Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 168-181.

Chronic inflammation has been linked to cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory lipid and one of the downstream products of 2 isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Although both COX isoforms have similar structure and function, they are encoded by different genes and show distinct expression patterns. COX-1 is expressed in most cells and tissues and remains constant under most physiologic conditions to play a housekeeping role whereas the COX-2 form is inducible and usually only expressed in response to various inflammatory stimuli. COX enzymes may be involved in both tumor establishment and maintenance of existing tumors. PGE2 exerts its effects on target cells by coupling to four subtypes of receptors which have been classified as EP1-4. Ovarian cancer is the most lethal gynecological malignancy and mainly occurs in older women. Prevention may be the best approach to reduce ovarian cancer. Ovarian cancer is the fifth leading cause of cancer death among women and the most lethal gynecological malignancy. There are at least 3 well established risk factors for ovarian cancer: age, family history and environmental factors. Ovarian cancer is mainly seen in older women when their ovaries are not reproductively functional. Close to half of the women with ovarian cancer (48%) are in the age group of 65 or older. Epidemiological and preclinical studies indicate that increased dietary intake of omega-3 fatty acids (OM-3FAs) reduces the incidence and growth of various cancers. Thus, increasing the consumption of OM-3FAs may be a nontoxic way to prevent or suppress ovarian cancer. Flaxseed is the richest vegetable source of omega-3 fatty acids which may be effective in the prevention of ovarian cancer. Fish oil is a source of OM-3FAs which may be effective in prevention of ovarian cancer. The main OM-6FA, Linoleic Acid (LA), is a direct precursor of the Arachidonic Acid (AA). Alpha-linolenic acid (ALA) is the main OM-3FA found in flax oil, whereas eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the OM-3FAs in fish oil. ALA is elongated to form EPA and DHA in the intestine. Celecoxib is a non-steroidal anti-inflammatory (NSAID) drug that selectively inhibits COX-2. There are evidences showing that Celecoxib has some anti-cancer properties. Progress in the treatment and prevention of ovarian cancer has been hampered due to the lack of an appropriate animal model and absence of effective chemo-prevention strategies. The domestic hens spontaneously develop ovarian adenocarcinomas that share similar histological appearance and symptoms such as ascites and metastasis with humans. Our first objective was to investigate the effect of flaxseed supplementation for one year on ovarian cancer and correlate its effects to expression of COX enzymes and concentrations of prostaglandins. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for one year. The severity of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 localization and protein and mRNA expression and PGE2 and PGE3 concentrations in ovaries were measured by Immunohistochemistry, western blot, quantitative real-time PCR and LC-MS-MS, respectively. The results demonstrated a significant reduction in late stage ovarian tumors in the flaxseed-fed hens compared with the control diet-fed hens. In correlation with decreased ovarian cancer severity, concentrations of PGE2 and expression of COX-2 were diminished in ovaries of flaxseed-fed hens. PGE3 concentrations were below the level of detection. The results demonstrated that in normal ovaries, COX-1 was localized to the granulosa cell layer surrounding the follicles and ovarian surface epithelium (OSE) whereas COX-2 protein was localized to the granulosa cell layer in the follicle. Extensive COX-1 and COX-2 protein expression was found throughout the ovarian carcinoma. Our findings suggest that the flaxseed-mediated reduction in the severity of ovarian cancer in hens is correlated to the reduction in PGE2 in the ovaries of flaxseed-fed hens. Since no effect on ovarian cancer incidence was detected after feeding the 2. 5 year old hens with 10% flaxseed for 1 year, we designed a long-term study using 6 month old hens. Our objectives were: 1) to examine the expression of COX enzymes and PGE2 levels in ovaries and correlate them to ovarian cancer and aging 2) to determine if long-term consumption of a flaxseed enriched diet decreases ovarian cancer severity and incidence in the laying hen and to investigate its potential correlation with the expression of COX enzymes and PGE2 concentration. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for 4 years. The severity and incidence of ovarian cancer were determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE2 concentrations in ovaries were measured by western blot, quantitative real-time PCR and ELISA, respectively. Our results indicated an increase in ovarian cancer incidence and expression of both COX enzymes in ovaries of older hens. In correlation with ovarian cancer incidence and COX enzymes expression, PGE2 concentrations were elevated with age. Ovaries with tumor had elevated COX-1 expression and PGE2 concentration compared to normal ovaries. Our findings suggest that the up-regulation of COX enzymes with age is the main contributing factor in the age associated increase in PGE2. Furthermore, elevated PGE2 in ovaries of hens concomitant with age suggests its important role in early stages of ovarian carcinogenesis. The results demonstrated that there was a reduction in ovarian cancer severity and incidence in hens fed flaxseed diet. In correlation with decreased ovarian cancer severity and incidence, concentration of PGE2 and expression of COX-2 were diminished in ovaries of hens fed flaxseed. Our findings suggest that the lower levels of COX-2 and PGE2 are the main contributing factors in the chemo-suppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer. Based on our previous findings, targeting COX expression and prostaglandin biosynthesis by dietary intervention using OM-3FAs and selective COX inhibitor can be an effective approach to prevent or suppress ovarian cancer. Thus, we conducted a series of studies to assess effect of fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined or flax oil and Celecoxib combined on COX-1 and COX-2 expression, PGE2 concentrations, proliferation and apoptosis in normal and cancerous ovaries of laying hens. This study had not been performed in hens before, thus the first step was to find the optimum doses. In order to do so, we utilized one year old hens, divided them to groups of 6 hens, and fed them different doses of fish oil (50, 100, 175, 375 and 700 mg/kg), flax oil (100, 250, 500, 1000 and 1500 mg/kg) or Celecoxib (35, 65 and 100 mg/kg) for three weeks. The OM3-FAs andomega-6 fatty acids contents of egg yolks were determined by gas chromatography. Proliferation, apoptosis,COX-1, COX-2 and prostaglandin receptor subtype 4 (EP4) protein and mRNA expression and PGE2 concentration in ovaries were measured by PCNA, TUNEL, western blot, quantitative real-time qPCR and ELISA, respectively. The results indicated that 100 mg/kg fish oil was the most effective dose in reducing COX enzymes and PGE2, and increased apoptosis and reduced proliferation in ovaries. The lower doses of fish oil incorporated more OM-3FAs into yolks, reduced OM-6FAs and increased the egg laying frequency but did not affect EP4 expression. Unlike fish oil, the highest dose of flax oil (1500 mg/kg) caused the most significant reduction in COX expression and PGE2 concentration. Celecoxib was not perfectly selective in targeting COX-2, however, treating the hens with 65 mg/kg Celecoxib resulted in the most significant amelioration of PGE2 levels in ovaries. Using the optimum doses of fish oil, flax oil and Celecoxib, we aimed to investigate if these components can alter ovarian cancer end-points in normal and cancerous hen ovaries. There is an adverse relation between ovulation and health of ovaries. Thus, 3-4 year old hens were monitored for egg laying frequency and the hens with the least ovulation rate were selected for health assessment. The hens presenting poor health were scanned using ultrasound and if tumor mass and/or ascites were detected, they were chosen for this study. The hens with normal and cancerous ovaries were divided to groups and were fed fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined, or flax oil and Celecoxib combined for 42 days. The results showed that fish oil and flax oil increased the incorporation of OM-3FAs into egg yolks in both normal and cancerous ovaries of hens. Fish oil reduced COX-1 and COX-2 in normal and cancerous ovaries. Fish oil, flax oil and Celecoxib reduced the COX-2 expression in ovaries. Combination of fish oil and Celecoxib and flax oil and Celecoxib decreased COX and PGE2 more than each of these treatments alone. The cancerous ovaries of hens treated with fish oil, flax oil, Celecoxib, and flax oil and Celecoxib combined increased the percentage of apoptotic cells compared to cancerous ovaries of control hens. The cancerous ovaries of hens treated with fish oil and Celecoxib had the highest number of apoptotic cells indicating that the combination of fish oil and Celecoxib is more effective than fish oil or Celecoxib alone. To our knowledge the present study provides the first insight into the efficacy of fish oil, flax oil, Celecoxib, alone or combined on the reduction of COX enzyme expression, PGE2 concentration and apoptosis in the normal and cancerous ovaries and further demonstrates the utility of the hen model for ovarian cancer. Our studies provided new insight into the potential mechanism of action of flaxseed, fish oil, flax oil and Celecoxib in the reduction of ovarian cancer and will establish the foundation for clinical trials to test the efficacy of dietary intervention for the prevention and suppression of ovarian cancer in women.

A osteoartrite (OA) é uma doença degenerativa que afeta grande parte da população e resulta em significativa morbidade e incapacidade. O presente estudo teve como objetivo investigar os efeitos periféricos da S(+) cetamina na expressão da ciclo-oxigenase 2 (COX-2). Foram utilizados modelos experimentais de osteoartrite em ratos. Inicialmente setenta e dois ratos foram utilizados no estudo. Foram divididos em três grupos de 24 animais cada. Em dois grupos foi induzida a OA através de 2mg de MIA (monoiodo acetato de sódio) por via intra-articular (i.a), em um volume máximo de 50μL e em um dos grupos não foi realizada a indução da OA. No sétimo dia após a indução, dois grupos, incluindo o sem OA, receberam injeção i.a de salina 0,9% em volume máximo de 50μL e o terceiro grupo recebeu injeção de S(+) cetamina na dose de 0,5mg/kg. Nos dias 7, 14, 21 e 28 os animais foram anestesiados e sacrificados para coleta da membrana sinovial e análise imuno-histoquímica da ciclo-oxigenase-2. Durante o estudo ocorreram 29 perdas do material a ser analisado, totalizando um n = 43. O protocolo adotado para a interpretação imuno-histoquímica foi a imunomarcação citoplasmática da COX-2 em células da membrana sinovial, tecido conjuntivo e adiposo, conforme a intensidade da coloração. A análise dos resultados foi realizada através do teste do quiquadrado. A reatividade da COX-2 foi positiva em 53,8% dos animais do grupo sem OA, em 60% do grupo OA com salina e em 80% dos animais do grupo OA com cetamina, sem diferença estatisticamente significante entre os grupos (p = 0,3069). Esse estudo sugeriu que a S(+) cetamina por via intra-articular não inibiu a expressão da COX-2 em modelos de osteoartrite em ratos. / Osteoarthritis (OA) is a degenerative disease that effects a large population and results in significant morbidity and disability. The objective of the present study was to investigate the peripheral effects of S(+) ketamine on the COX-2 expression. Experimental models of OA in rats were used. At first, 72 rats were used in the study. The animals were divided into three groups of 24 each. In two groups, OA was induced through intra-articular (i.a.) injection of 2mg of monoiodine acetate (MIA), at a maximum volume of 50μL, while one of the groups was not submitted to OA induction. On the seventh day following the induction, the animals of two groups, including those form the not-induced group, received an i.a. injection of saline at 0.9% at a maximum volume of 50μL, while the third group received and injection of S(+) ketamine at 0.5mg/kg. On days 7, 14, 21 and 28 the animals were anesthetized and sacrificed, and the synovial membrane was extracted and submitted to immunohistochemistry analysis of the cyclooxygenase-2. Throughout the study, there were 29 losses of materials that were to be analyzed, totaling an n = 43. The protocol used for the immunohistochemical interpretation was cytoplasmic immunostaining of COX-2 in cells of the synovial membrane, conjunctive and adipose tissue, according to the intensity of the stain. Results were analyzed by the chi-square test. COX-2 reactivity was positive in 53.8% of animals in the group without OA, in 60% of those of the OA group with saline, and in 80% of group OA with ketamine, with no statistically significant difference between the groups (p = 0.3069). Thus, the study implies that intra-articular injections of S(+) ketamine did not inhibit the COX-2 expression in osteoarthritis models in rats.

O Carcinoma de Pulmão de Células Não Pequenas (NSCLC) é uma doença freqüentemente letal e altamente resistente à terapia oncológica convencional, como por exemplo, o tratamento quimioterápico com cisplatina e paclitaxel. A superexpressão de Ciclooxigenase-2 (COX-2) é constantemente observada em pacientes com NSCLC, estando associada ao prognóstico ruim destes pacientes. Acredita-se que a alta expressão de COX-2 produz efeitos anti-apoptóticos, porém pouco é conhecido sobre os mecanismos de regulação desta enzima. Muitos sinais capazes de ativar COX-2 também induzem a proteína supressora de tumor p53, conhecida pelo seu papel fundamental no controle da proliferação celular e apoptose. Dados recentes indicam que a proteína p53 é um importante regulador da expressão de COX-2. O objetivo desta dissertação foi avaliar os efeitos da quimioterapia na expressão da enzima COX-2 em linhagens celulares com diferente status do gene TP53, e ainda, correlacionar a expressão de COX-2 e o status mutacional de TP53, com as características clínico-patológicas de pacientes com NSCLC. Como ferramentas experimentais foram usadas técnicas de biologia celular e molecular como interferência de RNA, PCR em tempo real, análise mutacional e imuno-histoquímica. Com os resultados obtidos, observamos que as linhagens celulares de câncer de pulmão que apresentam p53 na sua forma selvagem, quando expostas ao tratamento com cisplatina, apresentaram indução da expressão de COX-2 (RNAm e proteína), em adição ao aumento da síntese de Prostaglandina E2 (PGE2). Em contrapartida, a expressão de COX-2 não foi alterada após o tratamento com cisplatina nas linhagens celulares que apresentavam mutação no gene TP53. Ao avaliar o tratamento com paclitaxel, foi observado um aumento da expressão de COX-2 nas linhagens A549 e H460 (linhagens celulares do tipo selvagem para p53), entretanto não foi observada alteração nos níveis de PGE2. Em adição, o tratamento com paclitaxel induziu um aumento da expressão de COX-2 na linhagem com deleção em TP53, ACC LC-319. Em seguida, após silenciamento de p53 na linhagem celular A549, por interferência de RNA, a cisplatina passou a não ser mais capaz de induzir o aumento da expressão de COX-2. No tratamento com paclitaxel, o silenciamento de TP53 não mudou a expressão de COX-2, indicando assim um efeito independente de p53. Dessa maneira, sugerimos que a indução de COX-2, por cisplatina, em linhagens celulares NSCLC é dependente de p53. Na análise dos pacientes NSCLC, os resultados demonstram que 54% dos pacientes apresentam expressão positiva de COX-2. Mutações em TP53 foram observadas em 57% dos pacientes, incluindo 56% de fumantes correntes e 37% de ex-fumantes. Uma associação entre a expressão de COX-2 e o status selvagem de TP53 foi observada, entre os pacientes que apresentaram expressão positiva de COX-2, 80% apresentaram TP53 selvagem. Um número maior de pacientes é necessário para aumentar o poder estatístico e confirmar as tendências observadas nesse estudo / Non-Small Cell Lung Cancer (NSCLC) is a lethal disease and highly resistant to conventional chemotherapeutic agents, like cisplatin and paclitaxel. Overexpression of cyclooxygenase-2 (COX-2) is frequently found in NSCLC, and is associated with a bad prognosis. High COX-2 expression is thought to exert antiapoptotic effects in cancer, but little is known about COX-2 regulation. Many signals that activate COX-2 also induce the tumor suppressor protein p53, which is believed to play a pivotal role in controlling proliferation and apoptosis. Recent data indicate the tumor suppressor protein p53 as an important regulator of COX-2. The main objective of this work was to analyze the effect of chemotherapy on the expression of COX-2 according to TP53 status and also evaluate the relationship between COX-2 expression and TP53 mutational status, according to clinic pathological features in NSCLC patients. As experimental tools techniques of cell and molecular biology such as RNA interference, real time PCR, mutational analysis and immunohistochemistry were used. We report herein that lung cancer cell lines expressing wild-type p53 when exposed to cisplatin treatment induced COX-2 (mRNA and protein), with concurrent synthesis of prostaglandins (PGE2). In contrast, COX-2 expression was not detected or changed after cisplatin treatment of cells containing an inactive form of p53. For paclitaxel treatment, an increase in COX-2 mRNA expression was observed in H460 and A549 (wild-type p53 cell lines), although the same was not observed on PGE2 production. Moreover, paclitaxel treatment increased COX-2 expression in ACC-LC-319 cell lines (p53 null). Further, after silencing wild-type p53 expression in A549 cells by RNA interference, cisplatin was no longer able to induce COX-2 mRNA and protein expression. While in treatment with paclitaxel the silencing of wild type TP53 did not change COX-2 expression, showing a p53-independent effect. Therefore, we suggest that induction of COX-2 by cisplatin in NSCLC cell lines is dependent on p53. In the NSCLC patients analysis, the results showed a COX-2 positive staining in 54% patients. TP53 mutations were found in 57% of the patients, including 56% in current smokers and 37% in former smokers. There was an association between COX-2 expression and wild-type TP53. Among COX-2 positive patients, 80% were wild-type TP53. A higher number of patients are necessary to increase statistical power and confirm the tendencies observed in this study

O Carcinoma de Pulmão de Células Não Pequenas (NSCLC) é uma doença freqüentemente letal e altamente resistente à terapia oncológica convencional, como por exemplo, o tratamento quimioterápico com cisplatina e paclitaxel. A superexpressão de Ciclooxigenase-2 (COX-2) é constantemente observada em pacientes com NSCLC, estando associada ao prognóstico ruim destes pacientes. Acredita-se que a alta expressão de COX-2 produz efeitos anti-apoptóticos, porém pouco é conhecido sobre os mecanismos de regulação desta enzima. Muitos sinais capazes de ativar COX-2 também induzem a proteína supressora de tumor p53, conhecida pelo seu papel fundamental no controle da proliferação celular e apoptose. Dados recentes indicam que a proteína p53 é um importante regulador da expressão de COX-2. O objetivo desta dissertação foi avaliar os efeitos da quimioterapia na expressão da enzima COX-2 em linhagens celulares com diferente status do gene TP53, e ainda, correlacionar a expressão de COX-2 e o status mutacional de TP53, com as características clínico-patológicas de pacientes com NSCLC. Como ferramentas experimentais foram usadas técnicas de biologia celular e molecular como interferência de RNA, PCR em tempo real, análise mutacional e imuno-histoquímica. Com os resultados obtidos, observamos que as linhagens celulares de câncer de pulmão que apresentam p53 na sua forma selvagem, quando expostas ao tratamento com cisplatina, apresentaram indução da expressão de COX-2 (RNAm e proteína), em adição ao aumento da síntese de Prostaglandina E2 (PGE2). Em contrapartida, a expressão de COX-2 não foi alterada após o tratamento com cisplatina nas linhagens celulares que apresentavam mutação no gene TP53. Ao avaliar o tratamento com paclitaxel, foi observado um aumento da expressão de COX-2 nas linhagens A549 e H460 (linhagens celulares do tipo selvagem para p53), entretanto não foi observada alteração nos níveis de PGE2. Em adição, o tratamento com paclitaxel induziu um aumento da expressão de COX-2 na linhagem com deleção em TP53, ACC LC-319. Em seguida, após silenciamento de p53 na linhagem celular A549, por interferência de RNA, a cisplatina passou a não ser mais capaz de induzir o aumento da expressão de COX-2. No tratamento com paclitaxel, o silenciamento de TP53 não mudou a expressão de COX-2, indicando assim um efeito independente de p53. Dessa maneira, sugerimos que a indução de COX-2, por cisplatina, em linhagens celulares NSCLC é dependente de p53. Na análise dos pacientes NSCLC, os resultados demonstram que 54% dos pacientes apresentam expressão positiva de COX-2. Mutações em TP53 foram observadas em 57% dos pacientes, incluindo 56% de fumantes correntes e 37% de ex-fumantes. Uma associação entre a expressão de COX-2 e o status selvagem de TP53 foi observada, entre os pacientes que apresentaram expressão positiva de COX-2, 80% apresentaram TP53 selvagem. Um número maior de pacientes é necessário para aumentar o poder estatístico e confirmar as tendências observadas nesse estudo / Non-Small Cell Lung Cancer (NSCLC) is a lethal disease and highly resistant to conventional chemotherapeutic agents, like cisplatin and paclitaxel. Overexpression of cyclooxygenase-2 (COX-2) is frequently found in NSCLC, and is associated with a bad prognosis. High COX-2 expression is thought to exert antiapoptotic effects in cancer, but little is known about COX-2 regulation. Many signals that activate COX-2 also induce the tumor suppressor protein p53, which is believed to play a pivotal role in controlling proliferation and apoptosis. Recent data indicate the tumor suppressor protein p53 as an important regulator of COX-2. The main objective of this work was to analyze the effect of chemotherapy on the expression of COX-2 according to TP53 status and also evaluate the relationship between COX-2 expression and TP53 mutational status, according to clinic pathological features in NSCLC patients. As experimental tools techniques of cell and molecular biology such as RNA interference, real time PCR, mutational analysis and immunohistochemistry were used. We report herein that lung cancer cell lines expressing wild-type p53 when exposed to cisplatin treatment induced COX-2 (mRNA and protein), with concurrent synthesis of prostaglandins (PGE2). In contrast, COX-2 expression was not detected or changed after cisplatin treatment of cells containing an inactive form of p53. For paclitaxel treatment, an increase in COX-2 mRNA expression was observed in H460 and A549 (wild-type p53 cell lines), although the same was not observed on PGE2 production. Moreover, paclitaxel treatment increased COX-2 expression in ACC-LC-319 cell lines (p53 null). Further, after silencing wild-type p53 expression in A549 cells by RNA interference, cisplatin was no longer able to induce COX-2 mRNA and protein expression. While in treatment with paclitaxel the silencing of wild type TP53 did not change COX-2 expression, showing a p53-independent effect. Therefore, we suggest that induction of COX-2 by cisplatin in NSCLC cell lines is dependent on p53. In the NSCLC patients analysis, the results showed a COX-2 positive staining in 54% patients. TP53 mutations were found in 57% of the patients, including 56% in current smokers and 37% in former smokers. There was an association between COX-2 expression and wild-type TP53. Among COX-2 positive patients, 80% were wild-type TP53. A higher number of patients are necessary to increase statistical power and confirm the tendencies observed in this study

A osteoartrite (OA) é uma doença degenerativa que afeta grande parte da população e resulta em significativa morbidade e incapacidade. O presente estudo teve como objetivo investigar os efeitos periféricos da S(+) cetamina na expressão da ciclo-oxigenase 2 (COX-2). Foram utilizados modelos experimentais de osteoartrite em ratos. Inicialmente setenta e dois ratos foram utilizados no estudo. Foram divididos em três grupos de 24 animais cada. Em dois grupos foi induzida a OA através de 2mg de MIA (monoiodo acetato de sódio) por via intra-articular (i.a), em um volume máximo de 50μL e em um dos grupos não foi realizada a indução da OA. No sétimo dia após a indução, dois grupos, incluindo o sem OA, receberam injeção i.a de salina 0,9% em volume máximo de 50μL e o terceiro grupo recebeu injeção de S(+) cetamina na dose de 0,5mg/kg. Nos dias 7, 14, 21 e 28 os animais foram anestesiados e sacrificados para coleta da membrana sinovial e análise imuno-histoquímica da ciclo-oxigenase-2. Durante o estudo ocorreram 29 perdas do material a ser analisado, totalizando um n = 43. O protocolo adotado para a interpretação imuno-histoquímica foi a imunomarcação citoplasmática da COX-2 em células da membrana sinovial, tecido conjuntivo e adiposo, conforme a intensidade da coloração. A análise dos resultados foi realizada através do teste do quiquadrado. A reatividade da COX-2 foi positiva em 53,8% dos animais do grupo sem OA, em 60% do grupo OA com salina e em 80% dos animais do grupo OA com cetamina, sem diferença estatisticamente significante entre os grupos (p = 0,3069). Esse estudo sugeriu que a S(+) cetamina por via intra-articular não inibiu a expressão da COX-2 em modelos de osteoartrite em ratos. / Osteoarthritis (OA) is a degenerative disease that effects a large population and results in significant morbidity and disability. The objective of the present study was to investigate the peripheral effects of S(+) ketamine on the COX-2 expression. Experimental models of OA in rats were used. At first, 72 rats were used in the study. The animals were divided into three groups of 24 each. In two groups, OA was induced through intra-articular (i.a.) injection of 2mg of monoiodine acetate (MIA), at a maximum volume of 50μL, while one of the groups was not submitted to OA induction. On the seventh day following the induction, the animals of two groups, including those form the not-induced group, received an i.a. injection of saline at 0.9% at a maximum volume of 50μL, while the third group received and injection of S(+) ketamine at 0.5mg/kg. On days 7, 14, 21 and 28 the animals were anesthetized and sacrificed, and the synovial membrane was extracted and submitted to immunohistochemistry analysis of the cyclooxygenase-2. Throughout the study, there were 29 losses of materials that were to be analyzed, totaling an n = 43. The protocol used for the immunohistochemical interpretation was cytoplasmic immunostaining of COX-2 in cells of the synovial membrane, conjunctive and adipose tissue, according to the intensity of the stain. Results were analyzed by the chi-square test. COX-2 reactivity was positive in 53.8% of animals in the group without OA, in 60% of those of the OA group with saline, and in 80% of group OA with ketamine, with no statistically significant difference between the groups (p = 0.3069). Thus, the study implies that intra-articular injections of S(+) ketamine did not inhibit the COX-2 expression in osteoarthritis models in rats.

histoplasmose é uma doença granulomatosa crônica, cujo agente etiológico é o fungo dimórfico Histoplasma capsulatum. A infecção ocorre pela inalação de conídios ou pequenos fragmentos de micélio que alcançam os alvéolos, onde se transformam em leveduras que é responsável pela patogenia da doença. A imunidade celular do hospedeiro determina o grau das manifestações clínicas na histoplasmose, sendo a interação entre células T e macrófagos, fundamental para o controle da infecção e erradicação do H. capsulatum. Recentemente, nosso grupo de pesquisa demonstrou a participação de leucotrienos nos mecanismos de defesa do hospedeiro durante a histoplasmose. Neste trabalho descrevemos o papel das prostaglandinas, demonstramos que este mediador lipídico contribui para a patogênese da doença, pois sua inibição, com celecoxibe, resultou na sobrevivência de até 80% dos animais infectados com o inóculo letal de H. capsulatum, em contraste com 100% de mortalidade dos animais somente infectados. Além disso, a inibição das prostaglandinas resultou na diminuição (i) da síntese de citocinas pró-inflamatórias e da resposta imune celular e (ii) do recrutamento de neutrófilos e macrófagos para o espaço bronco-alveolar. Por outro lado, resultou no aumento (iii) de células TCD4+ no pulmão, (iv) na síntese de óxido nítrico por células do parênquima pulmonar, (v) na fagocitose de leveduras de H. capsulatum por macrófagos alveolares e (vi) da síntese de LTB4. Nossos resultados sugerem que prostaglandinas têm papel importante na patogênese na infecção por H. capsulatum, modulando a resposta imune do hospedeiro. / The Histoplasmosis is a chronic granulomatosas disease whose etiologic agent is pathogenic dimorphic fungus Histoplasma capsulatum. Infection occurs mainly by fungal inhalation that reaches the alveoli, where if transforms into leavenings that are responsible for pathogenic diseases. The cellular immunity of the host determines the degree of the clinical manifestations in histoplasmosis, being the interaction between cells T and macrophages, basic for the control of the infection and eradication of the H. capsulatum. Recently, our group of research demonstrated the participation of leukotrienes in the mechanisms of defense of the host during the Histoplasmosis. Beyond this important lipid mediator who participates in the immune reply against H. capsulatum. In this work, we describe another involved mediator, the prostaglandin. In the present work, we demonstrate that the prostaglandins contribute for pathogenic of the disease, being that during its inhibition with celecoxib it resulted in the survival of up to 80% of the infection-mice with inoculum lethal of H. capsulatum, in contrast with 100% of mortality infection-mice. Moreover, the inhibition of prostaglandins resulted in the reduction (i) of the synthesis of pro-inflammatory cytokines and the cellular immune response and (ii) in the migration of neutrophils and macrophages. For other hand, increased (iii) of cells TCD4+ in the lung, (iv) of the nitric oxide synthesis, (v) of phagocytosis of yeast of H. capsulatum for alveolar macrophages and (vi) of the synthesis of LTB4. Our results suggest that prostaglandins have important role in pathogenic in the infection for H. capsulatum, modulating the host immune response.

Orientadores: Sophie Françoise Mauricette Derchain, Luis Otávio Zanatta Sarian / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T22:51:42Z (GMT). No. of bitstreams: 1 Serra_KatiaPiton_M.pdf: 1719188 bytes, checksum: fd6b0e67d77fd1b8fce08cde3c09dd45 (MD5) Previous issue date: 2010 / Resumo: Introdução: evidências laboratoriais sugerem que a enzima ciclooxigenase-2 (COX-2), é um dos principais componentes da cascata inflamatória. A enzima é responsável pela conversão do ácido araquidônico em prostaglandinas e tromboxanos. Estudos sugerem que a expressão da COX-2 correlaciona-se diretamente com o potencial maligno dos tumores de mama, e essa relação é em parte explicada pelo papel desempenhado pela COX-2 no reforço da neoangiogênese e processos de imortalização celular. Pouco se sabe, no entanto, sobre a relação da expressão da COX-2 com outros marcadores prognósticos e preditivos de tumores de mama como HER-2, p53, e receptores de hormônios (estrogênio [RE] e progesterona [RP]). Objetivos: avaliar a relação entre a expressão da COX-2 e da p53, receptores de hormônios e HER-2 nas frações in situ e invasivo de carcinomas ductais da mama. Sujeitos e métodos: foram incluídas amostras de 87 mulheres com carcinoma invasivo da mama, que tivessem áreas de carcinoma intraductal associadas. A expressão da COX-2, p53 e receptores hormonais foi avaliada por imuno-histoquímica (IHQ), a expressão de HER-2 foi avaliada por IHQ e hibridização fluorescente in situ (FISH). Nas análises estatísticas, os níveis de confiança foram ajustados para 5% (p=0,05). Na análise univariada, qui-quadrados foram calculados para comparar a expressão dos marcadores tumorais nos componentes in situ e invasivo. Coeficiente de correlação intraclasse (ICC) e qui-quadrado foram calculados para avaliar a tabulação cruzada da expressão da COX-2 nos componentes intraductal e invasor. Qui-quadrados foram utilizados para comparar as proporções de tumores in situ e invasivos que expressaram cada um dos marcadores tumorais de acordo com a expressão da COX-2. Todas as tabulações foram novamente testadas de forma multivariada, utilizando modelos de regressão logística para avaliar especificamente a expressão dos marcadores nos componentes intraductal versus invasivos e nos grupos formados pela expresão da COX-2. Resultados: a COX-2 estava expressa em 44 (61%) dos componentes in situ e em 49 (58%) dos componentes invasivos; 44% dos casos expressaram COX-2 em ambos os componentes. Dos componentes invasivos com expressão da COX-2, 17% foram negativos para a enzima no componente intraductal. Em contrapartida, nos tumores que expressaram COX-2 no componente in situ, 17% apresentaram resultados negativos para a enzima em seu componente invasivo (ICC 0,29, p=0,02). Não houve diferença estatística na expressão da COX-2 ao comparar os componentes intraductal e invasivo dos tumores (p=0,80). A expressão da p53 foi maior no componente intraductal (52%), comparada ao invasor (33%) (p<0,01). O HER-2 estava superexpresso em 21% na fração in situ e 28% no componente invasivo (p=0,49); 69% dos componentes intraductais foram positivos para RE. Aproximadamente a mesma proporção (75%) dos tumores invasivos foram também positivos para RE (p=0,36). Houve um desequilíbrio marginal na expressão de RP, com maior prevalência deste na forma in situ (59% versus 46% no componente invasivo, p=0,08). No componente intraductal, houve uma diferença estatisticamente limítrofe da expressão da p53 em tumores que também expressaram COX-2 (66% versus 44% em amostras negativas para COX-2 p=0,07). No entanto, a proporção de tumores que expressaram HER-2 (p=0,73), RE (p=0,25) e RP (p=0,57) não diferiu em tumores que expressaram ou não a COX-2. Houve uma proporção ligeiramente maior (84% versus 67%) das amostras RE positivas no grupo de tumores invasivos que expressaram COX-2 (p=0,07). Em contrapartida, a expressão de RP não foi relacionada com a da COX-2 (p=0,22) na avaliação multivariada. Conclusões: a expressão da COX-2 foi semelhante nas frações intraductal e invasora das neoplasias de mama. A expressão da p53 foi marginalmente superior nas frações in situ que expressavam COX-2. Na fração invasora, houve maior proporção de tumores expressando receptores de estrógeno entre os que expressaram COX-2 / Abstract: Introduction: laboratorial evidence implicates the cyclooxygenase-2 (COX-2) enzyme as one of the major components of the inflammatory cascade. The enzyme is responsible for the conversion of aracdonic acid in prostaglandins and tromboxanes. Previous research suggests that COX-2 expression correlates directly with the malignant potential of breast tumors, and this relation is, at least in part explained by the role played by COX-2 in the enhancement of the neoangiogenesis and cell immortalization processes. Little is known, however, about the relation of COX-2 expression with other well-stablished breast tumor prognostic and predictive markers, e.g. HER-2, p53, and hormone (estrogen [ER] and progesterone [PR]) receptors. Objectives: to assess the relationship between the expression of COX-2 and that of p53, hormone receptors (estrogen (ER) and progesterone (PR)) and HER-2 in the in situ and invasive regions of ductal carcinomas of the breast. Subjects and methods: samples from 87 women with invasive carcinoma of the breast with areas of in situ carcinoma were included. The expressions of COX-2, p53 and hormone receptors were assessed with immunohistochemistry (IHC); the expression of HER-2 was assessed with IHC and Fluorescent in situ Hybridization (FISH). In statistical analyses, confidence levels were set to 5% (p 0.05). In univariate analysis, chi-squares were calculated to confront the expression of the tumor markers in the in situ and invasive components. The intraclass correlation coefficient (ICC) and chi-squares were calculated to assess the cross-tabulation of COX-2 expression in the in situ versus invasive components. Then, chi-squares were also used to compare the proportions of tumors expressing (individually for the in situ and invasive components) each of the tumor markers in the groups formed according to the COX-2 expression. All tabulations were then retested in a multivariate fashion, using logistic regression models fit specifically for the comparison of marker expression in the in situ versus the invasive components, and in the COX-2-positive and negative groups. Results: COX-2 was expressed in 44 (61%) of the in situ components and in 49 (58%) of the invasive components; 44% of the cases expressed COX-2 in both components. Of the tumors whose invasive components expressed COX-2, 17% were negative for the enzyme in the in situ component. By contrast, of the tumors that expressed COX-2 in the in situ component, 17% were negative for the enzyme in their invasive component (ICC 0.29; p=0.02). There was no statistical difference in COX-2 expression comparing the in situ and invasive components of the breast tumors (p 0.80). The p53 expression was higher in the in situ component (52%), contrasted to that in the invasive (33%) region of the tumors (p<0.01). HER-2 was expressed in 21% in the in situ component and 28% in the invasive component (p=0.49). Sixty-nine percent of the in situ components tested positive for ER, and approximately the same proportion (75%) of the invasive components were positive for ER (p=0.36). There was a marginal imbalance in PR expression, favoring the in situ component (59% versus 46% in the invasive component; p=0.08). In the in situ component, there was a statistically borderline increase in p53 expression in tumors that also expressed COX-2 (66% versus 44% in COX-2 negative specimens p=0.07). However, the proportions of tumors that expressed HER-2 (p=0.73), ER (p=0.25) and PR (p=0.57) did not differ in tumors that expressed or not COX-2 protein. There was a marginally increased proportion (83% versus 66%) of ER-positive specimens in the group of invasive tumors that expressed COX-2 (p=0.07). By contrast, PR expression was not related to that of COX-2 (p=0.22) in the multivariate assessment. Conclusions: the expression of COX-2 was similar in the in situ and invasive regions of the breast neoplasms. The expression of p53 was marginally higher in the in situ regions that were positive for COX-2. In the COX-2-positive invasive regions, there were a higher proportion of ER-positive tumors / Mestrado / Oncologia Ginecológica e Mamária / Mestre em Ciências da Saúde

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / COX-2 e Caderina-E participam de forma fundamental na manutenÃÃo do estado fisiolÃgico da mucosa gÃstrica e tÃm papel essencial na reaÃÃo inflamatÃria e reparo, e no cÃncer. O objetivo deste trabalho à avaliar a expressÃo das duas proteÃnas no carcinoma gÃstrico e metÃstases linfonodais e suas possÃveis participaÃÃes na progressÃo tumoral. Foram utilizados 97 casos de gastrectomias por carcinoma gÃstrico, 36 dos quais com linfonodos disponÃveis, dos arquivos do Hospital do CÃncer do CearÃ. Os casos foram classificados nos tipos intestinal (40 casos), difuso (34), mistos (16) e nÃo-classificados (7 casos) de acordo com a classificaÃÃo de Lauren (1965). Utilizou-se tÃcnica de tissue microarray associada à imunohistoquÃmica com anticorpo monoclonal anti-COX-2 e anti-Caderina-E e sistema de detecÃÃo universal estreptavidina-biotina-peroxidase. A expressÃo de COX-2 foi avaliada de acordo com os seguintes escores: Intensidade (I): 0=negativa; 1=discreta; 2= moderada; 3= acentuada; ExtensÃo (E) de cÃlulas coradas: 1= 0 a 25%; 2= >25 a 50%; 3= >50 a 75%; 4= >75 a 100%. Escore final: I x E, sendo considerados escores < 6 como COX-2 de baixa expressÃo e escores &#8805;6 de alta expressÃo. Classificou-se a expressÃo de Caderina-E nos escores: 0=negativo; 1=citoplasmÃtica; 2=citoplasmÃtica + membranar; 3= membranar-normal (Jawhari et al., 1997a). Foram comparadas expressÃo normal e anormal e membranar e nÃo membranar em cada histotipo de carcinoma, na sede primÃria e linfonodos. ExpressÃo positiva para COX-2 e anormal de Caderina-E predominaram nos diversos histotipos de carcinoma gÃstrico primÃrio, principalmente difusos e mistos. Observou-se maior expressÃo de COX-2 nas metÃstases linfonodais, em relaÃÃo Ãs lesÃes primÃrias, sobretudo nos carcinomas difusos. Carcinomas intestinais estavam associados à expressÃo membranar de Caderina-E enquanto tumores difusos se relacionaram com ausÃncia de expressÃo membranar, o que mostra a importÃncia da Caderina-E na diferenciaÃÃo do cÃncer gÃstrico. Carcinomas gÃstricos apresentam dois padrÃes de imunomarcaÃÃo citoplasmÃtica: granular (paranuclear), associado à expressÃo citoplasmÃtica exclusiva, que prevalece no componente difuso dos tumores mistos, e homogÃneo, em todo o citoplasma, correlacionado com expressÃo citoplasmÃtica-membranar, predominante nos outros histotipos. Carcinomas difusos apresentam expressÃo membranar de Caderina-E, que se expressa com maior freqÃÃncia nas metÃstases linfonodais do que nas lesÃes primÃrias e està presente em grupos celulares infiltrantes e cÃlulas isoladas, nas duas sedes anatÃmicas. Os dados sugerem que o carcinoma misto representa histotipo distinto de carcinoma gÃstrico, baseado nos aspectos peculiares da expressÃo citoplasmÃtica de Caderina-E aqui mostrados e em outros achados da literatura. NÃo houve associaÃÃo estatisticamente significativa entre expressÃo de COX-2 e de Caderina-E e demais parÃmetros clÃnico-patolÃgicos nesta amostra. Os dados aqui observados sugerem que COX-2 e Caderina-E sÃo importantes proteÃnas relacionadas com a progressÃo tumoral-linfonodal no cÃncer gÃstrico / Both COX-2 and E-Cadherin play important roles in physiological and pathological processes in the stomach, such as control of acid secretion, inflammation and cancer. The aim of this study was to analyze the relationship between COX-2 and E-Cadherin immunoexpression in human gastric adenocarcinomas and respective lymph node metastases and their possible action in tumoral progression. Tissue microarrays were prepared from paraffin embedded samples of 97 primary gastric cancers, included 36 with respective nodal metastases. Cases were classified according to Laurenâs classification as intestinal (n=40), diffuse (n=34), mixed (n=16) and undetermined (n=7). Immunoexpression of COX-2 was evaluated regarding intensity (0-absent; 1-mild; 2-moderate; 3-strong) and extension (0-negative or rare cells; 1-<25%; 2-25-50%; 3-50-75%; 4->75% immunoreactive neoplastic cells). A combined score was calculated (intensity x extension): 0-12. A cut-off of 6 was considered to classify COX-2 expression as low (<6) or high (&#8805;6). E-Cadherin expression was evaluated according to the system proposed by Jawhari et al. (Gastroenterology, 1997) as abnormal patterns of expression: 0-no expression; 1-cytoplasmic expression; 2-heterogeneous expression, both membranous and cytoplasmic) and normal membranous pattern (3). Membranous (scores 2 and 3) and Non-membranous (scores 0 and 1) were too compared. Overall, COX-2 positive and abnormal E-Cadherin expression predominate in all types of primary gastric carcinomas. COX-2 expression was higher in lymph node metastases than in primary tumors, with a significant difference for diffuse carcinoma. A positive relationship was observed between E-Cadherin membranous expression and intestinal tumors, and absence of membranous expression and diffuse ones, which indicates the importance of E-Cadherin to gastric cancer differentiation. Granular (paranuclear) cytoplasmic immunostaining pattern was basically associated with cytoplasmic E-Cadherin expression while homogeneous pattern is frequently seen in cytoplasmic-membranous expression. Diffuse carcinomas show membranar expression more frequently in lymph nodes metastases than in gastric primary tumors in both isolated and grouped cells. The data suggest that mixed carcinoma is a distinct hystotype, based on its peculiar cytoplasmic expression of E-Cadherin shown here and other features of literature. There was no significant association linking COX-2 and E-Cadherin expression to other clinicopathological parameters. The data show that COX-2 and E-Cadherin are important proteins related to tumoral progression in gastric cancer

Orientadores: Sophie Fraçoise Mauricette Derchain, Luís Otávio Zanatta Sarian / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T00:11:32Z (GMT). No. of bitstreams: 1 Serra_KatiaPiton_D.pdf: 3794654 bytes, checksum: ce5565714883e2e12d99e6f9ed0ca141 (MD5) Previous issue date: 2014 / Resumo: Introdução: Na última década, doferentes subtipos moleculares de cancer de mama foram propostos. A classificação clinic-patológicas dos subtipos vem comprovando ser estratégica para predizer sobrevida e resposta ao tratamento. Modificação recente da classificação considera a avaliação semiquantitativa da expressão dos RP no curso clínico e resposta ao tratamento. Embora exista associação apreciável com o prognóstico e indicação de terapia citotóxica e endócrina, os subtipos parecem falhar em explicar completamente o comçortamento da doença e a resposta ao tratamento. Moléculas como as da família das cicloxigenases (COX), composta por três entidades (COX 1, 2 e 3) vem demonstrando associação com a carcinogênese mamária, e a análise da expressão da p53 nos tumores de mama pode também oferecer informações adicionais para determinação do prognóstico. Objetivos: Foi avaliada a associação entre os subtipos clinic-patológicos do cancer de mama com o prognóstico e fatores preditivos em uma relativamente grande casuística de pacientes Brasileiras com câncer de mama, que foram acompanhadas por cerca de quatro anos. Foram discutidas as vantagens e possíveis ressalvas relacionadas à nova classificação. Também foi mensurada a expressão da COX2 e da p53 em relação aos subtipos clínico-patológicos e avaliada se a expressão destas molécular poderia explicar a variabilidade no prognóstico ainda encontrada entre os subtipos clínico-patológicos do câncer de mama. Metodologia: Um total de 183 amostras de cancer de mama foram obtidas de mulheres tratadas no Hospital da Mulher da Universidade Estadual de Campinas, Campinas, Brasil, entre Junho de 2008 e Janeiro de 2011. Tissue microarrays (TMA) foram construídos dos blocos originais de parafina para realização de imunoistoquímica (IQ) e hibridização fluorescente in situ (FISH). IQ foi realizada para detecção da expressão de RE, RP, ki67, COX2 e p53; o status do HER2 foi avaliado por FISH nas 183 amostras. Os tumores foram classificados em cinco categorias de acordo com a definição correspondente clinic-patológica dos dos subtipos intrínsecos do câncer de mama, definida durante a 13th St Gallen International Breast Cancer Conference (2013). As características clínicas e patológicas das pacientes e seus tumors e a sobrevida foi avaliada em relação aos subtipos clínico-patológicos, a COX2 e a p53. O tempo médio de seguimento foi 2,94 anos (90% faixa central = 0,93 a 4,1 anos). Resultados: Aproximadamente 75% dos tumors foram classificados como luminais-like. OS HER2 positivos (não luminais) somaram 9,3% dos casos e os Triplos-negativos 13,1%. Os Luminais B-like e HER2 positivos (não luminais) foram associados a alto grau histológico quando comparados aos Luminais A-like (p<0,01). Os Luminais A-like associaram-se significativamente com melhor sobrevida global e livre de doença quando comparados aos HER2 positivos (não luminais) e Triplos-negativos. Não houve tendência à expressão de COX2 relacionada aos subtipos de Luminal A-like a Triplo-negativo. Em contraste, a p53 se expressou em cerca de 67% dos tumores Luminais A-like, 50% dos Luminais B-like HER2 positivos, 60,9% dos Luminais B-like HER2 negativos, 82% dos HER2 positivos (não luminais) e 87% dos Triplos-negativos (p para tendências = 0.06). Houve uma significativa expressão de COX2 nos tumors (66,9%) quando a p53 eram também positive, comparada àqueles tumors que não expressavam p53 (em cujo caso apenas 18,0% dos tumores foram positivos para COX2; p<0,001). Nem a COX2, nem a p53 se relacionaram à sobrevida das pacientes. Conclusões: O critério mais estrito para definer os tumors Luminais A-like aumentou a acurácia da classificação para selecionar tumors que partilhem um bom prognóstico e respondam a terapia endócrina. Parece haver uma associação positive entre a expressão da COX2 e da p53. Por outro lado, nem a expressão da COX2 nem a da p53 se associaram aos subtipos clínico-patológicos, características clínicas e do tumor e ao prognóstico. Parece ser muito cedo para eleger a detecção de COX2 usando IQ como ferramenta de prognóstico ou preditiva, mas evidências incipientes apontam para um possível papel para o marcador / Abstract: Background: In the last decade, different molecular subtypes of breast cancer have been proposed. The clinico-pathological surrogate subtypes of breast cancer classification has been proven as straightforward strategy to predict patient survival and response to treatment. Recent modifications to the classification considered the semi quantitative evaluation of the expression of PR in the clinical course and response to treatment. Although displaying appreciable association with disease prognosis and the prognostic value of cytotoxic and endocrine therapeutic modalities, the subtypes seem to fail at completely explaining disease behavior and response to treatment. Molecules such as those of the cyclocooxigenase (COX) family, currently composed of three entities (COX 1, 2 and 3) have been shown to be associated with breast carcinogenesis, and the analysis of p53 expression in breast tumors may also offer some additional prognostic clues. Objectives: We tested the association of the current clinico-pathological surrogate subtypes of breast cancer with the main prognostic and predictive factors in a relatively large dataset of breast cancer Brazilian patients, which were followed up for almost four years. We discuss the advantages and possible caveats related to this new classification. Our study also assessed COX2 and p53 expression in these clinico-pathological subtypes, and evaluated whether the expression of these molecules could help further explain the variability in prognosis still found within the surrogate molecular groups of breast cancer. Methods: A total of 183 breast cancer samples were obtained from women treated at the Women's Hospital of Campinas State University, Campinas, Brazil, between June 2008 and January 2011. Tissue microarrays (TMA) were constructed from the original paraffin blocks for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses. Immunohistochemistry was performed to detect the expression of ER, PR, ki67, COX2, and p53; the HER2 status of the 183 specimens was assessed using FISH. Tumors were subtyped into five distinct categories according to the Clinico-Pathological surrogate definitions of intrinsic subtypes of breast cancer defined during the 13th St Gallen International Breast Cancer Conference (2013). Clinical and pathological features of patients and their tumors, and patients¿ survival were assessed in relation to the surrogate subtypes, COX2 and p53. Mean follow-up time was 2.94 years (90% central range = 0.93 to 4.1 years). Results: Approximately 75% of the tumors were classified as luminal-type-like. HER2 positive (non-luminal) tumors accounted for 9.3% of the cases and Triple-negative tumors for the remainder 13.1%. Luminal B-like and HER2 positive (non-luminal) tumors were associated with higher histological grades when compared to Luminal A-like tumors (p<0.01). Luminal A-like tumors were significantly associated with better disease free and overall survival when compared to HER2 positive (non-luminal) and Triple-negative tumors. There was no trend in COX2 overexpression from Luminal A to Triple-negative subtypes. By contrast, p53 was expressed in roughly 67% of the Luminal A-like tumors, 50% of the Luminal B-like HER2 positive tumors, 60.9% of the Luminal B-like HER2 negative, approximately 82% of the HER2 positive (non-luminal) and 87% of the Triple-negative tumors (p for trends = 0.06). There was a significantly higher proportion of COX2 positive tumors (66.9%) when p53 was also positive compared to when the tumor was negative for p53 (in which case only 18.0% of the tumors were positive for COX2; p<0.001). Neither COX2 nor p53 were found to be associated with patients¿ survival. Conclusions: The more strict criteria to define Luminal A-like tumors increased the accuracy of the classification by selecting tumors that share a good prognosis and response to endocrine therapy.There seems to be a positive association between the expressions of COX2 and p53. On the other hand, neither the expression of COX nor that of p53 was associated with clinic-pathological subtypes, tumor features and prognosis. It seems to be too early to elect the detection of COX2 using IHC as prognostic or predictive tool, but incipient evidence points towards a possible role for the marker / Doutorado / Oncologia Ginecológica e Mamária / Doutora em Ciências da Saúde