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An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma.

Ren W, Beebe SJ - Apoptosis (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

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NsPEF-induced cell death is correlated with the activation of caspase-3/7, -8, and -9 in an electric field amplitude-dependent manner. E4 cells were pulsed ten times at 300 ns and electric field strengths between 0 and 60 kV/cm. (Top panel) Cell viability was determined by counting trypan blue negative cells 24 h post pulse and normalizing values to the control. (Bottom panel) The percentage of cells with active caspase-3/7, -8, -9 was analyzed by flow cytometry using the cell permeable irreversible caspase inhibitors FAM-DEVD-fmk (caspase-3), FAM-LETD-fmk (caspase-8), FAM-LEHD-fmk (caspase-9) as described in Experimental Procedures. The values represent the mean ± SE of three independent determinations. * P < 0.05; ** P < 0.01
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Fig1: NsPEF-induced cell death is correlated with the activation of caspase-3/7, -8, and -9 in an electric field amplitude-dependent manner. E4 cells were pulsed ten times at 300 ns and electric field strengths between 0 and 60 kV/cm. (Top panel) Cell viability was determined by counting trypan blue negative cells 24 h post pulse and normalizing values to the control. (Bottom panel) The percentage of cells with active caspase-3/7, -8, -9 was analyzed by flow cytometry using the cell permeable irreversible caspase inhibitors FAM-DEVD-fmk (caspase-3), FAM-LETD-fmk (caspase-8), FAM-LEHD-fmk (caspase-9) as described in Experimental Procedures. The values represent the mean ± SE of three independent determinations. * P < 0.05; ** P < 0.01

Mentions: To determine cell viability after nsPEF treatment and whether nsPEFs activate caspase-associated cell death pathways in E4 SCC, cells were exposed to ten 300 ns pulses of 0–60 kV/cm electric field strengths. We chose ten, 300 ns pulses as parameters for this study so we could observe a complete range of electric field amplitude-dependent effects on cell survival from little of no cell death to maximal cell death. Thus, we could observe various cell responses as possible mechanisms that were coincident with cell death. Cell viability was determined by counting trypan blue negative cells 24 h post pulse (Fig. 1 top panel). Caspase-3/7, -8, -9 activations were determined 2 h after nsPEF administration by flow cytometry using a cell permeable, irreversible inhibitors with selectivity towards specific caspase isoforms (Fig. 1 bottom panel). Results indicate an electric field-dependent decrease in cell viability that was correlated with electric field amplitude-dependent increases in percentages of cells that exhibited active caspases. Analysis of caspase activation time courses indicated that they were activated between 20 and 60 min post pulse using the cell permeable, pan caspase irreversible inhibitor, z-VAD-fmk (data not shown). Therefore, nsPEF-induced E4 cell death is correlated with early caspase activation. Very few if any cells immediately lose plasma membrane integrity indicating that frank necrosis is not a mechanism of cell death.Fig. 1


An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma.

Ren W, Beebe SJ - Apoptosis (2011)

NsPEF-induced cell death is correlated with the activation of caspase-3/7, -8, and -9 in an electric field amplitude-dependent manner. E4 cells were pulsed ten times at 300 ns and electric field strengths between 0 and 60 kV/cm. (Top panel) Cell viability was determined by counting trypan blue negative cells 24 h post pulse and normalizing values to the control. (Bottom panel) The percentage of cells with active caspase-3/7, -8, -9 was analyzed by flow cytometry using the cell permeable irreversible caspase inhibitors FAM-DEVD-fmk (caspase-3), FAM-LETD-fmk (caspase-8), FAM-LEHD-fmk (caspase-9) as described in Experimental Procedures. The values represent the mean ± SE of three independent determinations. * P < 0.05; ** P < 0.01
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3066400&req=5

Fig1: NsPEF-induced cell death is correlated with the activation of caspase-3/7, -8, and -9 in an electric field amplitude-dependent manner. E4 cells were pulsed ten times at 300 ns and electric field strengths between 0 and 60 kV/cm. (Top panel) Cell viability was determined by counting trypan blue negative cells 24 h post pulse and normalizing values to the control. (Bottom panel) The percentage of cells with active caspase-3/7, -8, -9 was analyzed by flow cytometry using the cell permeable irreversible caspase inhibitors FAM-DEVD-fmk (caspase-3), FAM-LETD-fmk (caspase-8), FAM-LEHD-fmk (caspase-9) as described in Experimental Procedures. The values represent the mean ± SE of three independent determinations. * P < 0.05; ** P < 0.01
Mentions: To determine cell viability after nsPEF treatment and whether nsPEFs activate caspase-associated cell death pathways in E4 SCC, cells were exposed to ten 300 ns pulses of 0–60 kV/cm electric field strengths. We chose ten, 300 ns pulses as parameters for this study so we could observe a complete range of electric field amplitude-dependent effects on cell survival from little of no cell death to maximal cell death. Thus, we could observe various cell responses as possible mechanisms that were coincident with cell death. Cell viability was determined by counting trypan blue negative cells 24 h post pulse (Fig. 1 top panel). Caspase-3/7, -8, -9 activations were determined 2 h after nsPEF administration by flow cytometry using a cell permeable, irreversible inhibitors with selectivity towards specific caspase isoforms (Fig. 1 bottom panel). Results indicate an electric field-dependent decrease in cell viability that was correlated with electric field amplitude-dependent increases in percentages of cells that exhibited active caspases. Analysis of caspase activation time courses indicated that they were activated between 20 and 60 min post pulse using the cell permeable, pan caspase irreversible inhibitor, z-VAD-fmk (data not shown). Therefore, nsPEF-induced E4 cell death is correlated with early caspase activation. Very few if any cells immediately lose plasma membrane integrity indicating that frank necrosis is not a mechanism of cell death.Fig. 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus