title: Amphotericin B-induced apoptosis and cytotoxicity is prevented by the Na+, K+, 2Cl(-)-cotransport blocker bumetanide.
abstract: Amphotericin B is the most commonly used antifungal drug although it exhibits poor effectiveness and considerable toxicity during treatment. It acts as a ionophore inducing cellular potassium efflux. The efflux of potassium, which is necessary for cell shrinkage during apoptosis, is counteracted by increased inward pumping of potassium ions. Modulation of potassium pump activity could therefore interact with programmed cell death depending on the nature of the disruption of cellular potassium homeostasis and subsequently affect the cytotoxicity of various drugs. We explored the role of apoptosis in amphotericin B-induced cytotoxicity in a mesothelioma cell line (P31) and investigated the role of K+ influx inhibitors of Na+, K+, ATPase and Na+, K+, 2Cl(-)-cotransport in these processes. Clone formation was used to determine the cytotoxicity of amphotericin B, ouabain (Na+, K+, ATPase blocker), and bumetanide (Na+, K+, 2Cl(-)-cotransport blocker), alone or in combination. Apoptosis was estimated by quantifying free nucleosomes. Amphotericin B (3.2 micromol/L, 3 mg/L) per se reduced the percentage of surviving clones to 64% and increased the number of nucleosomes by 31% compared to untreated control. When ouabain (100 micromol/L) was added to amphotericin B a less than additive effect on clone formation was seen but no reduction of nucleosomes was noted. Bumetanide (100 micromol/L) per se was not cytotoxic but increased cellular nucleosome expression. Bumetanide eradicated amphotericin B-induced reduction of formed clones and generated nucleosomes. In conclusion, the induction of apoptosis seems to be of significant importance in amphotericin B-induced cytotoxicity. Amphotericin B-induced cytotoxicity and apoptosis was eradicated by the Na+, K+, 2Cl(-)-cotransport inhibitor bumetanide. The changes of cellular K+ fluxes induced by bumetanide combined with amphotericin B needs further elucidation. Bumetanide could possibly be used in antifungal therapy to increase amphotericin B effectiveness doses without increasing its adverse effects.
authors.affiliation: Department of Clinical Chemistry, Umeå University, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Marklund
authors.forename: R
authors.initials: R
authors.lastname: Henriksson
authors.forename: K
authors.initials: K
authors.lastname: Grankvist
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002034:Bumetanide / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D016923:Cell Death / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009707:Nucleosomes; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D004791:Enzyme Inhibitors; D009707:Nucleosomes; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D010042:Ouabain; D000666:Amphotericin B; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium