Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients

PLoS One. 2012;7(9):e42541. doi: 10.1371/journal.pone.0042541. Epub 2012 Sep 25.

Abstract

Background: Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+) entry (SOCE), which is activated by a depletion of the intracellular Ca(2+) pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+)-sensor, Stim1, and the plasmalemmal Ca(2+) channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+) influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.

Methodology/principal findings: The present study employed Ca(2+) imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+) and Gd(3+). Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca(2+) release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca(2+) buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.

Conclusions: SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Adult
  • Aged
  • Aged, 80 and over
  • Boron Compounds / pharmacology
  • Cadmium / pharmacology
  • Calcium Channels / genetics
  • Calcium Channels / metabolism
  • Carcinoma, Renal Cell / blood supply*
  • Carcinoma, Renal Cell / genetics
  • Carcinoma, Renal Cell / metabolism
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Endothelial Cells / pathology
  • Female
  • Gene Expression Regulation, Neoplastic* / drug effects
  • Humans
  • Indoles / pharmacology
  • Intracellular Calcium-Sensing Proteins
  • Kidney Neoplasms / blood supply*
  • Kidney Neoplasms / genetics
  • Kidney Neoplasms / metabolism
  • Lanthanum / pharmacology
  • Male
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Middle Aged
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism
  • Neoplastic Stem Cells / drug effects
  • Neoplastic Stem Cells / metabolism*
  • Neoplastic Stem Cells / pathology
  • Neovascularization, Pathologic
  • ORAI1 Protein
  • Primary Cell Culture
  • Signal Transduction / drug effects
  • Stromal Interaction Molecule 1
  • TRPC Cation Channels / genetics
  • TRPC Cation Channels / metabolism

Substances

  • Boron Compounds
  • Calcium Channels
  • Indoles
  • Intracellular Calcium-Sensing Proteins
  • Membrane Proteins
  • Neoplasm Proteins
  • ORAI1 Protein
  • ORAI1 protein, human
  • SARAF protein, human
  • STIM1 protein, human
  • Stromal Interaction Molecule 1
  • TRPC Cation Channels
  • transient receptor potential cation channel, subfamily C, member 1
  • Cadmium
  • Lanthanum
  • Adenosine Triphosphate
  • 2-aminoethoxydiphenyl borate
  • cyclopiazonic acid

Grants and funding

This work was supported by a grant from Ricerca Corrente, #674, IRCCS Policlinico San Matteo Foundation, Pavia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.