15-hydroxyprostaglandin dehydrogenase

Inhibition of 15-PGDH by CAY10397 in LoVo and HCA-7 Cell Lysates [1]
In contrast to LoVo cells, the HCA-7 cell line is known to express COX-2 and only trace amounts of COX-1 and 15-PGDH. This was confirmed by Western blot analysis (data not shown). LoVo (Figure 6a) or HCA-7 (Figure 6b) cell lysates were incubated with 500 mM NAD+, with or without the 15-PGDH inhibitor, CAY10397 (50 μM), for 10 min. Inhibition of 15-PGDH significantly abolished the formation of endogenous 11-oxo-ETE (Figure 6a) by 92%, along with the diminished formation of 15-oxo-ETE as well as 13,14-dihydro-15-oxo-PGE2 (Figure 6a) in the LoVo cell lysate. However, since the oxo-ETEs and 13,14-dihydro-15-oxo-PGE2 were undetectable in HCA-7 cell lysate, their precursors were quantified instead, and CAY10397 had no effect on their levels (Figure 6b).

Inhibition of 15-PGDH enzyme by CAY10397 significantly diminished the formation of endogenous 11-oxo-ETE in LoVo cell lysates (Figure 6a). In addition, the formation of 15-oxo-ETE as well as 13,14-dihydro-15-oxo-PGE2, the other two 15-PGDH-dependent metabolites, was also significantly reduced in LoVo cell lysates treated with CAY10397 (Figure 6a). In contrast, the three 15-PGDH products, namely, 11- and 15-oxo-ETE and 13,14-dihydro-15-oxo-PGE2, were undetectable in HCA-7 cells as these cells do not express any 15-PGDH.10,28 Instead, the corresponding precursors for these three metabolites were observed in the lysates. Furthermore, treatment with CAY10397 had no effect on levels of 11(R)-HETE, 15(S)-HETE or PGE2 (Figure 6b).

15-PGDH Inhibition in LoVo or HCA-7 Cell Lysates by CAY10397 [1]
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.

[1]. 11-Oxoeicosatetraenoic acid is a cyclooxygenase-2/15-hydroxyprostaglandin dehydrogenase-derived antiproliferative eicosanoid. Chem Res Toxicol. 2011 Dec 19;24(12):2227-36.

CAY10397 belongs to the azobenzene class of compounds. Its structure is as follows: one benzene ring has a 4-position substituted ethoxycarbonyl group, and the other benzene ring has a 3-position substituted carboxymethyl group and a 4-position substituted hydroxyl group. It is a potent and selective inhibitor of 15-hydroxyprostaglandin dehydrogenase (15-hydroxy-PGDH), belonging to EC 1.1.1.141 [15-hydroxyprostaglandin dehydrogenase (NAD(+))] inhibitors. It is a dicarboxylic acid monoester, ethyl ester, belonging to the azobenzene and phenolic classes. Previously, we confirmed that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) is an important cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. We used stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) to quantify COX-2-derived eicosate derivatives in a human colorectal adenocarcinoma (LoVo) epithelial cell line expressing COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Upon addition of AA, the secretion of 11(R)-HETE reached a peak concentration within minutes, followed by a rapid decline, indicating further metabolism. Surprisingly, recombinant 15-PGDH, which typically specifically oxidizes the 15(S)-hydroxyeicosate derivative, was found to convert 11(R)-HETE to 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (ETE). Furthermore, LoVo cell lysates converted 11(R)-HETE to 11-oxo-ETE, while inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxyphenylacetic acid (CAY10397) (50 μM) significantly suppressed the production of endogenous 11-oxo-ETE, while the level of 11(R)-HETE increased accordingly. These data confirm that COX-2 and 15-PGDH are the enzymes responsible for the biosynthesis of 11-oxo-ETE. Finally, the addition of AA to LoVo cells led to the rapid secretion of 11-oxo-ETE into the culture medium, reaching a peak within 20 minutes of incubation. Subsequently, the level of 11-oxo-ETE decreased sharply. LC-MS/MS analysis confirmed that in LoVo cells, glutathione (GSH) S-transferase (GST) metabolizes 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG) adduct. The human umbilical vein endothelial cell (HUVEC) proliferation assay based on bromodeoxyuridine (BrdU) showed that the half-maximal inhibitory concentration (IC50) of 11-oxo-ETE on HUVEC proliferation was 2.1 μM. These results indicate that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosate with similar ability to inhibit endothelial cell proliferation as 15d-PGJ2. [1]

CH3*Y

103.94042

328.105

C, 62.19; H, 4.91; N, 8.53; O, 24.36

78028-01-0

2601

Typically exists as solid at room temperature

1.3±0.1 g/cm3

578.0±50.0 °C at 760 mmHg

303.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.599

3.41

2

7

7

24

460

0

CCCOC(=O)C1=CC=C(C=C1)N=NC2=CC=C(C(=C2)CC(=O)O)O

YYBSDOZYJSGLTH-UHFFFAOYSA-N

InChI=1S/C17H16N2O5/c1-2-24-17(23)11-3-5-13(6-4-11)18-19-14-7-8-15(20)12(9-14)10-16(21)22/h3-9,20H,2,10H2,1H3,(H,21,22)

2-[5-[(4-ethoxycarbonylphenyl)diazenyl]-2-hydroxyphenyl]acetic acid

78028-01-0; 5-[2-[4-(Ethoxycarbonyl)phenyl]diazenyl]-2-hydroxybenzeneacetic acid; 5-(2-(4-(Ethoxycarbonyl)phenyl)diazenyl)-2-hydroxybenzeneacetic acid; cay10397; CK47A; 2-[3-[(4-ethoxycarbonylphenyl)hydrazinylidene]-6-oxo-1-cyclohexa-1,4-dienyl]acetic Acid; 2-[5-[(4-ethoxycarbonylphenyl)diazenyl]-2-hydroxyphenyl]acetic acid; SCHEMBL10834750;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)