Non-small cell lung cancer cycloxygenase activity and proliferation are inhibited by non-steroidal antiinflammatory drugs.

The effects of non-steroidal antiinflammatory drugs (NSAIDs) on non-small cell lung cancer (NSCLC) were investigated. Arachidonic acid (AA) was metabolized to prostaglandin E2 (PGE2) in NSCLC cells. NSAIDs such as aspirin or indomethacin reduced PGE2 levels in NCI-H157 and H1264 cells, and the decrease caused by PGE2 was reversed by epidermal growth factor (EGF). By RT-PCR, both cyclooxygenase (COX)-1 and COX-2 mRNAs are detected in NCI-H157 and H1264 cells. By Northern analysis, COX-2 mRNA was induced by EGF and phorbol ester. By immunocytochemistry, COX-1 and COX-2 enzymes were localized to NSCLC tumors. Aspirin, indomethacin and ibuprofen decreased NSCLC growth in vitro. Aspirin and indomethacin inhibited proliferation of NSCLC xenografts in nude mice. These data suggest that COX enzymes may be important regulatory components of NSCLC.

Systemic activation of 15-lipoxygenase in heart, lung, and vascular tissues by hypercholesterolemia: relationship to lipoprotein oxidation and atherogenesis.

There is evidence that oxidized lipoproteins are a major contributing factor in atherosclerosis. 15-Lipoxygenase is the principal mammalian enzyme that can oxidize polysaturated fatty acids present in intact lipoproteins, and in membrane phospholipids in situ. We, and others, have reported previously that levels of the enzyme are increased in aortas of cholesterol-fed and spontaneously atherosclerotic WHHL rabbits. In the present study, rabbits were fed an atherogenic diet containing 1% cholesterol for 14 weeks, and levels of [14C]arachidonate metabolizing enzymes in the excised tissues were measured by HPLC analysis. 15-Lipoxygenase levels in heart, aortic adventitia, and lung, but not in liver, were increased up to 100-fold above controls, without major significant changes in prostaglandin endoperoxide synthases or the 5- and 12-lipoxygenases. The induced 15-lipoxygenase activity in the aortic adventitia was approximately 15 times greater than that found in the vessel wall. Hypercholesterolemia and elevated 15-lipoxygenase were associated with a 40% lowering of blood hematocrit. The hemolytic agent phenylhydrazine duplicated the effects of hypercholesterolemia on hematocrit, and induced up to 100-fold increases in 15-lipoxygenase activity in tissues within 7 days. The induced 15-lipoxygenase activities in heart and lung were 4 and 8 times greater, respectively, than in reticulocytes, previously the richest known source of the enzyme. Direct measurements of hemoglobin content also demonstrated that contaminating reticulocytes were not the source of the tissue enzyme. A similar tissue-specific activation of 15-lipoxygenase was observed in rat heart and lung, but also not in liver. It is concluded that the elevated level of 15-lipoxygenase activity previously reported in atherosclerotic aorta is symptomatic of a generalized and massive induction of the enzyme in cardio-pulmonary tissues by hypercholesterolemia, which may be related to the membrane perturbation and increased hemolysis that is induced by cholesterol feeding.

Atherosclerosis: the eicosanoid connection.

This bird's eye view presents connections between the metabolically short-lived local hormones (collectively known as eicosanoids) and atherosclerotic cardiovascular disease. The discussion will be centered around an overview of coronary atherosclerosis with an emphasis on the sequences involved in the formation of atherosclerotic lesions; structure and historical background of oxygenated fatty acids cyclooxygenase and lipoxygenase products--eicosanoids; the generation of free radicals during the formation of endoperoxides by cyclooxygenase; the involvement of eicosanoids in the atherosclerotic inflammatory process, and finally, the effects of non-steroidal and steroidal anti-inflammatory drugs on the synthesis of eicosanoids and experimental atherosclerosis. Little is known about the exact role of eicosanoids in the genesis of atherosclerosis.

Relationship of vascular 15-lipoxygenase induction to atherosclerotic plaque formation in rabbits.

Aortas from atherosclerotic rabbits have increased levels of 15-lipoxygenase, but the relationship between induction of this enzyme and the atherosclerotic process has not been defined. We found that dietary administration of cortisone acetate significantly suppressed atherosclerotic plaque formation in both Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed WHHL/NZW heterozygous rabbits. There was, however, no corresponding decrease in the elevated 15-lipoxygenase activity. In addition, the elevated 15-lipoxygenase activity in atherosclerotic rabbit aortas was uniformly distributed throughout the aorta, and was not preferentially localized in the lesions. These results indicate that induction of the 15-lipoxygenase is not necessarily causally related to plaque development, and that plaques are not the major source of the increased enzyme activity. However, the results confirm that hypercholesterolemia is a necessary condition for both atherosclerosis and 15-lipoxygenase induction, suggesting that perhaps the 15-lipoxygenase may represent a protective response to the hyperlipidemic stress. This possibility is supported by the finding that the induced 15-lipoxygenase converts linoleic acid, which is the predominant essential fatty acid in aorta, to 13-hydroxyoctadecadienoic acid (13-HODE). This compound is a chemorepellant factor for platelets, inhibits platelet thromboxane synthesis, and stimulates prostacyclin synthesis by endothelial cells.

Antiplatelet constituents of garlic and onion.

We have identified three main antiplatelet constituents, namely adenosine, allicin and paraffinic polysulfides in both garlic and onion. Adenosine and allicin both inhibited platelet aggregation without affecting cyclooxygenase and lipoxygenase metabolites of arachidonic acid. The trisulfides inhibited platelet aggregation as well as thromboxane synthesis along with induction of new lipoxygenase metabolites. The data indicate that the observed in vivo antiplatelet effects of ingesting onion and garlic are attributable more to the adenosine than to the allicin and paraffinic polysulfide constituents.

The induced lipoxygenase in atherosclerotic aorta converts linoleic acid to the platelet chemorepellant factor 13-HODE.

Mammalian tissues contain 5-, 12- and 15-lipoxygenases. Only the 15-lipoxygenase can act on linoleic acid, the predominant essential fatty acid of tissues and plasma, producing 13-hydroxyoctadecadienoic acid (13-HODE). Intracellular production of 13-HODE renders endothelial cells resistant to platelet adhesion, while its hydroperoxy precursor, 13-HPODE, synergises with the platelet anti-aggregatory factor prostacyclin. We have found that a 15-lipoxygenase activity is induced in aortas of cholesterol-fed and Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Aortic tissue from WHHL rabbits incubated with 3H-linoleic acid produced a major metabolite identified as 13-HODE, which was formed with an efficiency comparable to the synthesis 15-HETE from arachidonic acid. These findings indicate that the increased aortic 15-lipoxygenase in vascular tissue is capable of producing 13-HODE in vivo. Since platelet adhesion is increased in atherogenesis, and thrombogenesis is a major complication of advanced atherosclerosis, it is suggested that induction of this enzyme may be a protective response to hypercholesterolemia.

Anti-inflammatory drugs in experimental atherosclerosis. 7. Spontaneous atherosclerosis in WHHL rabbits and inhibition by cortisone acetate.

The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for familial hypercholesterolemia, has a deficiency in low density lipoprotein (LDL) receptor binding and exhibits elevated plasma lipoprotein levels and spontaneous atherosclerosis. Since atherosclerotic plaque formation has a number of features in common with the inflammatory process, we have investigated the effect of dietary supplementation with an anti-inflammatory steroid (cortisone acetate, 5 mg daily for 3 months) on atherosclerosis using the WHHL rabbit as a model. Atherosclerotic plaque formation in cortisone-fed animals was reduced by about 60% compared to control WHHL rabbits. Steroid administration increased circulating cholesterol levels modestly and triglycerides were increased about 6-fold. While very low density lipoprotein (VLDL)-cholesterol was increased, LDL-cholesterol levels were decreased and the particle was more triglyceride-enriched as well as less dense. Steroid-fed animals also exhibited decreased platelet aggregation and increased aortic 15-lipoxygenase activity. The histological observations showed typical fibrous plaques in aortas of both control and cortisone-fed rabbits, with intima thickened by foamy macrophages and subcellular lipoproteinaceous debris covered by a fibrous cap. These findings thus indicate that steroids reduce the rate of plaque initiation or progression but do not significantly change the histological appearance of the lesion.

Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) as the predominant eicosanoid in aortas from Watanabe Heritable Hyperlipidemic and cholesterol-fed rabbits.

Atherosclerotic plaque formation is accompanied by hyperproliferative events which have many features of an inflammatory response. A high-performance liquid chromatography procedure was developed to analyze the inflammatory prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) produced by aortic segments. Normal rabbit aortas incubated with tritiated arachidonic acid synthesized 12-HETE as the principal lipoxygenase metabolite, and prostacyclin as the major cyclooxygenase product. In contrast, atherosclerotic aortas from both cholesterol-fed and Watanabe Heritable Hyperlipidemic rabbits showed major increases in synthesis of lipoxygenase-derived 15-HETE, which became the predominant eicosanoid in the aortas of both types of rabbit. No leukotrienes or other 5-lipoxygenase products were detected to the detection limit of 0.5 pmol/cm aorta. 15-HETE, which is chemotactic for smooth muscle cells, mitogenic for endothelial cells, and an inhibitor of prostacyclin synthesis may thus play a role in atherogenesis.

Corticosteroids suppress cyclooxygenase messenger RNA levels and prostanoid synthesis in cultured vascular cells.

Prostacyclin synthesis by cultured vascular smooth muscle cells was inactivated by aspirin. Recovery required serum factors replaceable by EGF plus TGF-beta and was blocked by cycloheximide but not by actinomycin D. Recovery of cyclooxygenase activity was prevented by preincubation with dexamethasone (0.1 to 2 microM), which also suppressed basal enzyme activity by up to 70%. A full length 2.8 Kb cDNA hybridization probe for human cyclooxygenase identified a cyclooxygenase messenger RNA of approximately 2.8 Kb in these cells. Cyclooxygenase mRNA levels were enhanced by EGF/TGF-beta, but suppressed completely by corticosteroids. It is concluded that inhibition of prostanoid synthesis by corticosteroids is mediated by suppressing cyclooxygenase messenger RNA. These observations provide a new molecular mechanism for the anti-inflammatory activity of the corticosteroids.

A platelet phospholipase inhibitor from the medicinal herb feverfew (Tanacetum parthenium).

Feverfew has been used since antiquity to treat fevers and other inflammatory conditions. Feverfew extracts were found to inhibit ADP, thrombin, or collagen-induced aggregation of human platelets, but significantly, did not affect aggregation induced by arachidonic acid. Synthesis of thromboxane B2 from exogenous 14C-arachidonic acid was also not inhibited. Washed platelets prelabelled with 14C-AA responded normally to thrombin by releasing 14C-TXB2. This was completely blocked by feverfew. A purified platelet phospholipase A2 was inhibited by the material with an I50 of 0.1 antiplatelet units. The pharmacological properties of feverfew may thus be due to an inhibitor of cellular phospholipases, which prevents release of arachidonic acid in response to appropriate physiological stimuli.

Effects of onion (Allium cepa) extract on platelet aggregation and thromboxane synthesis.

Oral administration of onion and garlic reportedly decreases platelet aggregation in both human and animal subjects. An oily chloroform extract of onion (Allium Cepa) was prepared and separated by column chromatography on silicic acid into six fractions by elution with solvents of increasing polarity. The least polar fraction contained most of the inhibitory activity towards platelet aggregation induced by either ADP or arachidonic acid. Further purification was afforded by thin-layer chromatography. The specific activity of this major active fraction (I50 per ml of PRP) was approximately 7 units per milligram. Platelets incubated in the presence of onion inhibitor and (1-14C)-arachidonic acid showed striking changes in the pattern of arachidonic acid metabolites formed. Thromboxane B2 synthesis was almost completely suppressed without significant decreases in total hydroxy fatty acid formation. It was concluded that the observed antiplatelet activity of onion relates to the presence of a non-polar, heat stable inhibitor of thromboxane synthesis. This appears to be the first demonstration of this type of inhibitor present in significant quantities in a common foodstuff.