Made available in DSpace on 2015-04-14T13:35:58Z (GMT). No. of bitstreams: 1 458593.pdf: 1436191 bytes, checksum: b126309dee58688d2fe23cb45be67db7 (MD5) Previous issue date: 2014-02-21 / The purinergic system can induce proliferation, differentiation and death on tumor cells. Different P2 purinergic receptors are involved in preventing the growth of these cells. Although the mechanisms are not completely known, they may represent interesting therapeutic targets to control tumors. The RT4 and T24 human tumor bladder cell lines have been used as low and high cancer malignancy phenotype models, respectively. Our research group has demonstrated that quercetin increases the ADP hydrolysis and inhibits ecto-5'-nucleotidase/ CD73, exerting an anti-proliferative effect on T24 cell lines. We also demonstrate that T24 cell lines expresses the purinergic enzymes CD39L4 and ecto-5'-nucleotidase / CD73, and have low hydrolytic activity of ATPase and ADPase in contrast to a high activity AMPase. These data suggest the involvement of the enzymes and purinergic receptors on progression the tumor of the bladder, as well as other tumor types. The objective of the present study was characterize the purinergic receptors P2X (1-7) expression profile on human bladder tumor cell lines and biopsies; and investigate the inflammatory genes that are up-regulated on high-grade human tumor bladder cells compared to low grade tumor bladder cells. Human bladder cells RT4 and T24 were obtained from the ATCC. Cells were cultured in DMEN and RPMI, respectively, supplemented with 10% of fetal bovine serum (FBS), under ideal conditions of cultivation Data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey Kramer test. To confirm the relevance of P2X (1-7) receptors in bladder cancer, we have also assessed their expression in human biopsies. For this purpose, upon approval by the local ethics committee, specimens of bladder tumors and normal tissues were obtained from the same patients, who have been undergone surgical resection at the Hospital S?o Lucas/PUCRS. All samples were collected and rapidly frozen in -80?C with RNA holder. Tissue specimens were ground and then sonicated in a TRIzol kit. The mRNA level was analyzed using RT-PCR analysis. Real-time PCR revealed that the mRNA expression of P2X purinergic receptors, particulary P2X4R was significantly up-regulated on both T24 and RT4 cell lines. Data from human bladder tumor biopsies comparing to bladder normal tissue, showed that P2X6 and P2X7 receptors appeared to be most often up-regulated on tumor tissues. Twelve inflammation related genes, CCL2, CCR2, TNF-α, MAPK3, IL-1β, IL-6, ADORA1, CD200, CD4, CHRNA4, EDNRA e ITGB2 were significantly up-regulated on the grade III bladder tumor cell line T24 when compared to grade I bladder tumor cell line RT4. Our results indicate that the inflammation genes overexpressed may play an important role in regulating urinary bladder cancer. We can conclude that P2X purinoreceptors and inflammation genes are differentially distributed among normal bladder, low and high tumor bladder, which could contribute to the different characteristics of these different types of cells. / O sistema purin?rgico pode induzir a prolifera??o, diferencia??o e morte em c?lulas tumorais. Diferentes receptores purin?rgicos P2, est?o envolvidos na inibi??o do crescimento tumoral. Embora os mecanismos n?o sejam completamente conhecidos, estes receptores podem representar alvos terap?uticos interessantes no tumor de bexiga. As linhagens celulares derivadas de tumor de bexiga humano RT4 e T24 t?m sido usadas como modelo de baixa e alta malignidade de tumor, respectivamente. Estudos realizados pelo nosso grupo de pesquisa t?m demonstrado o envolvimento do sistema purin?rgico no tumor de bexiga. A quercetina aumenta a hidr?lise do ADP e inibe a ecto - 5' - nucleotidase / CD73, exercendo um efeito anti - proliferativo sobre as c?lulas T24. Tamb?m demonstramos que as linhagens de c?lulas T24 expressam enzimas CD39L4 e ecto - 5' - nucleotidase / CD73, e t?m baixa atividade hidrol?tica de ATPase e ADP?sica com uma elevada atividade AMP?sica. Estes dados sugerem o envolvimento das enzimas e receptores purin?rgicos na progress?o do tumor de bexiga, bem como em outros tipos de tumor. O objetivo do estudo ? caracterizar a express?o dos receptores purin?rgicos P2X (1-7) em bi?psias de c?ncer de bexiga e linhagens celulares de tumor de bexiga humano, relacionado com o perfil de genes inflamat?rios mais expressos nestas amostras. Metodologia: As linhagens celulares de bexiga humanas RT4 e T24 foram obtidas a partir da ATCC. As c?lulas foram cultivadas em meio RPMI e DMEN, respectivamente, suplementado com 10 % de soro fetal bovino (SFB), sob condi??es ideais de cultivo. Para confirmar a relev?ncia dos receptores P2X (1-7) no tumor de bexiga, avaliou-se a express?o em bi?psias humanas. Para isso, ap?s aprova??o pelo comit? de ?tica local, amostras de tecidos normais e tumorais de bexiga foram obtidas dos mesmos pacientes, submetidos ? ressec??o cir?rgica no Hospital S?o Lucas / PUCRS. O RNA total dos tecidos foi isolado utilizando kit TRIzol . O n?vel de mRNA foi analisado utilizando an?lise RT - PCR . Resultados: O PCR em tempo real revelou que a express?o de mRNA de receptores purin?rgicos P2X, particularmente P2X4R foi significativamente up- regulada em ambas linhagens RT4 e T24. Dados a partir de bi?psias de tumores de bexiga humanos que comparam os resultados de express?o do tecido tumoral em rela??o ao tecido normal de bexiga, demonstrou que os receptores de P2X6 e P2X7 est?o superexpressos em tecidos tumorais. Doze genes relacionados com a inflama??o, CCL2, CCR2, TNF-α, MAPK3, IL-1β, IL-6, ADORA1, CD200, CD4, CHRNA4, EDNRA e ITGB2 foram significativamente superexpressos em c?lulas T24 derivadas de tumor de grau III quando comparadas com as c?lulas RT4 derivadas de tumor de grau I. Nossos resultados indicam que os genes superexpressos de inflama??o podem desempenhar um papel importante na regula??o do tumor de bexiga urin?ria. Conclus?o: Podemos concluir que purinoreceptores P2X e os genes inflamat?rios s?o diferencialmente distribu?dos entre bexiga normal e tumoral, o que pode contribuir para as diferentes caracter?sticas destes diferentes tipos de tumores.

Made available in DSpace on 2015-04-14T13:35:28Z (GMT). No. of bitstreams: 1 437983.pdf: 3242745 bytes, checksum: 9b67fa711fdfe3bdd518bb49b3f07fc0 (MD5) Previous issue date: 2012-03-19 / This study was designed to characterize, by means of functional and molecular approaches, the relevance of kinin B1 and B2 receptors in bladder cancer. Our data clearly shows that both B1 des-Arg9-BK and B2 BK receptor agonists were able to stimulate the proliferation of the grade 3-derived bladder cancer T24 cells. Furthermore, the incubation of B1 and B2 receptor antagonists, SSR240612 and HOE140, respectively, markedly inhibited the proliferation rate of T24 cells. In contrast, only higher concentrations of BK elicited the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg9-BK incubation completely failed to induce its proliferation. Interestingly, real time PCR experiments revealed that mRNA expression of B2, and mainly B1 receptors was found superior in T24 cells, in comparison to the low malignity grade RT4 cells. Furthermore, data obtained using bladder cancer human biopsies revealed that B1 receptor expression was visibly increased in all tumoral samples or under chronic inflammation of bladder. Concerning the signaling pathways related to the mitogenic effects of kinins, we bring novel evidence showing that pharmacological inhibition of PI3Kg with AS252424 concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg9-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Our results indicate that kinin B2, and especially B1 receptors appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential therapeutic alternatives for bladder cancer control. / O presente estudo teve por objetivo caracterizar, atrav?s de abordagens funcionais e moleculares, a relev?ncia dos receptores B1 e B2 para as cininas no c?ncer de bexiga. Os dados obtidos mostraram que tanto o agonista dos receptores B1, des- Arg9-BK, quanto do receptor B2, BK, foram capazes de estimular a prolifera??o das c?lulas de c?ncer de bexiga grau 3, denominadas T24. Al?m disso, a incuba??o dos antagonistas dos receptores B1 e B2, SSR240612 e HOE140, respectivamente, inibiram acentuadamente a prolifera??o das c?lulas T24. Por outro lado, apenas maiores concentra??es de BK levaram ? prolifera??o das c?lulas de c?ncer de bexiga grau 1 RT4; enquanto a incuba??o com des-Arg9-BK n?o induziu sua prolifera??o. De maneira similar, os resultados revelaram que a express?o de mRNA dos receptores B2 e, principalmente, dos receptores B1 foi superior em c?lulas T24, em compara??o com as c?lulas RT4, que apresentam baixo grau de malignidade. Al?m disso, os dados obtidos com bi?psias de c?ncer de bexiga humano revelaram que a express?o do receptor B1 foi claramente maior em todas as amostras tumorais ou, em uma bi?psia obtida de um quadro de inflama??o cr?nica de bexiga. Em rela??o ?s vias de sinaliza??o relacionadas com os efeitos mitog?nicos das cininas, os dados obtidos mostram que a inibi??o farmacol?gica da PI3Kg com AS252424 reduziu de maneira concentra??o-dependente a prolifera??o das c?lulas T24, quando est? foi induzida por BK ou des-Arg9-BK. Finalmente, a incuba??o das c?lulas T24 com agonistas dos receptores de cininas produziu uma acentuada ativa??o das vias de sinaliza??o PI3K/AKT e ERK 1/2, enquanto a via p38 MAP quinase permaneceu inalterada. Nossos resultados indicam que os receptores de cininas B2 e, especialmente, os receptores B1 parecem estar associados com a progress?o do c?ncer de bexiga. Dessa forma, ? poss?vel sugerir que antagonistas seletivos de receptores de cininas poderiam representar alternativas terap?uticas interessantes para o controle do c?ncer de bexiga.