Marilyn A. Isbell

Inhibition of Cytochrome P450ω-Hydroxylase A Novel Endogenous Cardioprotective Pathway

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&ohgr;-hydroxylase inhibitors and a CYP&ohgr;-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemia–reperfusion 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 CYP&ohgr;-hydroxylase 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) (19.6±1.7% [control], 8.4±2.5% [0.96 mg/kg miconazole], 5.9±2.2% [0.28 mg/kg 17-ODYA], and 10.8±1.8% [0.40 mg/kg DDMS], P<0.05, respectively). Conversely, exogenous 20-HETE administration significantly increased infarct size (26.9±1.9%, P<0.05). Several CYP&ohgr;-hydroxylase isoforms, which are known to produce 20-HETE such as CYP4A1, CYP4A2, and CYP4F, were demonstrated to be present in canine heart tissue and their activity was markedly inhibited by incubation with 17-ODYA. These results indicate an important endogenous role for CYP&ohgr;-hydroxylases and in particular their product, 20-HETE, in exacerbating myocardial injury in canine myocardium. The full text of this article is available online at http://circres.ahajournals.org.

2-Arachidonoylglycerol: A Novel Inhibitor of Androgen-Independent Prostate Cancer Cell Invasion

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.

Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion

The PC-3 Low Invasive cells and the PC-3 High Invasive cells were used to investigate the correlation of the COX-2 expression and its arachidonic acid metabolites, prostaglandins, with their invasiveness through Matrigel® using a Boyden chamber assay. The COX-2 expression in PC-3 High Invasive cells was approximately 3-fold higher than in PC-3 Low Invasive cells while the COX-1 expression was similar in both cell sublines. When incubated with arachidonic acid, PGE2 was the major prostaglandin produced by these cells. PC-3 High Invasive cells produced PGE2 approximately 2.5-fold higher than PC-3 Low Invasive cells. PGD2 was the second most abundant prostaglandin produced by these cells. Both indomethacin (a nonspecific COX inhibitor) and NS-398 (a specific COX-2 inhibitor) inhibited the production of prostaglandins and the cell invasion. PGE2 alone did not induce the cell invasion of PC-3 Low Invasive cells. However, PGE2 reversed the inhibition of cell invasion by NS-398 and enhanced the cell invasion of the PC-3 High Invasive cells. In contrast, PGD2 slightly inhibited the cell invasion. These results suggest that in the PC-3 Low Invasive cells, COX-2-derived PGE2 may not be sufficient to induce cell invasion while in the PC-3 High Invasive cells, PGE2 may be sufficient to act as an enhancer for the cell invasion. Further, PGD2 may represent a weak inhibitor and counteracts the effect of PGE2 in the cell invasion.

Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: Location, hydrolysis and 12-lipoxygenase metabolism

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 cells fate. Therefore, inhibitors of 2‐AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer.

Liquid chromatographic–mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2)) produced by cultured cells. Samples were separated on a C(18) column with water-acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2), respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10(-6) M) stimulated a marked increase in the production of 6-keto-PGF(1alpha), PGE(2), and PGF(2alpha) and a small increase of PGD(2) by ECs. 6-Keto-PGF(1alpha) was the major metabolite in these cells. The production of PGE(2) was threefold higher and PGD(2) was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.

12-Lipoxygenase in Opioid-Induced Delayed Cardioprotection: Gene Array, Mass Spectrometric, and Pharmacological Analyses

Abstract— 12-Lipoxygenase (12-LO) has been shown to be a factor in acute ischemic preconditioning (IPC) in the isolated rat heart; however, no studies have been reported in delayed PC. We characterized the role of 12-LO in an intact rat model of delayed PC induced by a &dgr;-opioid agonist SNC-121 (SNC). Rats were pretreated with SNC and allowed to recover for 24 hours. They were then treated with either baicalein or phenidone, 2 selective 12-LO inhibitors. In addition, SNC-pretreated rats had plasma samples isolated at different times after ischemia-reperfusion for liquid chromatographic–mass spectrometric analysis of the major metabolic product of 12-LO, 12-HETE. Similar studies were conducted with inhibitors. Gene array data showed a significant induction of 12-LO message (P <0.05) after opioid pretreatment. This induction in 12-LO mRNA was confirmed by real-time polymerase chain reaction, and 12-LO protein expression was enhanced by SNC pretreatment at 24 hours relative to vehicle treatment. Both baicalein and phenidone attenuated the protective effects of SNC pretreatment on infarct size (50±4% and 42±3% versus 29±2%, P <0.05, respectively). No significant differences were observed in 12-HETE concentrations between baseline control and SNC-treated rats. However, 12-HETE concentrations were increased significantly at both 15 minutes during ischemia and at 1 hour of reperfusion in the SNC-treated rats compared with controls. Baicalein and phenidone attenuated the increase in 12-HETE at 1 hour of reperfusion. These data suggest that SNC-121 appears to enhance message and subsequently the activity and expression of 12-LO protein during times of stress, resulting in delayed cardioprotection.

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.

Structural characterization of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids by ESI-FTICR

The fragmentation characteristics of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids were investigated by electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation (IRMPD). The fragmentation patterns of these compounds were associated with the number and positions of the hydroxyl substituents. The fragmentation is more complicated with increasing number of the hydroxyl groups of the compounds. In general, the major carbon-carbon cleavage of [M−H]− ions occurred at the α-position to the hydroxyl group, and the carbon-carbon cleavage occurred when there was a double-bond at the β-position to the hydroxyl group. SORI-CID and IRMPD produced some common fragmentation patterns; however, each technique provided some unique patterns that are useful for structural identification of these compounds. This study demonstrated the application of FTICR via the identification of regioisomers of trihydroxyeicosatrienoic acids in rabbit aorta samples.

Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H(2)O(2)), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H(2)O(2) causes vasoconstriction. To determine the physiological contribution of H(2)O(2), catalase is used to inactivate H(2)O(2). However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10-50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1-10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (V(max) = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase(-1)·min(-1), respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H(2)O(2) and EETs.