Micro- and Nanosecond Pulses Used in Doxorubicin Electrochemotherapy in Human Breast and Colon Cancer Cells with Drug Resistance

Abstract

(1) Background: Pulsed electric field (PEF) techniques are commonly used to support the delivery of various molecules. A PEF seems a promising method for low permeability drugs or when cells demonstrate therapy resistance and the cell membrane becomes an impermeable barrier. (2) Methods: In this study, we have used doxorubicin-resistant and sensitive models of human breast cancer (MCF-7/DX, MCF-7/WT) and colon cancer cells (LoVo, LoVoDX). The study aimed to investigate the susceptibility of the cells to doxorubicin (DOX) and electric fields in the 20-900 ns pulse duration range. The viability assay was utilized to evaluate the PEF protocols' efficacy. Cell confluency and reduced glutathione were measured after PEF protocols. (3) Results: The obtained results showed that PEFs significantly supported doxorubicin delivery and cytotoxicity after 48 and 72 h. The 60 kV/cm ultrashort pulses × 20 ns × 400 had the most significant cytotoxic anticancer effect. The increase in DOX concentration provokes a decrease in cell viability, affected cell confluency, and reduced GSSH when combined with the ESOPE (European Standard Operating Procedures of Electrochemotherapy) protocol. Additionally, reactive oxygen species after PEF and PEF-DOX were detected. (4) Conclusions: Ultrashort electric pulses with low DOX content or ESOPE with higher DOX content seem the most promising in colon and breast cancer treatment.

Keywords: doxorubicin; drug delivery; drug resistance; electroporation; human adenocarcinoma.

Figure 1

The waveform of (A) long- and (B) short-duration pulses and (C) graphical representation of the experimental setup.

Figure 2

The level of MDR proteins in human and colon and breast cancer (A), relative quantity level of MDR genes expression (B). Asterisk (*) corresponds to statistically significant (p < 0.05) difference.

Figure 3

The viability of human breast adenocarcinoma cells exposed to electrical pulses without and with doxorubicin (DOX, C_DOX = 2 µM) after 24 h for (A) MCF-7/WT and (C) MCF-7/DX cells, and after 72 h for (B) MCF-7/WT and (D) MCF-7/DX cells. The following treatment protocols were used: PEF1—10 kV/cm × 300 ns × 200; PEF2—40 kV/cm × 20 ns × 400; PEF3—60 kV/cm × 20 ns × 400; PEF4—1.2 kV/cm × 100 µs × 8 (ESOPE); DOX—DOX treated only. All the data are normalized to untreated control. Asterisk (*) corresponds to (p < 0.05) or (**) to (p < 0.005) statistically significant difference.

Figure 4

The viability of human colon adenocarcinoma cells exposed to electrical pulses without and with doxorubicin (DOX, C_DOX = 2 µM) after 24 h for (A) LoVo and (C) LoVoDX cells, and after 72 h for (B) LoVo and (D) LoVoDX cells. The following treatment protocols were used: PEF1—10 kV/cm × 300 ns × 200; PEF2—40 kV/cm × 20 ns × 400; PEF3—60 kV/cm × 20 ns × 400; PEF4—1.2 kV/cm × 100 µs × 8 (ESOPE); red bar DOX—DOX treated only. All the data are normalized to untreated control. Asterisk (*) corresponds to (p < 0.05) or (**) to (p < 0.005) statistically significant difference.

Figure 5

The viability of cells exposed to electrical pulses without (no DOX) and with doxorubicin (DOX, C_DOX = 2, 20 or 50 µM) after 24 h for MCF-7/WT (A) and MCF-7/DX (B) cells; 72 h for MCF-7/WT (C) and MCF-7/DX (D). The following treatment protocols were used: PEF1—10 kV/cm × 300 ns × 200; PEF4—1.2 kV/cm × 100 µs × 8 (ESOPE). All the data are normalized to untreated control. Asterisk (*) corresponds to (p < 0.05) or (**) to (p < 0.005) statistically significant difference.

Figure 6

The viability of cells exposed to electrical pulses without (no DOX) and with doxorubicin (DOX, C_DOX = 2, 20 or 50 µM) after 24 h for LoVo (A) and LoVoDX (B) cells; 72 h for LoVo (C) and LoVoDX (D) cells. The following treatment protocols were used: PEF1—10 kV/cm × 300 ns × 200; PEF4—1.2 kV/cm × 100 µs × 8 (ESOPE). All the data are normalized to untreated control. Asterisk (*) corresponds to statistically significant (p < 0.05) difference.

Figure 7

The analysis of the effects after exposure to electrical pulses without and with doxorubicin (DOX, C_DOX = 2 µM) on cells’ confluency analyzed during 44 h for breast cancer MCF-7/WT (a) and MCF-7/DX (b) cells, and colon cancer LoVo (c) and LoVoDX (d) cells; the evaluation of reduced glutathione (GSSH) in breast cancer (e) and colon cancer (f) cells. The following treatment protocols were used: PEF1 —10 kV/cm × 300 ns × 200; PEF4—1.2 kV/cm × 100 µs × 8 (ESOPE). All the data are normalized to untreated control. Asterisk (*) corresponds to statistically significant (p < 0.05) difference.

Figure 8

The visualization of mitochondria (green) and ROS (red) in colon and breast cancer cells after PEF1 and PEF4 with/without doxorubicin (DOX, C_DOX = 2 µM).