Efeito antitumoral de 22??-hidroxitingenona obtida de Salacia impressifolia (Miers) A.C. (Celastraceae) contra c??lulas de melanoma humano

Autor: Aranha, Elenn Suzany Pereira, 0000-0002-8758-5464
Přispěvatelé: Vasconcellos, Marne Carvalho, Lima, Emerson Silva, Souza, Afonso Duarte Le??o, Koolen, Hector Henrique Ferreira, Fujimoto, Luciana Botinelly Mendon??a
Jazyk: portugalština
Rok vydání: 2020
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFAM
Universidade Federal do Amazonas (UFAM)
instacron:UFAM
Popis: Submitted by Elenn Aranha (elenn_suzany@yahoo.com.br) on 2020-08-31T16:00:07Z No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE- ELENN SUZANY PEREIRA ARANHA.pdf: 7282675 bytes, checksum: 7b2fce80197b34a48dfb2405b59255ed (MD5) cartaencaminhamentoTEDE.pdf: 68701 bytes, checksum: a74752e3f84e6226bb4637bc7bdc2b16 (MD5) Approved for entry into archive by PPG BIONORTE (secestadualppgbionorte@uea.edu.br) on 2020-08-31T16:10:39Z (GMT) No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE- ELENN SUZANY PEREIRA ARANHA.pdf: 7282675 bytes, checksum: 7b2fce80197b34a48dfb2405b59255ed (MD5) cartaencaminhamentoTEDE.pdf: 68701 bytes, checksum: a74752e3f84e6226bb4637bc7bdc2b16 (MD5) Approved for entry into archive by PPG BIONORTE (secestadualppgbionorte@uea.edu.br) on 2020-08-31T16:10:43Z (GMT) No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE- ELENN SUZANY PEREIRA ARANHA.pdf: 7282675 bytes, checksum: 7b2fce80197b34a48dfb2405b59255ed (MD5) cartaencaminhamentoTEDE.pdf: 68701 bytes, checksum: a74752e3f84e6226bb4637bc7bdc2b16 (MD5) Approved for entry into archive by Divis??o de Documenta????o/BC Biblioteca Central (ddbc@ufam.edu.br) on 2020-08-31T18:43:41Z (GMT) No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE- ELENN SUZANY PEREIRA ARANHA.pdf: 7282675 bytes, checksum: 7b2fce80197b34a48dfb2405b59255ed (MD5) cartaencaminhamentoTEDE.pdf: 68701 bytes, checksum: a74752e3f84e6226bb4637bc7bdc2b16 (MD5) Made available in DSpace on 2020-08-31T18:43:41Z (GMT). No. of bitstreams: 3 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE- ELENN SUZANY PEREIRA ARANHA.pdf: 7282675 bytes, checksum: 7b2fce80197b34a48dfb2405b59255ed (MD5) cartaencaminhamentoTEDE.pdf: 68701 bytes, checksum: a74752e3f84e6226bb4637bc7bdc2b16 (MD5) Previous issue date: 2020-03-06 CAPES - Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior FAPEAM - Funda????o de Amparo ?? Pesquisa do Estado do Amazonas The search for new therapeutic options to melanoma remains one of the focus of scientific research around this type of cancer, including sources from natural products, such as 22??-hydroxytingenone, a quinonemethide triterpenes obtained from Salacia impressiofolia. The present thesis aimed to investigate the effect of the substance 22??-hydroxytingenone against melanoma cells. Initially, the effect of 22??-hydroxytingenone on properties related to tumor progression, such as proliferation, migration, cellular invasion and mechanisms of reprogramming of energy metabolism, through the enzymatic activity of lactate dehydrogenase (LDHA), was evaluated. Subsequently, the mechanism of cell death induced by 22-HTG was investigated through the analysis of morphological changes, following cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), mitochondrial membrane potential, production of reactive oxygen species (ROS) by flow cytometry, and fluorescence microscopy with acridine orange/ ethidium bromide assay (AO/BE) staining. RT-qPCR was performed to evaluate the expression of BRAF, NRAS, and NRAS genes and docking mocelular was executed. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin. With the results obtained, it was possible to identify the cytotoxic effect of 22-HTG against melanoma cells, with IC50 value 4.35, 3.72, and 3.29 ??M after 24, 48, and 72 h of incubation, respectively. After, was defined the time of 24 h and the concentrations of 1,0, 2,0 and 2,5 ??M were defined for the other tests, in which a reduction in cell viability, inhibition of new colony formation, reduction of LDHA gene expression, migration and invasion of melanoma cells, and decreased activity of metaloproteinases (MMP-2 and MMP-9), and LDHA. Regarding the investigation of cell death, treatment with 22-HTG caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Further investigation showed an increase in AO/BE-labelled cells and apoptosis was confirmed with Anexin/IP-labeling. Exposure to 22-HTG led to reduced mitochondrial membrane potential, which is not involved by increased oxidative stress. Using the 3D model of reconstructed human skin, 22-HTG reduced the ability of melanoma cells to invade the dermis, highlighting antimetastatic potential. Expression of BRAF, NRAS, and KRAS, important biomarkers in melanoma development, was reduced by 22-HTG treatment and was strong binding affinity with BRAF and NRAS in molecular docking, which may help explain the observed cellular events. According to the data from the thesis, it was possible to conclude that 22-HTG has anti-tumor effect on melanoma cells, with induction of apoptosis and reduction of events such as proliferation, migration, invasion, proteolytic action of MMPs. This study provide new insights for future work on investigating the utilization of 22-HTG in malignant melanoma treatment. A procura por novas op????es terap??uticas de combate ao melanoma, continua sendo um dos focos de investiga????es cient??ficas em torno deste c??ncer, incluindo fontes oriundas de produtos naturais, como, por exemplo, 22??-hidroxitingenona (22-HTG), um triterpeno quinonamet??deo obtido da esp??cie Salacia impressiofolia. A presente tese teve como objetivo investigar o efeito da subst??ncia 22-HTG contra c??lulas do melanoma. Inicialmente, foi avaliado o efeito de 22-HTG em propriedades relacionadas a progress??o tumoral, como prolifera????o, migra????o e invas??o celular, al??m de mecanismos de reprograma????o do metabolismo energ??tico, atrav??s da atividade enzim??tica de lactato desidrogenase (LDHA). Posteriormente, foi investigado o mecanismo de morte celular induzido por 22-HTG atrav??s da an??lise das altera????es morfol??gicas, do ciclo celular, o ensaio de morte celular pela marca????o com anexina V-FITC/iodeto de propidio (Anexina/IP), potencial de membrana mitocondrial, produ????o de esp??cies reativas de oxig??nio (EROs) por citometria de fluxo e microscopia de fluoresc??ncia com laranja de acridina/ brometo de et??dio (AO/BE). RT-qPCR tamb??m foi realizado para avaliar a express??o dos genes BRAF, NRAS e KRAS, assim como an??lise in silico de docking mocelular. O potencial anti-invasivo de 22-HTG foi avaliado usando um modelo tridimensional (3D) de pele humana reconstru??da com c??lulas de melanoma. Com os resultados obtidos foi poss??vel identificar o efeito citot??xico de 22-HTG contra c??lulas de melanoma, com valor de CI50 4,35; 3,72 e 3,29 ??M ap??s 24, 48 e 72 horas de exposi????o. A partir dessa informa????o foi definido o tempo de 24 horas e as concentra????es de 1,0; 2,0; e 2,5 ??M para os demais ensaios, nos quais observou-se redu????o da viabilidade celular, inibi????o da forma????o de novas col??nias, redu????o da express??o g??nica de LDHA, e ainda redu????o da migra????o, invas??o e atividade de metaloproteinases (MMP-2 e MMP-9). Quanto a investiga????o da morte celular, o tratamento com 22-HTG causou altera????es morfol??gicas as c??lulas de melanoma, como redu????o do volume celular, condensa????o da cromatina e fragmenta????o nuclear, caracter??sticas de apoptose. A continua????o da investiga????o mostrou aumento de c??lulas marcadas com AO/BE e a apoptose foi confirmada com a marca????o por Anexina/IP. A exposi????o a 22-HTG provocou redu????o do potencial de membrana mitocondrial, o que n??o est?? envolvido pelo aumento do estresse oxidativo, ou seja, o tratamento com 22-HTG reduziu a produ????o de EROs. Usando o modelo 3D de pele humana reconstru??da, 22-HTG reduziu a capacidade das c??lulas de melanoma invadirem a derme, relevando um potencial antimetast??tico. A express??o dos genes BRAF, NRAS e KRAS foi reduzida pela presen??a de 22-HTG e existe forte afinidade de liga????o entre a subst??ncia testada e as prote??nas BRAF e NRAS, o que pode ajudar a explicar os eventos celulares observados. Quando analisados os dados oriundos da tese, foi poss??vel concluir que 22-HTG possui efeito anti-tumoral em c??lulas de melanoma, com a indu????o de apoptose e redu????o de eventos como prolifera????o, migra????o, invas??o, a a????o proteol??tica de MMPs. Esse estudo fornece novas informa????es para trabalhos futuros acerca do uso de 22-HTG para o tratamento do melanoma. Deixar salvo na p??gina do Youtube a aula "Produtos e servi??os do SISTEBIB" ofertada durante o treinamento e oficinas do m??s de agosto de 2020.
Databáze: OpenAIRE
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