Mechanisms of Nanosecond Pulsed Electric Field (NsPEF)-Induced Cell Death in Cells and Tumors

The evolution of pulse power technology from high power physics to biology and medicine places nanosecond pulsed electric fields (nsPEFs) in positions for in vitro and in vivo applications as non-ligand agonists that not only bypass plasma membrane receptors for induction of intracellular signaling pathways, but also bypass intracellular oncogenic impasses to induce cell death by regulated mechanisms. Based on work reviewed here, a likely scenario for cell and tumor demise includes nsPEF-induced permeabilization of the plasma membrane, Ca2+ influx, dissipation of the mitochondrial membrane potential, which is likely due to events beyond permeabilization of the inner mitochondrial membrane, cytochrome c release and activation of caspase-dependent and -independent cell death mechanisms. In vivo, nsPEF-treated orthotopic rat N1-S1 hepatocellular carcinoma tumors exhibit caspase-9 and caspase-3 positive and –negative tumor cells, indicating intrinsic apoptotic and non-apoptotic cell death. Interestingly, after N1-S1 tumor ablation and clearance, rats are resistant to challenge injections of the same N1-S1 tumor cells, indicating a protective, vaccine-like effect that appears to be due to innate and/ or adaptive immune responses that are under further investigation. This provides additional impetus to further develop nsPEF ablation as a cancer therapy

Cell Responses Without Receptors and Ligands, Using Nanosecond Pulsed Electric Fields (nsPEFs)

Stephen J Beebe Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk VA, USAThe plasma membrane is a lipid bilayer that surrounds and shelters the living structural and metabolic systems within cells. That membrane is replete with transmembrane proteins with and without ligand binding sites, oligosaccharides, and glycolipids on the cell exterior. Information transfer across this structure is closely controlled to maintain homeostasis and regulate cell responses to external stimuli. The plasma membrane is contiguous with the endoplasmic reticulum (ER) and nuclear membranes. A number of proteins form ER–mitochondria junctions, allowing interorganelle communications, especially for calcium transport. Transport mechanisms across these membranes include nongated channels or pores; single-gated channels for ion transport; carrier molecules for facilitated diffusion; and pumps for active transport of ions and macromolecules. During the activation of these transport systems, "pores" are formed through protein structures that transiently connect the intracellular and extracellular milieu. These pores are nanoscale structures with diameters of 0.2−4.0 nm. However, there can also be maligned movements of molecules across the plasma membranes. Staphylococcus aureus protein α-toxin and Streptococcus pyogenes protein streptolysin O both create pores that allow unsolicited molecular transfer across membranes that disrupts vital functions. Cytotoxic T-cells permeabilize the invading cell membranes with perforin, creating pores through which granzymes can induce apoptosis. These pores have a lumen of 5–30 nm with the majority at 13–20 nm.

Bioelectrics in Basic Science and Medicine: Impact of Electric Fields on Cellular Structures and Functions

Bioelectrics is a new interdisciplinary field that investigates electric field effects on cell membranes and other cellular components. It incorporates four main technologies, including electroporation, nanosecond pulsed electric fields, picosecond pulsed electric fields and cold plasmas. The parent technology in Bioelectrics is electroporation, which uses milli- and/or micro-second electric pulses to permeabilize cells and tissues, for delivery of membrane impermeable molecules. It is now being used for electro-gene delivery, with vascular endothelial growth factor, for revascularization in wound healing and cardiovascular and peripheral vascular disease. Plasmids expressing IL-12 are being delivered for immune system activation in melanoma treatment, now in phase II clinical trials. DNA vaccine delivery by electroporation is also being investigated. More recently, electroporation has been extended to include nanosecond pulsed electric fields (nsPEFs), a pulse power technology that was originally designed for military applications. It stores intense levels of electric energy, and then unleashes nanosecond bursts of instantaneous power into cells and tissues, creating unique intracellular conditions of high power and low, non-thermal energy. It is presently being used for cancer ablation of skin and internal tumors, and for platelet activation for wound healing in injury and diabetes. An extension of nsPEFs is to make the pulses even shorter, using picosecond pulsed electric fields. This is being developed as an imaging system to detect cancer and other aberrant tissues, using an antenna. The fourth technology is cold plasmas or ionized gasses, a fourth state of matter. Applications of these ionized gases are being developed for decontaminating wounds, water, food and surfaces. Other possible applications that are of specific interest, but not yet fully investigated, and/or developed, are pain control, fat ablation and decontamination of indwelling catheters. This review will outline some applications of Bioelectrics, with greatest focus on nsPEF effects on cells in vitro and tumors in vivo

Nanosecond Pulsed Electric Fields: A New Stimulus to Activate Intracellular Signaling

When new technologies are introduced into the sci-entific community, controversy is expected and both ex-citement and disappointment enrich the lives of those who initiate the new ideas. It becomes the mission of the “inventors ” to embrace the burden of proof to estab-lish their ideas and convince the skeptics and disbeliev-ers who will undoubtedly temper their enthusiasm and test their patience. While open mindedness is generally a scientific motto, those who review patents, manuscripts, and grants do not always readily practice it, even when the evidence is convincingly presented; old ideas and concepts often die hard. So it has been and still is in many instances as engineers, physicists, biologists, and physicians pursue innovative ideas and novel technolo-gies. So what is “Bioelectrics”? It is the application of ultra-short pulsed electric fields to biological cells, tissues, and organs. More specifically, it is the analysis of how these bi-ological systems respond to high electric fields (10–100 s of kV/cm) when applied with nanosecond (1–300) dura-tions. Compressing electrical energy by means of pulsed power techniques allows the generation of ultrashort (bil-lionth of a second) electrical pulses [1]. Because the pulses are so short the energy density is quite low and there-fore nonthermal. However, the power is extremely high generating billions of watts. This can be compared to a coal power plant, which generates less than billion watts, but does it continuously. For example, for a 10 ns, 40 kV, 10Ω pulse generator, the power provided by the pulse is 160MW, however, the energy is only 1.6 J. Depositing thi

Nanosecond Pulsed Electric Field (nsPEF) Ablation as an Alternative or Adjunct to Surgery for Treatment of Cancer

Surgery as resection or transplantation remains a fundamental means for cancer treatment and often offers an opportunity for a cure. However, surgery is not always possible because of tumor proximity to blood vessels or ducts or when a patient is not healthy enough to undergo surgery. Application of nanosecond pulsed electric fields (nsPEFs) is a new approach to treat cancer using pulse power technology that was originally designed for military purposes. This novel approach deposits extremely short pulses of high power, low energy electric fields into malignant tissues using electrodes to encompass tumors. Pre-clinical studies show that treatments are effective and without local or systemic side effects, including absences of scarring. Pre-clinical trials for basal cell carcinoma are completed, but results have not been published. For treating internal tumors, electric fields can be delivered by catheter electrodes and laparoscopy procedures. Here we present a review of the literature using nsPEFs for cancer ablation and present some recent work from the author’s laboratory. We demonstrate efficacy for treatment of an ectopic mouse (Hepa-1- 6) and an orthotopic rat (N1-S1) Hepatocellular Carcinoma (HCC). NsPEFs eliminate tumors by mechanisms in the presence of active caspases (apoptosis) as well as in absences of active caspases (necrosis/necroptosis). Treatment also breaches small vessels, but spares larger vessels and ducts. NsPEF treatments also reduce angiogenesis as determined by decreases in Vascular Endothelia Growth Factor (VEGF). Microvascular density markers (CD-31, CD-34 and CD-105) are significantly decreased after treatment, limiting new blood vessel formation and reinforcing tumor cell demise. Furthermore, initial challenge studies show that mice are resistant to re-introduction of the same tumor cells after treatment, suggesting that nsPEFs induces immunogenic cell death and possible host cell immune responses after treatment. NsPEF ablation of cancer targets at least three hallmarks of cancer (evasion of apoptosis, angiogenesis maintenance and immune surveillance) and provides an effective alternative or adjunct therapy for cancers in skin and internal organs

Two Classes of cAMP Analogs Which Are Selective for the Two Different cAMP-Binding Sites of Type II Protein Kinase Demonstrate Synergism When Added Together to Intact Adipocytes

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their K(a) for protein kinase and their lipophilicity rather than with either parameters alone. Some of the most potent lipolytic analogs had high I50 values for the particulate low K(m) cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations some analogs were as effective as optimal concentrations of epinephrine in stimulating glycerol release. The regulatory subunit of protein kinase has two different intrachain cAMP-binding sites and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1 and analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2 (Rannels, S.R., and Corbin, J.D. (1980) J. Biol. Chem. 255, 7085-7088). Thus, C-8 and C-6 analogs were tested in combination to stimulate lipolysis in intact adipocytes and to activate protein kinase in vitro. Each process was stimulated synergistically by a combination of a C-6 and C-8 analog. Two C-8 analogs or two C-6 analogs added together did not cause synergism of either process. For both lipolysis and protein kinase activation, C-8 thio analogs acted more synergistically than C-8 amino analogs when incubated in combination with C-6 analogs, a characteristic of type II protein kinase. It is concluded that the observed synergism of lipolysis is due to binding of cAMP analogs to both intrachain sites and that it is the type II protein kinase isozyme which is responsible for the lipolytic response

Induction of Cell Death Mechanisms and Apoptosis by Nanosecond Pulsed Electric Fields (nsPEFs)

Pulse power technology using nanosecond pulsed electric fields (nsPEFs) offers a new stimulus to modulate cell functions or induce cell death for cancer cell ablation. New data and a literature review demonstrate fundamental and basic cellular mechanisms when nsPEFs interact with cellular targets. NsPEFs supra-electroporate cells creating large numbers of nanopores in all cell membranes. While nsPEFs have multiple cellular targets, these studies show that nsPEF-induced dissipation of DeltaPsim closely parallels deterioration in cell viability. Increases in intracellular Ca2+ alone were not sufficient for cell death; however, cell death depended of the presence of Ca2+. When both events occur, cell death ensues. Further, direct evidence supports the hypothesis that pulse rise-fall times or high frequency components of nsPEFs are important for decreasing DeltaPsim and cell viability. Evidence indicates in Jurkat cells that cytochrome c release from mitochondria is caspase-independent indicating an absence of extrinsic apoptosis and that cell death can be caspase-dependent and -independent. The Ca2+ dependence of nsPEF-induced dissipation of DeltaPsim suggests that nanoporation of inner mitochondria membranes is less likely and effects on a Ca2+-dependent protein(s) or the membrane in which it is embedded are more likely a target for nsPEF-induced cell death. The mitochondria permeability transition pore (mPTP) complex is a likely candidate. Data demonstrate that nsPEFs can bypass cancer mutations that evade apoptosis through mechanisms at either the DISC or the apoptosome

Type I cAMP-Dependent Protein Kinase Delays Apoptosis in Human Neutrophils at a Site Upstream of Caspase-3

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t 50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX ± 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had no effect on apoptosis in the absence of the analogue. In contrast, site-selective cAMP analogues that specifically activated the type I cAPK, but not type II cAPK, synergistically attenuated apoptosis. Exposure to 8-CPT-cAMP delayed, in parallel, the activity of caspase-3 (CPP-32β), whereas mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD98059, had no effect on CHX-induced apoptosis ± 8-CPT-cAMP. Together these results indicate that type I cAPK activation is necessary and sufficient to mediate cAMP-induced delay in human neutrophil apoptosis induced by several mechanisms and suggest that one of the major sites of cAPK action is upstream of caspase-3 (CPP-32β) activation