Intense Ultrashort Pulsed Fields

Technol Cancer Res Treat.  2012 Feb;11(1):83-93.

Long term survival of mice with hepatocellular carcinoma after pulse power ablation with nanosecond pulsed electric fields.

Chen X, Zhuang J, Kolb JF, Schoenbach KH, Beebe SJ.

Source

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk Virginia, 4211 Monarch Way, Norfolk, Virginia 23508, USA.

Abstract

Novel therapies are needed for treating hepatocellular carcinoma (HCC) without recurrence in a single procedure. In this work we evaluated anti-neoplastic effects of a pulse power ablation (PPA) with nanosecond pulsed electric fields (nsPEFs), a non-thermal, non-drug, local, regional method and investigated its molecular mechanisms for hepatocellular carcinoma tumor ablation in vivo. An ectopic tumor model was established using C57BL/6 mice with Hepa1-6 hepatocellular carcinoma cells. Pulses with durations of 30 or 100 ns and fast rise times were delivered by a needle or ring electrode with different electric field strengths (33, 50 and 68 kV/cm), and 900 pulses in three treatment sessions (300 pulses each session) or a single 900 pulse treatment. Treated and control tumor volumes were monitored by ultrasound and apoptosis and angiogenesis markers were evaluated by immunohistochemistry. Seventy five percent of primary hepatocellular carcinoma tumors were eradicated with 900 hundred pulses at 100 ns pulses at 68 kV/cm in a single treatment or in three treatment sessions without recurrence within 9 months. Using quantitative analysis, tumors in treated animals showed nsPEF-mediated nuclear condensation (3 h post-pulse), cell shrinkage (1 h), increases in active executioner caspases (caspase-3 > -7 > -6) and terminal deoxynucleotidyl transferase dUTP nick-end-labeling (1 h) with decreases in vascular endothelial growth factor expression (7d) and micro-vessel density (14d). NsPEF ablation eliminated hepatocellular carcinoma tumors by targeting two therapeutic sites, apoptosis induction and inhibition of angiogenesis, both important cancer hallmarks. These data indicate that PPA with nsPEFs is not limited to treating skin cancers and provide a rationale for continuing to investigate pulse power ablation for hepatocellular carcinoma using other models in pre-clinical applications and ultimately in clinical trials. Based on present treatments for specific HCC stages, it is anticipated that nsPEFs could be substituted for or used in combination with ablation therapies using heat, cold or chemicals.

PLos One.  2011;6(12):e28419. Epub 2011 Dec 2.

DNA electrophoretic migration patterns change after exposure of jurkat cells to a single intense nanosecond electric pulse.

Romeo S, Zeni L, Sarti M, Sannino A, Scarfì MR, Vernier PT, Zeni O.

Source

CNR – Institute for Electromagnetic Sensing of Environment, Naples, Italy.

Abstract

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.  2010 Oct;27(5):1128-32.

Focusing properties of picosecond electric pulses in non-invasive cancer treatment.

[Article in Chinese]
Long Z, Yao C, Li C, Mi Y, Sun C.

Source

State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China. longzaiquan@foxmail.com

Abstract

In the light of optical theory, we advanc an ultra-wideband impulse radiating antenna (IRA) which is composed of an ellipsoidal reflector and a cone radiator. The high-intensity ultra-short electric pulses radiated by IRA can be transferred into the deep target in tissue non-invasively and be focused effectively. With the focused picosecond electric pulses, the organelles (mitochondria) transmembrane potential shall change to collapse under which the tumor cells will be targetly induced to apoptosis, so the method of non-invasive treatment of tumors would be achieved. Based on the time-domain electromagnetic field theory, the propagation characteristics of picosecond electric pulses were analyzed with and without the context of biological tissue, respectively. The results show that the impulse characteristics of input pulse were maintained and the picosecond electric pulses can keep high resolution in target areas. Meanwhile, because of the dispersive nature of medium, the pulse amplitude of the pulses will attenuate and the pulse width will be broadened.

Bioelectrochemistry. 2010 Oct;79(2):257-60. Epub 2010 Mar 10.

Electroporation and alternating current cause membrane permeation of photodynamic cytotoxins yielding necrosis and apoptosis of cancer cells.

Traitcheva N, Berg H.

Institute of Plant Physiology “M. Popov,” Bulgarian Acad. of Sciences, Sofia, Bulgaria.

Abstract

In order to increase the permeability of cell membranes for low doses of cytostatic drugs, two bioelectrochemical methods have been compared: (a) electric pore formation in the plasma membranes by single electric impulses (electroporation), and (b) reordering of membrane structure by alternating currents (capacitively coupled). These treatments were applied to human leukemic K-562 cells and human lymphoma U-937 cells, yielding apoptotic and necrotic effects, determined by flow cytometry. Additional cell death occurs after exposure to light irradiation at wavelengths lambda > 600 nm, of cells which were electroporated and had incorporated actinomycin-C or daunomycin (daunorubicin). It is observed that drug uptake after an exponentially decaying electroporation pulse of the initial field strength Eo=1.4 kV/cm and pulse time constants in the time range 0.5-3 ms is faster than during PEMF-treatment, i.e., application of an alternating current of 16 kHz, voltage U<100 V, I=55 mA, and exposure time 20 min. However, at the low a.c. voltage of this treatment, more apoptotic and necrotic cells are produced as compared to the electroporation treatment with one exponentially decaying voltage pulse. Thus, additional photodynamic action appears to be more effective than solely drugs and electroporation as applied in clinical electrochemotherapy, and more effective than the noninvasive pulsed electromagnetic fields (PEMFs), for cancer cells in general and animals bearing tumors in particular.

Arch Biochem Biophys. 2010 May;497(1-2):82-9. Epub 2010 Mar 24.

Nanosecond pulsed electric fields stimulate apoptosis without release of pro-apoptotic factors from mitochondria in B16f10 melanoma.

Ford WE, Ren W, Blackmore PF, Schoenbach KH, Beebe SJ.

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA.

Abstract

Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.

Melanoma Res. 2009 Aug 26. [Epub ahead of print]

Histopathology of normal skin and melanomas after nanosecond pulsed electric field treatment.

Chen X, James Swanson R, Kolb JF, Nuccitelli R, Schoenbach KH.

Department of Hepatobiliary Surgery, the First Affiliated Hospital Zhejiang University School of Medicine, Hangzhou, Zhejiang, China Frank Reidy Research Center for Bioelectrics cDepartment of Biological Sciences, Old Dominion University, Norfolk, Virginia, USA.

Abstract

Nanosecond pulsed electric fields (nsPEFs) can affect the intracellular structures of cells in vitro. This study shows the direct effects of nsPEFs on tumor growth, tumor volume, and histological characteristics of normal skin and B16-F10 melanoma in SKH-1 mice. A melanoma model was set up by injecting B16-F10 into female SKH-1 mice. After a 100-pulse treatment with an nsPEF (40-kV/cm field strength; 300-ns duration; 30-ns rise time; 2-Hz repetition rate), tumor growth and histology were studied using transillumination, light microscopy with hematoxylin and eosin stain and transmission electron microscopy. Melanin and iron within the melanoma tumor were also detected with specific stains. After nsPEF treatment, tumor development was inhibited with decreased volumes post-nsPEF treatment compared with control tumors (P<0.05). The nsPEF-treated tumor volume was reduced significantly compared with the control group (P<0.01). Hematoxylin and eosin stain and transmission electron microscopy showed morphological changes and nuclear shrinkage in the tumor. Fontana-Masson stain indicates that nsPEF can externalize the melanin. Iron stain suggested nsPEF caused slight hemorrhage in the treated tissue. Histology confirmed that repeated applications of nsPEF disrupted the vascular network. nsPEF treatment can significantly disrupt the vasculature, reduce subcutaneous murine melanoma development, and produce tumor cell contraction and nuclear shrinkage while concurrently, but not permanently, damaging peripheral healthy skin tissue in the treated area, which we attribute to the highly localized electric fields surrounding the needle electrodes.

Anticancer Res. 2008 Jul-Aug;28(4B):2245-51.

Effect of steep pulsed electric field on proliferation, viscoelasticity and adhesion of human hepatoma SMMC-7721 cells.

Song G, Qin J, Yao C, Ju Y.

Department of Bioengineering, College of Bioengineering, Ministry of Education of China, Chongqing University, Chongqing, PR China.

song@cqu.edu.cn

Abstract

It has been proven that steep pulsed electric field (SPEF) can directly kill tumor cells and plays an important role in anticancer treatment. The biorheological mechanisms, however, that destroy tumor cells are almost unknown. To resolve this issue, here, an SPEF generator was used to assess the effects of high- and low-dose SPEF on the proliferation of human hepatoma SMMC-7721 cells by MTT assay, and on the viscoelasticity, adhesion of SMMC-7721 cells to endothelial cells by micropipette aspiration technique. Viability and proliferation of SPEF-treated SMMC-7721 cells were significantly inhibited. Cell cycle analysis indicated that SPEF arrested the cell cycle progression of SMMC-7721 cells at the G0/G1 transition to the S-phase. Viscoelastic data fitted by a standard linear solid model showed that viscoelasticity of SMMC-7721 cells changed after treatment with SPEF. Moreover, the adhesive force of low-dose SPEF-treated SMMC-7721 cells to endothelial cells markedly decreased compared to that of control cells. These results suggest that the suppressant effects of SPEF on the proliferation of SMMC-7721 cells appeared to be mediated, at least in part, through arresting cell cycle progression and altering the viscoelastic and adhesive properties of the cells, which provides a novel biorheological mechanism for the antitumor therapy of SPEF.

The Effect of Intense Subnanosecond Electrical Pulses on Biological Cells

 Schoenbach, K.H.   Shu Xiao   Joshi, R.P.   Camp, J.T.   Heeren, T.   Kolb, J.F.   Beebe, S.J.
Old Dominion Univ., Norfolk;

This paper appears in: Plasma Science, IEEE Transactions on
Publication Date: April 2008
Volume: 36,  Issue: 2, Part 1
On page(s): 414-422
Location: Eindhoven, Netherlands,
ISSN: 0093-3813
INSPEC Accession Number: 9921271
Digital Object Identifier: 10.1109/TPS.2008.918786
Current Version Published: 2008-04-08

AbstractNanosecond electrical pulses have been successfully used to treat melanoma tumors by using needle arrays as pulse delivery systems. Reducing the pulse duration of intense electric field pulses from nanoseconds into the subnanosecond range will allow us to use wideband antennas to deliver the electromagnetic fields into tissue with a spatial resolution in the centimeter range. To explore the biological effect of intense subnanosecond pulses, we have developed a generator that provides voltage pulses of 160 kV amplitude, 200 ps rise time, and 800 ps pulse width. The pulses are delivered to a cylindrical Teflon chamber with polished flat electrodes at either end. The distance between the electrodes is variable and allows us to generate electric fields of up to 1 MV/cm in cell suspensions. The pulses have been applied to B16 (murine melanoma) cells, and the plasma membrane integrity was studied by means of trypan blue exclusion. For pulse amplitudes of 550 kV/cm, approximately 50% of the cells took up trypan blue right after pulsing, whereas only 20% were taking it up after 1 h. This indicates that the plasma membrane in a majority of the cells affected by the pulses recovers with a time constant of about 1 h. The cells that show trypan blue uptake after this time suffer cell death through apoptosis. Evaluation of the experimental results and molecular dynamics modeling results indicate that with a pulse duration of 800 ps, membrane charging and nanopore formation are the dominant bioelectric effects on B16 cells. This information has been used in a continuum model to estimate the increase in membrane permeability and, consequently, the increase in pore size caused by repetitive pulsing.

Conf Proc IEEE Eng Med Biol Soc. 2008;2008:1044-7.

Experiment and mechanism research of SKOV3 cancer cell apoptosis induced by nanosecond pulsed electric field.

Yao C, Mi Y, Hu X, Li C, Sun C, Tang J, Wu X.

State Key Laboratory of Power Transmission Equipment & System Security and New Technology, Chongqing University, Chongqing 400044, China.

Abstract

This paper studies the apoptosis of human ovarian carcinoma cell Line (SKOV3) induced by the nanosecond pulsed electric field (10kV/cm, 100ns, 1 Hz) and its effect on intracellular calcium concentration ([Ca2+]i). These cells were doubly marked by Annexin V-FITC/PI, and the apoptosis rate was analyzed with flow cytometry. After AO/EB staining the morphological changes were observed under fluorescent microscope, and their ultrastructural changes were observed under scanning electron microscope (SEM). With Fluo-3/AM as calcium fluorescent marker, laser scanning confocal microscope (LSCM) was used to detect the effect of nsPEF on [Ca2+]i and the source of Ca2+. The results showed that the early apoptosis rate of the treatment group was (22.21+/-2.71)%, significantly higher than that of the control group (3.04+/-0.44)% (P<0.01). The typical features of apoptotic cell have been observed by fluorescent microscope and SEM. It is proved that nsPEF can induce apoptosis of SKOV3 cells and result in distinct increase in [Ca2+]i (P0.01), which was independent of extracellular calcium concentration (P>0.05). Since nsPEF can penetrate cell membrane due to its high frequency components, one of the mechanisms of nsPEF-induced apoptosis may be that activating intracellular calcium stores can increase the [Ca2+]i, and consequently, the apoptotic signal pathway can be induced.

Apoptosis. 2007 Sep;12(9):1721-31.

Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells.

Hall EH, Schoenbach KH, Beebe SJ.

Center for Pediatric Research, Children’s Hospital of the King’s Daughters, Department of Physiological Sciences, Eastern Virginia Medical School, PO Box 1980, Norfolk, VA 23501-1980, USA.

Abstract

Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53(+/+) and HCT116p53(-/-) colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53(+/+) and HCT116p53(-/-) cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2007 Feb;24(1):230-4.

Biological effects and their applications in medicine of pulsed electric fields.

[Article in Chinese]

Huang H, Song G, Wang G, Sun C.

Key Laboratory for Biomnechanics & Tissue Engineering of the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.

Abstract

Pulsed electric fields can induce various kinds of biological effects that are essentially different from the normal electric fields, especially the interactions of Nanosecond Pulsed electric field (nsPEF) with cells. The biological effects of different pulsed electric fields on cell membranes, cytoplasmic matrixes, cell growth are introduced in this paper. Based on these effects, some applications of pulsed electric fields in cancer therapy, gene therapy, and delivery of drugs are reviewed in details.

Biochem Biophys Res Commun. 2006 May 5;343(2):351-60. Epub 2006 Mar 10.

Nanosecond pulsed electric fields cause melanomas to self-destruct.

Nuccitelli R, Pliquett U, Chen X, Ford W, James Swanson R, Beebe SJ, Kolb JF, Schoenbach KH.

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. yaochenguo@cqu.edu.cn

Abstract

We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 micros. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 degrees C, ten degrees lower than the minimum temperature for hyperthermia effects.

Conf Proc IEEE Eng Med Biol Soc. 2006;1:6370-2.

Outlook for the use of focused shock waves and pulsed electric fields in the complex treatment of malignant neoplasms.

Garilevich BA, Andrianov YV, Olefir YV, Zubkov AD, Rotov AE.

Central Air Force Clinical Hosp., Moscow, Russia.

medic-air@mtu-net.ru

Abstract

The experimental studies the synchronous action of electric field microsecond range with amplitude within the range of 1-7 kV/sm and shock waves with pressure before 100 MPa on cells membrane permeability of the mouse’s ascitic tumors in vitro have shown the intensification the efficiency of the forming the irreversible pores under synchronous action. Thereby, enabling the electric field in the compression phase of shock wave pulse which can essentially reduce the electric field intensity required for breakdown cell membrane. In usual condition at amplitude of electric field, specified above, electric breakdown membrane carries basically reversible nature. At the same time in the pressure field tension phase of shock-wave pulse reversible pores, created by electric field, can grow before sizes, under which wholeness membrane is not restored. Under simultaneous action on cellular suspension the shock wave and electric field with moderate intensity cells survival is reduced in 5 once in contrast with occuring at different time’s action, and in 10 once in contrast with checking. The most sensitive to influence by under study fields are cells in phase of the syntheses DNA, preparation to fission and in phase of the mitosis. Thereby, continuation of the studies on use synchronous action shock waves and pulsed electric fields in complex treatment of the tumors introduces perspectiv

Clin Cancer Res. 2005 Oct 1;11(19 Pt 2):7093s-7103s.

Application of high amplitude alternating magnetic fields for heat induction of nanoparticles localized in cancer.

Ivkov R, DeNardo SJ, Daum W, Foreman AR, Goldstein RC, Nemkov VS, DeNardo GL.

Triton BioSystems, Inc., Chelmsford, Massachusetts 01824, USA. rivkov@tritonbiosystems.com

Abstract

OBJECTIVE: Magnetic nanoparticles conjugated to a monoclonal antibody can be i.v. injected to target cancer tissue and will rapidly heat when activated by an external alternating magnetic field (AMF). The result is necrosis of the microenvironment provided the concentration of particles and AMF amplitude are sufficient. High-amplitude AMF causes nonspecific heating in tissues through induced eddy currents, which must be minimized. In this study, application of high-amplitude, confined, pulsed AMF to a mouse model is explored with the goal to provide data for a concomitant efficacy study of heating i.v. injected magnetic nanoparticles.

METHODS: Thirty-seven female BALB/c athymic nude mice (5-8 weeks) were exposed to an AMF with frequency of 153 kHz, and amplitude (400-1,300 Oe), duration (1-20 minutes), duty (15-100%), and pulse ON time (2-1,200 seconds). Mice were placed in a water-cooled four-turn helical induction coil. Two additional mice, used as controls, were placed in the coil but received no AMF exposure. Tissue and core temperatures as the response were measured in situ and recorded at 1-second intervals.

RESULTS: No adverse effects were observed for AMF amplitudes of < or = 700 Oe, even at continuous power application (100% duty) for up to 20 minutes. Mice exposed to AMF amplitudes in excess of 950 Oe experienced morbidity and injury when the duty exceeded 50%.

CONCLUSION: High-amplitude AMF (up to 1,300 Oe) was well tolerated provided the duty was adjusted to dissipate heat. Results presented suggest that further tissue temperature regulation can be achieved with suitable variations of pulse width for a given amplitude and duty combination. These results suggest that it is possible to apply high-amplitude AMF (> 500 Oe) with pulsing for a time sufficient to treat cancer tissue in which magnetic nanoparticles have been embedded.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Aug;21(4):546-8.

Effects of steep pulsed electric fields on cancer cell proliferation and cell cycle.

[Article in Chinese]

Yao C, Sun C, Mi Y, Xiong L, Hu L, Hu Y.

Key Lab of High Voltage Engineering and Electrical New Technology, Ministry of Education, Chongqing University, Chongqing 400044, China.

Abstract

To assess study the cytocidal and inhibitory effects of steep pulsed electric fields (SPEFs) on ovarian cancer cell line SKOV3, the cancer cell suspension was treated by SPEFs with different parameters (frequency, pulse duration, peak value of voltage). Viability rate and growth curves of two test groups (high dosage and low dosage of SPEFs) and one control group were also measured. The DNA contents and cell cycle were analyzed by flow cytometry (FCM). Different dosing levels of SPEFs exerted obviously different effects on cancer cell viability. With the enhancement of each pulse parameter, the viability rate was promoted and the inhibitory effect on the proliferation of treated cells was more evident. The cells exposed to SPEFs grew slower than the control. The ratio of S+G2/M phase cells was decreased, which restrained the DNA synthesis and division, but the ratio of G0/G1 phase cells was increased in the treated groups. It was also indicated that the SPEFs blocked the cell transition from G0/G1 phase to S+G2/M phase. There was a significant difference in cell cycle between treated group and control group (P<0.01). Lethal effects of SPEFs were represented by inhibiting the cancer cell proliferation at the cell level and by influencing the cell cycle at the DNA level.

Physiol Meas. 2004 Aug;25(4):1077-93.

Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms.

Beebe SJ, Blackmore PF, White J, Joshi RP, Schoenbach KH.

Center for Pediatric Research, Eastern Virginia Medical School, Children’s Hospital for The King’s Daughters, Norfolk, VA, USA. sbeebe@chkd.com

These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004 Jun;21(3):433-5.

Effect of steep pulsed electric fields on survival of tumour-bearing mice.

[Article in Chinese]

Yao C, Sun C, Xiong L, Mi Y, Liao R, Hu L, Hu Y.

College of Electrical Engineering, Chongqing University, Chongqing, 400044, China.

To investigate the lethal effect of steep pulsed electric fields (SPEFs) on cancer cells and the life-prolonging effect of SPEFs on the survival of tumour-bearing mice, this study was carried out with the use of SPEFs to treat 40 BALB/C mice inoculated by cervical cancer. The lethal effect on cancer cells and the life-prolonging effect on tumour-bearing mice were examined and compared between the experiment group and control group. The survival periods of the experiment group and control group were 52.05 days and 33.03 days, respectively. There was a significant difference in survival curve between the two groups. The results confirmed he inhibitiory effect and lethal effect of SPEFs on cancer cells. SPEFs can prolong the survival period of tumour-bearing mice.

Technol Cancer Res Treat. 2002 Feb;1(1):71-82.

Enhancing the effectiveness of drug-based cancer therapy by electroporation (electropermeabilization).

Rabussay DP, Nanda GS, Goldfarb PM.

Genetronics, Inc., 11199 Sorrento Valley Road, San Diego CA 92121, USA. dietmarr@genetronics.com

Abstract

Many conventional chemotherapeutic drugs, as well as DNA for cancer gene therapy, require efficient access to the cell interior to be effective. The cell membrane is a formidable barrier to many of these drugs, including therapeutic DNA constructs. Electropermeabilization (EP, often used synonymously with “electroporation”) has become a useful method to temporarily increase the permeability of the cell membrane, allowing a broad variety of molecules efficient access to the cell interior. EP is achieved by the application of short electrical pulses of relatively high local field strength to the target tissue of choice. In cancer therapy, EP can be applied in vivo directly to the tumor to be treated, in order to enhance intracellular uptake of drugs or DNA. Alternatively, EP can be used to deliver DNA into cells of healthy tissue to achieve longer-lasting expression of cancer-suppressing genes. In addition, EP has been used in ex vivo therapeutic approaches for the transfection of a variety of cells in suspension. In this paper, we communicate results related to the development of a treatment for squamous cell carcinomas of the head and neck, using electropermeabilization to deliver the drug bleomycin in vivo directly into the tumor cells. This drug, which is not particularly effective as a conventional therapeutic, becomes highly potent when the intracellular concentration is enhanced by EP treatment. In animal model experiments we found a drug dose of 1 U/cm(3) tumor tissue (delivered in 0.25 mL of an aqueous solution/cm3 tumor tissue) and an electrical field strength of 750 V/cm or higher to be optimal for the treatment of human squamous cell tumors grown subcutaneously in mice. Within 24-48 hours, the majority of tumor cells are rapidly destroyed by this bleomycin-electroporation therapy (B-EPT). This raises the concern that healthy tissue may be similarly affected. In studies with large animals we showed that normal muscle and skin tissue, normal tissue surrounding major blood vessels and nerves, as well as healthy blood vessels and nerves themselves, are much less affected than tumor tissue. Normal tissues did show acute, focal, and transitory effects after treatment, but these effects are relatively minor under standard treatment conditions. The severity of these effects increases with the number of electric pulse cycles and applied voltage. The observed histological changes resolved 20 to 40 days after treatment or sooner, even after excessive EP treatment. Thus, B-EPT is distinct from other ablative therapies, such as thermal, cryo, or photodynamic ablation, which equally affect healthy and tumor tissue. In comparison to surgical or radiation therapy, B-EPT also has potential as a tissue-sparing and function-preserving therapy. In clinical studies with over 50 late stage head and neck cancer patients, objective tumor response rates of 55-58%, and complete tumor response rates of 19-30% have been achieved.

Laryngoscope. 2001 Jan;111(1):52-6.

Electroporation therapy for head and neck cancer including carotid artery involvement.

Allegretti JP, Panje WR.

Department of Otolaryngology, Rush-Presbyterian-St Luke’s Medical Center, Rush Medical College, Chicago, Illinois 60612, USA.

Abstract

OBJECTIVES: Electroporation therapy with intralesional bleomycin (EPT) is a novel, technically simple outpatient technique in which high-voltage electric impulses delivered into a neoplasm transiently increase cell membrane permeability to large molecules, including cytotoxic agents, causing localized progressive necrosis. Unlike many laser ablation methods, EPT can treat bulky tumors (>2 cm) with complete penetration. Our recent publication confirms an excellent response rate in the use of EPT in a clinical trial. STUDY

DESIGN, PATIENTS, AND METHODS: Following our initial prospective study report in 1998, we have followed our entire initial cohort (10 patients) of patients with head and neck cancer beyond 24-months follow-up. Additionally, we have used this approach to treat four additional patients (total: 9 males/5 females) with upper aerodigestive tract squamous cell carcinoma, including three with internal carotid artery (ICA) involvement up to or within the skull base. Two patients underwent preoperative balloon test occlusion with cerebral perfusion studies followed by carotid embolization. EPT was then done safely at least 2 weeks later to avoid the temporary hypercoagulable state.

RESULTS: Within the overall cohort (14 patients) 6 patients had a complete response, 6 had a partial response, and 2 did not respond (overall 85.7% response rate). Both patients with ICA involvement had a partial or complete response to treatment; neither patient had a hemorrhagic or neurologic complication. Overall, 13 of the 14 patients were treated for persistent or recurrent head and neck cancer. Two of the four patients with early recurrent stage tumors had no evidence of recurrence after EPT with an average follow-up of 31.5 months. The overall early stage tumor group had four complete responders out of five (80%). On the contrary, only 2 of 9 patients with advanced recurrent stage tumors were disease-free at 18 months. Morbidity was low for early stage tumors, but higher for advanced tumors with complications, including poor wound healing, dysphagia, and osteomyelitis. There were no treatment-related deaths.

CONCLUSION: We found EPT to be safe and efficacious in patients with head and neck cancer, even with internal carotid artery involvement. Patients with early stage recurrences have the potential for prolonged survival beyond 2 years without the morbidity of surgery and radiation or toxicity of systemic chemotherapy. Because of its superb access qualities even for bulky tumors, EPT is a potential method of delivery for other tumoricidal agents such as in genetic-altering schemes.

J Physiol Biochem. 1999 Jun;55(2):79-83.

Growth modification of human colon adenocarcinoma cells exposed to a low-frequency electromagnetic field.

Ruiz Gómez MJ, Pastor Vega JM, de la Peña L, Gil Carmona L, Martínez Morillo M.

Departamento de Radiología y Medicina Física, Facultad de Medicina, Universidad de Málaga, Teatinos, Spain. mjrg@uma.es

Abstract

The influence of variable low-intensity, low-frequency electromagnetic fields on culture cells is investigated. Human colon adenocarcinoma cells were exposed to a rectangular and variable magnetic field (1 and 25 Hz; 1.5 mT peak). Cultures were exposed to a dose for 15 and 360 minutes, and after 24 hours incubation, cell viability was measured with neutral red stain. The group treated for 15 minutes showed a statistically significant increase in cell growth with 1 Hz (p < 0.002) and 25 Hz (p < 0.003). In contrast, a significant decrease in cell growth was found in those cultures treated with 1 Hz for 360 minutes (p < 0.02). The effects reported could be influenced by the magnetic field frequency and the exposure time.

In Vivo. 1991 Jan-Feb;5(1):39-40.

Effect of a 9 mT pulsed magnetic field on C3H/Bi female mice with mammary carcinoma.  A comparison between the 12 Hz and 460 Hz frequencies.

Bellossi A, Desplaces A.

Laboratoire de Biophysique, Faculte de Medecine, Rennes, France.

In a previous experiment, the exposure of tumoral C3H/Bi female mice to a 9 mT, 460 Hz pulsed magnetic field led to an increase in the length of survival in the late period of the disease; this might be due to a hampered metastatic process. In the present study 27 controls and 52 exposed mice were treated with the same protocol (a 10-minute exposure, 3 non-consecutive days a week, from 2-3 weeks after the tumors appeared until death) but with a 12 Hz PMF. In this experiment the 12 Hz PMF appeared to increase length of survival times in the early period of the disease.

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