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 (IC 50 ¼ 8-10 mM) and EA (IC 50 ¼ 2-3 mM) suppressed 435S cell growth, but had less effect on human CD31 þ EC growth. Epigallocatechin gallate (IC 50 ¼ 11 mM) and EA (IC 50 ¼ 1 mM) also prevented CD31 þ EC tubulogenesis on Matrigelt. 435S cellconditioned media induced tubulogenesis in a cell number, time, and nucleotide-dependent manner. Ellagic acid (1 mM), but not equimolar EGCG, reduced cell number-dependent angiogenesis. P2Y 1 receptor activation by NDPK-generated nucleotide (100 mM ATP) or by 10 mM 2-methyl-thio-ATP (2MS-ATP) promoted tubulogenesis on collagen and was blocked by the P2Y 1 antagonist MRS2179 (10 mM). Physiological amounts of purified as well as 435S cell-secreted NDPK also promoted angiogenesis that was attenuated by NDPK depletion or 10 mM 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.
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 μ M 2-methyl-thio-ATP (2MS-ATP)) of angiogenesis is suppressed by the VEGFR-2 tyrosine kinase inhibitor, SU1498 (1 μ M ). Phosphorylation of VEGFR-2 by 0.0262 or 2.62 n M VEGF was comparable with 0.01 or 10 μ M 2MS-ATP stimulation of the P2Y 1 R. 2MS-ATP, and VEGF stimulation increased tyrosine phosphorylation at tyr1175. 2MS-ATP (0.1–10 μ M ) also stimulated EC tubulogenesis in a dose-dependent manner. The addition of sub-maximal VEGF (70 p M ) in the presence of increasing concentrations of 2MS-ATP yielded additive effects at 2MS-ATP concentrations <3 μ M , whereas producing saturated and less than additive effects at ⩾3 μ M . 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.
Including undergraduate research in STEM education is a well-supported and growing high-impact practice that has been made much more scalable through integrating these experiences into the classroom. Here we describe a new biochemistry Course-based Undergraduate Research Experience (CURE) that follows a design-to-data workflow with a strong connection to a worldwide community of protein modeling software developers. Analysis of psychosocial developments in association with participating in this CURE from the first set of students formally participating in the course suggest a beneficial effect on attributes associated with STEM persistence. To increase successful propagation, the design of the CURE's curriculum, supporting learning materials, and instructor resources are provided to make it facile for faculty at any institution to join this network and implement the CURE. With this foundation, we expect student participation and the data set to continue to grow.
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