Chemicals and Reagents
AA (peroxide-free), 11(R,S)-HETE, 15(R,S)-HETE, [2H8]-15(S)-HETE, [13C20]-15-oxo-ETE, 15-oxo-ETE, PGE2, [2H4]-PGE2, 13,14-dihydro-15-keto-PGE2, [2H4]-13,14-dihydro-15-keto-PGE2, 15d-PGJ2, CAY10397, protease inhibitor cocktail and recombinant human 15-PGDH were purchased from Cayman (Ann Arbor, MI). The Dess–Martin reagent [1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one], 2,3,4,5,6-pentafluorobenzyl (PFB) bromide, Trizma-HCl, lipoxidase from Glycine max (soybean), sodium borohydride, and NAD+ were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was from Gemini Bioproducts (West Sacramento, CA). F12K medium, DMEM medium, Medium 200 (M200), d-glucose, l-glutamine, low-serum growth supplement (LSGS) kit, penicillin, and streptomycin were supplied by Invitrogen (Carlsbad, CA). LC–MS grade water, hexane, methanol, isopropanol, acetonitrile and dichloromethane were obtained from Fisher Scientific (Pittsburgh, PA). Gases were supplied by The Linde Group (Munich, Germany). [13C20]-AA was obtained from Spectra Stable Isotopes (Columbia, MD).
Human colorectal adenocarcinoma LoVo cells (ATCC, Manassas, VA) were cultured in F12K medium supplemented with 10% FBS, 2 mM l-glutamine, 100,000 units/L penicillin and 100 mg/L streptomycin. Human colonic adenocarcinoma HCA-7 Colony 29 cells (Sigma-Aldrich, St. Louis, MO) were grown in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 110 mg/L sodium pyruvate, 100,000 units/L penicillin and 100 mg/L streptomycin. For lipidomics analysis, the culture medium was replaced with serum-free F12K or DMEM medium before the treatment. HUVECs were obtained from Invitrogen (Carlsbad, CA) and cultured on collagen I-coated tissue culture dishes in Medium 200 supplemented with LSGS kit. Cell proliferation assays were performed using HUVECs from passage 4.
A triple stage quadrupole (TSQ Quantum) mass spectrometer (Thermo Electron, San Jose, CA) equipped with an APCI source was used for the quantitative lipidomics analyses. Targeted chiral LC-ECAPCI/SRM/MS analysis was conducted using PFB derivatives of 7 lipids and 4 heavy isotope analogue internal standards. For the lipidomics profile, the instrument was operated in the negative ion mode, and unit resolution was maintained for both precursor and fragment ions. Operating conditions for the TSQ Quantum were as follows: vaporizer temperature at 450 °C; heated capillary temperature at 250 °C with the corona discharge needle set at 30 μA; nitrogen as sheath (25 psi) and auxiliary (5 arbitrary units) gas. Collision-induced dissociation (CID) was performed using argon as the collision gas at 2.7 mTorr in the rf-only quadrupole. The following SRM transitions were used: 11-oxo-ETE-PFB, m/z 317 → 165 (collision energy (CE), 25 eV); 15-oxo-ETE-PFB, m/z 317 → 113 (CE, 18 eV); [13C20]-15-oxo-ETE-PFB, m/z 337 → 120 (CE, 18 eV); 11(R)-HETE-PFB, m/z 319 → 167 (CE, 16 eV); [2H8]-15(S)-HETE-PFB, m/z 327 → 226 (CE, 13 eV); PGE2-PFB, m/z 351 → 271 (CE, 18 eV); [2H4]-PGE2-PFB, m/z 355 → 275 (CE, 18 eV); 13,14-dihydro-15-keto-PGE2-PFB, m/z 351 → 235 (CE, 22 eV); [2H4]-13,14-dihydro-15-keto-PGE2-PFB, m/z 355 → 239 (CE, 22 eV).
For GSH adduct analysis, the mass spectrometer was operated in the positive ion mode with an electrospray ionization (ESI) source. The operating conditions were as follows: spray voltage at 4 kV; capillary temperature at 350 °C; nitrogen as sheath (35 psi) and auxiliary (13 arbitrary units) gas. CID was performed using argon as the collision gas at 2.7 mTorr in the rf-only quadrupole. The following SRM transition (m/z 626 → 497) was monitored for 11-oxo-ETE-GSH (CE, 18 eV).
LC separations were conducted using a Waters Alliance 2690 HPLC system. A Chiralpak AD-H column (250 × 4.6 mm inner diameter, 5 μm; Daicel) was employed for normal phase separation (flow rate 1 mL/min) of PFB derivatives of eicosanoids. Gradient 1 was used for separating PFB-derivatives of HETEs and PGE2, whereas gradient 2 was used for PFB derivatives of oxo-ETEs. For gradient 1, solvent A was hexane, and solvent B was methanol/isopropanol (1:1; v/v). Gradient 1 was as follows: 2% B at 0 min, 2% B at 3 min, 3.6% B at 11 min, 8% B at 15 min, 8% B at 27 min, 50% B at 30 min, 50% at 35 min, and 2% B at 37 min. Separations were performed at 30 °C using a linear gradient. For gradient 2, solvent A was hexane, and solvent B was isopropanol/hexane (6:4; v/v). Gradient 2 was as follows: 2% B at 0 min, 2% B at 14.5 min, 12% B at 15 min, 23% B at 19 min, 90% B at 19.5 min, 90% B at 23.5 min, and 2% B at 24 min.
A Chiralpak AD-RH column (150 × 4.6 mm inner diameter, 5 μm; Daicel) was used for reversed phase (isocratic method 1, flow rate 0.5 mL/min) separation of the underivatized 11-oxo-ETE. The mobile phase for isocratic separations was methanol/water/formic acid (95:5:0.1; v/v).
Chemically synthesized 11-oxo-ETE was purified by normal-phase (isocratic method 2) preparative LC (Ultrasphere 250 × 10 mm, inner diameter, 5 μm; Beckman) using Waters Alliance 2690 HPLC system by monitoring the UV absorbance at 236 nm. The mobile phase for the isocratic method 2 (flow rate 2.5 mL/min) was hexane/isopropanol/acetic acid (98.5:1.5:0.1 ; v/v).
GSH adducts were separated by reversed phase using gradient 3 on Waters Alliance 2690 HPLC system. The separation employed a Phenomenex Synergi Hydro-RP column (150 × 4.6 mm inner diameters, 5 μm). Solvent A was 0.1% aqueous formic acid, and solvent B was methanol/acetonitrile (50:50; v/v). Gradient 3 was as follows: 2% B at 0 min, 2% B at 14 min, 30% B at 20 min, 42% B at 21 min, 65% B at 27 min, 80% B at 29 min, 90% B at 33 min, 90% B at 34 min, 2% B at 35 min. The flow rate was 0.4 mL/min. The separation was performed at ambient temperature using a linear gradient.
Eicosanoids were dissolved in 100 μL of acetonitrile and then reacted with 100 μL of PFB bromide in acetonitrile (1:19; v/v) and 100 μL of diisopropylethylamine in acetonitrile (1:9; v/v) at room temperature for 30 min. The derivatives were evaporated to dryness, dissolved in 100 μL of hexane/ethanol (95:5; v/v) and analyzed by chiral LC-ECAPCI/SRM/MS.
Enzymatic Conversion of 11(R)-HETE by 15-PGDH
Various concentrations of 11(R)-HETE (0, 2.3 μM, 4.6 μM, 6.9 μM, 9.2 μM and 23 μM) were incubated with 9 nM recombinant human 15-PGDH (52.2 ng, 1.8 pmol) and cofactor NAD+ (400 μM) in 50 mM Tris-Cl (pH 7.9) for 3.5 min at 37 °C. Each total reaction volume was 200 μL. After a 3.5 min incubation, the enzymatic reaction was quenched with 400 μL of ice cold methanol and [13C20]-15-oxo-ETE (8 ng) added as the internal standard. Eicosanoids were extracted with 1.2 mL of dichloromethane/methanol (8:1; v/v). The lower organic layer was then evaporated to dryness under nitrogen and reconstituted in methanol (100 μL). An aliquot (25 μL) was separated using the isocratic method 1 and analyzed by LC–ESI/MS as described above. The retention time for 11-oxo-ETE was 8.7 min. In separate experiments, the formation of 11-oxo-ETE was found to be linear for the first 5 min. Eicosanoids were quantified by interpolation from a standard curve prepared with 11-oxo-ETE using [13C20]-15-oxo-ETE as the internal standard.
Chemical Synthesis and Purification of 11-Oxo-ETE
The Dess–Martin reagent (2 mg, 4.7 μmol) was added to a solution of 11(R,S)-HETE (0.5 mg, 1.6 μmol) in dichloromethane (1.0 mL) and stirred for 2 h at room temperature. The reaction was monitored by LC–MS using gradient 1 as described above, after PFB derivatization until there was no starting material left. There was only one major product, which corresponded to 11-oxo-ETE. The reaction mixture was centrifuged twice at 3,400 rpm (10 min), and the supernatant was evaporated. The residue was dissolved in the mobile phase (800 μL) and purified by isocratic method 2 as described above. The retention time for 11-oxo-ETE was 13.1 min. High resolution accurate mass measurements were obtained using electrospray ionization on a Thermo LTQ-FT mass spectrometer at a resolution of 100,000 (data not shown). NMR spectra were obtained on a Bruker 500 MHz NMR instrument.
15-PGDH Inhibition in LoVo or HCA-7 Cell Lysates by CAY10397
LoVo or HCA-7 cells were grown to 90% confluence, washed with 10 mL of phosphate-buffered saline (PBS) buffer (2 times), and then gently scraped in 600 μL of lysis buffer containing 0.1 M Tris-HCl (pH 7.9) and the protease inhibitor. Cell suspension was transferred to 2 mL Eppendorf tubes and sonicated for 60 s on ice (power 5). Cell lysate was then incubated with or without the selective 15-PGDH inhibitor (CAY10397, 50 μM) and its cofactor (NAD+, 500 μM) for 10 min at 37 °C. The pH was then adjusted to 4 with 10% aqueous acetic acid (10 μL) followed by addition of the internal standard mix, [13C20]-15-oxo-ETE, [2H8]-15(S)-HETE and [2H4]-PGE2 (50 pg/μL, 20 μL). Diethyl ether (600 μL) was added, and samples were vortex-mixed and centrifuged (15000 rpm × 2 min). The organic layer was evaporated under nitrogen, and then the eicosanoids were derivatized with PFB bromide as mentioned above. Finally, samples were redissolved in hexane/ethanol (95:5; v/v, 100 μL) and analyzed (20 μL) by normal phase LC-ECAPCI/MS. The amounts of eicosanoids were normalized by protein concentrations of each lysate, which were determined by BCA assay.
Metabolism of AA by LoVo or HCA-7 Cells
LoVo or HCA-7 cells were grown to 90% confluence in 6-well plates as described above, and then fed fresh serum-free F-12K or DMEM medium. Cells were then incubated with AA (10 μM) for 0, 5, 10, and 30 min, 1 and 2.5 h at 37 °C. At each time point, 0.6 mL of medium was taken out, and 20 μL of 10% aqueous acetic acid was added to adjust pH to 3–4, together with 20 μL of internal standards mixture (50 pg/μL [13C20]-15-oxo-ETE, [2H8]-15(S)-HETE, [2H4]-PGE2 and [2H4]-13,14-dihydro-15-keto-PGE2-PFB). Then diethyl ether (1 mL) was added, and the mixture was vortex-mixed and centrifuged (15000 rpm × 2 min). The upper ether layer was evaporated under nitrogen, and PFB derivatives were synthesized as described above and analyzed by normal phase LC-ECAPCI/MS.
Standard Curves for Eicosanoid Quantification
To quantify eicosanoids excreted in the medium, Eppendorf tubes containing 0.6 mL of F12K medium were spiked with lipid standards, together with internal standards for [13C20]-15-oxo-ETE, [2H8]-15(S)-HETE, [2H4]-PGE2 and [2H4]-13,14-dihydro-15-keto-PGE2-PFB (1 ng each). To quantify eicosanoids in the cell lysate, lipid standards and internal standards mixture were spiked into 0.2 mL of Tris-HCl buffer. The extraction and PFB derivatization methods are the same as mentioned above.
Analysis of 11-OEG Adducts in LoVo cell Lysate
LoVo cells were grown to 90% confluence and then washed with PBS (10 mL). Cells were gently scraped in 600 μL of Tris-HCl buffer (0.1 M, pH = 7.9), containing protease inhibitor. Cell lysates were transferred to 2 mL Eppendorf tubes and sonicated for 60 s on ice (power 5). 11-Oxo-ETE (20 ng in ethanol) was added to the lysate together with 1 mM GSH. After incubation for 25 min at 37 °C, 10 μL of 10% acetic acid was added, and the sample was loaded onto an SPE column (Oasis HLB, 30 mg) preconditioned with methanol and then 0.1% formic acid. The column was washed with 1 mL of water and eluted with 250 μL of methanol, and then 20 μL was analyzed by reversed phase LC–ESI/MS using gradient 3, as described above.
Cell Proliferation Assay
BrdU incorporation in HUVECs was used to assess the effects of 11-oxo-ETE on cell proliferation. The BrdU assay was performed in a 96-well format using a commercially available colorimetric enzyme-linked immunosorbent assay (ELISA) kit (Roche), and also by immunofluorescence microscopy. Equal numbers of HUVECs in passage 4 were plated on either collagen-I coated 96-well plates (2000 cells/well) or collagen-I coated 8-chamber tissue culture glass slides (10000 cells/chamber). Cells were allowed to attach overnight in 0.25% DMSO containing medium. Eicosanoids were dissolved in DMSO, such that the final concentration of DMSO in cell medium was always 0.25% or lower. Cells were then treated for 24 h with either vehicle (0.25% DMSO), 11-oxo-ETE (1 nM to 100 μM), or 15d-PGJ2 (1 nM to 100 μM). After 18 h of treatment, BrdU (final concentration, 10 μM in 0.25% DMSO) was added to each treatment group for an additional 6 h.
For the colorimetric ELISA, the manufacturer’s protocol was followed to perform the assay. The absorbance at λ = 370 nm obtained from the assay was transformed to the cell numbers using a standard curve constructed by plating known number of HUVECs in triplicate. The IC50 values for eicosanoid inhibition of HUVEC proliferation were defined as the half maximal inhibitory concentration for endothelial cell proliferation over 24 h when compared with vehicle-treated cells. They were determined from the regression lines of the log inhibitor vs response curves using a least-squares fit.
For the immunofluorescence staining, cells were fixed with neutral buffered formalin for 10 min, permeabilized with methanol for 20 min, and then DNA was denatured by pressure cooking the slides in 10 mM citric acid buffer, pH 6 for 1 h. Cells were then incubated overnight with rat anti-BrdU antibody (1:1000, Accurate Chemical & Scientific Corp.) at 4 °C, followed by 30 min incubation at 37 °C with Cy3-conjugated donkey anti-rat secondary antibody (1:600, Jackson Immuno Research). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) and visualized using a Nikon E600 microscope equipped with differential interference contrast (Nomarski) optics and photographed (×200 magnification) with a Fast 1394 QICam (QImaging). Positive BrdU staining was quantified by image analysis using IVision Analysis Software (Biovision). The percentage of proliferating cells was determined by counting the BrdU-positive cells versus the total number of cells in randomly selected microscopic fields (10/replicate) for each treatment group.
All experiments were conducted in triplicate, unless otherwise indicated. Statistical significance (p value ≤0.05) was determined using a two-tailed unpaired t test employing GraphPad Prism software (v 5.01).