Induction of apoptosis by intracellular potassium ion depletion: using the fluorescent dye PBFI in a 96-well plate method in cultured lung cancer cells

Toxicol In Vitro. 2006 Sep;20(6):986-94. doi: 10.1016/j.tiv.2005.12.013. Epub 2006 Feb 17.


Depletion of intracellular potassium ions (K+) is necessary for cells to shrink, activate caspases and induce DNA fragmentation, events which are features of apoptosis. Here we describe a 96-well plate method using the cell permeable form of K+ binding benzofuran isophtalate (PBFI-AM) to measure intracellular K+ content in relation to untreated control. Cultured human pulmonary mesothelioma cells (P31) and small-cell lung cancer cells (U1690) were treated with K+ flux modulators in order to deprive the cells of intracellular K+. The combination of K+ influx inhibition with 10 micromol/L bumetanide plus 10 micromol/L ouabain and K+ efflux stimulation with 3 mg/L amphotericin B or 5 micromol/L nigericin efficiently reduced the intracellular K+ content after 3 h. Manipulation of K+ fluxes with subsequent intracellular K+ depletion induced apoptosis of lung cancer cells, as detected by caspase-3 activity after 3 h K+ depletion followed by 24 h proliferation and TUNEL positive staining after 48 h proliferation. We concluded that the PBFI-AM assay was a useful tool to determine intracellular K+ content in relation to untreated control, and that intracellular K+ depletion of lung cancer cells by clinically used drugs of relevant concentrations induced apoptosis. These findings may lead to novel therapeutic strategies in the treatment of lung cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects*
  • Benzofurans / pharmacology*
  • Caspase 3
  • Caspases / metabolism
  • Cell Line, Tumor
  • Ethers, Cyclic / pharmacology*
  • Humans
  • In Situ Nick-End Labeling
  • Lung Neoplasms / pathology
  • Potassium / analysis*
  • Potassium / metabolism


  • Benzofurans
  • Ethers, Cyclic
  • potassium-binding benzofuran-isophthalate acetoxymethyl ester
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Potassium