Glucocorticoids (GCs) are endogenous steroid hormones that regulate a number of critical physiological processes. Psychological stress increases the level of GCs in the circulating system. The biological effect of elevated GCs on the heart is not well understood. We found that GCs induced Cyclooxygenase-1 (COX-1) and COX-2 gene expression in cardiomyocytes. COX-1 or COX-2 encodes the rate-limiting enzyme in the biosynthesis of prostanoids, which modulate crucial physiological and pathophysiological responses. The present studies aim to elucidate the signaling transduction pathway and the mechanism underlying GC induced COX expression.Our data demonstrate that GCs activate COX-1 gene expression through transcriptional regulation. COX-1 gene promoter studies support a role of Sp binding site in CT induced COX-1 gene expression. The nuclear protein binding to this site appears to be Sp3 transcription factor. Co-immunoprecipitation assays indicated a physical interaction between GR and Sp3 protein. Silencing of Sp3 transcription factor with small interfering RNA suppressed CT-induced COX-1 promoter activation. These data suggest that the activated GR interacts with Sp3 transcription factor that binds to COX-1 promoter to up-regulate COX-1 gene expression in cardiomyocytes.We also found that administration of GC in adult mice increased the level of COX-2 in the ventricles. With isolated neonatal cardiomyocytes, corticosterone (CT) induces the transcription of COX-2 gene. This response appears to be cardiomyocyte cell type specific and GC receptor (GR)-dependent. CT causes activation of p38 MAPK and subsequently CREB phosphorylation that mediates COX-2 gene expression. Mifepristone, a GR antagonist, failed to inhibit p38 and CREB activation and p38 inhibition failed to prevent activation of GR. These data suggest that two parallel signaling pathways, GR and p38 MAPK, act in concert to regulate the expression of COX-2 gene in cardiomyocytes.In addition to the investigation of mechanism and signaling transduction pathway, I have explored pharmacological agents that modulate COX expression. LY294002, a commonly used PI3K inhibitor, inhibited COX-2 gene expression via a PI3K-independent mechanism. Whereas GSK-3 inhibitors, such as lithium chloride, upregulated COX-2 gene expression, but suppressed GC-induced COX-1 expression. These data have paved the foundation for pharmacological manipulation of COX-1 and COX-2 gene expression in the heart.

p. 1-78 / Submitted by Antonio Geraldo Couto Barreto (ppgms@ufba.br) on 2013-10-02T18:05:28Z No. of bitstreams: 1 Hugo Maia.pdf: 4715178 bytes, checksum: 067aa12a42b4bcfc56dd4ac8e4173ab8 (MD5) / Approved for entry into archive by Rodrigo Meirelles(rodrigomei@ufba.br) on 2013-10-02T22:07:53Z (GMT) No. of bitstreams: 1 Hugo Maia.pdf: 4715178 bytes, checksum: 067aa12a42b4bcfc56dd4ac8e4173ab8 (MD5) / Made available in DSpace on 2013-10-02T22:07:54Z (GMT). No. of bitstreams: 1 Hugo Maia.pdf: 4715178 bytes, checksum: 067aa12a42b4bcfc56dd4ac8e4173ab8 (MD5) Previous issue date: 2013 / A expressão de aromatase no endométrio eutópico é desencadeada pela constante exposição a mediadores inflamatórios, que são produzidos durante o período menstrual e proliferativo do ciclo menstrual. A presença de aromatase nas células endometriais é um dos fatores desencadeantes de endometriose na cavidade peritonial, miomas submucosos e intra-murais, pólipos endometriais e adenomiose. Diante disso, esta tese tem como objetivo investigar os efeitos da expressão de aromatase no endométrio, compreendendo a ação desta e como se evitar o desenvolvimento das patologias endometriais. Para isso, foram analisados resultados de biopsias de pacientes submetidas à histerectomia e laparoscopia, no período de janeiro de 2007 a março de 2009 de dois centros de tratamento da cidade de Salvador- Bahia, as quais apresentavam algumas das patologias citadas, seguindo os critérios da American Sciety of Reproductive Medicine. Por fim concluiu-se que a diminuição da expressão de aromatase induzida por progestínicos foi acompanhada por uma redução na expressão de enzimas como ciclooxigenase-2 (Cox-2) ou de fatores angiogênicos como VEGF no endométrio. A inflamação no endométrio também foi reduzida pela progesterona ou por progestínicos e este mecanismo envolveu a inibição da ativação do NF-kappa B. Estes achados sustentam a hipótese do papel que teriam os progestínicos como agentes anti-aromatase e anti-inflamatórios no manejo atual da endometriose e de outras patologias ginecológicas. E que o uso contínuo de contraceptivos orais combinados contendo gestodeno ou o uso de sistemas intra-uterinos liberadores de levonorgetsrel são efetivos na prevenção tanto da recorrência de endometriose, quanto da menorragia associada a miomas. / Salvador

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-10-01T21:53:22Z No. of bitstreams: 1 Carolina da Paz Zampier.pdf: 2134379 bytes, checksum: d822f378a88e2cf44b02c5ea60f52ea0 (MD5) / Made available in DSpace on 2013-10-01T21:53:22Z (GMT). No. of bitstreams: 1 Carolina da Paz Zampier.pdf: 2134379 bytes, checksum: d822f378a88e2cf44b02c5ea60f52ea0 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A caveolina-1 (Cav-1), uma proteína essencial para a formação de cavéolas, apresenta atividade na modulação da sinalização intracelular. Cav-1 é capaz de interagir com diversas proteínas através de seu domínio CSD (caveolin scaffolding domain) e, em geral, essa interação leva à inibição das proteínas associadas. O peptídeo CSD tem sido utilizado como um mimético de Cav-1 em relação à sua capacidade modulatória sobre a atividade de outras proteínas. Recentemente, tem sido mostrado que Cav-1 é capaz de modular a resposta inflamatória em diversos aspectos. Neste trabalho, examinamos o papel de Cav-1 na regulação da síntese de mediadores inflamatórios por macrófagos. O lipopolissacarídeo (LPS) de E.coli, um protótipo de estímulo inflamatório, foi capaz de induzir a expressão de Cav-1 e Cox-2 em macrófagos peritoneais in vitro. Estas proteínas são induzidas em um curso temporal semelhante, sendo detectadas por Western blot a partir de 3h com níveis de expressão crescentes até 18h. Por imunofluorescência, observamos que Cav-1 e Cox-2 apresentam um padrão de expressão mutualmente exclusivo em macrófagos estimulados com LPS. Mostramos por Western blot que a expressão de Cox-2 é induzida por LPS e que o tratamento com CSD leva à inibição da expressão de Cox- 2, mas não de Cox-1. Observamos, também, a redução parcial dos níveis de PGE2 no sobrenadante de macrófagos estimulados com LPS e tratados com CSD. O tratamento com o peptídeo CSD também foi capaz de reduzir os níveis de IL-1, IL- 6, e IL-12 induzidos por LPS. O LPS induz o aumento da expressão e fosforilação de STAT-1. A fosforilação de STAT-1 foi diminuída após o tratamento com CSD, indicando que Cav-1 modula negativamente a ativação de STAT-1. Estudos posteriores são necessários para complementar os dados obtidos até o momento para esclarecer os mecanismos de modulação da síntese de mediadores inflamatórios por Cav-1. Em conclusão, Cav-1 apresenta uma atividade inibitória sobre a expressão de Cox-2 e produção dos mediadores inflamatórios PGE2, IL1, IL-6, e IL-12 em macrófagos estimulados com LPS in vitro. O mecanismo de inibição possivelmente envolve inibição da ativação de STAT-1. / Caveolin-1 (Cav-1), a protein essential for the formation of caveolae, shows activity in the modulation of intracellular signaling. Cav-1 can interact with several proteins by its caveolin scaffolding domain (CSD) and, in general, this interaction leads to inhibition of associated proteins. The peptide CSD has been used as a Cav- 1 mimetic in relation to its capacity on the modulatory activity of other proteins. Recently, it has been shown that Cav-1 can modulate the inflammatory response in several respects. We examined the role of Cav-1 in regulating the synthesis of inflammatory mediators by macrophages. Lipopolysaccharide (LPS) from E. coli, a prototype of inflammatory stimulus, was able to induce the expression of Cav-1 and Cox-2 in peritoneal macrophages in vitro. These proteins are induced in a similar time course, being detected by Western blot at 3 hours with increasing levels of expression up to 18 hours. By immunofluorescence, we observed that Cav-1 and Cox-2 have a mutually exclusive pattern of expression in macrophages stimulated with LPS. Western blot analysis showed that the expression of Cox-2 is induced by LPS and that treatment with CSD leads to inhibition of Cox-2 but not Cox-1. We also observed the partial reduction of PGE2 levels in supernatants of macrophages stimulated with LPS and treated with CSD. Treatment with CSD peptide was also able to reduce the levels of IL1, IL-6 and IL-12 induced by LPS. LPS induces increased expression and phosphorylation of STAT-1. The phosphorylation of STAT- 1 was decreased after treatment with the CSD, indicating that a Cav-1 negatively modulates activation of STAT-1. Further studies are needed to supplement the data obtained so far to clarify the mechanisms of modulation of the synthesis of inflammatory mediators by Cav-1. In conclusion, Cav-1 shows an inhibitory activity on Cox-2 expression and production of the inflammatory mediators PGE2, IL1β, IL-6 and IL-12 in macrophages stimulated with LPS in vitro. The mechanism of inhibition possibly involves inhibition of STAT-1 activation.

Made available in DSpace on 2016-01-26T18:55:31Z (GMT). No. of bitstreams: 1 DISSERTACAO.pdf: 506827 bytes, checksum: 85183faa6a63e1b561cacb568f6cf77e (MD5) Previous issue date: 2011-02-24 / The anti-inflammatory nonsteroidal compounds have extremely widespread use in the therapy of small animals, due to their anti-inflammatory and analgesic properties. However, the use of these drugs can produce changes in kidney function. The present study was conducted to assess kidney function in healthy dogs undergoing therapy with anti-inflammatory nonsteroidal compounds. Thirty mongrel dogs, adults, males and females, clinically healthy, were divided randomly into 5 groups (G) of six animals each receiving the following therapies: Gceto ketoprofen, a 2 mg/kg dose (VO), every 24 hours, during 10 days; Gnime nimesulide, 5 mg/Kg, VO, every 24 hours, during 10 days; Gmelo- meloxican, 0.2 mg/Kg on the first day, followed by 0.1 mg/Kg, VO, every 24 hours, 7 days; Geto etodolac, 15 mg/Kg, VO, every 24 hours, 7 days; Gcele- celecoxibe, 5 mg/Kg, VO, every 12 hours, for 20 days. The physical examination and renal function (urinalysis, urinary GGT, creatinine and sodium, serum urea, creatinine, potassium and sodium, and endogenous creatinine clearance) were assessed prior to the treatment, on the 5th and 10th days (T0, T5 and T10) into the treatment in all groups, and also on the 20th day (T20) into the treatment in Gcele. Few changes were observed in urinalysis parameters, only with significant increase in the presence of renal cells in the urine in T5, in the nimesulide group. There was a significant reduction in sodium elimination in the animals urine in the nimesulide group. The clearance values showed significant decrease in the celecoxibe group in T20, in relation to T5. The enzyme GGT urinary showed no variation among groups or moments. Values of sodium, potassium, urea and creatinine serum remained within normal ranges in all times, in the different groups. In conclusion, minimal changes of renal function occur 10 days after therapy on set with NSAIDS nimesulide, after 10 days with ketoprofen, on the 20th days of therapy with celecoxibe in health dogs. / Os anti-inflamatórios não-esteroidais têm uso extremamente difundido na clínica de pequenos animais, devido às suas propriedades analgésicas e anti-inflamatórias. Entretanto, o uso desses fármacos pode produzir alterações da função renal. O presente estudo teve como objetivo avaliar a função renal de cães saudáveis, submetidos à terapia com anti-inflamatórios não-esteroidais não seletivos, COX-2 preferenciais e COX-2 seletivos. Foram utilizados 30 cães, sem raça definida, adultos, machos e fêmeas, clinicamente sadios, divididos aleatoriamente em 5 grupos (G) de 6 animais cada, que receberam as seguintes terapias: Gceto cetoprofeno, na dose de 2 mg/Kg, por via oral (VO), a cada 24 horas, durante 10 dias; Gnime nimesulida, 5 mg/Kg, VO, a cada 24 horas, durante 10 dias; Gmelo - meloxican, 0,2 mg/Kg no primeiro dia, seguido por 0,1 mg/Kg, VO, a cada 24 horas, por 10 dias; Geto etodolaco, 15 mg/Kg, VO, a cada 24 horas, 10 dias; Gcele - celecoxibe, 5 mg/Kg, VO, a cada 12 horas, por 20 dias. O exame físico e a função renal (urinálise; GGT, creatinina e sódio urinários; uréia, creatinina, sódio e potássio séricos; e clearance endógeno de creatinina) foram avaliados antes, aos 5, 10 dias (T0, T5 e T10) de tratamento em todos os grupos, e também aos 20 dias (T20) de tratamento no Gcele. Poucas alterações foram observadas na urinálise, apenas com aumento significativo da presença de células renais na urina no T5 e T10 em relação ao T0, no grupo nimesulida. Houve redução significativa da eliminação de sódio na urina, nos animais do grupo nimesulida, no T5. Os valores de clearance foram os mais baixos no grupo Cetoprofeno no T10, e revelaram diminuição significativa no grupo Celecoxibe no T20, em relação ao T5. A enzima GGT urinária não apresentou variação entre grupos ou momentos. Valores de sódio, potássio, uréia e creatinina séricos mantiveram-se dentro da normalidade em todos os momentos nos diferentes grupos. Conclui-se que, em cães hígidos, alterações mínimas da função renal ocorrem aos cinco dias de terapia com o AINE nimesulida, aos dez dias com cetoprofeno, e aos vinte dias de terapia com celecoxibe.

Made available in DSpace on 2016-07-18T17:53:08Z (GMT). No. of bitstreams: 1 DISSERTACAO.pdf: 506827 bytes, checksum: 85183faa6a63e1b561cacb568f6cf77e (MD5) Previous issue date: 2011-02-24 / The anti-inflammatory nonsteroidal compounds have extremely widespread use in the therapy of small animals, due to their anti-inflammatory and analgesic properties. However, the use of these drugs can produce changes in kidney function. The present study was conducted to assess kidney function in healthy dogs undergoing therapy with anti-inflammatory nonsteroidal compounds. Thirty mongrel dogs, adults, males and females, clinically healthy, were divided randomly into 5 groups (G) of six animals each receiving the following therapies: Gceto ketoprofen, a 2 mg/kg dose (VO), every 24 hours, during 10 days; Gnime nimesulide, 5 mg/Kg, VO, every 24 hours, during 10 days; Gmelo- meloxican, 0.2 mg/Kg on the first day, followed by 0.1 mg/Kg, VO, every 24 hours, 7 days; Geto etodolac, 15 mg/Kg, VO, every 24 hours, 7 days; Gcele- celecoxibe, 5 mg/Kg, VO, every 12 hours, for 20 days. The physical examination and renal function (urinalysis, urinary GGT, creatinine and sodium, serum urea, creatinine, potassium and sodium, and endogenous creatinine clearance) were assessed prior to the treatment, on the 5th and 10th days (T0, T5 and T10) into the treatment in all groups, and also on the 20th day (T20) into the treatment in Gcele. Few changes were observed in urinalysis parameters, only with significant increase in the presence of renal cells in the urine in T5, in the nimesulide group. There was a significant reduction in sodium elimination in the animals urine in the nimesulide group. The clearance values showed significant decrease in the celecoxibe group in T20, in relation to T5. The enzyme GGT urinary showed no variation among groups or moments. Values of sodium, potassium, urea and creatinine serum remained within normal ranges in all times, in the different groups. In conclusion, minimal changes of renal function occur 10 days after therapy on set with NSAIDS nimesulide, after 10 days with ketoprofen, on the 20th days of therapy with celecoxibe in health dogs. / Os anti-inflamatórios não-esteroidais têm uso extremamente difundido na clínica de pequenos animais, devido às suas propriedades analgésicas e anti-inflamatórias. Entretanto, o uso desses fármacos pode produzir alterações da função renal. O presente estudo teve como objetivo avaliar a função renal de cães saudáveis, submetidos à terapia com anti-inflamatórios não-esteroidais não seletivos, COX-2 preferenciais e COX-2 seletivos. Foram utilizados 30 cães, sem raça definida, adultos, machos e fêmeas, clinicamente sadios, divididos aleatoriamente em 5 grupos (G) de 6 animais cada, que receberam as seguintes terapias: Gceto cetoprofeno, na dose de 2 mg/Kg, por via oral (VO), a cada 24 horas, durante 10 dias; Gnime nimesulida, 5 mg/Kg, VO, a cada 24 horas, durante 10 dias; Gmelo - meloxican, 0,2 mg/Kg no primeiro dia, seguido por 0,1 mg/Kg, VO, a cada 24 horas, por 10 dias; Geto etodolaco, 15 mg/Kg, VO, a cada 24 horas, 10 dias; Gcele - celecoxibe, 5 mg/Kg, VO, a cada 12 horas, por 20 dias. O exame físico e a função renal (urinálise; GGT, creatinina e sódio urinários; uréia, creatinina, sódio e potássio séricos; e clearance endógeno de creatinina) foram avaliados antes, aos 5, 10 dias (T0, T5 e T10) de tratamento em todos os grupos, e também aos 20 dias (T20) de tratamento no Gcele. Poucas alterações foram observadas na urinálise, apenas com aumento significativo da presença de células renais na urina no T5 e T10 em relação ao T0, no grupo nimesulida. Houve redução significativa da eliminação de sódio na urina, nos animais do grupo nimesulida, no T5. Os valores de clearance foram os mais baixos no grupo Cetoprofeno no T10, e revelaram diminuição significativa no grupo Celecoxibe no T20, em relação ao T5. A enzima GGT urinária não apresentou variação entre grupos ou momentos. Valores de sódio, potássio, uréia e creatinina séricos mantiveram-se dentro da normalidade em todos os momentos nos diferentes grupos. Conclui-se que, em cães hígidos, alterações mínimas da função renal ocorrem aos cinco dias de terapia com o AINE nimesulida, aos dez dias com cetoprofeno, e aos vinte dias de terapia com celecoxibe.

Ovarian cancer is the fifth most lethal cancer in women (1) and the most lethal gynecological malignancy. In 2018, there were approximately 22,240 new diagnosed cases of ovarian cancer and 14,070 deaths in the United States alone (2). The lifetime risk for developing ovarian cancer in the United States is 1.3% or approximately 1 in 78 women. The five-year survival rate for women with ovarian cancer is a grim 47.6% (2) while the average five year survival rate for all cancers is about 68%. This dismal prognosis for ovarian cancer patients indicates the critical need for improved treatment options, efficient early detection methods and effective preventative measures for ovarian cancer (1). The objective of this study was to determine if DHA causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of NF-κB regulated transcription in the ovary. DHA is a 22 carbon long-chain omega-3 polyunsaturated fatty acid that is biologically derived from Alpha-linolenic acid (ALA) found in flaxseed. COX-2 is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. Prostaglandin E2 (PGE2) is a key regulator of inflammation which has been shown to be highly associated with ovarian cancer development and progression. Our laboratory studies ovarian cancer in the laying hen because it is the only known animal model to naturally develop ovarian cancer that both pathologically and histologically matches that of the human form of the disease. Dietary flaxseed is one of the richest vegetable sources of omega-3 polyunsaturated fatty acids. Our previous studies have shown that in laying hens, a long-term flaxseed supplemented diet reduces the incidence and severity of ovarian cancer and decreases COX-2 and PGE2. It was hypothesized that DHA, derived from ALA found in flaxseed, decreases inflammation in the ovaries by suppressing the activation of COX-2 and the production of PGE2 through inhibition of the NF-κB pathway. For this study, an NF-κB reporter plasmid was transfected into HEK293 cells. The reporter plasmid (“met-luc”) produces a secreted luciferase allowing sequential analysis of media from DHA and TNF-α treated cells to assess changes in NF-κB transcriptional activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB was used as a positive control. NF-κB activation was also assessed by measuring its nuclear translocation and cytoplasmic accumulation through immunocytochemistry (ICC) and western blot analysis. In a parallel study, immortalized ovarian surface epithelial (IOSE) cells were challenged with the same treatments of DHA and TNF-α. In these cells, COX-2 mRNA was assessed through RT-qPCR and COX-2 protein expression was analyzed through ICC and western blot.Our results indicate that DHA acts in a cell specific manner to reduce inflammation associated with cancer. We have found that in HEK293 cells DHA reduces TNFα induced NF-κB reporter activity. In contrast, ALA does not affect NF-κB reporter activity. HEK293 cells treated with TNFα alone indicated a dose-dependent increasing trend in nuclear translocation of the NF-κB p65 subunit and a decreasing trend in cytoplasmic p65, suggesting potential increased pathway activation. ICC suggests DHA treatment causes increased cytoplasmic sequestration of the NF-κB p65 subunits indicating inhibition of TNFα induced NF-κB activation. Western blot data also indicates a decreasing trend in nuclear NFκB p65 when cells are pretreated with DHA and subsequently challenged with TNF. The IOSE cells, were the only cells out of the cell lines tested (BG1, HEYC2, TOV112D, SKOV3, HEK293) to express COX-2. In these IOSE cells, TNFα alone showed a dose-dependent increasing trend in COX-2 protein (analyzed through ICC and western blot) and mRNA levels (analyzed through RT-qPCR). ICC analysis revealed that DHA reduces TNF induced COX-2 protein expression. However, the western blot did not further support this observation. Only a slight non-significant reduction with DHA treatment was observed. In addition, both DHA and TNFα, while also not significant, seemed to increase mRNA levels of COX-2 compared to control. This slight decreasing trend in COX-2 protein expression and increase in mRNA, could indicate a possible post-transcriptional mechanism of regulation of COX-2 by DHA independent of NF-κB in the IOSE cells. These data suggest that DHA could act via distinct mechanisms in a cell specific manner to potentially reduce COX-2 and subsequently PGE2 levels. DHA can act at the transcriptional level by reducing the nuclear translocation of NF-κB and transcriptional activation of NF-κB target genes such as COX-2 in some cell types. DHA also has the potential to work via a post-transcriptional mechanism to inhibit COX-2 and in turn reduce PGE2 levels. Both mechanisms ultimately have the potential to decrease the inflammation associated with ovarian cancer. This study describes the anti-inflammatory action of dietary flaxseed consumption, making flaxseed supplementation a promising preventive measure for reducing the risk of ovarian carcinogenesis.

No description available.

Diversos mecanismos celulares estão associados à patogênese do Carcinoma Epidermoide de Cabeça e Pescoço (CECP). Algumas dessas alterações envolvem proteínas pertencentes à via de sinalização do Akt, e o fator de transcrição NF-kB, o qual têm importante papel na fisiologia normal e no câncer. A proteína COX-2, descrita em processos inflamatórios, também participa da carcinogênese e está associada com a via de sinalização do Akt e com o NF-kB. Dendrímeros são uma forma única de nanotecnologia, surgindo como nanotransportadores com a capacidade de penetrar na célula tumoral liberando drogas quimioterápicas em seu interior. Os benefícios desta tecnologia são o aumento da eficicácia do princípio ativo utilizado e a redução dos seus efeitos secundários tóxicos. O Celecoxibe, antiinflamatório não esteroidal, inibidor seletivo da COX-2, tem se mostrado um importante agente anticarcinogênico, no entanto seu mecanismo de ação no CECP não é totalmente compreendido. Neste trabalho, um Dendrímero de Poliglicerol associado ao Celecoxibe (PGLD-celecoxibe) foi sintetizado e caracterizado por técnicas de espectroscopia ¹H-RMN, ¹³C-RMN, Maldi-Tof, TLC e DSC. Além disso, o conjugado foi testado in vitro em três linhagens celulares de CECP. O PGLD-Celecoxibe foi sintetizado com sucesso e promoveu a redução da dose capaz de inibir a proliferação celular, reduzindo o IC 50 do Celecoxibe de forma significativa em todas as linhagens celulares, se aproximando da dose sérica alcançada por este medicamento, resultado corroborado pelo Ensaio de Migração Celular. O mecanismo de morte celular observado foi a apoptose, associada a diminuição significativa da expressão de COX-2 ou por uma via alternativa independente. Alguns dos grupos tratados apresentaram alteração na expressão das proteínas pAkt e NF-kB. / Several cellular mechanisms are associated with the pathogenesis of Head and Neck Squamous Cell Carcinoma (HNSCC). Some of these alterations involve proteins in the Akt signaling pathway and the transcription factor NF-kB, which plays an important role in normal physiology and in cancer. COX-2 protein, described in inflammatory processes, and also involved in the carcinogenesis is associated with the Akt signaling pathway and the NF-kB. Dendrimers are a unique form of nanotechnology, emerging as nanocarriers with the ability to penetrate the tumor cell releasing chemotherapeutic. This technology increases the active substance efficiency and reduces its toxic side effects. Celecoxib, a nonsteroidal anti-inflammatory, selective inhibitor of COX-2 has been shown to be an important anticancer agent, but its action mechanism in HNSCC is not fully understood. A polyglycerol dendrimer linked to celecoxib (PGLD-Celecoxibe) was synthesized and characterized by NMR spectroscopy ¹H-NMR, ¹³C-NMR,TLD, DSC and Maldi-Tof techniques. In addition, in vitro assays were performed in three HNSCC cell lines The PGLD-Celecoxibe was successfully synthesized and provided a decrease in the dose able to inhibit cell proliferation reducing the IC 50 index of Celecoxib significantly in all cell lines, approaching to the serum dose achieved for this product, result supported by Wound Healing Assay. The cell death mechanism observed was apoptosis, which can be associated with significant reduction of expression of COX-2 also may be occurring by a COX-2 independent pathway. Some of the treated groups showed alterations in pAkt and NF-kB proteins expression.

Les cyclooxygénases (COXs) sont une famille d'enzymes impliquées dans la biosynthèse des prostaglandines. COX-2 est la forme inductible qui est induite pendant l'inflammation et qui est surexprimée dans plusieurs cancers. Plusieurs évidences suggèrent que COX-2 joue un rôle dans la prolifération cellulaire et l'apoptose. Ces évidences concernent surtout les tumeurs solides et les mécanismes impliqués ne sont pas complètement connus et surtout pour les cancers d'origine hématopoïétique. Pour notre étude, nous avons étudié l'effet d'inhibiteurs de COX-2 (nimésulide, NS-398 et célécoxib) sur la prolifération et l'apoptose de lignées cellulaires leucémiques et lymphoblastiques, Hel, Jurkat, Raji et U937. Nous avons montré que les différents inhibiteurs de COX-2 diminuent la prolifération des différentes lignées cellulaires. Les cellules U937 sont apparues comme les cellules les plus sensibles à ces inhibiteurs alors que les cellules K562 étaient les plus résistantes. Nous avons montré que cette modulation correspond à une accumulation des cellules en phase G0/G1 du cycle cellulaire, accompagnée d'une diminution précoce de l'expression de c-Myc et d'une augmentation de l'expression de marqueurs de différenciation dans les cellules U937 (CD15) et Hel (CD41a et CD61). Dans la deuxième partie de ce projet, nous avons étudié les effets des différents inhibiteurs de COX-2 sur l'apoptose induite par différents agents chimiothérapeutiques dans nos modèles cellulaires. Nous avons ainsi montré que les inhibiteurs de COX-2 inhibent fortement l'apoptose induite par plusieurs agents chimiothérapeutiques. Nous avons démontré que la prévention de l'apoptose se situe avant l'activation de Bax et de Bak. Par ailleurs, cet effet est caractérisé par une incapacité des agents chimiothérapeutiques à déclencher un stress apoptotique. Toutes nos données ont donc démontré un effet anti-apoptotique des inhibiteurs de COX-2 sur l?apoptose intrinsèque vs l'apoptose extrinsèque à un stade précoce de l'induction de l'apoptose. Ces données suggèrent des précautions quant à l'utilisation des inhibiteurs de COX-2 en combinaison avec la chimiothérapie. Dans la troisième partie de notre projet, nous avons étudié la combinaison des inhibiteurs de COX-2 avec la curcumine, une substance naturelle connue pour ses propriétés antitumorales. Nos travaux ont montré que la curcumine seule conduit à une accumulation des cellules U937 en phase G2/M du cycle cellulaire, suivie d'une induction d'apoptose. Cependant, le prétraitement des cellules U937 avec du célécoxib à des concentrations non-apoptogéniques contrecarre l'apoptose induite par la curcumine, suggérant ainsi que ce type de combinaison ne serait pas une bonne stratégie dans les cellules hématopoïétiques. L'utilisation chronique des inhibiteurs de COX-2 peut être associée à des effets secondaires importants consécutifs à l'inhibition de l'activité de COX-2. Dans la dernière partie de notre projet, nous avons démontré que le 2,5 diméthyl-célécoxib (DMC), un analogue du célécoxib qui n'inhibe pas l'activité de COX-2, induit une diminution de la prolifération cellulaire et induit l'apoptose des cellules U937 et K562. Par ailleurs, ces effets sont plus importants que ceux observés avec le célécoxib. Par conséquent, ce composé a démontré de meilleures propriétés antitumorales et représente une voie thérapeutique prometteuse contre les leucémies. Tous nos résultats soutiennent donc l'idée que les inhibiteurs de COX-2 présentent les effets anti-tumoraux les plus efficaces que lorsqu'ils sont administrés seuls. Les effets observés avec le DMC suggèrent que ce composé pourrait représenté une voie alternative aux inhibiteurs de COX-2 en thérapie anti-cancéreuse. / Cyclooxygenases (COXs) are a family of enzymes, which catalyze the rate-limiting step in prostaglandin biosynthesis. COX-2 is the inducible isoform, upregulated during inflammation and overexpressed in various cancers. There are evidences of a role for COX-2 in cell proliferation and apoptosis especially in solid tumors, whereas little is known for cancers of hematopoietic origin. In our study, we analyzed the effect of COX-2 inhibitors (nimesulide, NS-398 and celecoxib) on cell proliferation and apoptosis of a panel of leukemic and lymphoblastic cell lines, Hel, Jurkat, K562, K562, Raji and U937. We found that the different inhibitors slow down cell proliferation in the different hematologic cell lines tested. U937 cells appeared as the most sensitive, whereas K562 were the most resistant to this effect. We provide evidence that this modulation corresponds to an accumulation of the cells in G0/G1 paralleled by an early downregulation of c-Myc and the expression of cell type-specific differentiation markers in U937 (CD15) and Hel (CD41a and CD61). In the second part of our study, we investigate the effect of COX-2 inhibitors on apoptosis induced by chemotherapeutic agents in our cell models. We demonstrated that COX-2 inhibitors strongly prevent apoptosis induced by a panel of chemotherapeutic agents. We demonstrated an early prevention of apoptotic signaling, prior to Bax/Bak activation. The preventive effect is associated with an impairment of the ability of chemotherapeutic agents to trigger their apoptogenic stress. Altogether, our results demonstrate an anti-apoptotic effect of COX-2 inhibitors on intrinsic vs. extrinsic apoptosis at early steps of apoptosis commitment. These results suggest cautions in the use of COX-2 inhibitors with chemotherapy. In the third part of our project, we investigated the combination of COX-2 inhibitors with curcumin, a natural product known for its anti-tumor properties. Our findings show that curcumin alone leads to an accumulation of U937 cells in G2/M phase of cell cycle, followed by an induction of apoptosis. However, the pretreatment of U937 cells with celecoxib at non-apoptogenic concentrations, counteracted curcumin-induced apoptosis, thus showing that this combination is not a good anti-cancer strategy in our cell models. The chronic use of COX-2 inhibitors can be associated with severe side effects due to the inhibition of COX-2 enzyme. In the last part of our project, we demonstrated that 2,5 dimethyl-celecoxib (DMC), a structurally analogue of celecoxib, which is not able to inhibit COX-2 activity, induces an inhibition of cell proliferation and an induction of apoptosis in U937 and K562 cells. These effects are stronger than those observed with celecoxib. Thus, this compound demonstrated better anti-tumor properties and may represent a promising therapeutic approach against leukemia. Altogether, our study supports the idea that COX-2 inhibitors display anti-tumor effects in our cell models, but only when administrated alone. The effects observed with DMC suggest that this compound may represent an alternative approach to COX-2 inhibitors in cancer therapy.

Diversos mecanismos celulares estão associados à patogênese do Carcinoma Epidermoide de Cabeça e Pescoço (CECP). Algumas dessas alterações envolvem proteínas pertencentes à via de sinalização do Akt, e o fator de transcrição NF-kB, o qual têm importante papel na fisiologia normal e no câncer. A proteína COX-2, descrita em processos inflamatórios, também participa da carcinogênese e está associada com a via de sinalização do Akt e com o NF-kB. Dendrímeros são uma forma única de nanotecnologia, surgindo como nanotransportadores com a capacidade de penetrar na célula tumoral liberando drogas quimioterápicas em seu interior. Os benefícios desta tecnologia são o aumento da eficicácia do princípio ativo utilizado e a redução dos seus efeitos secundários tóxicos. O Celecoxibe, antiinflamatório não esteroidal, inibidor seletivo da COX-2, tem se mostrado um importante agente anticarcinogênico, no entanto seu mecanismo de ação no CECP não é totalmente compreendido. Neste trabalho, um Dendrímero de Poliglicerol associado ao Celecoxibe (PGLD-celecoxibe) foi sintetizado e caracterizado por técnicas de espectroscopia ¹H-RMN, ¹³C-RMN, Maldi-Tof, TLC e DSC. Além disso, o conjugado foi testado in vitro em três linhagens celulares de CECP. O PGLD-Celecoxibe foi sintetizado com sucesso e promoveu a redução da dose capaz de inibir a proliferação celular, reduzindo o IC 50 do Celecoxibe de forma significativa em todas as linhagens celulares, se aproximando da dose sérica alcançada por este medicamento, resultado corroborado pelo Ensaio de Migração Celular. O mecanismo de morte celular observado foi a apoptose, associada a diminuição significativa da expressão de COX-2 ou por uma via alternativa independente. Alguns dos grupos tratados apresentaram alteração na expressão das proteínas pAkt e NF-kB. / Several cellular mechanisms are associated with the pathogenesis of Head and Neck Squamous Cell Carcinoma (HNSCC). Some of these alterations involve proteins in the Akt signaling pathway and the transcription factor NF-kB, which plays an important role in normal physiology and in cancer. COX-2 protein, described in inflammatory processes, and also involved in the carcinogenesis is associated with the Akt signaling pathway and the NF-kB. Dendrimers are a unique form of nanotechnology, emerging as nanocarriers with the ability to penetrate the tumor cell releasing chemotherapeutic. This technology increases the active substance efficiency and reduces its toxic side effects. Celecoxib, a nonsteroidal anti-inflammatory, selective inhibitor of COX-2 has been shown to be an important anticancer agent, but its action mechanism in HNSCC is not fully understood. A polyglycerol dendrimer linked to celecoxib (PGLD-Celecoxibe) was synthesized and characterized by NMR spectroscopy ¹H-NMR, ¹³C-NMR,TLD, DSC and Maldi-Tof techniques. In addition, in vitro assays were performed in three HNSCC cell lines The PGLD-Celecoxibe was successfully synthesized and provided a decrease in the dose able to inhibit cell proliferation reducing the IC 50 index of Celecoxib significantly in all cell lines, approaching to the serum dose achieved for this product, result supported by Wound Healing Assay. The cell death mechanism observed was apoptosis, which can be associated with significant reduction of expression of COX-2 also may be occurring by a COX-2 independent pathway. Some of the treated groups showed alterations in pAkt and NF-kB proteins expression.