RESUMEN
The severity of the bladder carcinoma (BC) is directly linked to cell invasion and metastasis. Indoleamine 2,3-dioxygenase-1 (IDO-1) is an INF-γ-induced immunomodulating enzyme that has been linked to the cancer cell invasiveness. Because IDO1 is variable among the tumors, we analyzed its expression in the BC invasion using BC mice models and cell culture. MB49 cells were orthotopically or ectopically inoculated in C57Bl6 mice to evaluate IDO1 by immunohistochemistry. For in vitro experiments, expression of IDO1 and INF-γ was evaluated in grade-1 (RT4) and in grade-3 (T24) BC cell lines. Invading and non-invading T24 cells were separated using the Matrigel/Transwell system, of which total RNA was extracted immediately or after 2 weeks of subculture. Finally, IDO1 was silenced in T24 cells to verify its role on cell invasiveness. In both animal models, IDO1 was differentially expressed between non-invading and invading cells. In cell culture, T24 cells expressed more IDO1 than RT4 cells, independently of the INF-γ expression. IDO1 was differentially expressed between non-invading and invading T24 cells, a difference that was lost by long-time subculture. IDO1 silencing resulted in diminished cell invasiveness. In conclusion, IDO1 expression is changed during bladder carcinoma invasion, playing an important role in this process.
RESUMEN
The severity of the bladder carcinoma (BC) is directly linked to cell invasion and metastasis. Indoleamine 2,3-dioxygenase-1 (IDO-1) is an INF-γ-induced immunomodulating enzyme that has been linked to the cancer cell invasiveness. Because IDO1 is variable among the tumors, we analyzed its expression in the BC invasion using BC mice models and cell culture. MB49 cells were orthotopically or ectopically inoculated in C57Bl6 mice to evaluate IDO1 by immunohistochemistry. For in vitro experiments, expression of IDO1 and INF-γ was evaluated in grade-1 (RT4) and in grade-3 (T24) BC cell lines. Invading and non-invading T24 cells were separated using the Matrigel/Transwell system, of which total RNA was extracted immediately or after 2 weeks of subculture. Finally, IDO1 was silenced in T24 cells to verify its role on cell invasiveness. In both animal models, IDO1 was differentially expressed between non-invading and invading cells. In cell culture, T24 cells expressed more IDO1 than RT4 cells, independently of the INF-γ expression. IDO1 was differentially expressed between non-invading and invading T24 cells, a difference that was lost by long-time subculture. IDO1 silencing resulted in diminished cell invasiveness. In conclusion, IDO1 expression is changed during bladder carcinoma invasion, playing an important role in this process.
RESUMEN
Immunoglobulin M (IgM) is a pentamer of approximately 950kDa, consisting of two heavy chains of 75kDa each, two light chains of 25kDa and a J chain of 15kDa. IgM is a promising therapeutic candidate due to increasing evidence suggesting its potential as a tumor marker, anti-inflammatory and immunomodulatory activities. In order to exploit its full potential, it is important that large amounts of IgM are available at low cost. In this work, two purification steps that were already being developed by the laboratory were explored. Purification by metal affinity chromatography (IMAC) and cation exchange of the pool from plasma purification on Sepharose 4FF followed by ANX Sepharose FF was evaluated. In IMAC, the purification was evaluated at pHs 5.0, 6.0 and 7.0 and the results indicate that with pH 6.0 in IMAC-Co2+ we obtained IgM with better recovery and greater purity. By varying the NaCl concentration in the equilibrium buffer between 250mM and 500mM, we found that IgM was obtained with greater purity in the purification in which 250mM NaCl was used in the equilibrium buffer. Using the cation exchange column, the results obtained were not satisfactory. The purity of IgM, calculated by the ImageJ program, was approximately 75%. To increase the purity of IgM, purification on the Superdex 200 gel filtration column will be evaluated.
A imunoglobulina M é um pentâmero de aproximadamente 950kDa, sendo composto por duas cadeias pesadas de 75kDa cada, duas cadeias leves de 25kDa e uma cadeia J de 15kDa. IgM é um candidato terapêutico promissor devido ao aumento de evidências que sugerem seu potencial de marcador tumoral, atividades anti-inflamatória e imunomoduladoras. Com o intuito de que todo seu potencial seja explorado, é importante que grandes quantidades de IgM estejam disponíveis a baixo custo. Nesse trabalho foram exploradas duas etapas de purificação que já estavam sendo desenvolvida pelo laboratório. Aplicou-se cromatografia de afinidade ao metal (IMAC) na fração 350mM proveniente da ANX Sepharose FF, com o intuito de obter IgM com alto grau de pureza. Foi observado que a melhor faixa de trabalho foi em pH 6,0 utilizando como metal imobilizado o cobalto e solução contendo NaCl 250mM. Como segunda estratégia, aplicou-se cromatografia de troca catiônica na fração 350mM diluído 10 vezes com água purificada e ao contrário da IMAC, não obtivemos resultados satisfatórios em relação a pureza de IgM. Apesar dos resultados alcançados, ainda é necessário o desenvolvimento de novas estratégias e estudos para o aumento de IgM, a fim de se obter uma imunoglobulina mais pura para possível uso terapêutico.