POLYAMINES AS NEW POTENTIAL BIOMARKERS FOR DIFFERENTIAL DIAGNOSIS OF PROSTATE CANCER AND ESTIMATION OF ITS AGGRESSIVENESS.

BACKGROUND: Prostate cancer (PCa) is one of the most common malignancies in older men. The study of tissue markers of PCa can provide information about the state of proliferation and apoptosis in tumors, the susceptibility of tumor cells to metastasis and the mechanisms of resistance to therapy, which, in turn, can help predict the course of the disease and develop personalized treatment. Polyamines (PAs) spermine, spermidine, putrescine are of particular interest in terms of PCa tissue markers. AIM: To investigate the levels of basic and acetylated forms of PAs in the postoperative samples of malignant and benign tumors of the human prostate and evaluate the possibility of their use for differential diagnosis and assessment of the PCa aggressiveness. OBJECT AND METHODS: 57 postoperative tumor samples from patients with prostate adenocarcinoma of different Gleason score (GS) and clinical stage (T1-T4) and 20 samples of tumors from patients with benign prostate hyperplasia (BPH) were studied. The content of PAs was determined by high performance liquid chromatography. RESULTS: Among the studied PAs, the most significant difference between PCa and BPH was observed for spermine (Spm). The level of Spm in PCa samples was 16 times lower than in BPH samples (p < 0.01). We did not find a significant dependence of PAs levels, including Spm, on the clinical stage. The association between the Spm level and the GS was established. The indolent (GS6) tumors were characterized by the highest Spm level while in the most aggressive (GS9 and GS10) tumors Spm content was the lowest. CONCLUSIONS: A sharp decrease in Spm levels is probably a characteristic feature of prostate malignant tumors. The obtained results indicate an association of Spm levels in tumors with the GS. This may indicate Spm involvement in the formation of the aggressiveness of PCa. The results of the study can be further used for differential diagnosis of prostate tumors and for assessing the aggressiveness of PCa.

Extracellular stress stimuli alter galectin expression profiles and adhesion characteristics of HL-60 cells.

Galectins, a family of soluble ß-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 ß-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.

[The formation of stable aggrregates of human platelets induced by lectin from Solanum tuberosum].

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.

Migration-promoting role of VEGF-C and VEGF-C binding receptors in human breast cancer cells.

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 alpha9beta1. 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 alpha9beta1 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.

COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer.

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.

Immunotherapy of C3H/HeJ mammary adenocarcinoma with interleukin-2, mistletoe lectin or their combination. effects on tumour growth, capillary leakage and nitric oxide (NO) production.

Clinical application of interleukin (IL)-2-based immunotherapy of cancer has been limited by a major side-effect known as 'capillary leak syndrome', resulting from nitric oxide (NO) overproduction. A galactoside-specific lectin from Viscum album L. (VAA) has been reported to induce certain lymphokines and upregulate IL-2 receptors on lymphocytes. Present study was, therefore, designed to compare the effects of combination therapy with IL-2 (10(4) Cetus units/mouse, intraperitoneal (i.p). every 8 h, given as 5 day rounds per week, for one or two rounds) and VAA (1 ng/kg subcutaneous (s.c.), biweekly) with those of IL-2 or VAA therapy alone in C3H/HeJ female mice bearing s.c. transplants of a highly metastatic C3L5 mammary adenocarcinoma. IL-2 therapy alone reduced tumour growth and metastasis, but caused significant water retention indicative of capillary leakage in the kidneys after both rounds of therapy, whereas pleural effusion was only evident after the first round and not the second round. A sharp rise in the systemic NO levels after the first round, followed by a decline after the second round of IL-2 therapy suggested a causal relationship of increased NO levels to pleural effusion. A strong immunostaining for nitrotyrosine (a marker for the production of peroxynitrite) was noted in the renal tubules at the end of both rounds of therapy suggestive of a causal association of this toxic NO-metabolite with capillary leakage in the kidneys. Addition of VAA to IL-2 therapy had no effect on any of the above parameters. Unexpectedly, however, VAA therapy alone stimulated tumour growth as well as lung metastases. NO induction in the C3L5 cells by VAA was excluded as a possible reason for this stimulation. Present results suggest the need for exercising caution in the use of VAA as an immunoadjuvant in human cancer therapy.

Impact of MK-886 on H2O2 generation by human neutrophils and cell degranulation.

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 microM) 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.

Lectin (WGA)-dependent superoxide anion release by neutrophils as a prognostic factor in lung cancer.

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.

Cell-type dependence of stability modulation of lectin-initiated contacts by impairment of multivalent carbohydrate binding and intracellular signaling.

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.

Effect of signaling inhibitors on the release of lysozyme from human neutrophils activated by Sambucus nigra agglutinin.

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.

Dissection of the impact of various intracellular signaling pathways on stable cell aggregate formation of rat thymocytes after initial lectin-dependent cell association of using a plant lectin as model and target-selective inhibitors.

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-ethylmaleimide, nordihydroguaiaretic acid, and trifluoperazine the agents diacylglycerol kinase inhibitor II, emodin, D609, DPI, KT5720, KT5926, MK-886, bisindolylmaleimide I, and (+/-)methoxyverapamil were able to reduce aggregate stability in the presence of the haptenic sugar. Thus, various types of kinases including p561lck tyrosine kinase, lipoxygenases, phosphatidylcholine-specific phospholipase C as well as calmodulin and Ca(2+)-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 Ca(2+)-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, p56lck tyrosine kinase, leukotrienes and/or hydroxyeicosatetraenoic acids, phosphatidylcholine-specific phospholipase C and Ca(2+)-fluxes in events following initial binding of a galactoside-specific plant lectin to rat thymocytes which establish firm cell contacts.

Differential potency of two crosslinking plant lectins to induce formation of haptenic-sugar-resistant aggregates of rat thymocytes by post-binding signaling.

To evaluate the significance of post-binding events for stable aggregate formation, the aggregation/dissociation of rat thymocytes initiated by two crosslinking plant lectins, namely concanavalin A (Con A) and Solanum tuberosum agglutinin (STA), were comparatively studied. Despite intimate cell contacts in the aggregates only Con A led to establishment of haptenic-sugar-resistant (HSR) complexes. The presence of inhibitor II of diacylglycerol kinase, a dual calmodulin antagonist/protein kinase C inhibitor (trifluoperazine), and a sulfhydryl group reagent (N-ethylmaleimide) impaired this process. The obtained results indicate that the formation of HSR cellular contacts is not an automatic response to lectin-dependent cell association. In contrast to STA, Con A binding elicits this reaction with involvement of diacylglycerol kinase, protein kinase C and/or calmodulin as well as thiol level perturbation, as inferred by the application of target-selective inhibitors.

NO-dependent regulation of lectin- and menadione-induced H2O2 production by cells from pleural effusions of lung cancer patients and by immune cells.

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.

[Lectin-reactive immune complexes in plasma in ENT diseases]. / Lektin-reaktivnye immunnye kompleksy v plazme krovi pri LOR-zabolevaniiakh.

The content of circulating immune complexes (IC) interacting with plant lectins from Canavalia ensiformis (Con A), phaseolus vulgaris (PHA) and wheat germ (WGA) was examined in blood plasma of healthy donors and patients with ENT diseases (n = 36). It was found that the level of PHA- and WGA-reactive IC in patients with laryngeal carcinoma was significantly higher than in control, whereas only the level of WGA-reactive IC was elevated in patients with scleroma. The level of ConA-reactive IC was statistically uniform in control and both ENT diseases.

Formation of lactose-resistant aggregates of human platelets induced by the mistletoe lectin and differential signaling responses to cell contact formation by the lectin or thrombin.

Human platelets afford a suitable and physiologically relevant model to study receptor-dependent cell aggregation and ensuing biosignaling reactions. Since cell surface glycoconjugates can serve as ligands in recognitive protein--carbohydrate interactions, it is of interest to investigate the reactivity of such epitopes for a plant lectin and the elicited intracellular responses. Therefore, the galactose-specific lectin (Viscum album agglutinin, VAA) was employed as a tool for this purpose. It was found that VAA induced platelet aggregation at a concentration of 2.5 microgram/ml using 2.5. 108 cells/ml, composed of the formation of both lactose-sensitive (Lac+) and lactose-resistant (Lac-) intercellular contacts. Lac- aggregates were formed only by metabolically active platelets of about 70% of the samples from the group of studied volunteers. The requirement of metabolic activity for formation of these contacts which no longer depend on lectin--ligand recognition was underscored by the lack of their appearance in the presence of metabolic inhibitors such as nordihydroguaiaretic acid, trifluoperazine, N-ethylmaleimide and menadione. With respect to biosignaling, the effective aggregation of platelets did not affect the basal level of Ca2+ in cells and reduced the rate of the menadione-dependent generation of H2O2. In parallel series platelet aggregation induced by bovine thrombin (0.03 U/ml) triggered an increase in the cytoplasmic Ca2+ level and an enhancement of the H2O2 generation. Overall, these results imply metabolically controlled post-binding reactions which strengthen the lectin-induced cell association and demonstrate differential responses with respect to the Ca2+ level and H2O2-generation between lectin- or thrombin-mediated aggregation of human platelets.

Modification of the functional activity of neutrophils treated with acute phase response proteins.

The effects of alpha1-acidic glycoprotein (AGP) and its subfractions (glycoforms) with different affinity for concanavalin A on generation of H2O2 by human neutrophils exposed to stimulators of different nature, namely, galactose-specific mistletoe (Viscum album) lectin (VAA), digitonin, and N-formyl-Met-Leu-Phe, a chemotactic peptide (FMLP), were studied. Within the concentration range of 13-500 microgram/ml, AGP and its glycoforms produced dose-dependent inhibition of digitonin-induced cell responses. AGP also inhibited the VAA-induced generation of H2O2; however, this cell response was potentiated by low concentrations (50 microgram/ml) of AGP and AGP-A. FMLP induced the most consistent response of neutrophils, which changed only slightly in the presence of AGP and AGP-B; however, low concentrations of AGP-A inhibited this response. The presence of sialic acid in the terminal position of carbohydrate antennae of AGP is not necessary for its inhibitory effect on human neutrophil respiratory burst because asialo-AGP (250 microgram/ml) inhibited H2O2 generation by cells stimulated with agonists of the NADPH-oxidase system of phagocytes. In contrast to AGP, two other acute phase response proteins displaying a lectin activity (C-reactive protein and serum amyloid P component) within the concentration range of 10-100 microgram/ml produced no significant effect on H2O2 generation by stimulated neutrophils. These data suggest that AGP is an effector molecule responsible for feedback regulation of the functional activity of neutrophils.

Lectin-triggered superoxide/h(2)o (2) and granule enzyme release from cells.

The modulatory potency of lectins on cellular activities deserves attention from a cell biological and a clinical point-of-view. In addition to serving as tools to delineate signaling processes that follow carbohydrate-dependent cell binding, plant lectins can apparently affect certain characteristics of the host immune system in vitro and in vivo with potential clinical benefit (1,2). With respect to defense mechanisms the generation of reactive oxygen compounds such as the superoxide anion radical (O (2) (-)) and H(2)O(2) or the release of granule enzymes are supposed to play a notable role (3-6) Deliberate enhancement of these activities by lectins may increase the host's capacity to control growth of infectious organisms or malignant cells. Therefore, rigorous testing of plants agglutinins and endogenous lectins that have been isolated from human tissues may enable one to devise a rational lectin-mediated treatment modality This chapter describes the protocols for the respective assay procedures used to determine the effects of lectins on production of reactive oxygen compounds and release of granule enzymes.

Metabolic inhibitors as tools to delineate participation of distinct intracellular pathways in enhancement of lactose-induced dissociation of neutrophil and thymocyte aggregates formed by mediation of a plant lectin.

Signaling processes in the course of the formation of the lectin-mediated aggregates may partake in conveying enhanced stability to the cell clusters. To prove the validity of this reasoning in a model, we have studied the impact of addition of three metabolic inhibitors (N-ethylmaleimide, nordihydroguaiaretic acid, and trifluoperazine) on lactose-dependent dissociation of cell aggregates, formed in the presence of the galactoside-binding mistletoe lectin. Using both human neutrophils and rat thymocytes to avoid measurement of responses restricted to a single cell type, an enhanced dissociation of lectin-formed cell aggregates was observed, when lactose and an inhibitor were present. Among the tested inhibitors, nordihydroguaiaretic acid and N-ethylmaleimide were more potent enhancers of cell dissociation than trifluoperazine. These results suggest that biosignalling pathways connected with lipoxygenase activity as well as the level of intracellular sulfhydryl groups confer further stability to lectin-dependent cell aggregates. The systematic evaluation of inhibitors for defined activities is thus suggested as a tool to disclose the nature and the contribution of individual signaling mechanisms to post-binding effects following lectin-initiated cell contact formation.

Generation of H2O2 by human neutrophils and changes of cytosolic Ca2+ and pH of rat thymocytes in response to galactoside-binding proteins (lectins or immunoglobulins).

In contrast to plant agglutinins, biological activities of animal/human lectins are not well defined yet. Testing a panel of seven mammalian carbohydrate-binding proteins we have found that the dimeric lectin from chicken liver (CL-16) was a stimulator of H2O2 release from human neutrophils as well as effector for induction of cytosolic Ca2+ and pH increase in rat thymocytes. Activity of this lectin was comparable to potent galactoside-specific plant lectins such as Viscum album L. agglutinin. The activities of the tested plant lectins depended significantly on their nominal carbohydrate specificity as well as on the source. The results indicate that endogenous lectins may be involved in the regulation of neutrophil and lymphocyte functions by elicitation of selective biosignaling reactions.

Effects of metabolic inhibitors and lectins on the menadione-dependent generation of H2O2 by rat thymocytes.

The capacity of oxidative metabolism and its regulation is an important factor in disease control. Using scopoletin as a fluorescent substrate of peroxidase the extent of menadione-dependent production of H2O2 by rat thymocytes was determined. The reaction was inhibited by 2,4-dinitrophenol, papaverine, nordihydroguaiaretic acid and iodacetamide. The membrane-penetrating SH reagent N-ethylmaleimide primed the reaction, probably due to an inhibition of glutathione peroxidase. To delineate an influence of cell-surface protein-carbohydrate interactions by exogenous lectins, the impact of cell binding was analyzed for several plant lectins, namely concanavalin A, phytohemagglutinin, the lectins from Triticum vulgaris and from Sambucus nigra. Except for the alpha-NeuNAc(2-6)gal/galNAc-binding agglutinin, the other three plant proteins with specificities to different parts of N-linked oligosaccharides primed the reaction. This activity of lectins did not coincide with their ability to aggregate cells. The given results indicate that biosignaling pathways triggered by lectins are involved in the regulation of the intracellular reduction of menadione.