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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Cathepsin B; Cell Adhesion; Cell Adhesion Molecules; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glucose-6-Phosphate Isomerase; Humans; Linoleic Acids; Melanoma; Melanoma, Experimental; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Receptors, Autocrine Motility Factor; Receptors, Cytokine; Receptors, Vitronectin; Signal Transduction; Ubiquitin-Protein Ligases

The arachidonic acid metabolite of 12 lipoxygenase, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) promotes metastatic behavior of tumor cells (1). In this study we set out to identify 12(S)-HETE stimulated signaling pathways, and their contribution to cellular functions in A431 epidermoid carcinoma. 1) 12(S)-HETE signaling involves extracellular-regulated protein kinase (ERK1/2), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3 kinase) and Src kinase. 2) 12(S)-HETE stimulates cell migration on laminin, which is eliminated by PKC and PI3 kinase inhibitors, reduced by 50% with Src inhibitor, but unaffected by inhibition of ERK1/2. 3) 12(S)-HETE stimulated spreading on fibronectin relies on ERK1/2 and PI3 kinase activities, but not on PKC or Src. 4) Focal adhesion kinase, a key organizer of focal adhesions, is tyrosine phosphorylated in response of 12(S)-HETE treatment, which requires Src, but not PKC, PI3 kinase or ERK1/2 activity. 5) Inhibition of 12 lipoxygenase leads to apoptosis in serum starved A431 cells. 12(S)-HETE stimulated p90Rsk and Akt, key players in an ERK and a PI3 kinase (respectively) dependent anti apoptotic pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Carbazoles; Enzyme Inhibitors; Humans; Indoles; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Signal Transduction; Tumor Cells, Cultured

Megakaryocytic genes such as alphaIIbbeta3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed alphaIIbbeta3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of alphaIIbbeta3, PAC-1 and the physiological ligand, fibrinogen. Ligation of alphaIIbbeta3 induced down-regulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces approximately 50% increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of alphaIIbbeta3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon alphaIIbbeta3 ligation paralleled by downregulation of p125FAK and phoshotyrosine+ adhesion plaques and translocation of PKCalpha to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the alphaIIbbeta3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligation-induced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic alphaIIbbeta3 integrin may partially explain its role in tumor progression.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antibodies, Monoclonal; Arachidonate 12-Lipoxygenase; Cell Adhesion; Enzyme Inhibitors; Fibrinogen; Flow Cytometry; Melanoma; Mice; Microscopy, Confocal; Phosphorylation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Serine; Signal Transduction; Tumor Cells, Cultured

12-lipoxygenase (12-LOX) expression and function in the regulation of the metastatic phenotype was demonstrated in several murine melanoma lines before. Here we have provided novel evidences that, though at a low level (in max. 15% of the cell population), human melanoma lines (HT168, M1, HT199, HT18 and WM35) express the platelet-type isoform of 12-LOX both at mRNA and protein levels. 12-LOX expression was demonstrated in cultured tumor cells and in skin tumor xenografts. Comparison of the expression of 12-LOX in skin primary tumors and its lung metastases indicated a stable expression. The low level of 12-LOX expression in human melanoma cell lines suggests that other lipoxygenase(s) could also be responsible for the metabolism of arachidonic acid to 12-HETE breakdown products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Animals, Newborn; Arachidonate 12-Lipoxygenase; Gene Expression; Humans; Melanoma; Mice; Mice, SCID; Neoplasm Transplantation; Rats; RNA, Messenger; RNA, Neoplasm; Transplantation, Heterologous; Tumor Cells, Cultured

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid, has been shown to be involved in a wide variety of cellular activities (i.e., adhesion, spreading, motility, invasion) which promote metastasis to occur in tumor cells. In this study, several techniques (Western blotting, flow cytometry and DNase I assay) were performed to examine the alterations in the distribution of G- and F-actin expressed in B16a melanoma cells. Each of these methods independently revealed that 12(S)-HETE treatment (0.1 mM, 15 min) resulted in an increase in the F-actin content in the cytoskeletal preparations. Since the integrity of cytoskeletal networks (i.e., actin filaments) can be dynamically regulated through protein phosphorylation, we investigated the potential role of several protein kinases in the 12(S)-HETE-induced actin polymerization. By flow cytometric analysis, 12(S)-HETE was found to increase the actin filament contents. This effect could be inhibited by protein kinase C (PKC) inhibitors (calphostin C and staurosporine) as well as by protein tyrosine kinase (PTK) inhibitor (genistein) but not by protein kinase A inhibitor (H8), suggesting that the 12(S)-HETE effect involves PKC and PTK. This conclusion is consistent with the observations that phorbol 12-myristate-13-acetate (PMA) mimics the biological effect of 12(S)-HETE in promoting the F-actin formation in B16a cells. As a final analysis, direct protein phosphorylation studies indicate that 12(S)-HETE treatment led to enhanced phosphorylation of myosin light chain, which may contribute to the increased stress fiber formation following 12(S)-HETE stimulation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Actin Cytoskeleton; Actins; Animals; Dose-Response Relationship, Drug; Melanoma; Mice; Myosin Light Chains; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Integrins play an important role in mediating tumor cell-extracellular matrix (ECM) and tumor cell-endothelial cell interactions. The integrin alphaIIb beta3 (GPIIb-IIIa) is expressed on the surface of platelets in an inactive state and requires a conformational change to recognize extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and others. In this study, we questioned whether human melanoma cells express the alphaIIb beta3 integrin. Reverse transcription-PCR/Southern blotting, Northern blotting, and dot blotting demonstrated the presence of the platelet-type alphaIIb beta3 integrin in human melanoma WM 983B, WM 983A, and WM 35 cells. AP-2, a monoclonal antibody (mAb) to alphaIIb beta3, positively stained two human melanoma specimens, indicating expression of this integrin in vivo. Phorbol 12-myristate 13-acetate and 12(S)-hydroxyeicosatetraenoic acid, two activators of protein kinase C, stimulated adhesion of melanoma cells to immobilized fibronectin and PAC-1, a mAb to alphaIIb beta3. PAC-1 specifically recognizes the conformationally active form of platelet alphaIIb beta3. Phorbol 12-myristate 13-acetate-stimulated adhesion of WM 983B cells to PAC-1 was completely blocked by an RGD peptide, thus providing evidence that tumor cell adhesion to PAC-1 is mediated via the alphaIIb beta3 integrin but not the Fc receptor. Confocal immunofluorescent studies demonstrated that fibronectin-adherent melanoma cells possess an intracellularly localized pool of high-affinity alphaIIb beta3. Invasion of WM 983B cells through fibronectin was stimulated by 12(S)-hydroxyeicosatetraenoic acid, and this stimulated invasion was blocked by the mAb PAC-1. The data suggest that melanoma cells express the high-affinity alphaIIb beta3 integrin, which is involved in tumor invasion.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Antibodies, Monoclonal; Blotting, Northern; Blotting, Southern; Cell Adhesion; Epitopes; Female; Fibronectins; Fluorescent Antibody Technique, Indirect; Humans; Immunohistochemistry; Melanoma; Microscopy, Confocal; Neoplasm Invasiveness; Oligopeptides; Platelet Glycoprotein GPIIb-IIIa Complex; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A M(r) 55,000 tumor cell-secreted cytokine has been described which influenced the migration of the producing cells and was called autocrine motility factor (AMF). Activation of the cell surface receptor for AMF (gp78) was shown to stimulate production of a 12-lipoxygenase metabolite of arachidonic acid, 12-(S)-hydroxyeicosatetraenoic acid [12-(S)-HETE], in highly metastatic murine melanoma cells. AMF stimulated the motility of the high-metastatic (K1735-M1) but not the low-metastatic variant (K1735-Cl.11) of the K1735 murine melanoma and increased expression of the 12-lipoxygenase enzyme predominantly in the high-metastatic counterpart. The K1735-M1 cells responded to motile stimulation with increased endogenous 12-(S)-HETE production, and, reciprocally, exogenous 12-(S)-HETE up-regulated surface gp78 and caused gp78 translocation from an intracellular perinuclear pool to tubulovesicles which extended to the cell periphery in the K1735-M1 cells exclusively. These results suggest that differences in AMF responses may be due to alterations in the capacity of low-metastatic cells to transduce signals through 12-lipoxygenase or to involve downstream effector(s) of 12-(S)-HETE after gp78 activation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Cell Movement; Glucose-6-Phosphate Isomerase; Humans; Hydroxyeicosatetraenoic Acids; Melanoma; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Autocrine Motility Factor; Receptors, Cytokine; Signal Transduction; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

Several eicosanoids were tested for ability to inhibit proliferation of cells in culture. In rabbit aortic smooth muscle cells and mouse B16BL6 melanoma cells, order of potency was: 12-HETE greater than PGJ2 greater than PGA1 greater than or equal to PGE1 greater than PGE2 greater than or equal to PGD2 greater than or equal to PGA2. PGB1 was active in smooth muscle cells (greater than PGD2) but not in B16 cells. 5-HETE and Leukotriene B4 were weakly active in smooth muscle cells, and PGB2, PGF2 alpha and TXB2 were inactive in both cells types. In Swiss albino mouse 3T3 fibroblasts, PGJ2 and PGE1 showed much lower relative potency than in the other two cell lines, although the profile was otherwise similar. These findings may be relevant to the anti-atherosclerotic (and perhaps anti-tumor activity) of some eicosanoids.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arteriosclerosis; Cell Division; Cells, Cultured; Fibroblasts; Hydroxyeicosatetraenoic Acids; Melanoma; Mice; Muscle, Smooth, Vascular; Prostaglandins; Rabbits; Thromboxanes