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.
Subject(s)
Breast Neoplasms/blood supply , Breast Neoplasms/metabolism , Nucleoside-Diphosphate Kinase/metabolism , Nucleotides/metabolism , Breast Neoplasms/pathology , Cell Growth Processes/physiology , Cell Line, Tumor , Endothelial Cells/metabolism , Female , Humans , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Phosphorylation , Proto-Oncogene Mas , Signal Transduction , Tumor Cells, Cultured , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
P2Y purine nucleotide receptors (P2YRs) promote endothelial cell tubulogenesis through breast cancer cell-secreted nucleoside diphosphate kinase (NDPK). We tested the hypothesis that activated P2Y(1) receptors transactivate vascular endothelial growth factor receptor (VEGFR-2) in angiogenic signaling. P2Y(1)R stimulation (10 microM 2-methyl-thio-ATP (2MS-ATP)) of angiogenesis is suppressed by the VEGFR-2 tyrosine kinase inhibitor, SU1498 (1 microM). Phosphorylation of VEGFR-2 by 0.0262 or 2.62 nM VEGF was comparable with 0.01 or 10 microM 2MS-ATP stimulation of the P2Y(1)R. 2MS-ATP, and VEGF stimulation increased tyrosine phosphorylation at tyr1175. 2MS-ATP (0.1-10 microM) also stimulated EC tubulogenesis in a dose-dependent manner. The addition of sub-maximal VEGF (70 pM) in the presence of increasing concentrations of 2MS-ATP yielded additive effects at 2MS-ATP concentrations <3 microM, whereas producing saturated and less than additive effects at > or =3 microM. We propose that the VEGF receptor can be activated in the absence of VEGF, and that the P2YR-VEGFR2 interaction and resulting signal transduction is a critical determinant of vascular homoeostasis and tumour-mediated angiogenesis.
Subject(s)
Neovascularization, Pathologic/physiopathology , Receptors, Purinergic P2/physiology , Vascular Endothelial Growth Factor A/physiology , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Cardiovascular Physiological Phenomena , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Female , Flow Cytometry , Humans , Neovascularization, Pathologic/pathology , Phosphorylation , Receptors, Purinergic P2Y1 , Signal Transduction/physiology , Vascular Endothelial Growth Factor Receptor-2/physiologyABSTRACT
MDA-MB-435S human breast cancer cells (435S) secrete nucleoside diphosphate kinase (NDPK) that supports metastases and is inhibited by epigallocatechin gallate (EGCG) and ellagic acid (EA). We hypothesise that 435S cell-secreted NDPK-B supports tumour formation by modulating ATP levels locally to activate endothelial cell (EC) P2Y receptor-mediated angiogenesis. Epigallocatechin gallate (IC50=8-10 microM) and EA (IC50=2-3 microM) suppressed 435S cell growth, but had less effect on human CD31+ EC growth. Epigallocatechin gallate (IC50=11 microM) and EA (IC50=1 microM) also prevented CD31+ EC tubulogenesis on Matrigeltrade mark. 435S cell-conditioned media induced tubulogenesis in a cell number, time, and nucleotide-dependent manner. Ellagic acid (1 microM), but not equimolar EGCG, reduced cell number-dependent angiogenesis. P2Y 1 receptor activation by NDPK-generated nucleotide (100 microM ATP) or by 10 microM 2-methyl-thio-ATP (2MS-ATP) promoted tubulogenesis on collagen and was blocked by the P2Y 1 antagonist MRS2179 (10 microM). Physiological amounts of purified as well as 435S cell-secreted NDPK also promoted angiogenesis that was attenuated by NDPK depletion or 10 microM MRS2179, indicating a P2Y 1 receptor-mediated pathway. These results support the notion that secreted NDPK mediates angiogenesis via P2Y receptor signalling and suggests that novel inhibitors of NDPK may be useful as therapeutics.
