12-hydroxy-5-8-10-14-eicosatetraenoic-acid and Fibrosarcoma

We report here that the 12-lipoxygenase metabolite of arachidonic acid, 12-hydroxy-5Z, 8Z, 10E, 14Z, eicosatetraenoic acid (12-HETE), stimulates cAMP production in human fibroblasts among various cultured cell lines tested. Although 12-HETE seemed to stimulate the phospholipase C (PLC)-protein kinase C (PKC) system, inhibitors against PLC and PKC did not reduce the cAMP production induced by 12-HETE, indicating that the activation of PLC-PKC system is not positively coupled with the stimulation of cAMP production. On the other hand, the cAMP production induced by 12-HETE was dependent on the Ca2+/calmodulin system in the cells. The results suggest that 12-HETE specifically stimulates Ca2+/calmodulin-dependent adenylyl cyclase to increase cAMP level in the fibroblasts.

Topics: 1-Methyl-3-isobutylxanthine; 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cells, Cultured; Chelating Agents; Cyclic AMP; Egtazic Acid; Enzyme Inhibitors; Estrenes; Fibroblasts; Fibrosarcoma; HL-60 Cells; Humans; Indoles; Phosphodiesterase Inhibitors; Pyrrolidinones; Rats; Sulfonamides; Vasodilator Agents

Infiltrating macrophages elicit tumor-destructive reactions by releasing cytolytic factors including tumor necrosis factor alpha (TNF-alpha). Because platelets represent another major component of the cell infiltrate in tumors, we examined whether they could affect TNF alpha-induced cell death. Exposure of L-929 fibrosarcoma cells to human platelets reduced TNF alpha-induced cytotoxicity and cytolysis, as determined by 51Cr release assay and DNA fragmentation assay. This inhibitory effect, which depended on the concentration of platelets (0.1 to 10 x 10(6)/0.1 ml), was as high as 50%. The decrease in responsiveness to TNF-alpha reflected neither a degradation of TNF-alpha nor an inability of L-929 cells to bind TNF-alpha. Indeed, even though Scatchard analysis indicated the presence of 100 to 150 125I-TNF-alpha binding sites/platelet with a kd of 3.8 to 6.4 nM, addition of platelets up to 5 x 10(6)/0.1 ml did not compete with 125I-TNF-alpha binding to L-929 cells. Furthermore, addition of platelets 1 or 2 hours after that of TNF-alpha was still protective suggesting that platelets rather promoted hyporesponsiveness of L-929 cells to a postbinding effect of TNF-alpha Platelet-induced reduction of TNF-alpha response could be reproduced with supernatant fluids from platelets incubated at 37 degrees C for 24 hours. The platelet-derived factor responsible for this effect was found to be a lipid of low molecular weight with high affinity for albumin and charcoal. A role for 12(S) hydroxyeicosatetraenoic acid is proposed because this metabolite reduced TNF-alpha-induced cytolysis in a dose-dependent manner, whereas other platelet-derived lipids including thromboxane A2 and platelet activating factor were inactive. These observations indicate that the role of associated platelets has to be considered when analyzing the cytotoxic and cytolytic activity of macrophage-derived TNF-alpha on tumor cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Blood Platelets; Cell Death; Culture Media, Conditioned; Dose-Response Relationship, Drug; Fibrosarcoma; Hydroxyeicosatetraenoic Acids; Iodine Radioisotopes; Mice; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The omega 3 class of polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA, 20:5), has been shown to alter the patterns of arachidonic acid (20:4) metabolism in both in vitro and in vivo systems. To examine further the role of arachidonic acid conversion to prostaglandins (PG) in hypercalcemic mice bearing the PG-producing HSDM1 fibrosarcoma, we have performed experiments in which control and tumor-bearing animals were fed diets either low (0.1-0.2% of total fatty acid) or high (17%) in EPA. In all five experiments performed, tumor-bearing mice eating control diets had markedly elevated (average sixfold above control) plasma concentrations of 13,14-dihydro-15-keto-PGE2 (PGE2-M), while in mice bearing HSDM1 tumors and eating the EPA-enriched menhaden oil diet, the elevation was reduced to only twice control values. The increase in plasma calcium concentration (approximately 2.5 mg/dl above control) in tumor-bearing animals was also reduced significantly (P less than 0.05) to only 1.3 mg/dl above control in mice eating the diet enriched in EPA. Plasma immunoreactive hydroxy fatty acids (i12-HETE) and sulfidopeptide leukotrienes (iSRS) were not elevated in tumor-bearing mice and were unaffected by diet. The contents of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were lower in tumor tissue from animals eating the diet high in EPA, whereas the tissue contents of i12-HETE and iSRS were not altered by diet. Fatty acid analysis of liver and tumor tissue revealed marked increases in certain omega 3 fatty acids (20:5, 22:5, and 22:6) from animals eating the enriched diet. Body weights, tumor weights, and tumor histology were not significantly altered by diet. To determine whether dietary calcium played a role in the elevation of plasma calcium in mice bearing the HSDM1 tumor and the reduction of plasma calcium in animals fed EPA, we compared results in mice fed diets containing 0.80% (normal) and 0.015% (deficient) calcium. The increases in plasma calcium and PGE2-M observed in tumor-bearing mice were the same on both normal and very low calcium intakes. We conclude, in mice of the Swiss albino strain bearing the HSDM1 fibrosarcoma, that consumption of a diet enriched in EPA reduces the production of cyclooxygenase products of arachidonic acid metabolism and thereby reduces the elevation of plasma calcium concentration. Dietary enrichment with EPA did not alter the production of serologically determined lipoxygenase products of arachidonic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Body Weight; Calcium; Calcium, Dietary; Dietary Fats; Dinoprostone; Fatty Acids; Fibrosarcoma; Fish Oils; Hydroxyeicosatetraenoic Acids; Male; Mice; Oils; Prostaglandins; Prostaglandins E; SRS-A; Tissue Distribution