Characterization of thimet oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth.
BACKGROUND
Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading oligopeptidases which have been investigated.


RESULTS
Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-oligopeptidase inhibitor, JA-2. Thimet oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin - induced angiogenesis. A possible regulation of the homologous tumor enzyme in the perivascular microenvironment is suggested based on the observed rTOP inhibition by an S-nitrosothiol NO donor.


CONCLUSIONS
Data show that melanoma cells secrete endo-oligopeptidases which have an important role in tumor proliferation in vitro and in vivo. rTOP inhibited growth of subcutaneously injected B16F10-Nex2 cells in mice. TOP from tumor cells and bradykinin in endothelial cells are two antagonist factors that may control angiogenesis essential for melanoma growth. A regulatory role of NO or S-nitrosothiols is suggested.
Paschoalin
Thaysa
T
Department of Microbiology, Immunology and Parasitology, Experimental Oncology Unit (UNONEX), Federal University of São Paulo, São Paulo, Brazil. thaysa@ecb.epm.br
Carmona
Adriana K
AK
Rodrigues
Elaine G
EG
Oliveira
Vitor
V
Monteiro
Hugo P
HP
Juliano
Maria A
MA
Juliano
Luiz
L
Travassos
Luiz R
LR
D042501:Angiogenic Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D001920:Bradykinin / Q000378:metabolism; D002457:Cell Extracts; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D018920:Coculture Techniques; D003094:Collagen; D017077:Culture Media, Conditioned / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006868:Hydrolysis; D007797:Laminin; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008546:Melanoma, Experimental / Q000098:blood supply / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000201:enzymology* / Q000473:pathology / Q000517:prevention & control; D009496:Neurotensin / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011509:Proteoglycans; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D013379:Substrate Specificity
D042501:Angiogenic Proteins; D000970:Antineoplastic Agents; D002457:Cell Extracts; D017077:Culture Media, Conditioned; D004151:Dipeptides; D004338:Drug Combinations; C404521:JA 2 compound; D007797:Laminin; D020030:Nitric Oxide Donors; D009842:Oligopeptides; D010455:Peptides; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011509:Proteoglycans; C059485:matrigel; D009569:Nitric Oxide; D009496:Neurotensin; C092562:prolylisoleucine; D026423:S-Nitroso-N-Acetylpenicillamine; D003094:Collagen; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; C062316:neurolysin; D007930:Leucine; C012211:ubenimex; D001920:Bradykinin; C025205:1,10-phenanthroline