Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia, United States of America.
PLoS ONE (Impact Factor: 3.23). 07/2013; 8(7):e70278. DOI: 10.1371/journal.pone.0070278
Source: PubMed


High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity ("nanoelectroporation"), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1-2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6-24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.

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    • "Disruption of the lamina[41]and the nucleus could contribute substantially to such a decrease in cellular elasticity. Pakhomova et al. have shown that the rate of increase of internal osmotic pressure and calcium influx can explain the rates of intracellular membrane permeabilization and cell death[42,43]. Here, longer microscopy acquisition times and more cells are needed to accurately calculate the rate of changes of the nucleus for a given AD. "
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    ABSTRACT: Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus.
    Full-text · Article · Dec 2015 · Biochemical and Biophysical Research Communications
    • "Romeo et al. proved that a single 5 ns, 10 MV/m electric pulse permeabilizes all membranes in living cells [10]. Other authors showed that nanosecond electroporation can be applied independently to induce cancer cells damage [4] [11] [12] [13]. Additionally, one of the nsPEF advantages is the inhibition of blood flow in the vessels feeding the cancer cells which induces apoptotic pathways [4] [14]. "
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    ABSTRACT: Background: Nanosecond pulsed electric field (nsPEF) treatment is a new anti-cancer strategy with ultrashort pulse duration and high intensity of electric fields. The application of nsPEFs affects all intra- and extracellular membranes and independently initiates the process of apoptosis within cancer cells, leading the tumor to slowly auto-destruct without the use of toxic drugs. Methods: This study involves cells of gastric adenocarcinoma (EPG85-257P and EPG85-257RDB), metastatic melanoma (Me45), epidermal cancer (A431), normal keratinocytes (HaCaT), and macrophages (P388/D1). The influence of nanosecond pulses on the cellular structure and cellular proliferation was evaluated. The effect of nsPEF was determined by MTT and clonogenic assays and the efficiency was monitored by following the propidium iodide and Photofrin II(®) uptake using FACS analysis. The cell membranes state was visualized with DHCC marker. Results: nsPEFs (up to 60kV/cm) induced significant decrease of cellular viability in all cancer cells except the A431 cell line. Photodynamic reactions combined with nsPEFs induced the highest decrease of cellular viability in both gastric cell lines and skin derived cancer cells. Normal (HaCaT and P388/D1) cells were in contrary not significantly affected. Propidium iodide and Photofrin II(®) uptake, used as markers of membrane permeabilization, were the most efficient in gastric cells. Finally, the most disturbed morphology was observed in the latter. Conclusions: This is the first attempt of combining nsPEF with photodynamic reaction using Ph II(®) for selective destruction of cancer cells. The results indicate the potential of nsPEF for inducing cytotoxicity mainly in adenocarcinoma cells, while combined with Photofrin II(®) and irradiation.
    No preview · Article · Nov 2015 · Photodiagnosis and photodynamic therapy
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    • "Since nsPEFs do not exist in nature, eukaryotic cells evolved without such stimuli so cell responses to them are distinct from those induced by other known forms of cellular stresses [23]. NsPEF-induced cell death has been defined as intrinsic caspase-dependent and caspase-independent [24] as well as apoptotic and necrotic [25] [26] [27]. Studies show that nsPEFs affect multiple therapeutic targets, including plasma membranes and mitochondria [28] [29] and cancer hallmarks including resisting cell death [9] [10]. "
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    ABSTRACT: Strategies for treating liver cancer using radiation, chemotherapy combinations and tyrosine kinase inhibitors targeting specific mutations have provided longer survival times, yet multiple treatments are often needed and recurrences with new malignant phenotypes are not uncommon. New and innovative treatments are undoubtedly needed to successfully treat liver cancer. Over the last decade, nanosecond pulsed electric fields (nsPEFs) have shown promise in pre-clinical studies; however, these have been limited to treatment of skin cancers or xenographs in mice. In the present report, an orthotopic hepatocellular carcinoma (HCC) model is established in rats using N1-S1 HCC cells. Data demonstrate a response rate of 80-90% when 1000 pulses are delivered with 100ns durations, electric field strengths of 50kV/cm and repetition rates of 1Hz. N1-S1 tumours treated with nsPEFs expressed significant number of cells with active caspase-3 and caspase-9, but not caspase-8, indicating an intrinsic apoptosis mechanism(s) as well as caspase-independent mechanisms. Most remarkably, rats with successfully ablated tumours failed to re-grow tumours when challenged with a second injection of N1-S1 cells when implanted in the same or different liver lobe that harboured the original tumour. Given this protective effect, infiltration of immune cells and the presence of granzyme B expressing cells within days of treatment suggest the possibility of an anti-tumour adaptive immune response. In conclusion, NsPEFs not only eliminate N1-S1 HCC tumours, but also may induce an immuno-protective effect that defends animals against recurrences of the same cancer.
    Full-text · Article · Jul 2014 · European journal of cancer (Oxford, England: 1990)
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