Floating Electrode Dielectric Barrier Discharge Plasma in Air Promoting Apoptotic Behavior in Melanoma Skin Cancer Cell Lines
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- Fridman, G., Shereshevsky, A., Jost, M.M. et al. Plasma Chem Plasma Process (2007) 27: 163. doi:10.1007/s11090-007-9048-4
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Initiation of apoptosis, or programmed cell death, is an important issue in cancer treatment as cancer cells frequently have acquired the ability to block apoptosis and thus are more resistant to chemotherapeutic drugs. Targeted and perhaps selective destruction of cancerous tissue is desirable for many reasons, ranging from the enhancement of or aid to current medical methods to problems currently lacking a solution, i.e., lung cancer. Demonstrated in this publication is the inactivation (killing) of human Melanoma skin cancer cell lines, in vitro, by Floating Electrode Dielectric Barrier Discharge (FE-DBD) plasma. Not only are these cells shown to be killed immediately by high doses of plasma treatment, but low doses are shown to promote apoptotic behavior as detected by TUNEL staining and subsequent flow cytometry. It is shown that plasma acts on the cells directly and not by “poisoning” the solution surrounding the cells, even through a layer of such solution. Potential mechanisms of interaction of plasma with cells are discussed and further steps are proposed to develop an understanding of such systems.
KeywordsNon-thermal plasmaDielectric barrier discharges (DBDs)ApoptosisMelanoma cancer cellsCancer treatmentSkin diseases
Apoptosis, or programmed cell death, is a complex biochemical process of controlled self-destruction of a cell in a multicellular organism [1, 2]. This process plays an important role in maintaining tissue homeostasis, fetal development, immune cell “education”, development, and aging. Examples of apoptosis that occur during normal body processes include the formation of the outer layer of skin, the inner mucosal lining of the intestine, and the endometrial lining of the uterus, which is sloughed off during menstruation. During apoptosis, cellular macromolecules are digested into smaller fragments in a controlled fashion, and ultimately the cell collapses into smaller intact fragments that can be removed by phagocytosis without damaging the surrounding cells or causing inflammation. In contrast, during necrosis, also termed “accidental cell death”, the cell bursts and the cellular contents spill out into the extracellular space, which can cause inflammation. Necrosis is induced by cellular injury, for example, extreme changes in osmotic pressure or heat, that lead to adenosine tri-phosphate (ATP) depletion of the cell. With cancer cells, however, a problem arises with apoptosis as the tumor cells frequently “learn” how to turn off apoptosis as one of the processes they employ in evading the immune system and surviving under unfavorable conditions. For this reason, for example, chemotherapy as means of treatment of breast, colon, and lung cancer met limited success . In general, the employment of systemic chemotherapy drugs to induce apoptosis in cells that try to block it is not an easy task, as these drugs tend to affect all cells in the body [1, 2]. A way to target apoptosis development only in specific areas of the body is needed and a method to do so is offered in this paper where apoptotic behavior is promoted in Melanoma cancer cell lines following low doses of non-thermal plasma treatment insufficient to destroy the cell immediately.
In recent years, non-thermal plasma discharges have been gaining popularity in the materials processing industry for their ability to selectively modify a surface with minimal, if any, damage to this surface and practically no change to the bulk material. This way, for example, a surface of an implant may be made biologically compatible to the tissues and cells it will come in contact with, while the bulk material of the implant can be tailored to desirable mechanical properties like high strength, low weight, durability, fracture resistance, etc. Recently, the demand for sterilization and disinfection of various surfaces increased and non-thermal plasmas were found to be an effective solution. Many groups worldwide have successfully demonstrated plasma’s ability to treat, disinfect, and sterilize various targets. Plasmas are widely used in textile [3–5] and lighting industries [6–8], electronics [8–11], and in many other applications (see [8–12], for example). It is no surprise that biology and medicine also employ the “fourth state of matter” in materials processing [13–15], sterilization [10, 16–23], improvement of bio-compatibility [13, 24, 25], tissue engineering [26–28], to increase adhesion and wettability and for other surface modifications [29–34]. Medical applications focused on plasma treatment of living tissue, which are of growing interest these days, require treatments at atmospheric pressure since cells and tissues are not vacuum-compatible [16, 35]. Atmospheric pressure treatments can be separated into two major categories: where temperature is used as means of treatment [36–40], and where active species, radicals, or ultraviolet radiation generated by plasma are used for targeted chemical modification and catalysis [8, 10, 16, 41–48]. Thermal plasmas are widely used in medicine today both in attached arc mode where arc contacts the tissue directly [40, 49–51] and in a “jet” mode where gas (usually argon) is blown through the plasma but remains at high temperature [36, 37, 39, 52]. Both attached arc and thermal jet are known and used for their ability to rapidly coagulate blood [36–40, 50, 52]; however, they can cause significant thermal tissue desiccation, burning, and eschar formation1. Additionally, excessive smoke could be a problem during thermal treatment of tissues. During open-air treatments smoke can be removed by means of vacuum suction (though it is not a simple procedure); however, during endoscopic treatments smoke becomes a major issue where it is very difficult to remove and obstructs the view of the camera. For these reasons, development of non-thermal plasma methods of treatment where temperature does not exceed 60°C is needed.
Recently, the focus of the plasma community has shifted to applications where tissue damage and desiccation are minimized or eliminated . Many configurations have been proposed for treatment of biological surfaces, cells, and tissues. The “plasma needle,” for example, can possibly be used for selective cell detachment [28, 30, 45]. It involves a corona discharge igniting at the end of a sharp tip in helium upon application of radio frequency (∼13 MHz) electromagnetic excitation. This discharge operates at near room temperature, dissipating milliwatts in several cubic millimeters. Suggested applications for this technique include treatment of dental cavities and skin disorders. This plasma has been demonstrated to destroy cells and bacteria in a highly localized fashion without disrupting the nearby tissue . Recently, it was also shown that this plasma promotes inactivation in mouse fibroblast cells, where apoptosis-like behavior is observed after treatment––the cells appear to clump up and die . Another promising use of non-thermal plasmas is reversible pore formation for targeted drug delivery [28, 30] or irreversible pore formation [30, 54]. Pulsed Electron Avalanche Knife (PEAK) is one more plasma-based surgical tool where thermal damage to tissue is reduced by keeping the current pulses short (microseconds) and the electrodes thin (microns). Even though the device operates in the high current regime, timescales of treatment are not enough to heat up the tissue, resulting in non-damaging treatment [55, 56]. PEAK was successfully demonstrated in precise cutting with minimal damage. However, these systems are designed for precise treatment or cutting of very small areas and another system, capable of treating large areas of living tissue, is discussed in this paper. The Floating Electrode Dielectric Barrier Discharge (FE-DBD) system, constructed similarly to conventional dielectric barrier discharges (DBDs) is inherently non-thermal––it is able to operate at room temperature and pressure [10, 57]. This system operates at power densities of 0.1–2 W/cm2. FE-DBD was applied for complete skin sterilization without any damage to skin and blood coagulation without damage to surrounding tissue .
Presented in this paper is a way to treat cells where immediate destruction and necrosis is not desired or directly achieved by plasma. FE-DBD plasma treatment is shown to initiate apoptosis in Melanoma cancer cell lines––a threshold at which plasma treatment does not cause immediate necrosis but initiates complex cascade of biochemical processes leading to cell death many hours and even days following the treatment. Melanoma cells, treated by plasma at doses significantly below those required for cell destruction, survive the plasma treatment but develop apoptosis many hours post treatment and die (disintegrate) by themselves gracefully. This could potentially be an intriguing new idea for cancer treatment, especially if by manipulation of plasma parameters the treatment could be made selective to cancerous cells over healthy cells, as was demonstrated before for bacteria vs. healthy cells .
Following this introduction, Sect. 2 briefly discusses construction of the Floating Electrode DBD system used for the treatment. Sections 3 and 4 present details of the biological preparations and experiments performed on Melanoma cancer cell lines including growing these cells, their life path, treatment dynamics, apoptosis assays, and flow cytometry. Section 5 describes treatment of these cell lines for inactivation and necrosis, while Sect. 6 describes modes where the cells have not been inactivated by plasma but developed apoptosis many hours and days following the plasma treatment. This paper is concluded with ideas for future work to further analyze plasma-induced apoptosis mechanisms and their dependence on specific plasma-generated chemistry.
Floating Electrode Dielectric Barrier Discharge
Power deposited into plasma discharge gap was analyzed by measuring current passing through the discharge gap and the voltage drop in the gap. For current measurements, a magnetic core Pearson current probe was utilized (1 V/A +1/−0% sensitivity, 10 ns usable rise time, 35 MHz bandwidth). Voltage was measured using a wide bandwidth voltage probe (PVM-4 130 MHz 1000:1 <5% error, North Star High Voltage, Marana, AZ). Signals from the current and voltage probes were acquired and recorded by a Digital Phosphor Oscilloscope (DPS) (500 MHz bandwidth, 5 × 109 samples/sec, TDS5052B, Tektronix, Inc.). Acquired data was then processed using customized MATLAB code. In all experiments presented in this paper the plasma power was kept at 4 ±1 W, corresponding to power density of 0.8 ± 0.2 W/cm2.
Preparation and Treatment of Melanoma Skin Cancer Cells
For trypan blue exclusion, aliquots of the cell suspension were mixed with an equal volume of trypan blue (0.4% w/v in PBS, Cambrex Corp, Baltimore, MD) and transferred to a Hemocytometer slide. Live cells with intact cell membrane exclude the dye and are unstained. Total number of cells and percent of viable cells were determined by counting stained and unstained cells. Trypan Blue exclusion test was performed at different time periods after treatment: immediately following treatment, 1, 3, 24, 48, and 72 h following treatment.
Another group of experiments was performed, testing cells for the onset of apoptosis. Cell propagation and plasma treatment were performed as above, except that the dose of plasma for this experiment was 5 s, as this dose was found to inactivate the least number of cells. Following treatment, cells were harvested as above at 24, 48 and 72 h after treatment and DeadEnd™ Fluorometric TUNEL System apoptosis test was performed (Promega, Madison, WI) [59, 60]. This test detects apoptotic DNA fragmentation by catalytically incorporating fluorescein-12-dUTP(a) at 3′-OH DNA ends using the enzyme Terminal Deoxynucleotidyl Transferase (TdT), which forms a polymeric tail using the principle of the TUNEL (TdT-mediated dUTP Nick-End Labeling) assay. The fluorescein-12-dUTP-labeled DNA can then be visualized directly by fluorescence microscopy or quantified by flow cytometry [59, 60]. For fluorescence microscopy, cells were fixed on Lab-Tek slides for the TUNEL assay. Samples were analyzed in a fluorescence microscope using a standard fluorescence filter set to view the green fluorescence of Fluorescein at 520 ± 20 nm. Both control and treated cells were cultured in aluminum dishes to analyze the possibility of apoptosis developing from contact with aluminum. Minimum levels of cell death were detected in control dishes that were not subjected to plasma (see Fig. 2 for example).
For flow cytometry analysis, cell suspensions were treated with the DeadEnd™ Fluorometric TUNEL System (Promega US Co), following the manufacturer’s instructions. Counterstaining was done by incubating cells for 20 min at room temperature in the dark in phosphate-buffered saline containing 6 μg/mL RNAse (Roche Applies Sciences, Indianapolis, IN) and 5 μg/mL propidium iodide (Invitrogen, Carlsbad, CA) in PBS. Suspensions were washed once in PBS and resuspended in PBS for analysis. Flow cytometry was performed using a FACSORT flow cytometer (BD Biosciences, San Diego, CA) with 488 nm excitation from an argon ion laser at 15 mW. Forward scatter threshold was set to exclude small debris. Fluorescein fluorescence was captured on the FL1 channel (Fig. 11) equipped with a 530 nm wavelength filter with 30 nm bandwidth in log mode, and propidium iodide fluorescence with a 585 nm filter and 42 nm bandwidth in linear mode. Data acquisition was done using Lysis II software (version 2.0, BD Biosciences). Fluorescence spill-over was removed by compensation. Photomultiplier sensitivity was adjusted so that control cell FL1 fluorescence appeared in the first log, and PI fluorescence at approximately 200 counts (Fig. 11). At least ten thousand events were acquired per sample. Data analysis was performed using WinMDI software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA, available online from http://www.facs.scripps.edu). Apoptotic and necrotic cells were differentiated by plotting PI fluorescence over fluorescein in a bivariate plot. Quadrants were set to define TUNEL-negative cells with normal DNA content; TUNEL-positive cells were counted as apoptotic, TUNEL-negative cells with lower DNA content as necrotic.
Inactivation of Melanoma Cells by FE-DBD
Melanoma Cell Apoptotic Behavior
FE-DBD plasma was shown to kill Melanoma skin cancer cells through necrosis at higher treatment doses (15 s and over at 1.4 W/cm2 of plasma treatment) which are still below the threshold of damaging healthy tissue . Very low doses of FE-DBD (5 s at 0.8 W/cm2 of plasma treatment) where no cell necrosis was observed were shown to initiate apoptotic behavior, or programmed cell death in Melanoma cancer cells. During apoptosis, cells undergo a series of complex biochemical changes leading to cell death without causing inflammation. Apoptotic behavior was deduced from the fact that treated cells do not initially die but stop growth and die en masse 12–24 h following treatment, while untreated cells continue to grow and proliferate. Apoptotic behavior was confirmed through DeadEnd™ Fluorometric TUNEL System apoptosis staining with subsequent flow cytometry. It was shown that the plasma treatment initiates this behavior in cells not through poisoning of the growth media in which the cells reside or through interaction with the aluminum dishes the cells reside in, but through direct interaction with the cells.
Previously it was shown by authors  and by other groups (see [35, 63–71]) that plasma is able to destroy cells; however, it was also observed that plasma might be able to initiate or catalyze some biochemical processes in biological systems. This is an initial step toward understanding mechanisms by which non-thermal atmospheric pressure discharge in direct contact with cells is able to influence their activity. Previous and presented results may be promising, but quite a few unanswered questions remain. Deeper understanding of plasma-cell interaction and of the specific biochemistry is needed to answer how plasma promotes apoptotic behavior. It is important to separate all of the effects and constituents of plasma in direct contact with cells and to analyze these constituents individually as well as to study synergetic effects of and between different plasma components to potentially receive further insight into the plasma-chemical interaction mechanisms. Understanding of the apoptotic biochemical pathways invoked by plasma, which species generated by plasma are able to invoke these pathways, and how these mechanisms are invoked is also essential. Future work will primarily address fundamental understanding of plasma interaction with living tissue and physical and biochemical mechanisms thereof, for example effect of various plasma-generated excited species on the cell membrane and membrane proteins.
In medicine, the term “eschar” describes a slough or dry scab that forms on an area of skin that has been burnt or exposed to corrosive agents. The term “eschar” is commonly confused with “char.”
This work was supported in part by the Defense Advanced Research Projects Agency (DARPA) Award #W81XWH-05-2-0068, National Science Foundation (NSF) Grant #ECS-0304453. Assistance from DARPA program managers Drs. Rick Satava and Jay Lowell is greatly appreciated.