differential responses of human liver cancer and normal cells to atmospheric pressure...
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Differential responses of human liver cancer and normal cells to atmosphericpressure plasma
Bomi Gweon,1 Mina Kim,2 Dan Bee Kim,1,a) Daeyeon Kim,2 Hyeonyu Kim,2 Heesoo Jung,1
Jennifer H. Shin,2,3,b) and Wonho Choe1,b)
1Department of Physics, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu,Daejeon 305-701, South Korea2Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology,291 Daehak-ro, Yuseong-gu, Daejeon 305-701, South Korea3Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology,291 Daehak-ro, Yuseong-gu, Daejeon 305-701, South Korea
(Received 8 March 2011; accepted 22 May 2011; published online 10 August 2011)
When treated by atmospheric pressure plasma, human liver cancer cells (SK-HEP-1) and normal
cells (THLE-2) exhibited distinctive cellular responses, especially in relation to their adhesion
behavior. We discovered the critical threshold voltage of 950 V, biased at the electrode of the
micro-plasma jet source, above which SK-HEP-1 started to detach from the substrate while THLE-
2 remained intact. Our mechanical and biochemical analyses confirmed the presence of intrinsic
differences in the adhesion properties between the cancer and the normal liver cells, which provide
a clue to the differential detachment characteristics of cancer and normal cells to the atmospheric
pressure plasma. VC 2011 American Institute of Physics. [doi:10.1063/1.3622631]
Atmospheric pressure plasma (APP) has been suggested
as a biomedical tool for its simplicity in application and its
capability to generate abundant chemically reactive spe-
cies.1–3 While sterilization of medical devices and removal/
prevention of biofilm have been widely reported as the repre-
sentative applications of atmospheric pressure plasma,1–4
recent attempts have been made on the direct application of
plasma to wounded skin for enhancement of cellular healing
process5,6 and to dental cavities for sterilizing the infected
tissues.7 Recently, in vitro studies on a few mesenchymal
and epithelial cell types have indicated that APP induces cel-
lular necrosis, apoptosis, and detachment, suggesting its
potential use in cancer cell removal.8–11 However, induction
of such cellular changes would be meaningful in cancer ther-
apy practices if and only if removal was selective to cancer
cells leaving the normal cells unaffected.9 This requires a
parallel comparison of cancer and normal cells from the
same tissue under the controlled plasma condition. There-
fore, in the present study, the responses of cancer and normal
cells to the plasma treatment are compared to discover the
behavioral difference between two cell types in their detach-
ment characteristics from the substrate. We hypothesized
that either physical or bio-chemical stresses were responsible
for the plasma-induced changes in cells and performed sets
of experiments to elucidate the mechanism behind the dis-
tinct response between cancer and normal cells [Figs. 1(a)–
1(c)]. For instance, physical shear stress imposed by the gas
flow, as shown in Fig. 1(b), could possibly deliver a shearing
force to the adherent cells to peel them off from the sub-
strate. In addition, bio-chemical stress induced by reactive
oxygen species (ROS) can inflict damage on intracellular
proteins as well as surface proteins such as those involved in
focal adhesions (FAs), leading to the detachment of the cells
[Fig. 1(c)].
For the plasma applicator, we used a single pin electrode
type micro jet plasma, whose detailed description is reported
elsewhere12 and the additional information is provided in the
supplementary material.13
To study the effects of plasma on cellular viability, we
stained cells using live/dead assay13 following a brief expo-
sure of cells to APP (950-1200 V applied voltage, 50 kHz
driving frequency, 2 min treatment time with 2 slpm Helium
gas) and noticed the presence of differential responses
between two cell types. As shown in Figs. 2(a)–2(d), cells
begin to detach from the surface leaving a void above a criti-
cal voltage. At higher voltages, over 1000 V, three distinc-
tive regions are observed; the dead cell region at the center,
the live cell region at the periphery and the void region along
the interface between the live and dead zones [Figs. 2(c) and
2(d)]. The dead zone consists of necrotized cells, and the
void zone is the blank area of no cells, both of which collec-
tively refer to the plasma effective zone (PEZ) [Figs. 2(b)–
2(d)].
When the plasma above a critical voltage impinged on
the monolayer of the cells covered by phosphate buffered sa-
line solution, we observed cells starting to detach from the
FIG. 1. (Color online) The description of (a) an adherent cell to ECM
coated substrate through surface receptor protein integrins, (b) a cell detach-
ment forced by physical stress, and (c) by bio-chemical stress.
a)Present address: Korea Research Institute of Standards and Science, 267
Gajeong-ro, Yuseong-gu, Daejeon 305-340, South Koreab)Authors to whom correspondence should be addressed. Electronic
addresses: [email protected] and [email protected].
0003-6951/2011/99(6)/063701/3/$30.00 VC 2011 American Institute of Physics99, 063701-1
APPLIED PHYSICS LETTERS 99, 063701 (2011)
Author complimentary copy. Redistribution subject to AIP license or copyright, see http://apl.aip.org/apl/copyright.jsp
substrate along the periphery of the plasma edge while cells
at the center region became necrotized.14 At the lowest
applied voltage of 950 V, the liver metastatic cancer SK-
HEP-1 cells started to detach from the substrate as shown in
Fig. 2(b). Unlike the case for SK-HEP-1, the normal liver
THLE-2 cells remained intact at the same applied voltage of
950 V [Fig. 2(a)]. At the highest applied voltage of 1200 V,
SK-HEP-1 exhibited a much enlarged plasma effective zone
including both the dead and void zones as shown in Fig.
2(d). To quantify the distinctive response of these two differ-
ent cell types, we plotted the fraction of live cells as depicted
in Fig. 2(e). SK-HEP-1 cells, having a larger PEZ and a
smaller fraction of live cells, seem more susceptible to the
plasma than THLE-2 at all applied voltages. Also, the quan-
titative results in Fig. 2(f) clearly show that the SK-HEP-1
void zone is distinctively larger than that of the THLE-2
cells by more than 1.5 times, which led us to question the
possible existence of fundamental differences in the adhe-
sion strength and ability to respond to external disturbance
among different cell types.11,14
In our results with metastatic cancerous SK-HEP-1 and
normal THLE-2 cells, the important question was whether
the differential detachment characteristics arise from the
intrinsic distinction between cancer and normal cell types in
the liver epithelium. Based on our observations, we hypothe-
sized that SK-HEP-1 cells would have weaker adhesion
strength than THLE-2 cells resulting in easier detachment,
which would also be consistent with the characteristics of
highly motile metastatic cells compared to that of sessile
type normal cells. In this scenario, when certain plasma
properties, either physical or biochemical, trigger cells to
detach from the substrate, cancer cells would detach more
readily due to their weaker nature in adhesion. To investigate
which of the physical or biochemical stress would have a
dominant effect on cellular detachment, we performed a cou-
ple of experiments. Although our previous study on THLE-2
cells has shown that the gas flow alone does not inflict intra-
cellular changes,14 there still remains a possibility that the
drag force of the liquid medium induced by the impinging
gas flow can impose physical shear stress on the adherent
cells. To test the possibility of functional dominancy of
physical shear stress in the plasma induced detachment of
cells, we designed a set of experiments using commercial
micro-channels (l-Slide VI0.4 ibidi GmbH) and investigated
whether SK-HEP-1 and THLE-2 responded differentially to
shear stress.13
The results shown in Fig. 3(a) indicate no significant dif-
ference in the adhesion strength against physical shear stress
between SK-HEP-1 and THLE-2 in all tested magnitudes
from 10 to 40 dyn/cm2. The critical shear stress at which
only 50% of the cells remain attached reflects the physical
adhesion strength of each cell. In our case, the critical shear
stresses for SK-HEP-1 and THLE-2 were measured to be
25.62 dyn/cm2 and 25.87 dyn/cm2. Since our shear experi-
ment confirms that the difference in cellular detachment
characteristics induced by physical shear stress is insignifi-
cant between the two cell types, we conclude that the
observed differential responses in SK-HEP-1 and THLE-2
upon plasma treatment are unlikely due to physical shearing
imposed by the gas flow.
The next candidate for cell detachment is the biochemi-
cal stresses from various plasma species. Since the chemical
species in the plasma have been shown to play critical roles
to induce changes in intracellular cytoskeletal proteins as
well as surface adhesion proteins,8,11,14 we designed a set of
experiments to test the susceptibility of cells to biochemical
stresses causing them to detach from the surface. Because
the chemical species originated from the plasma are likely to
alter membrane proteins causing cellular detachment in such
a short term treatment of 5 min, the detachment experiment
due to biochemical stress was limited to the digestion of
integrins serving as the linkage between the extracellular ma-
trix (ECM) on the substrate and the cells. To realize the
appropriate experimental conditions, we treated the cells
with trypsin-ethylenediaminetetraacetic acid (EDTA), which
is the most well known clipper of biotic anchors including
integrins. As shown in Figs. 3(b) and S1(c), when cells were
FIG. 2. (Color online) (a)-(d) The live (green)/dead (red) assayed samples
after plasma treatment. THLE-2 treated at the applied voltage of (a) 950 V
and (c) 1200 V and SK-HEP-1 treated at (b) 950 V and (d) 1200 V, respec-
tively (white circle: PEZ). (e) Proportion of live cells of SK-HEP-1 and
THLE-2 after plasma treatment where No is number of live cells of control
sample and N is number of live cells of plasma treated sample. (f) Areal pro-
portion of void for SK-HEP-1 (-�-) and THLE-2 (-*-) formed by plasma
treatment: Ao is total area from plasma treated sample, and A is void area
from plasma treated sample. “IMAGE J” software was used.
FIG. 3. (Color online) Red (right column) for SK-HEP-1 and blue (left col-
umn) for THLE-2 (a) Fraction of adherent cells brought about by different
shear force. (b) Schematic of de-adhesion process: time interval between i to
ii is s1, which represents the biological adhesion strength, and time interval
between ii to iii is s2, which reflects the elasticity of the cell. (c) De-adhesion
time s1 of SK-HEP-1 and THLE-2 at the biochemical stress condition.
063701-2 Gweon et al. Appl. Phys. Lett. 99, 063701 (2011)
Author complimentary copy. Redistribution subject to AIP license or copyright, see http://apl.aip.org/apl/copyright.jsp
treated with this enzyme, cells started to round up (i-ii with
s1) by decreasing the contact area between the cell and the
substrate and finally get detached by contractility (ii-iii with
s2). The de-adhesion time s1 was obtained by plotting the
cell’s areal changes over time using the following sigmoid
function [Figs. S3(a)–S3(c), Ref. 15]: Areanormalized
¼ 1� 1=½1þ expððt� T1Þ=T2Þ�:Here, s1 represents the adhesion strength of the cell rela-
tive to the substrate. The longer the s1, the higher the adhesion
strength while s2 characterizes the elasticity (or contractility)
of the cell.15 In our study, s1 can be considered as the toler-
ance for cells to biochemical stress inflicted by trypsin-EDTA.
As shown in Fig. 3(c), cancerous SK-HEP-1 featured much
shorter detachment time s1 than normal THLE-2 cells. The
differential de-adhesion time s1 between the two cell types is
consistent with our findings that SK-HEP-1 is more suscepti-
ble to plasma exposure than THLE-2, creating larger void
area [Fig. 2(g)]. These distinctive de-adhesion properties may
have originated from the difference in intrinsic properties of
the FA. Consistent to these results, the immunofluorescence
images indicate stronger and more pronounced paxillin dots in
the THLE-2 cells when compared to those in SK-HEP-1
[Figs. 4(a) and 4(b)], implying more stable FA in normal
THLE-2 than cancerous SK-HEP-1. From the confocal
images, only the paxillin dots, whose long axis is larger than 2
lm and the ends are connected to actin stress fibers, are selec-
tively counted as mature FA and their lengths are measured.
As shown in Fig. 4(c), we find that there exists almost twice
the number of mature FA in a single THLE-2 cell than those
in a single SK-HEP-1 cell averaged over 30 cells in 10 differ-
ent images. Interestingly, the distribution of the lengths of
focal dots is similar in both cell types except that only THLE-
2 cells feature very large FA whose length is longer than 7 lm
[Fig. 4(d)], possibly indicating more mature and stronger
FA.16 To test whether these phenotypic differences in FA cor-
relate with their activity, western blot assay was performed
for focal adhesion kinase (FAK) and a5 integrin protein [Figs.
S4(a) and S4(b)]. As shown in Fig. S4(a), more FAK proteins
exist in THLE-2 than in SK-HEP-1. Moreover, the quantita-
tive value of a5 integrin protein, which is a part of FA, consis-
tently reveals that THLE-2 has stronger coupling to the ECM
by showing more a5 integrins on THLE-2 [Fig. S4(b)]. These
results from liver cells are consistent with our aforementioned
hypothesis, where the presence of intrinsic difference in adhe-
sion properties between cancer and normal cells leads to dif-
ferential detachment behavior upon application of the
atmospheric pressure plasma, and the detachment is achieved
by biochemical disruption of anchorage proteins rather than
physical tearing off from the substrate.
To support our hypothesis, the similar experiments
were performed on another set of cancer and normal cells
from the mammary gland epithelium (MDA-MB-231 vs.
MCF10A) [Figs. S5–S7]. These two cells also exhibited
consistent detachment characteristics where the void zone
of cancer cells formed after plasma treatment was larger
than that of normal cells [Fig. S5(b)].
In summary, we found that the cancer cells (SK-HEP-1)
detached more readily compared to normal cells (THLE-2)
upon a short treatment by atmospheric plasma. Based on the
results from the biophysical and biochemical assays, SK-
HEP-1 was found to feature weaker adhesion strength than
THLE-2, exhibiting different responses against plasma treat-
ment, which seems to inflict biochemical stress to disrupt the
integrin anchorage of cells at the cell-ECM interface. This
difference in cellular adhesion property between two cell
types attributes to the intrinsic physiological difference of
focal adhesions between the two cancer and normal cells in
hepatocytes.
The authors thank Wonjong Song, Sunghyun Kim,
Unghyun Ko Sukhyun Song, Se Youn Moon, and Sunhee
Kim for their initial contribution in experiments. This work
was in part supported by KAIST.
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FIG. 4. (Color online) Immunofluorescence image of paxillin dots (green)
and actin stress fibers (red) of (a) THLE-2 and (b) SK-HEP-1 (scale bar¼ 20
lm). Insets in (a) and (b) show the z axis-sectioned confocal microscopy
images (scale bar¼ 10 lm). (c) Number of FA of THLE-2 and SK-HEP-1
obtained by paxillin dots from the confocal images. (d) Distribution of focal
adhesion dots of THLE-2 and SK-HEP-1.
063701-3 Gweon et al. Appl. Phys. Lett. 99, 063701 (2011)
Author complimentary copy. Redistribution subject to AIP license or copyright, see http://apl.aip.org/apl/copyright.jsp