A novel activity of microsomal epoxide hydrolase: metabolism of the endocannabinoid 2-arachidonoylglycerol
2014; Elsevier BV; Volume: 55; Issue: 10 Linguagem: Inglês
10.1194/jlr.m051284
ISSN1539-7262
AutoresKasem Nithipatikom, Michael P. Endsley, Adam W. Pfeiffer, John R. Falck, William B. Campbell,
Tópico(s)Alcohol Consumption and Health Effects
ResumoMicrosomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway. Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway. Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a xenobiotic metabolizing enzyme that is functionally associated with the cytochrome P450 family. The commonly known function of EPHX1 is to metabolize xenobiotics including environmental chemicals and many therapeutic drugs. This enzymatic action converts lipophilic and sometimes reactive epoxides to more polar 1,2-diols. It also activates (pro)toxins and (pro)carcinogens (1Fretland A.J. Omiecinski C.J. Epoxide hydrolases: biochemistry and molecular biology.Chem. Biol. Interact. 2000; 129: 41-59Crossref PubMed Scopus (258) Google Scholar, 2Graham M.A. Riley R.J. Kerr D.J. Drug metabolism in carcinogenesis and cancer chemotherapy.Pharmacol. Ther. 1991; 51: 275-289Crossref PubMed Scopus (33) Google Scholar). In addition to xenobiotic metabolism, EPHX1 regulates endogenous steroid metabolism (3Fändrich F. Degiuli B. Vogel-Bindel U. Arand M. Oesch F. Induction of rat liver microsomal epoxide hydrolase by its endogenous substrate 16 alpha, 17 alpha-epoxyestra-1,3,5-trien-3-ol.Xenobiotica. 1995; 25: 239-244Crossref PubMed Scopus (15) Google Scholar), bile acid transportation (4Alves C. von Dippe P. Amoui M. Levy D. Bile acid transport into hepatocyte smooth endoplasmic reticulum vesicles is mediated by microsomal epoxide hydrolase, a membrane protein exhibiting two distinct topological orientations.J. Biol. 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The actions of 2-AG are tightly regulated by enzymatic hydrolysis, a major deactivation pathway. Monoacylglycerol lipase (MGL) (12Dinh T.P. Carpenter D. Leslie F.M. Freund T.F. Katona I. Sensi S.L. Kathuria S. Piomelli D. Brain monoglyceride lipase participating in endocannabinoid inactivation.Proc. Natl. Acad. Sci. USA. 2002; 99: 10819-10824Crossref PubMed Scopus (1131) Google Scholar, 13Dinh T.P. Freund T.F. Piomelli D. A role for monoglyceride lipase in 2-arachidonoylglycerol inactivation.Chem. Phys. Lipids. 2002; 121: 149-158Crossref PubMed Scopus (269) Google Scholar) and possibly FA amide hydrolase (FAAH) (14Goparaju S.K. Ueda N. Yamaguchi H. Yamamoto S. Anandamide amidohydrolase reacting with 2-arachidonoylglycerol, another cannabinoid receptor ligand.FEBS Lett. 1998; 422: 69-73Crossref PubMed Scopus (309) Google Scholar) are responsible for hydrolysis of 2-AG to free arachidonic acid (AA) and glycerol. Non-FAAH or non-MGL enzymes in porcine membranes (15Goparaju S.K. Ueda N. Taniguchi K. Yamamoto S. Enzymes of porcine brain hydrolyzing 2-arachidonoylglycerol, an endogenous ligand of cannabinoid receptors.Biochem. Pharmacol. 1999; 57: 417-423Crossref PubMed Scopus (199) Google Scholar) and mouse microglial cells (16Muccioli G.G. Xu C. Odah E. Cudaback E. Cisneros J.A. Lambert D.M. Lopez Rodriguez M.L. Bajjalieh S. Stella N. Identification of a novel endocannabinoid-hydrolyzing enzyme expressed by microglial cells.J. Neurosci. 2007; 27: 2883-2889Crossref PubMed Scopus (152) Google Scholar) have been demonstrated to hydrolyze 2-AG. Recent studies discovered two integral membrane enzymes, α/β-hydrolase fold 6 and 12, that contributed to ∼4% and 9% of total 2-AG hydrolysis, respectively, in mouse brain membrane (17Blankman J.L. Simon G.M. Cravatt B.F. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.Chem. Biol. 2007; 14: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar, 18Marrs W.R. 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Inhibition of microsomal epoxide hydrolases by ureas, amides, and amines.Chem. Res. Toxicol. 2001; 14: 409-415Crossref PubMed Scopus (52) Google Scholar, 24Morisseau C. Newman J.W. Wheelock C.E. Hill Iii T. Morin D. Buckpitt A.R. Hammock B.D. Development of metabolically stable inhibitors of Mammalian microsomal epoxide hydrolase.Chem. Res. Toxicol. 2008; 21: 951-957Crossref PubMed Scopus (30) Google Scholar, 25Spiegelstein O. Kroetz D.L. Levy R.H. Yagen B. Hurst S.I. Levi M. Haj-Yehia A. Bialer M. Structure activity relationship of human microsomal epoxide hydrolase inhibition by amide and acid analogues of valproic acid.Pharm. Res. 2000; 17: 216-221Crossref PubMed Scopus (13) Google Scholar). The results suggest that these compounds can bind to EPHX1 and may modify and/or interrupt the catalytic sites for the substrates. These results also suggest that ester compounds containing a long-chain unsaturated FA backbone may fit in a catalytic binding pocket and be metabolized by EPHX1. In this study, we demonstrate that the long-chain unsaturated FA glycerol ester, the endocannabinoid 2-AG, is metabolized by EPHX1 to free AA and glycerol. It is not known whether EPHX1 contributes to 2-AG hydrolysis in vivo. The results suggest potential roles of EPHX1 in regulating endocannabinoid signaling and free AA biosynthesis. Microsomes of control, vector-transfected, and EPHX1-transfected human lymphoblasts were obtained from BD Biosciences (San Jose, CA) and Sigma-Aldrich (St. Louis, MO). Human prostate carcinoma cells (PC-3 and LNCaP) and hepatocellular carcinoma cells (HepG2) were obtained from ATCC (Rockville, MD). 2-AG, [2H5]2-AG, [2H8]2-AG, [2H8]anandamide ([2H8]AEA), N-arachidonoyl dopamine (AA-dopamine), arachidonoyl serinol (AA-serinol), 4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), methoxy-arachidonyl fluorophosphate (MAFP), (Z)-octa-9-decenamide (oleamide), (3′-(aminocarbonyl) [1,1'-biphenyl]-3-yl)-cyclohexylcarbamate (URB597), cyclohexyl [1,1'-biphenyl]-3-ylcarbamate (URB602), MGL primary antibody, and FAAH primary antibody were obtained from Cayman Chemical Co. (Ann Arbor, MI). 2-Arachidonoyl-[3H]glycerol (2-AG*, 3H-labeled on the glycerol moiety) and [3H]AEA (AEA*, 3H-labeled on the ethanolamine moiety) were obtained from American Radiolabeled Chemical (St. Louis, MO). [14C]2-AG (labeled at C1 position) was synthesized by Dr. J. R. Falck. 10-Hydroxystearamide (10-HSA), 3-(dodecylthio)-1,1,1-trifluoropropan-2-one (DDTFP), 3-(decylthio)-1,1,1-trifluoropropan-2-one (DETFP), 3-(octylthio)-1,1,1-trifluoropropan-2-one (OTFP), and recombinant human soluble epoxide hydrolase (EPHX2; EC 3.3.2.10) were generously provided by Dr. Bruce Hammock. Primary antibody against human EPHX1 was obtained from BD Biosciences and Santa Cruz Biotechnologies (Santa Cruz, CA). Goat anti-rabbit and rabbit anti-mouse IgG-HRP secondary antibodies were obtained from Zymed Laboratories Inc. (South San Francisco, CA). Goat anti-mouse IgG-HRP secondary antibody and Pluronic F-127, the trade name of a nonionic surfactant polyol, were obtained from Invitrogen (Eugene, OR). pCMV6-XL4 vector and pCMV6-XL4 containing EPHX1 cDNA were obtained from Origene Technologies (Rockford, MD). EPHX1 siRNAs, a functional nontargeting control (siControl), and DharmaFECT1 transfection reagent were obtained from Dharmacon Inc. (Lafayette, CO). NADPH regenerating system was obtained from BD Biosciences. Primary antibodies against human β-actin, cSO, FA-free BSA, SDS, and CHAPS were obtained from Sigma-Aldrich. Primary monoclonal antibody against human pan-cadherin was obtained from Abcam (Cambridge, MA). ECL for Western blotting detection and BCA protein determination assay kits were obtained from Pierce (Rockville, IL). SDS-PAGE ReadyGels™ and Mini-PROTEAN TGX gels were obtained from BioRad (Hercules, CA). Distilled, deionized water was used in all experiments. HepG2 cells were maintained in essential modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, sodium pyruvate (100 mM), l-glutamine (2 mM), streptomycin (100 µg/ml), and penicillin (100 U/ml). PC-3 and LNCaP cells were maintained in RPMI 1640 medium as previously described (26Nithipatikom K. Endsley M.P. Isbell M.A. Falck J.R. Iwamoto Y. Hillard C.J. Campbell W.B. 2-Arachidonoylglycerol: a novel inhibitor of androgen-independent prostate cancer cell invasion.Cancer Res. 2004; 64: 8826-8830Crossref PubMed Scopus (119) Google Scholar). Cells were grown in 75 cm2 polystyrene tissue culture flasks at 37°C in 5% CO2 in air. [14C]2-AG (10 μM) was incubated with EPHX1 microsomes (30 μg) from BD Biosciences or Sigma-Aldrich in PBS buffer containing essentially FA-free BSA (1 mg/ml) at 37°C for 15 min at pH 7.4 or 9.0. Samples were extracted by solid phase extraction as previously described (27Nithipatikom K. Grall A.J. Holmes B.B. Harder D.R. Falck J.R. Campbell W.B. Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.Anal. Biochem. 2001; 298: 327-336Crossref PubMed Scopus (105) Google Scholar). Samples were separated by HPLC using a C18 reverse phase column (4.6 × 250 mm2, Nucleosil, Phenomenex) and water-acetonitrile containing 0.1% acetic acid as a mobile phase at a flow rate of 1.0 ml/min. The mobile phase started at 50% acetonitrile and linearly increased to 100% acetonitrile in 35 min. The eluent was collected at 5 fractions/min and counted for radioactivity. The retention times of the radioactive peaks of [14C]2-AG and [14C]AA in the samples were compared with the retention times of 2-AG and AA standards. 2-AG* was used as enzyme substrate for determination of 2-AG hydrolysis activity, and AEA* (3H-labeled on the ethanolamine moiety) was used as a substrate for determination of AEA hydrolysis activity. The percent hydrolysis of 2-AG* and AEA* was determined as previously described (26Nithipatikom K. Endsley M.P. Isbell M.A. Falck J.R. Iwamoto Y. Hillard C.J. Campbell W.B. 2-Arachidonoylglycerol: a novel inhibitor of androgen-independent prostate cancer cell invasion.Cancer Res. 2004; 64: 8826-8830Crossref PubMed Scopus (119) Google Scholar) and normalized to the hydrolysis of the control (as the relative hydrolysis activity). In this series of experiments, 2-AG was used as a substrate. In some cases, [2H5]2-AG was used as a substrate to make certain that there is no contribution from endogenous 2-AG in the samples. After the hydrolysis reaction, samples were added with [2H8]2-AG or [2H8]AEA as an internal standard, extracted by solid phase extraction, and analyzed by using LC-ESI/MS (Agilent 1100 LC-MSD, SL model) as previously described (27Nithipatikom K. Grall A.J. Holmes B.B. Harder D.R. Falck J.R. Campbell W.B. Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.Anal. Biochem. 2001; 298: 327-336Crossref PubMed Scopus (105) Google Scholar, 28Endsley M.P. Aggarwal N. Isbell M.A. Wheelock C.E. Hammock B.D. Falck J.R. Campbell W.B. Nithipatikom K. Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: location, hydrolysis and 12-lipoxygenase metabolism.Int. J. Cancer. 2007; 121: 984-991Crossref PubMed Scopus (45) Google Scholar). The detection was made in the positive ion mode. For quantitative measurement, m/z 379, 384, 387, and 356 were used for 2-AG, [2H5]2-AG, [2H8]2-AG, and [2H8]AEA, respectively. The concentrations of 2-AG (or [2H5]2-AG) were calculated by comparing their ratios of peak areas to the standard curves. The results were normalized to the protein content and compared with the control. After the incubation of cSO, a known substrate for EPHX1, with the membrane fractions of PC-3 cells overexpressing EPHX1, the amount of unhydrolyzed cSO was determined by GC-MS (H/P 5890 GC coupled to the H/P 5971 MS, Hewlett-Packard, Palo Alto, CA). The peak area of the selected m/z 167 was used to quantify cSO as compared with the standard curve. PC-3 cells were chosen for EPHX1 overexpression because of their low level of endogenous EPHX1 expression. PC-3 cells, at ∼70% confluence, were transfected with 5 μg of purified pCMV6-XL4 vector or purified pCMV6-XL4 containing EPHX1 cDNA using Lipofectamine (0.1%, v/v) in Opti-MEM reduced serum media. Concentrations of cDNA and transfection times were initially optimized. After 5 h of transfection, RPMI 1640 feed medium was replaced with 10% serum feed medium and incubated for an additional incubation of 24 h posttransfection. Cells were lysed, and samples were separated for membrane fractions by centrifugation at 100,000 g at 4°C for 60 min. Then, EPHX1 enzyme activity for 2-AG hydrolysis in the membrane fractions was determined using 2-AG or [2H5]2-AG as a substrate. In another set of experiments, the intact PC-3 cells were used after transfection for [2H5]2-AG incubation. In this case, control and transfected PC-3 cells were pretreated with JZL184 (100 nM), a known selective MGL inhibitor, to block the 2-AG hydrolysis by the highly abundant MGL for 10 min at 37°C. Then, [2H5]2-AG was added as a substrate and incubated for 30 min. Then, cells were lysed, and samples were analyzed for EPHX1 protein expression. Samples were extracted by solid phase extraction (27Nithipatikom K. Grall A.J. Holmes B.B. Harder D.R. Falck J.R. Campbell W.B. Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.Anal. Biochem. 2001; 298: 327-336Crossref PubMed Scopus (105) Google Scholar), and the remaining (unmetabolized) [2H5]2-AG was determined by LC/MS as described previously. HepG2 and LNCaP cells contain high EPHX1 expression and 2-AG hydrolysis activity. EPHX1 expression in HepG2 and LNCaP cells was knocked down using specific EPHX1 siRNA of four separate sequences. A functional nontargeting siRNA that was bioinformatically designed by Dharmacon Inc. to have ≥4 mismatches with known human genes was included as a control (siControl). Different siRNA concentrations and transfection times were optimized for the maximal suppression of EPHX1 expression. Cells were transfected at 37°C in antibiotic-free medium with DharmaFECT1 alone, siControl, or EPHX1 siRNA. At 5 h posttransfection, the transfection medium was replaced with RPMI 1640 feed medium for 24 h, and cells were harvested for Western immunoblot analysis and enzyme activity. Proteins in samples were electrophoretically separated by SDS-PAGE (Ready Gels) or Mini-PROTEAN TGX gels and transferred to a nitrocellulose membrane (BioRad). Blots were incubated with EPHX1 primary antibody (1:250) (BD Biosciences or Santa Cruz Biotechnology) or MGL primary antibody (1:200) or FAAH primary antibody (1:200), followed by HRP-conjugated secondary antibody. Protein concentrations and β-actin or pan-cadherin were used as loading controls. Detection was made by using ECL Western Blotting Substrate (Pierce) and captured by Fuji film X-ray (Tokyo, Japan). Band densities were analyzed using Image J software from the National Institutes of Health. The means of the measured values of each treatment group were compared using Student's t-test. Means were considered statistically different from one another if P < 0.05. The presence of EPHX1 and other major enzymes metabolizing 2-AG, MGL, and FAAH, in control microsomes, vector microsomes, and EPHX1 microsomes from BD Biosciences, were determined by Western immunoblotting. EPHX1 immunoreactive bands were detected at very low levels in the control and vector microsomes, while an intense immunoreactive band was detected in the EPHX1 microsomes (Fig. 1A, left panel). Immunoreactive bands for MGL and FAAH were not detected in these microsomes at 30 μg protein (Fig. 1A, middle and right panels). These results indicate that EPHX1 microsomes contained high EPHX1 protein but contained MGL and FAAH at levels below detection by Western blot analysis at 30 μg of microsomes. To test that EPHX1 microsomes metabolize 2-AG to free AA and glycerol (shown in Fig. 1B), two sets of experiments were performed. First, control and EPHX1 microsomes were incubated with [14C]2-AG in PBS buffer containing essentially FA-free BSA (1 mg/ml) pH 7.4 at 37°C for 15 min, and radioactive products separated on a reverse phase LC. Fractions were collected and counted for radioactivity. In the second set of experiments, microsomes were incubated with 2-AG under the same conditions, and 2-AG and its metabolite (AA) were analyzed by LC/MS. The left chromatogram of Fig. 1C indicates the radioactive peaks comigrated with 2-AG standard and a small peak comigrated with 1/3-AG, rearranged regioisomers of 2-AG. The chromatogram also shows the radioactive reaction product that comigrated with the AA standard. LC/MS analysis of the incubation of 2-AG with EPHX1 microsomes also indicates the production of AA (Fig. 1C, right panel). These results indicate that EPHX1 microsomes convert 2-AG to free AA and glycerol. 2-AG hydrolysis by EPHX1 microsomes was determined in various reaction conditions. EPHX1 microsomes from two suppliers were incubated with 2-AG* (3H-labeled on the glycerol moiety) as a substrate for 2-AG hydrolysis or AEA* (3H-labeled on the ethanolamine moiety) as a substrate for AEA hydrolysis. 2-AG hydrolysis by control microsomes from BD Biosciences was also determined. The reaction was performed at 37°C in PBS buffer containing essentially FA-free BSA (1 mg/ml) at pH 7.4 for various reaction times. The percent hydrolysis of these substrates was determined as previously described (29Endsley M.P. Thill R. Choudhry I. Williams C.L. Kajdacsy-Balla A. Campbell W.B. Nithipatikom K. Expression and function of fatty acid amide hydrolase in prostate cancer.Int. J. Cancer. 2008; 123: 1318-1326Crossref PubMed Scopus (76) Google Scholar). At these conditions, the hydrolysis of 2-AG increased with incubation time and reached the maximum after 30 min (Fig. 2A). The rate and extent of hydrolysis was similar with microsomes from the two suppliers (Fig. 2A). The hydrolysis of 2-AG also took place in the basic solutions, at pH 9.0 (Fig. 2A). In the presence of JZL184 (1–100 nM), a potent MGL-selective inhibitor (30Long J.Z. Li W. Booker L. Burston J.J. Kinsey S.G. Schlosburg J.E. Pavon F.J. Serrano A.M. Selley D.E. Parsons L.H. et al.Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects.Nat. Chem. Biol. 2009; 5: 37-44Crossref PubMed Scopus (749) Google Scholar), the 2-AG hydrolysis was not altered when compared with the control without JZL184 (data not shown), suggesting that the 2-AG hydrolysis in the EPHX1 microsomes was not from the activity of MGL (a major known enzyme hydrolyzing 2-AG). AEA was not hydrolyzed by the EPHX1 microsomes, further indicating that the 2-AG hydrolysis in EPHX1 microsomes was not from the activity of FAAH. 2-AG hydrolysis by EPHX1 microsomes was also determined in the presence of a NADPH regenerating system or under hypoxic conditions (<0.5% oxygen hypoxic chamber). The 2-AG hydrolysis by EPHX1 microsomes with the NADPH regenerating system was not significantly different from the control reaction of EPHX1 microsomes alone (Fig. 2B). The 2-AG hydrolysis by EPHX1 microsomes under the hypoxic conditions was not significantly different from the normoxic (control) conditions (Fig. 2B). These results suggest that the 2-AG hydrolysis by EPHX1 microsomes is not from other enzymes such as flavin-containing monooxygenases, cytochrome c reductase, cytochrome b5 reductase, or cytochrome P450 enzymes that might be present in the microsomes. Next, the effects of other substrates of EPHX1 or EPHX2 were tested on the 2-AG hydrolysis. 14,15-Epoxyexicosatrienoic acid (14,15-EET, a known substrate for EPHX2) at 1 and 10 μM was not hydrolyzed, and it did not significantly alter the 2-AG hydrolysis by EPHX1 microsomes (Fig. 2C). Styrene oxide (100 μM) reduced the 2-AG hydrolysis by ∼18%. 2-AG was not hydrolyzed by the recombinant human EPHX2 in the same reaction conditions (at 37°C for 30 min) used for EPHX1 microsomes (Fig. 2C). Interestingly, cSO, a known substrate for EPHX1, reduced the relative 2-AG hydrolysis in a concentration-dependent manner (Fig. 2D). In another set of experiments, the relative 2-AG hydrolysis in membrane fractions of the nontransfected (control) PC-3 cells or PC-3 cells overexpressing EPHX1 was carried out in the presence of BSA or detergents such as CHAPS (4 mM), SDS (7 mM), and Pluronic F-127 (1%). The hydrolysis by membrane fractions of PC-3 cells overexpressing EPHX1 in the presence of BSA was significantly higher than the nontransfected (control) cells. However, in the presence of detergents, the hydrolysis was significantly lower than the reaction in the presence of BSA and similar to the nontransfected (control) cells (Fig. 2E). These results suggest that detergents interfere with the 2-AG hydrolysis activity by EPHX1. Two approaches were used to alter the EPHX1 expression and the 2-AG hydrolysis. First, the EPHX1 siRNA was used to knockdown EPHX1 in HepG2 and LNCaP cells, cells that contain high basal EPHX1 expression. The expression of EPHX1 in HepG2 and LNCaP cells is shown in Fig. 3. For EPHX1 siRNA knockdown in HePG2 and LNCaP cells, the siRNA sense sequence -GAGGAAACUUUGCCACUUGUU and antisense sequence -CAAGUGGCAAAGUUUCCUCUU yielded the best knockdown, and the sense sequence -UGAAAGGCCUGCACUUGAACC and antisense sequence -UUCAAGUGCAGGCCUUUCAUU yielded the second-best result. Thus, the first duplex was subsequently used for all other experiments. EPHX1 siRNA markedly decreased EPHX1 protein expression in the membrane fractions of HepG2 cells (Fig. 3A, left panel) and LNCaP cells (Fig. 3B, left panel) as compared with the control and siControl. The 2-AG hydrolysis decreased in the membrane fractions of siEPHX1 HepG2 cells (Fig. 3A, right panel) and the membrane fractions of siEPHX1 LNCaP cells (Fig. 3B, right panel) as compared with the control and siControl membrane fractions. Second, PC-3 cells were transfected with the EPHX1 cDNA to increase EPHX1 expression. EPHX1 expression in the membrane fraction of PC-3 cells significantly increased after transfection with EPHX1 cDNA as compared with control and vector-transfected cells (Fig. 3C, left panel). The 2-AG hydrolysis in the membrane fractions of PC-3 cells overexpressing EPHX1 increased as compared with the membrane fractions of the control and vector-transfected cells (Fig. 3C, middle panel). Furthermore, the cSO hydrolysis by the membrane fractions was determined. The cSO hydrolysis in the overexpressed EPHX1 markedly increased above the control and vector-transfected membranes (Fig. 3C, right panel). These results indicated that transfection with EPHX1 cDNA markedly increased the EPHX1 protein and cSO hydrolysis activity. Despite a very low endogenous expression of EPHX1 protein in PC-3 cells, these cells have a relatively high 2-AG hydrolysis (28Endsley M.P. Aggarwal N. Isbell M.A. Wheelock C.E. Hammock B.D. Falck J.R. Campbell W.B. Nithipatikom K. Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: location, hydrolysis and 12-lipoxygenase metabolism.Int. J. Cancer. 2007; 121: 984-991Crossref PubMed Scopus (45) Google Scholar) in the control cells. Other enzymes metabolizing 2-AG contributed to 2-AG hydrolysis as reflected by the smaller increase of 2-AG hydrolysis than the increase of cSO hydrolysis (Fig. 3C). The 2-AG hydrolysis in intact PC-3 cells overexpressi
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