12-hydroxy-5-8-10-14-eicosatetraenoic-acid and Carcinoma--Squamous-Cell

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Biopsy; Carcinoma, Squamous Cell; Celecoxib; Chemoprevention; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Glucuronosyltransferase; Humans; Keratosis, Actinic; Lipoxygenase Inhibitors; Neoplasms, Radiation-Induced; Skin Neoplasms

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Arachidonate Lipoxygenases; Arachidonic Acid; Carcinoma, Squamous Cell; Cell Proliferation; Flavanones; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Nitrobenzenes; Salicylamides; Sulfonamides; Tumor Cells, Cultured; Umbelliferones

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Apoptosis; Arachidonate 12-Lipoxygenase; Blood Platelets; Carcinoma, Squamous Cell; Cell Membrane; Cell Survival; Flow Cytometry; Humans; Integrin alphaVbeta3; Integrins; Male; Prostatic Neoplasms; Receptors, Vitronectin; Transfection; Tumor Cells, Cultured

A murine model (C3H mice) of squamous cell carcinoma (SCCVII) has been used to investigate the role of arachidonic acid (AA) metabolites in head and neck cancer. Inhibition of tumor growth by cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors of AA metabolism has been associated with changes in levels of AA metabolites in tumor tissues and inflammatory cell infiltrates. To characterize this model further, the effects of exogenous AA metabolites on tumor growth in vitro and in vivo were investigated.. Following subcutaneous inoculation with SCCVII tumor cells, control (16 mice) and treatment (24 mice) groups were injected with peritumoral vehicle or AA metabolite. Peritumoral injections of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 12-hydroxyeicosatetraenoic acid (12-HETE) were performed for 16-21 days, and final excised tumor weights were measured. In vitro production of PGE2 and LTB4 was assayed in 2-5 day cultures of SCCVII. Exogenous PGE2 effects on tumor cell growth was assessed with the MTT assay in vitro.. Tumor growth was significantly inhibited (p =.03) following peritumoral injection of PGE2. Final tumor weights were not affected by LTB4 or 12-HETE. Tumor inhibition by PGE2 was associated with increased tumor tissue levels of LTB4 (p =.04). In vitro, SCCVII produced minimal amounts of PGE2 and LTB4, and PGE2 had minimal effect on growth.. In this model, tumor inhibition by exogenous PGE2 is primarily mediated by affecting host-tumor interactions, although there may be some direct effect on tumor cells. Changes in tumor tissue levels of LTB4 following peritumoral PGE2 administration may be attributable to negative feedback inhibition of the COX pathway with shunting into the LOX pathway. SCCVII cells are probably not a significant source of prostaglandins and leukotrienes in vivo. These data provide insight into the mechanism of action of inhibitors of AA metabolism on tumor growth.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; Dinoprostone; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Head and Neck Neoplasms; Injections, Intralesional; Leukotriene B4; Mice; Mice, Inbred C3H; Reference Values

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase Cgamma1 and activity of protein kinase Calpha (PKCalpha). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nm) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Carcinoma, Squamous Cell; Enzyme Activation; Epidermal Growth Factor; Humans; Isoenzymes; Kinetics; Lipoxygenase Inhibitors; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phospholipase C gamma; Phosphorylation; Protein Kinase C; Protein Kinase C-alpha; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Calcimycin; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Communication; Cell Line; Coculture Techniques; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lung Neoplasms; Models, Biological; Neutrophils; Phospholipases A; Phospholipases A2; Tumor Cells, Cultured

The investigation was undertaken to characterize the profile of arachidonic acid metabolites in different spontaneous and transplantable tumors in mice. The five metabolites via the cyclooxygenase pathway (PGE2, PGF2 alpha, PGD2, TxB2, 6-keto-PGF1 alpha), as well as the three lipoxygenase products (5-HETE, 12-HETE, and 15-HETE) were monitored by thin layer chromatography and high performance liquid chromatography after "ex vivo" metabolism of exogenous [1-C14]-arachidonic acid by homogenates of tumor tissues. It was shown that all tumors had a unique profile of eicosanoids. The most cyclooxygenase activity along with the significant synthesis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha was noted in lung tumors. The antitumor effect of indomethacin was directly related to the ability of tumors to produce PGE2. On the other hand, there were varying lipoxygenase activities in tumors. In some cases, the extremely high levels of 15- and 12-HETE synthesis in neoplastic tissue could indicate that there was a basic possibility of using lipoxygenase inhibitors for suppressing malignant tumors.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Animals; Arachidonic Acid; Carcinoma, Lewis Lung; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dinoprostone; Eicosanoids; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indomethacin; Leukemia L1210; Lipoxygenase Inhibitors; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred DBA; Neoplasm Transplantation; Neoplasms, Experimental; Skin Neoplasms

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amino Acid Sequence; Animals; Arachidonate 12-Lipoxygenase; Base Sequence; Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion; Chromatography, High Pressure Liquid; Extracellular Matrix; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms, Experimental; Polymerase Chain Reaction; Rats; RNA-Directed DNA Polymerase

The lipoxygenase inhibitor 2-phenylmethyl-1-naphthol (DuP 654) has been shown to be an active anti-inflammatory drug in a murine skin inflammation model. Since 12-HETE is assumed to have a pathophysiological role in inflammatory skin diseases, and epidermal cells possess high affinity binding sites for 12(S)-HETE, we studied the effect of DuP 654 on 12(S)-HETE binding to the human epidermal cell line SCL-II. DuP 654 antagonized 12(S)-HETE binding in a dose-dependent manner with a Ki of 3.41 +/- 0.23 nM. The antagonistic effect was reversible. After 1- and 24-hour preincubation, the drug had no more significant inhibitory effect at concentrations between 10(-10) and 10(-5) M on specific 12(S)-HETE binding (Bmax of 215,000 +/- 21,000 receptors per cell) or on receptor affinity (Kd of 3.25 +/- 0.42 nM). Our results show that DuP 654, in addition to its 5-lipoxygenase inhibitory activity, exhibits 12-HETE receptor antagonist effect, and therefore may be of benefit in skin diseases with elevated 12-HETE levels.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Carcinoma, Squamous Cell; Epidermal Cells; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Naphthols; Receptors, Cell Surface; Receptors, Eicosanoid; Skin Neoplasms; Tumor Cells, Cultured

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxyl]-3, 4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, a potent leukotriene-B4 (LTB4) receptor antagonist, inhibits in vivo 12-hydroxyeicosatetraenoic acid (12-HETE)-induced neutrophil infiltration, suggesting a potential 12-HETE receptor antagonist effect, as well. Since 12-HETE is assumed to have a pathophysiological role in inflammatory skin diseases, and epidermal cells possess high affinity binding sites for 12(S)-HETE, we studied the effect of SC-41930 on 12(S)-HETE binding to the human epidermal cell line, SCL-II. SC-41930 antagonized the 12(S)-HETE binding to SCL-II cells with a Ki of 480 nM. This Ki value is similar to that obtained for the inhibition of LTB4 binding to human neutrophils. Our results show that SC-41930, in addition to its LTB4 receptor antagonist effect, exhibits 12-HETE receptor antagonist effect as well, and therefore may be of benefit in skin diseases with elevated 12-HETE levels.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Benzopyrans; Carcinoma, Squamous Cell; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; Receptors, Immunologic; Receptors, Leukotriene B4; Tumor Cells, Cultured

We recently detected specific high-affinity binding sites for 12(S)-HETE, the main arachidonic acid metabolite in skin, on epidermal cells. The putative receptor is involved in keratinocyte chemotaxis toward 12(S)-HETE, which points to its participation in wound healing. In an effort to further characterize the 12(S)-HETE receptor, we investigated its regulation by various cytokines. Of the tested cytokines, only interferon (IFN)-gamma led to a massive induction of the 12(S)-HETE receptors. The effect was dose and time dependent and blocked by cycloheximide. The up-regulation of 12(S)-HETE receptors by IFN-gamma may represent an amplification mechanism of the assumed role of 12(S)-HETE in skin wound repair.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Binding Sites; Carcinoma, Squamous Cell; Cell Line; Cycloheximide; Dose-Response Relationship, Drug; Epidermal Cells; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Interferon-gamma; Keratinocytes; Receptors, Cell Surface; Receptors, Eicosanoid; Skin Neoplasms; Time Factors; Up-Regulation

The arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) is assumed to play an important role in skin physiology and pathophysiology. Specifically, it has recently been discussed as a growth promoting agent in keratinocytes. Our aim was to find out whether epidermal cells possess specific receptors for 12-HETE which would mediate the effects of this eicosanoid in skin, including the putative growth stimulating activity. We could identify specific binding sites for 12(S)-HETE on the human epidermal cell line SCL-II. The analysis of binding data revealed a single class of binding sites with a Kd of 2,6 nM and a Bmax of 216,000 sites per cell. The binding was saturable, readily reversible, and specific for 12(S)-HETE with lower affinities for other monoHETE. We failed to detect any significant proliferative activity of 12(S)-HETE in the same epidermal cell line, although we applied three independent methods for evaluation of cell growth and used a concentration of 12(S)-HETE which should enable an optimal receptor occupancy. Thus, epidermal cells possess high-affinity 12(S)-HETE binding sites which are likely to be involved in the effects of this eicosanoid in epidermis. However, biologic effects other than direct growth stimulation seem to be transduced by 12(S)-HETE receptors in epidermal cells which need further investigation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Binding Sites; Carcinoma, Squamous Cell; Cell Division; Cell Line; DNA; Growth; Humans; Hydroxyeicosatetraenoic Acids; Receptors, Drug; Skin; Tumor Cells, Cultured

We identified leukotriene B4 (LTB4)/12-hydroxyeicosatetraenoic acid (12-HETE) binding sites in a squamous cell cancer-derived human epidermal cell line. Analysis of the binding data revealed a single class of binding sites with a dissociation constant of 0.16 microM and a Bmax of 3.8 x 10(6) sites per cell. Competitive binding assays with various eicosanoids at 37 degrees C showed nearly equal binding of 12(S)-HETE, 12(R)-HETE and LTB4. 5(S)-HETE and LTB4-analogs bound with lesser affinity. Specific LTB4 binding at 37 degrees C could also be demonstrated in freshly isolated normal human keratinocytes. Since lipoxygenase-derived eicosanoids are thought to play an important role in hyperproliferative and inflammatory skin diseases, the identification of LTB4/12-HETE binding sites in keratinocytes could have implications for the development of new drugs controlling these disease processes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Binding Sites; Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Leukotriene B4

There is evidence suggesting a role of eicosanoids in the growth of certain tumors. In this study, tissue samples were collected from basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin. Both BCCs and SCCs contained more prostaglandin E2 and F2 alpha (PGE2 and PGF2 alpha) than normal epidermis. In vitro incubation of tumor samples with arachidonic acid also resulted in PGE2 and PGF2 alpha formation. Basal cell carcinomas exhibiting a histologically aggressive growth pattern contained higher levels of prostaglandins than those with a nonaggressive growth pattern, both in vivo and after in vitro incubation. Lipoxygenase products (12- and 15-hydroxyeicosatetraenoic acid) were present in smaller amounts than cyclo-oxygenase products (PGE2 and PGF2 alpha) in vivo. Compared with normal epidermis, SCCs and, particularly, BCCs produced smaller amounts of 12-hydroxyeicosatetraenoic acid during in vitro incubation with arachidonic acid. The levels of lipoxygenase products were not related to the tumor growth pattern. These results indicate that excessive prostaglandin levels in BCCs may be associated with an aggressive growth pattern.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Breast Neoplasms; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Dinoprost; Dinoprostone; Head and Neck Neoplasms; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Mice; Prostaglandins; Prostaglandins E; Prostaglandins F; Rabbits; Radioimmunoassay; Skin Neoplasms

The metabolism of [14C]arachidonic acid into cyclooxygenase and lipoxygenase products by homogenates of primary squamous carcinomas of head and neck in 12 patients was studied in vitro. The lipoxygenase pathway was predominant in all samples. The major metabolites were 12-hydroxy-5,8,11-14-eicosatetraenoic acid, (12-HETE) and 15-HETE. 5-HETE, 5,12-diHETE, 8-HETE and 9-HETE were also detected. The cyclooxygenase products detected were in the following order: PGE2 greater than PGF2 alpha greater than TxB2 greater than 15-keto-PGE2 greater than 6-keto-PGF1 alpha greater than PGD2. Literature review of the biological activities of these oxygenated metabolites of arachidonic acid suggest important modulatory roles in the pathophysiology of head and neck cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Prostaglandins