In vivo and in vitro study of endothelial-to-mesenchymal transition as a contributor mechanism for the reactive stroma associated with prostate cancer in TRAMP mice: influence of antiangiogenic and anti-inflammatory therapies

Abstract

Prostate cancer is the most common neoplastic disease in men and its etiology is directly related to senescence. The phenotypic and genotypic alterations of stromal cells in response to epithelial carcinoma have been collectively designated as reactive stroma and, in the prostate, the stromal reaction is characterized by increased amount of cancer associated fibroblasts (CAFs) and myofibroblasts. The secretory activity of these cells favors tumor progression, especially through angiogenesis induction and inflammatory cell influx. The mechanism known as endothelial-to-mesenchymal transition (EndMT) has been pointed out as a possible source of these reactive stroma cells in several tumor types. Thus, the aim of this study is to characterize, both in vivo and in vitro, the potential EndMT contribution to the recruitment of activated fibroblasts in the cancer-associated reactive stroma. We also intend to investigate the influence of antiangiogenic and anti-inflammatory therapies on this endothelial plasticity mechanism and on its regulatory signaling pathways. In vivo methods: Male TRAMP (12 weeks-old) mice will be submitted to the following treatments during 6 weeks: SU5416 (6 mg/kg, each other day), Celecoxib (15 mg/kg, twice daily) and SU5416 + Celecoxib. Dorsolateral prostate samples will be harvested to morphological, immunohistochemical and Western Blotting. In vitro methods: PC-3 cells will be treated with SU5416 (1µM) and Celecoxib (10µM) for 48 hours. Cell cultures and/or conditioned media will then undergo cell viability analysis, enzyme immunoassays and Western Blotting. In another series of experiments, HUVEC cells will receive the following treatments for 48 hours: TGF-²2 (10 ng/mL), for EndMT induction; TGF-²2 + BMP7 (10 ng/mL), for EndMT inhibition; TGF-²2 + SU5416 (1µM) and TGF-²2 + Celecoxib (20µM). Afterwards, the cultures will be submitted to cell viability analysis, immunofluorescent dual-staining, Western Blotting and PCR array. (AU)