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.
Department of Medical Biosciences, Clinical Chemistry, Building 6 M, Second Floor, Umeå University, S-901 85 Umeå, Sweden.
B
B
Andersson
V
V
Janson
P
P
Behnam-Motlagh
R
R
Henriksson
K
K
Grankvist
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