Mammary carcinogenesis in female rats: contribution to monitoring and treatment
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2017-06-14
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O cancro é um importante problema de saúde pública a nível mundial. De acordo com a Organização Mundial de Saúde, no ano de 2012, esta doença afetou 14 milhões de pessoas em todo o mundo. O cancro da mama é o mais frequente na mulher, tendo sido responsável por mais de meio milhão de mortes no ano de 2012. Este tipo de cancro é também o mais frequente nas cadelas não castradas e o terceiro mais frequente na gata. O desenvolvimento de cancro da mama encontra-se associado a diversos fatores de risco, como a idade, o sexo, a raça, fatores reprodutivos, a exposição aos estrogénios, a massa corporal, mutações genéticas e o estilo de vida.
Existem diversas opções médicas para o tratamento do cancro da mama, como a cirurgia, a radioterapia, a quimioterapia, a terapia hormonal e a imunoterapia. Contudo, a possível ineficácia, a imprevisibilidade da resposta do cancro à terapia instituída e os efeitos secundários associados à terapia constituem uma permanente preocupação no tratamento do cancro. Desta forma, é essencial investir na pesquisa de novas abordagens terapêuticas que possam melhorar o prognóstico e a qualidade de vida dos doentes oncológicos.
O modelo de cancro da mama quimicamente induzido no rato pela administração do agente carcinogénico N-metil-N-nitrosureia (MNU) é internacionalmente reconhecido para o estudo da carcinogénese mamária. Este modelo permite estudar diversos aspetos da carcinogénese mamária, como a patogenia, e as alterações genéticas e moleculares, bem como avaliar novas abordagens preventivas e terapêuticas.
Com esta tese pretendemos contribuir para a monitorização e tratamento do cancro da mama utilizando este modelo animal. Neste sentido, o crescimento e a vascularização das neoplasias foram monitorizados de forma não invasiva, recorrendo a métodos de diagnóstico por imagem, e foram avaliadas duas abordagens terapêuticas: o efeito do estilo de vida (prática de exercício físico) e a ação do fármaco anti-histamínico H1 de segunda geração e estabilizador dos mastócitos - cetotifeno.
Neste trabalho foram realizados dois protocolos experimentais, um para avaliar o efeito da prática de exercício físico moderado durante 35 semanas e outro para avaliar o efeito do cetotifeno na progressão e vascularização do cancro da mama. No primeiro protocolo, 50 ratos do sexo feminino da estirpe Sprague-Dawley foram aleatoriamente divididos em quatro grupos experimentais: MNU sedentário (n=15), MNU exercitado
(n=15), controlo sedentário (n=10) e controlo exercitado (n=10). Aos 50 dias de idade, os animais dos grupos MNU receberam uma injeção intraperitoneal do agente carcinogénico MNU. Após a administração, os animais foram exercitados num tapete rolante a uma velocidade de 20 m/min, 60 min/dia, 5 dias/semana, durante 35 semanas. No segundo protocolo experimental, 34 ratos do sexo feminino da estirpe Sprague-Dawley foram aleatoriamente divididos em cinco grupos experimentais: MNU (n=10), MNU + cetotifeno-1 (n=10), MNU + cetotifeno-2 (n=10), cetotifeno (n=2) e controlo (n=2). O desenvolvimento de cancro da mama foi induzido nos animais dos grupos MNU através da administração do agente carcinogénico MNU de acordo com as condições descritas no primeiro protocolo experimental. Os animais do grupo MNU + cetotifeno-1 receberam o fármaco anti-histamínico e inibidor da desgranulação dos mastócitos cetotifeno por via oral após a administração da MNU, durante 18 semanas. Os animais do grupo MNU + cetotifeno-2 apenas receberam o cetotifeno após o desenvolvimento da primeira neoplasia mamária. Os restantes grupos foram utilizados como controlos. Em ambos os protocolos experimentais, o crescimento das neoplasias e a vascularização foram monitorizados recorrendo a métodos não invasivos (utilização do paquímetro, ultrassonografia e termografia). No final dos ensaios, os animais sobreviventes foram sacrificados e foi realizada uma necrópsia completa.
De acordo com os nossos resultados, as neoplasias da mama induzidas pela MNU em ratos do sexo feminino crescem como uma superfície circular com pouca profundidade, assemelhando-se à forma de um esferoide oblato. A ultrassonografia é o método recomendado para a determinação do volume das neoplasias in vivo, enquanto o deslocamento de água e a determinação do volume com base na massa das neoplasias constituem as metodologias mais adequadas para determinar o volume após o sacrifício dos animais ou a excisão cirúrgica das neoplasias. A ultrassonografia e a termografia são métodos de imagem recomendados para a avaliação da vascularização das neoplasias. A análise das neoplasias por microscopia eletrónica revelou semelhanças entre as neoplasias induzidas pela MNU no rato e o cancro da mama da mulher.
A prática de exercício físico moderado durante 35 semanas inibiu a carcinogénese mamária, reduzindo a inflamação, o número total de neoplasias, o número de neoplasias por animal e a sua malignidade, e aumentou o período de latência. Adicionalmente, as neoplasias mamárias dos animas exercitados exibiram maior imunoexpressão dos recetores de estrogénios α, sugerindo uma maior diferenciação destas neoplasias e melhor resposta à terapia hormonal. Apesar de apresentarem menor malignidade, as neoplasias
dos animais exercitados exibiram maior vascularização quando comparadas com as neoplasias dos animais sedentários. Foi também observado que o cancro e a prática de exercício físico induzem algumas alterações ao nível do músculo esquelético. Nos animais com neoplasias (grupos MNU) foi observada uma menor ecogenicidade do músculo gastrocnémio sugestiva de uma menor infiltração de tecido adiposo, e níveis séricos de miostatina mais elevados provavelmente devido ao efeito catabólico do processo neoplásico no tecido muscular. O músculo gastrocnémio dos animais exercitados (MNU e controlo) exibiu menor ecogenicidade quando comparada com a ecogenicidade do músculo dos animais dos respetivos grupos sedentários devido à diminuição da infiltração de tecido adiposo nos grupos exercitados.
A administração do cetotifeno inibiu a desgranulação dos mastócitos. O principal efeito positivo desta inibição na carcinogénese mamária foi a redução da proliferação das neoplasias quando a desgranulação dos mastócitos foi inibida antes do desenvolvimento das mesmas.
De acordo com os nossos resultados, a prática de exercício físico moderado durante um longo período de tempo e a administração do cetotifeno são recomendadas para a prevenção e tratamento do cancro da mama.
Cancer is a major public health problem around the globe. According to the World Health Organization, 14 million of people were affected by this disease in the year 2012. Breast cancer is the most frequent cancer among women, being responsible for more than a half a million of deaths in 2012. It is also the most frequent cancer in intact female dogs and the third most frequent in female cats. Breast cancer development is intimately associated with several risk factors, namely age, sex, race, reproductive factors, estrogens exposure, body weight, genetic mutations and lifestyle. Several medical options are available for breast cancer treatment, such as surgery, radiotherapy, chemotherapy, hormone therapy and immunotherapy. However, the possible ineffectiveness, the distinct response of cancer to the therapies and the devastating effects of some of these therapies for patients are the major concerns in cancer treatment. So, it is crucial to search for new or at least adjuvant therapies that may improve the lifespan and quality of life of oncologic patients. The rat model of chemically-induced mammary cancer by the administration of the carcinogen N-methyl-N-nitrosourea (MNU) is internationally recognized for the study of mammary carcinogenesis. It allows to better understand several aspects of mammary carcinogenesis, namely pathogenesis, genetic and molecular basis, as well as to evaluate new preventive and therapeutic approaches. With this thesis, we intend to contribute to the monitoring and treatment of mammary cancer using this animal model. For this purpose, mammary tumor growth and vascularization were non-invasively evaluated by imaging modalities, and two distinct therapeutic approaches were tested: we evaluated the role of lifestyle (practice of physical exercise) and the effects of the second-generation H1-antihistamine and mast cell stabilizer drug - ketotifen. Two distinct experimental protocols were performed: one to evaluate the effects of long-term moderate exercise training and another to evaluate the effects of ketotifen in the progression and vascularization of mammary cancer. In the first experiment, 50 female Sprague-Dawley rats were randomly divided into four experimental groups: MNU sedentary (n=15), MNU exercised (n=15), control sedentary (n=10) and control exercised (n=10). At 50 days of age, animals from MNU groups received a single intraperitoneal injection of the carcinogen agent MNU. Then, animals were exercised on a treadmill running at a velocity of 20 m/min, 60 min/day, five days a week, for 35 weeks. In the second experiment, 34 female Sprague-Dawley rats were randomly divided into five experimental groups: MNU (n=10), MNU + ketotifen-1 (n=10), MNU + ketotifen-2 (n=10), ketotifen (n=2) and control (n=2). Mammary tumor development was induced in animals from MNU groups by the administration of MNU according to the conditions described above. Animals from MNU + ketotifen-1 group were orally administered with ketotifen immediately after MNU administration for 18 weeks, while each animal from MNU + ketotifen-2 group only received ketotifen after the development of the first mammary tumor. The remaining groups were used as controls. Mammary tumor growth and vascularization were non-invasively monitored throughout both experiments, using caliper, ultrasound and thermography. At the end of the experiments, all survived animals were humanely sacrificed and submitted to a complete necropsy. According to our results, MNU-induced mammary tumors in female rats grow as a circular surface with small depth, resembling the shape of an oblate spheroid. Ultrasonography is the recommended way to determine tumor volume in vivo, while water displacement or tumor weight are the best ways to assess tumor volume after animal sacrifice or tumor excision. Ultrasound and thermographic techniques are applicable to provide information on tumor vascularization. Electron microscopy analysis revealed similar features between MNU-induced mammary tumors in rats and women mammary tumors. Lifelong moderate exercise training inhibited the carcinogenic response, by reducing inflammation, multiplicity, burden and malignancy, and increasing the cancer latency. Additionally, mammary tumors from exercised animals exhibited higher immunoexpression of estrogen receptors α, which is an indicator of tumor differentiation and better response to hormone therapy. Surprisingly, despite low malignancy, mammary tumors from exercised animals were more vascularized when compared with mammary tumors from sedentary animals. It was also observed that cancer and the practice of exercise training promoted some changes in the skeletal muscle. A lower echogenicity of gastrocnemius muscle suggestive of lower fat infiltration and higher myostatin serum levels were observed in animals with cancer (MNU-exposed groups) probably due to cancer-associated catabolic effects on body tissues. Exercised groups (MNU and control) also exhibited lower echogenicity when compared with respective sedentary groups due to the reduction of muscular fat. Ketotifen inhibited mast cell degranulation. The main positive effect of this inhibition on mammary carcinogenesis was the reduction of mammary tumor proliferation when the mast cell degranulation was inhibited before tumor development. According to our results, the practice of moderate exercise training for a long period of time and the administration of ketotifen are recommended for mammary cancer prevention and treatment.
Cancer is a major public health problem around the globe. According to the World Health Organization, 14 million of people were affected by this disease in the year 2012. Breast cancer is the most frequent cancer among women, being responsible for more than a half a million of deaths in 2012. It is also the most frequent cancer in intact female dogs and the third most frequent in female cats. Breast cancer development is intimately associated with several risk factors, namely age, sex, race, reproductive factors, estrogens exposure, body weight, genetic mutations and lifestyle. Several medical options are available for breast cancer treatment, such as surgery, radiotherapy, chemotherapy, hormone therapy and immunotherapy. However, the possible ineffectiveness, the distinct response of cancer to the therapies and the devastating effects of some of these therapies for patients are the major concerns in cancer treatment. So, it is crucial to search for new or at least adjuvant therapies that may improve the lifespan and quality of life of oncologic patients. The rat model of chemically-induced mammary cancer by the administration of the carcinogen N-methyl-N-nitrosourea (MNU) is internationally recognized for the study of mammary carcinogenesis. It allows to better understand several aspects of mammary carcinogenesis, namely pathogenesis, genetic and molecular basis, as well as to evaluate new preventive and therapeutic approaches. With this thesis, we intend to contribute to the monitoring and treatment of mammary cancer using this animal model. For this purpose, mammary tumor growth and vascularization were non-invasively evaluated by imaging modalities, and two distinct therapeutic approaches were tested: we evaluated the role of lifestyle (practice of physical exercise) and the effects of the second-generation H1-antihistamine and mast cell stabilizer drug - ketotifen. Two distinct experimental protocols were performed: one to evaluate the effects of long-term moderate exercise training and another to evaluate the effects of ketotifen in the progression and vascularization of mammary cancer. In the first experiment, 50 female Sprague-Dawley rats were randomly divided into four experimental groups: MNU sedentary (n=15), MNU exercised (n=15), control sedentary (n=10) and control exercised (n=10). At 50 days of age, animals from MNU groups received a single intraperitoneal injection of the carcinogen agent MNU. Then, animals were exercised on a treadmill running at a velocity of 20 m/min, 60 min/day, five days a week, for 35 weeks. In the second experiment, 34 female Sprague-Dawley rats were randomly divided into five experimental groups: MNU (n=10), MNU + ketotifen-1 (n=10), MNU + ketotifen-2 (n=10), ketotifen (n=2) and control (n=2). Mammary tumor development was induced in animals from MNU groups by the administration of MNU according to the conditions described above. Animals from MNU + ketotifen-1 group were orally administered with ketotifen immediately after MNU administration for 18 weeks, while each animal from MNU + ketotifen-2 group only received ketotifen after the development of the first mammary tumor. The remaining groups were used as controls. Mammary tumor growth and vascularization were non-invasively monitored throughout both experiments, using caliper, ultrasound and thermography. At the end of the experiments, all survived animals were humanely sacrificed and submitted to a complete necropsy. According to our results, MNU-induced mammary tumors in female rats grow as a circular surface with small depth, resembling the shape of an oblate spheroid. Ultrasonography is the recommended way to determine tumor volume in vivo, while water displacement or tumor weight are the best ways to assess tumor volume after animal sacrifice or tumor excision. Ultrasound and thermographic techniques are applicable to provide information on tumor vascularization. Electron microscopy analysis revealed similar features between MNU-induced mammary tumors in rats and women mammary tumors. Lifelong moderate exercise training inhibited the carcinogenic response, by reducing inflammation, multiplicity, burden and malignancy, and increasing the cancer latency. Additionally, mammary tumors from exercised animals exhibited higher immunoexpression of estrogen receptors α, which is an indicator of tumor differentiation and better response to hormone therapy. Surprisingly, despite low malignancy, mammary tumors from exercised animals were more vascularized when compared with mammary tumors from sedentary animals. It was also observed that cancer and the practice of exercise training promoted some changes in the skeletal muscle. A lower echogenicity of gastrocnemius muscle suggestive of lower fat infiltration and higher myostatin serum levels were observed in animals with cancer (MNU-exposed groups) probably due to cancer-associated catabolic effects on body tissues. Exercised groups (MNU and control) also exhibited lower echogenicity when compared with respective sedentary groups due to the reduction of muscular fat. Ketotifen inhibited mast cell degranulation. The main positive effect of this inhibition on mammary carcinogenesis was the reduction of mammary tumor proliferation when the mast cell degranulation was inhibited before tumor development. According to our results, the practice of moderate exercise training for a long period of time and the administration of ketotifen are recommended for mammary cancer prevention and treatment.
Descrição
Tese de Doutoramento em Ciências Veterinárias - Clínica
Palavras-chave
Neoplasias mamárias animais , Carcinógenese química , Cetotifeno , Ultrassonografia mamária , Modelo animal , Rato , Uso terapêutico , N-metil-N-nitrosureia (MNU)