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

Hansen EL, Sozer EB, Romeo S, Frandsen SK, Vernier PT, Gehl J - PLoS ONE (2015)

Bottom Line: 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).Increasing extracellular calcium allows the use of a lower electric field.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Drug and Gene Electrotransfer, Department of Oncology, Copenhagen University Hospital Herlev, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0122973.g003: 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.

Mentions: After analysis with MATLAB curve fitting tool, we found that the best fitting function for the relation between cell survival after 24 hours and the electric field amplitude at fixed calcium concentrations, was a two-parameter exponential function, y(x) = a·exp(–b·x) where x is the electric field amplitude and y(x) is the percentage of cell survival. Results on SW780 cells are reported in Fig 3. The E50 parameter at each calcium concentration considered was extracted using the found function. The results, schematically reported in Table 1 for both the H69 and the SW780 cell line, allowed us to quantify the increased cell killing efficiency of electroporation in the presence of calcium. In both cell lines, and for all the calcium concentrations tested, the electric field required to halve cell survival at 24 hours after treatment in presence of extracellular calcium was two- to three times lower than in the case of electroporation alone. Different responses were obtained between the two cell lines. The H69 cell line required higher electric field amplitude (around 20%) than SW780 cells to obtain a 50% reduction in cell viability, with an extracellular concentration of calcium chloride from 1 to 5 mM. The SW780 cell line may be more sensitive to treatment with low doses of calcium than H69 cells in vitro.


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

Hansen EL, Sozer EB, Romeo S, Frandsen SK, Vernier PT, Gehl J - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390219&req=5

pone.0122973.g003: 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.
Mentions: After analysis with MATLAB curve fitting tool, we found that the best fitting function for the relation between cell survival after 24 hours and the electric field amplitude at fixed calcium concentrations, was a two-parameter exponential function, y(x) = a·exp(–b·x) where x is the electric field amplitude and y(x) is the percentage of cell survival. Results on SW780 cells are reported in Fig 3. The E50 parameter at each calcium concentration considered was extracted using the found function. The results, schematically reported in Table 1 for both the H69 and the SW780 cell line, allowed us to quantify the increased cell killing efficiency of electroporation in the presence of calcium. In both cell lines, and for all the calcium concentrations tested, the electric field required to halve cell survival at 24 hours after treatment in presence of extracellular calcium was two- to three times lower than in the case of electroporation alone. Different responses were obtained between the two cell lines. The H69 cell line required higher electric field amplitude (around 20%) than SW780 cells to obtain a 50% reduction in cell viability, with an extracellular concentration of calcium chloride from 1 to 5 mM. The SW780 cell line may be more sensitive to treatment with low doses of calcium than H69 cells in vitro.

Bottom Line: 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).Increasing extracellular calcium allows the use of a lower electric field.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Drug and Gene Electrotransfer, Department of Oncology, Copenhagen University Hospital Herlev, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus