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.
Andersson
B
B
Department of Medical Biosciences, Clinical Chemistry, Building 6 M, Second Floor, Umeå University, S-901 85 Umeå, Sweden.
Janson
V
V
Behnam-Motlagh
P
P
Henriksson
R
R
Grankvist
K
K
D017209:Apoptosis / Q000187:drug effects*; D001572:Benzofurans / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008175:Lung Neoplasms / Q000473:pathology; D011188:Potassium / Q000032:analysis* / Q000378:metabolism
D001572:Benzofurans; D004988:Ethers, Cyclic; C095549:potassium-binding benzofuran-isophthalate acetoxymethyl ester; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D011188:Potassium