l-663536 and esculetin

Various compounds that inhibit processing of arachidonic acid (AA) are being intensively tested for their antitumour activity. However, the mechanisms responsible for such activity remain rather elusive. To approach this issue, we examined the effects of several structurally different inhibitors of AA metabolism in the human keratinocyte HaCaT cell line.. Several parameters were determined in HaCaT cells exposed to increasing concentrations of the inhibitors for 24 and/or 48 h. These included (1) oxidoreductase activity, total protein mass and cell cycle distribution to assess cell proliferation, (2) degradation of PARP protein to assess apoptosis, and (3) cell morphology, distribution of F-actin and expression of cytokeratins and E-cadherin to evaluate changes in differentiation status.. While eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), esculetin and MK-886 reduced proliferation of HaCaT cells, the cyclooxygenase inhibitors indomethacin and piroxicam had no such effects. Esculetin and NDGA arrested cells in S phase, and ETYA and MK-886 delayed cell progression through G(1) phase. Higher concentrations of NDGA, MK886 and/or ETYA caused cleavage of PARP. No changes in the expression of cytokeratins and E-cadherin were observed upon treatment with any of the inhibitors. However, esculetin induced redistribution of F-actin accompanied by increased cell adhesion and size.. Our findings indicate that, in addition to their ability to inhibit cell proliferation and to induce apoptosis, lipoxygenase inhibitors and/or ETYA may also elicit other important physiological responses in HaCaT keratinocytes.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Arachidonic Acid; Cell Differentiation; Cell Division; Cell Line, Transformed; Cell Survival; Cyclooxygenase Inhibitors; Humans; Indoles; Keratinocytes; Lipoxygenase Inhibitors; Umbelliferones

It was clearly demonstrated that two structurally different inhibitors of 5-lipoxygenase (5-LPO) metabolism (leukotriene synthesis), i.e. MK-886 and esculetin, when combined with transforming growth factor-beta 1 (TGF-beta 1), significantly enhanced the differentiation but did not change proliferation (i.e. cell number and cell cycle parameters) of human leukemia HL-60 cells in vitro. Although cell morphology and measurement of cell surface antigens (CD11b, CD14, and CD66b) after 48 h of combined treatment with MK-886 and TGF-beta 1 suggested a shift of the HL-60 cell population into more differentiated stages of myelopoiesis, cells with a fully mature phenotype were not observed. The effects on differentiation were better distinguished in the functional parameters of differentiation, i.e. oxidative burst of cells as detected by chemiluminescence and the nitroblue tetrazolium reduction test. Detailed statistical analysis of these data proved a significant synergistic interaction between TGF-beta 1 and inhibitors of 5-LPO metabolism. The differentiation effects of TGF-beta 1 alone and especially of its combination with MK-886 were most pronounced when the cells were pretreated with dimethyl sulfoxide or all-trans-retinoic acid. The results imply that either the lack of 5-LPO metabolites or a certain type of mis-balance in arachidonic acid metabolism can modulate the TGF-beta 1 effect on myeloid differentiation.

Topics: Cell Differentiation; HL-60 Cells; Humans; Indoles; Lipoxygenase Inhibitors; Luminescent Measurements; Reactive Oxygen Species; Transforming Growth Factor beta; Tretinoin; Umbelliferones

Metastasis is a complex process, almost a cascade, involving multiple steps and activities. However, an important factor is that malignant cells are able to penetrate through the multiple basement membrane barriers surrounding tissues, blood vessels, nerves and muscle that would otherwise block their dissemination. Penetration of malignant tumor cells through basement membrane is an active process requiring proteolysis. We report here that inhibitors of both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism convert mouse melanoma and human fibrosarcoma cells to a non invasive state by reducing the production of MMP-2, an enzyme required for the degradation of basement membranes. Specific metabolites of each pathway, i.e. PGF2 alpha and 5-HPETE, are able to transcend the block and restore collagenase production, invasiveness in vitro and metastatic activity in vivo. These studies indicate a key role for arachidonic acid metabolites in metastasis and suggest novel therapeutic approaches for inhibiting the spread of cancer.

Topics: Animals; Arachidonic Acid; Caffeine; Collagen; Cyclooxygenase Inhibitors; Dinoprost; Drug Combinations; Extracellular Matrix; Fibrosarcoma; Gelatinases; Humans; Indoles; Indomethacin; Laminin; Leukotrienes; Lipoxygenase Inhibitors; Masoprocol; Matrix Metalloproteinase 2; Melanoma; Metalloendopeptidases; Mice; Neoplasm Metastasis; Proteoglycans; Tumor Cells, Cultured; Umbelliferones

The eicosanoid generating potential of the brain, gills, skin, ovary, muscle, eye, liver, spleen, heart, and alimentary canal in the rainbow trout, Oncorhynchus mykiss, was examined. All the organs/tissues examined synthesized the 12-lipoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), and 12-hydroxyeicosapentaenoic acid (12-HEPE), implying the widespread nature of this enzyme in trout. Both prostaglandin E and LTC were also found in variable amounts in the organs, with the greatest amount of PGE found in the gill. Leukotriene (LT) B4 and LTB5 were found in supernatants from calcium ionophore-challenged brain, skin, ovary, liver, spleen, and heart, but the lipoxins A4 and A5 were only present in brain, ovary, and spleen in relatively small amounts. As lipoxins have previously been shown to be synthesized by macrophages in rainbow trout [Pettitt et al., J. Biol. Chem. 266, 8720-8726 (1991)], and related cells (microglial cells) are found in the brain of mammals, the localization of macrophage-like cells in trout brain was investigated immunocytochemically. Monoclonal antibodies specific for trout leucocytes failed to identify any microglial-like cells in sections of the brain, although microvessels containing immuno-positive reaction products were observed. A number of distinct lipoxygenase products were found in supernatants of ionophore-challenged gill, including 14-hydroxydocosahexaenoic acid, 12-HETE, and 12-HEPE, and a large number of dihydroxy fatty acid derivatives with conjugated triene chromophores. One of these products was tentatively identified as 8(R),15(S)-dihydroxyeicosatetraenoic acid, a dual 12- and 15-lipoxygenase product, but apparently no LTB4 was generated by this tissue.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Brain; Captopril; Eicosanoids; Eicosapentaenoic Acid; Fatty Acids; Female; Gills; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene C4; Masoprocol; Oncorhynchus mykiss; Prostaglandins E; Tissue Distribution; Umbelliferones