12-hydroxy-5-8-10-14-eicosatetraenoic-acid and Lung-Neoplasms

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cytosol; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Lung Neoplasms; Mice; Molecular Structure; Substrate Specificity; Tumor Cells, Cultured

Immunotherapy of cancer is a burgeoning field of research since the realization that our immune system intrinsically has the capacity to restrict tumor occurrence and progression. Though strategies to maximize antitumor T-cell activation are well established, the efficacy of these therapies is limited by an insufficient knowledge of the intricate tumor microenvironment and its capacity to thwart antitumor immunity. Chen and colleagues now uncover a novel immunosuppressive pathway in non-small cell lung carcinoma. Overexpression of cytochrome P450F2 in cancer cells increases production of 20-hydroxyeicosatetraenoic acid, which instructs the expression of immunosuppressive molecules in cancer-associated fibroblasts by binding the GPR75 receptor and activating STAT3/c-Jun signaling. This work proposes several innovative therapeutic anchor points that may improve the efficacy of existing immunotherapies. See related article by Chen et al., p. 4016.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; Catalysis; Fibroblasts; Hot Temperature; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Lung Neoplasms; Phenotype; Stromal Cells; Tumor Microenvironment

12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Collagen; Disease Progression; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fibronectins; Focal Adhesion Protein-Tyrosine Kinases; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Signal Transduction; Transplantation, Heterologous; Tumor Cells, Cultured

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

A validated method is described for the simultaneous analysis of PGE2, 11-, 12-, and 5-HETEs from cultured cells using HPLC negative electrospray ionization tandem mass spectrometry (LC/MS/MS). This method permits quantification of selected individual arachidonic acid metabolites from cell extracts without derivatization, multiple purification steps, or lengthy separation times required by traditional GC-MS- or HPLC-UV -based methods. Accuracy assessments of values calculated using this method showed deviations from nominal values were < or =15%. An average relative deviation of 7% of mean calculated values was observed for values taken on separate days. The lower limit of detection for all metabolites was 1.3 pg. The method was used to quantify arachidonic acid metabolites present in various cancer cell lines after incubation with arachidonic acid and the selective cyclooxygenase-2 inhibitor celecoxib. Results showed that the presence of celecoxib in lung cancer A549 cells reduced production of both PGE2 and 11-HETE in a concentration-dependent manner.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Celecoxib; Chromatography, High Pressure Liquid; Cyclooxygenase 2; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Leukemia; Lipoxygenase; Lung Neoplasms; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Spectrometry, Mass, Electrospray Ionization; Sulfonamides; Tumor Cells, Cultured

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Neoplasm Metastasis

Cathepsins B and L are catabolic lysosomal enzymes but are likely candidates for extracellular proteolysis in normal and malignant processes. The signal mediator 12(S)-HETE selectively triggers a shot-gun release of cathepsin B. We have therefore investigated the intracellular distribution of cathepsins in unstimulated and 12(S)-HETE-stimulated tumor cells. Cathepsins B and L have only limited colocalization, which is found in the regions of synthesis and sorting (endoplasmic reticulum, Golgi, trans Golgi network). Treatment by 12(S)-HETE scatters cathepsin B but not cathepsin L and proform of cathepsin B. Colocalization with both mannose 6-phosphate receptors is very limited for both cathepsins. But extensive colocalization of cathepsin B and the endosomal/lysosomal marker CD63 (LIMP-I) documents the main fraction of the enzyme in these compartments. The supposed non-lysosomal fraction of cathepsin B is very likely the secretable material which follows a regulated secretory pathway. Storage and regulated secretion in tumor cells support extracellular proteolysis as a means in invasion which may lead to metastasis. But the mechanisms by which cells might acquire and eventually apply this means is still unknown.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antigens, CD; Biological Transport; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Endopeptidases; Endosomes; Humans; Lung Neoplasms; Lysosomes; Microscopy, Fluorescence; Microscopy, Immunoelectron; Platelet Membrane Glycoproteins; Protein Precursors; Receptor, IGF Type 2; Tetraspanin 30; Tumor Cells, Cultured

12(S)-HETE stimulates gene and cell surface expression of the integrin GPIIb/IIIa in carcinoma cells. The cells have high affinity binding sites for 12(S)-HETE. Analyses of the subcellular distribution and size of these sites showed that cytosol was the predominant location and that the apparent molecular weight was close to 670,000. Besides cytosol, mitochondria, nuclei, and plasma membranes also contained 12(S)-HETE binding sites. The mainly cytosolic location of the binding sites is different from that of other eicosanoid receptors which are G-protein coupled plasma membrane proteins of the rhodopsin gene family.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Binding Sites; Cell Fractionation; Cell Membrane; Cell Nucleus; Centrifugation, Density Gradient; Chromatography, Gel; Cytosol; Lung Neoplasms; Mice; Mitochondria; Receptors, Eicosanoid; Tumor Cells, Cultured

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Carcinosarcoma; Cell Adhesion; Cell Membrane; Cytoskeleton; Gene Expression Regulation, Neoplastic; Integrins; Lung Neoplasms; Melanoma, Experimental; Mice; Nocodazole; Organelles; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Processing, Post-Translational; Rats; Tumor Cells, Cultured

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenosine Triphosphate; Animals; Binding Sites; Cell-Free System; Cytosol; HSP70 Heat-Shock Proteins; Lipoxygenase; Lung Neoplasms; Macromolecular Substances; Mice; Substrate Specificity; Tumor Cells, Cultured

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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Carbon Radioisotopes; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Neoplasm Metastasis

Cathepsins B and L play roles in intracellular and extracellular proteolysis in normal and malignant processes. A directed extracellular proteolysis by regulated secretion could facilitate the process of invasion. We have therefore investigated the effect of the physiological signal mediator 12(S)-hydroxy-eicosatetraenoic acid on the release of cathepsins B and L in normal and malignant human lung cells. Quantitative determinations of cathepsin activities were done by flow cytometry and spectrofluorometry using synthetic dipeptidyl substrates coupled to fluorogens. Most interestingly, a difference in the secretion of cathepsins B and L was found: only release of active cathepsin B was detected. The effect was specific for 12(S)-hydroxy-eicosatetraenoic acid, 12(R)-hydroxy-eicosatetraenoic acid, and 5(S)-hydroxy-eicosatetraenoic acid were ineffective. The response was immediate but a substantial amount of nonreleasable activity remained cell bound. Alveolar macrophages, Wi-38 fibroblasts, and tumor cells derived from large cell carcinomas and adenocarcinomas were sensitive to 12(S)-hydroxy-eicosatetraenoic acid, but cells from undifferentiated squamous cell carcinomas were not. Sensitivity did not parallel malignancy but more likely the degree of differentiation of cells. The investigated tumor cell lines showed no detectable endogenous 12-lipoxy-genase activity to synthesize 12(S)-hydroxy-eicosatetraenoic acid from arachidonate; therefore, we assume a paracrine mechanism for 12(S)-hydroxy-eicosatetraenoic acid action. Protein kinase C alpha, a key enzyme involved in 12(S)-hydroxy-eicosatetraenoic acid-elicited responses, was expressed in all sensitive tumor cells, but insignificantly in a sensitive normal cell line and an insensitive tumor cell line. From our experiments we propose two separate intracellular pools of active cathepsin B: an unreleasable, lysosomal fraction and a fraction available for regulated secretion. Different processing and sorting mechanisms may be responsible for the generation of these cathepsin B-fractions in these pools.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Cathepsin B; Cathepsin L; Cathepsins; Cell Membrane; Cells, Cultured; Culture Media, Conditioned; Cysteine Endopeptidases; Cytosol; Endopeptidases; Enzyme Precursors; Fibroblasts; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Kinetics; Lung; Lung Neoplasms; Macrophages, Alveolar; Protein Kinase C; Tumor Cells, Cultured

We studied cellular interactions between human polymorphonuclear leukocytes (PMN) and lung cancer cell lines by investigating the influence of cancer cells on the production of leukotriene B4 (LTB4) and superoxide anion (O2-) by stimulated PMN. Of the nine cancer cell lines established from human lung cancers that we examined, H23 cells showed the highest LTA4 hydrolase activity. When PMN were stimulated by the calcium ionophore A23187 in the presence of H23 cells, the production of LTB4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), and 12(S)-hydroxyeicosatetraenoic acid (12-HETE) decreased in a dose-dependent manner. On the contrary, H23 did not inhibit O2- production by PMN. Two other cell lines (N417 and Q9) caused similar inhibition of LTB4 production by PMN. These three cancer cell lines alone did not generate any metabolites of the arachidonic acid (AA) lipoxygenase pathway or any O2- upon stimulation with A23187 alone. The addition of AA dose-dependently reversed the H23-induced inhibition of LTB4, 5-HETE, and 12-HETE production by PMN, suggesting inhibition at the phospholipase A2 (PLA2) level. Furthermore, addition of the cancer cell line Q9 inhibited 14C release from [14C]AA prelabeled PMN in a cell number-dependent manner in the buffer, with and without albumin. The supernatant of H23 cells also inhibited the production of LTB4 by PMN stimulated by A23187, as did the addition of H23 lysate or its 10(4) x g centrifugation supernatant. While neither the 10(5) x g supernatant (cytosol) nor the pellet (microsome) exhibited inhibitory activity, the combination of the separated cytosol and microsomal fractions restored the inhibitory activity. Furthermore, addition of the 10(4) x g supernatant of Q9 lysate to partially purified human cytosolic PLA2 inhibited PLA2 activity in a dose-dependent manner. Our results indicate that the lung cancer cell lines used in our study inhibit LTB4 production by human PMN through inhibition of phospholipase A2 activity, which may contribute to a predisposition to pulmonary infections in patients with lung cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Calcimycin; Calcium; Coculture Techniques; Cyclooxygenase Inhibitors; Dinoprostone; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Ionophores; Leukotriene B4; Lipoxygenase; Lung Neoplasms; Neutrophils; Phospholipases A; Phospholipases A2; Subcellular Fractions; Superoxides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

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

We have previously demonstrated that the metastatic potential of tumor cells can be increased by treatment with exogenous 12(S)hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid. However, the biosynthesis of the authentic lipid mediator by tumor cells, and especially the correlation of its biosynthesis to tumor cell metastatic capacity have not been characterized. In addition, a role for other mono HETEs in influencing tumor cell metastatic behavior has been suggested, but conclusive evidence is lacking. In this study, we analyzed the biosynthesis of mono HETEs from arachidonic acid in tumor cells of different metastatic ability and correlated biosynthesis to metastatic potential.. The biosynthesis of mono HETEs by low and high metastatic subpopulations of B16 amelanotic melanoma (B16a) cells was analyzed by high performance liquid chromatography (HPLC). The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry (GC/MS) and its stereochemical structure assigned by chiral phase HPLC. The effect of a lipoxygenase inhibitor on the biosynthesis of mono HETEs and its effect on metastatic behavior was examined.. HPLC analysis revealed that low (LM180) and high (HM340) metastatic B16a cells exhibited different profiles and efficiencies for conversion of arachidonic acid to mono HETEs. LM180 cells produced equal quantities of 12-HETE and 5-HETE. In contrast, HM340 cells synthesized predominantly 12-HETE and small amounts of 15-, 11- and 5-HETEs. At equal concentrations of substrate, four times more 12-HETE was synthesized by HM340 cells than by LM180 cells. The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry and chiral phase HPLC demonstrated that it was the S enantiomer. The biosynthesis of 12(S)-HETE, but not other HETEs, was significantly inhibited by a lipoxygenase inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide. N-benzyl-N-hydroxy-5-phenylpentanamide, in a dose-dependent manner, decreased the adhesion of HM340 cells to murine pulmonary microvessel endothelium in vitro and lung colony formation in vivo. Furthermore, re-introduction of 12(S)-HETE, but not other mono HETEs, to HM340 cells pretreated with N-benzyl-N-hydroxy-5-phenylpentanamide, increased their adhesion to endothelium.. Biosynthesis of 12(S)-HETE by tumor cells is a determinant of their metastatic potential and inhibition of 12(S)-HETE biosynthesis in tumor cells may be a crucial target for intervening in metastasis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Cell Adhesion; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Hydroxamic Acids; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Lung Neoplasms; Male; Melanoma, Amelanotic; Mice; Mice, Inbred C57BL; Molecular Conformation; Pentanoic Acids; Radioimmunoassay; Skin Neoplasms; Tumor Cells, Cultured

Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of tumor cell extracts identified 12(S)-HETE as the major lipoxygenase metabolite of arachidonic acid and tumor cell generation of 12(S)-HETE was specifically blocked by a select 12-lipoxygenase inhibitor N-benzyl-N-hydroxy-5-phenyl-pentamide. The identity and stereochemistry of tumor cell-derived 12-HETE was substantiated by gas chromatography-mass spectrometry analysis and chiral phase high-performance liquid chromatography, respectively. Lewis lung carcinoma cell adhesion to CD3 monolayers was accompanied by an enhanced 12(S)-HETE biosynthesis by tumor cells, which paralleled the tumor cell-induced endothelial cell retraction in a cell number-dependent manner. Pretreatment of tumor cells with N-benzyl-N-hydroxy-5-phenylpentamide inhibited both increased 12(S)-HETE biosynthesis and tumor cell-induced endothelial cell retraction. Highly metastatic variants of elutriated B16a cells which had been shown to produce large quantities of 12(S)-HETE induced significant CD3 cell retraction, while low metastatic subpopulations of B16a cells which synthesized no or little 12(S)-HETE did not induce endothelial cell retraction. These results suggest that 12(S)-HETE synthesis during tumor cell-endothelial cell interactions may represent a key contributory factor in cancer metastasis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amides; Animals; Benzylamines; Carcinoma; Cell Adhesion; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Tumor Cells, Cultured

We recently reported that the Ca(2+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), was involved in rat Walker carcinosarcoma cell adhesion to large-vessel endothelium. We extended our studies to explore the role of this kinase in the adhesion to small-vessel endothelium and lung colonization of murine B16 amelanotic melanoma (B16a). Subpopulations of B16a cells, which differ in lung-colonization potentials, were isolated by centrifugal elutriation from solid tumors. In this study, we demonstrate that cells from a high metastatic sub-population (HM340), when compared with cells from a low metastatic sub-population (LM180), exhibit elevated levels of total cellular as well as membrane-bound PKC. The increase in PKC in cells from the HM340 correlates positively to their increased ability to adhere to murine pulmonary-microvessel endothelial-cell monolayer, and to form pulmonary colonies in syngeneic mice. Calphostin C, a potent and selective PKC inhibitor, decreases in a dose-dependent manner the adhesion to endothelium and the lung colonization of cells from both the low and the high metastatic sub-populations with IC50 at sub-micromolar concentrations. In conclusion, our results suggest that PKC may be a key element in regulating tumor-cell metastasis and that PKC inhibitors may be anti-metastatic agents.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cell Adhesion; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Melanoma, Experimental; Mice; Naphthalenes; Polycyclic Compounds; Protein Kinase C

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) enhances tumor cell adhesion to endothelial cells [Honn et al. (1988) Proc. Soc. Exp. Biol. Med. 189, 130-135]. The effect is correlated to surface expression of an integrin receptor, GpIIb/IIIa. Here, we describe evidence for high-affinity binding of 12(S)-HETE to Lewis lung carcinoma cells. Scatchard plot analyses indicated a single class of sites with apparent Kd and Bmax values of 0.44 nM and 66,000 sites per cell, respectively. Competition experiments with unlabeled compounds shod d that the binding was reversible and saturable as well as stereo- and regiospecific. The 12(S)-HETE binding, demonstrated here, might be an important step in a series of events controlling surface expression of integrin receptors.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Binding Sites; Binding, Competitive; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Lung Neoplasms; Tumor Cells, Cultured

Lewis lung carcinoma cells express a plasma membrane receptor (i.e., IRGpIIb/IIIa) which is immunologically and functionally related to the platelet aggregation receptor complex (i.e., GpIIb/IIIa). Both fluorescence microscopy and flow cytometric analysis reveal that surface expression and/or activation of this tumor cell receptor is enhanced by a phorbol ester [i.e., 12-O-tetradecanoylphorbol-13-acetate (TPA)] and a lipoxygenase metabolite of arachidonic acid; 12-hydroxyeicosatetraenoic acid (i.e., 12-HETE). TPA-enhanced expression appears to be mediated by a lipoxygenase metabolite, as this effect can be reversed by lipoxygenase inhibitors but not by cyclooxygenase inhibitors. In parallel with these results both TPA and 12(S)-HETE [but not 12(R)-HETE] enhance tumor cell adhesion to endothelial cells, subendothelial matrix and fibronectin, but not to type IV collagen. TPA-enhanced adhesion can be reduced by lipoxygenase inhibitors but not by cyclooxygenase inhibitors and in addition, stimulated adhesion can be blocked by pretreatment of tumor cells with specific polyclonal or monoclonal antibodies which react against IRGpIIb/IIIa. 12(S)-HETE-enhanced adhesion can also be inhibited by these same antibodies. In contrast, a lipoxygenase product of linoleic acid, 13(S)-hydroxyoctadecadienoic acid, inhibited TPA and 12(S)-HETE-enhanced tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin. These results suggest that (a) IRGpIIb/IIIa is a multifunctional receptor which mediates tumor cell adhesion to a variety of biological substrata, (b) TPA enhances surface expression and/or activation of this receptor possibly via a lipoxygenase metabolite of arachidonic acid, and (c) these effects are opposed by a lipoxygenase metabolite of linoleic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antibodies; Antibodies, Monoclonal; Antigen-Antibody Complex; Arachidonic Acids; Cell Adhesion; Cell Membrane; Collagen; Endothelium, Vascular; Flow Cytometry; Fluorescent Antibody Technique; Hydroxyeicosatetraenoic Acids; Isomerism; Linoleic Acid; Linoleic Acids; Lipoxygenase; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Platelet Membrane Glycoproteins; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate