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.
Ren W, Sain NM, Beebe SJ (2012) Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells. Biochemical and biophysical research communications 421: 808–812.
Beebe SJ, Fox PM, Rec LJ, Somers K, Stark RH, et al. (2002) Nanosecond Pulsed Electric Field (nsPEF) Effects on Cells and Tissues: Apoptosis Induction and Tumor Growth Inhibition. IEEE Transactions on Plasma Science 30: 286–292.
Garon EB, Sawcer D, Vernier PT, Tang T, Sun Y, et al. (2007) In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies. Int J Cancer 121: 675–682.
Nuccitelli R, Chen X, Pakhomov AG, Baldwin WH, Sheikh S, et al. (2009) A new pulsed electric field therapy for melanoma disrupts the tumor’s blood supply and causes complete remission without recurrence. Int J Cancer 125: 438–445.
Chen X, Zhuang J, Kolb JF, Schoenbach KH, Beebe SJ (2012) Long term survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields. Technology in cancer research & treatment 11: 83–93.
Ibey BL, Roth CC, Pakhomov AG, Bernhard JA, Wilmink GJ, et al. (2011) Dose-dependent thresholds of 10-ns electric pulse induced plasma membrane disruption and cytotoxicity in multiple cell lines. PLoS One 6: e15642.
Stacey M, Stickley J, Fox P, Statler V, Schoenbach K, et al. (2003) Differential effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA damage, and cell cycle analysis. Mutat Res 542: 65–75.
Nesin OM, Pakhomova ON, Xiao S, Pakhomov AG (2011) Manipulation of cell volume and membrane pore comparison following single cell permeabilization with 60- and 600-ns electric pulses. Biochim Biophys Acta 1808: 792–801.
Wang J, Guo J, Wu S, Feng H, Sun S, et al. (2012) Synergistic effects of nanosecond pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro. PLoS One 7: e43213.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG (1993) Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 53: 3976–3985.
Manns J, Daubrawa M, Driessen S, Paasch F, Hoffmann N, et al. (2011) Triggering of a novel intrinsic apoptosis pathway by the kinase inhibitor staurosporine: activation of caspase-9 in the absence of Apaf-1. FASEB J 25: 3250–3261.