Indoleamine 2,3-Dioxygenase-1 Expression is Changed During Bladder Cancer Cell Invasion.

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.

Renal Subcapsular Space of Balb/c Nude Mice as a Route for Evaluating Subpopulations of Human Bladder Carcinoma Cells.

BACKGROUND/AIM: Subpopulations of bladder cancer (BC) cells have been found in tumors, with different abilities for malignancy and chemotherapy resistance. The BC cell line T24 has frequently been used to evaluate this phenomenon. Since technical limits exist in orthotopic procedures, we evaluated the renal subcapsular space as an alternative route for analyzing subpopulations of T24 BC cells in vivo. MATERIALS AND METHODS: Balb/c nude mice underwent renal subcapsular inoculation with T24 cells, suspended in two different volumes of PBS. Four weeks post-inoculation, histology and immunohistochemistry were carried out. RESULTS: In all the animals inoculated with a 10 µl volume of suspended cells, a pseudo-bladder structure in the renal subcapsular space was observed, with differential expression of mesenchymal and epithelial markers. T24 cells infiltrating the renal parenchyma towards the medulla and vessels were also observed. The volume used for inoculation was an important factor for the success of this technique. CONCLUSION: Renal subcapsular inoculation is an effective route for analyzing subpopulations and differentiation of T24 cells.

1-Methyl-D-tryptophan potentiates TGF-ß-induced epithelial-mesenchymal transition in T24 human bladder cancer cells.

Immune escape and metastasis are the hallmarks of several types of cancer including bladder cancer. One of the mechanisms involved in these processes has been linked to indoleamine 2,3-dioxygenase (IDO). Although IDO is classically recognized for its immunomodulatory property, it has presented nonimmunological effects in some tumors. TGF-ß1 is believed to contribute to carcinoma development by modulating immunossupressive molecules, including IDO. In addition, TGF-ß1 induces the epithelial-mesenchymal transition (EMT), which is a critical step in the tumor invasiveness and metastasis. We investigated the role of MT and IDO modulation in the induction of EMT by TGF-ß1 in T24 human bladder carcinoma cells. When T24 cells were incubated with the IDO inhibitor (MT, 1-methyl-D-tryptophan), with TGF-ß1, and with MT+TGF-ß1, a significant decrease of IDO expression and activity was observed. In addition, downregulation of e-cadherin and upregulation of n-cadherin and EMT transcription factors were induced by the treatments, confirming the induction of EMT. siRNA-mediated knockdown of IDO decreased e-cadherin expression, but had no effect on EMT transcription factors. In the scratch-wound assay, the heightened migration process was intensified when the cells were incubated with MT+TGF-ß1. These effects were associated with a robust inhibition of Akt activation. After inoculation of T24 cells under the kidney capsule of Balb/c nude, the cells were positive for IDO in the center of the cell infiltrate, being negative in the periphery, where EMT is high. In conclusion, inhibition of IDO by TGF-ß1 and MT is associated with EMT in T24 human bladder carcinoma cells. MT has potentiating effect in TGF-ß1-induced EMT, independently of IDO. This nonimmunological effect of MT should be considered if IDO is the target to avoid immune escape in bladder cancer.