30th Annual International IEEE EMBS Conference

Vancouver, British Columbia, Canada, August 20-24, 2008

Experiment and Mechanism Research of SKOV3 Cancer Cell

Apoptosis Induced by Nanosecond Pulsed Electric Field
Chenguo Yao, Yan Mi, Xiaoqian Hu, Chengxiang Li, Caixin Sun, Junying Tang, Xiaojuan Wu

nucleolus). After pulsing, in most cases, these pores will

Abstract—This paper studies the apoptosis of human reseal without any damage to cell. This physical procedure of
ovarian carcinoma cell Line (SKOV3) induced by the transient pores appearing at cell membrane is termed
nanosecond pulsed electric field (10kV/cm, 100ns, 1 Hz) and its Electroporation. The electroporation therapy, combining
effect on intracellular calcium concentration ([Ca2+]i). These PEF with chemotherapy, has been applied to treating
cells were doubly marked by Annexin V-FITC/PI, and the
tumor[4~6], such as head-neck cancer, skin cancer, pancreatic
apoptosis rate was analyzed with flow cytometry. After AO/EB
staining the morphological changes were observed under
cancer and liver cancer. But the cancer cells cannot be killed
fluorescent microscope, and their ultrastructural changes were directly, and the damage to patients resulting from chemical
observed under scanning electron microscope (SEM). With drug cannot be avoided.
Fluo-3/AM as calcium fluorescent marker, laser scanning When shortening pulse duration and increasing field
confocal microscope (LSCM) was used to detect the effect of intensity, PEF has a series of unique advantages on
nsPEF on [Ca2+]i and the source of Ca2+. The results showed nanosecond timescale [7~10]: 1) due to the very short duration,
that the early apoptosis rate of the treatment group was the energy transferred to issue and cell is extremely little,
(22.21±2.71)%, significantly higher than that of the control with negligible thermal effect; 2) the front edge (rise or fall
group (3.04±0.44)% (P<0.01). The typical features of apoptotic
edge) of pulse is quite steep, which can restrain chemical
cell have been observed by fluorescent microscope and SEM. It
is proved that nsPEF can induce apoptosis of SKOV3 cells and
reaction of issue and cell; 3) instantaneous high power pulse
result in distinct increase in [Ca2+]i (P<0.01), which was can cause much stronger nonthermal biological effects. In
independent of extracellular calcium concentration (P>0.05). cell interior (e.g. nucleus, endoplasmic reticulum,
Since nsPEF can penetrate cell membrane due to its high mitochondrial, etc), bioelectric effects very different from
frequency components, one of the mechanisms of electroporation phenomenon could be induced. Schoenbach,
nsPEF-induced apoptosis may be that activating intracellular Beebe et al [7-9] found that many functional changes in inner
calcium stores can increase the [Ca2+]i, and consequently, the membrane structure and cell signal transition were induced
apoptotic signal pathway can be induced. by nsPEF (typical parameters: 100kV/cm, 10ns). These
changes include perforation of nuclear membrane,
Index Terms—Nanosecond pulsed electric field (nsPEF); micronucleus formation, enhancement of gene expression,
SKOV3 cancer cell; apoptosis; flow cytometry; ultrastructure;
mitochondrial perforation that can cause release of
Ca2+
intracellular calcium ion, etc. Compared with the
electroporation, this phenomenon is termed intracellular
I. INTRODUCTION
electromanipulation (IEM).

R ecently, the pulsed electric fields (PEF) has been applied

to the fields of drug delivery, gene transfection, tumor
therapy, etc. Some researchers [1~3] found that when
From the above researches, the type of PEF and its
parameter selection have significant effect on structure
function and signal transduction of cell. If nsPEF with
exposed to PEF (typical parameters: 1kV/cm, 100µs), lipid suitable parameters can induce cell apoptosis, then it can be
bilayer in cell membrane could temporarily rearrange, applied to killing tumor cell. By far there’s no detailed report
leading to the formation of aqueous channels that are often on morphology such as apoptosis body.
called pores. Such changes will make cell membrane more In this study, the nsPEF (10kV/cm, 100ns) was applied to
permeable to a variety of hydrophilic molecules, while there inducing apoptosis of SKOV3 cancer cell. With the help of
is no effect on the intramembranous organelles (e.g. flow cytometry, fluorescent microscope, LSCM, and SEM,
and based on statistics, morphology and ultrastructure, it is
This work was supported by the National Natural Science Foundation of showed that this nsPEF can induce apoptosis of SKOV3
China (No: 50637020). cancer cell. Moreover, in order to better understand the
Chenguo Yao, Yan Mi, Chengxiang Li and Caixin Sun are with State Key
Laboratory of Power Transmission Equipment & System Security and New
mechanism of apoptosis induction, Ca2+, the important
Technology, Chongqing University, Chongqing, 400044 China (phone: second messenger of signal transfer in cell apoptosis, is
86-023-65112058; e-mail: yaochenguo@cqu.edu.cn) studied.
Xiaoqian Hu is with the College of Electronic Information and Its
Automation, Chongqing Institute of Technology, Chongqing, 400050
China
II. MATERIALS AND METHODS
Junying Tang, Xiaojuan Wu are with Chongqing University of Medical
Science, Chongqing 400010, China A. Cell culture

978-1-4244-1815-2/08/$25.00 ©2008 IEEE. 1044

SKOV3 cancer cells were cultured in RPMI-1640 substrate the rate of early apoptosis
(GIBCO Company), which contained 10% calf serum,
the total rate of late
100U/ml penicillin and 100U/ml streptomycin. Cultured in a apoptosis and necrosis
37, 5%CO2 humidified incubator, these cells bred one time
every 4-5days.
Before experiment, these SKOV3 cells in the logarithmic
growing period were digested with 0.25% pentazyme
(GIBCO Corporation) and centrifugated. Then the single cell
suspension was made by RPMI-1640 culture fluid, and its
concentration was adjusted to 1×106/ml. 1ml cell suspension Treatment group Control group
was taken and randomly divided into two groups: control Fig 1 statistics analysis of apoptosis of SKOV3 cancer cells
group and treatment group, which were respectively given (*P<0.05, **P<0.01, compared with control cells)
mock treatment and nsPEF (10kV/cm, 100ns, 1Hz, 5min) alcohol and sprayed gold in vacuum, the cells were observed
treatment. The pulse generator is developed by us[11]. under SEM (KYKY1000B Amray).
B. Detecting apoptosis cell by flow cytometry E. Detecting [Ca2+] i concentration by LSCM
Annexin-V-FITC and PI were used as fluorescent markers. 4h after nsPEF treatment, 1×106 cells were respectively
4h after nsPEF treatment, 1×106 cells were respectively collected from the control group and the treatment group.
collected from the control group and the treatment group. After centrifugal operation at 1000rpm for 5min, the cells
After being washed twice with cold PBS, these cells were were resuspended with serum-free substrate. Centrifugal
resuspended in 250ul Annexin combining with buffer operation was performed again at 1000rpm for 5min, and the
solution. 100ul cell suspension was taken and put into a 5ml cells were resuspended to 1ml with serum-free substrate.
cuvette, in which then 5 ul Annexin V FITC and 10ul PI were Fluo-3/AM (ANASPEC), the calcium ions probe, was added to
added. They were uniformly mixed and incubated for 15 min adjust final concentration to 5umol/L. Then the cell suspension
in the dark at room temperature. After that, 400ul PBS was
was incubated for 30min in the dark at 37 and centrifuged
added, and these cells were immediately detected by flow
at 1000rpm for 5min. After that, the cell suspension were
cytometry (FACSCalibur). Finally, early apoptosis, late
resuspended twice with PBS to adjust the concentration to
apoptosis or necrosis, damnified cells due to operation, and
1×106/ml, and then observed under LSCM (Leica TCS-SPZ,
normal cell were identified by Annexin +/ PI –, Annexin +/ PI
Germany). We used LSCM quantitative analysis software to
+, Annexin – / PI + and Annexin– / PI –, respectively.
analyze images and measure the relative fluorescence
C. Detecting cell morphological changes by AO/EB staining intensity of single SKOV3 cell. In each group the
Before nsPEF treatment, cells were collected from fluorescence intensities of calcium ions of 15 cells were
treatment group and control group and counted. The culture calculated and the mean value was computed.
fluid was diluted to 5×104/ml and dropped in sterile 24-pore F. Observing the effect of CaCl2 on [Ca ]i
2+

plate (each pore 1ml), which then was placed in CO2

4h after nsPEF treatment, 4ml cell suspension was
incubator. 4h after nsPEF treatment, 40ul AO/EB mixed
collected from the treatment group and its concentration was
liquid (100, 100 mg/L) was added in every pore. After the
adjusted to 1×106/ml. It was then divided into 4 groups (each
liquid was uniformly mixed, pictures were taken by
group 1ml). CaCl2 solution were added into these groups to
fluorescent microscope. AO can penetrate cell with intact
adjust their concentration 0.1, 0.2, 0.3 (mmol/L),
cytomembrane, insert nuclear DNA, and make it emit bright
respectively. 15min later, every group was loaded with
green fluorescence. In contrast, EB can only penetrate cell
Fluo-3/AM. Then the mean value of fluorescence intensity of
with damaged cytomembrane, insert nuclear DNA, and make
each group was measured.
it emit bright orange-red fluorescence. In this way, four
morphoses of cell can be distinguished. G. Statistics processing
D. Sampling and observation under SEM All data were expressed by mean±standard deviation, and
tested by SPSS12.0 software. The results of flow cytometry and
4h after nsPEF treatment, 1×106 cells were respectively
the effects of nsPEF on cell’s [Ca2+]i were made T-test. The
collected from the control group and the treatment group. 2+
effect of CaCl2 on cell [Ca ]i was analyzed by one-way
These cells were washed twice with PBS, centrifuged at
variance.
1500rpm for 5min, and fixed in 4% glutaraldehyde for 30min
at 4 ºC. Then they were soaked in PBS for 5min at the room
III. RESULTS
temperature, and this step was performed twice. After that,
these cells were fixed with 1% osmium tetroxide for 30min in A. Results measured by flow cytometry
the dark at 4. Alcohol gradient elution: these cells were
respectively dehydrated with 30%, 50%, 70%, 90%, 100% 4h after nsPEF treatment, with the help of Annexin
alcohol for 10min at 4. After being dried with tert-butyl V-FITC/PI, the rate of early apoptosis and the total rate of
late apoptosis and necrosis were analyzed by flow cytometry,
as shown in Fig.1. The early apoptosis rate of the treatment

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 The volume of the treated cell diminished significantly and
was approximately two-thirds of the untreated, as shown in
Fig.3. With intact cytomembrane and fewer microvilli,
vesicular protuberances of different sizes appeared on the
surface of the treated cell, which was forming apoptotic
bodies, indicated by arrows. The untreated cell was spherical,
with intact cytomembrane and stubby microvilli of different
types at the cell surface.
D. Effect of nsPEF on [Ca2+]i of SKOV3 cancer cell
A Control group (h200) B Treatment group (h200) From each group we randomly collected 15 SKOV3 cancer
Fig.2 Results observed after AO/EB staining
cells with clear structure and intact cytomembrane, and
measured their relative fluorescence intensity by LSCM.
Fluorescence intensity of the treatment group [Ca2+]i was
(12.80±2.10)%, higher than that of the control group
(9.22±1.63)%, P<0.01, as shown in Fig.4.
E. Effect of CaCl2 on [Ca2+]i of SKOV3 cancer cell
After nsPEF treatment combining with CaCl2, using
LSCM, relative fluorescent intensity of [Ca2+]i in each group
A Untreated cell (h2500) B Treated cell. (h5000) was obtained and given in Table 1. The difference between
Fig.3 SKOV3 cancer apoptosis observed under SEM two random groups had no statistical meaning (P>0.05). The
group was (22.21±2.71)%, higher than that of the control result shows that the [Ca2+]i change of SKOV3 cancer cell
group (3.04±0.44)% (P<0.01). The total rate of late only depends on nsPEF treatment, and is independent of
apoptosis and necrosis of the treatment group was concentration change of extracellular calcium ion.
(9.78±2.71)%, while that of the control group was TABLE1. EFFECT OF CaCl2 ON [Ca2+]i ( x ± s )
(0.45±0.32)%. The difference between them was meaningful Relative fluorescent
Group Repeat times/n
statistically (P<0.05). The early apoptosis rate of the intensity of cell [Ca2+]i
treatment group was much higher than its total rate of late nsPEF combining with
15 11.14±1.02
apoptosis and necrosis (P<0.01). 0mmol/L CaCl2 group
nsPEF combining with
15 10.73±1.60
B. Results observed after AO/EB staining 1mmol/L CaCl2 group
nsPEF combining with
There were much more apoptotic and necrotic cells in the 15 11.61±1.73
2mmol/L CaCl2 group
treatment group, as shown in Fig2. The living cell has intact nsPEF combining with
15 10.55±1.53
cytomembrane and uniform nuclear chromatin, and looks 3mmol/L CaCl2 group
green. The cell in early apoptosis has intact cytomembrane
but diminished cell body, and its nuclear chromatin is IV. DISCUSSION
yellowish green, condensed and bead-like. The cell in late Apoptosis is that under definite physiological or
apoptosis also has intact cytomembrane but diminished cell pathological conditions, cells kill themselves following their
body while its nuclear chromatin is orange-red, condensed own program. The mechanism of traditional cancer therapies
and bead-like. The necrotic cell, with orange-red nuclear is to kill tumor cell and restrain its growth and proliferation.
chromatin, has swelled cell body and broken cytomembrane, In contrast, apoptosis can make tumor tissue diminish and
and is disintegrated or close to disintegration. In the control even disappear, decreasing the side effects and the harm to
group were only fusiform cancer cells with regular morphosis, normal cells. In a word, apoptosis plays a significant role in
intact cytomembrane and the nucleus exhibiting uniform, cancer therapy. Nowadays, experiment and mechanism
strong and green fluorescence. research on cancer cell apoptosis induced by nsPEF have
C. Results observed by SEM become a research hotspot in the field of tumor therapy
internationally.
In this study, after nsPEF (10kV/cm, 100ns) treatment, the
Relative fluorescent intensity

SKOV3 cancer cells were doubly marked by Annexin

V-FITC/PI. For early apoptosis cell, the phosphatidyl serine
(PS) exposed on the cell membrane, exhibits green
fluorescence when combining with Annexin V-FITC. But its
cell membrane is intact, so the fluorescent dyestuff PI cannot
enter into the cell. In contrast, necrotic cells and secondary
necrotic cells in late apoptosis can be doubly marked by
Treatment group Control group Annexin V-FITC/PI. Therefore, the apoptosis can be
Fig.4 Effect of nsPEF on [Ca2+]i of SKOV3 cancer cell quantitatively analyzed by flow cytometry. The results
(**P<0.01, compared with control group) showed that the early apoptosis rate of the treatment group

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was significantly higher than that of the control group intracellular Ca2+, this study observed the effect of nsPEF on
(P<0.01), and that the early apoptosis rate of the treatment SKOV3 [Ca2+]i when extracellular suspension did and did not
group was much higher than its total rate of late apoptosis and contain calcium, respectively. There may come two results: 1)
necrosis (P<0.01). This study detected the specific if Ca2+ is only from the influx of extracellular calcium, or
transforms of apoptotic cells according to morphological and from both the influx of extracellular calcium and the release
ultrastructure changes. Typical apoptotic cells have been of intracellular calcium, the fluorescent intensity of calcium
observed by the fluorescent, scanning electron microscopes. ion may intensify with the increased concentration of
After nsPEF treatment, SKOV3 cancer cells were in the extracellular CaCl2; 2) if Ca2+ is only from the release of
change from early to middle apoptosis. In summary, these intracellular calcium, the fluorescent intensity of calcium ion
experiments quantitatively and qualitatively indicate that the may be independent of the concentration of extracellular
nsPEF (10kV/cm, 100ns) can induce apoptosis of SKOV3 CaCl2. The results showed that there was no significant
cancer cell. difference (P>0.05) in fluorescent intensity between the
Then how does nsPEF induce apoptosis of cancer cells? group only applying nsPEF and that combing nsPEF with
Considering that Ca2+ is a second cell messenger that CaCl2. It suggests that when exposed to nsPEF, the increase
participates in inducing cell apoptosis, the LSCM was used to in [Ca2+]i might only originate from the release of
observe the effect of nsPEF on Ca2+ concentration inside the intracellular calcium stores. The results show that one of the
SKOV3 cancer cell. Increase in intracellular [Ca2+]i can take mechanisms of nsPEF-induced apoptosis may be that
part in inducing cell apoptosis in the following ways: 1) activating intracellular calcium stores can increase the [Ca2+]i
activating protein kinase and/or phosphorylase depended by and induce the apoptotic signal pathway
Ca2+ can activate many apoptosis related genes; 2) activating According to bioelectricity characteristics of each part, a
caspase can lead to cracking of cell frame protein and laminin; cell can be regarded as conductive cytoplasm and organelle
3) activating endogenous endonuclease can result in the DNA (equivalent to resistance approximately), which are
specific fracture and the formation of the characteristic DNA surrounded by dielectric outer and inner membrane
fragment; 4) activating the enzyme that keeps symmetry of (equivalent to capacitance approximately). Considering the
plasma membrane phospholipids. The PS externalization and equivalent capacitance of cell membrane, nsPEF can
exposure is a Ca2 dependent process. Fluo-3/AM, a penetrate cell interior due to its high frequency components,
Ç

fluorescent marker for calcium, can enter into cell interior leading to a series of functional changes in mitochondria,
and combine with intracellular free Ca2+. Therefore, endoplasmic reticulum, nucleolus and cell signal transfer (e.g.
fluorescence measured by LSCM can reflect the level and the activation of intracellular calcium stores). This may be
distribution of Ca2+. This study found that [Ca2+]i of SKOV3 one of the bioelectric mechanisms of how nsPEF affects
cell in the treatment group increased significantly, and that SKOV3 cancer cells and then induces the cell apoptosis.
the intracellular [Ca2+]i fluorescence is non-uniformly
distributed, with a few cells’ interior exhibiting spot strong V. CONCLUSION
fluorescence. All these suggest that nsPEF affects the nsPEF can induce cell apoptosis, and consequently, wound
concentration and distribution of intracellular [Ca2+]i. and kill cancer cells. Compared with EPT, nsPEF can kill
Since the extremely short duration of nsPEF does not meet cancer cells without aids of toxic drug. Therefore, nsPEF can
the membrane opening time (microseconds) required for make up deficiencies of surgery, radiotherapy and
electroporation of cell membrane, Ca2+ cannot enter into the chemotherapy, and may be a promising new approach for
cell interior through the membrane pores. Therefore, under cancer therapy. Presently, researches on mechanism of
the condition of intact cell membrane, there are two sources nsPEF-induced apoptosis and relevant animal experiments
of increased Ca2+: one is the influx of extracellular Ca2+, are still ongoing and further studies are needed.
mainly resulting from the opening of membrane calcium
channel; the other is the Ca2+ released from intracellular
calcium stores. In order to study the source of increased
for DNA electrotransfer for gene therapy, Biochimicaet Biophysica
Acta. 1519(2000) 73-83.
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