An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma
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- Ren, W. & Beebe, S.J. Apoptosis (2011) 16: 382. doi:10.1007/s10495-010-0572-y
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Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55–60%) and calcium-dependent (40–45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.
KeywordsCell death Cytochrome c Mitochondria membrane potential Caspase Calpain Calcium Bcl2 Bclxl Phosphatidylserine externalization Propidium iodide Apoptosis pathways
The administration of nanosecond second pulsed electric fields (nsPEFs) is emerging as a novel stimulus for inducing cell death. Apoptosis can be activated by nsPEFs in various cancer cell lines in vitro [1, 2, 3], a fibrosarcoma tumor ex vivo [1, 4, 5, 6] and in B16f10 melanoma tumors in vivo . Apoptosis is a highly conserved mechanism in eukaryotes for eliminating superfluous, aberrant and damaged cells. It is a complex cascade of events morphologically characterized by phosphatidylserine externalization, cell shrinkage, chromatin condensation, nucleosomal DNA fragmentation, disruption of mitochondrial membrane potential (Δψm) resulting in plasma membrane blebbing and formation of apoptotic bodies . These morphological features are believed to be caused at least in part by activation of caspases, a family of cysteine proteases, which plays a crucial role to initiate and execute apoptosis [9, 10, 11], although other proteases may also be activated.
Caspases can be triggered through extrinsic death receptor pathways or intrinsic pathways depending on the cell type and stimulus [12, 13]. Extrinsic pathway are initiated by specific ligands, such as Fas and TRAIL, which aggregate their cognate death receptor, allowing assembly of death-inducing signaling complexes (DISC) on the cytoplasmic side of plasma membranes leading to initiator caspase-8 (and/or -10) activation. In type I cells, caspase-8 activates executioner caspases independent of mitochondria. In type II cell pathways, caspase-8 cleaves the BH3-only protein Bid to activate pro-apoptotic truncated Bid (t-Bid). Translocation of t-Bid triggers cytochrome c release from mitochondria and caspase-9 activation through formation of the apoptosome, which causes cell demise through activation of caspase-3.
Unlike conventional electroporation, which primarily affects plasma membranes [14, 15], nsPEFs also electroporate intracellular membranes . Modeling studies [14, 15, 17, 18] and evidence determined experimentally by patch clamp and fluorescent imaging studies [19, 20], indicated that nsPEFs with sufficiently short pulse durations and rapid rise times  induced nanopores in plasma membranes that were often smaller than propidium iodide (PI). This supra-electroporation involved all cell membranes going through cells, extensively penetrating all organellar membranes [14, 15]. Effects on intracellular vesicles were observed without measurable effects on plasma membranes [16, 21] with PI or calcein and on intracellular calcium mobilization from the endoplasmic reticulum and capacitative calcium entry in HL-60 cells , suggesting that these external electric fields can mimic ligand signaling . It is now know that the absence of effects on plasma membranes was due to the presence of nano pores that were too small to allow entry of the membrane markers or that detection systems were insufficiently sensitive to measure them [19, 20]. Nevertheless, modeling evidence suggests that under certain circumstances it is possible to have effects on intracellular membranes without effects on the plasma membrane .
Studies have suggested that nsPEF-induced apoptosis mechanisms are cell type-dependent. In Jurkat cells caspase activation and cytochrome c release were nearly coincident, making it difficult to differentiate between extrinsic and intrinsic mechanisms . In B16f10 cells cytochrome c was not released, implicating the extrinsic apoptosis pathway typical of type I cells . In HCT 116 cells, caspase activation occurred before cytochrome c release, suggesting activation of extrinsic pathways followed by mitochondria-mediated cytochrome c release through the intrinsic pathway, type II cell extrinsic apoptosis pathway and/or feed-forward amplification from the initial extrinsic signal . However, specific contributions from extrinsic or intrinsic mechanisms have not been determined.
Available data indicate pervasive effects of nsPEF on all cell membranes inducing apoptosis signaling in several cell types. However, their mechanisms, sites of action and unambiguous confirmation of apoptosis pathways contributing to cell death have not been clearly determined. In this study, we demonstrate direct activation of apoptosis signaling through multiple mechanisms and quantify relative contributions from several apoptosis pathways originating from both plasma membranes and intracellular membranes that exhibit both dependence and independence of caspases, calpains, calcium, and cytochrome c release. Several quantified nsPEF-induced biological functions exhibit different electric field sensitivities and kinetics that promote heterogeneous apoptosis characteristics in E4 squamous carcinoma cells (SCC). Diverse effects of nsPEFs on different E4 SCC death mechanisms and programmed pathways can induce cell death by multiple mechanisms bypassing apoptosis-deficient syndromes in SCC.
Cells and cell culture
The E4 squamous cells were obtained from Dr. Jian-Hua Mao (Lawrence Berkeley National Laboratories) and were maintained in Dulbecco’s modified Eagle’s medium (ATCC) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), 2 mM l-glutamine (Mediatech Cellgro), and 1% Penicillin–Streptomycin solution (Mediatech Cellgro). The cells were cultured in a humid atmosphere containing 5% CO2 and 95% air at 37°C until reaching 50–75% confluency with passage ranging between 11 and 19.
Induction of cell death by nanosecond pulsed electric fields
Cells were harvested with trypsin and re-suspended in fresh DMEM medium with 10% FBS to a concentration of 7.7 × 106 cells/ml. 130 μl of cell suspension (1 × 106 cells) were placed in 0.1 cm gap cuvettes (Biosmith) and exposed to ten pulses with 300 ns duration and electric field strength from 0 to 60 kV/cm. The pulse power devise was previously described . The energy absorbed or the dose (J/g) was calculated as previously described . For all cell responses, time courses were conducted to determine time for maximal responses. Cell responses at all electric fields were stable for at least 90 min.
Determination of 24 h cell survival
After treatment, E4 cells were seeded in 6-well plates (3.3 × 105 cells/well). Twenty-four hours later, cells were detached and trypan blue negative cells were counted. The cell numbers were normalized to control cells as 100%.
Assessment of caspases-3 & -7, -8, -9 activation by flow cytometry
Caspase-3 & -7,-8, -9 activation were assayed using a commercial method based on fluorochrome-labeled, irreversible caspase inhibitors (FLICA, Immunochemistry Technologies LLC). E4 cells were treated with different nsPEF conditions as mentioned above. One hour after treatment, cells were gently removed from cuvettes and re-suspended in DMEM without FBS. According to the manufacturer’s recommendations, cells were labeled with carboxyfluorescein caspase-3 & -7, -8, -9 irreversible inhibitors for 1 h at 37°C under 5% CO2 and protected from light as previously described . Cells were washed with buffer to remove the unbound reagent. Cell fluorescence was determined by Becton–Dickinson FacsAria. Ten thousand cells were acquired for analyses and expressed as percentage of cells showing positive fluorescence. As electric fields were increased, greater numbers of cells became caspase positive with homogeneous shifts of cells into the caspase positive gate, making cell percentages with active caspases the most accurate and meaningful quantification of active caspases as an apoptosis marker.
Evaluation of active pan caspase by flow cytometry
E4 cells were exposed to specific nsPEF conditions as previous described and incubated with 40 μM fluorescent-labeled, cell permeable, irreversible inhibitor FITC-VAD-fmk (CaspACE™, Promega) for 20 min. Cells were washed once with 1 ml PBS and resuspended in 500 μl PBS 1 h post pulse for flow cytometric analysis with Becton–Dickinson FACS Aria.
Measurement of cytochrome c release by two different assays
Cytochrome c release was measured by two different methods, which gave similar results (data not shown). In method I cytochrome c, which was used in all studies here, was assessed using the Innocyte Flow Cytometric Cytochrome c Release assay (Calbiochem). One hour after nsPEF treatment, cells were permeabilized, fixed and washed. Cells were then incubated with primary antibody overnight at 4°C and secondary antibody for 1 h at room temperature. Cells (10,000) were then analyzed by Becton–Dickinson FacsAria flow cytometer. Method II data is not shown but was carried out as previously described [2, 3]. Briefly, after treatment cells were incubated with a monoclonal anti-cytochrome c antibody for 30 min, washed with PBS, incubated with Alexa-Fluor-488 goat anti-mouse (Molecular Probes) for 30 min, washed again and analyzed flow cytometry.
Measurement of mitochondrial membrane potential (Δψm)
Depolarization of Δψm was detected using a fluorescent lipophilic cation, tetramethylrhodamine ethyl ester (TMRE) (Immunochemistry Technologies LLC). Cells were incubated with 200 nM TMRE for 10–20 min at 37°C 30 min post pulse. Twenty minute incubations were suggested by the manufacturer, but 10 min incubations, which gave similar results, were used in some experiments to determine the earliest response. At the end of incubation, cells were re-suspended in assay buffer. The average intensity of red fluorescence (10,000 cells) was analyzed on the FL-2 channel of a Becton–Dickinson FacsAria flow cytometer. When JC-1 was used, cells were pulsed and incubated with the JC-1, washed and analyzed by flow cytometry as previously described . For JC-1, red fluorescent aggregates change to green fluorescent monomers when Δψm decreases.
Determination of phosphatidylserine externalization
External phosphatidylserine exposure and plasma membrane permeability was evaluated by double-labeling cells with FITC-Annexin-V and PI using a detection kit (BD Pharmingen Biosciences). After administration of nsPEFs, cells were re-suspended in binding buffer and incubated with FITC-Annexin-V and PI according to the manufacture’s procedures. The fluorescence signal was quantified by flow cytometry using FL1 (Annexin-V) and FL2 (PI) channels 15 min post pulse.
Assessment of cytochrome c release with administration of z-VAD-fmk
Cells were preloaded with 100 μM of z-VAD-fmk for 30 min and followed by exposing cells to ten 300 ns pulses with 0 and 60 kV/cm electric fields. Cytochrome c monoclonal antibody (BD Pharmingen Biosciences) and Alexa-Fluor 488 goat anti-mouse antibody (Invitrogen) were added to cell suspensions 1 h after pulsing as described previously . Flow cytometric analysis of fluorescent intensity was done by using Becton–Dickinson FacsAria.
Immunoblot analysis for Bcl-2, Bcl-xl, Bid and t-Bid
For each antigen samples were taken for immunoblot analysis 0 15, 30 and 60 min after pulsing. Bcl2 and Bcl-xl data are not shown. At the indicated times E4 cells were washed twice with cold PBS and lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Pierce) for 30 min on ice. Then cell lysates were centrifuged at 15,000×g for 20 min at 4°C and protein content in the supernatant was measured using the BCA method (Pierce). Lysates were then boiled with 4× DualColor™ Protein Loading Buffer (Fermentas Life Sciences) for 5 min. Protein extracts (75 μg) were separated by electrophoresis on 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were transferred onto a PVDF membrane with pore size of 0.2 μm (Pierce, Rockford, IL). Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) for 1 h, and incubated overnight at 4°C with the respective primary antibody directed against Bcl-2 (1:200 mouse polyclonal, Santa Cruz Biotechnology), Bcl-xl (1:1000 rabbit monoclonal, Cell Signaling Technology), or Bid/t-Bid (1:100 mouse monoclonal, R&D System) diluted in blocking buffer. Membranes were washed three times with 0.1% Tween-20 in PBS and then incubated with an IRDye680-conjugated secondary antibody (LI-COR Biosciences) in blocking buffer for 1 h. Protein–antibody complexes conjugated with IRDye 680 were quantified on the Odyssey Infrared Imaging System (LI-COR Biosciences). β-Actin (1:1000; Li-Cor Biosciences) was used as loading control.
Assessment of inhibitory effects of z-VAD-fmk, EGTA and BAPTA on nsPEFs induced-Bid cleavage
E4 cells were preincubated with 100 μM z-VAD-fmk, 5 mM EGTA, and/or 20 μM BAPTA-AM, for 30 min followed by nsPEFs treatment. Cells were then quantified by immunoblot analysis using Odyssey for levels of Bid and t-Bid as described above. Caspase-dependence was determined by Bid cleavage that was inhibited by z-VAD-fmk. Calcium dependence was determined by Bid cleavage that was inhibited by EGTA and BAPTA-AM in the absence of z-VAD-fmk. Dependence on extracellular calcium was determined by Bid cleavage that was inhibited by EGTA in the presence of z-VAD-fmk. Dependence on total calcium was determined by Bid cleavage in the presence of EGTA and BAPTA-AM in the presence of z-VAD-fmk. Dependence on intracellular calcium was determined by the difference between total calcium and extracellular calcium responses.
Assessment of calpain activity in response to nsPEFs by fluorometric assay
Analysis of calpain catalytic activity was determined in total cell lysates (BioVision). Cell lysates were prepared and incubated with substrate (AC-LLY-AFC) and reaction buffer for 1 h at 37°C in the dark. Cleavage substrate fluorescence was measured by a microplate spectrofluorometer (ex/em = 488/520 nm) with SoftMax Pro 5 software (Molecular Devices). A calpain-specific inhibitor (z-LLY-fmk) was used to identify calpain-specific substrate hydrolysis. Recombinant calpain I was used as positive control (data not shown).
Determination of intracellular reactive oxygen species (ROS) accumulations
E4 Cells were preloaded with 1 μM CM-H2DCFDA (5-6-chloromethy-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester, Molecular Probes) in phenol red free DMEM media (Invitrogen) for 60 min, and then pulsed with nsPEF as described previously . Cells were analyzed by flow cytometry at 0, 5, 15, 30, and 60 min after pulsing. Cells incubated at 4°C for 1 h were used as positive controls. After removing from the cold, these cells were also sampled at 0, 5, 15, 30 and 60 min.
Experiments were performed at least three times independently in this study. All the data is expressed as mean ± standard error (SE). Statistical differences between control and treated groups were analyzed by student’s t test (two-tailed) with P < 0.05 regarded as statistically significant.
NsPEFs induce caspase-correlated cell death in E4 squamous carcinoma cells (SCC) in vitro in an electric field amplitude-dependent manner
Different sensitivities and heterogeneous responses in E4 cells to nsPEFs: electric field amplitude-dependent effects on plasma membrane indicators, Δψm, caspase activation and cytochrome c release
Caspase-dependent and caspase-independent cytochrome c release in response to nsPEFs
NsPEFs generate Bid cleavage to t-Bid
NsPEFs induce caspase-dependent and calcium-dependent Bid cleavage; repetitive mechanisms for cytochrome c release
NsPEFs induce calpain activity
It was possibility that calcium-dependent Bid cleavage in response to nsPEFs was due to calpain activation. To test this, E4 cells were treated with ten 300 ns pulses at 60 kV/cm in the presence or absences of BAPTA and EGTA, which inhibits intracellular and extracellular calcium-mediated events, respectively, and z-LLY-fmk, which specifically inhibits calpain activity. One hour after treatment cell extracts were prepared and assayed for calpain activity using the substrate AC-LLY-AFC. In the absence of these inhibitors/chelators, basal calpain activity was about 50 units and nsPEFs increased this activity 1.7-fold. In the presence of the calpain inhibitor, both basal and nsPEF-stimulated activities were decreased by about 50%. In the presence of BAPTA and EGTA, basal activity was not significantly affected, but nsPEF-stimulated activity was inhibited by >50%. When calcium chelators and the calpain inhibitor were present, basal activity was not inhibited any greater than when the calpain inhibitor was present alone. NsPEF-stimulated activity was inhibited by >80%. This data indicate that E4 cells exhibit calcium-insensitive basal calpain activity and calcium-sensitive nsPEF-stimulated activity. Although not tested here, these data suggest that other protease activities might be activated by nsPEFs.
NsPEFs induce cell death without ROS generation or changes in pro-survival protein Bcl2 and Bcl-xl
NsPEF have no significant effects of Bcl-2 or Bcl-xl levels
In a further analysis of nsPEF effects, levels of the anti-apoptotic proteins Bcl-2 and Bcl-xl were analyzed in the absence and presence of nsPEF treatment in E4 cells using quantitative immunoblot analysis with Odyssey imaging (data not shown). Both Bcl2 and Bcl-xl were present a single bands in control cells. There were no effects of ten 300 ns pulses at 60 kV/cm on either anti-apoptotic protein with the first hour after treatment with nsPEFs, suggesting that anti-apoptotic protein levels were unaffected while a pro-apoptotic protein, Bid was activated.
Evidence from E4 SCC gives new insight and understanding for cell death and apoptosis-like mechanisms and their origin in response to nsPEFs. In the experiments presented here we quantified a diverse set of cell responses to nsPEFs in E4 SCC. The results indicate that cell death can be dependent and/or independent of caspase activity, calpain activity, calcium from intracellular stores or extracellular media, cytochrome c release and decreases in Δψm. Thus, nsPEFs initiated charge-mediated events in plasma membranes and intracellular membranes through multiple signaling mechanisms inducing signatures of apoptosis and other forms of cell death. Even at the highest electric fields investigated, frank necrosis as defined by immediate plasma membrane rupture was not present, so cell death is driven by biological responses that appear to be, at least in part, programmed. Ibey et al.  found that apoptosis (DNA fragmentation of agarose gels) was present in U937 cells exposed to 600 pulses at 10 ns and 100 kV/cm (700 J/g); however, necrosis was a major form of cell death. However, in this study the cell type was different and the energy absorbed or dose (J/g) for the pulses was about seven times higher than in the present study (~700 vs. ~100 J/g). It should be pointed out that factors other than energy absorbed are likely involved to determine nsPEF effects .
After treatment with nsPEFs caspases are activated at earlier times and at lower electric fields than cytochrome c release, demonstrating initial activation of a type I cell extrinsic pathway. This is similar to results in nsPEF treated HCT-116 colon carcinoma cells , but different than B16f10 cells that did not release cytochrome c  and Jurkat cells with concomitant cytochrome c release and caspase activation . This clearly defines cell type-specific response to nsPEF-induced cell death. Unlike the other analyzes, here we show for the first time caspase-dependent and caspase-independent cytochrome c release, demonstrating the additional presence of a type II cell extrinsic pathway through Bid cleavage for cytochrome c release. Caspase-dependent (55–60%) and calcium-dependent (40–45%) Bid cleavage pathways were demonstrated with quantitative and reciprocal increases in t-Bid and decreases in Bid. Further, for calcium-dependent Bid cleavage, extracellular calcium (~70%) played a predominant role compared to calcium from intracellular stores (~30%). Caspase-independent Bid cleavage is most likely accomplished by charge-mediated interventions with calcium signaling from and/or nanoporation in the endoplasmic reticulum, capacitative calcium entry [5, 22] and/or calcium entry through nanopores in plasma membranes . Since caspase activation has been shown to be calcium-independent [2, 3], calcium-dependent Bid cleavage appears to be due to calpain activation. This was confirmed by showing calcium-sensitive calpain activity was specifically inhibited by the calpain inhibitor z-LLY-fmk. Taken together, these results show for the first time nsPEF-mediated at least two mechanisms for Bid cleavage and cytochrome c release—one caspase-dependent and one calpain-dependent. While there are reports of cross-talk and/or synergistic behaviors between calpains and caspases [29, 30], since the sum of caspase-dependent and calpain-dependent Bid cleavage activities, which were determined independently of the other, equal about 100% of total Bid cleavage. Generation of t-Bid, a critical point connecting extrinsic and intrinsic pathways, by at least two different protease families provides beneficial repetitive mechanisms to maximize cytochrome c release and apoptosis induction through mitochondria pathways. Thus, nsPEFs promote pro-apoptotic signals that overcome resistances to apoptosis, which are present in most if not all cancers, thereby potentially abolishing the universal cancer hallmark of apoptosis evasion .
By analyzing several different E4 cell responses to nsPEFs by flow cytometry, it was possible to establish several other characteristics for nsPEF-induced cell death. Under nsPEF conditions that eliminated ≥95% of E4 SCC, only 50% of cells exhibited cytochrome c release and decreases in Δψm, while 75–80% of cells exhibited active caspases. Moreover, each of these responses showed different thresholds or sensitivities among themselves, with cytochrome c release as the least sensitive response. Sensitivities for plasma and inner mitochondria membrane permeability appeared to be similar, although plasma membrane reactions as determined by PI uptake and PS externalization were overall more responsive than inner mitochondria membrane as determined by decreases in Δψm. These are most likely due to nanopore formation in the respective membranes. These results are consistent with an overall greater initial response from plasma membrane sensitivity leading to activation of extrinsic apoptosis mechanisms compared to intrinsic mechanisms from mitochondria membrane sensitivity. An extrinsic mechanism is also consistent with a greater response from extracellular calcium and a lesser response from intracellular calcium.
The predominance of extrinsic mechanisms with greater effects on plasma membranes and from extracellular calcium-dependent responses compared to intrinsic mechanisms measured by decreases in Δψm and intracellular calcium-dependent responses can possibly be explained by characteristics of electric fields pulses. Pulsed electric field requirements for intracellular membrane effects include a fast pulse rise time, high electric fields, and a pulse duration that is shorter than the relaxation time of intracellular membrane, which is on the order of ~100 ns . While pulses used here have sufficient rise times and electric fields (≤4 ns and 60 kV/cm, respectively), the relatively long 300 ns pulse duration may not be short enough to allow fully effective intracellular effects. While the mechanism(s) for caspase activation remains to be defined, nsPEF effects are due to membrane charging events. While PI uptake occurs in >95% of cells, and cytochrome c is released in about 50% of cells, caspases are activated in 75–80% of cells. This suggests that charging of the plasma membrane may be more closely tied to caspase activation than charging of intracellular membranes. This is consistent with caspase activation occurring before cytochrome c release, reminiscent of the type I cell extrinsic mechanism, and greater numbers of cells exhibiting PI uptake at lower electric fields than cells with decreased Δψm. At the highest electric field tested (60 kV/cm), all of these nsPEF-induced responses are coincident with cell death in >95% of cells indicating heterogeneous cell responses in nsPEF-induced cell death. Given that ~75–80% of cells express active caspases, caspase-associated cell death appears to be a predominant event. Thus, cell death occurs against different backgrounds of mitochondria responses in Δψm, cytochrome c release and caspase activation. While decreases in Δψm, caspase activation and cytochrome c release may all occur within given populations of cells, other cell populations may exhibit one or more of these, most notably through the cytochrome c-independent extrinsic pathway(s). Thus, nsPEF-induced apoptosis mechanisms are heterogeneous within E4 SCC populations as well as heterogeneous among cell types [1, 3].
Special consideration should be made for using PI uptake as a cell viability marker and phosphatidylserine externalization as an apoptosis marker in response to electric field stimulation. Using PI as a viability marker could be misleading given that electric fields have transient and reversible effects on membrane permeability. Some cells can survive after conventional electroporation as well as after sub-lethal nsPEF conditions . Thus, to avoid possible erroneous conclusions when evaluating effects of electric field on plasma membranes it is not advisable to use PI uptake as a viability marker. In the studies here, 300 ns pulse durations are long enough to allow plasma membrane pores to expand so they are large enough to include PI, which is on the order of about a nanometer. Nevertheless, the pulse durations are short and fast enough to induce intracellular membrane effects. Moreover, modeling evidence indicates that nsPEFs can “pull” phosphatidylserine through nanopores [33, 34, 35, 36, 37] acting as an apoptotic mimic that in the presence of macrophages was shown to cause phagocytosis of caspase-negative cells . Thus, to avoid possible erroneous conclusions, phosphatidylserine externalization should not be used as an apoptosis marker or used very cautiously when evaluating effects of electric fields on plasma membranes. Particularly, PI and Annexin-V should not be used alone as indicators for viability/necrosis and apoptosis, respectively, when evaluating effects of electric fields or other stimuli. In addition, defining apoptosis characteristic in vitro can be complicated by the presence of artifacts that are not present during apoptosis in vivo. In this study, events within the first 2 h were a primary focus to avoid or lessen these issues. While time courses for cell death are stimuli-dependent, nsPEF effects occur relatively rapidly in E4 cells. What can be discerned in this time fame, especially within the first hour(s), is reported in this study.
There may be a case made for a unique type of nsPEF-induced cell death. Cell death initiated by nsPEFs can be differentiated from TNF-induced necroptosis, H2O2-induced necrosis and anti-Fas-induced secondary necrosis  in that nsPEF, in contrast to cold exposure, did not exhibit ROS generation determined with CM-H2DCFDA and did not lead to hyperpolarization of Δψm as the other stimuli-induced cell death did. Thus, E4 SCC mechanisms that protect against DNA and other damage from ROS are not expected to be effective in response to nsPEFs. Given that electric fields with nsPEF characteristics do not exist in nature, nsPEFs provide a distinct cell death stimulus, which could account for the heterogeneity of nsPEF-induced responses in E4 SCC.
In E4 cells, new evidence is provided that nsPEFs act through multiple mechanisms to induce cell death at least in part due to apoptosis through extrinsic type I, extrinsic type II and/or intrinsic pathway(s). Based on several lines of evidence, the type I extrinsic pathway appears to be predominant among these. While calpain activation can be initiated by mobilization of calcium from intracellular and/or extracellular sources by nanopore formation, it remains to be determined how a presumed plasma membrane stimulus leads to caspase activation. The extrinsic pathway is activated by plasma membrane death receptors. It is possible that electric field interactions with the plasma membrane could activate these receptors either through nanopore formation or some other charging event that rearranges lipid bilayers. Alternatively, changes in membrane permeability could alter the intracellular milieu, such as apoptosis volume decrease , leading to caspase activation. Aside from caspase activation other mechanisms are likely to contribute to nsPEF-induced cell death. Since necrosis induced by immediate plasma membrane rupture is not a significant cause of E4 SCC cell death, nsPEFs must set into motion events that are mediated by biological mechanisms in response to nsPEF-mediated stresses that are at least in part programmed.
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