Studies of Cellular Pathways and Enzymatic Modulations of Prostanoids Using Mass Spectrometry

Cyclooxygenase (COX) enzymes catalyze conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2) that serves as the common substrate for five prostanoids, PGE2, PGD2, PGF2α, PGI2, and thromboxane A2 (TXA2). These prostanoids consist of a series of extracellular and intracellular messengers producing diverse physiologic functions on inflammation, asthma, cardiovascular diseases, cancer, etc. Non-steroidal anti-inflammatory drugs (NSAIDs) competitively interfere with binding of the AA substrate to COXs. Treatment with non-selective NSAIDs and selective COX-2 inhibitors (COXIBs) has been documented to show serious gastrointestinal toxicity and cardiovascular disease, respectively. This recognition has redirected efforts to examine the effect of interdiction in microsomal PGE synthase-1 (mPGES-1). However, it would be premature to consider mPGES-1 as a promising and safe new therapeutic target without further investigation and understanding of the effect of inhibition on the entire prostanoid cascade. COX-2 and its catalytic product PGE2 can contribute to carcinogenesis including tumor promotion, inhibition of apoptosis, angiogenesis and the tumor metastasis, and COXIBs have consistently shown potential in cancer chemoprevention. It appears that targeting the COX-2 pathway is a promising strategy in the chemoprevention and treatment of solid tumors without the serious side effects usually associated with cancer chemotherapy. Thus, further discovery and development of novel COXIBs are still needed. In this dissertation, (i) high performance liquid chromatography tandem mass spectrometry (HPLC-MS-MS) and ultra high pressure liquid chromatography (UHPLC)-MS-MS methods were developed and validated for the measurement of different prostanoids; (ii) how PGH2 metabolism is re-distributed by distal synthases or by blocking the major metabolic outlet were investigated, to help determine the relative benefits and risks resulting from interdiction in the prostanoid synthesis pathway; (iii) COX functional inhibition and pulsed ultrafiltration LC-MS assays were used for screening and to determine the inhibitory potency of lead compounds from botanicals and marine microbes; and finally, (iv) how the lipopolysaccharide-induced generation of PGD2 is functionally connected with NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) production was investigated in bone marrow-derived macrophages (BMDM).