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.

High CD39 expression is associated with the non-muscle-invasive phenotype of human bladder cancer.

BACKGROUND: An accurate prediction of progression is critical to define the management of bladder cancer (BC). The ectonucleotidases CD39 and CD73 play strategic roles in calibrating purinergic signals via an extracellular balance between ATP and adenosine. The altered expression of these enzymes plays a potential role in tumor invasion and metastasis, therefore, has been proposed to be used for prognosis of solid tumor. In BC this is not yet clear. OBJECTIVE: This study aimed to evaluate CD39 and CD73 expression in a cohort of patients with non-muscle-invasive (NMI) and muscle-invasive (MI) BC regard to its association with clinicopathological features. MATERIALS AND METHODS: Retrospective clinical follow-up data and primary urothelial BC specimens of 162 patients were used (87 from patients who underwent transurethral resection and 75 from cystectomized patients). Tissue microarrays were constructed, and immunohistochemistry for CD39 and CD73 was performed to make associations with clinicopathological data. RESULTS: Overall, 96 were NMI (59.3%) and 66 MI (40.7%). CD39 immunoreactivity in BC cells was found in 72% of the cases, while CD73 was found in 97%. High CD39 expression alone was more frequent in NMI BC (p < 0.001), while CD73 expression was not powerful to predict the stage of BC. The association of both markers confirmed that only CD39 has potential in BC prognosis. CONCLUSIONS: The altered expression of CD39 presented herein supports the idea that this ectonucleotidase may be involved in bladder tumorigenesis. High expression of CD39 in tumor cells is correlated with the early stage of BC.

1-Methyl-D-tryptophan activates aryl hydrocarbon receptor, a pathway associated with bladder cancer progression.

BACKGROUND: Indoleamine 2, 3-dioxygenase-1 (IDO1) is a promising target for immunotherapy in bladder cancer (BC). IDO1 breaks-down tryptophan to generate kynurenine derivatives, which may activate the aryl hydrocarbon receptor (AHR). AHR is an important target for carcinogens, but its association with BC progression was unknown. Two IDO1 inhibitors used in clinical trials are 1-methyl-D-tryptophan (MT) and INCB240360. Because MT is an aromatic hydrocarbon, it may be a ligand for AHR. We hypothesized that AHR could be associated with BC progression and that MT could activate AHR in BC. METHODS: BC patients (n = 165) were selected from the Gene Expression Omnibus database. A cut-off point for relative expression of AHR and cytochrome 450 enzymes (CYP1A1, CYP1A2, and CYP1B1; markers of AHR activation) was determined to compare with the grade, stage, and tumor progression. For in vitro experiments, RT4 (grade 1) and T24 (grade 3) BC cells were incubated with MT and INCB240360 to evaluate the expression of AHR and CYP1A1. RESULTS: AHR activation was associated with grade, stage, and progression of BC. T24 cells express more CYP1A1 than RT4 cells. Although IDO1 expression and kynurenine production are elevated in T24 cells concomitantly to CYP1A1 expression, IDO1 inhibitors were not able to decrease CYP1A1 expression, in contrast, MT significantly increased it in both cell lines. CONCLUSION: In conclusion, it is rational to inhibit IDO1 in BC, among other factors because it contributes to AHR activation. However, MT needs to be carefully evaluated for BC because it is an AHR pathway agonist independently of its effects on IDO1.

Proteinuria is Associated with Urinary Loss of Cubilin and Vitamin D-Binding Protein in Patients with Preeclampsia.

Women with preeclampsia (PE) form a vulnerable group for vitamin D3 deficiency. Reabsorption of vitamin D3 occurs in the proximal tubule after being endocytosed in combination with DBP (vitamin D binding protein) by the megalin/cubilin receptor. Because proteinuria promotes tubule injury and dysfunction, we hypothesized that the proteinuria present in PE could promote the loss of these components into the urine. Twenty preeclamptic patients and ten normal pregnant women with a gestational age greater than 20 weeks composed three groups: NC, normotensive control pregnant patients; PE, non-proteinuric preeclamptic patients; and PPE, preeclamptic patients with proteinuria. When proteinuria was absent, preeclampsia was diagnosed accordingly to the American College of Obstetricians and Gynecologists' (ACOG) guideline. The presence of 24-hour proteinuria equal to or greater than 300 mg was considered to form the PPE group. Urinary cubilin, megalin, and DBP were measured by ELISA and normalized by urinary creatinine. Regarding gestational age, there was no difference between the groups. NC group had arterial pressure within normal values, whereas PE and PPE groups had a significant increase (p < 0.01). As expected, PPE group presented elevated ACR (p < 0.05), accompanied by large amounts of cubilin and DBP in the urine (p < 0.05 vs. NC and PE). No difference was found in urinary megalin. PPE patients showed more chance of shedding cubilin into the urine compared to non-proteinuric patients (odds ratio 12.7 (1.2-136.3). In conclusion, this study further tightens the relationship between PE and vitamin D3 deficiency, since proteinuria present in PE induces the loss of molecules responsible for renal tubular vitamin D3 reabsorption for subsequent activation. Combined with other factors, the proteinuria may intensify vitamin D3 deficiency in PE.

Indoleamine 2, 3-dioxygenase (IDO) increases during renal fibrogenesis and its inhibition potentiates TGF-ß 1-induced epithelial to mesenchymal transition.

BACKGROUND: Indoleamine 2, 3-dioxygenase (IDO) is an immunomodulatory molecule that has been implicated in several biological processes. Although IDO has been linked with some renal diseases, its role in renal fibrosis is still unclear. Because IDO may be modulated by TGF-ß1, a potent fibrogenic molecule, we hypothesized that IDO could be involved in renal fibrosis, especially acting in the TGF-ß1-induced tubular epithelial-mesenchymal transition (EMT). We analyzed the IDO expression and activity in a model of renal fibrogenesis, and the effect of the IDO inhibitor 1-methyl-tryptophan (MT) on TGF-ß1-induced EMT using tubular cell culture. METHODS: Male Wistar rats where submited to 7 days of UUO. Non-obstructed kidneys (CL) and kidneys from SHAM rats were used as controls. Masson's Tricrome and macrophages counting were used to chatacterize the tissue fibrosis. The EMT was analysed though immunohistochemistry and qRT-PCR. Immunohistochemestry in tissue has used to show IDO expression. MDCK cells were incubated with TGF- ß1 to analyse IDO expression. Additionally, effects of TGF- ß1 and the inhibition of IDO over the EMT process was acessed by immunoessays and scrath wound essay. RESULTS: IDO was markedly expressed in cortical and medular tubules of the UUO kidneys. Similarly to the immunolocalizaton of TGF- ß1, accompanied by loss of e-cadherin expression and an increase of mesenchymal markers. Results in vitro with MDCK cells, showed that IDO was increased after stimulus with TGF-ß1, and treatment with MT potentiated its expression. MDCK stimulated with TGF-ß1 had higher migratory activity (scratch-wound assay), which was exacerbated by MT treatment. CONCLUSIONS: IDO is constitutively expressed in tubular cells and increases during renal fibrogenesis. Although IDO is induced by TGF-ß1 in tubular cells, its chemical inhibitor acts as a profibrotic agent.

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.