Role of Endocytosis Pathways in Electropermeablization of MCF7 Cells Using Low Voltage and High Frequency Electrochemotherapy

Abstract

Objective: The cell membrane is a major barrier for delivery of hydrophilic drugs and molecules into the cells. Although low voltage and high frequency electric fields (LVHF) are proposed to overcome the cell membrane barrier, the mechanism of membrane permeabilization is unclear. The aim of study is to investigate endocytosis pathways as a possible mechanism for enhancing uptake of bleomycin by LVHF.

Materials and methods: In this experimental study, MCF-7 cells were exposed to bleomycin or to electric fields with various strengths (10-80 V/cm), frequency of 5 kHz, 4000 electric pulse and 100 μs duration in the presence and absence of three endocytosis inhibitors-chlorpromazine (Cpz), amiloride (Amilo) and genistein (Geni). We determined the efficiency of these chemotherapeutic agents in each group.

Results: LVHF, depending on the intensity, induced different endocytosis pathways. Electric field strengths of 10 and 20 V/cm stimulated the macropinocytosis route. Clathrin-mediated endocytosis was observed at electric field intensities of 10, 30, 60 and 70 V/cm, whereas induction of caveolae-mediated endocytosis was observed only at the lowest electric field intensity (10 V/cm).

Conclusion: The results of this study imply that LVHF can induce different endocytosis pathways in MCF-7 cells, which leads to an increase in bleomycin uptake.

Keywords: Bleomycin; Electrochemotherapy; Endocytosis; Low Intensity Electric Field.

Fig.1

The effect of endocytosis inhibitors on cell viability and bleomycin (CT) uptake. The following concentrations of endocytosis inhibitors were used: 10 µM of chlorpromazine (Cpz), 400 µM of amiloride (Amilo) and 200 µM of genistein (Geni). The data are presented as mean cell viability ± standard deviation of the mean. P<0.05 indicates statistical significance.

Fig.2

The toxic effects of chlorpromazine (Cpz), genistein (Geni), and amiloride (Amilo) on the electric (E) field treated cells. In Graphs (A) to (C), the cell viability was measured 48 hours after cell treatment with electric field in the absence and presence of A. 10 μM chlorpromazine (Cpz), B. 400 μM amiloride (Amilo), and C. 200 μM genistein (Geni). The data represent mean cell viability ± standard deviation of the mean. P<0.05 indicates statistical significance.

Fig.3

Electrochemotherapy (ECT) effect in the presence and absence of endocytosis inhibitors. In graphs A-C, we measured cell viability 48 hours after treatment with ECT in the absence and presence of: A. 10 μM chlorpromazine (Cpz), B. 400 μM amiloride (Amilo), and C. 200 μM genistein (Geni). The data represent the mean cell viability ± standard deviation of the mean. *; P<0.05 indicates statistical significance.

Fig.4

The effect of electrochemotherapy (ECT) compared to bleomycin (CT) and electrical pulse (E) alone. The data represent mean cell viability ± standard deviation of the mean. P<0.05 indicates statistical significance.