Analysis of the effect of 1-methyl-D-tryptophan on signaling pathways mediated by indoleamine 2,3-dioxiogenase in bladder carcinoma: possible involvement in the determination of invasive phenotype

Abstract

Bladder cancer (BC) is among the most common cancers of the urinary tract. Although the most frequent is the non-muscle-invasive form, many of these cases evolve to the muscle-invasive form. Indoleamine 2,3-dioxygenase (IDO) is an enzyme expressed at sites where immunomodulation is required, including the maternal-fetal interface, where the presence of IDO protects the semi-halogen embryo. Because IDO is expressed in some types of cancer, it is believed to promote immune escape to the tumor. In BC, its role has not been elucidated, but we believe that IDO also participates in non-immunological events for tumor progression, such as mesenchymal to epithelial transition (MET). IDO exerts its effects through the depletion of tryptophan in the microenvironment, with consequent formation of kynurenine and its derivatives. Thus, three pathways are involved in the action of IDO: mTOR, GCN2 and AHR (aryl hydrocarbon receptor). The aim of the present study is to evaluate the expression of IDO and the activation of the mTOR, GCN2 and AHR pathways in non-muscle-invasive (RT4) and muscle-invasive (T24) CB cell lines and the effect of the IDO inhibitor ( MT, 1-methyl tryptophan) on these pathways and on the invasive cell phenotype.RT4 and T24 cells are cultured routinely in our laboratory. For the treatment of MT cells, the cells will be maintained in RPMI 1640 medium, supplemented with 10% BCS and antibiotics, containing or not MT at concentration of 500 µM. Real-time PCR will be used to evaluate the expression of IDO, CHOP (marker of GCN2 pathway activation), AHR, CYP1A1 (AHR receptor activation marker), and TEM markers. The mTOR pathway will be Western blotted. In addition, T24 cells with more invasive phenotype will be separated in Matrigel. The Scratch-wound assay will be used to evaluate the cell's migratory activity. As expected, this study may reveal whether IDO is differentially expressed in non-muscle-invasive and muscle-invasive CB cells, serving as a biomarker, and whether it has influence on the establishment of the invasive phenotype of these cells. (AU)