Dose-dependent ATP depletion and cancer cell death following calcium electroporation, relative effect of calcium concentration and electric field strength

Emilie Louise Hansen, Esin Bengisu Sozer, Stefania Romeo, Stine Krog Frandsen, P Thomas Vernier, Julie Gehl, Emilie Louise Hansen, Esin Bengisu Sozer, Stefania Romeo, Stine Krog Frandsen, P Thomas Vernier, Julie Gehl

Abstract

Background: Electroporation, a method for increasing the permeability of membranes to ions and small molecules, is used in the clinic with chemotherapeutic drugs for cancer treatment (electrochemotherapy). Electroporation with calcium causes ATP (adenosine triphosphate) depletion and cancer cell death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy.

Methods: In three human cell lines--H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability was determined after treatment with 1, 3, or 5 mM calcium and eight 99 μs pulses with 0.8, 1.0, 1.2, 1.4 or 1.6 kV/cm. Fitting analysis was applied to quantify the cell-killing efficacy in presence of calcium. Post-treatment intracellular ATP was measured in H69 and SW780 cells. Post-treatment intracellular ATP was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells.

Results: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p<0.05) and reduced viability. The 50% effective cell kill was found at 3.71 kV/cm (H69) and 3.28 kV/cm (SW780), reduced to 1.40 and 1.15 kV/cm (respectively) with 1 mM calcium (lower EC50 for higher calcium concentrations). Quinacrine fluorescence intensity of calcium-electroporated U937 cells was one third lower than in controls (p<0.0001).

Conclusions: Calcium electroporation dose-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field.

General significance: This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials.

Conflict of interest statement

Competing Interests: The authors declare the patent “Therapeutic applications of calcium electroporation to effectively induce tumor necrosis” (PCT/DK2012/050496, co-inventors SKF and JG) has been submitted. Other authors declare no conflict of interest. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Cellular ATP content in H69…
Fig 1. Cellular ATP content in H69 and SW780 cells after calcium electroporation.
Cellular ATP levels determined 1, 2, 4, and 8 hours after calcium electroporation treatment of two human cell lines (H69, a small cell lung cancer cell line (A); and SW780, a bladder cancer cell line (B)). Extracellular calcium concentrations of 0 mM, 1 mM, 3 mM, or 5 mM and applied electric field of 0.8 kV/cm, 1.0 kV/cm, or 1.2 kV/cm. Results are illustrated as percentage of control (no electroporation, no added calcium). Normalized to control at each time point (%), mean—S.D., n = 6.
Fig 2. Viability of H69 and SW780…
Fig 2. Viability of H69 and SW780 cells after calcium electroporation.
Viability, assessed using MTS assay, of H69, a human small cell lung cancer cell line (A); and SW780, a human bladder cancer cell line (B) 24 hours after treatment with calcium electroporation. Final extracellular calcium concentrations of 0 mM, 1 mM, 3 mM, or 5 mM and applied electric field of either 0.8 kV/cm, 1.0 kV/cm, or 1.2 kV/cm. Cells thrice thawed and frozen, then sonicated were used as control (dead cells). Results are illustrated as percentage of control (no electroporation, no added calcium), electroporation (EP), mean + S.D., n = 6.
Fig 3. SW780 viability as a function…
Fig 3. SW780 viability as a function of pulsed electric field and extracellular calcium concentration.
Cell survival (%) versus electric field (E) at calcium electroporation using 0, 1, 3, or 5mM calcium in SW780 human bladder cancer cells assessed using MTS assay 24 hours after treatment. Electric field amplitude of 0.8 kV/cm, 1.0 kV/cm, 1.2 kV/cm, 1.4 kV/cm, or 1.6 kV/cm was applied. Fitting curves were derived using MATLAB software. Results are illustrated as percentage of control (no electroporation, no added calcium), mean ± S.D., n = 6.
Fig 4. Scanning confocal images of U937…
Fig 4. Scanning confocal images of U937 15 min after calcium electroporation.
U937, a human leukaemia cell line. Final extracellular calcium chloride concentrations of 0 mM or 3 mM and applied electric field of 1.0 kV/cm. Quinacrine fluorescence (top row), 10 μM; propidium iodide fluorescence (middle row), 7.5 μM; phase contrast cell images (bottom row).
Fig 5. U937 cell response to calcium…
Fig 5. U937 cell response to calcium electroporation.
U937, a human leukaemia cell line. (A) Cellular quinacrine fluorescence intensity of U937 15 min after calcium electroporation. The extracellular quinacrine concentration was 10 μM during loading and electroporation. Final extracellular calcium concentrations of 0 mM or 3 mM and applied electric field of 1.0 kV/cm were used. Columns depict the average of 180 cells (9 experiments × 20 cells). Arbitrary units (AU), mean + S.D., n = 9. (B) U937 cell viability measured 24 h after calcium electroporation. Viability assessed for U937 using resazurin cell proliferation assay. Extracellular calcium concentrations of 0 or 3 mM. Applied electric field of 1.0 kV/cm. Results are illustrated as percentage of control (no electroporation, no added calcium), mean + S.D., n = 6.

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