Implications of the molecular basis of prostacyclin biosynthesis and signaling in pharmaceutical designs
ABSTRACT Prostacyclin (PGI(2)) is one of the major vascular protectors against thrombosis and vasoconstriction, caused by thromboxane A(2). Understanding the molecular mechanisms of PGI(2) biosynthesis and signaling is crucial to the development of therapeutic approaches to regulate PGI(2) functions. This review provides information regarding the most current advances in the findings of the molecular mechanisms for PGI(2) biosynthesis in the endoplasmic reticulum (ER) membrane through the coordination between PGI(2) synthase and its upstream enzymes, cyclooxygenase-1 (COX-1) or -2 (COX-2), and for PGI(2) signaling through its cell membrane receptors and nuclear peroxisome proliferator-activated receptors. The substrate presentation from the COXs to PGI(2) synthase and its cell membrane receptor/G protein coupling sites, as characterized by our group, are discussed in detail. The association between the regulation of the biosynthesis and signaling of PGI(2) with the pathophysiological processes of PGI(2)-related diseases is also discussed. The molecular knowledge of PGI(2) biosynthesis and signaling will help to design the next generation of drugs, specifically targeting the regulation of PGI(2) functions, which will undoubtedly provide advances in cardiovascular protection and the treatment of PGI(2)-related diseases.
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ABSTRACT: Prostaglandins exert a profound influence over the adhesive, migratory, and invasive behavior of cells during the development and progression of cancer. Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E(2) synthase-1 (mPGES-1) are upregulated in inflammation and cancer. This results in the production of prostaglandin E(2) (PGE(2)), which binds to and activates G-protein-coupled prostaglandin E(1-4) receptors (EP(1-4)). Selectively targeting the COX-2/mPGES-1/PGE(2)/EP(1-4) axis of the prostaglandin pathway can reduce the adhesion, migration, invasion, and angiogenesis. Once stimulated by prostaglandins, cadherin adhesive connections between epithelial or endothelial cells are lost. This enables cells to invade through the underlying basement membrane and extracellular matrix (ECM). Interactions with the ECM are mediated by cell surface integrins by "outside-in signaling" through Src and focal adhesion kinase (FAK) and/or "inside-out signaling" through talins and kindlins. Combining the use of COX-2/mPGES-1/PGE(2)/EP(1-4) axis-targeted molecules with those targeting cell surface adhesion receptors or their downstream signaling molecules may enhance cancer therapy.International Journal of Cell Biology 01/2012; 2012:723419. DOI:10.1155/2012/723419
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ABSTRACT: Our aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher "priority" to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs. COX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (½t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET. The Km for converting arachidonic acid (AA) to PGI2 by COX-2 coupled to PGIS is ~2.0μM; however, it was 3-fold more (~6.0μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~2.0μM and 20s, respectively, which were 2-5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS-CFP is shorter than that between COX-1-RFP and PGIS-CFP. The study provided strong evidence suggesting that the low Km, faster ½t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2.Life sciences 10/2010; 88(1-2):24-30. DOI:10.1016/j.lfs.2010.10.017 · 2.30 Impact Factor
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ABSTRACT: Abstract Objective: The purpose of this experiment was to investigate the possible toxic effects of Nepafenac, a nonsteroidal anti-inflammatory molecule, after its intravitreal application in various concentrations. Methods: Forty pigmented rabbits were randomly divided into 4 groups, each including 10 rabbits. The active ingredient Nepafenac was prepared to be applied in different doses, for intravitreal use. Under topical anesthesia, following pupil dilatation, 0.3, 0.5, 0.75, and 1.5 mg doses of Nepafenac was applied intravitreally into the right eye. In each rabbit, the right eye was considered to be the study group. Saline was injected intravitreally into the left eye of each rabbit, and these eyes were considered to be the control group. Immediately after the injection and at the 1st, 4th, and 8th weeks, fundus examination by indirect ophthalmoscopy and intraocular pressure measurement were conducted. Furthermore, electroretinographic (ERG) recordings were taken at the 4th and 8th weeks. At the end of the 8th week, eyes of the surviving 26 rabbits were enucleated, and then animals were sacrificed. Following necessary fixation procedures, histopathological investigations were conducted by using a light and electron microscope. In the histological cross sections, differences between the eyes with injection and the control group were evaluated, and total retinal thickness, inner nuclear layer thickness, and outer nuclear layer thickness were measured. Results: No pathology was found by clinical examination of either group. In the photopic and scotopic full-field ERG, conducted before the injection and in the 4th and 8th weeks after the injection, no statistically significant difference was determined between the study group and the control group. In the histological evaluation of the preparations, there were no statistically significant differences in the retina thickness of control and study groups. In the electron microscopic examinations, there were no toxicity findings in the eyes with injection. Conclusions: Our data show that intravitreal application of 0.3, 0.5, 0.75, and 1.5 mg doses of Nepafenac active substance is nontoxic to the rabbit retina.Journal of Ocular Pharmacology and Therapeutics 10/2014; 31(1). DOI:10.1089/jop.2014.0053 · 1.42 Impact Factor