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The DC electrical-field-induced Ca²⁺ response and growth stimulation of multicellular tumor spheroids are mediated by ATP release and purinergic receptor stimulation

Department of Neurophysiology, University of Cologne, Robert-Koch-Strasse 39, D-50931 Cologne, Germany

View larger version (70K): [in a new window]   Fig. 1. The electrical-field-induced Ca2+ response in multicellular prostate tumor spheroids. (A) Image gallery of representative multicellular tumor spheroids treated with a single electrical field pulse (750 Vm-1, 60 seconds). Images were recorded from the start point of the Ca2+ response, which occurred approximately 40 seconds after the onset of the electrical field. Tumor spheroids were loaded with the fluorescent Ca2+ indicator fluo-3. The Ca2+ response started at the anode-facing side and propagated towards the cathode-facing side. Images were recorded in 3 second intervals. Bar, 50 µm. (B) Representative tracings of the electrical-field-treated Ca2+ response recorded in single cells at the anode- as well as the cathode-facing side of the tumor spheroid. (C) Ca2+ responses in single cells of multicellular tumor spheroids after repetitive treatment with electrical field pulses (750 Vm-1, 60 seconds). Note that repetitive treatment with the electrical field decreased the amplitude as well as the duration of the Ca2+ response.

View larger version (18K): [in a new window]   Fig. 2. Involvement of purinergic receptor stimulation in the electrical-field-induced Ca2+ response. (A) Tumor spheroids were repetitively treated with 10 µM ATP, which elicited a Ca2+ response with declining amplitude during repetitive application of ATP. Subsequent application of an electrical field (750 Vm-1, 60 seconds) failed to raise [Ca2+]i, indicating interference with ATP-mediated signaling pathways. (B) Pretreatment of tumor spheroids for 60 minutes with 300 µM suramin, which inhibits purinergic receptor activation, blunted the electrical-field-induced Ca2+ response. (C) Inhibition of Ca2+ elevation was likewise achieved after incubation for 30 minutes with the ATP scavenger apyrase (2 U/ml). The tumor spheroids were treated with electrical field during the time indicated by the horizontal solid bar.

View larger version (23K): [in a new window]   Fig. 3. ATP release upon treatment of multicellular tumor spheroids with DC electrical fields. ATP release after electrical field treatment (750 Vm-1, 60 seconds) was evalulated by luciferin/luciferase-based bioluminescence. Between 30 and 50 tumor spheroids were treated with an electrical field pulse and subsequently an 150 µl aliquot of the supernatant was analyzed in the bioluminescence apparatus. Pretreatment for 30 minutes with the anion channel inhibitors niflumic acid (200 µM), NPPB (50 µM) and tamoxifen (50 µM) significantly inhibited the electrical-field-induced ATP release, indicating that ATP efflux occurs via anion channels.

View larger version (15K): [in a new window]   Fig. 4. Inhibition of the electrical-field-evoked Ca2+ response by anion channel inhibitors. Multicellular prostate tumor spheroids were pretreated for 60 minutes with the anion channel inhibitors tamoxifen (50 µM) (B), niflumic acid (200 µM) (C), and NPPB (50 µM) (D). Under these experimental conditions the electrical-field-induced [Ca2+]i response (A) was totally inhibited. Representative tracings recorded from single cells in multicellular tumor spheroids. The tumor spheroids were treated with electrical field during the time indicated by the horizontal solid bar.

View larger version (39K): [in a new window]   Fig. 5. Inhibition of DC electrical-field-induced growth stimulation of multicellular tumor spheroids by suramin and the anion channel antagonist niflumic acid (A), and effects of anion channel blockers on tumor spheroid growth induced by exogenous ATP (B). (A) Multicellular tumor spheroids were treated with a single electrical field pulse (750 Vm-1, 60 seconds) in the absence (control) or presence of either suramin (300 µM) or the anion channel inhibitor niflumic acid (200 µM). Subsequently they were immersed in cell culture medium in the absence of the compounds and cultivated for a further 6 days. Tumor spheroid volumes were determined immediately after electrical field treatment and after 6 days. (B) Tumor spheroids were treated with niflumic acid (200 µM), tamoxifen (50 µM) or NPPB (50 µM). After 30 minutes of incubation 10 µM ATP was added and tumor spheroids were incubated for a further 30 minutes. Subsequently, tumor spheroids were washed and tumor spheroid growth was evaluated after 24 hours. Tumor spheroid growth is presented as relative volume increase V/V0 where V0 is the spheroid volume at the beginning of the experiment and V is the spheroid volume at the end of the experiment.

View larger version (36K): [in a new window]   Fig. 6. Inhibition of DC electrical-field-induced c-Fos induction in multicellular tumor spheroids by suramin and anion channel antagonists. (A) Representative tumor spheroids immunolabeled with an antibody directed against Fos protein. Multicellular tumor spheroids remained untreated (control) (Aa) or were treated with a single electrical field pulse (750 Vm-1, 60 seconds) in the absence (Ab) or presence of either suramin (300 µM) (Ac) or the anion channel inhibitors niflumic acid (200 µM) (Ad), tamoxifen (50 µM) (Ae), and NPPB (50 µM) (Af). They were subsequently immersed in cell culture medium in the absence of the compounds and fixed after 1 hour of incubation. Bar, 75 µm. (B) Quantitative evalulation of Fos immunofluorescence in control and electrical-field-treated samples. Note that in the presence of suramin or anion channel blockers the induction of c-Fos following electrical field treatment was significantly inhibited.