20-hydroxyeicosatetraenoic acid (20-HETE), a Cytochrome (CYP) P450 metabolite of arachidonic acid, contributes to the metastatic features of human castration-resistant prostate cancer cells.

LAURA C. PANELO; CECILIA COLOMBRERO; PAULA SACCA; SUSANA NOWICKI; MONICA COSTAS

Orlando, Florida

Congreso; ENDO 2017, Abril 1 - 4 en Orlando, Florida; 2017

The Endocrine Society

The hydroxylation of arachidonic acid by 20-hydroxylases of the cytochrome P450 family (CYP4F2 and CYP4A11) renders 20-HETE, which has been implicated in oncogenic characteristics in several types of tumors. Previous results from our group have evidenced the expression of both, CYP4F2 and CYP4A11, in two human prostate cancer (PCa) cell lines: LNCaP (androgen-sensitive) and PC-3 (androgen-insensitive) and have shown that the inhibition of 20-HETE synthesis reduces the viability only in androgen-sensitive PCa cells.The aim of this study was to evaluate the role of 20-HETE on the metastatic features in vitro in PCa cells.LNCaP and PC-3 cells were incubated with either 20-HETE or HET0016 (a selective inhibitor of 20-HETE synthesis). Scratch wound assay (cell migration), and colony growth in soft agar assay (anoikis resistance) were performed. Also, E-cadherin and vimentin protein expression (epithelial-mesenchymal transition, EMT) were assessed by Western Blot. Additionally, the release of matrix metalloproteinase-2 (MMP-2), involved in the degradation of extracellular matrix, was assessed by zymography assay. Intracellular localization and structure of actin, tubulin and vimentin were determined by immunofluorescence.PC-3 cells migration in control conditions was 38±2%, this was reduced to 31±2%* and 25±1%*** by HET0016 1 and 10uM, respectively; whereas incubation with 20-HETE 100nM increased cell migration to 54±6%***. The analysis of cytoskeletal proteins distribution revealed that HET0016 disorganized the actin filaments throughout PC-3 cells, while the tubulin filaments remained unchanged. Furthermore, HET0016 reduced by 45%** the ability of PC-3 cells to form colonies in soft agar. Interestingly, the analysis of proteins involved in EMT showed that HET0016 increased the expression of E-cadherin in PC-3, as well as, reduced the expression of vimentin, without modifying its intracellular distribution. Conversely, 20-HETE decreased the expression of E-cadherin in PC-3 cells and increased the expression of vimentin. Moreover, 20-HETE 10 and 100nM increased by 100% the release of MMP-2 to the conditioned medium. As for LNCaP cells, the incubation with HET0016 or 20-HETE did not affect cell migration nor the expression of EMT-related proteins.Our results suggest that 20-HETE availability is necessary in the steps involved in the cell processes that contribute to metastasis in the highly aggressive prostate cancer cell line PC-3. *p