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Low doses of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, stimulate angiogenesis by regulating expression of urokinase type plasminogen activator and matrix metalloprotease 2.

Caldini R, Fanti E, Magnelli L, Barletta E, Tanganelli E, Zampieri M, Chevanne M - (2011)

Bottom Line: In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells.Our results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy.Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Pathology and Oncology, University of Florence, viale G,B, Morgagni 50, 50134 Florence, Italy. marta.chevanne@unifi.it.

ABSTRACT

Background: Poly(ADP-Ribose) polymerase (PARP) activity has been demonstrated fundamental in many cellular processes, including DNA repair, cell proliferation and differentiation. In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells. In recent times, PARP inhibitors have entered in clinical trials to potentiate cancer treatments by preventing DNA repair, but little is known about the effects performed by different drug concentrations on neoangiogenesis, an essential step in tumor growth.

Methods: Human umbilical vein endothelial cells were treated with 3 aminobenzamide (3ABA), a PARP inhibitor, and tested for several different cellular parameters.

Results: Here we present in vitro evidence that a low concentration of 3ABA (50 μM), stimulates angiogenesis by decreasing fibrinolytic activity, carried out by urokinase-type plasminogen activator (uPA), and by enhancing matrix metalloprotease-2 (MMP-2) gelatinolytic activity, in fibroblast growth factor-2-stimulated endothelial cells. These unbalanced pathways modify in vitro angiogenic steps, inhibiting chemoinvasion and stimulating tubulogenic activity.

Conclusions: Our results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy. Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

No MeSH data available.


Related in: MedlinePlus

Effect of 3ABA on MMPs gelatinolytic activity. (a) Representative zymogram, showing MMP-2 and MMP-9 activity in medium from untreated cells (Control), in the presence of FGF2 (10 ng/ml), of FGF2 plus 3ABA (50 μM), and of 3ABA. HT1080 human fibrosarcoma cell conditioned medium was used as a marker of molecular weight. The areas of protease activity appeared as dark bands. (b) On the basis of image analysis of zymogram reported in Figure 5a, the percent amount of MMP-2 active form (MMP-2) over the summation (Σ) of pro-MMP-2 plus MMP-2 amounts was calculated. (c) Quantification of MMP-2 activity. Densitometric analysis of MMP-2 activity in medium from cells incubated in the absence (Control), in the presence of FGF2, of FGF2 plus 3ABA, and of 3ABA alone. Exposure of FGF2-stimulated HUVEC to 3ABA induced an increase in the active form of MMP-2 compared to both untreated cells (Control), and FGF2 stimulated cells. Values are expressed as percent of control and are mean ± SEM of three experiments. Multiple comparisons were performed by the Student-Newman-Keuls test, after Kruskal-Wallis analysis. (* p < 0.05 compared to control; # p < 0.05 compared to FGF2 treated cells).
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Figure 5: Effect of 3ABA on MMPs gelatinolytic activity. (a) Representative zymogram, showing MMP-2 and MMP-9 activity in medium from untreated cells (Control), in the presence of FGF2 (10 ng/ml), of FGF2 plus 3ABA (50 μM), and of 3ABA. HT1080 human fibrosarcoma cell conditioned medium was used as a marker of molecular weight. The areas of protease activity appeared as dark bands. (b) On the basis of image analysis of zymogram reported in Figure 5a, the percent amount of MMP-2 active form (MMP-2) over the summation (Σ) of pro-MMP-2 plus MMP-2 amounts was calculated. (c) Quantification of MMP-2 activity. Densitometric analysis of MMP-2 activity in medium from cells incubated in the absence (Control), in the presence of FGF2, of FGF2 plus 3ABA, and of 3ABA alone. Exposure of FGF2-stimulated HUVEC to 3ABA induced an increase in the active form of MMP-2 compared to both untreated cells (Control), and FGF2 stimulated cells. Values are expressed as percent of control and are mean ± SEM of three experiments. Multiple comparisons were performed by the Student-Newman-Keuls test, after Kruskal-Wallis analysis. (* p < 0.05 compared to control; # p < 0.05 compared to FGF2 treated cells).

Mentions: MMP-2 and MMP-9 activity levels in the medium were determined by gelatin zymography. Cell-free aliquots of serum-free culture medium (conditioned medium) were loaded on 8% SDS-PAGE gels containing 1 mg/ml gelatin. The loading volume of each sample was adjusted in proportion to the protein content in the culture well from which the sample was taken, and ranged from 8 to 25 μl. After electrophoresis, the gel was rinsed with 2.5% Triton X-100 to remove SDS and restore enzyme activity, incubated 16-20 h at 37°C in substrate buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% dodecylpolyethyleneglycolether), stained with 0.1% Comassie Brilliant Blue R-250 dye (Sigma Co.) in 40% methanol/7% acetic acid for 1 h, and destained with 40% methanol/7% acetic acid until bands were apparent: the location of gelatinolytic activity was detected as clear bands, and shown in Figure 5a as inverted dark bands for a better visual prominence. HT1080 human fibrosarcoma cell conditioned medium was used as a marker of molecular weight. Arbitrary density of an individual cleavage band was determined by scanning densitometry using ImageJ software.


Low doses of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, stimulate angiogenesis by regulating expression of urokinase type plasminogen activator and matrix metalloprotease 2.

Caldini R, Fanti E, Magnelli L, Barletta E, Tanganelli E, Zampieri M, Chevanne M - (2011)

Effect of 3ABA on MMPs gelatinolytic activity. (a) Representative zymogram, showing MMP-2 and MMP-9 activity in medium from untreated cells (Control), in the presence of FGF2 (10 ng/ml), of FGF2 plus 3ABA (50 μM), and of 3ABA. HT1080 human fibrosarcoma cell conditioned medium was used as a marker of molecular weight. The areas of protease activity appeared as dark bands. (b) On the basis of image analysis of zymogram reported in Figure 5a, the percent amount of MMP-2 active form (MMP-2) over the summation (Σ) of pro-MMP-2 plus MMP-2 amounts was calculated. (c) Quantification of MMP-2 activity. Densitometric analysis of MMP-2 activity in medium from cells incubated in the absence (Control), in the presence of FGF2, of FGF2 plus 3ABA, and of 3ABA alone. Exposure of FGF2-stimulated HUVEC to 3ABA induced an increase in the active form of MMP-2 compared to both untreated cells (Control), and FGF2 stimulated cells. Values are expressed as percent of control and are mean ± SEM of three experiments. Multiple comparisons were performed by the Student-Newman-Keuls test, after Kruskal-Wallis analysis. (* p < 0.05 compared to control; # p < 0.05 compared to FGF2 treated cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117742&req=5

Figure 5: Effect of 3ABA on MMPs gelatinolytic activity. (a) Representative zymogram, showing MMP-2 and MMP-9 activity in medium from untreated cells (Control), in the presence of FGF2 (10 ng/ml), of FGF2 plus 3ABA (50 μM), and of 3ABA. HT1080 human fibrosarcoma cell conditioned medium was used as a marker of molecular weight. The areas of protease activity appeared as dark bands. (b) On the basis of image analysis of zymogram reported in Figure 5a, the percent amount of MMP-2 active form (MMP-2) over the summation (Σ) of pro-MMP-2 plus MMP-2 amounts was calculated. (c) Quantification of MMP-2 activity. Densitometric analysis of MMP-2 activity in medium from cells incubated in the absence (Control), in the presence of FGF2, of FGF2 plus 3ABA, and of 3ABA alone. Exposure of FGF2-stimulated HUVEC to 3ABA induced an increase in the active form of MMP-2 compared to both untreated cells (Control), and FGF2 stimulated cells. Values are expressed as percent of control and are mean ± SEM of three experiments. Multiple comparisons were performed by the Student-Newman-Keuls test, after Kruskal-Wallis analysis. (* p < 0.05 compared to control; # p < 0.05 compared to FGF2 treated cells).
Mentions: MMP-2 and MMP-9 activity levels in the medium were determined by gelatin zymography. Cell-free aliquots of serum-free culture medium (conditioned medium) were loaded on 8% SDS-PAGE gels containing 1 mg/ml gelatin. The loading volume of each sample was adjusted in proportion to the protein content in the culture well from which the sample was taken, and ranged from 8 to 25 μl. After electrophoresis, the gel was rinsed with 2.5% Triton X-100 to remove SDS and restore enzyme activity, incubated 16-20 h at 37°C in substrate buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% dodecylpolyethyleneglycolether), stained with 0.1% Comassie Brilliant Blue R-250 dye (Sigma Co.) in 40% methanol/7% acetic acid for 1 h, and destained with 40% methanol/7% acetic acid until bands were apparent: the location of gelatinolytic activity was detected as clear bands, and shown in Figure 5a as inverted dark bands for a better visual prominence. HT1080 human fibrosarcoma cell conditioned medium was used as a marker of molecular weight. Arbitrary density of an individual cleavage band was determined by scanning densitometry using ImageJ software.

Bottom Line: In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells.Our results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy.Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Pathology and Oncology, University of Florence, viale G,B, Morgagni 50, 50134 Florence, Italy. marta.chevanne@unifi.it.

ABSTRACT

Background: Poly(ADP-Ribose) polymerase (PARP) activity has been demonstrated fundamental in many cellular processes, including DNA repair, cell proliferation and differentiation. In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells. In recent times, PARP inhibitors have entered in clinical trials to potentiate cancer treatments by preventing DNA repair, but little is known about the effects performed by different drug concentrations on neoangiogenesis, an essential step in tumor growth.

Methods: Human umbilical vein endothelial cells were treated with 3 aminobenzamide (3ABA), a PARP inhibitor, and tested for several different cellular parameters.

Results: Here we present in vitro evidence that a low concentration of 3ABA (50 μM), stimulates angiogenesis by decreasing fibrinolytic activity, carried out by urokinase-type plasminogen activator (uPA), and by enhancing matrix metalloprotease-2 (MMP-2) gelatinolytic activity, in fibroblast growth factor-2-stimulated endothelial cells. These unbalanced pathways modify in vitro angiogenic steps, inhibiting chemoinvasion and stimulating tubulogenic activity.

Conclusions: Our results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy. Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

No MeSH data available.


Related in: MedlinePlus