Melanotransferrin induces human melanoma SK-Mel-28 cell invasion in vivo

The expression of melanotransferrin (MTf), a membrane-bound glycoprotein highly expressed in melanomas, is correlated with tumor vascularization and progression, suggesting a proinvasive function associated with MTf in malignant tumors. To test this hypothesis, we silenced MTf in human melanoma SK-MEL-28 cells using small interfering RNA (siRNA) and examined the plasmin activity and invasiveness of MTf-silenced melanoma. In vitro, the siRNA-mediated MTf knockdown inhibited by 58% the cell surface activation of plasminogen into plasmin. In addition, decreased expression of MTf in melanoma cells reduced cell migration. In vivo, we used a nude mice invasion model in which tissue factor (TF) induces vascular [125I]-fibrin deposition following injection. Using this metastasis model, the invasive potential of MTf-silenced cells into the lungs was reduced by fivefold. Altogether, these findings strongly suggest that MTf overexpression in melanoma cells contributes to tumor progression by stimulating plasmin generation as well as cell migration and invasion.     

Melanotransferrin stimulates t-PA-dependent activation of plasminogen in endothelial cells leading to cell detachment

Tissue plasminogen activator (t-PA) is an extracellular serine protease that converts the proenzyme plasminogen into the broad-spectrum substrate serine protease, plasmin. Plasmin, one of the most potent pro-angiogenic factors, is a key element in fibrinolysis, cell migration, tissue remodeling and tumor invasion. In the present investigation, we assessed the impact of the truncated form of soluble melanotransferrin (sMTf) on plasminogen activation by t-PA and subsequent endothelial cell detachment. Co-treatment of human endothelial microvessel cells with plasminogen, t-PA and sMTf significantly increased plasmin formation and activity in the culture medium. Plasmin generated in the presence of sMTf also led to a 30% reduction in fibronectin detection within cell lysates and to a 9-fold increase within the corresponding cell medium. Moreover, the presence of sMTf increases EC detachment by 6-fold compared to cells treated only with plasminogen and t-PA. Although the addition of alpha(2)-antiplasmin completely prevented plasmin formation and EC detachment, epigallocatechin gallate, GM6001 and a specific antibody directed against MMP-2 prevented cellular detachment without interfering with plasminogen activation. Overall, these data suggest that the anti-angiogenic properties of sMTf may result from local overstimulation of plasminogen activation by t-PA, thus leading to subsequent degradation of the Fn matrix and EC detachment.     

Inhibition of melanoma brain metastasis by targeting melanotransferrin at the cell surface

Brain metastases are a common feature of malignant melanoma and are associated with poor prognosis. Melanotransferrin (MTf), one of several antigens associated with the surface of melanoma cells, has been demonstrated to promote cell invasion. In this study, we investigated the role of membrane‐bound MTf in several of the steps leading to the development of melanoma brain metastasis. Our results indicated that MTf‐positive cells were detected in the brains of nude mice injected intravenously with human melanoma SK‐Mel 28 cells. Moreover, administration of a single dose of a monoclonal antibody (L235) directed against human MTf significantly reduced the development of human melanoma brain metastases in nude mice. The ability of melanoma cells to cross the blood–brain barrier (BBB) in vitro is correlated with their MTf expression levels at the cell surface. Overall, our results indicated that membrane‐bound MTf is a key element in melanoma cell transmigration across the BBB and subsequent brain metastasis. Thus, these data suggest MTf as an attractive target and demonstrate the therapeutic potential of an anti‐MTf mAb for preventing metastatic melanoma.     

The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.     

Plasminogen-dependent internalization of soluble melanotransferrin involves the low-density lipoprotein receptor-related protein and annexin II

We investigated the effect of plasminogen (Plg) on the internalization of recombinant soluble melanotransferrin (sMTf) using U87 human glioblastoma cells and murine embryonic fibroblasts (MEF) deficient in the low-density lipoprotein receptor-related protein (LRP). Using biospecific interaction analysis, both Glu- and Lys-Plg were shown to interact with immobilized sMTf. The binding of sMTf at the cell surface increased in the presence of both forms of Plg in control and in LRP-deficient MEF cells, whereas the uptake was strongly stimulated only by Lys-Plg in control MEF and U87 cells. In addition, in the presence of Lys-Plg, the internalization of sMTf was a saturable process, sensitive to temperature and dependent on the integrity of lysine residues. The addition of the receptor-associated protein, lactoferrin and aprotinin, as well as a monoclonal antibody (mAb) directed against LRP, inhibited the Lys-Plg-dependent uptake of sMTf. These results suggest an important role for LRP in this process. In addition, using binding and uptake assays in the presence of anti-annexin II mAb, we showed that annexin II might be responsible for the initial binding of sMTf in the presence of Plg. Our results suggest a Plg-mediated internalization mechanism for the clearance of sMTf via annexin II and LRP.     

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Inhibition of tumor growth by a truncated and soluble form of melanotransferrin

Melanotransferrin is a glycoprotein expressed at the cell membrane and secreted in the extracellular environment. Recombinant truncated form of membrane-bound melanotransferrin (sMTf) was reported to exert in vitro anti-angiogenic properties. Here we show that sMTf treatment leads to a 50% inhibition of neovascularization in Matrigel implants when stimulated by growth factors. Using a glioblastoma xenograft model, we demonstrate that sMTf delivery at 2.5 and 10 mg/kg/day by micro-osmotic pump inhibits tumor growth by 73% and 91%, respectively. In a lung carcinoma xenograft model, sMTf treatment at 2.5 and 10 mg/kg/day impeded tumor growth by 87% and 97%. Furthermore, subcutaneous glioblastoma and lung carcinoma tumors from mice treated with 10 mg/kg/day of sMTf present insignificant growth toward the study. In association with a reduction in endoglin mRNA expression, the hemoglobin content decreased by half in sMTf-treated glioblastoma tumors. In vitro experiments revealed that NCI-H460 cells treated with sMTf display an inhibition in their invasive capabilities with a concomitant reduction in the expression of the low-density lipoprotein receptor protein and urokinase plasminogen activator receptor. Altogether, our results demonstrate that sMTf exerts anti-cancer and anti-angiogenic activities, suggesting that its administration may provide novel therapeutic strategies for the treatment of cancer.     

The novel plasminogen receptor, plasminogen receptor(KT) (Plg-R(KT)), regulates catecholamine release

Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.     

The Ig VH complementarity-determining region 3-containing Rb9 peptide,inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the V_HCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca²⁺ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.     

Expression of active plasminogen activator inhibitor-1 reduces cell migration and invasion in breast and gynecological cancer cells

Urokinase-type (uPA) plasminogen activator is regulated by serine protease inhibitors (serpins), especially plasminogen activator inhibitor-1 (PAI-1). In many cancers, uPA and PAI-1 contribute to the invasive phenotype. We examined the in vitro migration and invasive capabilities of breast, ovarian, endometrial, and cervical cancer cell lines compared to their plasminogen activator system profiles. We then overexpressed active wild-type PAI-1 and an inactive "substrate" P14 form of PAI-1 (T333R) using stable transfection and adenoviral gene delivery. We also upregulated endogenous uPA and PAI-1 in these cells by treatment with transforming growth factor-beta. Some breast and ovarian cancer cell lines with natural expression of uPA, PAI-1, and urokinase receptor showed substantial migration and invasion compared to other cell lines that lack expression of these proteins. However, overexpression of active wild-type PAI-1, but not P14-PAI-1 (T333R), in these cell lines showed reduced migration and invasion. Since vitronectin binding by both forms of PAI-1 is equivalent, these results imply that PAI-1-vitronectin interactions are less critical in altering migration and invasion. Our results show that the in vitro migratory and invasive phenotype in these breast and ovarian cancer cell lines is reduced by active PAI-1 due to its ability to inhibit plasminogen activation.     

Inhibition of endothelial cell movement and tubulogenesis by human recombinant soluble melanotransferrin: involvement of the u-PAR/LRP plasminolytic system

We have previously demonstrated that human recombinant soluble melanotransferrin (hr-sMTf) interacts with the single-chain zymogen pro urokinase-type plasminogen activator (scu-PA) and plasminogen. In the present work, the impact of exogenous hr-sMTf on endothelial cells (EC) migration and morphogenic differentiation into capillary-like structures (tubulogenesis) was assessed. hr-sMTF at 10 nM inhibited by 50% the migration and tubulogenesis of human microvessel EC (HMEC-1). In addition, in hr-sMTf-treated HMEC-1, the expression of both urokinase-type plasminogen activator receptor (u-PAR) and low-density lipoprotein receptor-related protein (LRP) are down-regulated. However, fluorescence-activated cell sorting analysis revealed a 25% increase in cell surface u-PAR in hr-sMTf-treated HMEC-1, whereas the binding of the urokinase-type plasminogen activator (u-PA)*plasminogen activator inhibitor-1 (PAI-1) complex is decreased. This reduced u-PA-PAI-1 binding is correlated with a strong inhibition of the HMEC-1 plasminolytic activity, indicating that exogenous hr-sMTf treatment alters the internalization and recycling processes of free and active u-PAR at the cellular surface. Overall, these results demonstrate that exogenous hr-sMTf affects plasminogen activation at the cell surface, thus leading to the inhibition of EC movement and tubulogenesis. These results are the first to consider the potential use of hr-sMTf as a possible therapeutic agent in angiogenesis-related pathologies.     

Iron uptake by melanoma cells from the soluble form of the transferrin homologue,melanotransferrin

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that can also exist in a soluble form (sMTf). Considering the high homology of MTf to Tf, it is possible to suggest that sMTf could bind to the high affinity transferrin receptor 1 (TfR1) or lower affinity TfR2. We have used sMTf labelled with 59Fe to examine its ability to donate Fe to cells. Our experiments demonstrate that sMTf is far less effective than Tf at donating Fe to cells and this does not occur via specific receptors. Indeed, the uptake of sMTf by cells occurred via a non-specific process (e.g. adsorptive pinocytosis).     

Nuclear factor inducing kinase: A key regulator in osteopontin- induced MAPK/IκB kinase dependent NF-κB-mediated promatrix metalloproteinase-9 activation

Osteopontin (OPN) is a secreted, non-collagenous, sialic-acid rich, glycosylated adhesive phospho- protein. Several highly metastatic transformed cells synthesized a higher level of OPN compared with non-tumorigenic cells. We have recently reported that OPN induces nuclear factor-κB (NF-κB)-mediated promatrix metalloproteinase-2 activation through IκBα/IKK signaling pathways. However, the molecular mechanism(s) by which OPN regulates pro-matrix metalloproteinase-9 (pro-MMP-9) activation and involvement of upstream kinases in regulation of these processes that ultimately control cell motility and tumor growth in murine melanoma cells are not well defined. Here we report that OPN induces αvβ3 integrin-mediated phosphorylation and activation of nuclear factor inducing kinase (NIK) and enhances the interaction between phosphorylated NIK and IκBα kinase α/β (IKKα/β) in B16F10 cells. Moreover, NIK is involved in OPN-induced phosphorylations of MEK-1 and ERK1/2 in these cells. OPN induces NIK-dependent NF-κB activation through ERK/IKKα/β-mediated pathways. Furthermore, OPN enhances NIK-regulated urokinase-type plasminogen activator (uPA) secretion, uPA-dependent pro-MMP-9 activation, and cell motility. Pretreatment of cells with anti-MMP-2 antibody along with anti-MMP-9 antibody drastically inhibited the OPN-induced cell migration and chemoinvasion, whereas cells pretreated with anti-MMP-2 antibody had no effect on OPN-induced pro-MMP-9 activation suggesting that OPN induces pro-MMP-2 and pro-MMP-9 activations through two distinct pathways. Taken together, NIK acts as crucial regulator in OPN-induced MAPK/IKK-mediated NF-κB-dependent uPA secretion and MMP-9 activation thereby controlling melanoma cell motility and chemoinvasion. An erratum to this article is available at .     

The uptake of inorganic iron complexes by human melanoma cells

The human melanoma cell line, SK-MEL-28, expresses high levels of melanotransferrin. The uptake of inorganic iron (Fe) complexes compared to transferrin-bound Fe by these cells has been investigated to determine whether melanotransferrin has a role in Fe uptake. The mechanisms of Fe uptake have been characterised using 59Fe complexes of citrate, nitrilotriacetate, desferrioxamine, and 59Fe added to Eagle's minimum essential medium (MEM) and compared with human transferrin (Tf) labelled with 59Fe and iodine-125. Iron uptake from the Fe complexes of citrate, nitrilotriacetate and MEM were similar, and far greater than that from Tf at the same Fe concentration (2.5 microM). Ammonium chloride and a monoclonal antibody to the transferrin receptor (42/6), had no effect on the uptake of Fe from inorganic Fe complexes, suggesting that receptor-mediated endocytosis of Tf was not involved. The monoclonal antibody, 96.5, specific for melanotransferrin did not alter total Fe uptake but slightly increased the proportion of Fe internalised, possibly due to the modulation of the antigen by the antibody. However, from the time required for modulation to occur (approximately 2 h), the small increase in internalisation observed and the fact that no increase in total cell Fe occurred, it is suggested that melanotransferrin has little role in Fe uptake.     

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis in vivo

Melanotransferrin is a member of the transferrin family, which is comprised of serum transferrin, lactoferrin and ovotransferrin, and is highly expressed on melanoma cells compared to normal melanocytes. Since melanoma is an highly vascularized tumour that expresses melanotransferrin at high levels, we tested purified recombinant melanotransferrin for its capability to induce angiogenesis in the chick chorioallantoic membrane. Macroscopic and microscopic evaluation of the vascular density demonstrated that melanotransferrin exerts an angiogenic response quantitatively similar to that elicited by fibroblast growth factor-2. Overexpression of vascular endothelial growth factor-receptor-2 was observed in newly formed vessels, suggesting that the angiogenic activity of melanotransferrin may depend on activation of endogenous vascular endothelial growth factor. In addition, when antibodies against vascular endothelial growth factor were included in the assay, the angiogenic response was inhibited by 50%. In a Boyden chamber assay purified recombinant melanotransferrin induced chemotactic migration of vascular cells, which was decreased in the the presence of anti-vascular endothelial growth factor antibodies suggesting an involvement of vascular endothelial growth factor present in endothelial cells also in this assay. However, melanotransferrin was found not to directly bind to integrin alphavbeta3 or the vascular endothelial growth factor-receptor-2 as assessed in a BlAcore assay. A possible correlation between vascularization occurring during melanoma progression and the expression of melanotransferrin and vascular endothelial growth factor was established by immunolocalization of the two factors in sections of melanoma at different clinical steps of melanoma progression. These latter data strongly imply that melanotransferrin may participate in the vascularization of solid tumours and that inhibition of melanotransferrin could form the basis for intervention in tumours which use this pathway.