Preferential induction of apoptotic cell death in melanoma cells as ...

research paper

Research paper

Cancer Biology & Therapy 13:13, 1299–1306; November 2012; © 2012 Landes Bioscience

Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch Shoshanna N. Zucker,1,2,* Jennifer Zirnheld,3 Archis Bagati,1 Thomas M. DiSanto,3 Benjamin Des Soye,1 Joseph A. Wawrzyniak,2 Kasra Etemadi,3 Mikhail Nikiforov2 and Ronald Berezney1 Department of Biological Sciences; University at Buffalo; Buffalo, NY USA; 2Department of Cell Stress Biology; Roswell Park Cancer Institute; Buffalo, NY USA; 3 Department of Electrical Engineering; University at Buffalo; Buffalo, NY USA

Keywords: plasma, melanoma, keratinocytes, apoptosis, plasma torch, gap junctions, bystander effect Abbreviations: NTP, non-thermal plasma; GJ, gap junctions; DBD, dielectric barrier discharge

Selective induction of apoptosis in melanoma cells is optimal for therapeutic development. To achieve this goal, a nonthermal helium plasma torch was modified for use on cultured cells in a temperature-controlled environment. Melanoma cells were targeted with this torch (1) in parallel cultures with keratinocytes, (2) in co-culture with keratinocytes and (3) in a soft agar matrix. Melanoma cells displayed high sensitivity to reactive oxygen species generated by the torch and showed a 6-fold increase in cell death compared with keratinocytes. The extent of cell death was compared between melanoma cells and normal human keratinocytes in both short-term (5 min) co-culture experiments and longer assessments of apoptotic cell death (18–24 h). Following a 10 sec plasma exposure there was a 4.9-fold increase in the cell death of melanoma vs. keratinocytes as measured after 24 h at the target site of the plasma beam. When the treatment time was increased to 30 sec, a 98% cell death was reported for melanoma cells, which was 6-fold greater than the extent of cell death in keratinocytes. Our observations further indicate that this preferential cell death is largely due to apoptosis. In addition, we report that this non-thermal plasma torch kills melanoma cells growing in soft agar, suggesting that the plasma torch is capable of inducing melanoma cell death in 3D settings. We demonstrate that the presence of gap junctions may increase the area of cell death, likely due to the “bystander effect” of passing apoptotic signals between cells. Our findings provide a basis for further development of this non-invasive plasma torch as a potential treatment for melanoma.

Introduction In recent years, atmospheric pressure non-thermal plasmas have extensively been deployed in various medical applications.1 Currently, this type of plasma is the focus of investigations for its potential role in wound healing,2 tissue incision,3 protein destruction, cell and tissue modification,4 bacterial inactivation5 as well as cancer treatment.6,7 Non-thermal plasma (NTP) is an ionized gas with electron densities of 1011–1014 (1/cm3) and typical energies of 1–5 (eV). Despite its high energy, NTP exhibits near room temperature characteristics due to the low number density of electrons. This makes the device suitable for biological applications such as cell and tissue processing. The plasma torch is also a good source of reactive species which have been shown to induce apoptosis.8 Recent studies have shown that NTP can induce apoptosis to a limited extent.9 However, the device that is currently being tested for melanoma treatment is dielectric barrier discharge (DBD). DBD requires that the target tissue be placed between two metal plates that are used as classical type electrodes.9,10

In an attempt to improve upon the versatility of the NTP device and to aim to achieve a higher level of apoptosis induction, we developed a NTP device utilizing a plasma torch to induce reactive oxygen species (ROS). The atmospheric pressure nonthermal helium plasma torch that we utilize was based on the model by Stoffels et al.11 and has been modified by our group as previously described.12 In our plasma torch, the torch itself forms the first electrode and the second electrode is the target material (or whatever the plasma is interacting with—fluid, cells, etc.) or simply the atmosphere in the case of when the plasma jet is not interacting with a target. The primary advantage of using a plasma torch is the enhanced versatility for practical applications such as the potential to treat tumors virtually anywhere on the skin and possibly for the development of internal measuring devices. To our knowledge, we are the only group using the plasma torch (as opposed to other NTP tools) to target cancer cells. This study compares the extent of apoptosis induced in cultured melanoma cells to co-cultured skin keratinocytes. Melanoma is the most rapidly increasing malignancy in

*Correspondence to: Shoshanna N. Zucker; Email: [email protected] Submitted: 06/07/12; Revised: 07/16/12; Accepted: 08/08/12 http://dx.doi.org/10.4161/cbt.21787 www.landesbioscience.com

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©2012 Landes Bioscience. Do not distribute

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Results

Figure 1. Plasma torch and selective killing effects. (A) The plasma torch directly treating cells in culture on an inverted microscope. (B) A scanned image of the markings that were stamped on the cell culture plate to assay the target site (a), adjacent site (b), and distal site (c). (C) The two torches utilized in these experiments (a) Figures 1 and 2 and (b) Figure 4. (D) Images of 1205Lu melanoma cells following stable transfection and cell sorting. We measured 100% transfection efficiency, (a) phase contrast and (b) fluorescence microscopy. (E) Co-cultures of 1205Lu melanoma cells expressing GFP and keratinocytes seeded at a 1:25 ratio. Propidium iodide (stains dead cells in red) and Hoechst 33342 (labels DNA in cell nuclei blue) were added just prior to treatment. The GFP-expressing melanoma cell is shown within the red circle and demarcated with a white arrow (enlarged in box inset). The images were taken at time points indicated (a) untreated, (b) after 1 min post plasma treatment, and (c) after 5 min post-plasma treatment. These experiments were repeated several times with consistent results.

the United States, accounting for 75% of all skin cancer associated deaths.13 An estimated 160,000 new cases of melanoma are diagnosed worldwide each year, resulting in approximately 48,000 melanoma related deaths.14 Melanoma progresses from the benign nevus, to the radial growth phase (RGP), to the vertical growth phase (VGP), to metastasis. Survival rates in patients with VGP melanoma are directly related to the vertical diameter of the tumor.15 Despite many years of intensive laboratory and clinical research and new drugs that extend survival,16 the sole effective cure is surgical resection of the primary tumor before it achieves a Breslow thickness greater than 1 mm.17 Although surgical excision is still the best way to remove primary melanoma, there is still a need to target those cells that escape surgical resection. In a recent study of melanoma patients at stage II and stage III, the percent recurrence at the primary site was greater than 10%.18 Although the exact mechanisms underlying melanoma recurrence are not known, the escape of even a few cells from the surgical treatment could contribute to the resurgence of the

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Direct imaging of cells with the NTP torch. In an attempt to improve the existing plasma treatment of melanoma cells, we modified a non-thermal plasma (NTP) torch. The original plasma torch design has been outlined previously12 and generates NTP using two different models, the first design based on Stoffels model.11 In this study, the size of the torch was reduced enabling direct treatment of the cells under a temperature-controlled inverted microscope. The plasma beam was directly visualized on the computer monitor, showing that it made contact with the culture dish at the intended site. An image of the plasma torch treating cells in culture can be seen in Figure 1A. The automated microscopic stage enabled us to scan the plate and return to the same (x, y) coordinates for the “target site,” the “adjacent site,” and the “distal site” (Fig. 1B). For all experimental analysis, images of the cells were taken before plasma treatment at the three sites. For short-term experiments (up to 30 min), we continuously imaged through the three sites to determine cell death by staining with propidium iodide which was added prior to the pretreatment imaging. Since the plasma consistently showed no effect at the distal site, this region was used as an internal experimental control. Figure 1C shows the torch designs utilized for this study, which emphasizes the utility of the smaller design to accommodate direct microscopy treatment. Our initial experiments (Figs. 1E and 2) were performed with torch model 1 at a frequency of 112–117 kHz with a flow rate of 3–4.7 L/min and a minimal fluid level of media in the dish (0.2 ml) and a treatment time of 10 sec. All experiments in Figures 3–6 were performed with the second torch at 87 kHz with a 3 L/min flow rate and 1 mm of media. Although these torch models had distinct properties, in both cases we found similarly increased rates of cell death for the melanoma cells as compared with human epidermal keratinocytes (HEK). Increased cell death in melanoma cells as compared with keratinocytes. Our earlier preliminary analysis of short-term effects in separate culture dishes, suggested a preferential effect of plasma treatment on the killing of melanoma 1205Lu vs. normal human keratinocytes cells.12 To more rigorously evaluate the apparent differential effect of the plasma treatment on


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disease. Thus, there is a clinical need to develop better therapy for both primary and recurrent melanoma. In an attempt to increase the area that the plasma beam can target, the effect of gap junctions was considered. Melanoma cells usually have reduced expression of gap junctions which transfer molecules up to 1 kDa between cells.19 The “bystander effect” is the promotion of cell death through the passage of apoptosis-inducing signals through gap junctions.20 The bystander effect has been reported for UV radiation,21 photodynamic therapy,22 heat,23 chemotherapy agents24 and radiation.20 In this report we demonstrate that gap junctions may also play a role in promoting cell death in melanoma cells by NTP.

cell death, we used co-cultures of GFP-expressing melanoma cells with HEK cells that did not express fluorescent markers (Fig. 1D). In this way both cell types are exposed to the same treatment conditions. As shown in Figure 1E, the selective killing of melanoma cells can be observed after 5 min of exposure to NTP. This selective killing effect demonstrates the potential of the plasma for targeting melanoma cells that are surrounded by keratinocytes. Following a 10 sec plasma treatment and 15 min incubation, the preferential targeting of the melanoma cells (59%) compared with the keratinocytes (8%) was measured (Table 1). Examination of the extent of cell death indicated that cell death was limited to the target site with no significant cell death in the adjacent site or the distal site under any of the plasma conditions utilized thus far. Figure 4A, panel e shows the border between treated and untreated cells where cells above the white line are untreated. In order to compare the effects of pre-metastatic to metastatic melanoma, we utilized paired cell lines (1) WM793B which was derived from a vertical growth phase pre-metastatic melanoma and (2) 1205Lu which was derived from a lung metastasis of WM793B from the same patient. We assessed the comparative effects of the plasma torch on WM793B and 1205Lu in Figure 2A. While the plasma torch demonstrated effectiveness in both cell lines, we repeatedly observed more extensive cell death with the metastatic cell line, 1205Lu.

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Figure 3. Plasma induced cell surface membrane blebbing and apoptosis. (A) 1205Lu cell untreated (a) or 15 min after 10 sec plasma treatment (b), where membrane surface blebbing is readily observed (red arrows). (B) Untreated (a) and plasma treated (c) melanoma cells along with untreated (b) and treated (d) keratinocytes were stained with the TUNEL assay 18 h after plasma treatment to identify fragmented DNA diagnostic for apoptotic cells. Background non-specific red nucleolar staining was observed in keratinocytes. (C) Percent apoptotic cells in melanoma vs. HEK cells. Results are plotted for a representative experiment counting triplicate samples with greater than 150 cells per cell line (p = 0.00076).

A previous study 9 reported that plasma-induced free radicals affect the pH of the media and that the resulting acidity in the culture media may contribute to the induction of cell death. In order to reduce the possibility of generating this pH effect, we maintained the cells in Epilife media with 20 mM HEPES as a buffering agent. Measurement of the pH over a time course of plasma treatment up to 180 sec confirmed that the pH remained constant at 7.3 (Fig. 2B). The plasma torch induces apoptosis in melanoma cells. While the mechanism of cell death observed in these studies could be necrosis rather than apoptosis, which can occur within the short time frame of 5 min,25 we also investigated whether


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Figure 2. A comparison of the cytotoxic effects of NTP on premetastatic and metastatic melanoma under stable pH conditions. (A) WM793B and 1205Lu cells expressing the GFP vector were subjected to treatment with NTP for 10 sec and a time course in cell death was followed up to 17 min at the target site [p = 0.0006 (1.5 min), 0.0340 (7 min), 0.0920 (17 min)]. (B) The pH remained constant at 7.3 during the time course following NTP in 2A and in subsequent experiments.

Table 1. Summary of comparative cell death of melanoma cells and HEK by NTP Time post plasma treatment

Percent melanoma cell death

Percent HEK cell death

Percent melanoma membrane blebbing

Percent HEK membrane blebbing

15 min (10s NTP)

59.5 ± 6.40

8.3 ± 0.23

> 80%

< 5%

18 h (10s NTP)

90.0 ± 14.1

19.9 ± 6.20

NA

NA

24 h (10s NTP)

88.3 ± 3.00

18.3 ± 6.30

NA

NA

24 h (30s NTP)

97.9 ± 0.25

16.4 ± 0.13

NA

NA

Figure 4. Cell death following plasma treatment. (A) Identical phase contrast microscope fields for (a) untreated, (c) 10 sec plasma treated keratinocytes, (b) untreated and (d) 10 sec plasma treated melanoma cells after 24 h. The demarcation between treated and untreated cells is shown as a white line in (e). (B) These results are from a representative experiment with duplicate plates averaging 500–700 cells per plate prior to plasma treatment. *10 sec p = 0.0054), **30 sec p = 6 × 10 -6.

non-thermal plasma could selectively induce apoptotic cell death as previously reported.9,10 Fifteen minutes following a 10 sec treatment with plasma in melanoma cell cultures, induced extensions

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of the cell surface (Fig. 3A) resembling membrane blebbing (see red arrows), which is an early indication of apoptosis.26 This effect was only observed on melanoma cells and not keratinocytes. Scoring of the cells revealed blebs on > 80% of the melanoma cells with < 5% on normal keratinocytes (Table 1). Prompted by these observations, a TUNEL assay was performed to assess apoptosis 18 h after plasma treatment in cocultured melanoma and HEK cells. Multiple foci of fragmented DNA were observed in plasma treated but not control melanoma cells (Fig. 3B, panels a, c, red staining), The red staining of the nucleoli in the control and plasma treated keratinocytes (Fig. 3B, panels b and d) is due to non-specific staining with the secondary antibody (data not shown). Based on this assay, the percent of apoptotic cells was ~90% for 1205Lu melanoma cells and ~20% for HEK cells (Fig. 3C). We, therefore, conclude that the plasma treatment preferentially induces apoptosis in the melanoma cells. As a first step toward evaluating the potential value of this approach for cancer treatment, we investigated the long-term effects of NTP exposure on melanoma cells. The 1205Lu or HEK cells were treated with either a 10 or 30 sec continuous pulse of plasma. Images of the target site at 18 h after a 10 sec treatment are presented in Figure 4A. Both conditions showed a significant difference in cell death between the melanoma cells and the keratinocytes. However, while the effects were striking for the 10 sec treatment (88 vs. 18%), there was almost complete killing of the melanoma cells after a 30 sec treatment (98% vs. 16%) (Fig. 4B). We conclude that the increased time of plasma treatment resulted in increased cell death of the melanoma cells, whereas the HEK cells show no significant change in this time course within the range of experimental error. Targeting melanoma cells grown in soft agar. During the course of our experimentation, we observed a greater extent of beam spread on solid surfaces compared with liquids and we documented those findings in Figure 5A which shows the increase in spreading and area of penetration through a solid surface (Fig. 5Ab) compared with the cell culture media (Fig. 5Aa). To further test whether this increase in area of treatment could have potential to eliminate cells in 3D settings, we assayed for the ability of the plasma torch to target melanoma cells embedded within a matrix of soft agar. Single cells were plated in the agar matrix and the amount of cell colonies was counted following 8 d. A decrease in colony formation of 2.7-fold was reported when the torch was held stationary and a decrease of 4.5-fold when the torch was rotated in concentric circles (Fig. 5B). The increased killing effect with the moving beam suggests that the target site


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*Each value is the percent ± SD.

Discussion Throughout our experimental analysis, we have performed modifications of the plasma torch and altered conditions such as the frequency, duration of treatment, and presence of media in the dish. It is interesting to note that while the experiments were conducted under different plasma torch designs and conditions, the effects were very similar. This illustrates the consistency of the melanoma cell killing effect. In addition, this is the first report that directly compares the killing effects of non-thermal plasma on metastatic melanoma cells to human keratinocytes in co-culture. It is particularly significant because the keratinocytes, which are only slightly affected by the plasma, constitute the primary component of the human epidermis.28 While the well-established uses for non-thermal plasma include wound healing2 and sterilization through selective killing of bacteria,5 recent uses for plasma as an antitumor agent are currently under investigation. A jet-type cold plasma causes cell death of liver cancer cells29 and extensive studies have been done with DBD plasma therapy on melanoma cells including direct treatment in mice whereby multiple rapid pulses are given to cause electrodeformation of the cells which opens pores and disrupts cellular membranes. This leads to 90% shrinkage of the tumor within 2 weeks.30,31 DBD has also been shown to cause apoptotic cell death with 25% apoptosis by 24 h and 72% following 3 d with cellular changes such as membrane blebbing.2 The NTP torch modified and used in this study has the advantage of increased versatility


Figure 5. NTP-induced melanoma cell death within a soft agar matrix. (A) Images of the plasma torch making contact with either (a) the surface of cell culture media (4 ml Epilife media in a 35 mm culture dish) or (b) on the surface of an empty culture dish. Both images were taken under identical torch conditions (frequency: 97 kHz; flow rate: 6.0 L/min). (B) Soft agar colony forming assay to compare the number of colonies in control, untreated wells to wells treated with a motionless beam for 30 sec as well as a beam that was moved in concentric circles for 30 sec. The results are from a representative experiment and were repeated under the same plasma conditions (flow rate of 5.8 L/min) as well as at higher flow rates up to 11 L/min with similar results. The Student’s t-test shows high significance between the control and the still (p = 4.6 x 10 -8) or moving beam (p = 1.4 × 10 -9) conditions as well as significance between the still vs. moving conditions (p = 0.00135).

and can be used on the surface of the skin. The small size of the torch could enable precise targeting of tumor cells. Direct use of the plasma torch in the temperature-controlled inverted microscope is ideal for treating and tracking cells in situ to observe killing effects throughout a time course for short-term experiments (Figs. 1 and 2)12 as well as for long-term experiments (Figs. 3 and 4). Moreover, our observations of membrane blebbing for short periods (e.g., 15 min) after plasma treatment (Fig. 2A), suggest that the predominant mode of cell death is likely to be apoptosis. This was confirmed by direct measurements of apoptosis at 18 h post-plasma treatment (Fig. 2C). Additionally, the increased total cell death after 18 h of 90% as compared with 60% after 15 min is consistent with the time course of apoptotic cell death. This finding bodes well for the future development of plasma treatment for cancer therapy, since apoptosis is characterized by a lack of an inflammation response in the surrounding tissues.32 It has previously been reported that non-thermal plasma generated by helium gas in a method similar to our experiments produces a total of 45 species including reactive oxygen species (ROS) and free radicals such as NO, H2O2 and OH.33 ROS may lead to apoptosis when the antioxidant capacity of the cells is surpassed, inducing alterations in cellular macromolecules: lipid peroxidation, DNA damage, and enzyme activation.8 Studies have shown that melanoma cells may have an increased susceptibility


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can be substantially expanded. The significant decrease in colony formation following a 30 sec treatment with the torch indicates that the plasma torch can penetrate at increased depth below the surface (of at least 5 mm). This finding is significant because it provides evidence that non-thermal plasma may be an effective tool for targeting tumor cells that may escape surgical resection. Gap junctions increase the target area of the plasma torch. In our studies the region in which the torch effectively induced cell death was limited to the target site with no extensive killing in the adjacent or distal regions. However, in order to test whether the expression of gap junctions may play a role the spreading of cell death signals, the expression was altered in melanoma cells. The bystander effect is the promotion of cell death through the passage of apoptosisinducing signals through gap junctions which has previously been reported under various conditions.20 The WM793B and 1205Lu melanoma cells were transfected with wild type (Cx43) or a dominant negative Cx43 (T154A).27 Following two rounds of selection, we obtained 100% GFP expressing cells as shown in 1D. The cells were further characterized to demonstrate GJIC in 60–70% of the wild type cells and less than 10% for the T154A expressing cells (unpublished data). Based on approximately 3-fold increase in cell death, our data demonstrates that we can increase the extent of cell spreading with wild type Cx43 as compared with the dominant negative (Fig. 2B). This could have important implications for expanding the target area of NTP.

to apoptotic cell death due to high intracellular levels of superoxide anion.34-36 These mechanisms are likely to explain the results demonstrated in our studies with a preferential killing of melanoma cells. Therefore, non-thermal plasma provides a means to utilize the propensity of melanoma cells for apoptotic cell death to achieve potential therapeutic treatment. By inducing apoptosis through plasma treatment, we may be able to develop a mechanistic based therapy that can selectively target melanoma cells with minimal effects on normal tissue. Many studies have demonstrated that apoptosis-inducing signals such as calcium and ATP can pass through gap junctions and promote cell death.37 In melanoma, the connexin protein, Cx43, has also been shown to be suppressed.38 Our studies demonstrate that induced expression of Cx43 may help to increase the target area of plasma therapy which may occur through the bystander effect. Possible future gene-therapy approaches may help to mediate the efficacy of the bystander effect. Further development of non-thermal plasma as a clinical tool may have future implications for both primary and recurrent melanoma therapy. Materials and Methods Plasma torch design. The plasma torch works by applying a large ac voltage to a fine-tip metal electrode which allows helium gas to be mixed with atmospheric gases. This helium-air mixture becomes ionized near the tip of the electrode due to the intensity of the electric field. The length of the plasma jet can vary depending on the flow rate of the helium working gas and ranges between less than 1 mm to over 7 mm. The plasma gas spreads out upon contact with the liquid or solid surface ranging from 2–30 mm2. Two different plasma torch designs (Fig. 1C) are described in detail12,39 (and in Supplemental Material). For torch 1 (Fig. 1C, panel a), the frequency was 112–117 kHz with a flow rate of 3–4.7 L/min and a minimal fluid level of media in the dish (0.2 ml) and a treatment time of 10 sec. Torch model 2 was 87 kHz with a 3 L/ min flow rate. However, due to the increase in chemical interactions with the tissue culture dish for torch 2 (Fig. 1C, panel b), we increased the level of fluid in the dish to 1 ml (1 mm depth).

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Figure 6. The area of cell death can be increased, through the passage of signals between gap junctions. Comparison of the cell death by NTP in WM793B cells expressing either a wild type or dominant negative Cx43 (T154A). p = 0.0058.

Cell culture. Premetastatic WM793B melanoma cells were utilized as well as a cell line established from a lung metastasis of these cells, 1205Lu. Both cell lines were obtained from the Herlyn laboratory.40 Primary human epidermal keratinocytes (HEK) were isolated from neonatal foreskins and obtained from Invitrogen. The HEK cells were used between passages 3 and 8 and maintained in Epilife media plus keratinocyte supplement (Invitrogen). 1205Lu cells were maintained in complete melanoma media (a 4:1 ratio of MDCB153 to L-15 (Sigma) with the addition of 2 mM CaCl2, 5 μg/ml insulin, 1% pen/strep, and 2% FBS). All experiments and cocultures were performed in Epilife media plus 20 mM Hepes. The parental cells were transfected with the pBMNIGFP construct (a kind gift of Dr Garry Nolan, Stanford University). This construct is a retroviral vector that links the gene of interest to GFP via an internal ribosome entry site (IRES) to assure that all GFP expressing cells have the target gene. Cell lines were also stably transfected with wild type connexin 43 (Cx43) or a dominant negative Cx43 (T154A), both in the pBMNIGFP vector. Briefly, the cells were transfected with lipofectamine and stable transfectants were generated by two rounds of cell sorting with a flow cytometer. Cells were routinely checked for 100% transfection efficiency as monitored for GFP expression. Cells were plated overnight at 3 × 105 cells per 35 mm tissue culture dish. The plasma beam targets an area with a 2 mm diameter. Assuming a plating density of approximately 80%, we estimate that approximately 800 cells were in the direct path of the plasma for each treatment and 240,000 cells were in the surrounding area on the surface of the 35 mm plate. Cells were either treated in the absence of media, which was replenished immediately after treatment (Figs. 1 and 2), or in the presence of 1 ml (1 mm depth) of media per dish throughout the duration of the experiment (Fig. 3). Prior to plasma treatment and in parallel control experiments, the media was replaced with Epilife media, containing 1.25 μg/ml propidium iodide (Sigma), to stain dead cells in red and 20 μM Hoechst 33342 dye (Sigma) to label the DNA of nuclei in blue. In addition, the melanoma cells can be distinguished by their green color due to their GFP expression. The pH was monitored at several time points post plasma treatment and was shown to remain constant at 7.3. Microscopy analysis and experimentation. Live and fixed cell imaging was performed on an Olympus 1X70 inverted epifluorescence microscope, equipped with a temperature-controlled stage incubator, a Cooke Sensicam CCD camera, and z-axis control. Optical sections of 0.5 microns were collected and stored digitally using Slidebook software (Intelligent Imaging Innovations, Inc.). Experiments were performed in duplicate using duplicate or triplicate samples per experimental condition and always included an additional plate as a control under identical experimental conditions without plasma treatment. When treating the cells in the tissue culture plates with plasma, the distance between the culture dish and the plasma torch was fixed at 2 mm from the bottom of the dish to the torch nozzle for all experiments. However, when media was present in the culture dish during treatment, the distance from the torch to the surface of the media was 1 mm, with a 1 mm depth of media. Cells were treated with plasma for 10 or 30 sec and then observed immediately under the inverted

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Anchorage-independent colony forming assay. Cells were plated at 104/well in a 6-well plate in semi-solid medium containing 0.3% agarose (2 ml) on a basal layer of medium containing 0.5% agarose (2 ml). The two layers were covered with media and grown 2–4 weeks or until visible colonies appeared. The plasma torch was applied close to the soft agar surface with a constant distance of 2–3 mm. Based on the surface area of the beam spreading to the solid surface area (approximately 28 mm2) as compared with the area of the dish (110 mm2), we can estimate that 25% of the colonies were in the direct target of the beam. This value was increased to approximately 100% of the colonies in the direct target of the beam when the beam was rotated in concentric circles. Colonies were counted for 10 random fields per well in triplicate wells. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

This work was supported in part by NIH grant GM 072131 awarded to R.B. Supplemental Materials

Supplemental materials may be found here: http://www.landesbioscience.com/journals/cbt/article/21787/

10. Sensenig R, Kalghatgi S, Cerchar E, Fridman G, Shereshevsky A, Torabi B, et al. Non-thermal plasma induces apoptosis in melanoma cells via production of intracellular reactive oxygen species. Ann Biomed Eng 2011; 39:674-87; PMID:21046465; http://dx.doi. org/10.1007/s10439-010-0197-x. 11. Stoffels E, Kieft IE, Sladek REJ, van den Bedem LJM, van der Laan EP, Steinbuch M. Plasma needle for in vivo medical treatment: recent developments and perspectives. Plasma Sources Sci Technol 2006; 15:S16980; http://dx.doi.org/10.1088/0963-0252/15/4/S03. 12. Zirnheld J, Zucker SN, DiSanto TM, Berezney R, Etemadi K. Non-Thermal Plasma Needle: Development and Targeting of Melanoma Cells. IEEE Trans Plasma Sci 2010; 38:948-52; http://dx.doi. org/10.1109/TPS.2010.2041470. 13. Lucas R, McMichael T, Armstrong BK, Smith W. Global burden of disease of solar ultraviolet radiation, Environmental burden of disease series. News Release, World Health Organization 2006; 13. 14. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005; 55:74-108; PMID:15761078; http://dx.doi.org/10.3322/canjclin.55.2.74. 15. Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted therapy. Nature 2007; 445:851-7; PMID:17314971; http://dx.doi. org/10.1038/nature05661. 16. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al.; BRIM-3 Study Group. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364:2507-16; PMID:21639808; http://dx.doi. org/10.1056/NEJMoa1103782. 17. Breslow A. Thickness, cross-sectional areas and depth of invasion in the prognosis of cutaneous melanoma. Ann Surg 1970; 172:902-8; PMID:5477666; http:// dx.doi.org/10.1097/00000658-197011000-00017.


18. Meyers MO, Yeh JJ, Frank J, Long P, Deal AM, Amos KD, et al. Method of detection of initial recurrence of stage II/III cutaneous melanoma: analysis of the utility of follow-up staging. Ann Surg Oncol 2009; 16:941-7; PMID:19101766; http://dx.doi.org/10.1245/s10434008-0238-y. 19. Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev 2003; 83:1359-400; PMID:14506308. 20. Prise KM, O’Sullivan JM. Radiation-induced bystander signalling in cancer therapy. Nat Rev Cancer 2009; 9:351-60; PMID:19377507; http://dx.doi. org/10.1038/nrc2603. 21. Bagdonas S, Dahle J, Kaalhus O, Moan J. Cooperative inactivation of cells in microcolonies treated with UVA radiation. Radiat Res 1999; 152:174-9; PMID:10409327; http://dx.doi. org/10.2307/3580091. 22. Dahle J, Bagdonas S, Kaalhus O, Olsen G, Steen HB, Moan J. The bystander effect in photodynamic inactivation of cells. Biochim Biophys Acta 2000; 1475:27380; PMID:10913826; http://dx.doi.org/10.1016/ S0304-4165(00)00077-5. 23. DeVeaux LCDL, Durtschi LS, Case JG, Wells DP. Bystander effects in unicellular organisms. Mutat Res 2006; 597:78-86; PMID:16413587; http://dx.doi. org/10.1016/j.mrfmmm.2005.06.033. 24. Alexandre J, Hu Y, Lu W, Pelicano H, Huang P. Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species. Cancer Res 2007; 67:3512-7; PMID:17440056; http://dx.doi. org/10.1158/0008-5472.CAN-06-3914. 25. Challa S, Chan FK-M. Going up in flames: necrotic cell injury and inflammatory diseases. Cell Mol Life Sci 2010; 67:3241-53; PMID:20532807; http://dx.doi. org/10.1007/s00018-010-0413-8.

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microscope for the initial 30 min at the site of treatment and adjacent sites using the automated return to fixed (x, y) coordinates in the Slideboo software. For longer term studies cells were imaged, treated with plasma, and stored overnight in the 37°C CO2 incubator before examination and imaging after 18 or 24 h. As an internal control for each plate, a stamp was used to demarcate the “target site,” “adjacent site” and “distal site.” Once verified that the plasma did not have an effect at the distal site, this region was used as an internal control and showed no significant increased cell death during the short-term time courses or the long-term experiments. Cell death was calculated by propidium iodide staining for short-term experiments (1–30 min after plasma treatment) and as cell number post- vs. pre-plasma treatment for long-term experiments (18 or 24 h after plasma treatment) using a Student’s t-test to determine the significance of the difference at p values of 0.001 and 0.01. Apoptosis assay. The TUNEL assay was performed using the ApopTag Red In Situ Apoptosis Detection kit (Chemicon) according to the manufacturer’s instructions. Cells were plated onto glass coverslips and fixed in ethanol:acetic acid, 2:1 (v:v) at 18 h after plasma treatment. The percent apoptotic cells were calculated by examining DNA fragmentation, stained in red. Experiments were performed in triplicate slides in duplicate experiments using the Student’s t-test to determine the significance as described above.

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31. Hu Q, Sridhara V, Joshi RP, Kolb JF, Schoenbach KH. Molecular Dynamics Analysis of High Electric Pulse Effects on Bilayer Membranes Containing DPPC and DPPS. IEEE Trans Plasma Sci 2006; 34:1405-11; http://dx.doi.org/10.1109/TPS.2006.876501. 32. Rock KL, Latz E, Ontiveros F, Kono H. The sterile inflammatory response. Annu Rev Immunol 2010; 28:321-42; PMID:20307211; http://dx.doi. org/10.1146/annurev-immunol-030409-101311. 33. Sakiyama Y, Grave DB. Neutral gas flow and ringshaped emission profile in non-thermal RF-excited plasma needle discharge at atmospheric pressure. Plasma Sources Sci Technol 2009; 18:25022-33; http:// dx.doi.org/10.1088/0963-0252/18/2/025022. 34. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 2006; 160:140; PMID:16430879; http://dx.doi.org/10.1016/j. cbi.2005.12.009. 35. Meyskens FL Jr., McNulty SE, Buckmeier JA, Tohidian NB, Spillane TJ, Kahlon RS, et al. Aberrant redox regulation in human metastatic melanoma cells compared to normal melanocytes. Free Radic Biol Med 2001; 31:799-808; PMID:11557318; http://dx.doi. org/10.1016/S0891-5849(01)00650-5.


36. Locatelli C, Leal PC, Yunes RA, Nunes RJ, CreczynskiPasa TB. Gallic acid ester derivatives induce apoptosis and cell adhesion inhibition in melanoma cells: The relationship between free radical generation, glutathione depletion and cell death. Chem Biol Interact 2009; 181:175-84; PMID:19577552; http://dx.doi. org/10.1016/j.cbi.2009.06.019. 37. Kandouz M, Batist G; M K. Gap junctions and connexins as therapeutic targets in cancer. Expert Opin Ther Targets 2010; 14:681-92; PMID:20446866; http://dx.doi.org/10.1517/14728222.2010.487866. 38. Haass NK, Smalley KSM, Herlyn M. The role of altered cell-cell communication in melanoma progression. J Mol Histol 2004; 35:309-18; PMID:15339050; http:// dx.doi.org/10.1023/B:HIJO.0000032362.35354.bb. 39. Zirnheld J, DiSanto TM, Burke KM, Zucker SN, Etemadi K. Development of an atmospheric pressure non-thermal plasma needle for melanoma research. IEEE Puls Pwr Conf 2009:1429-32. 40. Li G, Satyamoorthy K, Herlyn M. Dynamics of cell interactions and communications during melanoma development. Crit Rev Oral Biol Med 2002; 13:62-70; PMID:12097238; http://dx.doi. org/10.1177/154411130201300107.

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26. Nusbaum P, Lainé C, Seveau S, Lesavre P, HalbwachsMecarelli L. Early membrane events in polymorphonuclear cell (PMN) apoptosis: membrane blebbing and vesicle release, CD43 and CD16 down-regulation and phosphatidylserine externalization. Biochem Soc Trans 2004; 32:477-9; PMID:15157165; http://dx.doi. org/10.1042/BST0320477. 27. Beahm DL, Oshima A, Gaietta GM, Hand GM, Smock AE, Zucker SN, et al. Mutation of a conserved threonine in the third transmembrane helix of α- and β-connexins creates a dominant-negative closed gap junction channel. J Biol Chem 2006; 281:79948009; PMID:16407179; http://dx.doi.org/10.1074/ jbc.M506533200. 28. Proksch E, Brandner JM, Jensen JM. The skin: an indispensable barrier. Exp Dermatol 2008; 17:106372; PMID:19043850; http://dx.doi.org/10.1111/ j.1600-0625.2008.00786.x. 29. Kim DGB, Kim DB, Choe W, Shin JH. A feasiblility study for the cancer therapy using cold plasma. ICBME Proc 2008; 23:355-7. 30. Nuccitelli R, Pliquett U, Chen X, Ford W, James Swanson R, Beebe SJ, et al. Nanosecond pulsed electric fields cause melanomas to self-destruct. Biochem Biophys Res Commun 2006; 343:35160; PMID:16545779; http://dx.doi.org/10.1016/j. bbrc.2006.02.181.