The hydrolysis of endocannabinoids has profound effects on the function of the endocannabinoid signaling system in the regulation of prostate carcinoma cells. Prostate carcinoma cells exhibit a wide range of hydrolysis activity for 2-arachidonoylglycerol (2-AG), the major endocannabinoid. However, enzyme(s) responsible for 2-AG hydrolysis and their functions in prostate cancer have not been characterized. In this study, we demonstrated that fatty acid amide hydrolase (FAAH) was differentially expressed in normal and prostate carcinoma cells. In PC-3 cells, overexpression of FAAH resulted in increased FAAH protein, 2-AG hydrolysis, cell invasion and cell migration. Conversely, small-interfering RNA (siRNA) knockdown of FAAH in LNCaP cells decreased FAAH protein, 2-AG hydrolysis and cell invasion. Furthermore, CAY10401, a FAAH inhibitor, decreased cell invasion and it enhanced the reduction of invasion in FAAH siRNA-transfected LNCaP cells. Immunohistochemistry staining of commercial tissue microarrays (TMAs) demonstrated FAAH staining in 109 of 157 cores of prostate adenocarcinomas but weak staining in 1 of 8 cores of normal prostate tissues. These results suggest that FAAH regulates 2-AG hydrolysis and invasion of prostate carcinoma cells and is potentially involved in prostate tumorigenesis.

Endocannabinoids have been implicated in cancer. Increasing endogenous 2-arachidonoylglycerol (2-AG) by blocking its metabolism inhibits invasion of androgen-independent prostate cancer (PC-3 and DU-145) cells. Noladin ether (a stable 2-AG analog) and exogenous CB1 receptor agonists possess similar effects. Conversely, reducing endogenous 2-AG by inhibiting its synthesis or blocking its binding to CB1 receptors with antagonists increases the cell invasion. 2-AG and noladin ether decrease protein kinase A activity in these cells, indicating coupling of the CB1 receptor to downstream effectors. The results suggest that cellular 2-AG, acting through the CB1 receptor, is an endogenous inhibitor of invasive prostate cancer cells.

Abstract-Cytochrome P450s (CYP) and their arachidonic acid (AA) metabolites have important roles in regulating vascular tone, but their function and specific pathways involved in modulating myocardial ischemia-reperfusion injury have not been clearly established. Thus, we characterized the effects of several selective CYP-hydroxylase inhibitors and a CYP-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemiareperfusion injury in canine hearts. During 60 minutes of ischemia and particularly after 3 hours of reperfusion, 20-HETE was produced at high concentrations. A nonspecific CYP inhibitor, miconazole, and 2 specific CYPhydroxylase inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly inhibited 20-HETE production during ischemia-reperfusion and produced a profound reduction in myocardial infarct size (expressed as a percent of the area at risk Key Words: arachidonic acid metabolites Ⅲ cytochrome p450 Ⅲ 20-HETE Ⅲ ischemia Ⅲ reperfusion I t has long been recognized that ischemia-reperfusion of the canine heart results in an accumulation of unesterified arachidonic acid (AA). 1,2 AA can be metabolized by cytochrome P450 (CYP) expoxygenases to 4 regioisomeric epoxyeicosatrienoic acids (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) and by CYP-hydroxylases to 20-hydroxyeicosatetraenoic acid (20-HETE). 3-6 Subsequently, EETs can be further metabolized to their corresponding dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolases. 7,8 Although EETs relax many blood vessels, including coronary, cerebral, renal, and pial arteries, and hyperpolarize vascular smooth muscle cells possibly by activation of calcium activated potassium (Kca) channels, 9 DHETs produce relaxation only in some vessels. 10,11 However, 20-HETE is a potent vasoconstrictor 12 that activates L-type Ca 2ϩ channels leading to vasoconstriction of renal afferent arterioles. 13,14 As noted, roles of these eicosanoids in the regulation of peripheral vascular tone have been extensively investigated. However, their functions during ischemia and after reperfusion in the heart and coronary circulation, as well as mechanisms responsible for their vascular and myocardial properties are not well-understood. It is known that CYP metabolites are involved in AA-induced relaxation of canine coronary arteries. 15 Both EETs and DHETs have been shown to be potent vasodilators in the canine coronary microcirculation. 16 The production of EETs and DHETs was increased in stenosed canine coronary arteries. 17 Recently, nonspecific CYP inhibitors such as chloramphenicol, cimetidine, and sulfaphenazole have been reported to reduce ischemia-reperfusion injury, as measured by recovery of contractile function and reduction of infarct size, in rat and rabbit hearts. 18 However, because the drugs used to inhibit CYP were not selective inhibitors, it was difficult to determine the precise pathway or metabolite responsible.Previously, we found that high plasma concentrations of CYP ...

Endogenous 2-arachidonoylglycerol (2-AG) inhibits invasion of androgen-independent prostate cancer cells. Blocking cellular hydrolysis of 2-AG to increase its endogenous concentration results in a decrease in cell invasion. A series of compounds containing a trifluoromethyl ketone (TFK) moiety or the methyl analog (known to inhibit carboxylesterases) were investigated for their ability to inhibit 2-AG hydrolysis and prostate cancer cell invasion. Compounds containing a thioether beta to a TFK moiety inhibited 2-AG hydrolysis as well as cell invasion in a concentration-dependent manner. Inhibition of 2-AG hydrolysis increased concomitantly with inhibitor alkyl chain length from 4- to 12-carbons while inhibition of cell invasion exhibited a maximum at 8- to 10-carbons of the compounds. These results demonstrate a new series of 2-AG hydrolysis inhibitors as a potential therapeutic approach for prostate cancer.

Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190 -202 and 260 -275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and ␤1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-␤1 integrin mega-complex that regulates mammary epithelial cell adhesion.Maspin is a non-inhibitory serine protease inhibitor (serpin) 4 that was originally identified as a type II tumor suppressor protein in mammary epithelial cells (1). One major tumor suppressor function of maspin is suppression of tumor cell motility, as it inhibits tumor cell migration/invasion in vitro and suppresses metastasis in mouse models (1-7). Several studies show that pericellular maspin inhibits cell motility by enhancing cell adhesion (2,3,8,9). In addition to its tumor suppressing functions, our laboratory showed that maspin is also essential for normal fetal development as maspin knock-out mice are embryonic lethal during the peri-implantation stage partially due to disrupted visceral endodermal cell adhesion (10). The underlying molecular mechanism by which maspin regulates cell adhesion is currently unknown and under intense investigation. To date, there are two proposed pathways utilized by maspin to increase cell-extracellular matrix (ECM) adhesion; that is, the plasminogen activation system and ␤1 integrin signaling (9, 11-13).The plasminogen activation system is believed to be a central player in several different processes important for tumor progression and metastasis (14 -16). In this system urokinase-type plasminogen activator (uPA), a serine protease, binds to its glycosylphosphatidylinositol-anchored receptor (uPAR) and readily activates plasminogen to initiate a protease cascade resulting in localized ECM degradation for the purpose of cell migration (17, 18). It has been suggested that maspin integrates into the plasminogen activation system. Maspin inhibits prostate carcinoma cell migration and invasion by strengthening mature focal adhesion contacts, reducing uPA activity by internalizing the maspin-uPA-uPAR complex and by binding to pro-uPA, thus blocking its activation (12). Although maspin is classified as a serpin and decreases pericellular uPA activity, maspin does not directly inhibit uPA proteolytic activity (19 -21). Together, these studies demonstrated that maspin can reduce prostate carcinoma cell migration and invasion by internalization of cell surface maspin-uPA-uPAR complexes.The second pro...

Endogenous 2‐arachidonoylglycerol (2‐AG) is antiinvasive in androgen‐independent prostate carcinoma (PC‐3) cells. Invasion of PC‐3 cells is also inhibited by exogenously added noladin ether, a non‐hydrolyzable analog of 2‐AG. In contrast, exogenous 2‐AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous 2‐AG. In PC‐3 cells, arachidonic acid (AA) and 12‐hydroxyeicosatetraenoic acid (12‐HETE) concentrations increased along with exogenously added 2‐AG, and 12‐HETE concentrations increased with exogenously added AA. Invasion of PC‐3 cells also increased with exogenously added AA and 12(S)‐HETE but not 12(R)‐HETE. The exogenous 2‐AG‐induced invasion of PC‐3 cells was inhibited by 3‐octylthio‐1,1,1‐trifluoropropan‐2‐one (OTFP, an inhibitor of 2‐AG hydrolysis) and baicalein (a 12‐LO inhibitor). Western blot and RT‐PCR analyses indicated expression of 12‐HETE producing lipoxygenases (LOs), platelet‐type 12‐LO (P‐12‐LO) and leukocyte‐type 12‐LO (L‐12‐LO), in PC‐3 cells. These results suggest that exogenous 2‐AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 12‐LO of AA to 12(S)‐HETE, a promoter of PC cell invasion. The results also suggest that PC‐3 cells and human prostate stromal (WPMY‐1) cells released free AA, 2‐AG, and 12‐HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The 2‐AG hydrolysis and concentration of 2‐AG in microenvironment are critical for PC cell's fate. Therefore, inhibitors of 2‐AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer. © 2007 Wiley‐Liss, Inc.

Metastatic outcomes depend on the interactions of metastatic cells with a specific organ microenvironment. Our previous studies have shown that triple-negative breast cancer (TNBC) MDA-MB-231 cells passaged in astrocyte-conditioned medium (ACM) show proclivity to form brain metastases, but the underlying mechanism is unknown. The combination of microarray analysis, qPCR, and ELISA assay were carried out to demonstrate the ACM-induced expression of angiopoietin-like 4 (ANGPTL4) in TNBC cells. A stable ANGPTL4-knockdown MDA-MB-231 cell line was generated by ANGPTL4 short-hairpin RNA (shRNA) and inoculated into mice via left ventricular injection to evaluate the role of ANGPTL4 in brain metastasis formation. The approaches of siRNA, neutralizing antibodies, inhibitors, and immunoprecipitation were used to demonstrate the involved signaling molecules. We first found that ACM-conditioned TNBC cells upregulated the expression of ANGPTL4, a secreted glycoprotein whose effect on tumor progression is known to be tumor microenvironment- and tumor-type dependent. Knockdown of ANGPTL4 in TNBC MDA-MB-231 cells with shRNA decreased ACM-induced tumor cell metastatic growth in the brain and attributed to survival in a mouse model. Furthermore, we identified that astrocytes produced transforming growth factor-beta 2 (TGF-β2), which in part is responsible for upregulation of ANGPTL4 expression in TNBC through induction of SMAD signaling. Moreover, we identified that tumor cells communicate with astrocytes, where tumor cell-derived interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) increased the expression of TGF-β2 in astrocytes. Collectively, these findings indicate that the invading TNBC cells interact with astrocytes in the brain microenvironment that facilitates brain metastases of TNBC cells through a TGF-β2/ANGPTL4 axis. This provides groundwork to target ANGPTL4 as a treatment for breast cancer brain metastases.

Cytochrome P-450 (CYP) omega-hydroxylases and their arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), produce a detrimental effect on ischemia-reperfusion injury in canine hearts, and the inhibition of CYP omega-hydroxylases markedly reduces myocardial infarct size expressed as a percentage of the area at risk (IS/AAR, %). In this study, we demonstrated that a specific CYP omega-hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly reduced 20-HETE production during ischemia-reperfusion and reduced myocardial infarct size compared with control [19.5 +/- 1.0% (control), 9.6 +/- 1.5% (0.40 mg/kg DDMS), 4.0 +/- 2.0% (0.81 mg/kg DDMS), P < 0.01]. In addition, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE, a putative 20-HETE antagonist) significantly reduced myocardial infarct size from control [10.3 +/- 1.3% (0.032 mg/kg 20-HEDE) and 5.9 +/- 1.9% (0.064 mg/kg 20-HEDE), P < 0.05]. We further demonstrated that one 5-min period of ischemic preconditioning (IPC) reduced infarct size to a similar extent as that observed with the high doses of DDMS and 20-HEDE, and the higher dose of DDMS given simultaneously with IPC augmented the infarct size reduction [9.9 +/- 2.8% (IPC) to 2.5 +/- 1.4% (0.81 mg/kg DDMS), P < 0.05] to a greater degree than that observed with either treatment alone. These results suggest an important negative role for endogenous CYP omega-hydroxylases and their product, 20-HETE, to exacerbate myocardial injury in canine myocardium. Furthermore, for the first time, this study demonstrates that the effect of IPC and the inhibition of CYP omega-hydroxylase synthesis (DDMS) or its actions (20-HEDE) may have additive effects in protecting the canine heart from ischemia-reperfusion injury.