Ação de compostos bioativos em câncer de tireoide
Data
2022-10-31
Autores
Rodrigues, Mariana Teixeira 
Orientadores
Rubió, Ileana Gabriela Sánchez
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A maioria dos casos de câncer de tireoide são carcinomas papilíferos (90 a 95%) e possuem um bom prognóstico, porém por volta de 10% desses casos desenvolvem maior agressividade e, juntamente com os carcinomas anaplásico, correspondem a mais de 50% das mortes. Para estes casos, há alguns inibidores disponíveis, mas estes possuem baixa eficácia e muitos efeitos colaterais, levando o paciente a abandonar o tratamento. Neste cenário, a busca por novos tratamentos é essencial. Atualmente, alguns dos quimioterápicos são ou derivam de compostos bioativos e, portanto, pesquisar a atividade antineoplásica de compostos que posam ser captados pela tireoide, que mostraram ter atividade antineoplásica em outros modelos de câncer, ou que são moléculas novas é muito relevante. Objetivo: Este estudo teve como objetivo fazer uma bioprospecção dos compostos bioativos Iodeto de sódio, Caleína C, Calealactona B, derivado de Caleína C, R- e S- Nuciferina, Tricina, Salcolinas A e B, Selênio-L-Metionina e Lisicamina, os quais poderiam apresentar atividade anti-neoplásica em linhagens de carcinoma anaplásico e papilífero da tireoide. Métodos: Foram utilizadas duas linhagens de carcinoma anaplásico (KTC2 e HTH83) e duas linhagens de carcinoma papilífero da tireoide (BCPAP e TPC1). Após o tratamento com os compostos, foram avaliadas a viabilidade celular por meio do ensaio de PrestoBlue™ , seguido do cálculo do IC50, migração celular por wound healing, morte celular por anexina V-FITC na presença de inibidores específicos, ativação de caspase 3/7 pelo ensaio CellEvent e geração de espécies reativas de oxigênio com o reagente DCF-DA. Ensaios de Western Blot foram feitos para determinar a modulação das vias de ERK/MAPK e PI3K/AKT, que foi complementado com estudo in sílico com as ferramentas PASS, SEA e GO Biologial Process. Foi utilizada a técnica de hanging drop para formar esferoides das linhagens, os quais foram submetidos ao tratamento crônico com os compostos e a viabilidade foi mensurada por PrestoBlue™. Resultados: Todos os compostos, com exceção do iodeto de sódio, reduziram a viabilidade celular em 72h. A Tricina e Salcolinas A e B tiveram um efeito anti-clonogênico, em HTH83 os três compostos induziram morte por necrose, enquanto em TPC1 a Tricina induziu apoptose. A Selênio-L-metionina reduziu a formação de colônias e induziu morte por apoptose confirmada por ativação de caspase 3/7. Ainda, a Selênio-L-Metionina também aumentou a geração de ROS em HTH83 e TPC1, que quando diminuído, levou a redução de apoptose em HTH83. O tratamento com Selênio-L-Metionina reduziu pAKT e pERK em KTC2. A Lisicamina também apresentou efeito anti-clonogênico e induziu morte por necrose nas quatro linhagens, aumentou a geração de ROS em HTH83 e TPC1, mas sua diminuição não afetou morte celular. Em KTC2, verificou-se que a inibição de RIPK1/RIPK3 diminuiu a morte celular, um indicativo de necroptose. Os esferoides das quatro linhagens foram mais resistentes ao tratamento com Selenio-L-Metionina e Lisicamina em comparação com a cultura em monocamada, sendo necessário adotar o tratamento crônico de até 14 dias e, em alguns casos, utilizar o dobro da concentração do IC50. Conclusões: Este estudo mostrou todos os bioativos estudados, exceto o iodeto de sódio, modulam a viabilidade celular, com certa seletividade, em células de câncer de tireoide, ainda, mostrou o potencial antitumoral da Se-L-Met e da Lisicamina, que podem modular vias associadas a stress oxidativo e morte celular. Porém, sua atividade foi diminuída no modelo de esferoide, destacando a importância de utilizar outros modelos além da cultura em monocamada.
Introduction: Most cases of thyroid cancer are papillary thyroid carcinomas (90 to 95%) and have a good prognosis, but around 10% of these cases develop more aggressiveness and, together with anaplastic carcinomas, correspond to more than 50% of deaths. For these cases, there are some inhibitors available, but these have low efficacy and many side effects, leading the patient to abandon the treatment. In this scenario, the search for new treatments is essential. Currently, some of the chemotherapeutics are, or are derived, from bioactive compounds and, therefore, researching the antineoplastic activity of compounds that can be uptake by the thyroid or that have been shown to have antineoplastic activity in other cancer models or that are new molecules is very relevant. Objective: This study aimed to carry out bioprospecting of the bioactive compounds Sodium Iodide, Calein C, Calealactone B, a derivative of Calein C, R- and S-Nuciferin, Tricine, Salcholines A and B, Selenium-L-Methionine and Lysicamine in anaplastic and papillary thyroid carcinoma cell lines. Methods: Two anaplastic carcinoma strains (KTC2 and HTH83) and two papillary thyroid carcinoma strains (BCPAP and TPC1) were used. After treatment with the compounds, cell viability was evaluated using the PrestoBlue™ assay, followed by IC50 calculation, cell migration by wound healing, cell death by annexin V-FITC in the presence of specific inhibitors, activation of caspase 3/7 by the CellEvent assay and the generation of ROS with the DCF-DA reagent. Western blot assays were performed to determine the modulation of the ERK/MAPK and PI3K/AKT pathways, which was complemented with an in silico study with the PASS, SEA, and GO Biological Process tools. The hanging drop technique was used to form spheroids from the strains, which were submitted to chronic treatment with the compounds, and the viability was measured by PrestoBlue™ . Results: All compounds except for sodium iodide reduced cell viability in 72h. Tricin and Salcolin A and B had an anti-clonogenic effect, and in HTH83 the three compounds induced death by necrosis, while in TPC1 Tricin induced apoptosis. Selenium-L-methionine reduced colony formation and induced death by apoptosis confirmed by caspase 3/7 activation. Furthermore, Selenium-L-Methionine also increased ROS generation in HTH83 and TPC1, and when decreased, led to reduced apoptosis in HTH83. Treatment with Selenium-L-Methionine reduced pAKT and pERK in KTC2. Lysicamine also showed an anti-clonogenic effect and induced death by necrosis in the four strains, increasing the generation of ROS in HTH83 and TPC1, but its decrease did not affect cell death. In KTC2, inhibition of RIPK1/RIPK3 decreased cell death, indicative of necroptosis. The spheroids of the four strains were more resistant to treatment with Selenium-L-Methionine and Lisicamine compared to the monolayer culture, requiring a chronic treatment of up to 14 days and, in some cases, using twice the IC50 concentration. Conclusions: This study showed that all bioactive evaluated except sodium iodide modulate cell viability, with selectivity, in thyroid cancer cell lines, and also reported the antitumor potential of Se-L-Met and Lisicamine, which can modulate pathways associated with oxidative stress and cell death. However, its activity decreased in the spheroid model, highlighting the importance of using other models in addition to the monolayer culture.
Introduction: Most cases of thyroid cancer are papillary thyroid carcinomas (90 to 95%) and have a good prognosis, but around 10% of these cases develop more aggressiveness and, together with anaplastic carcinomas, correspond to more than 50% of deaths. For these cases, there are some inhibitors available, but these have low efficacy and many side effects, leading the patient to abandon the treatment. In this scenario, the search for new treatments is essential. Currently, some of the chemotherapeutics are, or are derived, from bioactive compounds and, therefore, researching the antineoplastic activity of compounds that can be uptake by the thyroid or that have been shown to have antineoplastic activity in other cancer models or that are new molecules is very relevant. Objective: This study aimed to carry out bioprospecting of the bioactive compounds Sodium Iodide, Calein C, Calealactone B, a derivative of Calein C, R- and S-Nuciferin, Tricine, Salcholines A and B, Selenium-L-Methionine and Lysicamine in anaplastic and papillary thyroid carcinoma cell lines. Methods: Two anaplastic carcinoma strains (KTC2 and HTH83) and two papillary thyroid carcinoma strains (BCPAP and TPC1) were used. After treatment with the compounds, cell viability was evaluated using the PrestoBlue™ assay, followed by IC50 calculation, cell migration by wound healing, cell death by annexin V-FITC in the presence of specific inhibitors, activation of caspase 3/7 by the CellEvent assay and the generation of ROS with the DCF-DA reagent. Western blot assays were performed to determine the modulation of the ERK/MAPK and PI3K/AKT pathways, which was complemented with an in silico study with the PASS, SEA, and GO Biological Process tools. The hanging drop technique was used to form spheroids from the strains, which were submitted to chronic treatment with the compounds, and the viability was measured by PrestoBlue™ . Results: All compounds except for sodium iodide reduced cell viability in 72h. Tricin and Salcolin A and B had an anti-clonogenic effect, and in HTH83 the three compounds induced death by necrosis, while in TPC1 Tricin induced apoptosis. Selenium-L-methionine reduced colony formation and induced death by apoptosis confirmed by caspase 3/7 activation. Furthermore, Selenium-L-Methionine also increased ROS generation in HTH83 and TPC1, and when decreased, led to reduced apoptosis in HTH83. Treatment with Selenium-L-Methionine reduced pAKT and pERK in KTC2. Lysicamine also showed an anti-clonogenic effect and induced death by necrosis in the four strains, increasing the generation of ROS in HTH83 and TPC1, but its decrease did not affect cell death. In KTC2, inhibition of RIPK1/RIPK3 decreased cell death, indicative of necroptosis. The spheroids of the four strains were more resistant to treatment with Selenium-L-Methionine and Lisicamine compared to the monolayer culture, requiring a chronic treatment of up to 14 days and, in some cases, using twice the IC50 concentration. Conclusions: This study showed that all bioactive evaluated except sodium iodide modulate cell viability, with selectivity, in thyroid cancer cell lines, and also reported the antitumor potential of Se-L-Met and Lisicamine, which can modulate pathways associated with oxidative stress and cell death. However, its activity decreased in the spheroid model, highlighting the importance of using other models in addition to the monolayer culture.