The Impact of Extracellular Ca2+ and Nanosecond Electric Pulses on Sensitive and Drug-Resistant Human Breast and Colon Cancer Cells

Abstract

(1) Background: Calcium electroporation (CaEP) is based on the application of electrical pulses to permeabilize cells (electroporation) and allow cytotoxic doses of calcium to enter the cell. (2) Methods: In this work, we have used doxorubicin-resistant (DX) and non-resistant models of human breast cancer (MCF-7/DX, MCF-7/WT) and colon cancer cells (LoVo, LoVo/DX), and investigated the susceptibility of the cells to extracellular Ca²⁺ and electric fields in the 20 ns-900 ns pulse duration range. (3) Results: We have observed that colon cancer cells were less susceptible to PEF than breast cancer cells. An extracellular Ca²⁺ (2 mM) with PEF was more disruptive for DX-resistant cells. The expression of glycoprotein P (MDR1, P-gp) as a drug resistance marker was detected by the immunofluorescent (CLSM) method and rhodamine-123 efflux as an MDR1 activity. MDR1 expression was not significantly modified by nanosecond electroporation in multidrug-resistant cells, but a combination with calcium ions significantly inhibited MDR1 activity and cell viability. (4) Conclusions: We believe that PEF with calcium ions can reduce drug resistance by inhibiting drug efflux activity. This phenomenon of MDR mechanism disruption seems promising in anticancer protocols.

Keywords: calcium ions; electroporation drug resistance; human adenocarcinoma; membrane permeabilization.

Figure 1

Waveforms of the pulses applied in the study; red color represents signal of 100 ns pulse (15 kV/cm) and navy blue corresponds to 20 ns pulse (40 kV/cm).

Figure 2

The fraction of YO-PRO-1 permeable cells 10 min post electric field treatment evaluated using flow cytometry, where (A) doxorubicin-sensitive (MCF-7/WT) and (B) doxorubicin-resistant (MCF-7/DX) human breast adenocarcinoma cells; (C) doxorubicin-sensitive (LoVo) and (D) doxorubicin-resistant (LoVo/DX) human colon cancer cells. Asterisk (*) corresponds to statistically significant (p < 0.05) difference versus untreated control. The percentage of permeabilized cells shows the fraction of permeabilized cells compared to all the cells in each sample.

Figure 3

The fraction of YO-PRO-1 permeable cells 10 min post nanosecond-electric field treatment evaluated using flow cytometry in MCF-7/WT, MCF-7/DX, LoVo, and LoVo/DX cells. Asterisk (*) corresponds to statistically significant (p < 0.05) difference between two types of cells (MCF and LoVo). The percentage of permeabilized cells shows the fraction of permeabilized cells compared to the whole cells’ portion in each sample.

Figure 4

The viability of cells (A) MCF-7/WT and MCF-7/DX; (B) LoVo and LoVo/DX exposed to electroporation and calcium ions measured my MTT assay depending on the treatment protocol, where EP1–10 kV/cm × 300 ns × 200; EP2–40 kV/cm × 20 ns × 400; EP360 kV/cm × 20 ns × 400; EP4–1.2 kV/cm × 100 µs ns × 8 (ESOPE); CTRL–untreated control. All the data is normalized to untreated control. Asterisk (*) corresponds to statistically significant difference (p < 0.05) between protocols (EP1–EP4) with and without calcium; (x) corresponds to statistically significant difference (p < 0.05) between non-resistant cells and DX-resistant cells.

Figure 5

Immunofluorescent analysis of MDR1 protein double-stained with CellMask membrane marker in human breast adenocarcinoma cells, (a) MCF-7/WT; (b) MCF-7/DX and human colon carcinoma cells; (c) LoVo; (d) LoVo/DX. The following parameters were applied without/with 2 mM Ca²⁺: EP1–10 kV/cm × 300 ns × 200; EP2–40 kV/cm × 20 ns × 400; EP3–60 kV/cm × 20 ns × 400; EP4–1.2 kV/cm × 100 µs ns × 8 (ESOPE). Red fluorescence corresponds to the DeepRed^®CellMask cell membrane marker, green fluorescence corresponds to MDR1 protein.White scale bar corresponds to 10 µm.

Figure 6

The analysis of fluorescent P-gp (MDR1) signal from breast cancer cells (A) MCF-7/WT; (B) MCF-7/DX, and colon cancer cells (C) LoVo and (D) LoVo/DX. The following parameters were applied without/with 2 mM Ca²⁺: EP1–10 kV/cm × 300 ns × 200; EP2–40 kV/cm × 20 ns × 400; EP3–60 kV/cm × 20 ns × 400; EP4–1.2 kV/cm × 100 µs ns × 8 (ESOPE). Dotted line represents the control level. Signal was examined by ImageJ software. Grey fields indicate samples with calcium ions. Asterisk (* and #) corresponds to statistically significant (p < 0.05) difference. (*) in relation to the untreated control cells; (#) in relation to calcium-treated control.

Figure 7

Rhodamine 123 accumulation assay and viability in breast adenocarcinoma (a,b) sensitive and (c,d) resistant cells after 24 h post electroporation with/without CaCl₂. Results were normalized to control untreated cells (E/E0 where E0 = Ctrl, E–(MFI) mean fluorescence intensity of the appropriate sample). Exemplary dot plots and histograms are shown in (e), where X axis corresponds to FSC (forward scatter-diameter of the cells), and Y axis to SSC (side scatter-internal complexity of the cells. The following parameters were applied: EP1–10 kV/cm × 300 ns × 200; EP2–40 kV/cm × 20 ns × 400; EP3–60 kV/cm × 20 ns × 400; EP4–1.2 kV/cm × 100 µs ns × 8 (ESOPE), * p ≤ 0.05. E/E0 (Rod-123 uptake) and MFI results are taken from Gate 2 (blue); Gate 1 indicates dead or damaged cells; Gate 3 (purple) separates the analyzed cells from the debris.

Figure 8

Rhodamine 123 accumulation assay and viability in colon carcinoma (a,b) sensitive and (c,d) resistant cells after 24 h post electroporation with/without CaCl₂. Results were normalized to control untreated cells (E/E0 where E0 = Ctrl, E–(MFI) mean fluorescence intensity of the appropriate sample). Exemplary dot plots and histograms are shown in (e), where X axis corresponds to FSC (forward scatter-diameter of the cells), and Y axis to SSC (side scatter-internal complexity of the cells. The following parameters were applied: EP1–10 kV/cm × 300 ns × 200; EP2–40 kV/cm × 20 ns × 400; EP3–60 kV/cm × 20 ns × 400; EP4–1.2 kV/cm × 100 µs ns × 8 (ESOPE), * p ≤ 0.05. E/E0 (Rod-123 uptake) and MFI results are taken from Gate 2 (blue); Gate 1 indicates dead or damaged cells; Gate 3 (purple) separates the analyzed cells from the debris.