Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Ian A. Blair

Abstract

Inflammatory diseases and multiple human cancers are associated with increased cyclooxygenase-2 (COX-2) expression together with decreased expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH). This has been hypothesized to lead to a milieu of bioactive lipids modulating angiogenesis and cellular proliferation. Further insight into these processes were gained by traditional cell and molecular biology approaches, which were combined with cutting edge liquid chromatography mass spectrometry (LC-MS) methods to elucidate the function, disposition, and metabolism of a major COX-2/15-PGDH derived metabolite, 11-oxo-eicosatetraenoic acid (11-oxo-ETE). 11-oxo-ETE was found to possess significant anti-proliferative activity (IC50 2 microM) in human umbilical endothelial cells (HUVECS), which was 5 times more potent than isomeric 15-oxo-ETE. The corresponding methyl ester derivatives were even more potently anti-proliferative than the free acids. The gold standard method of quantification, stable isotope dilution (SID) LC-MS, was applied to determine the absolute intra- and extra- cellular levels of oxo-ETEs in multiple cell lines. Maximal intracellular concentrations of 11-oxo-ETE were 0.02 ng/4x10^5 cells in human colon adenocarcinoma (LoVo) cells versus 0.58 ng/4x10^5 cells in HUVECs. Its methyl ester derivative increased the intracellular concentration of free 11-oxo-ETE 3-fold. Pharmacological inhibition of relevant transporter proteins increased anti-proliferative activity and the intracellular pool of 11-oxo-ETE. Consistent with an observed structural homology to other bioactive fatty acids, 11-oxo-ETE reduced canonical nuclear factor-kappa B signaling activity. LC-MS revealed a mechanistic explanation of this activity via adduction to critical cysteine or histidine residues of the p50 subunit of the p50/p65 transcription factor. LC- high-resolution MS/MS approaches revealed that the oxo-ETEs underwent substantial intracellular metabolism. Two of the double bonds were reduced and novel coenzyme A (CoA) thioesters were formed followed by reduction of all four of their double bonds. The identification of novel CoA thioester metabolites could have important implications to detection and function of the oxo-ETEs as well as other electrophilic polyunsaturated fatty acids in normal physiology as well as in inflammation and cancer.

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