OMICS International welcomes submissions that are original and technically so as to serve both the developing world and developed countries in the best possible way. OMICS Journals are poised in excellence by publishing high quality research. OMICS International follows an Editorial Manager® System peer review process and boasts of a strong and active editorial board. Editors and reviewers are experts in their field and provide anonymous, unbiased and detailed reviews of all submissions. The journal gives the options of multiple language translations for all the articles and all archived articles are available in HTML, XML, PDF and audio formats. Also, all the published articles are archived in repositories and indexing services like DOAJ, CAS, Google Scholar, Scientific Commons, Index Copernicus, EBSCO, HINARI and GALE. For more details please visit our website: http://omicsonline.org/Submitmanuscript.php OMICS Journals are welcoming Submissions
Transcript
OMICS International welcomes submissions that are original
and technically so as to serve both the developing world and
developed countries in the best possible way.
OMICS Journals are poised in excellence by publishing high
quality research. OMICS International follows an Editorial
Manager® System peer review process and boasts of a strong
and active editorial board.
Editors and reviewers are experts in their field and provide
anonymous, unbiased and detailed reviews of all submissions.
The journal gives the options of multiple language translations
for all the articles and all archived articles are available in
HTML, XML, PDF and audio formats. Also, all the published
articles are archived in repositories and indexing services like
DOAJ, CAS, Google Scholar, Scientific Commons, Index
Copernicus, EBSCO, HINARI and GALE.
For more details please visit our website: http://omicsonline.org/Submitmanuscript.php
Nanosecond Pulsed Electric Field (NSpef)-Induced Mechanisms that Bypass Cancer Mutations and cause Cell Death in Cells
and Tumors
Stephen J. Beebe, PhD
Frank Reidy Research Center for Bioelectrics
Old Dominion University Norfolk VA
Journal of Molecular and Genetic Medicine
nsPEFs Abolish Rat HCC and Disable
Evasion of Apoptosis and Immune Surveillance
nsPEF Waveforms, Calcium, Mitochondria (ΔΨm)
and Effects on Proteins
Some Introduction and Concepts
Some Perspective about Cancer
nsPEFs Conquer Evasion of Apoptosis
Journal of Molecular and Genetic Medicine
Using Pulse Power Technology
Cell Manipulations by Pulsed Electric Fields Using Different Pulse Durations
This includes:
Conventional Plasma Membrane Electroporation
Milli- second, Micro- second pulses
Sub-MicroSecond Pulsed Electric Fields
Nanosecond and Pico-second
Journal of Molecular and Genetic Medicine
Pulse Power w/ nsPEFs - Concept 1
Electric Power -stored and released instantaneously into cells and tissues
This produces High Power, low energy, non-thermal conditions
If 1 joule of energy is released all at once in :
1 second = 1 watt
1 microsecond = 1 megawatt
1 nanosecond = 1 gigawatt
100 nanosecond = 10 megawatts
Journal of Molecular and Genetic Medicine
Pulse Power w/ nsPEFs - Concept - 2 Nanopores
High Density Nanoscale Pores in all Cell Membranes
Stewart et al., IEEE Trans Plasma Sci. 2004;32:1696-1708; Gowrishankar et al., BBRC 2006;341:266-1276; Vernier et al., BMC Cell Biol. 2006;7:37; Pakhomov et al., BBRC 2009;385:181-186.
Journal of Molecular and Genetic Medicine
0
10
20
30
40
50
60
0 2.5 5 7.5 10 20 30 40 50 60 70 80
% C
ell
s w
/ F
luo
resc
en
ce
Electric Field (kV/cm)
PI
Fluo-4
* +
+
+
+ + + + +
*
* *
Ca2+ and PI Permeabilization in Jurkat Cells (10 min post-pulse)
PI
Ca2+ (Fluo-4) Ca2+ w/ EGTA-BAPTA
Beebe et al., Cells 2013; 2: 136-162
Pulse Power w/ nsPEFs - Concept - 3 Hypothesis
Fast Rise Time (<~70 ns) or High Frequency Component of Sub-Microsecond Pulses
Provides Greater Possibilities for Intracellular Effects
Schoenbach et al., Bioelectromagnetics 2001; 22 :440-448. Beebe et al., PLoS One 2012 ;7 :e51349. Beebe et al., Cells 2012; 2: 136-162. Beebe et al., J Nanomedic Nanotechnol. 2013 ;4: 163.
Journal of Molecular and Genetic Medicine
0
20
40
60
80
100
0 2.5 5 7.5 10 20 30 40 50 60 70 80
% E
xp
ress
ion
kV/cm
TMRE
Fluo-4
cell viability
Electric Field (kV/cm)
+
Fast Rise-Fall Time (15 ns)
0
20
40
60
80
100
0 2.5 5 7.5 10 20 30 40 50 60 70 80
% E
xp
ress
ion
kV/cm
TMRE
Fluo-4
cell viability
Electric Field (kV/cm)
+
#
Slow Rise-Fall Time (150 ns)
#
*
A
B
*
Vo
lta
ge
(k
V)
Fast Slow
Time (µs)
C % C
ell
s w
/ F
luo
resc
en
ce
% C
ell
Via
bil
ity
% C
ell
s w
/ F
luo
resc
en
ce
%
Ce
ll V
iab
ilit
y
Beebe et al., PLoS One 2012; 7: 51349
1 pulse, 600 ns
-2
0
2
4
6
8
0 1 2 3
A
+
0
20
40
60
80
100
0 2 4 6 8 15 23 30 38 45 53 60
Electric Field (kV/cm)
TMRE
Fluo-4
cellviability
% C
ell
s w
/ F
luo
resc
en
ce
/
% V
iab
ilit
y
-4
-2
0
2
4
6
8
0 1 2 3
Vo
lta
ge
(k
V)
Time (µs)
B
Beebe et al., Cells 2012; 2: 136-162.
0 kV/cm 10 kV/cm
72%
1%
12%
15%
20 kV/cm
73%
2%
9%
16%
61%
3%
19%
17%
40 kV/cm 60 kV/cm 80 kV/cm
53%
7%
23%
18%
27%
25%
11%
37%
13%
34%
5%
48%
Fluo-4 Calcium Influx
Fluo-4 Calcium Influx
TM
RE
ΔΨ
m
TM
RE
ΔΨ
m
Fast Rise-Fall Time, Matched Load
Sain and Beebe, unpublished
79%
1%
10%
10%
69%
1%
20%
10%
55%
1%
35%
9%
43%
2%
45%
10%
41%
2%
47%
10%
41%
2%
46%
11%
Fluo-4 Calcium Influx
Fluo-4 Calcium Influx
Slow Rise-Fall Time, Unmatched Load
0 kV/cm 7.5 kV/cm 15 kV/cm
30 kV/cm 45 kV/cm 60 kV/cm
TM
RE
ΔΨ
m
TM
RE
ΔΨ
m
Sain and Beebe, unpublished
0
10
20
30
40
50
60
70
80
90
100
Control
EGTA
BAPTA/EGTA
0 10 20 30 40 50 60
Electric Fields kV/cm
% C
ell
w/
TM
RE
Flu
ore
sce
nce
10 min post-pulse
Figure Unpublished Beebe et al., Cells 2013; 2: 136-
162
NsPEF-induced Decrease in ΔΨm is Ca2+ Dependent [(1) Effects on Proteins (2) Not Poration of Inner Mitochondria Membrane]
Jurkat Cells
*
+
#
0
20
40
60
80
100
0kv/cm
40kv/cm
60kv/cm
Control +EGTA
Pe
rce
nt
Ce
lls
w/
TM
RE
F
luo
resc
en
ce
A
+
*
#
0
20
40
60
80
100
Control +EGTA
Pe
rce
nt
Ce
lls
w/
TM
RE
F
luo
resc
en
ce
B
Rat N1-S1 Hepatocellular Carcinoma Cells (Ca2+ dependent decrease in ΔΨm – not poration effect)
1 pulse, 600 ns
10 pulses, 600 ns
Sain, Harlow and Beebe, Unpublished
ΔΨm Ca2+
Protein(s) ?
EP IMM
Non-EP IMM
0
10
20
30
40
50
60
70
80
90
100
0 60
Pe
rce
nt
PI
Up
tak
e
Electric field kV/cm
With EGTA @60ns
With EGTA @600ns
Without EGTA @60ns
0 Ca2+ @ 10p 60 ns
0 Ca2+ @ 1p 600 ns
5mM Ca2+ @ 10p 60 ns 5mM Ca2+ @ 1p 600 ns
Plasma Membrane Permeabilization
is not Ca2+ Dependent
Beebe, Bioelectrochemistry, in press
Journal of Molecular and Genetic Medicine
Yuan and Kroemer Genes & Dev 2010; 24:2592-2602
Signaling Complexes induced by TNFα mediate NfκB activation, apoptosis and necroptosis
[Ca2+] i
Calpain
Cytochrome c
DNA Damage
BID t-BID
PM
FAS-R
FAS-L
FADD
ΔΨm
I
Bak DIS
C
Ap
op
toso
me Caspase-3, 6,
7
Extrinsic or Intrinsic Apoptosis
APAF-1
Beebe et al., Cells 2013; 2: 136-162. Caspase-Dependent
Cell Death
[Ca2+] i
Calpain
Cytochrome c
DNA Damage
BID t-BID
[Na+ ]o / [Ca2+
]o? PM
FAS-R
FAS-L
FADD
ΔΨm
I II
IV
V
Bak DIS
C
Ap
op
toso
me Caspase-3, 6,
7
Extrinsic or Intrinsic Apoptosis
III
APAF-1
1
2
3 Bcl-2/xl
Beebe et al., Cells 2013; 2: 136-162. Caspase-Dependent
Cell Death
0
20
40
60
80
100
120
0 10 20 30 40 50 60
% c
ell
via
bil
ity
nsPEFs (kV/cm)
A3
ΔFADD
Δcaspase-8
24hr cell viability (60ns, 10 pulses)
nsPEF-Induced Cell Death Does Not Require
The DISC (ΔCaspase-8 / Δ FADD)
FAS-L
FAS-R
DISC
FADD
Bypasses Cancer
Mutations @ Death
Receptors
Ren et al., BBRC 2012; 421: 808-112.
1
0
20
40
60
80
100
120
0 10 20 30 40 50 60
Pe
rce
nt
Via
bil
ity
nsPEFs (kV/cm)
pSUPER-neo
sh-Apaf-1 RNA
Cytochrome c
- APAF-1
APAF-1 Deficient Jurkat Cells
Require Higher Electric Fields to Induce Cell Death
Apoptosome
Bypasses Cancer Mutations @
IAPs Ren et al., BBRC 2012; 421: 808-112.
Caspase-Dependent CD
2
0
20
40
60
80
100
0 10 20 30 40 50 60
Ce
lls
w/
TM
RE
E
xp
ress
ion
(P
erc
en
t o
f C
on
tro
l)
Electric Field (kV/cm)
psFFV-neo/E6.1
psFFV-BCL-XL
0102030405060708090
100110
0 10 20 30 40 50 60
Via
bil
ity
(P
erc
en
t o
f C
on
tro
l)
Electric Field (kV/cm)
psFFV-neo/E6.1
psFFV-BCL-XL
A
B
1.77 11.89
E6.1 Bcl-xl
27 kDa Fluorescence A.U.
ΔΨm
Cancer Mutations
Ren, Harlow and Beebe, Unpublished
Ca2+
Protein(s)
3
neo control neo treated
Apaf-1 Control Apaf-1 (6h) treated
Histone 2AX Phosphorylation
Histone 2AX Phosphorylation
Fo
rwa
rd L
igh
t S
catt
er
nsPEF-induced DNA Damage is Caspase-dependent
Ren and Beebe, unpublished
[Ca2+]i
Calpain
Cytochrome c
DNA Damage
(H2AX/TUNEL)
Fragmentation
BID
[Ca2+
] o [Na+ ]o
FAS-R
FAS-L
FADD
APAF-1
ΔΨm
I
III
IV
V
Bclxl
Bak
DIS
C
Ap
op
toso
me
Caspase-3
[Puma / Noxa]
Intrinsic Cell Death Mechanisms
X
Caspase-Dependent
Cell Death
X
II
ATP
t-Bid
Caspase-9
Caspase-8
Caspase-Independent
Cell Death
?
X
X X
?
Adapted f/ Beebe et al., Cells 2013; 2: 136-162.
In Vitro Summary and Conclusions
Pulse Shape is a Determinant of Effects on ΔΨm and Viability
NsPEF-induced Decrease in ΔΨm is Ca2+ Dependent
Influx of Ca2+ Necessary, but not Sufficient, for Drop in ΔΨm and CD
Primary Decrease in ΔΨm is Not Due to Poration of IMM
Decrease in ΔΨm May be Due to Effects on Protein(s)
nsPEF-induced DNA Damage is Caspase-Dependent
nsPEF Bypass Cancer Mutations @ DR, Caspases and Mitochondria
A Criminal Gang Cancer Within the local community Microenvironment Coerces the local population Supporting host cells Uses their resources Growth, vascularization Thwart the authorities Evades immune surveillance The gang’s criminal activity The tumor as an organ
A Concept for Cancer
Expanded f/Drake (Neuberg S.) Nat Med 2011;17:757; Beebe SJ, J Nanomed & Biotherapeutic Discovery 4:e134
Not an Invading Army – Cancer Comes from Within Us
Electrode Design: 5 Needle Array
Kolb JF, unpublished
Journal of Molecular and Genetic Medicine
NsPEF Ablation of Rat N1S1 Orthotopic HCC
Conditions:
Pulse Duration: 100 ns
Electric Field: 50 kV/cm
Pulse Number: 100, 300, 500 or 1000
Treatments: 1
Electrodes: 5 Needle Array Chen R et al., Eur J Cancer 2014, in press
Journal of Molecular and Genetic Medicine
-5.000E+00 5.000E+00X (mm)
-5.000E+00
5.000E+00
Y (mm)
File prefix: Needle_4.EOU
Plot type: Element
Quantity: |E|(V/m)
Minimum value: 0.000E+00
Maximum value: 2.165E+07
6.019E+05
1.806E+06
3.009E+06
4.213E+06
5.417E+06
6.621E+06
7.824E+06
9.028E+06
1.023E+07
1.144E+07
1.264E+07
1.384E+07
1.505E+07
1.625E+07
1.745E+07
1.866E+07
1.986E+07
2.107E+07
Current view
window
5 Needle Array
Electric Field Simulation
Zhuang J, Kolb JF, Chen X, Beebe SJ, Unpublished
Journal of Molecular and Genetic Medicine
NADH activity using Nitro Blue Tetrazolium Viable: Purple Non-viable: Pink
Histopathological Analysis of Porcine Liver
30 pulses, 100ns and 10-12kV/cm
Long G et al., IEEE EMB, 2011; 58: 2161-2167
Day -1
5 6 7
Day +6
5 6 7
3.45 p/s 1.55 cm2
8.37 p/s 2.1 cm2
1.54 p/s 0.96 cm2
9.23 p/s 2.83 cm2
0 p/s 0 cm2
100p
Luminescence of N1S1/Luciferase Cells in Rat Liver
Before and After Treatment with 100 ns, 50 kV/cm
0 p/s 0 cm2
300p 1000p 100p 300p 1000p
Sain NM and Beebe SJ, unpublished
Sham
1000X
Day 6 Day 15
0
50
100
150
200
250
300
6 11 15 21
3-D
vo
lum
e (
mm
3)
Days after N1-S1 injection
N1-S1 Tumor volume
1000X RAT#1
1000X RAT#2
SHAM
1000XRAT#3
1000XRAT#4
Orthotopic Rat N1S1 HCC
Chen R et al., Eur J Cancer 2014, in press
0.00
1000.00
2000.00
3000.00
4000.00
5000.00
6000.00
7000.00
1 2 3 4 5
* *
* *
We
igh
t (m
g)
Number of Pulses
0 100 300 500 1000
100ns, 50kV/cm, 1Hz
2 weeks post-treatment
NsPEFs Eliminate Orthotopic Rat N1-S1 HCC Tumors
With 1000 pulses
Chen R et al., Eur J Cancer 2014, in press
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
0 300 500 1000% p
ost
-tre
atm
en
t vo
lum
e
vs.
co
ntr
ol
Tumor Volumes 6 weeks Post-Treatment: % Change vs. Sham Treatment
* *
**
Number of Pulses
Orthotopic Rat N1S1 Hepatocellular Carcinoma Treated with nsPEFs [100 ns, 50 kV/cm, 1 Hz]
Tumor Volumes 6 Weeks Post-Treatment
Percent Change vs. Sham Treatment
Chen R et al., Eur J Cancer 2014, in press
Journal of Molecular and Genetic Medicine
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
No Response
Stable
Partial Response
Complete
1000 pulses, 1Hz, 100 ns, 50kV/cm
1000p Sham
Pe
rce
nt
of
To
tal
NsPEFs Eliminate 80-90% of Rat N1-S1 HCC Tumors
n=36 n=23
Chen R et al., Eur J Cancer 2014, in press
Normal liver
Tumor
Before Treatment After Treatment 1000p x 100ns x 50kV/cm
nsPEFs Decrease Blood Flow – Laser Doppler
~60 % decrease
Before After
Chen R, Sain NM and Beebe SJ, unpublished
Journal of Molecular and Genetic Medicine
before treatment after treatment7 days post-treatment
Sham (n=3) 100.00 115.48 81.97
1000X (n=7) 100.00 44.85 123.73
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00%
Ch
an
ge
in
Blo
od
Flo
w
* #
Transient, Tumor-Specific Blood Flow Change
R Chen et al., Surgery: Current Research 2013, S12: 005.
Journal of Molecular and Genetic Medicine
Sham 6h 1000X 1h 1000X 2h 1000X 4h 1000X 6h
Caspase-3 (Cleaved)
Caspase-9 (Cleaved)
Caspase-8 (Cleaved)
Intrinsic Caspase Activation in N1S1 Tumors
Chen R et al., Eur J Cancer 2014, in press
Active Caspase-9
Active Caspase-3
Chen R et al., Eur J Cancer 2014, in press
challenge
NsPEF Ablation Induces a Vaccine-Like Protective Effects Against N1-S1 HCC
