5-Lipoxygenase Abstracts 1

© 2013

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

         (Boudreau, Maillet et al. 2012) Download

BACKGROUND: 5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases. METHODOLOGY/PRINCIPAL FINDINGS: Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 microM. CAPE inhibited 5-LO activity (IC(50) 0.13 microM, 95% CI 0.08-0.23 microM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC(50) 3.5 microM, 95% CI 2.3-5.4 microM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC(50) values in the low microM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis. CONCLUSIONS: CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.


5-lipoxygenase as an endogenous modulator of amyloid beta formation in vivo

         (Chu and Pratico 2011) Download

OBJECTIVE: The 5-lipoxygenase (5-LO) enzymatic pathway is widely distributed within the central nervous system, and is upregulated in Alzheimer's disease. However, the mechanism whereby it may influence the disease pathogenesis remains elusive. METHODS: We evaluated the molecular mechanism by which 5-LO regulates amyloid beta (Abeta) formation in vitro and in vivo by pharmacological and genetic approaches. RESULTS: Here we show that 5-LO regulates the formation of Abeta by activating the cAMP-response element binding protein (CREB), which in turn increases transcription of the gamma-secretase complex. Preventing CREB activation by pharmacologic inhibition or dominant negative mutants blocks the 5-LO-dependent elevation of Abeta formation and the increase of gamma-secretase mRNA and protein levels. Moreover, 5-LO targeted gene disruption or its in vivo selective pharmacological inhibition results in a significant reduction of Abeta, CREB and gamma-secretase levels. INTERPRETATION: These data establish a novel functional role for 5-LO in regulating endogenous formation of Abeta levels in the central nervous system. Thus, 5-LO pharmacological inhibition may be beneficial in the treatment and prevention of Alzheimer's disease.

Arachidonic acid stimulates prostate cancer cell growth: critical role of 5-lipoxygenase

         (Ghosh and Myers 1997) Download

Arachidonic acid (5,8,11,14-eicosatetraenoic acid), a member of the omega-6 poly-unsaturated fatty acids, was found to be an effective stimulator of human prostate cancer cell growth in vitro at micromolar concentrations. Selective blockade of the different metabolic pathways of arachidonic acid (e.g. ibuprofen for cyclooxygenase, SKF-525A for cytochrome P-450, baicalein and BHPP for 12-lipoxygenase, AA861 and MK886 for 5-lipoxygenase, etc.) revealed that the growth stimulatory effect of arachidonic acid is inhibited by the 5-lipoxygenase specific inhibitors, AA861 and MK886, but not by others. Addition of the eicosatetraenoid products of 5-lipoxygenase (5-HETEs) showed stimulation of prostate cancer cell growth similar to that of arachidonic acid, whereas the leukotrienes were ineffective. Moreover, the 5-series of eicosatetraenoids could reverse the growth inhibitory effect of MK886. Finally, prostate cancer cells fed with arachidonic acid showed a dramatic increase in the production of 5-HETEs which is effectively blocked by MK886. These experimental observations suggest that arachidonic acid needs to be metabolized through the 5-lipoxygenase pathway to produce 5-HETE series of eicosatetraenoids for its growth stimulatory effects on human prostate cancer cells.

Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells

         (Ghosh and Myers 1998) Download

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2-4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-L-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase-programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.

Modulation of arachidonic acid metabolism by curcumin and related beta-diketone derivatives: effects on cytosolic phospholipase A(2), cyclooxygenases and 5-lipoxygenase

         (Hong, Bose et al. 2004) Download

Aberrant arachidonic acid metabolism is involved in the inflammatory and carcinogenic processes. In this study, we investigated the effects of curcumin, a naturally occurring chemopreventive agent, and related beta-diketone derivatives on the release of arachidonic acid and its metabolites in the murine macrophage RAW264.7 cells and in HT-29 human colon cancer cells. We also examined their effects on the catalytic activities and protein levels of related enzymes: cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenases (COX) as well as 5-lipoxygenase (5-LOX). At 10 micro M, dibenzoylmethane (DBM), trimethoxydibenzoylmethane (TDM), tetrahydrocurcumin (THC) and curcumin effectively inhibited the release of arachidonic acid and its metabolites in lipopolysaccharide (LPS)-stimulated RAW cells and A23187-stimulated HT-29 cells. Inhibition of phosphorylation of cPLA(2), the activation process of this enzyme, rather than direct inhibition of cPLA(2) activity appears to be involved in the effect of curcumin. All the curcuminoids (10 micro M) potently inhibited the formation of prostaglandin E(2) (PGE(2)) in LPS-stimulated RAW cells. Curcumin (20 micro M) significantly inhibited LPS-induced COX-2 expression; this effect, rather than the catalytic inhibition of COX, may contribute to the decreased PGE(2) formation. Without LPS-stimulation, however, curcumin increased the COX-2 level in the macrophage cells. Studies with isolated ovine COX-1 and COX-2 enzymes showed that the curcuminoids had significantly higher inhibitory effects on the peroxidase activity of COX-1 than that of COX-2. Curcumin and THC potently inhibited the activity of human recombinant 5-LOX, showing estimated IC(50) values of 0.7 and 3 micro M, respectively. The results suggest that curcumin affects arachidonic acid metabolism by blocking the phosphorylation of cPLA(2), decreasing the expression of COX-2 and inhibiting the catalytic activities of 5-LOX. These activities may contribute to the anti-inflammatory and anticarcinogenic actions of curcumin and its analogs.


Spice phenolics inhibit human PMNL 5-lipoxygenase

         (Prasad, Raghavendra et al. 2004) Download

A wide variety of phenolic compounds and flavonoids present in spices possess potent antioxidant, antimutagenic and anticarcinogenic activities. We examined whether 5-lipoxygenase (5-LO), the key enzyme involved in biosynthesis of leukotrienes is a possible target for the spices. Effect of aqueous extracts of turmeric, cloves, pepper, chili, cinnamon, onion and also their respective active principles viz., curcumin, eugenol, piperine, capsaicin, cinnamaldehyde, quercetin, and allyl sulfide were tested on human PMNL 5-LO activity by spectrophotomeric and HPLC methods. The formation of 5-LO product 5-HETE was significantly inhibited in a concentration-dependent manner with IC(50) values of 0.122-1.44 mg for aqueous extracts of spices and 25-83 microM for active principles, respectively. The order of inhibitory activity was of quercetin>eugenol>curcumin>cinnamaldehyde>piperine>capsaicin>allyl sulfide. Quercetin, eugenol and curcumin with one or more phenolic ring and methoxy groups in their structure showed high inhibitory effect, while the non-phenolic spice principle allyl sulfide showed least inhibitory effect on 5-LO. The inhibitory effect of quercetin, curcumin and eugenol was similar to that of synthetic 5-LO inhibitors-phenidone and NDGA. Moreover, the inhibitory potency of aqueous extracts of spice correlated with the active principles of their respective spices. The synergistic or antagonistic effect of mixtures of spice active principles and spice extracts were investigated and all the combinations of spice active principles/extracts exerted synergistic effect in inhibiting 5-LO activity. These findings clearly suggest that phenolic compounds present in spices might have physiological role in modulating 5-LO pathway.

Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells

         (Raghavenra, Diwakr et al. 2006) Download

Polymorphonuclear leukocytes (PMNL) play an important role in the modulation of inflammatory conditions in humans. PMNL cells recruited at the site of inflammation, release inflammatory mediators such as leukotrienes, proteolytic enzymes and reactive oxygen species. Among these, leukotrienes are implicated in pathophysiology of allergic and inflammatory disorders like asthma, allergic rhinitis, arthritis, inflammatory bowel disease and psoriasis. 5-lipoxygenase (5-LO) is the key enzyme in biosynthetic pathway of leukotrienes. Our earlier studies showed that spice phenolic active principles significantly inhibit 5-LO enzyme in human PMNLs. In this study we have further characterized the inhibitory mechanism of eugenol, the active principle of spice-clove on 5-LO enzyme and also its effect on leukotriene C((4)) (LTC(4)). Substrate dependent enzyme kinetics showed that the inhibitory effect of eugenol on 5-LO was of a non-competitive nature. Further, eugenol was found to significantly inhibit the formation of LTC(4) in calcium ionophore A23187 and arachidonic acid (AA) stimulated PMNL cells. These data clearly suggest that eugenol inhibits 5-LO by non-competitive mechanism and also inhibits formation of LTC(4) in human PMNL cells and thus may have beneficial role in modulating 5-LO pathway in human PMNL cells.

Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon

         (Sarveswaran, Thamilselvan et al. 2011) Download

Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCepsilon), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCepsilon and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCepsilon), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCepsilon) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCepsilon. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCepsilon, a pro-survival serine/threonine kinase.


Boswellia serrata, a potential antiinflammatory agent: an overview

         (Siddiqui 2011) Download

The resin of Boswellia species has been used as incense in religious and cultural ceremonies and in medicines since time immemorial. Boswellia serrata (Salai/Salai guggul), is a moderate to large sized branching tree of family Burseraceae (Genus Boswellia), grows in dry mountainous regions of India, Northern Africa and Middle East. Oleo gum-resin is tapped from the incision made on the trunk of the tree and is then stored in specially made bamboo basket for removal of oil content and getting the resin solidified. After processing, the gum-resin is then graded according to its flavour, colour, shape and size. In India, the States of Andhra Pradesh, Gujarat, Madhya Pradesh, Jharkhand and Chhattisgarh are the main source of Boswellia serrata. Regionally, it is also known by different names. The oleo gum-resins contain 30-60% resin, 5-10% essential oils, which are soluble in the organic solvents, and the rest is made up of polysaccharides. Gum-resin extracts of Boswellia serrata have been traditionally used in folk medicine for centuries to treat various chronic inflammatory diseases. The resinous part of Boswellia serrata possesses monoterpenes, diterpenes, triterpenes, tetracyclic triterpenic acids and four major pentacyclic triterpenic acids i.e. beta-boswellic acid, acetyl-beta-boswellic acid, 11-keto-beta-boswellic acid and acetyl-11-keto-beta-boswellic acid, responsible for inhibition of pro-inflammatory enzymes. Out of these four boswellic acids, acetyl-11-keto-beta-boswellic acid is the most potent inhibitor of 5-lipoxygenase, an enzyme responsible for inflammation.


On the interference of boswellic acids with 5-lipoxygenase: mechanistic studies in vitro and pharmacological relevance

         (Siemoneit, Pergola et al. 2009) Download

Boswellic acids are pharmacologically active ingredients of frankincense with anti-inflammatory properties. It was shown that in vitro 11-keto-boswellic acids inhibit 5-lipoxygenase (5-LO, EC 1.13.11.34), the key enzyme in leukotriene biosynthesis, which may account for their anti-inflammatory effectiveness. However, whether 11-keto-boswellic acids interfere with 5-LO under physiologically relevant conditions (i.e., in whole blood assays) and whether they inhibit 5-LO in vivo is unknown. Inhibition of human 5-LO by the major naturally occurring boswellic acids was analyzed in cell-free and cell-based activity assays. Moreover, interference of boswellic acids with 5-LO in neutrophil incubations in the presence of albumin and in human whole blood was assessed, and plasma leukotriene B(4) of frankincense-treated healthy volunteers was determined. Factors influencing 5-LO activity (i.e., Ca(2+), phospholipids, substrate concentration) significantly modulate the potency of 11-keto-boswellic acids to inhibit 5-LO. Moreover, 11-keto-boswellic acids efficiently suppressed 5-LO product formation in isolated neutrophils (IC(50)=2.8 to 8.8 muM) but failed to inhibit 5-LO product formation in human whole blood. In the presence of albumin (10 mg/ml), 5-LO inhibition by 11-keto-boswellic acids (up to 30 muM) in neutrophils was abolished, apparently due to strong albumin-binding (>95%) of 11-keto-boswellic acids. Finally, single dose (800 mg) oral administration of frankincense extracts to human healthy volunteers failed to suppress leukotriene B(4) plasma levels. Our data show that boswellic acids are direct 5-LO inhibitors that efficiently suppress 5-LO product synthesis in common in vitro test models, however, the pharmacological relevance of such interference in vivo seems questionable.


References

Boudreau, L. H., J. Maillet, et al. (2012). "Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes." PLoS One 7(2): e31833 PMID: 22347509

Chu, J. and D. Pratico (2011). "5-lipoxygenase as an endogenous modulator of amyloid beta formation in vivo." Ann Neurol 69(1): 34-46 PMID: 21280074

Ghosh, J. and C. E. Myers (1997). "Arachidonic acid stimulates prostate cancer cell growth: critical role of 5-lipoxygenase." Biochem Biophys Res Commun 235(2): 418-23 PMID: 9199209

Ghosh, J. and C. E. Myers (1998). "Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells." Proc Natl Acad Sci U S A 95(22): 13182-7 PMID: 9789062

Hong, J., M. Bose, et al. (2004). "Modulation of arachidonic acid metabolism by curcumin and related beta-diketone derivatives: effects on cytosolic phospholipase A(2), cyclooxygenases and 5-lipoxygenase." Carcinogenesis 25(9): 1671-9 PMID: 15073046

Prasad, N. S., R. Raghavendra, et al. (2004). "Spice phenolics inhibit human PMNL 5-lipoxygenase." Prostaglandins Leukot Essent Fatty Acids 70(6): 521-8 PMID: 15120715

Raghavenra, H., B. T. Diwakr, et al. (2006). "Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells." Prostaglandins Leukot Essent Fatty Acids 74(1): 23-7 PMID: 16216483

Sarveswaran, S., V. Thamilselvan, et al. (2011). "Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon." Biochim Biophys Acta 1813(12): 2108-17 PMID: 21824498

Siddiqui, M. Z. (2011). "Boswellia serrata, a potential antiinflammatory agent: an overview." Indian J Pharm Sci 73(3): 255-61 PMID: 22457547

Siemoneit, U., C. Pergola, et al. (2009). "On the interference of boswellic acids with 5-lipoxygenase: mechanistic studies in vitro and pharmacological relevance." Eur J Pharmacol 606(1-3): 246-54 PMID: 19374837