Calbindin-D28k and calcium sensing receptor cooperate in MCF-7 human breast cancer cells

Int J Oncol. 2004 May;24(5):1111-9.


The metastases of breast cancer cells to bone result in osteolysis and release of Ca2+. Ca2+ as a primary signal transducer can regulate the expression patterns of cell signaling systems. The extracellular calcium ion concentration sensing receptor CaR is a 123 kDa G-protein coupled membrane protein that resides within caveolin-rich regions as a dimer. CaR is involved in regulating several cellular processes such as proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a 28 kDa high affinity calcium-binding protein and it is involved in regulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to extracellular calcium ion concentration, [Ca2+]o, and neomycin sulfate, and spatial interactions of CaR with calbindin-D28k in MCF-7 human breast cancer cells were studied. Fura-2 loaded MCF-7 cells were exposed to increasing concentrations of CaCl2 or neomycin sulfate and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 or neomycin sulfate caused an increase in [Ca2+]i. The normalized dose response curves fitting yielded Hill co-efficient values of 4.32+/-0.63 and 1.49+/-0.14 for Ca2+ and neomycin sulfate respectively, thus indicating highly co-operative, 4-5 binding sites for Ca2+ and 1-2 binding site(s) for neomycin sulfate on CaR. The EC50 values were 21+/-1.6 mM and 43+/-3.5 micro M for CaCl2 and neomycin sulfate respectively. The confocal microscopy data, obtained by using a highly sensitive tyramide signal amplification technology for immunofluorescence detection, showed CaR and calbindin-D28k were co-localized when cells were exposed to 200 micro M neomycin sulfate, whereas in control cells there was no co-localization of these two proteins. We hypothesize that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / pathology
  • Calbindin 1
  • Calbindins
  • Calcium / metabolism*
  • Calcium Chloride / pharmacology
  • Calcium Signaling*
  • Cytosol
  • Female
  • Fluorescent Dyes
  • Fura-2
  • Humans
  • Microscopy, Confocal
  • Neomycin / pharmacology
  • Protein Binding
  • Protein Synthesis Inhibitors / pharmacology
  • Receptors, Calcium-Sensing / metabolism*
  • S100 Calcium Binding Protein G / metabolism*
  • Tumor Cells, Cultured


  • CALB1 protein, human
  • Calbindin 1
  • Calbindins
  • Fluorescent Dyes
  • Protein Synthesis Inhibitors
  • Receptors, Calcium-Sensing
  • S100 Calcium Binding Protein G
  • Neomycin
  • Calcium Chloride
  • Calcium
  • Fura-2