Cis-polyunsaturated fatty acids stimulate beta1 integrin-mediated adhesion of human breast carcinoma cells to type IV collagen by activating protein kinases C-epsilon and -mu.

We have investigated the effects of various fatty acids (FAs) on integrin-mediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects required cis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCepsilon and PKCmu, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCalpha, as well as the atypical isozymes, PKCzeta and PKCiota, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for alpha1, alpha2, and beta1, integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to alpha3 and alpha5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these beta1, integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of beta1 integrins.

Arachidonic acid activates mitogen-activated protein (MAP) kinase-activated protein kinase 2 and mediates adhesion of a human breast carcinoma cell line to collagen type IV through a p38 MAP kinase-dependent pathway.

Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid-stimulated adhesion; this was demonstrated by the observation that 12-O-tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2)-terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV.

The role of protein glycosylation inhibitors in the prevention of metastasis and therapy of cancer.

Oligosaccharide moieties of cell-surface glycoproteins are thought to be involved in recognition events during cancer metastasis and invasion. Swainsonine, an inhibitor of the Golgi alpha-mannosidase II, has been shown to block pulmonary colonization by tumor cells and stimulate components of the immune system. Swainsonine also abrogates much of the toxicity of chemotherapeutic agents and stimulates bone marrow hematopoietic progenitor cells, suggesting additional therapeutic applications. We are currently characterizing the ability of swainsonine to modify cell growth in human and murine bone marrow progenitor cells. Furthermore, we are examining crucial steps in metastasis that depend upon cell surface molecules that play a role in cell-matrix interactions. Our work shows that tumor cell adhesion to collagen IV in vitro is rapidly stimulated by cis-polyunsaturated fatty acids and is dependent on protein kinase C activity. We are investigating the hypothesis that integrins are critical components of this adhesion and are examining potential signal transduction pathways that lead to the modulation of cell adhesion.

Characteristics of leukotriene biosynthesis by human granulocytes in presence of plasma.

The formation of leukotriene B4 (LTB4) by neutrophils stimulated with the ionophore A23187 or physiological stimuli in heparinized plasma was investigated. In comparison with neutrophils stimulated (A23187) in a protein-free buffered salt solution, neutrophils stimulated in plasma produced only trace amounts of LTB4. The addition of human recombinant LTA4-hydrolase or erythrocytes to plasma prior to A23187 stimulation strongly and selectively stimulated (> 4-fold) the formation of LTB4 supporting that neutrophils activated in plasma with A23187 release in the extracellular milieu most of LTA4 formed by the cells, and indicating that plasma proteins drastically slow down the further metabolism of LTA4 released by neutrophils. The formation of LTB4 was then investigated in GM-CSF-primed neutrophils stimulated with fMLP in plasma; levels of synthesis were very low and the addition of erythrocytes prior to stimulation strongly enhanced LTB4 synthesis, demonstrating that agonist-stimulated neutrophils also release most of LTA4 generated in the extracellular milieu. Investigations on the fate of LTA4 in plasma revealed that LTA4 was slowly degraded through an unknown process, i.e. not through the previously described non-enzymic hydrolysis resulting in the formation of dihydroxy derivatives of LTA4. Using neutrophils labeled with tritiated arachidonate, we also demonstrated that neutrophils stimulated in plasma with fMLP or A23187, almost exclusively use endogenous arachidonate, as opposed to plasma arachidonate, to generate 5-lipoxygenase products. Finally, experiments performed with purified eosinophils indicated that contrary to neutrophils, the eosinophils do not release LTA4, but directly release LTC4.

Regulation of the adhesion of a human breast carcinoma cell line to type IV collagen and vitronectin: roles for lipoxygenase and protein kinase C.

We have investigated the regulation of adhesion of metastatic human breast carcinoma cells to various protein substrates in the presence or absence of the protein kinase C (PKC) activator, 12-tetradecanoyl phorbol 13-acetate (TPA) or calcium ionophore A23187 (A23187). Both TPA and A23187 dramatically enhanced MDA-MB-435 cell adhesion to type IV collagen (collagen IV), vitronectin, and, to some extent, fibronectin and laminin. Adhesion to BSA and polylysine were not affected. TPA and A23187 induced substantial dose-dependent effects that were apparent after 30- and 60-min incubations, respectively, whereas a phorbol ester, which does not activate PKC, had no effect. A23187, but not TPA, induced a release of arachidonic acid (AA) from MDA-MB-435 cells. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, prevented A23187 and exogenous AA, but not TPA, from stimulating cell adhesion to collagen IV. In contrast, the increase in adhesion to vitronectin induced by A23187 and AA was, at best, only partially inhibited by nordihydroguaiaretic acid treatment. Calphostin C, a PKC inhibitor, blocked the stimulation of adhesion by A23187, exogenous AA, and TPA to both collagen IV and vitronectin. Together, these results suggest that calcium mobilization activates the release of AA and its metabolism through a lipoxygenase pathway leading to a rapid increase of MDA-MB-435 cell adhesion to collagen IV, whereas other mechanisms regulate adhesion to vitronectin. Finally, PKC activation, occurring downstream from calcium mobilization or the AA effects, is a key event involved in the regulation of adhesion to both proteins.

Priming for the synthesis of 5-lipoxygenase products in human blood ex vivo by human granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

BACKGROUND: Previous studies reported the priming effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF alpha) on leukotriene synthesis by isolated polymorphonuclear leukocytes; however, little is known as of yet of these biologic effects of the two cytokines in a physiologic environment. EXPERIMENTAL DESIGN: In this study, we investigate the effects of GM-CSF and TNF alpha on the synthesis of 5-lipoxygenase (5-LO) products in heparinized blood stimulated ex vivo, using reverse phase high performance liquid chromatography analysis of deproteinized plasma samples. RESULTS: Stimulation of blood with f-Met-Leu-Phe resulted in the accumulation of up to 30 pmol of 5-LO products/ml of plasma. Preincubation of blood with 100 pM GM-CSF or 1.2 nM (200 units/ml) TNF alpha for 30 minutes at 37 degrees C before stimulation with f-Met-Leu-Phe resulted in a marked enhancement (> 5-fold) of the synthesis of leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid, which were formed in equivalent amounts. GM-CSF and TNF alpha priming activities were detectable at concentrations as low as 3 pM and 6 pM (1 unit/ml), respectively. The preincubation times required for optimal priming by GM-CSF and TNF alpha were different (40 and 10 minutes, respectively), and the effects of the two cytokines on leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid synthesis were additive, suggesting different priming mechanisms. The synthesis of 5-LO products in primed blood was also induced by platelet-activating factor, the complement fragment C5a, the particulate stimulus zymosan, and the ionophore A23187, but not by interleukin-8. Polymorphonuclear leukocytes and mononuclear cells accounted for 80% and 20% of the synthesis of 5-LO products, respectively. CONCLUSIONS: These data demonstrate that GM-CSF and TNF alpha exert very potent priming effects on the biosynthesis of 5-LO products in whole blood stimulated by various stimuli and strongly support that these cytokines could be important modulators of lipid mediator synthesis in physiologic and pathophysiologic conditions.

Reverse-phase high-performance liquid chromatography analysis of arachidonic acid metabolites in plasma after stimulation of whole blood ex vivo.

Following the stimulation of heparinized blood ex vivo, aliquots of plasma were denatured with organic solvents containing the internal standards prostaglandin (PG) B2 and 19-hydroxy-PGB2. Precipitated material was removed by centrifugation and the supernatants were directly analyzed by reverse-phase HPLC with on-line extraction and uv detection. Stimulation of blood with A23187 lead to the formation of both leukocyte and platelet arachidonic acid metabolites as the 5-lipoxygenase products leukotriene (LT) B4, 5-hydroxy-eicosatetraenoic acid (5-HETE), 20-hydroxy-LTB4 and 20-carboxy-LTB4, the 12-lipoxygenase product 12-HETE, and the cyclooxygenase product 12-hydroxy-heptadecatrienoic acid (HHTrE) were detected in plasma; in some plasma samples LTC4 and/or LTE4 were also detected. Stimulation of blood with zymosan lead to the synthesis of LTB4 and 5-HETE as major products and of 12-HETE. Recoveries of 20-hydroxy-LTB4, LTB4, 5-HETE, 12-HETE, and HHTrE added to plasma were high (> or = 90%) while those of LTC4 and LTE4 were lower (50-70%); however, washing of the precipitated protein pellet resulted in > 90% recoveries for all metabolites including the cysteinyl-leukotrienes. Levels of arachidonic acid metabolites in native plasma samples stored at -20 degrees C were stable for at least 28 days, while significant loss of material was observed over the same period of time in denatured plasma samples. Finally, we made the critical observation that the capacity for A23187- (but not zymosan-, ionomycin-, or LPS and FMLP-) induced arachidonic acid metabolite synthesis in blood decreased by 80% within 1 h of blood collection.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen.

Stimulation of heparinized blood with 1 microM formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in the formation of < 30 pmol/ml plasma of 5-lipoxygenase (5-LO) products. The preincubation of blood with 1 microgram/ml of lipopolysaccharide (LPS) (Escherichia coli 0111-B4) for 30 min before stimulation with FMLP resulted in the accumulation of 250-300 pmol of 5-LO products per ml plasma. The major products detected were leukotriene B4 and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid which were produced in equivalent amounts. The priming activity was detectable with as little as 1-10 ng LPS per ml blood and was optimal using 1-10 micrograms LPS/ml blood. The priming for 5-LO product synthesis was optimal after 20-30 min of preincubation with LPS and declined at preincubation times > 30 min. The priming effect of LPS was also observed using the complement fragment C5a or interleukin 8 as agonists. Polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells accounted for 80 and 20% of the synthesis of 5-LO products, respectively. The ability of LPS to prime isolated PMN was dependent on the presence of plasma and was inhibited by the anti-CD14 antibody IOM2, indicating a CD14-dependent priming mechanism. The priming of whole blood with tumor necrosis factor alpha (TNF-alpha) and LPS was additive and the presence of mononuclear cells did not enhance the ability of LPS to prime PMN, indicating that the priming activity of LPS is independent of LPS-induced TNF-alpha synthesis. The mechanism by which LPS enhance 5-LO product synthesis in PMN was investigated. Treatment of PMN with LPS strongly enhanced the release of arachidonic acid after stimulation with FMLP. The release of arachidonic acid was optimal 2-3 min after stimulation with FMLP, attaining levels 5-15-fold greater than those observed in unprimed cells stimulated with FMLP. These results demonstrate that LPS dramatically increases the ability of blood to generate 5-LO products, and support the putative role of leukotrienes in pathological states involving LPS.

Human platelets release a paf-acether: acetylhydrolase similar to that in plasma.

Intact washed human platelets aggregated in response to paf-acether (paf) and did not metabolize [3H]paf at concentrations up to 10 nM. However, when platelets were lysed by exposure to pH 9.5, resulting in 37.5 +/- 2.5% (mean +/- SD, n = 3) lactic dehydrogenase (LDH) release, 20.5 +/- 5.7% of the radioactivity was detected as labeled lyso paf and 5.7 +/- 3.1% as labeled alkylacylglycerophosphocholine. When platelets were aggregated with 0.5 IU/mL thrombin or high concentrations of paf (100 nM), they released a part of their acetylhydrolase without releasing LDH. In supernatants obtained from aggregated platelets, 21 +/- 2% or 10 +/- 2% (n = 3), respectively, of the total platelet acetylhydrolase activity was detected vs. none in supernatants of resting cells. The release of acetylhydrolase was concentration- and time-dependent and paralleled the release of PF 4, a marker for alpha-granules. The acetylhydrolase affinity for paf (Km) measured in sonicates of resting and thrombin-activated platelets was 8.3 +/- 1.5 microM vs. 10.6 +/- 1.5 microM, n = 5, n.s. in a "Mann Whitney" test. The latter Km was slightly but significantly different (P < 0.05, n = 5) from that of the thrombin-released acetylhydrolase (7.9 +/- 1.5 microM) and that of the latter was itself different from plasma acetylhydrolase (5.3 +/- 0.5, P < 0.05, n = 5). Addition of plasma (acid-treated to inactivate acetylhydrolase) decreased the Km value of supernatant acetylhydrolase to 6.1 +/- 1.4 microM. All preparations of acetylhydrolase exhibited similar pH requirements and sensitivity to various inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin-activated platelets promote leukotriene B4 synthesis in polymorphonuclear leucocytes stimulated by physiological agonists.

1. The addition of 2 x 10(8) human platelets to 8 x 10(6) polymorphonuclear leucocytes (PMNL) incubated in presence of 2.5 u ml-1 thrombin and 0.1 microM N-formyl-Met-Leu-Phe (FMLP) (or C5a or PAF) led to enhancement of leukotriene B4 (LTB4) synthesis by the PMNL (measured by h.p.l.c. as 20-hydroxy- and 20-carboxy-LTB4) from 4 +/- 1 pmol (in absence of platelets) to 26 +/- 4 pmol (mean +/- s.e.mean, n = 9). Platelets and thrombin were both essential for the enhancement of LTB4 synthesis. 2. Platelets also caused enhancement of LTB4 synthesis from (30 +/- 12 to 134 +/- 25 pmol, n = 6) when PMNL pretreated with granulocyte-macrophage colony-stimulating factor were used in similar experiments. 3. Enhancement of LTB4 synthesis was also observed (from 5 +/- 1.5 to 26.5 +/- 5 pmol, n = 9) when the supernatants of thrombin-activated platelet suspensions were added to FMLP-stimulated PMNL. 4. Supernatants of platelet suspensions activated by thrombin in presence of cyclo-oxygenase and 12-lipoxygenase inhibitors led to greater enhancement (from 5 +/- 3 to 153.5 +/- 27.5 pmol, n = 3) of LTB4 synthesis by FMLP-stimulated PMNL, suggesting that arachidonic acid itself, rather than its metabolites was responsible for the effects of platelets. 5. Addition of arachidonic acid to FMLP-stimulated PMNL at a concentration comparable to that measured in thrombin-activated platelet supernatants (0.2 +/- 0.025 microM, n = 6) mimicked the effect of platelets or platelet supernatants on LTB4 synthesis in FMLP-activated PMNL. 6. The present data indicate that under conditions of cell activation by physiological agonists, platelets can significantly increase the formation of the proinflammatory compound LTB4 in PMNL by providing arachidonic acid. These data lend support to the concept that platelet-PMNL interactions could modulate the inflammatory process.

Biosynthesis of paf-acether. XIV. Paf-acether output in murine peritoneal macrophages is regulated by the level of acetylhydrolase.

Paf-acether (paf) synthesis was previously shown to be impaired in 24 hr-adherent and Bacillus Calmette-Guérin-activated murine peritoneal macrophages as compared to resident macrophages. We report here that the induction of acetylhydrolase was responsible for the decreased paf output in 24 hr-adherent macrophages. The kinetic analysis of the enzymes derived from 2 hr-, 24 hr- and BCG-activated adherent macrophages and from plasma revealed that the Km for paf was similar whatever the source of the acetylhydrolase whereas the Vmax was five-fold increased in 24 hr-cultured macrophages. The acetylhydrolase activity was Ca2+-independent and was not inhibited by addition of alkyl-acyl (long chain)-glycero-phosphocholine suggesting that the enzyme was not a phospholipase A2.