Histopathology and immunohistochemistry characterization of mammary lesions chemically-induced by 7,12-dimethylbenz(a)anthracene and 1-methyl-1-nitrosourea in female Sprague-Dawley rats
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2018-01-24
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As neoplasias de mama quimicamente induzidas pelos agentes carcinogénicos 7,12-dimetilbenz (a) antraceno (DMBA) e 1-metil-1-nitrosureia (MNU) em ratos têm sido frequentemente utilizadas como modelo para o estudo do cancro de mama humano, devido às semelhanças em termos da sua histopatologia e dependência hormonal. O receptor de estrogénio α (ERα), o receptor de progesterona (PR) e a proteína Ki-67 são considerados bons marcadores de prognóstico e fatores preditivos no cancro da mama. Este estudo teve como objetivo avaliar o comportamento biológico das lesões da mama induzidas pela MNU e pelo DMBA em ratos do sexo feminino da estirpe Sprague-Dawley, através da imunoexpressão dos marcadores de prognóstico ERα, PR e Ki-67, de modo a identificar o modelo mais adequado para o estudo do cancro da mama na mulher. Neste estudo também foram avaliados os efeitos da prática de exercício físico durante 35 semanas na imunoexpressão dos fatores de prognóstico ERα e Ki-67, no índice de atividade mitótica (MAI), e no desenvolvimento de metástases em neoplasias mamárias induzidas pela MNU em ratos do sexo feminino da estirpe Sprague-Dawley.
Para este estudo, um grupo de animais do sexo feminino da estirpe Sprague-Dawley recebeu uma única dose do agente carcinogénico DMBA (65 mg/kg) por gavage aos 50 diasde idade. Outro grupo de animais foi dividido aleatoriamente em quatro grupos experimentais: MNU sedentário, MNU exercitado, controlo sedentário e controlo exercitado. Aos 50 dias de idade, os animais dos grupos MNU receberam uma única injeção intraperitoneal do agente carcinogénico MNU (50 mg/kg). Os animais dos grupos controlo receberam uma injeção intraperitoneal do veículo (solução salina a 0,9%). No dia seguinte à administração, os animais dos grupos exercitados (grupos MNU e controlo) foram treinados num tapete rolante a uma velocidade de 20 m/min, 60 min/dia, 5 dias/semana durante 35 semanas. Os animais do grupo DMBA foram sacrificados 27 semanas após a administração do agente carcinogénico, enquanto os animais dos grupo MNU e controlo foram sacrificados 35 semanas após da administração do agente carcinogénico. As neoplasias mamárias induzidas pelos agentes carcinogénicos foram classificados por histopatologia; os órgãos das cavidades torácica e abdominal foram avaliados para o estudo das metástases. A expressão dos marcadores de prognóstico ERα, PR e índice de proliferação Ki-67 (Ki-67 PI) foi avaliada por imuno-histoquímica. O ERα-H-Score e o MAI também foram avaliados.
Assim, para um dos estudos desta tese foram analisadas 28 neoplasias mamárias induzidas pela MNU no grupo sedentário e 16 neoplasias mamárias induzidas pelo DMBA. Em cada neoplasia mamária foi identificado mais de um padrão histológico, assim apenas os carcinomas mamários foram analisados de modo a uniformizar as amostras. Todos os carcinomas mamários induzidos pela MNU e pelo DMBA foram positivos para o ERα e o PR (ER+/PR+), com maior expressão do ERα quando comparado com a expressão do PR. O peso das neoplasias, a expressão do ERα, do PR, o Ki-67 PI e o MAI foram mais elevados nos carcinomas mamários induzidos pela MNU quando comparados com os carcinomas induzidos pelo DMBA. Foram observadas diferenças estatisticamente significativas entre os grupos para ERα, o PR e o MAI (p<0,05).
No trabalho seguinte foram analisadas e comparadas as neoplasias mamárias induzidas pela MNU nos grupos exercitados e sedentários. Todas as neoplasias de ambos grupos foram positivas para o ERα com um H-score 20. Embora não tenham sido observadas diferenças estatisticamente significativas no ERα H-Score, no Ki-67 PI e no MAI entre os grupos, o valor absoluto do ERα H-Score foi maior no grupo exercitado, enquanto o Ki-67 PI e o MAI foram mais elevados no grupo sedentário. Finalmente, no estudo das metástases observou-se que os animais exercitados não desenvolveram qualquer metástase, enquanto que duas metástases pulmonares foram observadas no grupo sedentário. À semelhança dos carcinomas mamários, as metástases foram positivas para o ERα e para o PR, indicando uma elevada sensibilidade hormonal.
Os elevados valores do KI-67 PI e do MAI em carcinomas mamários induzidos pela MNU são indicadores de uma maior agressividade destes carcinomas quando comparados com os induzidos pelo DMBA, sugerindo uma pior resposta à terapia e um pior prognóstico. Estes dados sugerem que o modelo de cancro da mama em ratos induzidos pela MNU é aconselhado em protocolos experimentais que tenham como objectivo o estudo de carcinomas mamários mais agressivos dentro do grupo dos carcinomas mamários positivos para ambos os recetores hormonais (ER+/PR+). As neoplasias do grupo MNU exercitado foram menos proliferativas e mais diferenciadas quando comparadas com as neoplasias mamárias do grupo MNU sedentário, sugerindo que a prática de exercício físico durante um longo período de tempo teve efeitos positivos na carcinogénese mamária. Finalmente, foi possível concluir que a prática de exercício físico durante um longo período de tempo diminuíu o risco de formação de metástases do cancro da mama, este fenómeno ocorreu na presença de níveis de estrogénio
aumentados, com lesões primárias e metastáticas sensíveis a hormonas esteróides, indicando que este efeito foi independente da estimulação hormonal. A prática de exercício físico durante um longo período de tempo parece ser benéfica para doentes com cancro de mama, através do aumento da diferenciação das neoplasias mamárias, da diminuição da proliferação e da inibição da formação de metástases.
Chemically-induced mammary tumors in rats by the carcinogens 7,12-dimethylbenz(a)anthracene (DMBA) and 1-metil-1-nitrosourea (MNU) are the most widely used models for studies on mammary human carcinogenesis because of their similar histopathology and hormone dependence. Estrogen receptor α (ERα), progesterone receptor (PR) and Ki-67 protein are considered strong prognostic and predictive markers in breast cancer. This study aimed to assess the biological behavior of MNU and DMBA-induced mammary tumors in female Sprague-Dawley rats, through the immunoexpression of prognostic markers ERα, PR and Ki-67, in order to know the model that best suits to woman breast cancer. The effects of 35 weeks of exercise training on the immunoexpression of prognostic factors ERα, Ki-67 and the mitotic activity index (MAI), and the development of metastasis in mammary tumors MNU-induced in female Sprague-Dawley rats were also evaluated. For this study, a group of animals received a single dose of the carcinogen DMBA (65 mg/kg) by gavage at 50 days of age. Other group of animals was randomly divided into four experimental groups: MNU sedentary, MNU exercised, control sedentary and control exercised. The MNU groups received a single intraperitoneal injection of the carcinogen MNU (50 mg/kg) at 50 days of age. Control groups received an intraperitoneal injection of the vehicle (saline solution 0.9%). After this, animals from exercised groups (MNU and control groups) were exercised in a treadmill at a velocity of 20 m/min, 60 min/day, 5 days/week for 35 weeks. Animals from DMBA group were sacrificed 27 weeks after the carcinogen administration, while the animals from MNU and control groups were sacrificed 35 weeks after the carcinogen administration. Mammary tumors induced by both carcinogens were histologically classified; the organs from thoracic and abdominal cavities were evaluated for the metastasis study. The expression of the prognostic markers ERα, PR and Ki-67 proliferation index (Ki-67 PI) were assessed by immunohistochemistry. ERαH-Score and MAI were also evaluated. Thus for one of the studies of this these, 28 MNU-induced mammary tumors from sedentary group and 16 DMBA-induced mammary tumors were analyzed. More than one histological pattern was identified in each mammary tumor, so in order to homogenize the samples only mammary carcinomas were analyzed. All MNU and DMBA-induced mammary carcinomas were ERα and PR positive (ER+/PR+), with a higher expression of ERα when compared with PR. Tumor weight, the expression of ERα, PR, Ki-67 PI and MAI were higher in MNU-induced mammary carcinomas when compared with the DMBA-induced ones. Statistically significant differences between groups were observed for ERα, PR and MAI (p<0.05). In the following study, MNU-induced mammary tumors in exercised and sedentary groups were analyzed and compared. All neoplasms from both groups were ERα-positive with an H-score 20. Although statistically significant differences were not found in the ERα H-score, Ki-67 PI and MAI between groups, the absolute value of ERα H-score was higher in exercised group, while the Ki-67 PI and MAI were higher in sedentary group. Finally, in the metastasis study, it was observed that exercised animals did not develop any metastasis while two pulmonary metastases were observed in the sedentary group. Like the mammary carcinomas, their metastases were positive for ERα and PR, indicating high hormonal sensitivity. The higher KI-67 PI and MAI in MNU-induced mammary carcinomas was indicative of a higher aggressiveness of these carcinomas when compared with the DMBA-induced ones, suggesting a worse response to the therapy and a worse prognosis. These data suggest that the rat model of MNU-induced mammary tumors is advised in experimental protocols aiming to study more aggressive mammary carcinomas within the group of double-positive mammary tumors (ER+/PR+). Tumors from MNU exercised group were less proliferative and more differentiated when compared with mammary tumors from MNU sedentary group, suggesting that long-term exercise training had positive effects on mammary carcinogenesis. Finally, it was possible to conclude that long-term exercise training may decrease the risk of mammary cancer metastasis; this occurred in the presence of enhanced oestrogen levels, with hormone-sensitive primary and metastatic lesions, indicating that the effect was hormone-independent. The practice of exercise training during a long period of time seems to be beneficial for breast cancer patients, through the increase of mammary neoplasms differentiation, decrease of proliferation and inhibition of metastasis.
Chemically-induced mammary tumors in rats by the carcinogens 7,12-dimethylbenz(a)anthracene (DMBA) and 1-metil-1-nitrosourea (MNU) are the most widely used models for studies on mammary human carcinogenesis because of their similar histopathology and hormone dependence. Estrogen receptor α (ERα), progesterone receptor (PR) and Ki-67 protein are considered strong prognostic and predictive markers in breast cancer. This study aimed to assess the biological behavior of MNU and DMBA-induced mammary tumors in female Sprague-Dawley rats, through the immunoexpression of prognostic markers ERα, PR and Ki-67, in order to know the model that best suits to woman breast cancer. The effects of 35 weeks of exercise training on the immunoexpression of prognostic factors ERα, Ki-67 and the mitotic activity index (MAI), and the development of metastasis in mammary tumors MNU-induced in female Sprague-Dawley rats were also evaluated. For this study, a group of animals received a single dose of the carcinogen DMBA (65 mg/kg) by gavage at 50 days of age. Other group of animals was randomly divided into four experimental groups: MNU sedentary, MNU exercised, control sedentary and control exercised. The MNU groups received a single intraperitoneal injection of the carcinogen MNU (50 mg/kg) at 50 days of age. Control groups received an intraperitoneal injection of the vehicle (saline solution 0.9%). After this, animals from exercised groups (MNU and control groups) were exercised in a treadmill at a velocity of 20 m/min, 60 min/day, 5 days/week for 35 weeks. Animals from DMBA group were sacrificed 27 weeks after the carcinogen administration, while the animals from MNU and control groups were sacrificed 35 weeks after the carcinogen administration. Mammary tumors induced by both carcinogens were histologically classified; the organs from thoracic and abdominal cavities were evaluated for the metastasis study. The expression of the prognostic markers ERα, PR and Ki-67 proliferation index (Ki-67 PI) were assessed by immunohistochemistry. ERαH-Score and MAI were also evaluated. Thus for one of the studies of this these, 28 MNU-induced mammary tumors from sedentary group and 16 DMBA-induced mammary tumors were analyzed. More than one histological pattern was identified in each mammary tumor, so in order to homogenize the samples only mammary carcinomas were analyzed. All MNU and DMBA-induced mammary carcinomas were ERα and PR positive (ER+/PR+), with a higher expression of ERα when compared with PR. Tumor weight, the expression of ERα, PR, Ki-67 PI and MAI were higher in MNU-induced mammary carcinomas when compared with the DMBA-induced ones. Statistically significant differences between groups were observed for ERα, PR and MAI (p<0.05). In the following study, MNU-induced mammary tumors in exercised and sedentary groups were analyzed and compared. All neoplasms from both groups were ERα-positive with an H-score 20. Although statistically significant differences were not found in the ERα H-score, Ki-67 PI and MAI between groups, the absolute value of ERα H-score was higher in exercised group, while the Ki-67 PI and MAI were higher in sedentary group. Finally, in the metastasis study, it was observed that exercised animals did not develop any metastasis while two pulmonary metastases were observed in the sedentary group. Like the mammary carcinomas, their metastases were positive for ERα and PR, indicating high hormonal sensitivity. The higher KI-67 PI and MAI in MNU-induced mammary carcinomas was indicative of a higher aggressiveness of these carcinomas when compared with the DMBA-induced ones, suggesting a worse response to the therapy and a worse prognosis. These data suggest that the rat model of MNU-induced mammary tumors is advised in experimental protocols aiming to study more aggressive mammary carcinomas within the group of double-positive mammary tumors (ER+/PR+). Tumors from MNU exercised group were less proliferative and more differentiated when compared with mammary tumors from MNU sedentary group, suggesting that long-term exercise training had positive effects on mammary carcinogenesis. Finally, it was possible to conclude that long-term exercise training may decrease the risk of mammary cancer metastasis; this occurred in the presence of enhanced oestrogen levels, with hormone-sensitive primary and metastatic lesions, indicating that the effect was hormone-independent. The practice of exercise training during a long period of time seems to be beneficial for breast cancer patients, through the increase of mammary neoplasms differentiation, decrease of proliferation and inhibition of metastasis.
Descrição
Tese de Doutoramento em Ciências Veterinárias
Palavras-chave
Recetores estrogénicos , Recetores de progesterona , Neoplasias mamárias animais , Proteína Ki-67 , Carcinogénese química , Rato , Modelo animal , Imuno-histoquímica , Índice mitótico