Characterization of the Mechanisms of Action of the Intravesical Immunotherapy with P-MAPA Involving the Toll-like Receptors (TLRs) 2 and 4 Signaling Pathways in the Non-Muscle Invasive Bladder Cancer

Abstract

Immunotherapy represents one of the approaches for the treatment of cancer. Compounds which are able to act as toll-like receptors (TLRs) agonists may represent promising candidates to be developed as medicines cancer. The high rates of non muscle invasive bladder cancer (NMIBC) recurrence may be related to low effects of these therapies on the mechanisms of tissue repair, involving TLRs signaling pathway. Nowadays, the most effective therapy to NMIBC is the Bacillus Calmette-Guerin (BCG) immunotherapy associated with transurethral resection (TUR). However, BCG causes side effects of different intensities from irritative symptoms to serious systemic reaction, which contributes to treatment interruption besides increasing cancer index recurrence after treatment, up to 30%. In this scenario, we highlight the immunomodulator P-MAPA, which for its great versatility and minimal cytotoxicity opens a new perspective to treatment of some cancers types, including NMIBC. Thus, the aims of this study are to characterize the histopathological and molecular effects of the P-MAPA intravesical immunotherapy involving TLRs 2 and 4 signaling pathways in the treatment of chemically induced CBNMI in wild-type mice (WT), TLR4-knockout (KO) mice and MyD88-KO mice, as well as to establish the possible mechanisms of action of this immunomodulator relating the TLRs 2 and 4 with other cellular repair factors (NF-kB, p53, PI3K, PTEN, Akt). The P-MAPA effects will be compared with BCG treatment. A total of 40 female mice of the C57Bl/6 (WT) strain, 40 female mice of the TLR4-KO strain and 40 female mice of the MyD88-KO strain are going to be used. For induction of NMIBC, 90 (30 animals per strain) animals are going to receive a 1.5 mg/kg of N-methyl-N-nitrosourea (MNU) dose dissolved in 0.3 mL of sodium citrate (1M pH 6.0), every other week (weeks 0, 2, 4, 6), totaling 4 doses. The other 30 animals (10 animals per strain) will be considered as Control Groups (CTL). After the last dose of MNU, the animals will be examined by abdominal computerized tomography to evaluate the occurrence of tumor and subsequently divided in 4 groups (10 animals per group) for each strain: Control Group (CTL - Group 1): is going to receive intravesical dose of 0.3 mL of 0.9% saline for consecutive 6 weeks; MNU Group (Cancer - Group 2): is going to receive the same treatment in according Group 1; MNU-BCG Group (Group 3): is going to receive intravesical dose of 106 CFU - 40 mg of BCG diluted in 0.3 mL of 0.9% saline for consecutive 6 weeks, MNU-P-MAPA Group (Group 4): is going to receive intravesical dose of 5 mg/Kg of P-MAPA dissolved in 0.3 ml of 0.9% saline for consecutive 6 weeks. After 14 weeks of experiments, samples of the urinary bladder are going to be collected and submitted to histopathological, immunohistochemistry and Western blotting analysis. It is expected to obtain more effective therapy with fewer side effects and that can supplement or replace traditional therapies such as BCG. Besides the expected scientific / technological progress, the project will contribute to the realization of fundamental research and advanced, that will occur with publications in specialized journals, and based on ability to train specialized human resources. Finally, the implementation of this project will provide integration between researchers from different areas and different institutions.