Assessing 12(S)-lipoxygenase inhibitory activity using colorectal cancer cells overexpressing the enzyme.

12(S)-Lipoxygenase (LOX) is regarded as a pro-tumorigenic enzyme and as a potential target for therapy and prevention of cancer so that the search for specific 12(S)-LOX inhibitors is part of drug development strategies. To facilitate the identification of specific 12(S)-LOX inhibitors we have created an assay cell line by introducing a12(S)-LOX expression vector into SW480 colorectal cancer cells. When arachidonic acid was supplied in the medium both transiently and stably overexpressing cells produced 12(S)-hydroxytetraenic acid (HETE) originating from the transfected gene at 4-5-fold the amount obtained from control transfectants. 12(S)-HETE production was 1913.7+/-17.2pg/ml and reached a steady state level 24h after addition of arachidonic acid. To demonstrate the models suitability of 12(S)-LOX overexpressing SW480 cells they were used to measure the inhibitory activity of the plant phenols baicalein, kaempferol, quercetin, nordihydroguaretic acid and resveratrol which are known for their chemopreventive as well as LOX-inhibitory activity in different tumour models. All 5 compounds inhibited 12(S)-HETE production at concentrations below those necessary for growth inhibition.     

Enzymatically modified low-density lipoprotein upregulates CD36 in low-differentiated monocytic cells in a peroxisome proliferator-activated receptor-gamma-dependent way

Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been suggested to upregulate CD36. Since free oxidized polyunsaturated fatty acids are PPARgamma ligands, we studied the effects of LDL modified by the simultaneous action of sPLA2 and 15-lipoxygenase (15LO) on CD36 expression and PPARgamma activation in monocytic cells. Exposure of MM6 cells, which do not express CD36 or other scavenger receptors, to such enzymatically modified LDL (enzLDL) resulted in upregulation of CD36 surface protein and mRNA expression. Similar effects were observed with free 13-hydroperoxyoctadecadienoic acid but not its esterified counterpart. Less pronounced effects were observed with LDL modified by 15LO alone. Upregulation of CD36 was inversely correlated to the state of cell differentiation, as showed by lower response to enzLDL of the scavenger receptor-expressing MM6-sr and THP1 cells. Importantly, LDL modified by sPLA2 and 15LO did not efficiently induce upregulation CD36 in PPARgamma-deficient macrophage-differentiated embryonic stem cells confirming a role of PPARgamma in CD36 expression in cells stimulated with enzLDL. Our data show that LDL modified with physiologically relevant enzymes stimulates CD36 expression in non-differentiated monocytes and that this process involves PPARgamma activation. These effects of enzLDL can be considered pro-atherogenic in the context of early atherosclerosis.     

20-HETE is associated with unfavorable outcomes in subarachnoid hemorrhage patients

Emerging evidence has suggested that patients experiencing aneurysmal subarachnoid hemorrhage (aSAH) develop vascular dysregulation as a potential contributor to poor outcomes. Preclinical studies have implicated the novel microvascular constrictor, 20-hydroxyeicosatetraenoic acid (20-HETE) in aSAH pathogenesis, yet the translational relevance of 20-HETE in patients with aSAH is largely unknown. The goal of this research was to determine the relationship between 20-HETE cerebrospinal fluid (CSF) levels, gene variants in 20-HETE synthesis, and acute/long-term aSAH outcomes. In all, 363 adult patients (age 18 to 75) with aSAH were prospectively recruited from the University of Pittsburgh Medical Center neurovascular Intensive Care Unit. Patients were genotyped for polymorphic variants and cytochrome P450 (CYP)-eicosanoid CSF levels were measured over 14 days. Outcomes included delayed cerebral ischemia (DCI), clinical neurologic deterioration (CND), and modified Rankin Scores (MRS) at 3 and 12 months. Patients with CND and unfavorable 3-month MRS had 2.2- and 2.7-fold higher mean 20-HETE CSF levels, respectively. Patients in high/moderate 20-HETE trajectory groups (35.7%) were 2.5-, 2.1-, 3.1-, 3.3-, and 2.1-fold more likely to have unfavorable MRS at 3 months, unfavorable MRS at 12 months, mortality at 3 months, mortality at 12 months, and CND, respectively. These results showed that 20-HETE is associated with acute and long-term outcomes and suggest that 20-HETE may be a novel target in aSAH.     

Inhibition of superoxide anion production in non-stimulated guinea pig peritoneal exudate cells by anti-inflammatory drugs.

Inhibitory effects of anti-inflammatory drugs on the production of superoxide anion (·O₂^? by isolated non-treated guinea pig peritoneal exudate cells (PEC) was studied spectro-photometrically using NADH and lactate dehydrogenase (LDH). Values of ID₅₀ were; diclofenac sodium (2 × 10^?5M), indomethacin (3 × 10^?5M), oxyphenbutazone (8 × 10^?5M), fenamic acid (1 × 10^?4M), ibuprofen (1 × 10^?4M), benzydamine (3 × 10^?4M), aspirin (10^?3M<) and dexamethasone (10^?3M<). The mechanism of inhibition seemed to block plasma membrane associated NAD(P)H oxidase(s) activity which produces ·O₂^? ID₅₀ values of other drugs; superoxide dismutase (SOD, 2 × 10^?8M), cytochalasin B(1 × 10^?7M) and NEM (6 × 10^?6M). d-Mannitol radical scavenger), 1,3-diphenyl-isobenzofuran (singlet oxygen scavenger) and sodium azide (mitochondrial electron transport inhibitor and singlet oxygen scavenger) were negative.Superoxide radical itself or oxygen-centered radical(s) derived from ·O₂^? is supposed recently as a rate-limiting factor for prostaglandin (PG) synthetase. Whether the inhibition of non-steroidal anti-inflammatory drug (NSAID) on ·O₂^? production is linked directly to PG biosynthesis or not, ·O₂^? was already demonstrated in our laboratory to make a role for the development of rat carageenan foot oedema. It may serve as a new in vitro sceening method of NSAID, to check the inhibitory potency of a compound on ·O₂^? production by guinea pig PEC.     

Modulation of Vascular O2 Responses by Cytochrome 450‐4A ω‐Hydroxylase Metabolites In Dahl Salt‐Sensitive Rats

Objective: This study evaluated the role of the 20‐HETE/cytochrome P450‐4A ω‐hydroxylase (CYP450‐4A) system in microvascular regulation in the skeletal muscle circulation following short‐term (three‐day) exposure to a high‐salt (HS) diet in Dahl salt‐sensitive (SS) rats. Methods: The effects of inhibiting CYP450‐4A on resting diameter, O₂‐induced constriction, and vasodilator responses to acetylcholine (ACh) and the nitric oxide (NO) donor, sodium nitroprusside (SNP), were evaluated in cremasteric arterioles of SS rats fed a low‐ (LS; 0.4% NaCl) or high‐salt (HS; 4% NaCl) diet for three days. Results: The HS diet upregulated CYP450‐4A mRNA expression and led to an enhanced constriction of arterioles in response to elevated PO₂ in SS rats, which could be blocked by inhibiting CYP450‐4A enzymes with dibromododecenyl methylsulfimide (DDMS). DDMS also inhibited resting tone significantly in SS rats fed the HS, but not the LS, diet, despite similar resting diameters and active tone in the two groups. Arteriolar dilations in response to ACh and SNP were similar in SS rats fed the LS vs. the HS diet and were unaffected by DDMS. Conclusions: These findings suggest that CYP450‐4A enzymes contribute to resting tone and to an enhanced response to elevated PO₂ in arterioles of Dahl‐SS rats fed the HS diet.     

The Influence of Supplementation with Zinc in Micro and Nano Forms on the Metabolism of Fatty Acids in Livers of Rats with Breast Cancer

The aim of this study was to investigate the effect of zinc supplementation (in the form of nano or microparticles) on the profile and metabolism of fatty acids in the liver microsomes of rats with induced breast cancer. The activity of desaturases (Δ5, Δ6, Δ9) and the level of cholesterol and its oxidized derivatives were measured. The aim of this study was also to determine the effect of various forms of zinc supplements on rats that were on 5-, 12- and 15-hydroxyeicosatetraenoic (5-, 12- and 15-HETE) and hydroxyoctadecadienoic (HODE) acids, and the level of prostaglandin E2 (PGE2). Female Spraque-Dawley rats (n = 24) were divided into 2 groups that were supplemented with zinc in the micro form (342 nm) or nano form (99 nm) particles, respectively, and a group with a standard diet (control group). All animals received 7,12-dimethylbenz[a]anthracene twice for the induction of breast cancer. Dietary nano-Zn supplementation increased vaccenic acid content (p = 0.032) and decreased Δ6-desaturase activity (p = 0.006), whereas micro-Zn increased cholesterol (p = 0.006), ∑COPs (total cholesterol-oxidation products) (p = 0.019) and PGE2 (p = 0.028) content. Dietary enrichment with Zn microparticles resulted in lower concentrations of the metabolites 15-, 12- and 5-HETE and HODE. Our study indicates that the effect of zinc supplementation on the metabolism of fatty acids in the liver microsomes under neoplastic conditions depends on the form in which it is administered.     

Modulation of cytochrome-derived epoxyeicosatrienoic acids pathway: a promising pharmacological approach to prevent endothelial dysfunction in cardiovascular diseases?

Progress in methods of investigating endothelial function in humans has led to the demonstration that endothelial dysfunction is an early process involved in the pathophysiology of cardiovascular diseases, and represents a new independent therapeutic target that may help to improve patient health. The administration of antioxidant, anti-hypertensive, lipid lowering or antidiabetic agents appear insufficient to fully restore the normal functions of the vascular endothelium and specific therapeutic strategies are still lacking. In this context, one emerging promising pharmacological approach to prevent endothelial dysfunction is to restore epoxyeicosatrienoic acids (EETs) pathway. EETs are eicosanoids synthesized by endothelial cytochrome epoxygenases that contribute to the regulation of endothelium-dependent dilatation, vascular inflammation, cell proliferation, angiogenesis and hemostasis. Moreover, it has been shown in vivo in humans that EETs act as endothelium-derived hyperpolarizing factors to regulate the vascular tone in both resistance and conduit arteries. In various cardiovascular disorders such as arterial hypertension, a decrease in EETs availability, due to an increased degradation by soluble epoxide hydrolase (sEH), is a deleterious mechanism that contributes to endothelial dysfunction and promotes cardiovascular inflammation and remodeling. Subsequently, the use of sEH inhibitors, which have been shown to decrease blood pressure, limit ischemic injury and prevent hypertrophy in various animal models, appears to be an attractive opportunity to restore endothelial function. Future research will be necessary to demonstrate that sEH inhibitors can prevent endothelial dysfunction in human arteries, which may help to prevent the development of cardiovascular complications and improve cardiovascular prognosis in patients.     

Role of 5-lipoxygenase pathway in the regulation of RAW 264.7 macrophage proliferation

Arachidonic acid (AA) metabolites control cell proliferation, among other physiologic functions. RAW 264.7 macrophages can metabolise AA through the cyclooxygenase and lipoxygenase (LOX) pathways. We aimed to study the role of AA-metabolites derived from 5-LOX in the control of RAW 264.7 macrophage growth. Our results show that zileuton, a specific 5-LOX inhibitor, and nordihydroguaiaretic acid (NDGA), a non-specific LOX inhibitor, inhibit cell proliferation and [(3)H]-thymidine incorporation in a concentration-dependent fashion. Growth inhibition induced by NDGA can be explained by an apoptotic process, while zileuton does not seem to induce apoptosis. Moreover, these treatments delay the cell cycle, as analysed by flow cytometry. On the other hand, the leukotriene (LT) B(4) receptor antagonist U-75302, the LTD(4) receptor antagonists LY-171883 and MK-571, and the cysteinyl-LT receptor antagonist REV-5901 also inhibit cell proliferation and [(3)H]-thymidine incorporation in a concentration-dependent manner, and delay the RAW 264.7 cell cycle. However, these antagonists did not induce annexin V staining, caspase activation or DNA fragmentation. Furthermore, we demonstrated that exogenous addition of LTB(4) or LTD(4) revert the cell growth inhibition induced by zileuton or the leukotriene receptor antagonists mentioned above. Finally, we observed that LTB(4) and LTD(4), in the absence of growth factors, have pro-proliferative effects on macrophages, and we obtained preliminary evidences that this effect could be through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, our results show that the interaction between LTB(4) and LTD(4) with its respective receptor is involved in the control of RAW 264.7 macrophage growth.     

Synthesis of tritium labelled 3(R)‐HETE and 3(R),18(R/S)‐DiHETE through a common synthetic route

An efficient procedure for the synthesis of 3‐hydroxyoxylipins labelled with tritium on all double bond positions is reported. The synthetic scheme involves a joint route for the formation of tetraacetylenic precursors followed by stereoselective reduction of the triple bonds either with hydrogen or tritium. The final tritiated products were obtained with specific activities ranging from 1.65 to 1.80 Ci/mmol. Copyright © 2003 John Wiley & Sons, Ltd.     

A short-term increase in dietary cholesterol and fat intake affects high-density lipoprotein composition in healthy subjects

Background and Aims

High-cholesterol and high-fat diets alter biochemical composition and anti-oxidant properties of high-density lipoproteins (HDL) in animals. Whether this occurs in humans is unknown. Therefore, we examined the effect of a short-term elevation in dietary cholesterol and fat intake on HDL composition in healthy subjects.