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ORIGINAL ARTICLE
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Effective proliferation control of MCF7 breast cancer using microsecond duration electrical pulse


1 Department of Electrical Engineering Science, Homi Bhabha National Institute, Mumbai, Maharashtra, India
2 Department of Electrical Engineering, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
3 Department of Electrical Engineering Science, Homi Bhabha National Institute; Accelerator and Pulse Power Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Date of Submission11-Mar-2021
Date of Decision11-May-2021
Date of Acceptance30-May-2021
Date of Web Publication25-Apr-2022

Correspondence Address:
Gyanendra Kumar,
Department of Electrical Engineering Science, Homi Bhabha National Institute, Mumbai - 400 085
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_414_21

 > Abstract 


Background: Electro-permeablization using a pulse generator is a novel non-invasive approach for cancer therapy. It serves as a cell permeability enhancing agent for cancer treatment.
Objective: In this article in vitro investigation of the effect of 1.0 kV/cm, 1.5 kV/cm and 2.0 kV/cm, 50 μs duration pulsed electric field on MCF-7 cell line has been done. Furthermore, combinational therapy of curcumin and electrical pulses has been also investigated.
Material and Method: A variable voltage (100 V-1200 V, 100 V step) and 50 μs duration pulse generator has been designed, which is further used for the investigation of electroporation and destructive electrical field intensity. Investigation of the effect of electrical pulses on cancer cells has been performed using Trypan Blue Exclusion Test, MTT Assay and Clonogenic Assay.
Results: It has been observed that electrical field intensity of 2 kV/cm, 50 μsec duration, 10 pulses at repetition rate of 1 pulse per second corresponding to total energy of 4 J is more than enough for causing necrotic cell death due to permanent damage of cell membrane of the cancer cell. Also, it has been observed that electrical pulse application enhances curcumin uptake by cells.
Conclusion: Electrical pulses can effectively inhibit the cancer cell growth and proliferation. Furthermore, observation shows that electroporation enhances the curcumin uptake, therefore, it can be used for therapeutic purposes.

Keywords: Curcumin, electrical field, electroporation, field exposure, microsecond pulse



How to cite this URL:
Kumar G, Sarathi R, Sharma A. Effective proliferation control of MCF7 breast cancer using microsecond duration electrical pulse. J Can Res Ther [Epub ahead of print] [cited 2022 Dec 8]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=343919




 > Introduction Top


In the past few decades, treatment of cancer using pulsed electric field has gained extensive attention among scientists because of having the virtue of its medical and biological applications, such as gene delivery,[1],[2],[3],[4],[5] electrochemotherapy,[6],[7],[8],[9],[10],[11] and cancer therapy.[12],[13],[14],[15],[16],[17],[18] The advantage of pulsed electric field treatment, making it different from conventional and other physical techniques, is the ability to destroy cancer cells in a nonthermal manner[19],[20] by inducing apoptosis. Consequently, pulsed electric field treatment can make it possible to preserve sensitive tissues intact, such as blood vessels and axons.[21],[22] Moreover, this minimally invasive technique allows the possibility of regeneration with healthy cells and tissues in the treatment region and leaves almost no scar.[23] With the aid of ultrasound, CT, or MRI, pulsed electric field treatment could be monitored in real time, which helps improve the treatment efficacy immensely.[24],[25],[26]

An electric field having sufficient magnitude causes reversible (microsecond to millisecond and few hundred volts per centimeter) and irreversible changes (microsecond and further short having tenths of kilovolts per centimeter) in cell membranes. The first paper reporting the reversible breakdown of cell membranes when electric fields are applied was published in 1958.[27] The first report on the increase in permeability of the plasma membrane of a biological cell, an effect that is known as “electroporation,” appeared in 1972.[28] The electric fields that are required to achieve electroporation depend on the duration of the applied pulse. Typical pulses range from tens of milliseconds with amplitudes of several 100s V/cm to pulses of a few microseconds and several kV/cm. Mathematical modeling and analysis optimistically show this result. Microsecond and larger pulses cause an effect on the membrane and shorter pulses from nanosecond completely on intracellular domain while in the range between few nanoseconds to microsecond cause effect on both membrane and intracellular region.[29] For a substantial effect on the eukaryotic cell, the electric field needs to be 0.5–1 V on the cell membrane depends upon the duration of the electric field. Activated caspase-3, which is highly required in nanosecond pulsed electric field (nsPEF) treatment, increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h posttreatment. This increase is nonlinear and peaks at 15–20 J/mL for all field strengths.[30]

The required time to charge the surface membrane is depending on the electrical parameters of both the cell and the suspension medium. For a spherical cell having a surface membrane that is an ideal dielectric with no leakage currents, the charging time constant is given by:[31]

[19]

Where Cm is capacitance of the surface membrane per unit area, D the cell diameter, the resistivity of the cytoplasm, and ra the resistivity of the medium in which the cell is suspended, for a cell with a diameter of 10 μm, cytoplasm and medium resistivity of 100 Ω-cm, and a membrane capacitance of 1 μF/cm2, τc is 75 ns.[32] It needs to be mentioned that the charging time constant, τc, is defined as the time to charge the membrane to 63% of its final value. To charge it to 95%, the voltage needs to be applied for a time of 3τc (membrane charging time constant). During the charging of the plasma membrane, the nucleus and other organelles are also exposed to the electric field. The smaller the organelle diameter, the faster the subcellular membranes will be charged.[33] When the voltage across these membranes reaches critical values, poration is expected. Targeting of subcellular structures with unipolar pulses requires the pulse rise time to be much less than the charging time of the plasma membrane. Moreover, the pulse duration itself should be kept shorter than the time required for the onset of electroporation of the outer membrane. Experiments conducted with HL-60 type cells and Jurkat type cells have proven that, for sub-microsecond pulse durations, the probability of interactions with internal structures increases, whereas the outer cell membrane stays intact.[34]

In this report, observation of the effect of 1.0, 1.5, and 2.0 kV/cm, 50 μs-duration electrical field exposure to MCF-7 breast cancer cell lines has been reported. We have performed trypan blue exclusion test, MTT assay, and clonogenic assay to investigate the effect. Furthermore, the effect of the combination of curcumin and electrical pulse also has been investigated. In addition, our study investigates the minimum energy that causes necrotic cell death.


 > Materials and Methods Top


Pulse generator

To generate microsecond duration electrical pulse, capacitor C is charged by DC power supply (HV4800E, ECIL India) and discharged (through load) with the help of IGBT (SKM800GA176D, SEMIKRON) switch. Since load here also works as a capacitor, therefore, we require C? to transfer the energy from the charging capacitor to the load capacitor. To fulfill this requirement, a charging capacitor of value 100 μF is selected. IGBT is triggered with a square low-voltage pulse generator, to vary the voltage, the capacitor is charged with a variable DC voltage source (here 100 V-1200 V, since the IGBT has a maximum rating of 1200 V). To vary the duration of pulse variable duration, low-voltage square pulse generator will be required to trigger the IGBT. Here, voltage applied is 200 V, 300 V, and 400 V to 2 mm electroporation to produce electrical field intensity of 1, 1.5, and 2 kV/cm. The pulse duration of 50 μs is kept constant at 1 Hz of pulse repetition rate. [Figure 1] shows the experimental setup and [Figure 2] shows the electrical circuit diagram (a) and output voltage waveform at 300V of charging voltage.
Figure 1: Setup showing electrical field exposure to MCF-7 cells in electroporation cuvette

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Figure 2: Pulse generator circuit (a) and (b) output voltage under load (at 300 V)

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Cell culture preparation

MCF-7 cell line is used in the present study. Cells were harvested in a tissue culture flask (25 cm2, HiMedia), in Dulbecco's Modified Eagle Medium (DMEM, HiMedia) at 37°C in humidified, 5% CO2 incubator. DMEM contains 10% fetal bovine serum (FBS, GIBCO) and 1% antibiotics penicillin–streptomycin–amphotericin which support cell growth. After achieving 90% confluency, cultured cells adhering to the bottom surface of the flask were washed twice with phosphate-buffered saline (PBS, Wako) and detached from the flask bottom using PBS-based 0.025% trypsin and 0.01% ethylenediaminetetraacetic acid. The cell suspension was centrifuged at 1200 rpm for 5 min and re-suspended with a fresh DMEM medium to form a required cell concentration of 1 million/ml.

Pulse electric field application

Electrical pulse parameters were selected as 0, 5, 10, 15, 20, and 30 pulses at 1 Hz for 200, 300, and 400 V each. Cancer cells' suspension (in complete media) was taken in a 2 mm electroporation cuvette (BioRad, Inc., Hercules, CA). Exposure of 200 V, 300 V, and 400 V pulse produces electrical fields of 1, 1.5, and 2 kV/cm in between cuvette electrodes, respectively. Current through the load measured using current shunt (R = 0.005056Ω) is ~10A at 200 V, ~15 A at 300 V, and ~20 A at 400 V. For 50 μs duration, energy exposed to load (VIt) per pulse is ~0.1 J at 200 V, ~0.225 J at 300 V, and ~0.4 J at 400 V. Maximum energy exposed to cancer cells is 3 J, 7.5 J, and 12 J at 200 V, 300 V, and 400 V, respectively.

Trypan blue exclusion test

The trypan blue exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that living cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin, or propidium, whereas dead cells do not. 10 μl cell suspension is mixed with trypan blue (0.4% solution in PBS) and then visually examined to determine whether cells take up or exclude dye. A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.

MTT assay

Post-exposure of electrical pulses cells was seeded in 96 well plate at a concentration of 5 × 104 cells per well. The total volume of the well is made at 120 μl and cells are incubated for 24 h in a 5% CO2 incubator at 37°C. After 24 h incubation, media is removed carefully from the 96-well plate. MTT solution in DMEM (0.5 mg/ml) 100 μl is filled in each well and again incubated for 3–4 h. After 3–4 h, MTT solution is replaced with 100 μl of dimethyl sulfoxide (DMSO) +1% glacial acetic acid is filled in each well. The plate was shaken for 10 min by an orbital shaker. It is ready for quantitative analysis under 96-well plate spectrophotometer (microplate reader) at 570 nm wavelength. The experiment is performed in triplicate.



Clonogenic assay

The clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division. The clonogenic assay is the method to determine cell reproductive death after treatment with ionizing radiation but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retain the capacity to produce colonies. Before or after treatment, cells are seeded out with appropriate dilutions to form colonies in 10–15 days. Colonies are fixed with methanol and acetic acid (3:1), stained with crystal violet (0.5% w/v), and then counted.

Combination of curcumin and electrical pulse

Curcumin (>99% was received as a gift from Win Herbal Care, India) is a polyphenol derived from the herbal remedy and dietary spice turmeric. It possesses diverse anti-inflammatory and anticancer properties following oral or topical administration. Curcumin is a potent antioxidant and it initiates several cells signaling pathways at multiple levels. It effects on cellular enzymes including cyclooxygenase and glutathione S-transferases, immuno-modulation and effects on angiogenesis and cell-cell adhesion. It has the ability to affect gene transcription and to induce apoptosis in preclinical models. Curcumin is of low systemic bioavailability; therefore, its oral dosing may limit access of sufficient concentrations for pharmacological effect in certain tissues; furthermore, its particular relevance to cancer chemoprevention and chemotherapy in patients the attainment of biologically active levels in the gastrointestinal tract has been demonstrated in animals and humans.[35] We have used curcumin dose of 10 μM, 20 μM and 30 μM (stock solution 5mM in DMSO) in combination with 5, 10 and 15 electrical pulses of intensity 1.5 kV/cm, 50 μs. Effect of combination of curcumin doses on cancer cells was compared with the effect of curcumin doses 10 μM, 20 μM, 30 μM and effect of 5, 10, 15 electrical pulses.


 > Results and Discussion Top


Exposure of 5, 10, 15, 20, 30 pulses of 1, 1.5, and 2 kV/cm, 50 μs electrical pulses at the rate of 1 Hz to MCF-7 shows significant destructive effect. Trypan blue test shows immediate death of cells by applying these electrical pulses [Figure 3]. It has been observed that 10 pulses of 2 kV/cm field ~80% cells have up taken the dye which indicates immediate cell death, 10 pulses of 1 kV/cm field do not produce substantial killing effect, but 20 pulses of 1 kV/cm affect ~40% cells. Effect of 1.5 kV/cm field is in between 1 kV/cm and 2 kV/cm for >15 pulses. Fifteen or more pulses of 1.5 kV/cm affect the cells as 2 kV/cm. More than 10 electrical pulses of electrical field intensity of 2 kV/cm, 50 μs are highly lethal and cause permanent damage of cells, i.e., irreversible electroporation (IRE).
Figure 3: Trypan blue exclusion test (a) at 1, 1.5, and 2 kV/cm electrical fields and figure showing trypan blue up taken by cells

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Viability test using MTT shows that growth inhibition due to pulse treatment [Figure 4]. It was observed that exposure of 10 pulses of 2 kV/cm reduces the cell viability drastically from ~85% to 25%, same as in the trypan blue exclusion test where trypan blue positive cells increase ~25% to 85% and later on, it saturates. Fifteen pulses of 1.5 kV/cm reduce the cell viability from ~80% to ~30%, same as in trypan blue exclusion test, trypan blue positive cells increase ~25%% to 85%. Although, 1 kV/cm does not reduce the cell viability below ~23%, as observed in the MTT assay. The above observations show that most cell death is because of necrosis.
Figure 4: MTT assay result at 1, 1.5, and 2 kV/cm electrical field

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Observation of individual cell survival (colony formation assay) [Figure 5] at the exposure of electric field intensity of 1.5 kV/cm shows further reduction of individual cell survival compared to untreated cells. At this electric field, reduction of cell survival indicated the induction of late apoptosis.
Figure 5: Colony formation assay at 1 and 1.5 kV/cm electrical field

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The combination of curcumin and pulses drastically reduces cell survival, in comparison to curcumin or pulse alone [Figure 6]. At 10 μM, 20 μM, and 30 μM of curcumin dose, viability is ~90%, 88%, and ~85%, respectively. At 5, 10, and 15 pulses of 1.5 kV/cm electric field viability are ~90%, 87%, and 84%, respectively. Although 5 pulses and 10 μM of curcumin dose reduce the viability up to ~70% However when 10 pulses and 10 μM curcumin dose are used in combination, viability reduces up to ~38%. Fifteen pulses and 10 μM further reduce up to ~35%. Significant reduction in viability at 10 pulses and 10 μM of curcumin dose is because of electroporation. Under the electrical field, the formation of pores on the cell membrane is known as electroporation. This result infers that at 10 pulses of 1.5 kV/cm, 50 μs causes IRE.
Figure 6: MTT assay result showing the effect of curcumin, electrical pulse (1.5 kV/cm, 50 μs), and combination of both curcumin and electrical pulse

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Electroporation is a threshold phenomenon. The field strength necessary for molecule delivery must exceed a threshold value. Moderate increases in the applied field strength result in delivery; however, larger increases in the applied field from this threshold result in tissue damage.[36]

Curcumin exerts anticancer effects in various cancer cells at concentrations generally higher than 10 μM, and such concentrations can cause the cytotoxic effect on normal cells. Moreover, it is well known that curcumin has the problem of low bioavailability because of its poor absorption. Previous reports have shown that the effectiveness of curcumin could be increased in human breast cancer MCF-7 cells and human leukemia HL-10 cells when electroporation was applied.[35],[36] This effect has been investigated in this report.

The combination of curcumin and electrical pulses works synergistically to reduce cell viability. This is possible if uptake of curcumin is increasing with the application of electrical pulses. Duration is kept only 50 μs compared to Gothelf A et al.[11], Camarillo IG et al.[37] and Yanpeng Lv et al.[38] to reduce the thermal effect to prevent necrosis. In researches bleomycin[10],[11] has been used which may have side effect but herb curcumin has almost no side effect.

Our study includes the preliminary study of cell survival to cell survival in isolation. Death cause and triggered pathways were not included in this study which require further investigation through flow cytometry, western blot, etc. This is to only investigate minimum electrical field strength to trigger cell death through necrosis and threshold pulse number and field strength at 50 μs duration for safe electroporation. More methods are required to investigate the targeted protein causing cell death through the apoptosis pathway.


 > Conclusion Top


Here in this report, the effect of exposure of 50 μ duration electrical pulses at 1 Hz and electric field intensities of 1, 1.5, and 2 kV/cm has been investigated. Immediate death, viability after 24 h, and growth in isolation of cells have been examined with trypan blue, MTT assay, and clonogenic assay, respectively. It has been also found that exposure of 10 pulses of 2 kV/cm, 50 μs corresponding to the energy of ~4 J is highly destructive to the cells. The combination of curcumin and electrical pulses shows a synergistic effect on cell viability because of electroporation. Activated protein in cells causing reduction of cell viability through the combinational effect of electrical pulses and curcumin requires further analysis. This preliminary result shows the promising destructive effect of electrical pulses on cancer cells.

Financial support and sponsorship

This work was supported by funding from Bhabha Atomic Research Centre, Government of India.

Conflicts of interest

There are no conflicts of interest.



 
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