| In this study, we tested the hypothesis that 2-AG hydrolyzing enzymes are expressed and regulate the motility of prostate carcinoma cells . We characterized and determined the functions of fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), and microsomal epoxide hydrolase (EPHX1) as a novel enzyme involved in 2-AG hydrolysis, in normal prostate epithelial cells (PrECs), androgen-dependent (LNCaP), androgen-independent (DU-145 and PC-3) prostate carcinoma cells.;Our first aim determined the expression of FAAH and its regulation on prostate carcinoma cell motility. FAAH was expressed in prostate cancer tissues at a higher level compared with control tissues, suggesting that FAAH may be involved with the onset and/or progression of prostate cancer. In prostate carcinoma cells, FAAH expression and activity was differentially expressed. In comparison to PrECs, LNCaP cells had marked elevation of FAAH expression and activity whereas PC-3 cells had very low or absent FAAH expression and activity. Since LNCaP cells had high levels of FAAH expression, FAAH protein levels were decreased using small-interfering RNA (siRNA) knockdown. Reduced FAAH protein levels by siRNA significantly blocked 2-AG hydrolysis, increased 2-AG concentration, and inhibited LNCaP cell invasion. Conversely, overexpression of FAAH in PC-3 cells produced increased 2-AG hydrolysis, decreased 2-AG concentrations and increased cell invasion and migration. Together, these results suggested that FAAH regulates endogenous 2-AG concentrations thereby controlling prostate carcinoma cell motility.;In our second aim, we determined the expression and role of MGL in prostate carcinoma cell motility. DU-145 cells had the highest level of MGL mRNA expression compared to the other three cell types. MGL protein was highly expressed in the cytosolic fractions of DU-145 and PC-3, while only DU-145 cells had noticeable membrane expression. Moreover, inhibition of MGL in DU-145 cells significantly reduced 2-AG hydrolysis in both cytosol and membrane fractions with corresponding reduction in cell invasion. PC-3 cells overexpressing MGL had increased 2-AG hydrolysis in both the cytosol and membrane fractions. Furthermore, PC-3 cell invasion was increased, which could be blocked by URB602, a selective-MGL inhibitor. These results suggest that elevated MGL expression levels increased 2-AG hydrolysis and prostate carcinoma cell motility, particularly in DU-145 cells since they have elevated MGL levels in both the cytosolic and membrane fractions.;Our third aim set to identify and characterize EPHX1 as a novel 2-AG hydrolyzing enzyme that regulates prostate carcinoma cell motility. First, we characterized a novel function of EPHX1, hydrolysis of 2-AG to AA. 2-AG hydrolysis was increased in microsomes overexpressing EPHX1 compared to controls. Conversely, siRNA knockdown of EPHX1 decreased 2-AG hydrolysis compared to controls. These effects were independent of both MGL and FAAH activity. All three prostate carcinoma cell lines had higher basal EPHX1 mRNA and protein expression compared to PrECs. Using LNCaP cells, siRNA knocked down of EPHX1 expression reduced 2-AG hydrolysis and cell invasion. EPHX1 overexpression in PC-3 cell had the opposite effect, increased 2-AG hydrolysis and cell invasion, which was blocked by 10-hydroxysteramide, a specific EPHX1 inhibitor. Collectively, EPHX1 is a novel enzyme for 2-AG hydrolysis as well as a regulator of prostate carcinoma cell motility.;In summary, these 2-AG hydrolyzing enzymes were identified and characterized as mediators of prostate carcinoma cell motility. Elevated levels of 2-AG hydrolyzing enzymes increased 2-AG hydrolysis, production of other pro-invasive eicosanoids, and prostate carcinoma cell motility. These results corroborate our previous findings that regulation of 2-AG hydrolysis can determine the invasive phenotype of prostate carcinoma cells. These studies have advanced our understanding of how 2-AG and its hydrolyzing enzymes are able to regulate prostate carcinoma cells motility. All together, these studies implicate 2-AG hydrolyzing enzymes in controlling of prostate cancer metastasis and use of selective 2-AG hydrolase inhibitors may be a novel therapeutic approach for the treatment of metastatic prostate cancer. (Abstract shortened by UMI.)... |
