Efeito da inibição da indoleamina 2,3 dioxigenase associada ao BCG sobre células MB49 de carcinoma de bexiga – estudo in vitro e em modelo animal

Abstract

The transurethral resection of the bladder tumor (TUR), associated with Bacilo Calmette-Guérin (BCG) immunotherapy, is the first step in the treatment of noninvasive bladder cancer (BC). Although it is considered gold standard, this therapy seems to have limited benefits, since around 50% of the cases return to invasive forms in the first year after TUR, suggesting that there is some tumor escape, even in the presence of immunotherapy. Concerning that the presence of BCG induces the release of interferon-gamma, a potent inducer of indoleamine-2,3-dioxygenase (IDO), a molecule recognized as a tumor escape promoter, it is likely that IDO plays a role in the mechanisms triggered by BCG In CB. The objective was to verify the possible involvement of the enzyme IDO in BC, in order to evaluate its chemical inhibition with methyl tryptophan (MT) as a therapeutic possibility. In the first step, assays with culture of MB49 murine cells were performed. Cells were incubated with BCG, evaluating: cell viability, ability to incorporate BCG and expression of IDO. In addition, MB49 cells were cultured with mononuclear cells and incubated with BCG to verify the expression of IDO. The effect of MT on the viability of MB49 cells was also evaluated. For the second step, the orthotopic BC model was reproduced in order to evaluate treatment with MT and BCG. In this model, MB49 cells were transurethrally inoculated, and the tumor was diagnosed by the presence of hematuria and ultrasound tumor mass detection. After tumor establishment, the treatments with MT, BCG and the association of BCG with MT were evaluated. Over a period of 35 days, the animals were evaluated for survival, tumor mass and volume, IDO expression and number of intratumor T lymphocytes. In addition, we evaluated the expression of IDO in normal bladder and instilled with BCG. As in vitro results, incubation with BCG decreases the viability of MB49 cells and there was internalization of the bacillus by cells. MB49 cells constitutively express the IDO and incubation with BCG increased this expression. We observed no effect of BCG on increasing IDO expression in mononuclear cells and in coculture. There was a decrease in cell viability by inhibiting IDO with MT, but the association of MT with BCG did not potentiate this effect. Regarding the in vivo results, inoculations with 250,000 and 100,000 MB49 cells provided exacerbated tumor growth, leading us to decrease the number to 50,000 cells. Even so, the animals did not reach the pre-established protocol of 35 days. No effects of BCG, MT and BCG + MT treatments on the proposed parameters were observed. IDO expression is present in normal bladder and increases significantly in MB49-induced tumor. Normal bladder does not increase IDO after 24 hours of incubation with BCG. In conclusion, MB49 cells express IDO in culture and incubation with BCG enhances this expression. An inhibition of IDO with MT decreases the viability of MB49 cells. The MB49 cell tumor has higher IDO expression when compared to the normal bladder. The lack of effect of BCG and MT treatment on the evolution of animals with MB49 cell tumor does not preclude the possibility of these treatments influencing CB, since in this study this orthotopic model was very aggressive, offering low survival due to an exacerbated tumor growth.