Table 1.
Cytotoxicity of Irvalec in a panel of 25 human cancer cell lines.
Figure 1.
Irvalec induces a necrotic cell death.
(A) Representative images of A549 and HeLa cells treated with Irvalec (1 and 10 µM) and examined by phase contrast video-microscopy; pictures were taken 1 and 5 min after treatment. (B) Effects of 10 µM Irvalec at the cell membrane A549 cells pre-treated with AlexaFluor 488-conjugated β-subunit of the cholera toxin to fluorescently label the plasma membrane; white arrows indicate the formation of giant vesicles (C) Dose-response cytotoxicity curves to analyze the activity of Irvalec after 30 min (▪) and 72 h (□) incubation times using the SRB method. Results represent the mean±SD of at least three different experiments.
Figure 2.
Irvalec induces a concentration-dependent rapid membrane permeabilization.
(A) Representative images of A549 cells exposed to Irvalec 10 µM for 2 min. In the presence of propidium iodide (PI), unpermeabilized cells show intact nuclei (I) while permeabilized cells show PI stained nuclei (II); in pre-loaded with calcein AM cells, the intracellular fluorescence (III) rapidly vanished from permeabilized cells upon Irvalec treatment (IV) (scale bar: 50 µm) (B) Time-course of Irvalec-dependent membrane permeabilization of A549 cells using different concentrations of the drug (ranging from 1 to 6 µM) as assessed by plate fluorimetry using PI nuclear staining; (C) Time-course of Irvalec-dependent membrane permeabilization of A549 cells using different concentrations of the drug (ranging from 1 to 6 µM) as assessed LDH release.
Figure 3.
Effects of Irvalec on cytosolic Ca2+ concentrations and cell membrane conductivity in A549 cancer cells.
(A) Representation of cytosolic Ca2+ concentration imaging records (F340/F380 ratio) in A549 cells. (I) Alteration in cytosolic Ca2+ after treatment with Irvalec 1 µM (n = 12); the panel corresponds to the mean ± SEM values of [Ca2+]cyt in all cells for each experiment each microscopic field (II) Representation of cytoplasmic Ca2+ concentration records in A549 cells after treatment with Irvalec 0.5 µM (n = 29); the panel corresponds to the mean ± SEM values of [Ca2+]cyt in all cells for each experiment (III) Effects of removal of extracellular Ca2+ on the rise in cytosolic Ca2+ induced by 0.5 µM Irvalec; the graph corresponds to the mean ± SEM values of [Ca2+]cyt in all cells (n = 31) for two different experiments (IV) Effects of depletion of intracellular Ca2+ stores with 1 µM thapsigargin during 10 min on the rise in cytosolic Ca2+ induced by 500 nM Irvalec in Ca2+-free medium and after re-addition of extracellular calcium; the panel corresponds to the mean ± SEM values of [Ca2+]cyt in all treated cells (n = 29). (B) Electrophysiological characteristics of A549 cells; left panel shows the current-voltage relationship obtained after applying the pulse protocol described in Experimental Procedures. Amplitude of the potassium currents were measured at the end of 250 ms depolarizing pulses and were represented versus membrane potential. Right panel shows the ion current elicited after applying a ramp pulse protocol from −100 mV to +120 mV during 500 ms from a holding potential of −80 mV (C) Electrophysiological effects of 1 µM Irvalec; left panels shows the amplitude of the maximum current at the end of the ramp. Note that the downward of the current observed in the left panel is reflected in a plateau phase in the increase of the current; right panel shows original records after applying a ramp pulse protocol from −100 mV to +120 mV during 500 ms; white arrows shows a downward in the current.
Figure 4.
Zinc protects tumor cells against Irvalec cytotoxicity.
(A) Representative images of A549 cells treated with 5 µM Irvalec alone or in combination with 10 mM ZnCl2, in the presence of PI. Cell permeabilization was followed by fluorescence microscopy (red nuclear staining) (scale bar: 50 µm) (B) Quantification of Irvalec induced cell permeabilization in real time, using plate fluorimetry. A549 cells were treated with 10 µM Irvalec alone or in combination with different concentrations of ZnCl2 (ranging from 0.1 mM to 10 mM) in the presence of PI. Nuclear staining was measured at intervals of 1 min and represented as relative signal from control fluorescence (C) Effect of ZnCl2 on the electrophysiological recording obtained in A549 cells treated with Irvalec; cells were treated with the following solutions in a sequence, I: control conditions, II: ZnCl2 (10 mM), III: 1 µM Irvalec in the presence of 10 mM ZnCl2 and IV: ZnCl2-free and Irvalec-free external solution.
Figure 5.
Characterization of Irvalec-resistant A549-Irv cells.
(A) Dose-response cytotoxicity curves to analyze the activity of Irvalec in A549-Irv after exposure of 30 minutes (I) and 72 h (II); results represent the mean±SD of at least three different experiments (B) Effects of Irvalec treatment in cell membrane permeabilization; representative images of A549-Irv cells exposed to vehicle or to 10 µM Irvalec for 10 min are shown. Resistant cells kept cell morphology, did not show propidium iodide staining and retained preloaded calcein-AM in the cytoplasm (scale bar: 50 µm) (C) Effects of Irvalec treatment on cytosolic Ca2+; the left panel shows the effects of 0.5 µM Irvalec on cytosolic Ca2+ concentration (n = 27); the right panel represents the fura2 F340/F380 ratio in the presence of Mn2+ after treatment with 0.5 µM Irvalec (n = 16) (D) Effects of Irvalec treatment on cell membrane conductivity; left panel shows original records after applying a ramp pulse protocol from −100 mV to +120 mV during 500 ms. Right panel shows the amplitude of the maximum current at the end of the ramp.
Figure 6.
Interactions between Irvalec and the plasma cell membrane.
For these experiments, A549 cells were grown on LabTek chambered slides; 2P-FLIM images are representative of different experiments. (A) Visualization of changes in the plasma cell membrane organization in A549 cells without (I and II) or after treatment (III and IV) with 2 µM Irvalec. A549 cells were labelled with 1 µM PA-DPH and excited with polarized light microscopy. The direction of the linear polarized excitation beam is shown in the figure with red arrows: Horizontal (I and III) and Vertical (II and IV); Emission filter: 483/32; I–II: (XY section, Z = 0), III–IV (XY section, Z = 5 µm) (B) Localization of Irvalec in the cell membrane; A549 cells were treated with Irv-OG488 or Irv-A555 and analyzed by two-photon time-resolved fluorescence microscopy; I: untreated cells (XY section, Z = 0); II: cells treated with 0.6 µM of Irv-OG488 (XY section, Z = 0); III: cells treated with 2.2 µM of a mix of Irv-OG488 and Irvalec (1∶4.5) (XY image, Z = 0); IV and V: cells treated with 0.4 µM of a mix of Irv-OG488 and Irvalec (1∶3) (IV: XY image, Z = 5 µm; V: XZ section, Y position defined by white arrow in IV); VI: cells treated with 2 µM of a mix of Irv-A555 and Irvalec (1∶4). (XY section, Z = 10 µm); emission bandpass filters; I–V: FF01-520/35; VI: FF01-607/36; Total intensity scale in arbitrary units: 0–1(I, IV–VI); 0–2(II); 0–50 (III). (C) Representative example of time-resolved fluorescence resonance energy-transfer (FLIM-FRET) experiments in A549 cell exposed to a mix of 0.6 µM Irv-OG488, 1.8 µM Irv-A555 and 2.4 µM Irvalec; I (XY section, Z = 0) and II (YZ section, X position defined by white arrow in I): donor channel image (OG488); III (XY section, Z = 0) and IV (YZ section, X position defined by white arrow in III): acceptor channel image (A555); donor filter: FF01 520/35; acceptor filter: FF01 685/40; dichroic beam splitter: FF560-Di01; total intensity scale in arbitrary units: 0–7 (I–II); 0–1 (III–IV). Scale bar: 10 µm.