Purinergic regulation of angiogenesis by human breast carcinoma-secreted nucleoside diphosphate kinase

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 mu M) and EA (IC50 = 2-3 mu M) suppressed 435S cell growth, but had less effect on human CD31(+) EC growth. Epigallocatechin gallate (IC50 = 11 mu M) and EA (IC50 = 1 mu M) also prevented CD31(+) EC tubulogenesis on Matrigel(TM). 435S cell-conditioned media induced tubulogenesis in a cell number, time, and nucleotide-dependent manner. Ellagic acid (1 mu M), but not equimolar EGCG, reduced cell number-dependent angiogenesis. P2Y(1) receptor activation by NDPK-generated nucleotide (100 mu M ATP) or by 10 mu M 2-methyl-thio-ATP (2MS-ATP) promoted tubulogenesis on collagen and was blocked by the P2Y1 antagonist MRS2179 (10 mu M). Physiological amounts of purified as well as 435S cell- secreted NDPK also promoted angiogenesis that was attenuated by NDPK depletion or 10 mu M MRS2179, indicating a P2Y1 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.