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A role for nucleotides in support of breast cancer angiogenesis: heterologous receptor signalling.

Yokdang N, Tellez JD, Tian H, Norvell J, Barsky SH, Valencik M, Buxton IL - Br. J. Cancer (2011)

Bottom Line: We examined sNDPK secretion and its effects on human endothelial cells.Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor.Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Centre for Molecular Medicine, University of Nevada School of Medicine, Mail Stop 573, Reno, NV 89557, USA.

ABSTRACT

Background: Human breast carcinoma cells secrete an adenosine 5'-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells.

Methods: Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration.

Results: A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk(1/2), both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK(1/2) by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y(1)-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y(1), Src or VEGFR-2 antagonists.

Conclusion: sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

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Related in: MedlinePlus

Extracellular NDPK-B stimulates HEC growth and NDPK-B transphosphorylation inhibitors block the growth stimulatory effects of NDPK-B. HEC were maintained in low serum medium containing 2% FBS for 24 h before treatment with increasing concentrations of (A) VEGF, (B) NDPK-B or (C) catechin (EA/EGCG) for an additional 24 h in 2% FBS containing 0.01% (v/v) DMSO diluent in EBM media. (A) VEGF protein and (B) NDPK-B protein (C) treatment of cells with the NDPK-B antagonist catechins EA or EGCG at increasing concentrations. (D) Twenty units of NDPK-B in combination with increasing concentrations of EA. (E) Twenty units of NDPK-B in combination with increasing concentrations of EGCG. (F) Increasing concentrations of NDPK-B in combination with 10 or 30 μ concentrations of EGCG or EA. (G) The drug is removed and cells are returned to growth conditions. Data are mean±s.e.m., n=3, *P<0.05, ***P<0.001 vs no drug, +P<0.05 vs 10 μ EA. Micrographs are representative of results, n=4.
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fig5: Extracellular NDPK-B stimulates HEC growth and NDPK-B transphosphorylation inhibitors block the growth stimulatory effects of NDPK-B. HEC were maintained in low serum medium containing 2% FBS for 24 h before treatment with increasing concentrations of (A) VEGF, (B) NDPK-B or (C) catechin (EA/EGCG) for an additional 24 h in 2% FBS containing 0.01% (v/v) DMSO diluent in EBM media. (A) VEGF protein and (B) NDPK-B protein (C) treatment of cells with the NDPK-B antagonist catechins EA or EGCG at increasing concentrations. (D) Twenty units of NDPK-B in combination with increasing concentrations of EA. (E) Twenty units of NDPK-B in combination with increasing concentrations of EGCG. (F) Increasing concentrations of NDPK-B in combination with 10 or 30 μ concentrations of EGCG or EA. (G) The drug is removed and cells are returned to growth conditions. Data are mean±s.e.m., n=3, *P<0.05, ***P<0.001 vs no drug, +P<0.05 vs 10 μ EA. Micrographs are representative of results, n=4.

Mentions: Secreted NDPK-B induces angiogenesis as measured by endothelial cell tubule-like formation (Rumjahn et al, 2007). Here, we further investigated the role of NDPK-B in endothelial angiogenesis. We hypothesised that the activation of P2Y1R by extracellular NDPK-B would promote the growth of endothelial cells in vitro, and that inhibiting the function of NDPK-B would reduce endothelial cell proliferation. Human cord blood endothelial colony forming cells were stimulated for 24 h with 1–100 ng/ml VEGF (positive control) or 0.3–20 units of NDPK-B. Extracellular NDPK-B was as effective as VEGF in stimulating HEC proliferation (Figures 5A and B) over the 24-h period. The concentration range of NDPK employed here (0.3–30 units=0.5–500 ng ml−1) correlates well with the activity of sNDPK measured in breast cancer cell conditioned media (Ave. 272 ng ml−1). If the effect of NDPK-B relies on ATP generation, catechin compounds known to inhibit NDPK activity would prevent NDPK effects on cell proliferation (Figure 5C). Ellagic acid was more effective than EGCG in inhibiting cell growth consistent with its effect as an NDPK inhibitor (Buxton, 2008) and blocker of the effect of NDPK to promote angiogenesis (Rumjahn et al, 2007). To examine the effect of extracellular NDPK-B protein and its inhibitors in primary HEC proliferation, HEC were incubated with various concentrations of EA and EGCG (1–30 μ) with and without 20 units of NDPK-B protein for 24 h (Figures 5D and E). The activity of NDPK-B to induce angiogenesis via cell proliferation was diminished to the basal level in the presence of 10 μ EA (Figure 5D). The results indicate that 10 and 30 μ EGCG inhibit HEC proliferation (Figures 5C and E), but when combined with 20 units of extracellular NDPK-B, EGCG inhibition is trivial (Figure 5E). Cells were also treated with various amounts of NDPK-B (0, 5, 10 and 20 units) in the presence of EGCG or EA (10 or 30 μ) (Figure 5F). The effects of EA and EGCG were dose dependent (Figures 5D and E) and confirmed that EA is more efficacious than EGCG in preventing NDPK-mediated cell proliferation (Figures 5C–F). Control experiments confirmed that EA was non-toxic to the cells and show that the drug acts in a reversible manner (Figure 5G).


A role for nucleotides in support of breast cancer angiogenesis: heterologous receptor signalling.

Yokdang N, Tellez JD, Tian H, Norvell J, Barsky SH, Valencik M, Buxton IL - Br. J. Cancer (2011)

Extracellular NDPK-B stimulates HEC growth and NDPK-B transphosphorylation inhibitors block the growth stimulatory effects of NDPK-B. HEC were maintained in low serum medium containing 2% FBS for 24 h before treatment with increasing concentrations of (A) VEGF, (B) NDPK-B or (C) catechin (EA/EGCG) for an additional 24 h in 2% FBS containing 0.01% (v/v) DMSO diluent in EBM media. (A) VEGF protein and (B) NDPK-B protein (C) treatment of cells with the NDPK-B antagonist catechins EA or EGCG at increasing concentrations. (D) Twenty units of NDPK-B in combination with increasing concentrations of EA. (E) Twenty units of NDPK-B in combination with increasing concentrations of EGCG. (F) Increasing concentrations of NDPK-B in combination with 10 or 30 μ concentrations of EGCG or EA. (G) The drug is removed and cells are returned to growth conditions. Data are mean±s.e.m., n=3, *P<0.05, ***P<0.001 vs no drug, +P<0.05 vs 10 μ EA. Micrographs are representative of results, n=4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101911&req=5

fig5: Extracellular NDPK-B stimulates HEC growth and NDPK-B transphosphorylation inhibitors block the growth stimulatory effects of NDPK-B. HEC were maintained in low serum medium containing 2% FBS for 24 h before treatment with increasing concentrations of (A) VEGF, (B) NDPK-B or (C) catechin (EA/EGCG) for an additional 24 h in 2% FBS containing 0.01% (v/v) DMSO diluent in EBM media. (A) VEGF protein and (B) NDPK-B protein (C) treatment of cells with the NDPK-B antagonist catechins EA or EGCG at increasing concentrations. (D) Twenty units of NDPK-B in combination with increasing concentrations of EA. (E) Twenty units of NDPK-B in combination with increasing concentrations of EGCG. (F) Increasing concentrations of NDPK-B in combination with 10 or 30 μ concentrations of EGCG or EA. (G) The drug is removed and cells are returned to growth conditions. Data are mean±s.e.m., n=3, *P<0.05, ***P<0.001 vs no drug, +P<0.05 vs 10 μ EA. Micrographs are representative of results, n=4.
Mentions: Secreted NDPK-B induces angiogenesis as measured by endothelial cell tubule-like formation (Rumjahn et al, 2007). Here, we further investigated the role of NDPK-B in endothelial angiogenesis. We hypothesised that the activation of P2Y1R by extracellular NDPK-B would promote the growth of endothelial cells in vitro, and that inhibiting the function of NDPK-B would reduce endothelial cell proliferation. Human cord blood endothelial colony forming cells were stimulated for 24 h with 1–100 ng/ml VEGF (positive control) or 0.3–20 units of NDPK-B. Extracellular NDPK-B was as effective as VEGF in stimulating HEC proliferation (Figures 5A and B) over the 24-h period. The concentration range of NDPK employed here (0.3–30 units=0.5–500 ng ml−1) correlates well with the activity of sNDPK measured in breast cancer cell conditioned media (Ave. 272 ng ml−1). If the effect of NDPK-B relies on ATP generation, catechin compounds known to inhibit NDPK activity would prevent NDPK effects on cell proliferation (Figure 5C). Ellagic acid was more effective than EGCG in inhibiting cell growth consistent with its effect as an NDPK inhibitor (Buxton, 2008) and blocker of the effect of NDPK to promote angiogenesis (Rumjahn et al, 2007). To examine the effect of extracellular NDPK-B protein and its inhibitors in primary HEC proliferation, HEC were incubated with various concentrations of EA and EGCG (1–30 μ) with and without 20 units of NDPK-B protein for 24 h (Figures 5D and E). The activity of NDPK-B to induce angiogenesis via cell proliferation was diminished to the basal level in the presence of 10 μ EA (Figure 5D). The results indicate that 10 and 30 μ EGCG inhibit HEC proliferation (Figures 5C and E), but when combined with 20 units of extracellular NDPK-B, EGCG inhibition is trivial (Figure 5E). Cells were also treated with various amounts of NDPK-B (0, 5, 10 and 20 units) in the presence of EGCG or EA (10 or 30 μ) (Figure 5F). The effects of EA and EGCG were dose dependent (Figures 5D and E) and confirmed that EA is more efficacious than EGCG in preventing NDPK-mediated cell proliferation (Figures 5C–F). Control experiments confirmed that EA was non-toxic to the cells and show that the drug acts in a reversible manner (Figure 5G).

Bottom Line: We examined sNDPK secretion and its effects on human endothelial cells.Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor.Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Centre for Molecular Medicine, University of Nevada School of Medicine, Mail Stop 573, Reno, NV 89557, USA.

ABSTRACT

Background: Human breast carcinoma cells secrete an adenosine 5'-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells.

Methods: Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration.

Results: A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk(1/2), both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK(1/2) by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y(1)-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y(1), Src or VEGFR-2 antagonists.

Conclusion: sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

Show MeSH
Related in: MedlinePlus