An atmospheric-pressure cold plasma leads to apoptosis in Saccharomyces cerevisiae by

accumulating intracellular reactive oxygen species and calcium

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IOP PUBLISHING JOURNAL OF PHYSICS D: APPLIED PHYSICS

J. Phys. D: Appl. Phys. 46 (2013) 285401 (8pp) doi:10.1088/0022-3727/46/28/285401

An atmospheric-pressure cold plasmaleads to apoptosis in Saccharomycescerevisiae by accumulating intracellularreactive oxygen species and calciumR N Ma1, H Q Feng2, Y D Liang2, Q Zhang1, Y Tian1, B Su1, J Zhang1,2

and J Fang1,2

1 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871,People’s Republic of China2 College of Engineering, Peking University, Beijing 100871, People’s Republic of China

E-mail: zhangjue@pku.edu.cn

Received 8 April 2013, in final form 13 May 2013Published 24 June 2013Online at stacks.iop.org/JPhysD/46/285401

AbstractA non-thermal plasma is known to induce apoptosis of various cells but the mechanism is notyet clear. A eukaryotic model organism Saccharomyces cerevisiae was used to investigate thecellular and biochemical regulations of cell apoptosis and cell cycle after anatmospheric-pressure cold plasma treatment. More importantly, intracellular calcium (Ca2+)

was first involved in monitoring the process of plasma-induced apoptosis in this study. Weanalysed the cell apoptosis and cell cycle by flow cytometry and observed the changes inintracellular reactive oxygen species (ROS) and Ca2+ concentration, cell mitochondrialmembrane potential (�ψm) as well as nuclear DNA morphology via fluorescence stainingassay. All experimental results indicated that plasma-generated ROS leads to the accumulationof intracellular ROS and Ca2+ that ultimately contribute to apoptosis associated with cell cyclearrest at G1 phase through depolarization of �ψm and fragmenting nuclear DNA. This workprovides a novel insight into the physical and biological mechanism of apoptosis induced by aplasma which could benefit for promoting the development of plasmas applied tocancer therapy.

(Some figures may appear in colour only in the online journal)

1. Introduction

Atmospheric-pressure cold plasmas have shown promisingapplications in the biomedical field, thus leading to theemergence of ‘plasma medicine’ which includes bacteriainactivation, blood coagulation, wound healing, toothwhitening and tooth root canal sterilization [1–6]. Recently,several researchers in plasma medicine have studied thepossible applications in cancer therapy, as some pioneeringworks have shown that plasma exerts anti-tumour effectson a wide variety of cancer cells [7–16]. Compared withconventional anticancer therapies, e.g. ionizing radiation andchemotherapy, plasmas can kill cancer cells by triggering

apoptosis of cells with high efficiency and fewer side-effectson the surrounding healthy cells, which has been consideredas a prospective approach for cancer therapy [17, 18].

The molecular mechanisms responsible for the apoptosiscaused by plasmas are not yet fully understood. However,based on efforts over the past several years, many groupshave realized that reactive oxygen species (ROS) producedby plasmas can stimulate oxidative stress in cells andconsequently result in DNA damage, cell cycle modificationand cell apoptosis [7–16]. Actually, the effect of ROS oncells is dose-dependent. There exists a series of complicatedcellular responses to the plasma. A low dose of ROS is ableto drive cell mutagenesis, proliferation and differentiation,

0022-3727/13/285401+08$33.00 1 © 2013 IOP Publishing Ltd Printed in the UK & the USA

J. Phys. D: Appl. Phys. 46 (2013) 285401 R N Ma et al

Figure 1. Pictures of He/O2 PMJ sustained in (a) ambient air and (b) quasi-steady gas cavity in water. (c) Schematic diagram of the plasmatreatment in water.

while a high dose can trigger apoptotic and necrotic celldeath [15, 19, 20].

Furthermore, it is commonly recognized that apart fromROS, intracellular Ca2+ signal also plays a critical role in theapoptosis process [21]. As the simplest and most versatilesecond messenger in biology, Ca2+ regulates various cellularbehaviours ranging from synaptic transmission, musclecontraction, hormone secretion, cell locomotion, cell survivalto cell cycle regulation, mainly stored within the endoplasmicreticulum (ER) [22]. Moreover, the relationship between Ca2+

and ROS is complicated and close, either as a crucial partnerin regulating the redox status of cells, determining cell fate,or in signalling in response to a number of physiological andstress conditions [23].

Considering the above factors, both intracellular ROS andCa2+ are studied in this experiment to investigate plasma-induced cell death effects and the underlying molecularmechanisms using a well-studied eukaryotic model organismSaccharomyces cerevisiae (S. cerevisiae) of high homologywith humans [24]. In this study, the cell apoptosis andcell cycle were evaluated by flow cytometry and the changesin nuclear DNA morphology, cell mitochondrial membranepotential (�ψm) as well as intracellular ROS and Ca2+

concentrations following plasma treatment were detected viafluorescence staining assay. Moreover, we detected theROS generated by plasmas in water by oxidation–reductionpotential (ORP) and optical emission spectroscopy (OES).

2. Experimental setup and methods

2.1. Experimental setup and apparatus

Figure 1(c) is a schematic diagram of the experimental setup.The atmospheric-pressure cold plasma is produced via adirect current (dc) micro-hollow cathode discharge (MHCD)based plasma microjet (PMJ) device [25]. The plasmadevice comprises two copper tubes as electrodes separatedby a ceramic tube and is driven by a dc negative-polarityhigh-voltage power supply (Matsusada AU5R120) through a5 k� ballast resistor. The two metal electrodes are separated

from each other by approximately 0.5 mm. The openingsin the two electrodes are about 0.8 mm in diameter and thedepth of the exit opening is nearly 1 mm. Typical photographsof the PMJ sustained in ambient air and water are shown infigures 1(a) and (b). A He/O2 (2 vol%) premix was used as theworking gas at a flow rate of 3 standard litres per minute (slm).When sustained in air, the applied operating current and voltagewere 30 mA and 0.56 kV, respectively. When immersed inwater, the PMJ was sustained in a quasi-steady gas cavity withapproximately the same sustaining voltage. More details ofthe operation of the plasma can be found in [26, 27].

2.2. Yeast strains and culture media

Three types of yeast S. cerevisiae strains, namely a wild-type strain (BY4741 (ATCC 201388)) and two anti-oxygengene overexpression mutant strains p-Sod1 and p-Sod2 wereused in the experiment. The two overexpression mutantstrains p-Sod1 and p-Sod2 were constructed by respectivelyinserting the anti-oxidative genes superoxide dismutase 1and 2 (Sod1 and Sod2), which encode cytoplasmic Cu/Zn-SOD and mitochondrial Mn-SOD into a yeast expressionplasmid pACT2, and then by transducing the wild-type strainwith the recombinant plasmids, whose detailed description hasbeen reported previously [28]. SOD catalyses dismutation ofsuperoxide anion (O−·

2 ) to less harmful (hydrogen peroxide)H2O2, which is then decomposed by Catalase into H2O andO2. SOD in concert with Catalase forms the first and mostimportant line of antioxidant defense [29]. Therefore, the twomutant strains could produce more intracellular anti-oxidantsagainst external oxidative stress compared with the wild-typestrain. All yeast strains were routinely cultured in yeastextract-peptone-adenine-dextrose (YPAD) medium, which iscomposed of 1% yeast extract, 2% bactopeptone, 2% glucoseand 100 µg ml−1 adenine at 30 ◦C.

2.3. Plasma treatment of yeast cells

PMJ was sustained in a quasi-steady gas cavity in water foryeast cell treatment, and the distance between the end of thenozzle and the liquid level was fixed at 1 cm (figure 1(c)).

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The wide-type and overexpression strains were cultured to theexponential growth phase and harvested at a concentration of2.5×107 cells per ml. 1 ml cells were then resuspended in 5 mlautoclaved deionized water and treated with the PMJ for 0, 1,2, 3, 4 and 5 min. For subsequent cell apoptosis and cell cycleanalysis, DNA fragmentation assay, ��m measurement, andintracellular ROS and Ca2+ detection, the plasma-treated cellswere centrifuged and then incubated in 20 ml YPAD mediumfor 2 h after the plasma treatment to allow enough time forthe cells to respond to plasma exposure [30]. All experimentswere repeated three times for statistical analysis.

3. Analysis procedure

3.1. Measurement of major excited ROS generated by theplasma

To identify the major excited reactive species generated bythe He/O2 plasma in air and water, OES was employed inthe 200–1000 nm range along the axial direction of the PMJwith the AvaSpec-2048-8 Fibre Optic Spectrometer (Avantes,USA). One end of the fibre optics cable was used to acquirethe light signals at the bottom of the quartz tube at a distanceapproximately 5 mm away from the exit nozzle. The presenceof the end of the fibre optics cable at the bottom of the quartztube did not influence the plasma operation. The dispersedemission spectra were recorded by a 2048 pixel charge-coupleddevice (CCD) detector array. Detailed OES detection of thePMJ can be found in our earlier paper [28, 31].

Different from OES, which can accurately detect theROS, immediate ORP was utilized to evaluate the globallevels of ROS in the solution following plasma exposure [32].ORP is an indicator of the ability of a solution to oxidizeand is related to the concentration of oxidizers and theiractivity or strength. 5 ml deionized water and cell suspensionsof the three yeast strains were treated by the plasma fordifferent times ranging from 0 to 5 min. The ORP in eachsample was measured immediately after the treatment usinga redox sensitive electrode on the Microprocessor pH/ORP-meter (HANNA pH213 Instruments, USA), since most ROSare short lived.

3.2. Cell viability assay

To evaluate cell viability of the three strains exposed tothe plasma for a variety of treatment durations, XTTcolorimetric-assay (Sigma, St Louis, MO) was performed asan alternative way to colony-forming unit counting [33]. Thecell suspensions were treated with the plasma as describedearlier. Then 20 µl of the samples were added to 100 µl ofXTT working solution in a well of a 96-well plate, each samplerun in duplicate. Following, the yeast cells were culturedwith XTT for 8 h at 30 ◦C to yield the water soluble orangederivatives, which were then used to evaluate the numbers ofliving cells by measuring the optical density (OD) at 450 nm ona SPECTROstar Omega absorbance plate reader with a RapidUV/Vis spectrometer (BMG, Germany).

3.3. Cell apoptosis evaluation by flow cytometry

In order to confirm apoptotic activity, cells were stained withAnnexin-V and propidium iodide (PI) prior to flow cytometryanalysis. An Annexin V-fluorescein isothiocyanate (FITC)apoptosis detection kit (BD Biosciences Pharmingen) wasused for assessing apoptosis after different plasma treatmentsranging from 0 to 5 min. According to the manufacturer’sprotocol, plasma-treated cells were collected, washed withphosphate buffer saline (PBS) and resuspended in 1× bindingbuffer containing Annexin-V and PI. After incubation for20 min at 25 ◦C in the dark at room temperature, fluorescence-activated cells were detected by FACSauto flow cytometry(Becton Dickinson, USA), and the data were analysed withthe FlowJo 7.6.5 analysis software. For each measurement, atleast 10 000 cells were analysed by flow cytometry.

3.4. Analysis of cell cycle

Cell cycle analysis was detected by flow cytometry. Threeyeast strains were cultured overnight to the exponential growthphase, followed by 0, 1, 2, 3, 4 and 5 min plasma exposures.After the treatment, cells were harvested at about 107 cellsper ml and fixed by 70% (v/v) ethanol at 4 ◦C overnight.The fixed cells were rinsed twice with PBS and resuspendedin PBS containing 50 µg ml−1 PI and 0.1 mg ml−1 RNaseA(Beyotime, China). After incubation for 30 min at 37 ◦C inthe dark, the samples were filtered on a nylon mesh prior toanalysis using FACSauto flow cytometry (Becton Dickinson,USA). The distribution of cells in the G1, S and G2/M phases ofthe cell cycle was determined using the FlowJo 7.6.5 analysissoftware. For each measurement, at least 10 000 cells wereanalysed by flow cytometry.

3.5. Observation of nuclear DNA morphology

For nuclear DNA staining, three different yeast strains after theplasma treatment were harvested and fixed with 70% ethanolfor 1 h on ice, and resuspended in PBS containing 1 µg ml−1

4’,6-diamidino-2-phenylindole (DAPI) (Sigma, USA) and1 mg ml−1 antifade p-phenylenediamine (Sigma, USA) for30 min [34]. The cells were then washed twice in PBSand observed in a Ti-E fluorescence microscope (Nikon,Japan). In all cases two or three different fields wereobserved, containing about 10–100 cells. The cells of thesame fields were also visualized by bright field (BF) phasemicroscopy.

3.6. Detection of cell mitochondrial membranepotential (�ψm)

�ψm was analysed using tetramethylrhodamine methyl ester(TMRM) (Sigma, USA), a mitochondrial membrane-specificfluorescent dye [35]. Following plasma exposure, all yeaststrains were harvested, washed twice with PBS, and thenincubated at 30 ◦C for 30 min in PBS containing 20 nMTMRM. After incubation, the cells were washed three timesand then resuspended in PBS for observing in a Ti-Efluorescence microscope (Nikon, Japan).

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Figure 2. End-on optical emission spectra of He/O2 PMJ ranging from 200 to 1000 nm (a) operated in air and (b) operated in water.

3.7. Assays of intracellular ROS

Intracellular ROS was measured with the probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA) (Sigma, USA).DCFH-DA passively diffuses into the cell during vortex and isdeacetylated by esterases to form the nonfluorescent species2’,7’-dichlorofluorescein (DCFH). In the presence of ROS,DCFH is oxidized to the fluorescent product DCF [36]. Allstrains cells after plasma exposure were harvested, washedtwice with PBS, and then incubated at 30 ◦C for 30 min in PBScontaining 10 µM DCFH-DA. After incubation, the cells werewashed three times and then resuspended in PBS for observingin a Ti-E fluorescence microscope (Nikon, Japan).

3.8. Assessment of intracellular Ca2+

Intracellular Ca2+ was measured with the calcium-specificfluorescent dye, Fluo-4-acetoxymethyl ester (fluo-4 AM)(Sigma, USA) [37]. Plasma-treated cells were washed twicewith PBS, and then incubated at 30 ◦C for 30 min in PBScontaining 10 µM fluo-4 AM. After incubation, the cells werewashed three times and then resuspended in PBS for observingin a Ti-E fluorescence microscope (Nikon, Japan).

4. Results and discussion

Generally, the PMJ can produce chemically active species[38, 39], thus OES was used to investigate the active speciesgenerated in the He/O2 plasma ranging from 200 to 1000 nm.In the end-on spectra of the He/O2 PMJ operated in airat an operating current of 30 mA and voltage of 0.56 kV(figure 2(a)), strong atomic oxygen emission at 777 and844 nm were detected in the near IR region, while the emissionspectrum ranging from 440 to 800 nm was dominated by Helines (587.6, 667.8 and 706.6 nm). As shown in figure 2(b),when the He/O2 PMJ was submerged in water with thesame operating current and voltage, helium and oxygen peaksobserved in air still dominated the emission spectrum, but witha lower emission intensity. Interestingly, OH (A → X) band(306–309 nm) and the Hα line (656 nm) emissions occurredbecause of the water disassociated and excited by the plasma.The results of OES indicated that a high concentration ofexcited atomic oxygen is produced in water by the plasma,which can be easily converted to other ROS in the liquid, such

Figure 3. ORP of pure water and yeast cell suspensions after theplasma treatment.

as hydroxyl radical (·OH), singlet oxygen (1O2), superoxideanion (·O−

2 ) and hydrogen peroxide (H2O2) due to its highreactivity [43]. Furthermore, various kinds of ROS wereproved to exist in the water by electron spin resonance (ESR)diagnosis in our earlier papers [31, 38, 39].

Moreover, ORP was detected to evaluate the overall levelsof ROS in the solution treated by the plasma. In figure 3,ORP of pure water and cell suspensions all increased linearlywith exposure time. The ORP of pure water increased fromaround 350 mV to approximately 470 mV, indicating that largeamounts of ROS was produced by the plasma underwater,which was consistent with the results of OES. However, theORP values of the three different cell suspensions (wild-type,p-Sod1 and p-Sod2) are lower than that of pure water after theplasma treatment, demonstrating that the cell could undertakea part of the oxidative stress in water through interacting withthe ROS generated by the plasma.

Next, the effects of plasma on proliferation of the threeyeast strain cells were determined using XTT assay. The XTTassay detects mitochondrial dehydrogenase activity, whichproduces the resulting formazan in living cells. Therefore,OD represents the living cell numbers, and the survival ratesof each strain are calculated by normalizing to their respectivenon-treated samples. As figure 4 represents, there was nosignificant difference among the three strains for the 1 minplasma treatment; the survival rates all decreased extensivelyto around 50%, indicating that the cells of all the three strains

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Figure 4. Survival rates of three strains after 0, 1, 2, 3, 4 and 5 minplasma treatment following 8 h culture.

Figure 5. Flow cytometric analysis using Annexin-V and PI doublestaining. The analysis was obtained in the He/O2 PMJ under thefollowing different conditions: wild type with 0 (control), 1, 3 and5 min PMJ treatment as well as p-Sod1 and p-Sod2 strains with5 min PMJ treatment. The percentages of apoptotic cells (early andlate apoptosis) are also shown as indicated.

succeeded in repairing DNA damage after a low-dose plasmatreatment. After 5 min plasma exposure, the survival rateof p-Sod1 and p-Sod2 reached nearly 40%, while the wildstrain almost showed no surviving cells, suggesting that thetwo overexpression strains were more resistant to subsequentlethal exposure than the wild type probably through activatinga programme of anti-oxidative genes expression to alleviateintracellular oxidative stress induced by the high-dose plasmatreatment.

Furthermore, numbers of apoptotic and necrotic cellswere determined by Annexin-V and PI double staining.Phosphatidylserine is largely located in the cell membraneand the molecules are orientated towards the cytoplasm.Phosphatidylserine exposure to the outer side of the membraneserves as a sensitive marker for early stages of apoptosis,which can bind to Annexin-V staining with high affinity [40].Quadrants Q1, Q2, Q3 and Q4 denote necrotic cell (AnnexinV−/PI+), late apoptotic cell (Annexin V+/PI+), viable cell(Annexin V−/PI−) and early apoptotic cell (Annexin V+/PI−)regions, respectively. The movement of cells through earlyand late stages of apoptosis both confirms apoptosis. As forthe wild strain (figure 5), the plasma caused a time-dependent

Figure 6. Flow cytometry analysis for cell cycle distribution of thethree strains after different PMJ treatment, wild type with 0(control), 1, 3 and 5 min PMJ treatment as well as p-Sod1 andp-Sod2 strains with 5 min PMJ treatment. The percentages of cellsin G1, S and G2/M are also shown as indicated.

manner in the apoptosis. As time increased, even more cellsunderwent apoptosis, further reaching 66.1% of apoptoticcells after 5 min plasma treatment versus 0.1% in the non-treated group, suggesting that most cells underwent apoptosisinstead of necrosis when subjected to the plasma treatment.In addition, early apoptosis was induced markedly after theplasma treatment among all of the three strains. In particular,in the case of 5 min plasma treatment, the apoptotic rate of wildstrain reached 51.9%, and the two overexpression strains werelower, only close to 30%, indicating that induction of apoptosisby the plasma was likely mediated through the formation ofintracellular ROS.

To explore the possible mechanism of the apoptosisinduced by the plasma, the cell cycles of the three yeaststrains were examined. The experimental results show thatthe plasma-treated wild-type strain increased significantly inthe number of G1 phase cells, with a concurrent decrease inthe number of G2/M phase cells compared with a non-treatedwild-type sample (figure 6). Moreover, this increase in G1phase cells of wild strain was dependent on the exposuretime, which reached three fold of control at 5 min plasmatreatment. In contrast, the two overexpression strains showedlittle difference on the cell cycle distribution. After 5 minplasma exposure, the percentage of cells entering into theG1 phase was approximately 40%, significantly lower thanthe wild type. Given above results, the plasma-treated cellsmay experience DNA damage, leading to activation of G1/Scheckpoint. Thereby the cells are arrested at the G1 phaseto allow enough time for DNA repair to maintain genomestability. Moreover, if unrepaired, an apoptotic process isinitiated to prevent cell mutations [41].

To further determine whether DNA was damaged duringthe plasma treatment, the nuclear DNA was stained with DAPI.As figure 7(a) shows, DAPI staining of different cells displayeddistinctive morphology after the plasma treatment. The non-treated wild strain showed a typical nucleus with intact DNAas a single round spot in the cell, while the DNA of the treatedwild strain was fragmented into pieces, uniformly distributed in

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Figure 7. Cells were stained with (a) DAPI observed under fluorescence microscopy (1000×), (b) TMRM, (c) DCFH-DA and (d) fluo-4AM and observed under fluorescence microscopy (400×) with the following different conditions: wild-type strain with 0 (control), 1, 3 and5 min PMJ treatment as well as p-Sod1 and p-Sod2 strains with 5 min PMJ treatment. The upper panels of each figure represent therespective BF images.

the whole cell. In addition, the fluorescence intensity becamestronger following long-time exposure, indicating that moresmall DNA fragments were generated by the plasma. Andthe two overexpression strains after 5 min treatment had lowerfluorescence intensity compared with that of the wild type. Itis concluded that the plasma caused DNA fragmentation inthe nucleus, and the inability of the cells to repair the inducedDNA damage may ultimately lead to induction of apoptosisand cell cycle arrest at the G1 phase as observed.

For further considerations that mitochondria are wellknown as the primary coordinators of apoptotic processesto control the intrinsic apoptotic pathway [42], we testedwhether the plasma led to induction of apoptosis alsoby initiating a mitochondria-mediated pathway except forDNA damage. �ψm is an indicator of the mitochondrialmembrane integrity, which would depolarize when themembrane is perturbed, combining with less fluorescenceprobe. Figure 7(b) shows that in wild-type cells, comparedwith the untreated cells, the right fluorescence density oftreated cells decreased with increasing treatment time. Andthe fluorescence intensity of the two overexpression cellsat 5 min plasma treatment was close to the wild-type cellat 1 min plasma treatment. Our results present that theplasma treatment can induce depolarization of �ψm, causingthe opening of the mitochondrial permeability transitionpore (mPTP) and mitochondrial swelling, which leads tothe dysfunction of mitochondria, eventually resulting in cellapoptosis.

In addition, it is commonly recognized that two importantintracellular signal molecules, ROS and Ca2+, are regarded asthe initiator and executor of apoptosis progress [23], therebywe monitored the concentration of intracellular ROS and Ca2+.As shown in figure 7(c), in wild-type cells, the fluorescenceintensity as well as the amount of stained cells increased withlonger plasma treatment time. Meanwhile, a comparison of thefluorescence panels with the BF panels showed that essentially100% of the wild-type cells stained for ROS after 5 min plasmatreatment. By contrast, in overexpression strains, only a few

cells were stained green and the intensities were much lowerthan the wild type at 5 min plasma treatment. Furthermore, forthe result of intracellular Ca2+ level (figure 7(d)), the patternwas in accordance with the intracellular ROS measurement.However, a small difference existing between these tworesults was that the two overexpression strains significantlydecreased the fluorescence intensity of ROS, while onlypartially reducing that of Ca2+. Considering the above results,we found that the overexpression strains could not onlymarkedly reduce the intracellular ROS accumulation, but alsoinhibit the intracellular Ca2+ accumulation to a certain extentthrough elevating Sod1 and Sod2 genes expression activatedby long-time plasma exposure.

From the results presented here, it is clear that the plasmais able to produce a large concentration of excited atomicoxygen in water, which can be easily converted to other long-lived and freely diffusible ROS derivatives due to its highreactivity (figure 8(a)) [43]. ROS produced by the plasmaextracellularly may move across the cell membrane by activetransport across the bilayer, transient opening of pores in themembrane or activation of signalling pathways that form theaccumulation of intracellular ROS [44]. On the other hand,external oxidative stress also could induce intracellular Ca2+

release probably by opening an array of ryanodine receptor(RyR) Ca2+ release channels in the ER (figure 8(b)) [22].Moreover, excessive intracellular ROS and Ca2+ could inducemitochondrial swelling and opening of mPTP, which causes thedepolarization of �ψm and potential release of proapoptoticfactors, including cytochrome C (Cyt-c), leading to activationof a caspase cascade and subsequent cell apoptosis [23]. Inparticular, the intracellular ROS also could oxidize DNA inthe nucleus, causing the fragmentation of DNA. And themassive DNA damage could not only induce cell cycle arrestat the G1 phase through activating the checkpoint signal, butalso initiate apoptosis by a p53-dependent signalling cascade(figure 8(c)) [13]. Another important aspect is that the twooverexpression strains mitigated the apoptotic effects of theplasma by reducing the accumulation of intracellular ROS and

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Figure 8. Schematic diagram of the plasma-induced apoptosisprocess divided into three stages: (a) plasma-generated ROS inwater, (b) plasma-generated ROS induced the accumulation ofintracellular ROS and Ca2+, and (c) intracellular ROS and Ca2+ ledto dysfunction of mitochondria and fragmentation of nuclear DNA.

Ca2+, which further potentiate that plasma-induced apoptosismay be mediated by intracellular ROS and Ca2+. Nevertheless,we note that in another study [45], atmospheric-pressuredielectric barrier discharge (DBD) exposure could induceintracellular generation of electric fields, severely damagingthe cell membrane, resulting in electroporation on the cell,which may lead to cell apoptosis different from that caused bythe plasma treatment studied here.

5. Conclusion

Taken together, the plasma-generated ROS remarkablyelevates the concentration of intracellular ROS and Ca2+ ona time-dependent manner, which both function as surrogateapoptotic signals by targeting �ψm and nuclear DNA, thuscausing the dysfunction of mitochondria and fragmentationof nuclear DNA, consequently leading to apoptotic cell deathaccompanied by cell cycle arrest at the G1 phase in yeastcells. This work unravels the mechanism of plasma-inducedapoptosis in eukaryotic model organism yeast cells, whichmight be helpful in explaining the killing effect of plasmason cancer cells and eventually promoting the developmentof plasmas applied to cancer therapy. Moreover, we foundthat intracellular ROS and Ca2+ could serve as valuablebiomarkers for the oxidative stress produced by plasmas.In addition, as mentioned above, the cellular responsesare governed by the dose of plasma treatment [19, 20].Therefore, in future studies, we will measure the concentrationof intracellular ROS and Ca2+ by flow cytometry afterdifferent doses of plasma treatment by changing the plasmaparameters (e.g. the operating current and voltage, gas flowand treatment time). After obtaining the relationship betweenintracellular ROS and Ca2+ concentration and their relatedplasma parameters, intracellular ROS and Ca2+ may be used asalternative indicators for comprehensively measuring the doseof plasmas generated by different devices in future clinicalapplications.

Acknowledgment

This research is sponsored by Bioelectrics Inc. (USA), and thePeking University Biomed-X Foundation.

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