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

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.