O sarcoma de Ewing (SE) é um tumor caracterizado por possuir células indiferenciadas, de origem neuroepitelial. É o segundo tumor ósseo maligno mais comum em crianças e adolescentes. O tratamento consiste na aplicação de quimioterapia neoadjuvante, seguida da cirurgia e a quimioterapia adjuvante ou radioterapia. Contudo, a disseminação deste tumor é muito intensa, e mesmo em pacientes onde a metástase não ocorreu, podem haver células tumorais circulantes. Além disso, a sobrevida de 5 anos que cerca de 70% dos pacientes que não apresentam metástase no momento do diagnóstico atingem, cai para 25% nos pacientes que apresentam metástase. A migração e invasão celular dos tecidos vizinhos e vasos sanguíneos, a adesão celular e a proliferação são processos essenciais para que a metástase tumoral aconteça. Dentre as reguladoras da mobilidade, adesão celular e proliferação estão as GTPases da família Rho, que possuem entre suas quinases efetoras, ROCK1 e ROCK2. Assim, as proteínas ROCK induzem inúmeras respostas celulares que envolvem a regulação de muitas proteínas associadas ao citoesqueleto. Estudos evidenciam que respostas sinalizadas por ROCK agravam fenótipos associados ao câncer e outras doenças. O presente trabalho teve como objetivo verificar a expressão dos genes ROCK1 e ROCK2 em amostras tumorais e linhagens celulares de SE, e os efeitos in vitro da inibição farmacológica de ambas as quinases. O estudo da expressão gênica de ROCK1 e ROCK2 em amostras de pacientes com SE (n=18) revelaram uma hipoexpressão de ambos genes, tanto nas amostras de pacientes com SE quanto nas linhagens celulares SK-ES-1 e RD-ES. Não foi encontrada nenhuma relação das expressões gênicas com os dados clínicos. Contudo, a presença do gene de fusão EWS-FLI1 parece estar associada com menor expressão de ROCK1. Verificou-se que a proliferação celular, a capacidade clonogênica e o ciclo celular não se alteraram significativamente nas linhagens celulares de SE após os diferentes tratamentos com as drogas GSK429286 (inibidor de ROCK1), SR3677 (inibidor de ROCK2) e Hidroxifasudil (pan-inibidor). Porém, apesar de não significativo, observou-se um tênue aumento na migração e invasão celular após o tratamento com as drogas. Nossos dados indicam que ROCK1 e ROCK2 podem possuir um papel na tumorigênese do SE, e ainda estar relacionadas com os mecanismos de migração e invasão celular, processos importantes para a metástase tumoral. / Ewing\'s sarcoma (ES) is characterized by undifferentiated cells of neuroepithelial origin. It is the second most common malignant bone tumor in children and adolescents. Standard treatment consists of preoperative chemotherapy, followed by surgery and postoperative chemotherapy and/or radiotherapy. However, the spread of the tumor is very high and even in patients where the metastasis has not occurred, there may be circulating tumor cells. Furthermore, the 5-year survival which about 70% of patients without metastases at diagnosis achieve, falls to 25% in patients with metastases. Migration and invasion into adjacent tissues and blood vessels, cell adhesion and proliferation are essential for tumor metastasis to occur. Among the regulators of the processes are the Rho family of GTPases, which have as their effector kinases, ROCK1 and ROCK2. Thus, ROCK induce numerous cellular responses involving the regulation of many proteins associated with the cytoskeleton. Studies show that responses regulated by ROCK aggravate phenotypes associated with cancer and other diseases. This study aimed to verify the expression of ROCK1 and ROCK2 genes in ES tumor samples and to evaluate the effects of their pharmacological inhibition in vitro. Gene expression analysis showed lower expression of ROCK in both, patient samples (n=18) and the SK-ES- 1 and RD-ES cell lines. There was no relation of gene expression with clinical data. Nonetheless, the presence of EWS-FLI1 fusion appears to be associated with lower expression of ROCK1. Cell proliferation, clonogenic capacity and the cell cycle were not affected after different treatments with GSK429286 (ROCK1 inhibitor), SR3677 (ROCK2 inhibitor) and Hydroxyfasudil (pan-inhibitor). However, although not significant, there was a tenuous increase in cell migration and invasion following treatment with the drugs. Our data suggest that ROCK1 and ROCK2 might have a role in ES tumorigenesis and may be related im part to migration and invasion cell mechanisms, important processes for tumor metastasis.

Cardiac hypertrophy is a compensatory response to increased work load or stress on the heart, but over time can lead to heart failure and death. The molecular mechanisms underlying this disease are still not completely understood, however the Rho/Rho kinase pathway has been shown to play a role. N-[11C]-methyl-hydroxyfasudil, a PET radiotracer, binds to active Rho kinase and could be a possible tracer for hypertrophy. Hypertrophy was induced in vitro using the β-adrenergic receptor agonist isoproterenol to evaluate optimal Rho kinase activity. Rho kinase activity data was correlated to N-[11C]-methyl-hydroxyfasudil binding. Cardiac hypertrophy was verified with an increase in nuclear size (1.74 fold) and cell size (~2 fold), activation of hypertrophic signalling pathways, and increased Rho kinase activity (1.64 fold). This correlated to a 10.3% increase in N-[11C]-methyl-hydroxyfasudil binding. This data suggests that N-[11C]-methyl-hydroxyfasudil may be useful as a radiotracer for detecting cardiac hypertrophy and merits further in vivo investigation.

Cardiac hypertrophy is a compensatory response to increased work load or stress on the heart, but over time can lead to heart failure and death. The molecular mechanisms underlying this disease are still not completely understood, however the Rho/Rho kinase pathway has been shown to play a role. N-[11C]-methyl-hydroxyfasudil, a PET radiotracer, binds to active Rho kinase and could be a possible tracer for hypertrophy. Hypertrophy was induced in vitro using the β-adrenergic receptor agonist isoproterenol to evaluate optimal Rho kinase activity. Rho kinase activity data was correlated to N-[11C]-methyl-hydroxyfasudil binding. Cardiac hypertrophy was verified with an increase in nuclear size (1.74 fold) and cell size (~2 fold), activation of hypertrophic signalling pathways, and increased Rho kinase activity (1.64 fold). This correlated to a 10.3% increase in N-[11C]-methyl-hydroxyfasudil binding. This data suggests that N-[11C]-methyl-hydroxyfasudil may be useful as a radiotracer for detecting cardiac hypertrophy and merits further in vivo investigation.

O sarcoma de Ewing (SE) é um tumor caracterizado por possuir células indiferenciadas, de origem neuroepitelial. É o segundo tumor ósseo maligno mais comum em crianças e adolescentes. O tratamento consiste na aplicação de quimioterapia neoadjuvante, seguida da cirurgia e a quimioterapia adjuvante ou radioterapia. Contudo, a disseminação deste tumor é muito intensa, e mesmo em pacientes onde a metástase não ocorreu, podem haver células tumorais circulantes. Além disso, a sobrevida de 5 anos que cerca de 70% dos pacientes que não apresentam metástase no momento do diagnóstico atingem, cai para 25% nos pacientes que apresentam metástase. A migração e invasão celular dos tecidos vizinhos e vasos sanguíneos, a adesão celular e a proliferação são processos essenciais para que a metástase tumoral aconteça. Dentre as reguladoras da mobilidade, adesão celular e proliferação estão as GTPases da família Rho, que possuem entre suas quinases efetoras, ROCK1 e ROCK2. Assim, as proteínas ROCK induzem inúmeras respostas celulares que envolvem a regulação de muitas proteínas associadas ao citoesqueleto. Estudos evidenciam que respostas sinalizadas por ROCK agravam fenótipos associados ao câncer e outras doenças. O presente trabalho teve como objetivo verificar a expressão dos genes ROCK1 e ROCK2 em amostras tumorais e linhagens celulares de SE, e os efeitos in vitro da inibição farmacológica de ambas as quinases. O estudo da expressão gênica de ROCK1 e ROCK2 em amostras de pacientes com SE (n=18) revelaram uma hipoexpressão de ambos genes, tanto nas amostras de pacientes com SE quanto nas linhagens celulares SK-ES-1 e RD-ES. Não foi encontrada nenhuma relação das expressões gênicas com os dados clínicos. Contudo, a presença do gene de fusão EWS-FLI1 parece estar associada com menor expressão de ROCK1. Verificou-se que a proliferação celular, a capacidade clonogênica e o ciclo celular não se alteraram significativamente nas linhagens celulares de SE após os diferentes tratamentos com as drogas GSK429286 (inibidor de ROCK1), SR3677 (inibidor de ROCK2) e Hidroxifasudil (pan-inibidor). Porém, apesar de não significativo, observou-se um tênue aumento na migração e invasão celular após o tratamento com as drogas. Nossos dados indicam que ROCK1 e ROCK2 podem possuir um papel na tumorigênese do SE, e ainda estar relacionadas com os mecanismos de migração e invasão celular, processos importantes para a metástase tumoral. / Ewing\'s sarcoma (ES) is characterized by undifferentiated cells of neuroepithelial origin. It is the second most common malignant bone tumor in children and adolescents. Standard treatment consists of preoperative chemotherapy, followed by surgery and postoperative chemotherapy and/or radiotherapy. However, the spread of the tumor is very high and even in patients where the metastasis has not occurred, there may be circulating tumor cells. Furthermore, the 5-year survival which about 70% of patients without metastases at diagnosis achieve, falls to 25% in patients with metastases. Migration and invasion into adjacent tissues and blood vessels, cell adhesion and proliferation are essential for tumor metastasis to occur. Among the regulators of the processes are the Rho family of GTPases, which have as their effector kinases, ROCK1 and ROCK2. Thus, ROCK induce numerous cellular responses involving the regulation of many proteins associated with the cytoskeleton. Studies show that responses regulated by ROCK aggravate phenotypes associated with cancer and other diseases. This study aimed to verify the expression of ROCK1 and ROCK2 genes in ES tumor samples and to evaluate the effects of their pharmacological inhibition in vitro. Gene expression analysis showed lower expression of ROCK in both, patient samples (n=18) and the SK-ES- 1 and RD-ES cell lines. There was no relation of gene expression with clinical data. Nonetheless, the presence of EWS-FLI1 fusion appears to be associated with lower expression of ROCK1. Cell proliferation, clonogenic capacity and the cell cycle were not affected after different treatments with GSK429286 (ROCK1 inhibitor), SR3677 (ROCK2 inhibitor) and Hydroxyfasudil (pan-inhibitor). However, although not significant, there was a tenuous increase in cell migration and invasion following treatment with the drugs. Our data suggest that ROCK1 and ROCK2 might have a role in ES tumorigenesis and may be related im part to migration and invasion cell mechanisms, important processes for tumor metastasis.

Cardiac hypertrophy is a compensatory response to increased work load or stress on the heart, but over time can lead to heart failure and death. The molecular mechanisms underlying this disease are still not completely understood, however the Rho/Rho kinase pathway has been shown to play a role. N-[11C]-methyl-hydroxyfasudil, a PET radiotracer, binds to active Rho kinase and could be a possible tracer for hypertrophy. Hypertrophy was induced in vitro using the β-adrenergic receptor agonist isoproterenol to evaluate optimal Rho kinase activity. Rho kinase activity data was correlated to N-[11C]-methyl-hydroxyfasudil binding. Cardiac hypertrophy was verified with an increase in nuclear size (1.74 fold) and cell size (~2 fold), activation of hypertrophic signalling pathways, and increased Rho kinase activity (1.64 fold). This correlated to a 10.3% increase in N-[11C]-methyl-hydroxyfasudil binding. This data suggests that N-[11C]-methyl-hydroxyfasudil may be useful as a radiotracer for detecting cardiac hypertrophy and merits further in vivo investigation.