais, em comparação com as células normais
Title in English
Antiproliferative potential of monophosphoester 2-aminoethyl dihydrogen phosphate associated with tumor penetrating peptide BR2 in triple-negative breast tumor cells
Keywords in English
Breast neoplasms
Monofosfoester
Nanocarriers
Peptides
Abstract in English
Breast cancer is the most common type of cancer among women in the world. Most breast cancer-related deaths are due to the rapid growth of tumors and the formation of metastases. Current therapies are limited and resistance to chemotherapy is an important obstacle to successful treatment. 2-Aminoethyl dihydrogen phosphate (2-AEH2P) is a monophosphoester that is involved in phospholipid turnover, with antiproliferative effects in a wide variety of tumor cell lines. The BR2 peptide is a non-specific cell penetration derivative derived from buforin IIb, having shown greater specificity to tumor cells. The effects of toxicity were evaluated and the MTT colorimetric test determined inhibitory concentration (IC50%). The morphological and structural changes involved in the apoptosis process were evaluated by marking the cytoskeleton with Fhalloidin and mitochondria with the MitoRED probe. The expression of markers involved in the pathways of cell death, proliferation, as well as mitochondrial electrical potential was assessed by flow cytometry. The results obtained showed that the BR2 peptide and the association 2-AEH2P+BR2 promoted greater toxicity in tumor lines, in comparison to the monophosphoester 2-AEH2P, in addition, the BR2 peptide promoted stop in the S phase for MDA tumor cell MB-231 and G0/G1 for the 4T1 tumor cell, while the association 2-AEH2P+BR2 caused a stop in the G0/G1 phases, in the 4T1 and MDA MB-231 tumor cells. MDA MB-231 tumor cells were sensitive to all treatments. The treatments led to changes in the morphology of the tumor cells, mainly in the mitochondria, which lose their integrity and reorganized in the perinuclear region, increasing the percentage of cells with inactive mitochondria. The treatments with the BR2 peptide and the association 2-AEH2P+BR2 were effective in inducing cell death, modulating the intrinsic pathway of apoptosis and promoting: reduced expression of CD44, CD34, CD24, cyclin D1, and Bcl-2 markers, increased p53, p21, Bax, and active 8 and 3 caspases and release of cytochrome c in tumor cells MDA MB-231. There was a considerable decrease in the proliferative response of tumor cells after treatments, corroborating the reduction in the expression of the PCNA marker. Reduction of the expression of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) was observed after treatments for tumor cells MDA MB-231. The association 2- AEH2P+BR2 proved to be more effective, providing modulation of proteins involved in regulated cell death and senescence, as well as selective cytotoxicity for tumor cells, compared to normal cells
 
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Publishing Date
2021-08-18
 
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