Cisplatin-induced apoptosis of mesothelioma cells is affected by potassium ion flux modulator amphotericin B and bumetanide

Abstract

Key words: amphotericin B; apoptosis; bumetanide; carboplatin; cisplatin; cytotoxicity; potassium channelsThe efflux of ions, primarily potassium ions, plays an important role during the induction of apoptosis. Apoptosis with ensuing reduction in cell volume is enhanced under conditions when the intracellular K ϩ concentration is diminished, and only shrunken cells with reduced K ϩ exhibit DNA fragmentation and elevation of caspase-3-like activity-hallmarks of apoptosis. 1 Increased inward pumping of potassium ions counteracts apoptosis. 2 The major transport system accumulating ions in shrunken cells is NaϪ -cotransport. 3-5 Modulation of K ϩ efflux or influx rates could thus decrease or increase apoptosis due to the nature of the disruption of cellular potassium homeostasis. Because the mechanism by which several of the chemotherapeutic anti-cancer agents exert their effect involve induction of apoptosis, the tumoricidal effects could subsequently be altered. 6 -8 The platinum-based drug cisplatin (cis-diammine-dichloroplatinum) and its less toxic analogue carboplatin (cis-diammine-1,1-cyclobutanedicarboxyplatinum) are widely used in cancer treatment, alone or in combination with other anti-tumor drugs, against several solid tumors. Like most chemotherapeutic agents, cisplatin and carboplatin exert their action by inducing apoptosis, 9,10 probably mediated through caspase-3 activation. [11][12][13] To explore if enhanced potassium efflux could affect cisplatin and/or carboplatin induction of apoptosis and cytotoxicity we used amphotericin B as K ϩ ionophore. Amphotericin B is a polyene anti-fungal antibiotic known to potentiate cisplatin cytotoxicity in normal and cisplatinresistant cells, 14,15 by inducing cell membrane pores leading to potassium efflux. 16 To inhibit NaϪ -cotransport-dependent potassium influx, we used the clinically used diuretic drug bumetanide, a specific NaϪ -cotransport blocker. The effects of K ϩ flux modulators on cisplatin-and carboplatin-induced apoptosis and cytotoxicity were studied on a pulmonary mesothelioma cell line (P31), which to a large extent expresses NaϪ -cotransport activity. 17 MATERIAL AND METHODS Cell cultureHuman pulmonary mesothelioma cells (P31) were grown as monolayer culture in Eagle's minimal essential medium (MEM) in Earl's saline supplemented with 10% fetal calf serum and 200 mol/L L-glutamine. Analysis of DNA integrityEndonuclease-mediated nucleosome excision, observable as a DNA ladder in agarose gels was first used as a qualitative screening assay for apoptosis. Following a 6-hr incubation with 0, 0.05, 0.5, 5 or 50 mg/L cisplatin, P31 cells that had become nonadherent were suspended in phosphate-buffered bovine serum albumin (pBSA), centrifuged (3,000 g) and then resuspended in digestion buffer (100 mmol/L sodium chloride, 10 mmol/L TRISHCl pH 8, 0.5% sodium dodecyl sulfate and 0.1 mmol/L proteinase K), incubated for 12 hr at 50°C, followed by treatment with DNAse-free RNAse (0.5 mg/ml) for 1 hr. Loading buffer was added to each sample before loading on...

Acquisition of Cisplatin-resistance in Malignant Mesothelioma Cells Abrogates Na^+,K^+,2Cl^-;-cotransport Activity and Cisplatin-induced Early Membrane Blebbing

“…Not any of the exposures induced membrane blebbing or protrusion in the P31res1.2 cells. These concentrations also have similar inhibitory effects on NKCC1 activity in the P31 cells [16,19,35] demonstrating that there was little risk of non-specific effects of bumetanide regardless if 10 or 100 µmol/L bumetanide were used in the experiments.…”

Human ether-�-go-go-related gene expression is essential for cisplatin to induce apoptosis in human gastric cancer

“…Recent studies have provided substantial evidence that potassium channels are involved in the regulation of tumor cell proliferation, acquirement of invasive, metastatic ability, apoptosis, cell cycle and even drug resistance (4,6,25,26). Among these potassium channels, HERG has demonstrated the strongest relationship with human cancer, and been considered as a potential therapeutic target (25,27).…”

“…Like most chemotherapeutic drugs, cisplatin exerts its anti-cancer effect by inducing apoptosis, probably mediate through caspase-3 activity (3). Cytotoxicity of cisplatin, and its ability to induce cancer cell apoptosis is influenced by modulators of potassium ion flux (4).…”

Isoosmotic shrinkage by self-stimulated outward Na-K-Cl cotransport in quail erythrocytes

“…More importantly, bumetanide, a specific inhibitor of NKCC, can inhibit the evolution of the apoptotic process [18,23,24]. Moreover, Bortner et al [3] showed that an increase in intracellular Na + precedes the apoptotic volume decrease, and intracellular Na + is a potent stimulus of outward cotransport [11].…”

“…More recently NKCC has been implicated in other physiological phenomena such as cell division [5,28,29,30] and apoptosis [18,23,24], where a volume increase or decrease respectively seems necessary to allow such processes to occur.…”