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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

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).
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pone.0122973.g004: 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).

Mentions: We used confocal fluorescence imaging of quinacrine-labelled cells to investigate changes in intracellular levels and distribution of ATP [27–30]. Fig 4 shows selected representative images taken 15 minutes after treatment. The average quinacrine fluorescence of calcium electroporated samples was about 30% lower than in the calcium-only samples (p<0.0001), schematically shown in Fig 5A. The electric field needed to kill half the cells (E50) under the explored conditions in presence of 3 mM calcium, was estimated at 0.70 kV/cm.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122973.g004: 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).
Mentions: We used confocal fluorescence imaging of quinacrine-labelled cells to investigate changes in intracellular levels and distribution of ATP [27–30]. Fig 4 shows selected representative images taken 15 minutes after treatment. The average quinacrine fluorescence of calcium electroporated samples was about 30% lower than in the calcium-only samples (p<0.0001), schematically shown in Fig 5A. The electric field needed to kill half the cells (E50) under the explored conditions in presence of 3 mM calcium, was estimated at 0.70 kV/cm.

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