Alexander V. Timoshenko

Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.

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.

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 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.

PGE2‐mediated upregulation of iNOS in murine breast cancer cells through the activation of EP4 receptors

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.

Relationship between cyclooxygenase-2 and human epidermal growth factor receptor 2 in vascular endothelial growth factor C up-regulation and lymphangiogenesis in human breast cancer

Both cyclooxygenase (COX)‐2 and human epidermal growth factor receptor (HER)‐2 promote breast cancer progression; however, the relationship between the two molecules remains unclear. We utilized human breast cancer tissues and cell lines to examine whether COX‐2 and HER‐2 played independent or interdependent roles in vascular endothelial growth factor (VEGF)‐C up‐regulation and lymphangiogenesis. A paired correlation of immunodetectable levels of COX‐2, VEGF‐C, and HER‐2 proteins and lymphovascular density (LVD; D2‐40‐immunolabeled) in 55 breast cancer specimens revealed a positive correlation between COX‐2 and HER‐2 irrespective of clinicopathological status. However COX‐2 alone positively correlated with LVD. In 10 independent specimens, mRNA levels showed a positive correlation between HER‐2 and COX‐2 or VEGF‐C but not LYVE‐1 (lymphovascular endothelial marker). These findings implicate COX‐2, but not HER‐2, in breast cancer–associated lymphangiogenesis. Manipulation of the COX‐2 or HER‐2 genes in breast cancer cell lines varying widely in COX‐2 and HER‐2 expression revealed a direct role of COX‐2 and an indirect COX‐2 dependent role of HER‐2 in VEGF‐C up‐regulation: (i) high VEGF‐C expression in high COX‐2/low HER‐2 expressing MDA‐MB‐231 cells was reduced by siRNA‐mediated down‐regulation of COX‐2, but not HER‐2; (ii) integration of HER‐2 in these cells simultaneously up‐regulated COX‐2 protein as well as VEGF‐C secretion; and (iii) low VEGF‐C secretion by high HER‐2/low COX‐2 expressing SK‐BR‐3 cells was stimulated by COX‐2 overexpression. These findings of the primary role of COX‐2 and the COX‐2‐dependent role of HER‐2, if any, in VEGF‐C up‐regulation and lymphangiogenesis suggest that COX‐2 inhibitors may abrogate lymphatic metastasis in breast cancer irrespective of HER‐2 status. (Cancer Sci 2010)

Co-Expression of α9β1 Integrin and VEGF-D Confers Lymphatic Metastatic Ability to a Human Breast Cancer Cell Line MDA-MB-468LN

Introduction and Objectives Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. Results A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells. Conclusion Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.

Viscum album agglutinin-induced aggregation of blood cells and the lectin effects on neutrophil function.

Extracts from mistletoe enjoy a large popularity in central Europe as an unconventional treatment modality for cancer, warranting scientific efforts with defined components to delineate any potential benefit. The galactose-specific lectin from Viscum album (VAA), known to exhibit immunomodulatory and ensuing antitumoral capacities in animal model systems, was shown to aggregate human blood cells in the following order: neutrophils, mononuclear cells--thrombocytes and erythrocytes. To contribute to the analysis of lectin effects on individual aspects of the host defence system, two parameters of neutrophils were quantitatively assessed, namely the aggregating activity of VAA as a measure of strength of interaction with cell surface ligands and the effect of lectin on oxidative metabolism (H2O2 release) of these cells. It was found that whole lectin and its carbohydrate-binding B-subunit possessed the capacity to induce cell aggregation and H2O2 release, which were blocked by D-galactose and lactose. Both effects displayed similar dependence on the lectin concentration in the range 0.1-25 micrograms/ml. The toxic A-subunit displayed detectable activity only in high doses (50 micrograms/ml) while the bovine heart galaptin (14 kDa; galectin-1) failed to affect neutrophils. The role of oxidative metabolism in regulation of neutrophil aggregation induced by VAA was studied using metabolic inhibitors and controlled heating at 46 degrees C leading to inhibition of plasma membrane NADPH-oxidase system. Trifluoperazine and menadione inhibited the neutrophil aggregation in a dose-dependent manner in comparison with such inhibitors as amiloride and theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Dissection of the impact of various intracellular signaling pathways on stable cell aggregate formation of rat thymocytes after initial lectin-dependent cell association 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-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.

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.