Suhong Zhang

11-Oxoeicosatetraenoic acid is a cyclooxygenase-2/15-hydroxyprostaglandin dehydrogenase-derived antiproliferative eicosanoid.

Previously, we established that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) was a significant cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. Stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) was used to quantify COX-2-derived eicosanoids in the human colorectal adenocarcinoma (LoVo) epithelial cell line, which expresses both COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 11(R)-HETE secretion reached peak concentrations within minutes after AA addition before rapidly diminishing, suggesting further metabolism had occurred. Surprisingly, recombinant 15-PGDH, which is normally specific for oxidation of eicosanoid 15(S)-hydroxyl groups, 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 and inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) (50 μM) significantly suppressed endogenous 11-oxo-ETE production with a corresponding increase in 11(R)-HETE. These data confirmed COX-2 and 15-PGDH as enzymes responsible for 11-oxo-ETE biosynthesis. Finally, addition of AA to the LoVo cells resulted in rapid secretion of 11-oxo-ETE into the media, reaching peak levels within 20 min of starting the incubation. This was followed by a sharp decrease in 11-oxo-ETE levels. Glutathione (GSH) S-transferase (GST) was found to metabolize 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG)-adduct in LoVo cells, as confirmed by LC–MS/MS analysis. Bromodeoxyuridine (BrdU)-based cell proliferation assays in human umbilical vein endothelial cells (HUVECs) revealed that the half-maximal inhibitory concentration (IC50) of 11-oxo-ETE for inhibition of HUVEC proliferation was 2.1 μM. These results show that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosanoid, which inhibits endothelial cell proliferation with a potency that is similar to that observed for 15d-PGJ2.

Serum apolipoprotein A-1 quantification by LC–MS with a SILAC internal standard reveals reduced levels in smokers

Background: Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassays and LC–MS can provide misleading results. Results: Recombinant ApoA-1 internal standard was prepared using stable isotope labeling by amino acids in cell culture with [13C615N2]-lysine and [13C915N1]-tyrosine in human cells. A stable isotope dilution LC–MS method for serum ApoA-1 was validated and levels analyzed for 50 nonsmokers and 50 smokers. Conclusion: The concentration of ApoA-1 in nonsmokers was 169.4 mg/dl with an 18.4% reduction to 138.2 mg/dl in smokers. The validated assay will have clinical utility for assessing effects of smoking cessation and therapeutic or dietary interventions in high-risk populations.

Cellular uptake and antiproliferative effects of 11-oxo-eicosatetraenoic acid

Cyclooxygenases (COX) metabolize arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETE), which can then be oxidized by dehydrogenases, such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH), to oxo-eicosatetraenoic acids (ETE). We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) and 15-oxo-ETE are COX-2/15-PGDH-derived metabolites. Stable isotope dilution (SID) chiral liquid chromatography coupled with electron capture atmospheric pressure chemical ionization (ECAPCI) single reaction monitoring (SRM) MS has been used to quantify uptake of 11-oxo-ETE and 15-oxo-ETE in both LoVo cells and human umbilical vein endothelial cells (HUVEC). Intracellular 11-oxo- and 15-oxo-ETE concentrations reached maximum levels within 1 h and declined rapidly, with significant quantitative differences in uptake between the LoVo cells and the HUVECs. Maximal intracellular concentrations of 11-oxo-ETE were 0.02 ng/4 × 105 cells in the LoVo cells and 0.58 ng/4 × 105 cells in the HUVECs. Conversely, maximal levels of 15-oxo-ETE were 0.21 ng/4 × 105 in the LoVo cells and 0.01 ng/4 × 105 in the HUVECs. The methyl esters of both 11-oxo- and 15-oxo-ETE increased the intracellular concentrations of the corresponding free oxo-ETEs by 3- to 8-fold. 11-oxo-ETE, 15-oxo-ETE, and their methyl esters inhibited proliferation in both HUVECs and LoVo cells at concentrations of 2–10 μM, with 11-oxo-ETE methyl ester being the most potent inhibitor. Cotreatment with probenecid, an inhibitor of multiple drug resistance transporters (MRP)1 and 4, increased the antiproliferative effect of 11-oxo-ETE methyl ester in LoVo cells and increased the intracellular concentration of 11-oxo-ETE from 0.05 ng/4 × 105 cells to 0.18 ng/4 × 105 cells. Therefore, this study has established that the COX-2/15-PGDH-derived eicosanoids 11-oxo- and 15-oxo-ETE enter target cells, that they inhibit cellular proliferation, and that their inhibitory effects are modulated by MRP exporters.

Metabolism of a Representative Oxygenated Polycyclic Aromatic Hydrocarbon (PAH) Phenanthrene-9,10-quinone in Human Hepatoma (HepG2) Cells

Exposure to polycyclic aromatic hydrocarbons (PAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. Phenanthrene is a representative PAH present in crude oil, and it undergoes biological transformation, photooxidation, and chemical oxidation to produce its signature oxygenated derivative, phenanthrene-9,10-quinone. We report the downstream metabolic fate of phenanthrene-9,10-quinone in HepG2 cells. The structures of the metabolites were identified by HPLC–UV–fluorescence detection and LC–MS/MS. O-mono-Glucuronosyl-phenanthrene-9,10-catechol was identified, as reported previously. A novel bis-conjugate, O-mono-methyl-O-mono-sulfonated-phenanthrene-9,10-catechol, was discovered for the first time, and evidence for both of its precursor mono conjugates was obtained. The identities of these four metabolites were unequivocally validated by comparison to authentic enzymatically synthesized standards. Evidence was also obtained for a minor metabolic pathway of phenanthrene-9,10-quinone involving bis-hydroxylation followed by O-mono-sulfonation. The identification of 9,10-catechol conjugates supports metabolic detoxification of phenanthrene-9,10-quinone through interception of redox cycling by UGT, COMT, and SULT isozymes and indicates the possible use of phenanthrene-9,10-catechol conjugates as biomarkers of human exposure to oxygenated PAH.

Pretreatment Red Blood Cell Total Folate Concentration Is Associated With Response to Pemetrexed in Stage IV Nonsquamous Non–Small-cell Lung Cancer

Introduction Pemetrexed inhibits folate‐dependent enzymes involved in pyrimidine and purine synthesis. Previous studies of genetic variation in these enzymes as predictors of pemetrexed efficacy have yielded inconsistent results. We investigated whether red blood cell (RBC) total folate, a phenotypic rather than genotypic, marker of cellular folate status was associated with the response to pemetrexed‐based chemotherapy in advanced nonsquamous non–small‐cell lung cancer (NSCLC). Materials and Methods We conducted a prospective cohort study of patients with stage IV nonsquamous NSCLC receiving first‐line chemotherapy containing pemetrexed. The pretreatment RBC total folate level was quantified using liquid chromatography mass spectrometry. We then compared the objective response rate (ORR) between patients with RBC total folate concentrations greater than and less than an optimal cutoff value determined from the receiver operating characteristic curve. A logistic regression model was used to adjust for age, sex, and the use of bevacizumab. Results The ORR was 62% (32 of 52 patients). Receiver operating characteristic analysis was used to establish that a RBC total folate cutoff value of 364.6 nM optimally discriminated between pemetrexed responders and nonresponders. Patients with RBC total folate < 364.5 nM had an ORR of 27% compared with 71% for patients with RBC total folate > 364.5 nM (P = .01). This difference persisted after adjusting for age, sex, and the use of bevacizumab (odds ratio, 0.07; 95% confidence interval, 0.01‐0.57; P = .01). Conclusion A low pretreatment RBC total folate was associated with an inferior response to pemetrexed‐based chemotherapy in stage IV nonsquamous NSCLC. Larger, multicenter studies are needed to validate RBC total folate as a predictive marker of pemetrexed response. Micro‐Abstract We aimed to determine the role of red blood cell (RBC) total folate in predicting the pemetrexed response in advanced nonsquamous non–small‐cell lung cancer. Patients with low pretreatment RBC total folate concentrations had an inferior response rate to pemetrexed‐based chemotherapy. Our study results suggest the potential use of RBC total folate, a widely available and low‐cost assay, for predicting the pemetrexed response.

Potential Metabolic Activation of a Representative C2-Alkylated Polycyclic Aromatic Hydrocarbon 6-Ethylchrysene Associated with the Deepwater Horizon Oil Spill in Human Hepatoma (HepG2) Cells

Exposure to polycyclic aromatic hydrocarbons (PAHs) is the major human health hazard associated with the Deepwater Horizon oil spill. C2-Chrysenes are representative PAHs present in crude oil and could contaminate the food chain. We describe the metabolism of a C2-chrysene regioisomer, 6-ethylchrysene (6-EC), in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. 6-EC-tetraol isomers were identified as signature metabolites of the diol-epoxide pathway. O-Monomethyl-O-monosulfonated-6-EC-catechol, its monohydroxy products, and N-acetyl-l-cysteine(NAC)-6-EC-ortho-quinone were discovered as signature metabolites of the ortho-quinone pathway. Potential dual metabolic activation of 6-EC involving the formation of bis-electrophiles, i.e., a mono-diol-epoxide and a mono-ortho-quinone within the same structure, bis-diol-epoxides, and bis-ortho-quinones was observed as well. The identification of 6-EC-tetraol, O-monomethyl-O-monosulfonated-6-EC-catechol, its monohydroxy products, and NAC-6-EC-ortho-quinone supports potential metabolic activation of 6-EC by P450 and AKR enzymes followed by metabolic detoxification of the ortho-quinone through interception of its redox cycling capability by catechol-O-methyltransferase and sulfotransferase enzymes. The tetraols and catechol conjugates could be used as biomarkers of human exposure to 6-EC resulting from oil spills.

Abstract LB-9: Cyclooxygenase-2/15-prostaglandin dehydrogenase derived endogenous canonical NF-κB inhibitors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL In colon, breast, and lung cancers, up-regulation of cyclooxygenase-2 (COX-2) and down-regulation of 15-prostaglandin dehydrogenase (15-PGDH) has been hypothesized to lead to a pro-proliferative, angiogenic, and chronically inflamed state. Prostaglandin E2 has been thought to drive the feed forward aspect of this pathology; however, other endogenous lipids may be involved in this process. COXs also mediate the metabolism of arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETEs), which can then be further oxidized by dehydrogenases to the corresponding keto-products. We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) is a major COX-2/15-PGDH derived metabolites formed from human cells expressing COX-2 treated with AA. 11-oxo-ETE has been identified from human isolates yet currently has no known receptors or known physiologic function. Earlier studies had indicated that molecules possessing a similar α,β-unsaturated ketone moiety may act as canonical Nuclear Factor-κB (NF-κB) inhibitors. Therefore, the present study was aimed to investigate the signaling of this novel COX-2/15-PGDH-derived eicosanoid. The stability and uptake of 11-oxo-ETE into human umbilical vein endothelial cells (HUVECs) was quantified with stable isotope dilution chiral liquid chromatography coupled with electron capture atmospheric pressure chemical-ionization/mass spectrometry. Tumor necrosis factor (TNF)-α induced NF-κB p50/p65 subunit nuclear translocation and consensus DNA binding was reduced by pre-incubation with 11-oxo-ETE. Dosage and reduction in NF-κB were consistent with the levels for the prostaglandin analog 15-deoxy-prostaglandin J2 (15d-PGJ2). Using a HEK293 line stably expressing a firefly luciferase reporter driven by a 5x-NF-κB consensus sequence repeat, 11-oxo-ETE and its methyl ester were shown to significantly inhibit NF-kB signaling induced by TNF-α treatment. Ethacrynic acid (EA) was used in an additional experiment to inhibit cellular glutathione-S-transferases, and this increased the ability of 11-oxo-ETE to inhibit NF-κB signaling in the reporter assay. In vitro biochemical studies also revealed inhibition of the activating kinase Inhibitor of nuclear factor-κB kinase subunit β (≥KKβ) by 11-oxo-ETE. In summary, the present study has established that the COX-2/15-PGDH derived 11-oxo-ETE can enter target endothelial cells and inhibit NF-κB p50/p65 mediated signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-9. doi:1538-7445.AM2012-LB-9

Abstract LB-10: Distribution and potency of anti-proliferative arachidonic acid metabolites

Eicosanoids participate in the dysregulation of cell proliferation during tumorigenesis. The up-regulation of cyclooxygenase-2 (COX-2) and down-regulation of 15-prostaglandin dehydrogenase (15-PGDH) have been linked to the switch for a pro-proliferative and pro-angiogenic tumor microenvironment. Previous studies have characterized the effects of increased prostaglandin E 2 in tumor progression but the role of many other endogenous lipid mediators has yet to be determined. COX-2 can metabolize arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETEs), which in turn can be further oxidized by dehydrogenases into oxo-eicosatetraenoic acids (oxo-ETEs). We have previously reported that 11-oxo-ETE and 15-oxo-ETE are major COX-2/15-PGDH derived metabolites formed from human cells expressing COX-2 treated with AA. Although no known G-protein coupled receptor has been reported for either of these metabolites, both 11-oxo- and 15-oxo-ETE have been described from human clinical samples. This study provides a description of the uptake and distribution of 11-oxo-ETE and 15-oxo-ETE as well as their anti-proliferative activity in both human umbilical vein endothelial cells (HUVECs) and LoVo human colon adenocarcinoma cells. Quantification of the intracellular and extracellular levels of 11-oxo- and 15-oxo-ETE was conducted using liquid chromatography electron capture atmospheric pressure chemical ionization mass spectrometry with [ 13 C]-labeled 15-oxo-ETE as an internal standard. The intracellular concentrations of 11-oxo- and 15-oxo-ETE reached peak amounts within 1h followed by a rapid decline. Earlier studies have indicated that glutathione (GSH)-S-transferase further metabolized 11-oxo-ETE to form the 11-oxo-ETE-GSH (OEG)-adduct. In additional experiments, pretreatment with ethacrynic acid (EA), a known inhibitor of GSTs, increased recovery from the cellular extract. In addition, the methyl-ester of both 11-oxo- and 15-oxo-ETE was used for the targeted intracellular delivery of these COX-2/15-PGDH derived eicosanoids. Finally, we have shown that both 11-oxo- and 15-oxo-ETE as well as their methyl ester derivatives significantly inhibit cell proliferation as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Bromodeoxyuridine (BrdU) incorporation in multiple human cell lines. The present study has established the cellular uptake of the short-lived COX-2/15-PGDH derived eicosanoids 11-oxo- and 15-oxo-ETE and potent inhibition of new DNA synthesis and proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-10. doi:1538-7445.AM2012-LB-10

Abstract 856: Association of high levels of folate with increased risk for colitis-associated colorectal neoplasia

Low folate levels are known to be a risk factor for multiple human diseases including colorectal cancer. However, the relationship between folate levels and the prevention or development of colorectal cancer remains controversial. Patients with ulcerative colitis often possess low levels of folate due to inadequate intake and malabsorption of nutrients. Thus, folic acid supplementation is highly recommended despite very limited information regarding the effect of folic acid on the progression of colitis-associated colorectal cancer. The major objectives of the present study are to evaluate folate levels during the progression of colitis-associated colon cancer and to assess the potential functional role of folic acid supplementation on the growth of colon cancer cells. To study the effect of inflammation on levels of folate, acute and chronic colitis were induced within the mouse colon by administering 2 cycles of 3% dextran sulfate sodium (DSS) following one injection of azoxymethane (AOM; 7.4 mg/kg); a regimen that ultimately leads to the formation of colitis-associated dysplasias and carcinomas. At 10+, 12 and 19 wks of age, corresponding to acute colitis, chronic colitis, and colitis-associated dysplasia, respectively, blood samples were collected retro-orbitally and plasma folate levels were measured using stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry. At sacrifice, the histopathology of the colon was correlated with plasma folate levels. Levels of plasma folate were reduced 33% (78.4 + 7.2 nM vs. 116.2 + 15.6 nM, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 856. doi:10.1158/1538-7445.AM2011-856