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Construção de Cell Microarrays como estratégia para avaliar alterações na expressão de proteínas induzidas por tratamentos em linhas celulares de cancro do ovário
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Abstract(s)
O Cancro do Ovário é uma das principais causas de morte por cancro ginecológico, caracterizando-se frequentemente por um diagnóstico tardio devido à ausência de sintomas específicos em fases iniciais. A elevada taxa de mortalidade associada deve-se, em grande parte, à resistência aos tratamentos convencionais, como a quimioterapia baseada em agentes como a carboplatina e o paclitaxel. A investigação sobre novos compostos e estratégias terapêuticas, como a utilização da ivermectina, tem vindo a ganhar destaque na tentativa de contornar este desafio.
O presente trabalho foi desenvolvido no âmbito de um estágio de natureza profissional realizado no Laboratório de Patologia Toxicológica da CESPU, integrado na unidade de investigação UCIBIO, com a duração de 375 horas. Durante o estágio, tive a oportunidade de adquirir competências laboratoriais relevantes, tais como interpretação e apresentação
de resultados científicos, escrita de protocolos e relatórios técnicos, cumprimento de boas praticas laboratoriais e trabalhar em equipa multidisciplinar. Assim como, adquirir diferentes técnicas laboratoriais tais como, cultura celular em 2D e 3D, incluindo organoides, avaliação de viabilidade celular, em histologia: processamento histológico, imunocitoquímica, H&E, colorações especiais tais como Tricrómio de Masson e Sirius Red e construção de cell microarray. Este contexto de formação prática e investigação científica culminou na concretização do presente estudo, cujo objetivo foi avaliar a eficácia da ivermectina, isolada e em combinação com carboplatina e paclitaxel, na inibição da viabilidade celular em modelos de cancro do ovário, bem como investigar alterações na expressão de biomarcadores associados à quimiorresistência.
Foram utilizadas duas linhas celulares tumorais: uma resistente à carboplatina (OVCAR8) e outra resistente à carboplatina e paclitaxel (OVCAR8 PTX R C). As células foram expostas a diferentes regimes terapêuticos e analisadas por imunocitoquímica, integrando-se as amostras em cell microarrays para avaliação da expressão de marcadores moleculares relevantes.
Os resultados demonstraram uma redução significativa na viabilidade celular quando a ivermectina foi utilizada em combinação com os quimioterápicos, sugerindo um potencial efeito sinérgico destas combinações. Além disso, a expressão de determinados biomarcadores, nomeadamente a P-gp e a Mesotelina, indicou uma possível reversão do fenótipo resistente, abrindo novas perspetivas para futuras abordagens terapêuticas.
Em conclusão, este estudo, desenvolvido no contexto de um estágio laboratorial, contribui para o aprofundamento do conhecimento sobre o cancro do ovário e reforça a importância da investigação translacional na identificação de novas estratégias terapêuticas para contornar a quimiorresistência.
Ovarian cancer is one of the main causes of death from gynaecological cancer and is often characterized by late diagnosis due to the absence of specific symptoms in the early stages. The associated high mortality rate is largely due to resistance to conventional treatments, such as chemotherapy based on agents like carboplatin and paclitaxel. Research into new compounds and therapeutic strategies, such as the use of ivermectin, has been gaining prominence in an attempt to overcome this challenge. This work was carried out as part of a professional internship at CESPU's Toxicological Pathology Laboratory, as part of the UCIBIO research unit, which lasted 375 hours. During the internship, I had the opportunity to acquire relevant laboratory skills, such as interpreting and presenting scientific results, writing protocols and technical reports, complying with good laboratory practices and working in a multidisciplinary team. As well as acquiring different laboratory techniques such as 2D and 3D cell culture, including organoids, cell viability assessment, histology (histological processing, immunocytochemistry, H&E, special stains such as Masson's Trichrome and Sirius Red and cell microarray construction). This context of practical training and scientific research culminated in this study, the aim of which was to assess the efficacy of ivermectin, alone and in combination with carboplatin and paclitaxel, in inhibiting cell viability in models of ovarian cancer, as well as to investigate changes in the expression of biomarkers associated with chemoresistance. Two tumor cell lines were used: one resistant to carboplatin (OVCAR8) and the other resistant to carboplatin and paclitaxel (OVCAR8 PTX R C). The cells were exposed to different therapeutic regimens and analyzed by immunocytochemistry, integrating the samples into cell microarrays to assess the expression of relevant molecular markers. The results showed a significant reduction in cell viability when ivermectin was used in combination with chemotherapy, suggesting a potential synergistic effect of these combinations. In addition, the expression of certain biomarkers, namely P-gp and Mesothelin, indicated a possible reversal of the resistant phenotype, opening up new perspectives for future therapeutic approaches. In conclusion, this study, carried out in the context of a laboratory internship, contributes to furthering our knowledge of ovarian cancer and reinforces the importance of translational research in identifying new therapeutic strategies to overcome chemoresistance.
Ovarian cancer is one of the main causes of death from gynaecological cancer and is often characterized by late diagnosis due to the absence of specific symptoms in the early stages. The associated high mortality rate is largely due to resistance to conventional treatments, such as chemotherapy based on agents like carboplatin and paclitaxel. Research into new compounds and therapeutic strategies, such as the use of ivermectin, has been gaining prominence in an attempt to overcome this challenge. This work was carried out as part of a professional internship at CESPU's Toxicological Pathology Laboratory, as part of the UCIBIO research unit, which lasted 375 hours. During the internship, I had the opportunity to acquire relevant laboratory skills, such as interpreting and presenting scientific results, writing protocols and technical reports, complying with good laboratory practices and working in a multidisciplinary team. As well as acquiring different laboratory techniques such as 2D and 3D cell culture, including organoids, cell viability assessment, histology (histological processing, immunocytochemistry, H&E, special stains such as Masson's Trichrome and Sirius Red and cell microarray construction). This context of practical training and scientific research culminated in this study, the aim of which was to assess the efficacy of ivermectin, alone and in combination with carboplatin and paclitaxel, in inhibiting cell viability in models of ovarian cancer, as well as to investigate changes in the expression of biomarkers associated with chemoresistance. Two tumor cell lines were used: one resistant to carboplatin (OVCAR8) and the other resistant to carboplatin and paclitaxel (OVCAR8 PTX R C). The cells were exposed to different therapeutic regimens and analyzed by immunocytochemistry, integrating the samples into cell microarrays to assess the expression of relevant molecular markers. The results showed a significant reduction in cell viability when ivermectin was used in combination with chemotherapy, suggesting a potential synergistic effect of these combinations. In addition, the expression of certain biomarkers, namely P-gp and Mesothelin, indicated a possible reversal of the resistant phenotype, opening up new perspectives for future therapeutic approaches. In conclusion, this study, carried out in the context of a laboratory internship, contributes to furthering our knowledge of ovarian cancer and reinforces the importance of translational research in identifying new therapeutic strategies to overcome chemoresistance.
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Keywords
Cancro de ovário Ivermectina Carboplatina Placlitaxel Inibição celular Biomarcadores Quimiorresistência Biotecnologia