Microenvironment of prostatic adenocarcinoma: in vivo and in vitro effect of tempol on inflammatory, oxidative stress, proliferation markers and miRNAs

Author(s)

Isabela Maria Urra Rossetto

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2023-09-22

Examining board members

Valéria Helena Alves Cagnon; Rejane Maira Góes; Patricia Simone Leite Vilamaior; Gabriela Bortolança Chiarotto; Cynthia Aparecida de Castro

• Advisor: Valéria Helena Alves Cagnon; Murilo Vieira Geraldo

Abstract

Prostate cancer (PCa) is the second leading cause of cancer deaths worldwide. Among the aspects of the pathogenesis and progression of prostate cancer, inflammation and oxidative stress can be pointed out as fundamental factors, leading to the repeated process of tissue damage and regeneration and the imbalance of the glandular microenvironment. Tempol, an antioxidant, has been shown to be a promising adjuvant treatment in several types of cancers, with important effects on the inflammatory process and on oxidative stress. However, studies evaluating its actions in the prostate are scarce and contradictory. The aim of the present study was to evaluate the effects of different tempol treatment doses, in vitro and in vivo, on the microenvironment of prostate adenocarcinoma at different stages of progression of this disease, analyzing tissue proliferation, inflammation, oxidative stress, mitochondrial response, and expression of miRNAs. In vitro, the response of the PC-3 and LNCaP cell lines was verified in different times and tempol treatment doses. In vivo, ventral lobe prostate responses in the TRAMP model were verified at two stages of disease progression: 8-12 weeks of age (early stage; CT12 and TPL12) and 16-20 weeks of age (advanced stage; CT20, TPL20-I and TPL20-II). Transgenic mice were treated by gavage with 50 or 100 mg/Kg tempol doses diluted in water, for four weeks, with 5 treatment days per week. After the end of the experimental period, the ventral lobe was processed for morphological, immunohistochemical, protein level (western blotting), enzyme activity and gene expression (RT-PCR) analyses. The results showed that tempol showed antiproliferative action, decreasing the cell viability of tumor cell lines and delaying the appearance of lesions in the TRAMP model. Tempol presented a preponderant role in reducing inflammation both in vitro and in vivo. For all experimental conditions, there was attenuation of inflammatory signaling via toll-like receptors, differential increase of NF?B inhibitors and decrease of inflammatory markers. Tempol also altered parameters related to cell survival, such as STAT-3 and BCL2, showing tissue protective action mainly in the early stage. This nitroxide acted as an antioxidant, increasing endogenous SOD2 levels and acting as a catalase-like agent. Complementary analyses of mitochondrial parameters demonstrated that at the advanced stage, tempol led to decreased components of mitochondrial oxidative phosphorylation (OXPHOS) and decreased PGC-1?, preventing the tissue from matching the increased energy demands of the cell at this stage. By and large, tempol increased the expression of the miRNAs let-7c-5p, 26a-5p and 155-5p under the conditions studied, and differentially modulated NF?B pathway genes, both in vivo and in vitro. Particularly in the advanced stage, the tempol treatment dose-response was not evidenced for protein levels, since the double dose offered to the animals did not lead to effects superior to those observed with the lower treatment dose. However, if the molecular aspects of gene expression are considered, tempol led to a significant increase in the levels of miRNAs let-7c-5p, miR-26a-5p and miR-155-5p for the TPL20-II group when compared to the CT20 and TPL20-I groups. Systemically in the TRAMP model, tempol decreased the levels of miR-let-7c-5p and miR-26a-5p in both treatment stages, and these same miRNAs increased their expression at twice the treatment dose in the advanced stage. Thus, it was found that the actions of tempol were effective, but diverse, in the microenvironment of prostate adenocarcinoma, indicating differential responses in vitro, according to the hormonal dependence of the cell lines and, in vivo, based on the stage of progression of the lesions in the tissue. Through the present study, of an exploratory nature of the possible actions of tempol in CaP, it was concluded that its anti-inflammatory role was one of the most relevant and uniform components of its action, highlighting the changes in elements of the NF?B pathway after treatment. The decrease in inflammation reported here is intrinsically related to the findings of decreased cell survival and proliferation. Finally, this preclinical study suggests that tempol can be considered as a potential therapy for CaP. However, due to its multifaceted action, it highlights the need to consider its use after an individualized analysis of the stage of progression of the prostate lesion, as well as the degree of androgen dependence (AU)

• FAPESP's process: 18/21647-6 - Microenvironment of Prostatic Adenocarcinoma: in vivo and in vitro effect of tempol on inflammatory markers, oxidative stress, proliferation and miRNAs expression
• Grantee: Isabela Maria Urra Rossetto
• Support Opportunities: Scholarships in Brazil - Doctorate