Characterization of thimet oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth

Abstract

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.

Conclusion: 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.

Figure 1

Effect of BK and B16F10-Nex2 tumor cells on pro-angiogenic closed structures formed by sprouting of endothelial cells. HUVECs were plated on Matrigel in medium supplemented with 0.2% of FCS in the presence of BK, B16F10-Nex2 supernatant, cell membrane or lysate and irradiated B16F10-Nex2 whole cells. The number of pro-angiogenic structures was counted after 18 h. One representative picture of four different treatments is shown with the respective counts. * p < 0.005 vs control; ** p < 0.0005 vs control.

Figure 2

Effect of JA-2 on in vitro Matrigel angiogenesis assay. HUVECs were plated on Matrigel in medium supplemented with 0.2% of FCS in the presence of BK, B16F10-Nex2 supernatant and JA-2 (thimet oligopeptidase inhibitor). The number of pro-angiogenic structures was counted after 18 h. * p < 0.05 vs control; ** p < 0.005 vs control.

Figure 3

HPLC analysis of FRET peptides degradation by the B16F10-Nex2 supernatant. Abz-GFSPFR-EDDnp (A, C) or Abz-GFSPFRQ-EDDnp (B, D) were incubated with recombinant oligopeptidase TOP (A, B), or B16F10-Nex2 supernatant (C, D) in 50 mM Tris-HCl pH 7.4 at 37°C. Reaction products were separated by HPLC and were identified by mass spectrometry. Chromatograms developed by fluorescence detection at λ_em. = 420 nm and λ_ex. = 320 nm.

Figure 4

HPLC analysis of neurotensin (NT) degradation by B16F10-Nex2 supernatant. Neurotensin (20 μM) was incubated for 1 h at 37°C with recombinant TOP (A), neurolysin (B) or B16F10-Nex2 supernatant (D) in 50 mM Tris-HCl, pH 7.4. The reaction products were separated by HPLC. HPLC profile of melanoma supernatant without NT is shown on panel C. The neurotensin fragments were determined by mass spectrometry.

Figure 5

HPLC analysis of neurotensin (NT) degradation by B16F10-Nex2 supernatant in the presence of bestatin. Neurotensin (20 μM) was incubated for 1 h at 37°C with recombinant TOP (A), neurolysin (B) or B16F10-Nex2 supernatant (D) in the presence of bestatin in 50 mM Tris-HCl, pH 7.4. The reaction products were separated by HPLC. HPLC profile of melanoma supernatant in the presence of bestatin without NT is shown on panel C. The neurotensin fragments were determined by mass spectrometry.

Figure 6

HPLC analysis of BK degradation by B16F10-Nex2 supernatant. BK (20 μM) was incubated for 1 h at 37°C with recombinant TOP (A) or B16F10-Nex2 supernatant (B) in 50 mM Tris-HCl, pH 7.4. The reaction products were separated by HPLC. Bradykinin fragments were determined by mass spectrometry.

Figure 7

B16F10-Nex2 proliferation assay in the presence of JA-2 and/or bestatin inhibitors. 5 × 10³ B16F10-Nex2 cells were cultivated in 96-well plates, incubated for 12, 24 and 48 h with JA-2 (3 μM) and/or bestatin (50 μM) inhibitors, and the cell proliferation was measured using MTT in comparison with Controls. *p < 0.05.

Figure 8

Effect of active and inactive rTOP on tumor cell development and animal survival after subcutaneous implantation of B16F10-Nex2 melanoma cells. 5 × 10⁴ viable cells were injected subcutaneously with 8 μg of active rTOP (A, B, open circle), inactive rTOP (C, D, open circle), and PBS (control, solid circle) in C57Bl/6 mice (4–5 animals per group). The tumor volume was measured every 2–3 days and a maximal volume of 3 cm³ was allowed before sacrifice. (B, D), tumor volume of individual animals; (A, C), survival plots. Statistical analysis of survivals was performed using Kaplan-Meier test.

Figure 9

Effect of rTOP on in vitro Matrigel angiogenesis assay. HUVECs were plated on Matrigel in medium supplemented with 0.2% of FCS in the presence of BK (1 μM), rTOP (specific activity: 231 μM/min/mg), BK+ rTOP, NT (1 μM) and A-II (1 μM). The number of pro-angiogenic structures was counted after 18 h. * p < 0.05 vs control.