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

Abstract

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 μmol/L bumetanide plus 10 μmol/L ouabain and K + efflux stimulation with 3 mg/L amphotericin B or 5 μmol/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.