Differential responses of human liver cancer and normal cells to ...

APPLIED PHYSICS LETTERS 99, 063701 (2011)

Differential responses of human liver cancer and normal cells to atmospheric pressure 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)

1 Department of Physics, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, South Korea 2 Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, South Korea 3 Department 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 THLE2 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 C 2011 American Institute of Physics. [doi:10.1063/1.3622631] pressure plasma. V

Atmospheric pressure plasma (APP) has been suggested as a biomedical tool for its simplicity in application and its capability to generate abundant chemically reactive species.1–3 While sterilization of medical devices and removal/ prevention of biofilm have been widely reported as the representative 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 cellular 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 therapy 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. Therefore, 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 detachment 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 distinct 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 substrate. 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 exposure 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 critical voltage. At higher voltages, over 1000 V, three distinctive 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 collectively 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 saline solution, we observed cells starting to detach from the

a)

Present address: Korea Research Institute of Standards and Science, 267 Gajeong-ro, Yuseong-gu, Daejeon 305-340, South Korea b) Authors to whom correspondence should be addressed. Electronic addresses: [email protected] and [email protected]. 0003-6951/2011/99(6)/063701/3/$30.00

FIG. 1. (Color online) The description of (a) an adherent cell to ECM coated substrate through surface receptor protein integrins, (b) a cell detachment forced by physical stress, and (c) by bio-chemical stress.

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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, respectively (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 proportion 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.

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 SKHEP-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 different 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 quantitative 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 adhesion 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 hypothesized 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 couple of experiments. Although our previous study on THLE-2 cells has shown that the gas flow alone does not inflict intracellular changes,14 there still remains a possibility that the drag force of the liquid medium induced by the impinging

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FIG. 3. (Color online) Red (right column) for SK-HEP-1 and blue (left column) 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.

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 difference 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 experiment confirms that the difference in cellular detachment characteristics induced by physical shear stress is insignificant 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 biochemical 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 matrix (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


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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 bar ¼ 20 lm). Insets in (a) and (b) show the z axis-sectioned confocal microscopy images (scale bar ¼ 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.

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=½1 þ expððt T1 Þ=T2 Þ: Here, s1 represents the adhesion strength of the cell relative 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 tolerance 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 susceptible 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 selectively 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 different images. Interestingly, the distribution of the lengths of focal dots is similar in both cell types except that only THLE2 cells feature very large FA whose length is longer than 7 lm [Fig. 4(d)], possibly indicating more mature and stronger

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FA.16 To test whether these phenotypic differences in FA correlate 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 quantitative value of a5 integrin protein, which is a part of FA, consistently 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 adhesion properties between cancer and normal cells leads to differential 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, SKHEP-1 was found to feature weaker adhesion strength than THLE-2, exhibiting different responses against plasma treatment, 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. 1

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