Effect of calcium electroporation in combination with metformin in vivo and correlation between viability and intracellular ATP level after calcium electroporation in vitro

Abstract

BackgroundCalcium electroporation is a new experimental anti-cancer treatment where calcium is internalized into cells by application of short, high voltage pulses. Calcium electroporation has been shown to induce tumor necrosis associated with ATP depletion while the effect on normal fibroblasts was limited when investigated in a 3D in vitro spheroid model. We aimed to investigate the effect of calcium electroporation in combination with metformin, a drug that affects intracellular ATP level. We also aimed to study the relationship between the viability and intracellular ATP levels after calcium electroporation in vitro.MethodsIn this study, we investigated the effect of calcium electroporation with metformin on NMRI-Foxn1nu mice in vivo on tumor size, survival, and intracellular ATP. We further investigated viability and intracellular ATP level in vitro after calcium electroporation in two human cancer cell lines: Breast (MDA-MB231) and colon (HT29), and in normal human fibroblasts (HDF-n), as well as investigating viability in human bladder cancer cells (SW780) and human small cell lung cancer cells (H69) where we have previously published intracellular ATP levels.ResultsCalcium electroporation significantly reduced the size and ATP level of bladder cancer tumors treated in vivo but no increased effect of metformin combined with calcium electroporation was shown on neither tumor size, survival, nor ATP level. Calcium electroporation in vitro significantly decreased viability compared with calcium alone (p<0.0001 for calcium concentrations from 0.5 mM for H69, HDF-n, and MDA-MB231; p<0.0001 for calcium concentrations from 1 mM for HT29 and SW780). Intracellular ATP levels decreased significantly after calcium electroporation (p<0.05), however no correlation between intracellular ATP level and viability after treatment was observed.ConclusionCalcium electroporation caused reduced tumor size, increased survival, and acute ATP depletion in vivo. This effect was not augmented by metformin. Calcium electroporation is a possible novel anti-cancer treatment that has been shown to cause cell death associated with acute ATP depletion in vitro and in vivo.

A Comprehensive Review of Calcium Electroporation—A Novel Cancer Treatment Modality

“…Similar effects can be reached with calcium electroporation as with electrochemotherapy using bleomycin, but no synergistic effect is seen when combining calcium and bleomycin before electroporation [84]. There was no effect of treatment with calcium alone in concentrations up to 5 mM [18,77,[80][81][82] and even up to 20 mM in the two cell lines where such high concentrations were tested [78]. This is unlike treatment with bleomycin alone where some effect is seen; however, this effect is much less than when combined with electroporation [81,84].…”

“…There seems to be a similar effect of calcium electroporation when treating with calcium concentrations between 100-500 mM (injected in a volume equivalent to 50% of the tumor volume) and with injection volumes from 20% to 80% of the tumor volume (with 168 mM CaCl 2 ) [61]. The calcium concentrations used in vivo [18,61] are much higher than the concentrations inducing cell death in vitro [77,80], which is also seen when using bleomycin in combination with electroporation [79,84]. This is likely due to the smaller extracellular volume (ECV) in tissue compared with cells in suspension in vitro, which is also supported by a study on spheroids, a 3D in vitro model [85], where the drug concentrations (calcium or bleomycin) were similar to the in vivo studies and the ECV of spheroids is comparable to the ECV of tissue.…”

“…Calcium electroporation has been tested in vitro in 18 different cell lines (12 cancer cell lines and six normal cell lines of which five are immortalized cells and one is primary cells) [18,[77][78][79][80][81][82][83]. In all tested cell lines, calcium electroporation induced cell death in a dose-dependent manner [18,77,78,80,81] with an IC 50 ranging from 0.4-5.0 mM calcium (in combination with electroporation) in the studies where IC 50 was determined [80,81]. Similar effects can be reached with calcium electroporation as with electrochemotherapy using bleomycin, but no synergistic effect is seen when combining calcium and bleomycin before electroporation [84].…”

Evaluation of Calcium Electroporation for the Treatment of Cutaneous Metastases: A Double Blinded Randomised Controlled Phase II Trial

“…Ca-EP is a novel anticancer treatment that has been used successfully with tumours exhibiting various histologies. Preclinical studies provided the first support for the efficacy across cancer histologies, as well as an explanation of the mechanisms of action [11,13,14,[20][21][22][23][24][25][26][27]. The results of the first clinical trials suggested that Ca-EP is safe and efficient at the local level for tumours of different types, including cutaneous metastases from breast cancer and malignant melanoma and recurrent head and neck cancer [11,20].…”

Effects of extracellular medium conductivity on cell response in the context of sub-microsecond range calcium electroporation

“…The 2 mM concentration was selected based on the available knowledge on calcium electroporation. It is known that a threshold in concentration exists when the calcium www.nature.com/scientificreports www.nature.com/scientificreports/ electrotransfer starts to be effective, while 2 mM is an optimal dose across many cell lines 20 . The resultant conductivity of the medium during the 2 mM Ca 2+ electroporation procedure was 0.08 S/m.…”