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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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A caspase inhibitor (z-VAD-fmk) partially decreases nsPEF-induced cytochrome c release. E4 cells were pre-incubated with 100 μM z-VAD-fmk, a pan caspase inhibitor, for 30 min and then pulsed ten times with 300 ns pulses at 60 kV/cm or sham treated. Cells were analyzed by flow cytometry for cytochrome c release 1 h post pulse using Method I as described in Experimental Procedures. a Representative contour plots are shown with forward light scatter (FSC-A) on the y-axis and cytochrome c fluorescence (FITC-A) on the x-axis. The top panels show sham treated control cells in the absence (left) and presence (right) of z-VAD-fmk. The bottom panels show cells treated with nsPEFs in the absence (left) and presence (right) of z-VAD-fmk. A decrease in cytochrome c release is shown as a decrease in fluorescent intensity (shift from lower right quadrant to lower left quadrant) after preincubation with 100 μM z-VAD-fmk. b The percentage of cells exhibiting cytochrome c release (n = 3) is presented as mean ± SE. ** P < 0.01
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Fig3: A caspase inhibitor (z-VAD-fmk) partially decreases nsPEF-induced cytochrome c release. E4 cells were pre-incubated with 100 μM z-VAD-fmk, a pan caspase inhibitor, for 30 min and then pulsed ten times with 300 ns pulses at 60 kV/cm or sham treated. Cells were analyzed by flow cytometry for cytochrome c release 1 h post pulse using Method I as described in Experimental Procedures. a Representative contour plots are shown with forward light scatter (FSC-A) on the y-axis and cytochrome c fluorescence (FITC-A) on the x-axis. The top panels show sham treated control cells in the absence (left) and presence (right) of z-VAD-fmk. The bottom panels show cells treated with nsPEFs in the absence (left) and presence (right) of z-VAD-fmk. A decrease in cytochrome c release is shown as a decrease in fluorescent intensity (shift from lower right quadrant to lower left quadrant) after preincubation with 100 μM z-VAD-fmk. b The percentage of cells exhibiting cytochrome c release (n = 3) is presented as mean ± SE. ** P < 0.01

Mentions: To determine whether cytochrome c is released through the intrinsic pathway or type II cell extrinsic pathway, E4 cells were pretreated with 100 μM z-VAD-fmk, a general caspase inhibitor, and then pulsed ten times at 300 ns, 60 kV/cm in the presence of the inhibitor (Fig. 3). Inclusion of 1 mM z-VAD-fmk gave identical results (data not shown), indicating that caspase activity was completely inhibited at 100 μM z-VAD-fmk. Cytochrome c release was determined 1 h post pulse by flow cytometry using Method I (see Experimental Procedures). Figure 3a shows a contour plot of a typical experiment with forward scatter on the Y-axis (FSC-A) and cytochrome c fluorescence on the X-axis (FITC-A). Cells that exhibit cytochrome c release appear in the bottom right quadrant. Figure 3b shows the combined results of three separate experiments with statistical comparisons. In sham treated control cells, very few cells show cytochrome c release (7.7 ± 1.5%) and the caspase inhibitor has no significant effect (9.5 ± 2.2%). When cells were treated with ten 300 ns pulses at 60 kV/cm, 46.2 ± 2.5% of cells exhibited cytochrome c release and the caspase inhibitor decreased that to 26.2 ± 4.6%. Three separate determinations gave similar results (Fig. 3b). Thus, based on the mean values in these experiments 43.3% of cytochrome c release was caspase-dependent and 56.7% was caspase-independent, indicating more than one mechanism for cytochrome c release in response to nsPEFs.Fig. 3


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

Ren W, Beebe SJ - Apoptosis (2011)

A caspase inhibitor (z-VAD-fmk) partially decreases nsPEF-induced cytochrome c release. E4 cells were pre-incubated with 100 μM z-VAD-fmk, a pan caspase inhibitor, for 30 min and then pulsed ten times with 300 ns pulses at 60 kV/cm or sham treated. Cells were analyzed by flow cytometry for cytochrome c release 1 h post pulse using Method I as described in Experimental Procedures. a Representative contour plots are shown with forward light scatter (FSC-A) on the y-axis and cytochrome c fluorescence (FITC-A) on the x-axis. The top panels show sham treated control cells in the absence (left) and presence (right) of z-VAD-fmk. The bottom panels show cells treated with nsPEFs in the absence (left) and presence (right) of z-VAD-fmk. A decrease in cytochrome c release is shown as a decrease in fluorescent intensity (shift from lower right quadrant to lower left quadrant) after preincubation with 100 μM z-VAD-fmk. b The percentage of cells exhibiting cytochrome c release (n = 3) is presented as mean ± SE. ** P < 0.01
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Fig3: A caspase inhibitor (z-VAD-fmk) partially decreases nsPEF-induced cytochrome c release. E4 cells were pre-incubated with 100 μM z-VAD-fmk, a pan caspase inhibitor, for 30 min and then pulsed ten times with 300 ns pulses at 60 kV/cm or sham treated. Cells were analyzed by flow cytometry for cytochrome c release 1 h post pulse using Method I as described in Experimental Procedures. a Representative contour plots are shown with forward light scatter (FSC-A) on the y-axis and cytochrome c fluorescence (FITC-A) on the x-axis. The top panels show sham treated control cells in the absence (left) and presence (right) of z-VAD-fmk. The bottom panels show cells treated with nsPEFs in the absence (left) and presence (right) of z-VAD-fmk. A decrease in cytochrome c release is shown as a decrease in fluorescent intensity (shift from lower right quadrant to lower left quadrant) after preincubation with 100 μM z-VAD-fmk. b The percentage of cells exhibiting cytochrome c release (n = 3) is presented as mean ± SE. ** P < 0.01
Mentions: To determine whether cytochrome c is released through the intrinsic pathway or type II cell extrinsic pathway, E4 cells were pretreated with 100 μM z-VAD-fmk, a general caspase inhibitor, and then pulsed ten times at 300 ns, 60 kV/cm in the presence of the inhibitor (Fig. 3). Inclusion of 1 mM z-VAD-fmk gave identical results (data not shown), indicating that caspase activity was completely inhibited at 100 μM z-VAD-fmk. Cytochrome c release was determined 1 h post pulse by flow cytometry using Method I (see Experimental Procedures). Figure 3a shows a contour plot of a typical experiment with forward scatter on the Y-axis (FSC-A) and cytochrome c fluorescence on the X-axis (FITC-A). Cells that exhibit cytochrome c release appear in the bottom right quadrant. Figure 3b shows the combined results of three separate experiments with statistical comparisons. In sham treated control cells, very few cells show cytochrome c release (7.7 ± 1.5%) and the caspase inhibitor has no significant effect (9.5 ± 2.2%). When cells were treated with ten 300 ns pulses at 60 kV/cm, 46.2 ± 2.5% of cells exhibited cytochrome c release and the caspase inhibitor decreased that to 26.2 ± 4.6%. Three separate determinations gave similar results (Fig. 3b). Thus, based on the mean values in these experiments 43.3% of cytochrome c release was caspase-dependent and 56.7% was caspase-independent, indicating more than one mechanism for cytochrome c release in response to nsPEFs.Fig. 3

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