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Luciana Maria Silva Lopeshttp://lattes.cnpq.br/0162558710954506Frederico Marianetti SorianiAline Brito de Limahttp://lattes.cnpq.br/1440605647884016Nikole Gontijo Gonçales2024-02-26T16:53:44Z2024-02-26T16:53:44Z2020-06-05http://hdl.handle.net/1843/64704O câncer de ovário apresenta a maior taxa de mortalidade entre os tumores ginecológicos. O tratamento quimioterápico a base de platina e taxanos é a primeira linha de tratamento oferecido às pacientes, mas apesar da resposta inicial, 60-80% apresentarão recidiva da doença associada à quimiorresistência. O ligante indutor de apoptose relacionado ao fator de necrose tumoral (TRAIL) e seus receptores (TRAIL-R) têm sido alvo de estudos desde a descoberta de que esta proteína induz a morte em células tumorais, mas não em células normais. Elucidar o mecanismo pelo qual TRAIL atua nos diferentes tipos tumorais pode direcionar o seu uso corretamente na prática clínica. Sendo assim, este trabalho tem como objetivo avaliar a participação de alvos relacionados ao sistema TRAIL/TRAIL-R na quimiorresistência na linhagem celular de tumor de ovário SKOV-3. A caracterização celular da linhagem SKOV-3 para os receptores TRAIL-R2 e TRAIL-R3 foi realizada através de citometria de fluxo e imunofluorescência. Os resultados mostraram que estes receptores não se encontram dispostos na membrana celular ou no interior das células. Já no nível de transcritos, foi possível detectar a presença de mRNA para ambos os alvos, podendo-se inferir que uma regulação pós-transcricional destes receptores está ocorrendo nessa linhagem celular. Nos ensaios de viabilidade celular realizados pelo método MTT foram determinadas as concentrações de 0,015mg/mL de cisplatina, 100ng/mL de rhTRAIL e 0,025mg/mL;100ng/mL de cisplatina e rhTRAIL em combinação para tratar as células e realizar a extração de RNA. A avaliação do perfil de expressão dos genes TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RIPK1, MLKL, NFKB1, RELA, TP53, CASP8 e CFLAR foi avaliada por RT-qPCR. Nos três tratamentos foi observado um perfil de expressão diferencial. No tratamento com cisplatina foi observado um aumento na expressão de TP53. No tratamento com rhTRAIL, houve aumento de NFKB1 e RELA, indicativos de que a via de NF-kB pode ter sido iniciada. Já no tratamento com a combinação das drogas cisplatina e rhTRAIL o maior aumento na expressão foram dos alvos RIPK1 e MLKL, indicando que o mecanismo de necroptose pode ter sido desencadeado frente ao tratamento. Estes resultados mostram como o tratamento quimioterápico nem sempre culmina somente em apoptose, mas também regula transcricionalmente vias de sobrevivência e morte inflamatória.Ovarian cancer presents the highest rate of mortality among gynecological tumors. Chemotherapy based on platin and taxane compounds is the first line of treatment available for patients, therefore the initial response, about 60-80% will present relapse of the disease-associated to chemoresistance. TNF-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R) have become a target for cancer therapy since the discovery that this protein can induce death in tumoral cells and spare normal cell. Draw the mechanisms by which TRAIL acts in different tumoral types can orientate its right use in clinical practice. Thus, this work aims to evaluate the participation of targets related to the TRAIL/TRAIL-R system and chemoresistance in the ovarian cancer cell line SKOV-3. Characterization of the SKOV-3 cell line regarding TRAIL-R2 and TRAIL-R3 receptors was accomplished by flow cytometry and immunofluorescence. The results showed that these receptors were not present in the surface of the cell membrane, cytoplasm, or nucleus. At the transcriptional level, both mRNAs of TRAIL-R2 and TRAIL-R3 were detected, and by that, it can be assumed that post-transcriptional regulation of these receptors is happening in this cell line. Viability assays were performed by MTT and the concentrations found to cisplatin were 0.015mg/mL, 100ng/mL for rhTRAIL, and 0.025mg/mL;100ng/mL for cisplatin and rhTRAIL combinations to treat cells and perform the RNA extraction. Evaluation of gene expression profile of TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, RIPK1, MLKL, NFKB1, RELA, TP53, CASP8 and CFLAR was performed by RT-qPCR. Three differential responses in the profile of gene expression were observed. Cisplatin's treatment up-regulated the expression of TP53. rhTRAIL's treatment up-regulated NFKB1 and RELA gene expression, suggesting that the NF-kB pathway may have initiated. Already in the combination of both drugs, RIPK1 and MLKL were up-regulated, indicating that death by necroptosis may have activated. These results demonstrate that chemotherapy treatment not always reaches apoptosis but also transcriptionally regulates survival pathways and inflammatory death.CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em GenéticaUFMGBrasilICB - INSTITUTO DE CIÊNCIAS BIOLOGICAShttp://creativecommons.org/licenses/by-nc-sa/3.0/pt/info:eu-repo/semantics/openAccessGenéticaNeoplasias OvarianasAntineoplásicosResistência a Medicamentos AntineoplásicosCâncer de ovárioCisplatinaTrailQuimiorresistênciaAnálise de diferentes programas de morte e sobrevivência celular, relacionadas ao mecanismo de quimiorresistência na linhagem celular de câncer de ovário SKOV-3info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALAnálise de diferentes programas de morte e sobrevivência celular, relacionadas ao mecanismo de quimiorresistência na linhagem celular de câncer de ovário SKOV-3.pdfAnálise de diferentes programas de morte e sobrevivência celular, relacionadas ao mecanismo de quimiorresistência na linhagem celular de câncer de ovário SKOV-3.pdfapplication/pdf2028622https://repositorio.ufmg.br/bitstream/1843/64704/1/An%c3%a1lise%20de%20diferentes%20programas%20de%20morte%20e%20sobreviv%c3%aancia%20celular%2c%20relacionadas%20ao%20mecanismo%20de%20quimiorresist%c3%aancia%20na%20linhagem%20celular%20de%20c%c3%a2ncer%20de%20ov%c3%a1rio%20SKOV-3.pdfe752040e64d558b9a8f65cc186750986MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-81037https://repositorio.ufmg.br/bitstream/1843/64704/2/license_rdfd434b2e45b27c6ef831461f4412a9d4eMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82118https://repositorio.ufmg.br/bitstream/1843/64704/3/license.txtcda590c95a0b51b4d15f60c9642ca272MD531843/647042024-02-26 13:53:44.696oai:repositorio.ufmg.br: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ório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2024-02-26T16:53:44Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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