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Apoptosis induction with electric pulses — A new approach to cancer therapy with drug free
Biochemical and Biophysical Research Communications 390 (2009) 1098–1101
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mini Review
Apoptosis induction with electric pulses — A new approach to cancer therapy with drug free q Liling Tang a,b,c,*, Chenguo Yao a, Caixin Sun a a
State Key Laboratory of Power Transmission Equipment & System and New Technology, Chongqing University, Chongqing 400044, China Key Laboratory of Biorheological Science and Technology, Chongqing University, Ministry of Education, Chongqing 400044, China c College of Bioengineering, Chongqing University, Chongqing 400044, China b
a r t i c l e
i n f o
Article history: Received 14 October 2009 Available online 22 October 2009 Keywords: Electric pulse Apoptosis Cancer therapy Drug free
a b s t r a c t Electrical pulses have been widely used in biomedical fields, whose applications depend on the parameters such as durations and electric intensity. Conventional electroporation (0.1–1 kV/cm, 100 ls) has been used in cell fusion, transfection and electrochemotherapy. Recent studies with high-intensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact, resulting in an application of intracellular manipulation. The most recent development is the finding that parameters between those two ranges could be used to induce apoptosis of cancer cells. Proposal of apoptosis induction and tumor inhibition has advantages to pursue the treatment of cancer free of cytotoxic drugs. Ó 2009 Elsevier Inc. All rights reserved.
Electric pulses have been widely used in the biological and medical fields, including killing microorganisms, cell fusion, gene transfection, cancer therapy since 1980s [1–8]. For cancer therapy, radiofrequency and microwave devices can heat the tumor to greater than 43 °C to kill the cells via hyperthermia [5,6]. And the most advanced procedure is electrochemotherapy (ECT), which is developing rapidly after the first reports by Mir et al. [8] on 1988. The application of electric pulses to cells leads to the transient permeabilization of the cell membrane and increase the delivery of drugs (e.g. bleomycin) into cells. Effectiveness of ECT depends on the effective distribution of the cytotoxic drug in the tumor and the permeabilization of the vast majority of the tumor cells [7]. Although the ECT reduces the total dosage of the drug per patient considerably, it could not completely deplete the side effect of cytotoxic drugs on human body. Developments of pulsed power technology provide the possibility to find a method to kill cancer cells without the aids of cytotoxic drugs. Nowadays electric pulses can be applied in a wide parameter range with duration as short as nanosecond and electric intensity up to hundreds of kilovolts. The rising time of pulses can be controlled in several nanoseconds. It provides the necessary technical supports for the further application of electric pulses in cancer therapy. Responses of cells to electric pulses mainly depend on the parameters such as electric field intensity and pulse durations. q
Grant sponsor: Natural Science Foundation of China (No. 50637020). * Corresponding author. Address: College of Bioengineering, Chongqing University, Chongqing 400044, China. E-mail address: [email protected] (L. Tang). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.092
Electric pulses of 0.1–1 kV/cm and 100 ls normally result in electroporation of cells. Electroporation (EP) is termed as the physical procedure of transient pores on plasma membrane in response to electric pulses. EP brings new properties to the cell membrane. It becomes permeable to drugs, gene materials, proteins and other large molecules. The pores will recover from electric pulses and therefore EP is a reversible procedure [9]. On the contrary, highintensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact. Such effect is called intracellular manipulation (IEM) [10]. As so far, the observation of EP has resulted in the application of electrochemotherapy, and the IEM makes it possible to regulate the intracellular structures and functions. Recent studies on high-intensity pulsed electrical fields have led to a significant observation: it is possible to induce cell apoptosis [11–14]. Apoptotic cells exhibit features including externalization of phosphatidylserine (PS) on the plasma membrane, caspase activation and chromosomal condensation. Such electric effect is different from the EP and IEM. We term it as the apoptosis induction with electric pulses (AIEP), because cells are led to apoptosis and can not recover from the electric pulses. It can be used to introduce apoptosis of cancer cells without the aids of cytotoxic drug. Therefore, the proposal of AIEP and the determination of threshold values for electric pulses are significantly important. It will provide a new approach to treat cancer by electric pulses with drug free. In the current review, we discuss the effects of electric pulses with different parameters on tumor cells and the new approach to cancer therapy by inducing cell apoptosis with electric pulses.
L. Tang et al. / Biochemical and Biophysical Research Communications 390 (2009) 1098–1101
Electroporation The first use of electroporation to deliver DNA to mammalian cells was reported by Neumann’s group in the early 1980s. Since then electroporation has become a relatively standard laboratory technique to transfect cells (plants and animal cells) in culture [15,16]. Theoretically, cells in an electric field can be modeled as an electric circuit with cell membrane as capacitance and the cytoplasm as resistance. Application of microsecond or longer duration electric fields to cells causes large build-ups of oppositely charged ions on either side of the cell surface. If too much charge gathers at the cell membrane, the electric field there breaks the membrane down. Large holes (or pores) form in the membrane and allow ions to pour across. This effect is called electroporation [17]. Electroporation occurs when the transmembrane potential difference (Dw) reaches threshold values. The transmembrane potential (Dwi) is a complex function. Dwi = f g(k) r E cos h, in which k is the conductivities of the membrane, r is the cell radius, E is the field intensity, h is the angle between the direction of the normal to the membrane at the considered point on the cell surface and field direction, and f is a shape factor. Dw is the sum between the resulting value of cell membrane Dw0 and the electroinduced value Dwi [18]. The most important parameters for effective electroporation are the electric field strength and pulse length (duration). When the applied pulse achieves a threshold voltage of approximately 1 V across a membrane, the membrane becomes permeable and allows passage of materials through the membrane [9,19]. If the field strength is too low, the breakdown transmembrane potential is not achieved; similarly, if the duration is too short, the membrane cannot be charged enough to reach the electroporation membrane potential [19]. Electroporation enables efficient delivery of drugs or synthetic short interfering RNA (siRNA) into some organs and tumors, thus it can be used to treat various diseases [20,21]. Optimal parameters differ for different molecules and cell types. For the delivery of low molecular weight drugs into mammalian cells, typically fields of 1000 V/cm and durations of 100 ls are effective, whereas lower fields (>50 V/cm) and longer pulses (21 ms) are better for the delivery of genes [19]. When electric pulses of 100 ls are applied to deliver bleomycin in vivo, the threshold value is between 300 and 500 V/cm in tumors. Four hundred and fifty volts per centimeter is the threshold for 51Cr-EDTA uptake in skeletal muscle by electroporation [22]. In summary, 0.1–1 kV/cm electric pulses are usually applied with durations (100 ls–21 ms) to result in reversible electroporation. Electric pulses with higher intensity (2500 V/cm, 100 ls) than that of traditional EP cause irreversible electroporation in tumor cells in vivo [23].
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following features: (1) a rise time that is short relative to the charging time of intracellular organelle membranes, (2) a duration that is long enough to allow charging of organelle membranes but too short to achieve cell surface membrane changing, and (3) an intensity adequate to achieve organelle membrane charging during the pulse application. Such process is referred as IEM. For electroporation effect induced by electric pulses with durations at microsecond to millisecond, cells can be modeled as a capacitance, in which cell membrane envelopes the cytoplasm. However, for electric pulses with shorter duration, intracellular membrane including membrane of organelles and nucleus must be considered [26] (shown in Fig. 1). The effect of nsPEF treatment on cells also depends on two parameters: electric field intensity and pulse duration. In response to the applied electric fields, ions in the cell interior will move in the field direction and charge the membrane until they experience no further force. If the duration of nsPEF is shorter than the plasma membrane charging time, sPM (0.2–1 ls), the interior charges cannot redistribute enough to counter act the imposed field. Electric pulses will penetrate into the cell and charge every organelle membrane [27]. The force exerted on charges depends on the strength of the electric field. In Chen et al.’s [26] experiments with HL-60 cells, PI (propidium iodide) was used as a tracer for plasma membrane integrity. When the cell membrane is broken, PI enters the cell and can be visualized through its dark red fluorescence. Results have shown that 10-ns, 65-kV/cm pulses cannot result in electroporation in plasma membrane, while 60-ns, 26-kV/cm pulses (approximately same energy density as provided by the 10-ns pulse) break the plasma membrane. Analysis with a cell model shows a similar result [28]. For the 11-ns pulse with E = 25 kV/cm, the inner membrane transmembrane potential could easily reach the 1 V threshold, which means pulses are more likely to porate inner organelles, while leaving the outer membrane intact. Thus, electric pulses with duration of 10 ns–100 kV/cm are usually applied in IEM. Because of the special features of IEM in the selective electroporation of intracellular organelles membrane, it can induce a series of physiological responses including the release of calcium from intracellular calcium stores, changes in gene expression, DNA conformation and nucleus. IEM can be used for selective/generalized cell or tissue ablation, growth stimulation and tissue remodeling [9]. Furthermore, ultrashort pulses can be applied after the conven-
Intracellular electromanipulation (IEM) With the developments of electric engineering, researchers could apply electric fields with higher electric intensity and shorter duration to cells. Nanosecond pulsed electric field (nsPEF), ultrashort pulsed electric field (usPEF), and submicrosecond, intense pulsed electric field (sm/I-PEF) with intensity at 10–1000 kV/cm and duration less than 1 ls have been applied by different researchers [24,25]. Compared to traditional electroporation, nsPEF can result in quite different effects, which is called intracellular electromanipulation (IEM). Intracellular electromanipulation means the achievement of intracellular physiological effects by electric field application. According to the views of Buescher et al. [9,25], permeability/poration effects are predicted to occur at organelle/intracellular membranes if the pulse characteristics are modified to achieve
Fig. 1. An equivalent circuit cell model. A cell can be considered as a circuit made up of capacitors and resistors. Cell membrane and organelles’s membranes act as capacitors. The cytosol and nucleoplasm is conductive and so can be modeled as resistors. ‘‘C” refers to the capacitor and ‘‘R” is the resistor.
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L. Tang et al. / Biochemical and Biophysical Research Communications 390 (2009) 1098–1101
tional electroporation pulses. Effects of nsPEF on subcellular membrane change the nuclear membrane and facilitate the delivery of genes into nucleus. Studies have shown that a combination of a classical long pulse and a nanosecond pulse can increase gene expression in nucleus [29].
Apoptosis induction with electric pulses (AIEP) Apoptosis induced by electric pulses A recent finding in the application of electric pulses is based on the finding that apoptosis could be induced by electric pulses with certain parameters. Apoptosis is the morphological manifestation of programmed cell death in cells. It can be considered as the cell suicide. Beebe et al. [30] and Vernier et al. [11] have reported that cell apoptosis could be induced by electric pulses in vitro. Different cell types also have different responses to electrical pulses applications. Studies on Jurkat cells and HL-60 cells have shown that the former is more susceptible to electrical pulses [11,28,31]. Killing cancer cells by inducing cell apoptosis can prevent the inflammatory responses in vivo. The observation of cell apoptosis induced by electrical pulses seems to provide a bright future for cancer therapy without aids of drugs. Selective parameters of electric pulses play major roles in the type of electroeffect on stimulated cells. As described above, electric pulses of IEM with duration at 10 ns, intensity at 100 kV/cm induce intracellular effects, such as intracellular calcium release, DNA changes, poration on organelles membrane, while cell membrane is intact. For 100 ls, 1 kV/cm electric pulses, electroporation on cell membrane occurs to facilitate large molecular materials delivery. In this procedure, cell function is not affected and electroporation is reversible. It is interesting to investigate what will happen to cells when subjected electric pulses with parameters between IEM and EP. Significant apoptosis of several types of cells (SKOV3 human ovarian carcinoma cells, HCT116 colon carcinoma cells, HL-60 cells and Jurkat cells) has been observed when treated by such electric pulses [32–34]. We name the procedure of apoptosis of cells induced by electric pulse with parameters between electroporation and intracellular electromanipulation as ‘‘apoptosis induction with electric pulses (AIEP).” That is to say, the apoptotic effect of electric pulses on cells is irreversible. Thus we enlarge the parameter range for bioelectric applications proposed by Schoenbach et al. [25]. Possible parameters are shown in Fig. 2 according to results of our group and other studies. The observed apoptotic behavior appears to depend on the pulse duration. For cells subjected to external electric fields at a similar energy input, much stronger apoptosis markers were observed only for the longer (300 ns) pulses, less at the shorter (60 ns) durations, and almost negligible effects for a short 10 ns pulses [35]. The apoptotic trends are consistent with the extent of poration on cell surface membrane, in which levels of poration of 300 ns pulses larger than that of 60 ns, while a 10 ns pulse caused insignificant poration [30]. The results also suggest the integrity of plasma membrane plays a role in cell apoptosis induced by electric pulses. There are different characteristics of electroporation: poration on cell surface membrane, poration on organelles membrane or poration on all membranes, by which to distinguish EP, IEM and AIEP. Conventional EP causes electroporation on plasma membrane, while the IEM only electroporates the intracellular membrane. Some researchers viewed the permeabilization of plasma membrane as a secondary effect caused by changes of cell functions; however, apoptosis induced by electric pulses is always coupled to the poration of cell membrane. The extent of apoptotic cells is related to the extent of poration [30,36]. Apoptosis signals can arise from the
Fig. 2. Different electric pulses results from different electric parameters. Electric pulses of 0.1–1 kV/cm and 100 ls normally result in electroporation of cells, which are widely used in gene transfer, electroporation and transdermal drug delivery. Electric pulses of intensity at 10–1000 kV/cm and duration less than 100 ns are used to intracellular electromanipulation, which can regulate intracellular behavior such as release of calcium ion, improvement of gene expression, change of cell cycle and thus inhibiting the tumor growth. Electric pulses between these two parameters are found to bring force some special effects including irreversible apoptosis, antiangiogenesis, anti-lymphatic capillaries around cancer tissue, immune effects and so on.
plasma membrane or from intracellular structures, including the nucleus, mitochondria, or endoplasmic reticulum [28]. In other words, although intracellular mechanism influences the apoptosis of cells subjected by electric pulses, effect on plasma membrane at least play a supporting role in it. The possibility of using electric pulses independently to induce cell apoptosis in cancer therapy also relies on the different responses between cancers cells and normal cells. Cancer cells have a higher apoptosis percentage than normal cells when exposed to the same electric field [37]. A widely accepted theory is that cancer cells are more susceptible to electric field than normal cells, because cancer cells have higher dielectric constant and larger nucleus. Applications of AIEP Effectiveness of electric pulses on inhibiting cancer cells and tissues independently has been proved by many experiments. In vitro experiment showed a significant apoptosis in human ovarian carcinoma cell line (SKOV3) after treating with 10 kV/cm, 100 ns electric pulses [32]. Nuccitelli et al. [27] discovered that pulsed electric fields greater than 21 kV/cm with durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink. Melanomas (a kind of skin cancer) shrink by 90% within two weeks following a cumulative field exposure time of 121 ls. A most recent report has shown that the application of 40 kV/cm, 300 ns electric pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission [38]. Furthermore, electric pulses have additional effects on the migration of cancer cells. It was found that electric pulses had the potential to destroy lymphatic capillaries around cancer tissue and thus decreased the possibility of post-treatment lymphatic metastasis [39]. It is an important effect as that functional lymphatics in the tumor margin alone are sufficient for lymphatic metas-
L. Tang et al. / Biochemical and Biophysical Research Communications 390 (2009) 1098–1101
tasis and should be target therapeutically [40]. The electric pulses can reduce blood flow to the tumor [38]. The expression of vascular endothelial growth factor (VEGF), an important growth factor to vascular growth, was decreased by electric fields in vascules nearby the cancer tissues [39]. Although there is no experiment in human body as so far, we predict that electric pulses with defined parameters could be applied to human body without significant side effects. Conclusions In spite of many kinds of therapies for cancer, it is still a difficult task to treat cancer as well as keep the high living quality of patients. Proposal of AIEP has advantages to pursue the treatment of cancer free of cytotoxic drugs. The potential to achieve selective killing tumor cells by inducing cancer cells apoptosis would provide a method for cancer therapy. The low energy input of electric pulses because of short durations prevents damage to tissues and cells nearby the tumor. Thus the applications for AIEP present a broad and bright future. References [1] J. Mosqueda-Melgar, R.M. Raybaudi-Massilia, O. Martin-Belloso, Combination of high-intensity pulsed electric fields with natural antimicrobials to inactivate pathogenic microorganisms and extend the shelf-life of melon and watermelon juices, Food Microbiol. 25 (2008) 479–491. [2] N.W. Karja, T. Otoi, P. Wongsrikeao, R. Shimizu, M. Murakami, B. Agung, M. Fahrudin, T. Nagai, Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer, Theriogenology 66 (2006) 1237–1242. [3] J.W. Henshaw, D.A. Zaharoff, B.J. Mossop, F. Yuan, Electric field-mediated transport of plasmid DNA in tumor interstitium in vivo, Bioelectrochemistry 71 (2007) 233–242. [4] H. Aihara, J. Miyazaki, Gene transfer into muscle by electroporation in vivo, Nat. Biotechnol. 16 (1998) 867–870. [5] S. Kuriyama, Improved survival benefits with radiofrequency ablation for liver cancer, Cancer Treat. Rev. 31 (2005) 408–412. [6] J.L. Huang, Z.G. Zhang, Y.B. Chi, T.H. Yang, M.G. Xu, Cancer Lett. 82 (1994) 199– 202. [7] S. Satkauskas, D. Batuskatite, S. Salomskaite-Davalgiene, M.S. Venslauskas, Effectiveness of tumor electrochemotherapy as a function of electric pulse strength and duration, Bioelectrochemistry 65 (2005) 105–111. [8] L.M. Mir, H. Banoun, C. Paoletti, Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol, Exp. Cell Res. 175 (1988) 15–25. [9] E.S. Buescher, K.H. Schoenbach, Effects of submicrosecond, high intensity pulsed electric fields on living cells — intracellular electromanipulation, IEEE Trans. Dielect. Elect. Insul. 10 (2003) 788–794. [10] L.M. Mir, Therapeutic perspectives of in vivo cell electropermeabilization, Bioelectrochemistry 53 (2001) 1–10. [11] P.T. Vernier, A. Li, L. Marcu, C.M. Crait, M.A. Gundersen, Ultrashort pulsed electric fields induce membrane phospholipid translocation and caspase activation: differential sensitivities of Jurkat T lymphoblasts and rat glioma C6 cells, IEEE Trans. Dielect. Elect. Insul. 10 (2003) 795–809. [12] P.T. Vernier, Y. Sun, L. Marcu, C.M. Craft, M.A. Gunderson, Nanoelectropulseinduced phosphatidylserine translocation, Biophys. J. 86 (2004) 4040–4088. [13] K. Schoenbach, R. Joshi, J. Kolb, S. Buescher, S. Beebe, Subcellular effects of nanosecond electrical pulses, Conf. Proc. IEEE Eng. Med. Biol. Soc. 7 (2004) 5447–5450. [14] P.T. Vernier, M.J. Ziegler, Y. Sun, M.A. Gundersen, D.P. Tieleman, Nanoporefacilitated voltage-in cells and in silico, Phys. Biol. (2–3) (2006) 233–247. [15] R. Heller, The development of electroporation, Science 295 (2002) 277. [16] M. Fromm, L. Taylor, Stable transformation of maize after gene transfer by electroporation, Nature 319 (1986) 791–793.
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[17] K.H. Schoenbach, R. Nuccitelli, S.J. Beebe, Extreme voltage could be a surprisingly delicate tool in the fight against cancer, IEEE Spectr. 43 (2006) 20–26. [18] M. Golzio, M.P. Rols, J. Teissie, In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression, Methods 33 (2004) 126–135. [19] S.B. Dev, D.P. Rabussay, G. Widera, G.A. Hofmann, Medical applications of electroporation, IEEE Trans. Plasma Sci. 28 (2000) 206–223. [20] G. Sersa, M. Cemazar, J.M. Snoj, Electrochemotherapy of tumours, Curr. Oncol. 16 (2009) 34–35. [21] O. Mazda, T. Kishida, Molecular therapeutics of cancer by means of electroporation-based transfer of siRNAs and EBV-based expression vectors, Front. Biosci. 1 (2009) 316–331. [22] J. Gehl, T.H. Sorensen, K. Nielsen, P. Raskmark, S.L. Nielsen, T. Skovsgaard, L.M. Mir, In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution, Biochim. Biophys. Acta 1428 (1999) 233–240. [23] B. Al-Sakere, F. André, C. Bernat, E. Connault, P. Opolon, R.V. Davalos, B. Rubinsky, L.M. Mir, Tumor ablation with irreversible electroporation, Plos ONE 2 (2007) e1135. [24] P.S. Hair, K.H. Schoenbach, E.S. Buescher, Sub-microsecond, intense pulsed electric field applications to cells show specificity of effects, Bioelectrochemistry 61 (2003) 65–72. [25] K.H. Schoenbach, S. Katsuki, R.H. Stark, E.S. Buescher, S.J. Beebe, Bioelectrics — new applications for pulsed power technology, IEEE Trans. Plasma Sci. 30 (2002) 293–300. [26] N. Chen, K.H. Schoenbach, J. Kolb, A.L. Gamer, J. Yang, J.R. Swanson, S. Beebe, Leukemic cell intracellular responses to increased nanosecond electric fields, Biochem. Biophys. Res. Commun. 317 (2004) 422–427. [27] R. Nuccitelli, U. Pliquett, X. Chen, W. Ford, R. James Swanson, S.J. Beebe, J.F. Kolb, K.H. Schoenbach, Nanosecond pulsed electric fields cause melanomas to self-destruct, Biochem. Biophys. Res. Commun. 343 (2006) 351–360. [28] K.H. Schoenbach, R.P. Joshi, J.F. Kolb, N.Y. Chen, M. Stacey, E.S. Buescher, S.J. Beebe, P. Blackmore, Ultrashort electrical pulses open a new gateway into biological cells, Proc. IEEE 92 (2004) 1123–1134. [29] S.J. Beebe, J. White, P.F. Blackmore, Y. Deng, K. Somers, K.H. Schoenbach, Diverse effects of nanosecond pulsed electric fields on cells and tissues, DNA Cell Biol. 22 (2003) 785–796. [30] S.J. Beebe, P.M. Fox, L.J. Rec, K. Somers, R.H. Stark, K.H. Schoenbach, Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition, IEEE Trans. Plasma Sci. 30 (2002) 286–292. [31] S.J. Beebe, P.M. Fox, J. Rec, E. Lauren, K. Wills, K.H. Schoenbach, Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells, FASEB J. 17 (2003) 493–1495. [32] C. Yao, Y. Mi, X. Hu, C. Li, C. Sun, J. Tang, X. Wu, Experiment and mechanism research of SKOV3 cancer cell apoptosis induced by nanosecond pulsed electric field, Conf. Proc. IEEE Eng. Med. Biol. Soc. (2008) 1044–1047. [33] E.H. Hall, K.H. Schoenbach, S.J. Beebe, Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells, Apoptosis 12 (2007) 1721–1731. [34] S.J. Beebe, P.F. Blackmore, J. White, R.P. Joshi, K.H. Schoenbach, Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms, Physiol. Meas. 25 (2004) 1077–1093. [35] Q. Hu, S. Viswanadham, R.P. Joshi, K.H. Schoenbach, S.J. Beebe, P.F. Blachmore, Simulations of transient membrane behavior in cells subjected to a highintensity ultrashort electric pulse, Phys. Rev. E 71 (2005) 031914. [36] F. Hofmann, H. Ohnimus, C. Scheller, W. Stripp, U. Zimmermann, C. Jassoy, Electric field pulses can induce apoptosis, Membr. Biol. 169 (1999) 103–109. [37] L.L. Tang, C. Sun, H. Liu, Y. Mi, C.G. Yao, C.X. Li, Steep pulsed electric fields modulate cell apoptosis through the change of intracellular calcium concentration, Colloids Surf. B Biointerfaces 57 (2007) 209–214. [38] R. Nuccitelli, X. Chen, A.G. Pakhomov, W.H. Baldwin, S. Sheikh, J.L. Pomicter, W. Run, C. Osgood, R.J. Swanson, J.F. Kolb, S.J. Beebe, K.H. Schoenbach, A new pulsed electric field therapy for melanoma disrupts the tumor’s blood supply and causes complete remission without recurrence, Int. J. Cancer 125 (2009) 438–445. [39] L. Xiong, C. Sun, C. Yao, Y. Mi, S. Wang, X. Luo, L. Hu, Vascular effect and immunity effect of steep pulse electric field on Walker 256-bearing Wistar mice, Conf. Proc. IEEE Eng. Med. Biol. Soc. 7 (2004) 5009–5012. [40] T.P. Padera, A. Kadambi, E. Tomaso, C.M. Carreira, E.B. Brown, Y. Boucher, N.C. Choi, D. Mathisen, J. Wain, E.J. Mark, L.L. Munn, R.K. Jain, Lymphatic metastasis in the absence of functional intratumor lymphatics, Science 296 (2002) 1883– 1886.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mini Review
Apoptosis induction with electric pulses — A new approach to cancer therapy with drug free q Liling Tang a,b,c,*, Chenguo Yao a, Caixin Sun a a
State Key Laboratory of Power Transmission Equipment & System and New Technology, Chongqing University, Chongqing 400044, China Key Laboratory of Biorheological Science and Technology, Chongqing University, Ministry of Education, Chongqing 400044, China c College of Bioengineering, Chongqing University, Chongqing 400044, China b
a r t i c l e
i n f o
Article history: Received 14 October 2009 Available online 22 October 2009 Keywords: Electric pulse Apoptosis Cancer therapy Drug free
a b s t r a c t Electrical pulses have been widely used in biomedical fields, whose applications depend on the parameters such as durations and electric intensity. Conventional electroporation (0.1–1 kV/cm, 100 ls) has been used in cell fusion, transfection and electrochemotherapy. Recent studies with high-intensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact, resulting in an application of intracellular manipulation. The most recent development is the finding that parameters between those two ranges could be used to induce apoptosis of cancer cells. Proposal of apoptosis induction and tumor inhibition has advantages to pursue the treatment of cancer free of cytotoxic drugs. Ó 2009 Elsevier Inc. All rights reserved.
Electric pulses have been widely used in the biological and medical fields, including killing microorganisms, cell fusion, gene transfection, cancer therapy since 1980s [1–8]. For cancer therapy, radiofrequency and microwave devices can heat the tumor to greater than 43 °C to kill the cells via hyperthermia [5,6]. And the most advanced procedure is electrochemotherapy (ECT), which is developing rapidly after the first reports by Mir et al. [8] on 1988. The application of electric pulses to cells leads to the transient permeabilization of the cell membrane and increase the delivery of drugs (e.g. bleomycin) into cells. Effectiveness of ECT depends on the effective distribution of the cytotoxic drug in the tumor and the permeabilization of the vast majority of the tumor cells [7]. Although the ECT reduces the total dosage of the drug per patient considerably, it could not completely deplete the side effect of cytotoxic drugs on human body. Developments of pulsed power technology provide the possibility to find a method to kill cancer cells without the aids of cytotoxic drugs. Nowadays electric pulses can be applied in a wide parameter range with duration as short as nanosecond and electric intensity up to hundreds of kilovolts. The rising time of pulses can be controlled in several nanoseconds. It provides the necessary technical supports for the further application of electric pulses in cancer therapy. Responses of cells to electric pulses mainly depend on the parameters such as electric field intensity and pulse durations. q
Grant sponsor: Natural Science Foundation of China (No. 50637020). * Corresponding author. Address: College of Bioengineering, Chongqing University, Chongqing 400044, China. E-mail address: [email protected] (L. Tang). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.092
Electric pulses of 0.1–1 kV/cm and 100 ls normally result in electroporation of cells. Electroporation (EP) is termed as the physical procedure of transient pores on plasma membrane in response to electric pulses. EP brings new properties to the cell membrane. It becomes permeable to drugs, gene materials, proteins and other large molecules. The pores will recover from electric pulses and therefore EP is a reversible procedure [9]. On the contrary, highintensity (MV/cm) electric field applications with durations of several tens of nanoseconds can affect intracellular signal transduction and intracellular structures with plasma intact. Such effect is called intracellular manipulation (IEM) [10]. As so far, the observation of EP has resulted in the application of electrochemotherapy, and the IEM makes it possible to regulate the intracellular structures and functions. Recent studies on high-intensity pulsed electrical fields have led to a significant observation: it is possible to induce cell apoptosis [11–14]. Apoptotic cells exhibit features including externalization of phosphatidylserine (PS) on the plasma membrane, caspase activation and chromosomal condensation. Such electric effect is different from the EP and IEM. We term it as the apoptosis induction with electric pulses (AIEP), because cells are led to apoptosis and can not recover from the electric pulses. It can be used to introduce apoptosis of cancer cells without the aids of cytotoxic drug. Therefore, the proposal of AIEP and the determination of threshold values for electric pulses are significantly important. It will provide a new approach to treat cancer by electric pulses with drug free. In the current review, we discuss the effects of electric pulses with different parameters on tumor cells and the new approach to cancer therapy by inducing cell apoptosis with electric pulses.
L. Tang et al. / Biochemical and Biophysical Research Communications 390 (2009) 1098–1101
Electroporation The first use of electroporation to deliver DNA to mammalian cells was reported by Neumann’s group in the early 1980s. Since then electroporation has become a relatively standard laboratory technique to transfect cells (plants and animal cells) in culture [15,16]. Theoretically, cells in an electric field can be modeled as an electric circuit with cell membrane as capacitance and the cytoplasm as resistance. Application of microsecond or longer duration electric fields to cells causes large build-ups of oppositely charged ions on either side of the cell surface. If too much charge gathers at the cell membrane, the electric field there breaks the membrane down. Large holes (or pores) form in the membrane and allow ions to pour across. This effect is called electroporation [17]. Electroporation occurs when the transmembrane potential difference (Dw) reaches threshold values. The transmembrane potential (Dwi) is a complex function. Dwi = f g(k) r E cos h, in which k is the conductivities of the membrane, r is the cell radius, E is the field intensity, h is the angle between the direction of the normal to the membrane at the considered point on the cell surface and field direction, and f is a shape factor. Dw is the sum between the resulting value of cell membrane Dw0 and the electroinduced value Dwi [18]. The most important parameters for effective electroporation are the electric field strength and pulse length (duration). When the applied pulse achieves a threshold voltage of approximately 1 V across a membrane, the membrane becomes permeable and allows passage of materials through the membrane [9,19]. If the field strength is too low, the breakdown transmembrane potential is not achieved; similarly, if the duration is too short, the membrane cannot be charged enough to reach the electroporation membrane potential [19]. Electroporation enables efficient delivery of drugs or synthetic short interfering RNA (siRNA) into some organs and tumors, thus it can be used to treat various diseases [20,21]. Optimal parameters differ for different molecules and cell types. For the delivery of low molecular weight drugs into mammalian cells, typically fields of 1000 V/cm and durations of 100 ls are effective, whereas lower fields (>50 V/cm) and longer pulses (21 ms) are better for the delivery of genes [19]. When electric pulses of 100 ls are applied to deliver bleomycin in vivo, the threshold value is between 300 and 500 V/cm in tumors. Four hundred and fifty volts per centimeter is the threshold for 51Cr-EDTA uptake in skeletal muscle by electroporation [22]. In summary, 0.1–1 kV/cm electric pulses are usually applied with durations (100 ls–21 ms) to result in reversible electroporation. Electric pulses with higher intensity (2500 V/cm, 100 ls) than that of traditional EP cause irreversible electroporation in tumor cells in vivo [23].
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following features: (1) a rise time that is short relative to the charging time of intracellular organelle membranes, (2) a duration that is long enough to allow charging of organelle membranes but too short to achieve cell surface membrane changing, and (3) an intensity adequate to achieve organelle membrane charging during the pulse application. Such process is referred as IEM. For electroporation effect induced by electric pulses with durations at microsecond to millisecond, cells can be modeled as a capacitance, in which cell membrane envelopes the cytoplasm. However, for electric pulses with shorter duration, intracellular membrane including membrane of organelles and nucleus must be considered [26] (shown in Fig. 1). The effect of nsPEF treatment on cells also depends on two parameters: electric field intensity and pulse duration. In response to the applied electric fields, ions in the cell interior will move in the field direction and charge the membrane until they experience no further force. If the duration of nsPEF is shorter than the plasma membrane charging time, sPM (0.2–1 ls), the interior charges cannot redistribute enough to counter act the imposed field. Electric pulses will penetrate into the cell and charge every organelle membrane [27]. The force exerted on charges depends on the strength of the electric field. In Chen et al.’s [26] experiments with HL-60 cells, PI (propidium iodide) was used as a tracer for plasma membrane integrity. When the cell membrane is broken, PI enters the cell and can be visualized through its dark red fluorescence. Results have shown that 10-ns, 65-kV/cm pulses cannot result in electroporation in plasma membrane, while 60-ns, 26-kV/cm pulses (approximately same energy density as provided by the 10-ns pulse) break the plasma membrane. Analysis with a cell model shows a similar result [28]. For the 11-ns pulse with E = 25 kV/cm, the inner membrane transmembrane potential could easily reach the 1 V threshold, which means pulses are more likely to porate inner organelles, while leaving the outer membrane intact. Thus, electric pulses with duration of 10 ns–100 kV/cm are usually applied in IEM. Because of the special features of IEM in the selective electroporation of intracellular organelles membrane, it can induce a series of physiological responses including the release of calcium from intracellular calcium stores, changes in gene expression, DNA conformation and nucleus. IEM can be used for selective/generalized cell or tissue ablation, growth stimulation and tissue remodeling [9]. Furthermore, ultrashort pulses can be applied after the conven-
Intracellular electromanipulation (IEM) With the developments of electric engineering, researchers could apply electric fields with higher electric intensity and shorter duration to cells. Nanosecond pulsed electric field (nsPEF), ultrashort pulsed electric field (usPEF), and submicrosecond, intense pulsed electric field (sm/I-PEF) with intensity at 10–1000 kV/cm and duration less than 1 ls have been applied by different researchers [24,25]. Compared to traditional electroporation, nsPEF can result in quite different effects, which is called intracellular electromanipulation (IEM). Intracellular electromanipulation means the achievement of intracellular physiological effects by electric field application. According to the views of Buescher et al. [9,25], permeability/poration effects are predicted to occur at organelle/intracellular membranes if the pulse characteristics are modified to achieve
Fig. 1. An equivalent circuit cell model. A cell can be considered as a circuit made up of capacitors and resistors. Cell membrane and organelles’s membranes act as capacitors. The cytosol and nucleoplasm is conductive and so can be modeled as resistors. ‘‘C” refers to the capacitor and ‘‘R” is the resistor.
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tional electroporation pulses. Effects of nsPEF on subcellular membrane change the nuclear membrane and facilitate the delivery of genes into nucleus. Studies have shown that a combination of a classical long pulse and a nanosecond pulse can increase gene expression in nucleus [29].
Apoptosis induction with electric pulses (AIEP) Apoptosis induced by electric pulses A recent finding in the application of electric pulses is based on the finding that apoptosis could be induced by electric pulses with certain parameters. Apoptosis is the morphological manifestation of programmed cell death in cells. It can be considered as the cell suicide. Beebe et al. [30] and Vernier et al. [11] have reported that cell apoptosis could be induced by electric pulses in vitro. Different cell types also have different responses to electrical pulses applications. Studies on Jurkat cells and HL-60 cells have shown that the former is more susceptible to electrical pulses [11,28,31]. Killing cancer cells by inducing cell apoptosis can prevent the inflammatory responses in vivo. The observation of cell apoptosis induced by electrical pulses seems to provide a bright future for cancer therapy without aids of drugs. Selective parameters of electric pulses play major roles in the type of electroeffect on stimulated cells. As described above, electric pulses of IEM with duration at 10 ns, intensity at 100 kV/cm induce intracellular effects, such as intracellular calcium release, DNA changes, poration on organelles membrane, while cell membrane is intact. For 100 ls, 1 kV/cm electric pulses, electroporation on cell membrane occurs to facilitate large molecular materials delivery. In this procedure, cell function is not affected and electroporation is reversible. It is interesting to investigate what will happen to cells when subjected electric pulses with parameters between IEM and EP. Significant apoptosis of several types of cells (SKOV3 human ovarian carcinoma cells, HCT116 colon carcinoma cells, HL-60 cells and Jurkat cells) has been observed when treated by such electric pulses [32–34]. We name the procedure of apoptosis of cells induced by electric pulse with parameters between electroporation and intracellular electromanipulation as ‘‘apoptosis induction with electric pulses (AIEP).” That is to say, the apoptotic effect of electric pulses on cells is irreversible. Thus we enlarge the parameter range for bioelectric applications proposed by Schoenbach et al. [25]. Possible parameters are shown in Fig. 2 according to results of our group and other studies. The observed apoptotic behavior appears to depend on the pulse duration. For cells subjected to external electric fields at a similar energy input, much stronger apoptosis markers were observed only for the longer (300 ns) pulses, less at the shorter (60 ns) durations, and almost negligible effects for a short 10 ns pulses [35]. The apoptotic trends are consistent with the extent of poration on cell surface membrane, in which levels of poration of 300 ns pulses larger than that of 60 ns, while a 10 ns pulse caused insignificant poration [30]. The results also suggest the integrity of plasma membrane plays a role in cell apoptosis induced by electric pulses. There are different characteristics of electroporation: poration on cell surface membrane, poration on organelles membrane or poration on all membranes, by which to distinguish EP, IEM and AIEP. Conventional EP causes electroporation on plasma membrane, while the IEM only electroporates the intracellular membrane. Some researchers viewed the permeabilization of plasma membrane as a secondary effect caused by changes of cell functions; however, apoptosis induced by electric pulses is always coupled to the poration of cell membrane. The extent of apoptotic cells is related to the extent of poration [30,36]. Apoptosis signals can arise from the
Fig. 2. Different electric pulses results from different electric parameters. Electric pulses of 0.1–1 kV/cm and 100 ls normally result in electroporation of cells, which are widely used in gene transfer, electroporation and transdermal drug delivery. Electric pulses of intensity at 10–1000 kV/cm and duration less than 100 ns are used to intracellular electromanipulation, which can regulate intracellular behavior such as release of calcium ion, improvement of gene expression, change of cell cycle and thus inhibiting the tumor growth. Electric pulses between these two parameters are found to bring force some special effects including irreversible apoptosis, antiangiogenesis, anti-lymphatic capillaries around cancer tissue, immune effects and so on.
plasma membrane or from intracellular structures, including the nucleus, mitochondria, or endoplasmic reticulum [28]. In other words, although intracellular mechanism influences the apoptosis of cells subjected by electric pulses, effect on plasma membrane at least play a supporting role in it. The possibility of using electric pulses independently to induce cell apoptosis in cancer therapy also relies on the different responses between cancers cells and normal cells. Cancer cells have a higher apoptosis percentage than normal cells when exposed to the same electric field [37]. A widely accepted theory is that cancer cells are more susceptible to electric field than normal cells, because cancer cells have higher dielectric constant and larger nucleus. Applications of AIEP Effectiveness of electric pulses on inhibiting cancer cells and tissues independently has been proved by many experiments. In vitro experiment showed a significant apoptosis in human ovarian carcinoma cell line (SKOV3) after treating with 10 kV/cm, 100 ns electric pulses [32]. Nuccitelli et al. [27] discovered that pulsed electric fields greater than 21 kV/cm with durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink. Melanomas (a kind of skin cancer) shrink by 90% within two weeks following a cumulative field exposure time of 121 ls. A most recent report has shown that the application of 40 kV/cm, 300 ns electric pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission [38]. Furthermore, electric pulses have additional effects on the migration of cancer cells. It was found that electric pulses had the potential to destroy lymphatic capillaries around cancer tissue and thus decreased the possibility of post-treatment lymphatic metastasis [39]. It is an important effect as that functional lymphatics in the tumor margin alone are sufficient for lymphatic metas-
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tasis and should be target therapeutically [40]. The electric pulses can reduce blood flow to the tumor [38]. The expression of vascular endothelial growth factor (VEGF), an important growth factor to vascular growth, was decreased by electric fields in vascules nearby the cancer tissues [39]. Although there is no experiment in human body as so far, we predict that electric pulses with defined parameters could be applied to human body without significant side effects. Conclusions In spite of many kinds of therapies for cancer, it is still a difficult task to treat cancer as well as keep the high living quality of patients. Proposal of AIEP has advantages to pursue the treatment of cancer free of cytotoxic drugs. The potential to achieve selective killing tumor cells by inducing cancer cells apoptosis would provide a method for cancer therapy. The low energy input of electric pulses because of short durations prevents damage to tissues and cells nearby the tumor. Thus the applications for AIEP present a broad and bright future. References [1] J. Mosqueda-Melgar, R.M. Raybaudi-Massilia, O. Martin-Belloso, Combination of high-intensity pulsed electric fields with natural antimicrobials to inactivate pathogenic microorganisms and extend the shelf-life of melon and watermelon juices, Food Microbiol. 25 (2008) 479–491. [2] N.W. Karja, T. Otoi, P. Wongsrikeao, R. Shimizu, M. Murakami, B. Agung, M. Fahrudin, T. Nagai, Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer, Theriogenology 66 (2006) 1237–1242. [3] J.W. Henshaw, D.A. Zaharoff, B.J. Mossop, F. Yuan, Electric field-mediated transport of plasmid DNA in tumor interstitium in vivo, Bioelectrochemistry 71 (2007) 233–242. [4] H. Aihara, J. Miyazaki, Gene transfer into muscle by electroporation in vivo, Nat. Biotechnol. 16 (1998) 867–870. [5] S. Kuriyama, Improved survival benefits with radiofrequency ablation for liver cancer, Cancer Treat. Rev. 31 (2005) 408–412. [6] J.L. Huang, Z.G. Zhang, Y.B. Chi, T.H. Yang, M.G. Xu, Cancer Lett. 82 (1994) 199– 202. [7] S. Satkauskas, D. Batuskatite, S. Salomskaite-Davalgiene, M.S. Venslauskas, Effectiveness of tumor electrochemotherapy as a function of electric pulse strength and duration, Bioelectrochemistry 65 (2005) 105–111. [8] L.M. Mir, H. Banoun, C. Paoletti, Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol, Exp. Cell Res. 175 (1988) 15–25. [9] E.S. Buescher, K.H. Schoenbach, Effects of submicrosecond, high intensity pulsed electric fields on living cells — intracellular electromanipulation, IEEE Trans. Dielect. Elect. Insul. 10 (2003) 788–794. [10] L.M. Mir, Therapeutic perspectives of in vivo cell electropermeabilization, Bioelectrochemistry 53 (2001) 1–10. [11] P.T. Vernier, A. Li, L. Marcu, C.M. Crait, M.A. Gundersen, Ultrashort pulsed electric fields induce membrane phospholipid translocation and caspase activation: differential sensitivities of Jurkat T lymphoblasts and rat glioma C6 cells, IEEE Trans. Dielect. Elect. Insul. 10 (2003) 795–809. [12] P.T. Vernier, Y. Sun, L. Marcu, C.M. Craft, M.A. Gunderson, Nanoelectropulseinduced phosphatidylserine translocation, Biophys. J. 86 (2004) 4040–4088. [13] K. Schoenbach, R. Joshi, J. Kolb, S. Buescher, S. Beebe, Subcellular effects of nanosecond electrical pulses, Conf. Proc. IEEE Eng. Med. Biol. Soc. 7 (2004) 5447–5450. [14] P.T. Vernier, M.J. Ziegler, Y. Sun, M.A. Gundersen, D.P. Tieleman, Nanoporefacilitated voltage-in cells and in silico, Phys. Biol. (2–3) (2006) 233–247. [15] R. Heller, The development of electroporation, Science 295 (2002) 277. [16] M. Fromm, L. Taylor, Stable transformation of maize after gene transfer by electroporation, Nature 319 (1986) 791–793.
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[17] K.H. Schoenbach, R. Nuccitelli, S.J. Beebe, Extreme voltage could be a surprisingly delicate tool in the fight against cancer, IEEE Spectr. 43 (2006) 20–26. [18] M. Golzio, M.P. Rols, J. Teissie, In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression, Methods 33 (2004) 126–135. [19] S.B. Dev, D.P. Rabussay, G. Widera, G.A. Hofmann, Medical applications of electroporation, IEEE Trans. Plasma Sci. 28 (2000) 206–223. [20] G. Sersa, M. Cemazar, J.M. Snoj, Electrochemotherapy of tumours, Curr. Oncol. 16 (2009) 34–35. [21] O. Mazda, T. Kishida, Molecular therapeutics of cancer by means of electroporation-based transfer of siRNAs and EBV-based expression vectors, Front. Biosci. 1 (2009) 316–331. [22] J. Gehl, T.H. Sorensen, K. Nielsen, P. Raskmark, S.L. Nielsen, T. Skovsgaard, L.M. Mir, In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution, Biochim. Biophys. Acta 1428 (1999) 233–240. [23] B. Al-Sakere, F. André, C. Bernat, E. Connault, P. Opolon, R.V. Davalos, B. Rubinsky, L.M. Mir, Tumor ablation with irreversible electroporation, Plos ONE 2 (2007) e1135. [24] P.S. Hair, K.H. Schoenbach, E.S. Buescher, Sub-microsecond, intense pulsed electric field applications to cells show specificity of effects, Bioelectrochemistry 61 (2003) 65–72. [25] K.H. Schoenbach, S. Katsuki, R.H. Stark, E.S. Buescher, S.J. Beebe, Bioelectrics — new applications for pulsed power technology, IEEE Trans. Plasma Sci. 30 (2002) 293–300. [26] N. Chen, K.H. Schoenbach, J. Kolb, A.L. Gamer, J. Yang, J.R. Swanson, S. Beebe, Leukemic cell intracellular responses to increased nanosecond electric fields, Biochem. Biophys. Res. Commun. 317 (2004) 422–427. [27] R. Nuccitelli, U. Pliquett, X. Chen, W. Ford, R. James Swanson, S.J. Beebe, J.F. Kolb, K.H. Schoenbach, Nanosecond pulsed electric fields cause melanomas to self-destruct, Biochem. Biophys. Res. Commun. 343 (2006) 351–360. [28] K.H. Schoenbach, R.P. Joshi, J.F. Kolb, N.Y. Chen, M. Stacey, E.S. Buescher, S.J. Beebe, P. Blackmore, Ultrashort electrical pulses open a new gateway into biological cells, Proc. IEEE 92 (2004) 1123–1134. [29] S.J. Beebe, J. White, P.F. Blackmore, Y. Deng, K. Somers, K.H. Schoenbach, Diverse effects of nanosecond pulsed electric fields on cells and tissues, DNA Cell Biol. 22 (2003) 785–796. [30] S.J. Beebe, P.M. Fox, L.J. Rec, K. Somers, R.H. Stark, K.H. Schoenbach, Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition, IEEE Trans. Plasma Sci. 30 (2002) 286–292. [31] S.J. Beebe, P.M. Fox, J. Rec, E. Lauren, K. Wills, K.H. Schoenbach, Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells, FASEB J. 17 (2003) 493–1495. [32] C. Yao, Y. Mi, X. Hu, C. Li, C. Sun, J. Tang, X. Wu, Experiment and mechanism research of SKOV3 cancer cell apoptosis induced by nanosecond pulsed electric field, Conf. Proc. IEEE Eng. Med. Biol. Soc. (2008) 1044–1047. [33] E.H. Hall, K.H. Schoenbach, S.J. Beebe, Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells, Apoptosis 12 (2007) 1721–1731. [34] S.J. Beebe, P.F. Blackmore, J. White, R.P. Joshi, K.H. Schoenbach, Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms, Physiol. Meas. 25 (2004) 1077–1093. [35] Q. Hu, S. Viswanadham, R.P. Joshi, K.H. Schoenbach, S.J. Beebe, P.F. Blachmore, Simulations of transient membrane behavior in cells subjected to a highintensity ultrashort electric pulse, Phys. Rev. E 71 (2005) 031914. [36] F. Hofmann, H. Ohnimus, C. Scheller, W. Stripp, U. Zimmermann, C. Jassoy, Electric field pulses can induce apoptosis, Membr. Biol. 169 (1999) 103–109. [37] L.L. Tang, C. Sun, H. Liu, Y. Mi, C.G. Yao, C.X. Li, Steep pulsed electric fields modulate cell apoptosis through the change of intracellular calcium concentration, Colloids Surf. B Biointerfaces 57 (2007) 209–214. [38] R. Nuccitelli, X. Chen, A.G. Pakhomov, W.H. Baldwin, S. Sheikh, J.L. Pomicter, W. Run, C. Osgood, R.J. Swanson, J.F. Kolb, S.J. Beebe, K.H. Schoenbach, A new pulsed electric field therapy for melanoma disrupts the tumor’s blood supply and causes complete remission without recurrence, Int. J. Cancer 125 (2009) 438–445. [39] L. Xiong, C. Sun, C. Yao, Y. Mi, S. Wang, X. Luo, L. Hu, Vascular effect and immunity effect of steep pulse electric field on Walker 256-bearing Wistar mice, Conf. Proc. IEEE Eng. Med. Biol. Soc. 7 (2004) 5009–5012. [40] T.P. Padera, A. Kadambi, E. Tomaso, C.M. Carreira, E.B. Brown, Y. Boucher, N.C. Choi, D. Mathisen, J. Wain, E.J. Mark, L.L. Munn, R.K. Jain, Lymphatic metastasis in the absence of functional intratumor lymphatics, Science 296 (2002) 1883– 1886.