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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 induces HEC Erk1/2 phosphorylation. HEC were maintained in low serum medium containing 2% FBS for 24 h before pretreatment with antagonists followed by agonists. (A) Representative western blots showing both Erk and phospho-Erk (pErk1/2). (B) Average data from multiple experiments. All treatments gave significantly lower pErk1/2 (P<0.001) compared with addition of NDPK-B in the presence of phosphoryl donor and acceptor. Data are mean±s.e.m. normalised to total Erk expression by densitometry, n=3. (C) Representative confocal images of pErk fluorescence in HEC stimulated as shown (control 0.01% DMSO). (Bar=20 μm).
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fig3: Extracellular NDPK-B induces HEC Erk1/2 phosphorylation. HEC were maintained in low serum medium containing 2% FBS for 24 h before pretreatment with antagonists followed by agonists. (A) Representative western blots showing both Erk and phospho-Erk (pErk1/2). (B) Average data from multiple experiments. All treatments gave significantly lower pErk1/2 (P<0.001) compared with addition of NDPK-B in the presence of phosphoryl donor and acceptor. Data are mean±s.e.m. normalised to total Erk expression by densitometry, n=3. (C) Representative confocal images of pErk fluorescence in HEC stimulated as shown (control 0.01% DMSO). (Bar=20 μm).

Mentions: Erk1/2 phosphorylation via the Ras-RAF-Erk pathway induces cell proliferation contributing to the activation of angiogenesis and metastatic tumour growth. We hypothesised that extracellular NDPK-B induces exogenous mitogenic signalling from endothelial P2Y1R to MAPK. Stimulation of HEC with NDPK-B in the presence of phosphoryl donor and acceptor produced high Erk1/2 phosphorylation (Figure 3A, lanes 2 and 7, B and C). With addition of NDPK-B or GTP/ADP alone, Erk1/2 phosphorylation was reduced by 60% compared with maximum stimulation (Figure 3B), indicating that they produced partial activation of Erk1/2 consistent with sub-maximal P2Y stimulation. The effect of NDPK-B was inhibited by 10 μ EA (Figure 3A, lane 5) which diminished Erk1/2 phosphorylation back to near control levels (Figure 3). VEGF165 was employed as positive control and gave a 11-fold increase above the negative control (data not shown). Suramin (100 μ), a non-specific endothelial P2Y receptor inhibitor (Beindl et al, 1996), blocked 70% of the effect of 10 units NDPK-B plus donor/acceptor (Figure 3). Measurement of Erk1/2 phosphorylation by immunofluorescence confirmed that extracellular NDPK-B induced Erk1/2 phosphorylation, which was inhibited by the NDPK transphosphorylation inhibitor (EA) and non-specific P2Y receptor inhibitor suramin (Figure 3C).


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 induces HEC Erk1/2 phosphorylation. HEC were maintained in low serum medium containing 2% FBS for 24 h before pretreatment with antagonists followed by agonists. (A) Representative western blots showing both Erk and phospho-Erk (pErk1/2). (B) Average data from multiple experiments. All treatments gave significantly lower pErk1/2 (P<0.001) compared with addition of NDPK-B in the presence of phosphoryl donor and acceptor. Data are mean±s.e.m. normalised to total Erk expression by densitometry, n=3. (C) Representative confocal images of pErk fluorescence in HEC stimulated as shown (control 0.01% DMSO). (Bar=20 μm).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101911&req=5

fig3: Extracellular NDPK-B induces HEC Erk1/2 phosphorylation. HEC were maintained in low serum medium containing 2% FBS for 24 h before pretreatment with antagonists followed by agonists. (A) Representative western blots showing both Erk and phospho-Erk (pErk1/2). (B) Average data from multiple experiments. All treatments gave significantly lower pErk1/2 (P<0.001) compared with addition of NDPK-B in the presence of phosphoryl donor and acceptor. Data are mean±s.e.m. normalised to total Erk expression by densitometry, n=3. (C) Representative confocal images of pErk fluorescence in HEC stimulated as shown (control 0.01% DMSO). (Bar=20 μm).
Mentions: Erk1/2 phosphorylation via the Ras-RAF-Erk pathway induces cell proliferation contributing to the activation of angiogenesis and metastatic tumour growth. We hypothesised that extracellular NDPK-B induces exogenous mitogenic signalling from endothelial P2Y1R to MAPK. Stimulation of HEC with NDPK-B in the presence of phosphoryl donor and acceptor produced high Erk1/2 phosphorylation (Figure 3A, lanes 2 and 7, B and C). With addition of NDPK-B or GTP/ADP alone, Erk1/2 phosphorylation was reduced by 60% compared with maximum stimulation (Figure 3B), indicating that they produced partial activation of Erk1/2 consistent with sub-maximal P2Y stimulation. The effect of NDPK-B was inhibited by 10 μ EA (Figure 3A, lane 5) which diminished Erk1/2 phosphorylation back to near control levels (Figure 3). VEGF165 was employed as positive control and gave a 11-fold increase above the negative control (data not shown). Suramin (100 μ), a non-specific endothelial P2Y receptor inhibitor (Beindl et al, 1996), blocked 70% of the effect of 10 units NDPK-B plus donor/acceptor (Figure 3). Measurement of Erk1/2 phosphorylation by immunofluorescence confirmed that extracellular NDPK-B induced Erk1/2 phosphorylation, which was inhibited by the NDPK transphosphorylation inhibitor (EA) and non-specific P2Y receptor inhibitor suramin (Figure 3C).

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