Galectins, a family of soluble β-galactoside-binding proteins, are involved in the regulation of various cellular functions, which are essential for adaptive cellular stress responses (CSRs). Although expression patterns of galectins and galectin-binding glycans change during tissue development and cancer, the requirement and role of galectin networks in the CSRs are not completely understood. In this study, we report that the treatment of human promyelocytic HL-60 cells with stimuli mimicking hypoxia (CoCl2), inducing the endoplasmic reticulum stress (tunicamycin), and stimulating cell differentiation, result in stress-specific differential expression of galectin transcripts. In addition, we show that CoCl2 increases the expression of cell surface glycans recognized by both β-galactoside- and GlcNAc-binding lectins. Thus, microenvironmental stress changes the glycobiological status of cells representing expression profiles of endogenous lectins and corresponding glycans. These findings introduce a novel classification of galectins in HL-60 cells, which suggests diverse functions of galectin members in CSRs.

Halogenated lipids formed in the reactions with myeloperoxidase (MPO)-derived species may contribute to the regulation of the functional activity of cells. In the present study we have investigated the effects of chloro- and bromohydrins formed in the HOCl and HOBr reactions, respectively, with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on three different functional responses of human neutrophils: H2O2 generation, degranulation (MPO exocytosis), and cell aggregation. It was shown that POPC chloro- and bromohydrins (POPC-Cl and POPC-Br) induced the priming of neutrophils, resulting in significant upregulation of cell responses to neutrophil stimulators such as N-formyl-Met-Leu-Phe and lectin from Solanum tuberosum. The stimulating effects of POPC-Cl and POPC-Br were observed at low micromolar concentrations (liposomal concentration of POPC, 0.5–5 μM; the content of POPC-Cl or POPC-Br, 38 ± 3% of total lipids) after a short exposure (about 5 min) of the neutrophils to POPC-Cl or POPC-Br. These results suggest that halogenated lipids formed in vivo via MPO-dependent reactions may be considered as a new class of biologically active substances that are potentially able to contribute to the priming of myeloid cells in the sites of inflammation and serve as inflammatory response modulators.

The GlcNAc-specific lectin from Solanum tuberosum is shown to induce haptenic-sugar-resistant contacts in platelet aggregation but not to induce stable neutrophil and lymphocyte aggregation. The formation of such contacts in platelets was significantly hindered by the inhibitors of cAMP phosphodiesterase (papaverine) or arachidonic acid metabolism (indomethacin, aristolochic acid, or MK-886) and by a sulfhydryl reagent (N-ethylmaleimide). This lectin can be useful in studying the mechanisms of stable platelet aggregation, drug screening for antithrombotic activity, and developing the cell engineering techniques.

Vascular endothelial growth factor C (VEGF-C) is a lymphangiogenic factor over-expressed in highly metastatic, cyclooxygenase (COX)-2 expressing breast cancer cells. We tested the hypothesis that tumour-derived VEGF-C may play an autocrine role in metastasis by promoting cellular motility through one or more VEGF-C-binding receptors VEGFR-2, VEGFR-3, neuropilin (NRP)-1, NRP-2, and integrin α9β1. We investigated the expression of these receptors in several breast cancer cell lines (MDA-MB-231, Hs578T, SK-BR-3, T-47D, and MCF7) and their possible requirement in migration of two VEGF-C-secreting, highly metastatic lines MDA-MB-231 and Hs578T. While cell lines varied significantly in their expression of above VEGF-C receptors, migratory activity of MDA-MB-231 and Hs578T cells was linked to one or more of these receptors. Depletion of endogenous VEGF-C by treatments with a neutralising antibody, VEGF-C siRNA or inhibitors of Src, EGFR/Her2/neu and p38 MAP kinases which inhibited VEGF-C production, inhibited cellular migration, indicating the requirement of VEGF-C for migratory function. Migration was differentially attenuated by blocking or downregulation of different VEGF-C receptors, for example treatment with a VEGFR-2 tyrosine kinase inhibitor, NRP-1 and NRP-2 siRNA or α9β1 integrin antibody, indicating the participation of one or more of the receptors in cell motility. This novel role of tumour-derived VEGF-C indicates that breast cancer metastasis can be promoted by coordinated stimulation of lymphangiogenesis and enhanced migratory activity of breast cancer cells.

It has been demonstrated for the first time that GlcNAc-specific lectin from Solanum tuberosum induces the formation of haptenic sugar-resistant intercellular contacts (HSR-contacts) in platelet aggregation and does not induce stable neutrophil and lymphocyte aggregation. The formation of HSR-contacts in platelets was significantly impaired by the inhibitors of cAMP phosphodiesterase (papaverine) and arachidonic acid methabolism (indomethacin, aristolochic acid, and MK-886) as well as by the sulfhydryl reagent N-ethylmaleimide. The results obtained indicate that STA can be used to study the mechanisms of stable platelet aggregation, to screen drugs with potential antithrombotic activity, and to develop new cell engineering techniques.

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (EP) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. EP1 as well as EP4 receptor antagonists inhibited VEGF-C production indicating the roles of EP1 and EP4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by EP1/EP4 receptors.

Context: The root cause of preeclampsia in the human lies in the placenta, where a subpopulation of cytotrophoblast cells called extravillous trophoblasts (EVT), known to be involved in the invasion of the uterine endometrium and utero-placental arteries, become less invasive, resulting in poor perfusion of maternal blood into placenta.Objectives: Because EVT migrate into the prostaglandin (PG) E-2-rich decidua, we tested the roles of PGE(2) and PGE(2)-mediated signaling in EVT migration, using our well-characterized EVT line HTR-8/Svneo as well as first trimester villus explants in culture.Design: mRNA expression of different PGE(2) receptors (EPs) in HTR-8/Svneo cells was studied using RT-PCR. To characterize the functional significance of EP receptors in EVT, different EP receptor agonists and antagonists were used in our migration assay systems and in the measurements of intracellular concentration of Ca2+ ([Ca2+](i)) and calpain activity.Results: Exogenous PGE(2) stimulated EVT migration both in vitro and in the villus explant cultures. Although EVT expressed mRNA for all EP receptors (EP 1-4), a functional predominance of EP1 and EP4 was demonstrated in migration assays using specific EP agonists and antagonists. EP1-receptor-mediated signaling events such as activation of phospholipase C and elevation of cytosolic free [Ca2+](i) were confirmed by the following findings: 1) exogenous PGE2 or an EP1 agonist, but not an EP4 agonist, increased [Ca2+](i), which could be blocked with an EP1 antagonist as well as BAPTA and thapsigargin; 2) phospholipase C inhibitor U73122, BAPTA, and thapsigargin inhibited PGE(2)-mediated migratory response of EVT; and 3) PGE(2)-mediated EVT migration was shown to be dependent on a class of Ca2+-dependent proteases called calpains, known to be involved in cell detachment from substratum during migratory responses. The presence of PGE2 stimulated calpain activity, whereas two calpain inhibitors, calpastatin and N-Ac-Leu-Leu-methioninal (ALLM), blocked EVT migration.Conclusion: PGE(2) stimulates EVT migration by signaling through EP1 receptors, increasing [Ca2+](i), and activating calpain.

We report here that endogenous prostaglandin E2 (PGE2) resulting from cyclooxygenase (COX)‐2 expression in a highly metastatic murine breast cancer cell line C3L5 upregulates IFN‐γ + LPS‐induced nitric oxide (NO) synthase (iNOS) expression and NO production. This action of PGE2 is mediated through the EP4 receptor in a cAMP‐dependent manner. Both nonselective and selective COX‐2 inhibitors suppressed IFN‐γ + LPS‐induced NO production, which was largely restored by exogenous PGE2 or EP4 receptor agonist PGE1 alcohol. EP4 antagonist AH‐23848B inhibited NO production with a concomitant downregulation of iNOS mRNA in IFN‐γ + LPS‐stimulated cells. cAMP dependence of NO production by cells under inducible conditions was demonstrated by the use of known modulators of intracellular cAMP. Since both COX‐2 and iNOS are implicated in breast cancer progression, our findings of EP4 receptor‐mediated upregulation of iNOS in COX‐2‐expressing breast cancer cells suggest that blocking COX‐2 and/or EP4 may provide a simple therapeutic modality in this tumor model. © 2003 Wiley‐Liss, Inc.

To assess whether cancer-induced pleurisy is associated with an alteration of nitric oxide (NO)-synthase activity, the levels of nitrate/nitrite (NOx) were measured in blood serum (BS) and pleural effusion (PE) of 35 cancer patients (secondary pleural metastases and mesotheliomas), eight patients with benign lung diseases, and in BS of nine healthy donors. It was found that (1) BS of patients with secondary pleural metastases had an elevated level (P < 0.015) of NOx (59.7 +/- 24.4 mu M, n = 28) in comparison with control level of BS for healthy donors (43.4 +/- 13.5 mu M, n = 9); (2) BS of mesotheliomas (32.1 +/- 12.2 mu M, n = 4) had significantly (P < 0.05) lower level of NOx compared to BS of benign patients (61.2 +/- 28.8, n = 6); (3) differences in mean levels of NOx in BS and same PE of examined patient groups did not reach statistical significance, excepting sub-group of patients with primary mammary carcinoma; (4) significant interindividual differences of NOx in all groups of patients were revealed; (5) fluids from about 11% of cancer patients contained extremely high levels of NOx over 100 muM; (6) a significant elevation of apparent NOx level in BS and PE of patients with secondary pleural metastases in comparison with those in BS of healthy donors was revealed when the native, i.e. protein-contained, samples were managed with Griess reagent.The results described here, point up the diverse role of NOx in cancer patients. Its role is far from being clear but it seems that NOx acts as a signaling mediator during the formation of pleural metastases and might be considered as a non-specific marker in the corresponding PE. Furthermore, NOx could be used to give rationale of proper application of anticancer drugs affecting diversely NO-synthase activity in cells. Besides, a casual effectiveness of NOx measurements in native samples from cancer patients using Griess reagent needs additional elaboration. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Metabolic inhibitors can clearly affect different aspects of the functional activity of cells. This property was studied in the present work with respect to MK-886, a well-known inhibitor of the 5´-lipoxygenase-activating protein. It was found that this inhibitor in a micromolar concentration range (2-20 µM) induced in a dose-dependent manner H2O2 generation by human neutrophils and the release of lysozyme from the cells. The MK-886-induced activation of neutrophils was accompanied by a significant decrease in N-(1-pyrene)maleimide-accessible SH-groups in the cells. According to its activity, MK-886 can be considered an agonist that causes up-regulation of inherent neutrophil functions. In summary, the results indicate that during the application of MK-886 as a 5´-lipoxygenase inhibitor in neutrophils, the impact of the compound on the functional activity of the cells should be taken into consideration.

This study was performed to explore whether lectin-induced generation of superoxide anions by neutrophils, lectin-induced degranulation of these cells and NO-synthase activity in pleural effusions can be used as prognostic factors of primary lung cancer patients. Among the activities tested, increased generation of superoxide by Triticum vulgaris agglutinin (WGA)-activated neutrophils showed prognostic relevance. Thus, this lectin-dependent reactivity of immune cells warrants further investigation in addition to the histochemical analysis.

Carbohydrate-bearing polymers of biologically inert design are versatile tools to delineate functional aspects of oligosaccharides. Binding of synthetic N-substituted polyacrylamide (PAA) conjugates of histo-blood group (Adi, Atri, Bdi, Btri, Hdi, SiaLea, and SiaLex) to human polymorphonuclear leukocytes (PMNs), and effects on H2O2 generation elicited by different agonists such as digitonin, N-formyl-Met-Leu-Phe (FMLP) and the galactoside-specific lectin from Viscum album L. (VAA) were assessed. PMNs expressed binding sites for blood group-related neoglycoconjugates in the range of N∼106–107/cell with KD-values in the μM range. Treatment of PMNs (2×106cells/ml) with PAA-probes (50μg/ml) for 5min did not activate the “respiratory burst”. However, it led to suppression (range 20–70%) of H2O2 generation of cells in the presence of elicitors. In detail, the FMLP-induced response was significantly decreased by Adi, Atri, Btri, Hdi, SiaLea, and SiaLex conjugates, whereas for digitonin one only by Adi, Atri, Btri. All the seven tested PAA-probes were found to inhibit significantly VAA-mediated release of H2O2 from PMNs. In this case, interference can take place already, at the stage of initial binding, especially for B- and H-epitopes, but less prominently for A- and SiaLe-epitopes. These results support the notion that PAA-immobilized histo-blood group oligosaccharides can serve as effector molecules with the ability to reduce the H2O2-generation of PMNs, warranting further studies on the involved reaction pathway.

The effect of alpha-NeuAc(2-->6)Gal/GalNAc-specific lectin from Sambucus nigra (SNA) on the release of lysozyme from human neutrophils was studied in vitro. Interaction of cells with the lectin was accompanied by dose-dependent release of lysozyme, which was increased in the presence of cytochalasin B. The involvement of intracellular signaling pathways in the lectin-induced degranulation of neutrophils was determined using a panel of specific inhibitors tested at concentrations in the range of 10-100 microM. Aristolochic acid (a phospholipase A2 inhibitor), indomethacin (a cyclooxygenase inhibitor), neomycin sulfate (a phospholipase C inhibitor), trifluoperazine (a calmodulin antagonist/protein kinase C inhibitor), N-ethylmaleimide (a sulfhydryl reagent), and guanosine-5;-O-(2-thiodiphosphate) (a G-protein inhibitor) were found to reduce SNA-induced lysozyme release from neutrophils by 20-45%. The treatment of cells with bisindolylmaleimide (a protein kinase C inhibitor), H-8 (an inhibitor of protein kinases A, C, G and of myosin light chain kinase), PD 98059 (a MAP kinase inhibitor), and (+/-)-methoxyverapamil (a Ca2+-channel blocker) failed to affect the release of lysozyme. These results indicate that only selective intracellular pathways associated with activation of G-proteins and phospholipid metabolism as well as the thiol-dependent signaling systems are apparently involved in the realization of the SNA-induced degranulation response of human neutrophils.

Initial ligand selection and the intermolecular spatial arrangement of glycan-lectin complexes are assumed to be essential to induce formation of stable cell aggregates by a lectin. To distinguish effects of these two processes, the tetrameric mistletoe lectin and its isolated B-chain were used. A reduced impact of multivalency for Ehrlich ascites tumor cells in contrast to rat thymocytes was revealed. Signaling is thus initiated in a cell-type-dependent manner. Using selective metabolic inhibitors to reduce signal transfer for aggregate stability, decrease in cellular SH-group level was shown to be a common effect accompanying suppression of lectin-dependent aggregate stability. The results underscore an intrinsic variability in the relative importance of lectin-dependent glycan aggregation on the cell surface for triggering post-binding lectin effects.

Bivalent lectins as bridging molecules between cells or cell surface lectins as docking points are involved in mediation of cell adhesion by specific recognition of suitable glycoconjugates on an opposing surface. The initial contact formation by a lectin can lead to intracellular post-binding events which effect stable cell association even in the presence of the haptenic sugar. To delineate the participation of intracellular signaling pathways in the cascade of reactions to establish firm association, reagents with proven inhibitory capacity on certain biochemical targets provide suitable tools. Using this approach with rat thymocytes and the galactoside-binding lectin from mistletoe (Viscum album L. agglutinin, VAA) as a model, a panel of 27 inhibitors with impact on e.g. several types of kinases, tyrosine phosphatases, NO synthases, G proteins, enzymes of arachidonate and cyclic nucleotide metabolism and calmodulin was systematically tested with respect to their capacity to impair the formation of lactose-resistant cell aggregates. In addition to the recently reported effectiveness of N-ethyimaleimide, nordihydroguaiaretic acid, and trifluoperazine the agents diacyigiycerol kinase inhibitor II, emodin, D609, DPI, KT5720, KT5926, MK-886, bisindolyimaleimide I, and (±)methoxyverapamil were able to reduce aggregate stability in the presence of the haptenic sugar. Thus, various types of kinases including p56 tyrosine kinase, lipoxygenases, phosphatidylcholine-specific phospholipase C as well as calmodulin and Ca2+-currents, but not modulators of the metabolism of cyclic nucleotides, NO synthases, MAP kinases, tyrosine phosphatases and phospholipase A (preferentially group II) and C can play a role in eliciting contact stability. More than one principal signaling pathway appears to be linked to the measurable parameter, since inhibitory substances show additive properties in co-incubation assays and differentially affect two lectin-elicited cellular activities, i.e. intracellular movement of Ca2+-ions and H2O2-generation, which can accompany cell adhesion and aggregation. Pronounced differences in the extent of modulation of H2O2-generation in human neutrophils by the same set of substances emphasizes that general conclusions on the post-binding effects for a certain lectin in different cell types are definitely precluded. In aggregate, the approach to employ inhibitors with target selectivity intimates an involvement of protein kinases A, C, Ca2+/calmodulin-dependent protein kinase II, p56 tyrosine kinase, leukotrienes and/or hydroxyeicosatetraenoic acids, phosphatidylcholine-specific phospholipase C and Ca2+-fluxes in events following initial binding of a galactoside-specific plant lectin to rat thymocytes which establish firm cell contacts.

Molecular mechanisms of interplay between reactive oxygen (superoxide, hydroxyl radical, H2O2 etc.) and nitrogen (nitric oxide - NO, ONOO-, NO2-, NO3- etc.) forms are proposed to be of key importance for cell and tumor biology. Considering NO as a signal molecule we have studied the impact of NO release on processes of generation of H2O2 in different experimental systems including pleural effusions (PE) of lung cancer patients, human polymorphonuclear leukocytes (PMNs), and rat thymocytes. It was found that PE of lung cancer patients contain a high level of [NO2-+NO3-], i.e. 43.4 25.6 microM (n=15), and PE cells could effectively generate H2O2 in response to lectins from Viscum album (VAA), Phaseolus vulgaris (PHA), and Pisum sativum (PSA) as well as to menadione. A positive correlation between the [NO2-+NO3-] concentration and menadione-induced H2O2 generation (r=0.1964) was found, whereas the [NO2-+NO3-] concentration and lectin-induced H2O2 generation (PHA, r=-0.4099; PSA, r=-0.3949; VAA, r=-0.3225) were negatively correlated. Notably, an increase of H2O2 generation by PE cells was determined in the range of 20-35 microM [NO2-+NO3-]. When PMNs and rat thymocytes were treated with a donor of NO (sodium nitroprusside), the release of H2O2 in response to lectins or menadione was decreased in a dose-dependent manner. The end products of NO biochemistry, assayed as KNO2 and KNO3, were not able to affect significantly the H2O2 generation processes. In conclusion, the data indicate that the potential for triggered H2O2-generation of cells is modulated markedly by the presence of NO or derived reaction compounds. This relation may play an important role in the pathogenesis of PE malignancies with potential relevance for therapeutic strategies.