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Eicosanoid Biosynthesis Flashcards | Quizlet

Targeted proteomics of the eicosanoid biosynthetic pathway completes an integrated genomics–proteomics–metabolomics picture of cellular metabolism.

The activity of ALAS is the rate-limiting step in the haem biosynthetic pathway.

The steps within the haem biosynthetic pathway which have been used to measure effect are: 1) inhibition of delta-aminolaevulinic acid dehydratase (ALAD); 2) urinary excretion of delta-aminolaevulinic acid (ALAU); (3) the accumulation of zinc protoporphyrin (ZPP) in erythrocytes arising from the inhibition of the enzyme ferrochelatase or the iron transport system.

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White JM & Harvey DR (1972) Defective synthesis of alpha and ß globin chains in lead poisoning.

Technicians ensured conformance with theprostatectomy specimen map delineating individual tumor foci. Sevencore biopsy specimens containing tumor and three without tumor wereused to test for the expression of COXs and LOXs; seven core biopsyspecimens without tumor and 19 with tumor were used for evaluatingeicosanoid profiles. In this proof-of-principle analysis, thelimited amount of tissue prevented the testing of matchingspecimens across all two-sample types and evaluating intrapatientas well as interpatient variabilities.

For analysis of eicosanoids in the xenografttissues, each frozen tissue specimen (25–50 mg) was ground to afine powder in a liquid nitrogen-cooled mortar (Fisher). Sampleswere then transferred to microcentrifuge tubes. Ice-cold PBSbuffer, triple each sample’s volume and containing 0.1% BHT and 1mM EDTA, was added, and the tubes were sealed. The mixtures werethen homogenized using an ultrasonic processor (Misonix) at 0°C for3 min. A 100-μl aliquot of each homogenate was transferred to aglass tube (13×100 mm), and the eicosanoids were extracted usingthe method of Yang ().

COX enzymes involved in eicosanoid production are saturated by EPA ..

Lead-induced anaemia can be a direct consequence of inhibition of haem biosynthesis; it is not necessarily associated with iron deficiency.

We next measured the AA metabolites in the humanprostate core biopsy samples using a modification of the method ofYang (). In brief,the frozen specimens were mixed with 150 μl of homogenizationbuffer [500 mM Tris-HCl (pH 7.2), 0.5 M sucrose, 200 M EDTA, 100 mMEGTA, 0.4 M NaF, 10% Triton X-100, and 10 mM sodium orthovanadate(Sigma)] and incubated at 0°C for 30 min. The samples were thenhomogenized and processed for eicosanoid extraction as describedabove.

Shappell () found in human samples from radicalprostatectomy specimens that expression of COX-2 was elevated onlyin high-grade tumors. By comparison, the reduction of 15-LOX-2 and15-HETE formation is the most characteristic alteration of AAmetabolism in prostate cancer (). We also reported that 15-LOX-2expression was lost in all the prostate cancer cell lines we tested(including PC-3, LNCaP, and DU145 cells), yet easily detectablelevels of 15-LOX-2 were expressed in normal human prostate cells(,). Not surprisingly, eicosanoidprofiles differ between cancer and normal prostate cells andbetween mouse and human prostate tissues.

Colombo A (1985) The underdefined nature of the blood lead-air lead relationship from biological and statistical grounds.
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Rate limiting step of synthesis of prostaglandins

Expression of the eicosanoid enzymes(top panel) and their relevant products in human prostate mousexenograft tissues (bottom panel). Tumor tissues were collected whenthe tumor volume was approximately 1 cm3. Protein levelswere detected by Western blotting with relevant antibodies (toppanel). Flash-frozen tissues were pulverized and extracted for theendogenous eicosanoid levels by LC/MS/MS. The levels of eicosanoidswere normalized by protein concentrations (bottom panel, A–D). Dataare presented as the means ± SDs in each group (n≥5).*p

term:eicosanoids = include leukotrienes and …

To explore the role of eicosanoids in prostatecancer progression, we further investigated the expression of COX-2and various LOX proteins in human prostate xenograft tissues(). As thetumorigenicity of the MDA PCa 2a cells was extremely low, weevaluated the protein expression of eicosanoid enzymes in only theother four human prostate cancer xenograft tissues. The proteinexpression of the COX-2 and 5-LOX enzymes was highest in the PC-3xenografts, whereas the protein levels of 12-LOX were higher inboth PC-3 and PCa 2b cells. In line with the protein expression of15-LOX-2 in the cells , its expression wasconsistently highest in the two tumor tissues derived from MDA PCa2b xenografts ().

rate limiting step of eicosanoid synthesis

It has been suggested () that the 5-LOX and 12-LOX pathwaysare even more potent than the COX-2 pathways and that inhibitingthese LOX pathways would prove a more effective therapeuticstrategy than inhibiting COX-2 pathways. Our findings agree. Amongour five prostate cancer cell lines and their four xenograftmodels, only PC-3 cells and the related xenograft models showedrelatively high levels of COX-2 expression and formation of itsmetabolite PGE. Furthermore, the COX-2 andPGE levels in the core biopsy specimens containingtumor were similar to those of the core biopsy specimens withouttumor. The expression of mRNA and protein of COX-2 correlated wellwith the level of PGE in the prostate cancer cells, therelated xenograft tumors, and the human prostate biopsy specimenswith tumor. By contrast, the LOX enzyme expression and metabolitelevels in the different materials (i.e., cultured cells, xenograftmodels, and core biopsy specimen cores with tumors) were highlyelevated in a number of the prostate cancer cell lines tested. Thenotable variation in LOX expression and formation of its productsunder different microenvironmental conditions suggested thatmodulation of COX and LOX enzyme pathways differs, depending ontheir microenvironments and whether the source of AA is exogenousor endogenous. This, in turn, suggests that therapeuticinterventions targeting these pathways will be more effective thaninhibiting a specific enzyme. For example, the mRNA levels of 5-,12-, and 15-LOXs appear to be positively correlated with theendogenous levels of their relevant metabolites, whereas theexogenous levels of LOX products were proportional to those of theLOX protein (–). This discord might be due to thesensitivity of antibodies related to each LOX of interest; thecofactors needed for production of eicosanoids, and the relativeaffinities of each enzyme for their substrate, AA. Thus, these datasuggest that evaluating the expression of these eicosanoid enzymesas well as their relevant metabolites in the human biopsy samplesis important for delineating the role of these lipid mediators inthe development and progression of prostate cancer.

The rate limiting step in cholesterol synthesis occurs ..

The mRNA and protein levels of each eicosanoidenzyme were differently regulated in the five prostate cancer celllines we tested, which suggested that the relevant metabolites ofthe eicosanoids may also differ. Therefore, we measured bothendogenous and exogenous eicosanoid levels in those cell lines. Asshown in , levels of bothendogenous and exogenous PGE, a COX-2 metabolite, werehighest in the PC-3 cells, suggesting a high capacity to producethis pro-inflammatory eicosanoid. The level of exogenousPGE in PC-3 cells was almost 15 times higher than thatproduced endogenously.

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