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.
References
[1]
Schoenbach KS, Hargrave B, Joshi RP, Kolb J, Osgood C, et al. (2007) Bioelectric Effects of Nanosecond Pulses. IEEE Transactions on Dielectrics and Electrical Insulation 14: 1088–1109.
[2]
Ren W, Beebe SJ (2011) An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma. Apoptosis 16: 382–393.
[3]
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.
[4]
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.
[5]
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.
[6]
Yin D, Yang WG, Weissberg J, Goff CB, Chen W, et al. (2012) Cutaneous papilloma and squamous cell carcinoma therapy utilizing nanosecond pulsed electric fields (nsPEF). PLoS One 7: e43891.
[7]
Yang W, Wu YH, Yin D, Koeffler HP, Sawcer DE, et al. (2011) Differential sensitivities of malignant and normal skin cells to nanosecond pulsed electric fields. Technol Cancer Res Treat 10: 281–286.
[8]
Nuccitelli R, Pliquett U, Chen X, Ford W, James Swanson R, et al. (2006) Nanosecond pulsed electric fields cause melanomas to self-destruct. Biochem Biophys Res Commun 343: 351–360.
[9]
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.
[10]
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.
[11]
Walker K 3rd, Pakhomova ON, Kolb J, Schoenbach KS, Stuck BE, et al (2006) Oxygen enhances lethal effect of high-intensity, ultrashort electrical pulses. Bioelectromagnetics 27: 221–225.
[12]
Ibey BL, Pakhomov AG, Gregory BW, Khorokhorina VA, Roth CC, et al. (2010) Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells. Biochim Biophys Acta 1800: 1210–1219.
[13]
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.
[14]
Beebe SJ, Fox PM, Rec LJ, Willis EL, Schoenbach KH (2003) Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells. Faseb J 17: 1493–1495.
[15]
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.
[16]
Pakhomov AG, Miklavcic D, Markov MS, editors (2010) Advanced Electroporation Techniques in Biology in Medicine. Boca Raton: CRC Press. 528 p.
[17]
Andre FM, Rassokhin MA, Bowman AM, Pakhomov AG (2010) Gadolinium blocks membrane permeabilization induced by nanosecond electric pulses and reduces cell death. Bioelectrochemistry 79: 95–100.
[18]
Hall EH, Schoenbach KH, Beebe SJ (2005) Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells. DNA Cell Biol 24: 283–291.
[19]
Beebe SJ, Blackmore PF, White J, Joshi RP, Schoenbach KH (2004) Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms. Physiol Meas 25: 1077–1093.
[20]
Bevers EM, Comfurius P, Dekkers DW, Zwaal RF (1999) Lipid translocation across the plasma membrane of mammalian cells. Biochim Biophys Acta 1439: 317–330.
[21]
Zhao J, Zhou Q, Wiedmer T, Sims PJ (1998) Level of expression of phospholipid scramblase regulates induced movement of phosphatidylserine to the cell surface. J Biol Chem 273: 6603–6606.
[22]
Vernier PT, Sun Y, Marcu L, Craft CM, Gundersen MA (2004) Nanoelectropulse-induced phosphatidylserine translocation. Biophys J 86: 4040–4048.
[23]
Vernier PT, Ziegler MJ, Sun Y, Gundersen MA, Tieleman DP (2006) Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipid bilayers–in cells and in silico. Phys Biol 3: 233–247.
[24]
Pakhomov AG, Shevin R, White JA, Kolb JF, Pakhomova ON, et al. (2007) Membrane permeabilization and cell damage by ultrashort electric field shocks. Arch Biochem Biophys 465: 109–118.
[25]
Pakhomov AG, Pakhomova ON (2010) Nanopores: A distinct transmembrane passageway in electroporated cells. In: Pakhomov AG, Miklavcic D, Markov MS, editors. Advanced Electroporation Techniques in Biology in Medicine. Boca Raton: CRC Press. 178–194.
[26]
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.
[27]
Deng J, Schoenbach KH, Buescher ES, Hair PS, Fox PM, et al. (2003) The effects of intense submicrosecond electrical pulses on cells. Biophys J 84: 2709–2714.
[28]
Pakhomov AG, Phinney A, Ashmore J, Walker K, Kolb JF, et al. (2004) Characterization of the cytotoxic effect of high-intensity, 10-ns duration electrical pulses. IEEE Transactions on Plasma Science 32: 1579–1585.
[29]
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.
[30]
Pakhomova ON, Gregory BW, Khorokhorina VA, Bowman AM, Xiao S, et al. (2011) Electroporation-induced electrosensitization. PLoS One 6: e17100.
[31]
Schoenbach KH, Beebe SJ, Buescher ES (2001) Intracellular effect of ultrashort electrical pulses. Bioelectromagnetics 22: 440–448.
[32]
Casiano CA, Ochs RL, Tan EM (1998) Distinct cleavage products of nuclear proteins in apoptosis and necrosis revealed by autoantibody probes. Cell Death Differ 5: 183–190.
[33]
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.
[34]
Dunnet CW (1955) A multiple comparison procedure for comparing several treatments with a control. J of the American Statistical Association 50: 1096–1121.
[35]
Winer BJ (1971) Statistical Principles in Experimental Design. New York: McGraw-Hill Book Company.
[36]
Kinosita K Jr, Tsong TY (1977) Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature 268: 438–441.
[37]
Okada Y (2004) Ion channels and transporters involved in cell volume regulation and sensor mechanisms. Cell Biochem Biophys 41: 233–258.
[38]
Kinosita K Jr, Tsong TY (1977) Voltage-induced pore formation and hemolysis of human erythrocytes. Biochimica et biophysica acta 471: 227–242.
[39]
Bowman AM, Nesin OM, Pakhomova ON, Pakhomov AG (2010) Analysis of plasma membrane integrity by fluorescent detection of Tl(+) uptake. J Membr Biol 236: 15–26.
[40]
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.
[41]
Vernier PT, Sun Y, Gundersen MA (2006) Nanoelectropulse-driven membrane perturbation and small molecule permeabilization. BMC Cell Biol 7: 37.
[42]
Idziorek T, Estaquier J, De Bels F, Ameisen JC (1995) YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J Immunol Methods 185: 249–258.
[43]
Beebe SJ, White J, Blackmore PF, Deng Y, Somers K, et al. (2003) Diverse effects of nanosecond pulsed electric fields on cells and tissues. DNA Cell Biol 22: 785–796.
[44]
Kepp O, Galluzzi L, Martins I, Schlemmer F, Adjemian S, et al. (2011) Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy. Cancer Metastasis Rev 30: 61–69.