- Email: [email protected]
Radiosensitization of oral tongue squamous cell carcinoma by nanosecond pulsed electric fields (nsPEFs)
Bioelectrochemistry 113 (2017) 35–41
Contents lists available at ScienceDirect
Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Radiosensitization of oral tongue squamous cell carcinoma by nanosecond pulsed electric fields (nsPEFs) Jinsong Guo a, Yu Wang b, Jing Wang c,⁎, Jue Zhang a,b,⁎⁎, Jing Fang a,b a b c
College of Engineering, Peking University, Beijing, 100871, China Academy of Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China Department of Oral Medicine, School of Stomatology, Lanzhou University, Lanzhou, Gansu, China
a r t i c l e
i n f o
Article history: Received 10 November 2015 Received in revised form 16 September 2016 Accepted 20 September 2016 Available online 22 September 2016 Keywords: Radiosensitization nsPEFs TSCC G2/M arrest Nitric oxide Apoptosis
a b s t r a c t Nanosecond pulsed electric fields (nsPEFs) are a non-thermal and non-toxic technology that induce a myriad of biological effects. They have been proven to be effective in tumor shrinkage, but few studies focus on its radiosensitization in oral tongue squamous cell carcinoma. The purpose of this research was to study the radiosensitization effect of nsPEFs on a human oral tongue cancer cell line Tca8113 and to investigate the potential antitumor mechanism. A Tca8113 cell line was tested respectively by MTT assay, clonogenic assay, flow cytometry assay, annexin V-FITC/PI assay, mitochondrial potential assay and total nitric oxide assay. Our results showed that nsPEFs had a time and field strength dependent inhibition effect on Tca8113 cells. The sensitization enhancement ratio (SER) of nsPEFs was 1.453 ± 0.038. Furthermore, radiation induced G2/M arrest was augmented by treatment with nsPEFs. We observed many more Tca8113 cells showing early apoptosis after nsPEFs combined with radiotherapy. Additionally, the NO concentration was significantly increased after nsPEFs treatment. These findings indicate that combination of nsPEFs with radiotherapy can enhance the radiosensitivity of Tca8113 cells and nsPEFs could be a potential radiosensitizer for oral tongue squamous cell carcinoma. © 2016 Published by Elsevier B.V.
1. Introduction Oral tongue squamous cell carcinoma (TSCC) is one of the most common malignancies of the head and neck region. Recent survey data suggests that the incidence of TSCC is actually increasing in young and middle age populations [1,2]. Despite advances in cancer treatment, the 5-year survival rate has remained at about 50% over the past several decades [3]. Usually, operation and irradiation are the mainstay treatments for TSCC patients. However, surgical treatment for patients often produces a significant adverse effect on speech function, chewing and swallowing ability. Further, there is a high recurrence rate after surgery [4]. Thus, how to further improve the sensitivity of oral tongue cancer cells to radiation has become a research hotspot. Recently, looking for new, safe and effective radiosensitizers has become a novel strategy in oral tongue cancer treatment. Since the 1960s chemical sensitization in radiotherapy has already been studied [5]. Up until now, we have obtained some stable and high radiosensitizing effect radiosensitizers, such as misonidazole (MISO), taxanes and sirolimus. With advances in new radiosensitizers, the survival of TSCC patients have improved significantly [6–8]. ⁎ Corresponding author. ⁎⁎ Correspondence to: J. Zhang, College of Engineering, Peking University, Beijing 100871, China. E-mail addresses: [email protected] (J. Wang), [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.bioelechem.2016.09.002 1567-5394/© 2016 Published by Elsevier B.V.
However, it is difficult to avoid the toxicity effects induced by the chemical radiosensitizers, especially for the advanced cancer patients and those with TSCC recurrence after radiation therapy. Overall, we need to find an effective sensitization technology for radiotherapy in TSCC treatment without toxic side effects. Typically, the effects of pulsed electric fields on biological cells have been investigated since the late 1950s. More recently, the duration of the electric fields has been shortened to nanoseconds [9]. Many previous studies have found that nanosecond pulsed electric fields (nsPEFs) are able to induce a series of medical and biological effects. The pertinent areas include tumor shrinkage, wound healing, sterilization, and plant growth promoting [10–13]. Meanwhile, the present work describes in detail the anticancer activity of nsPEFs, including cell apoptosis, cytochrome C release, calcium release and DNA damage [14]. Additionally, some studies have also showed that nsPEFs have synergistic effects on low concentration chemotherapy drugs in oral cancer cell lines [15,16]. In particular, Nuccitelli R completed the first-in-human safety trials of nsPEFs in basal cell carcinoma (BCC). The results indicated that nsPEFs are safe and may offer a fast and scarless alternative to cancer treatment [17]. Also, Shan Wu, et al. found that nsPEFs treatment did not create permanent damage to the skin or normal tissues and only left shallow marks on the skin that faded completely in two weeks [18]. Moreover, we concluded that nsPEFs can effectively induce cell cycle arrest at G2/M phase [19,20]. Moreover, we inferred that NO was positively involved in the early growth effects of Haloxylon ammodendron seed
36
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
Fig. 1. a.) The photo of 60 ns nsPEFs generator used in this experiment. b.) Typical oscillogram of 60 ns pulse generator. c.) Circuit diagram of the basic Blumlein pulse forming system. Two cable length of 12 m and one cable length of 6 m were chosen in order to generate 60 ns pulses.
after nsPEFs exposure [13]. However, so far, there is no study focusing on the effect of nsPEFs for radiosensitization in oral tongue squamous cell carcinoma. The present study aimed to investigate the proliferation, apoptosis and cell cycle effects of nsPEFs combined with radiation in a human tongue squamous cell carcinoma Tac8113 cell line, providing a new theoretical basis of nsPEFs for radiosensitization in oral tongue squamous cell carcinoma. 2. Materials and methods 2.1. Cell line and cell culture Human squamous cell carcinoma Tca8113 cells were cultured in RPMI-1640 medium (GIBCO BRL, Rockville, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). No antibiotics were used in the preparation of the medium. Cells were grown under 5% CO2 in a humidified atmosphere at 37 °C. Cells in the logarithmic growth phase were used to perform the experiments described follows. 2.2. nsPEFs treatment In this study, the nsPEF generator was applied as previously described [21,22]. A Blumlein line pulse generator (Fig. 1A.) produced nearly rectangular 60 ns pulses that were delivered to the Tca8113 cells by a 2 mm cuvette. The electric field strength varied from 10 kV/cm to 50 kV/cm. A digital phosphor oscilloscope (DPO4054, Tektronix) with a probe (P6015A, Tektronix) was utilized to monitor the voltage waveform (Fig. 1B.). Tca8113 cells were counted with a hemocytometer, and 2.0 × 106 cells suspended in 500 μL culture medium were added to 2 mm gap cuvettes (Biosmith, San Diego, CA). Then the experimental
cuvettes were exposed to 20 pulses of nsPEFs of 60 ns duration at an electric field strength of 10, 20, 30 and 50 kV/cm, respectively. Cuvettes that did not undergo nsPEF treatment served as the control group. The time between each pulse was about 1 s. 2.3. Radiation platform To simulate clinical conditions, Tca8113 cells were irradiated with Xrays at 6 MV at room temperature using a linear accelerator (Elekta Precise, Stockholm, Sweden) under the source-to-skin distance (the distance from the radiation source to the central surface of the plate – 100 cm), and the dose rate was 2.0 Gy/min. 2.4. Cell proliferation assay The effects of nsPEFs on Tca8113 cells were determined with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The MTT assay uses a MTT dye which acts as a substrate for viable cell reductase enzyme. This enzyme reduces MTT yellow dye to purple colour formazan which is in proportional to viable cell eventually [23]. When cells began to grow exponentially, they were harvested and resuspended at a concentration of 2.0 × 106 cells/ml. A 500 μl cell suspension (1.0 × 106 cells) was placed in a 2 mm gap cuvette (Biosmith) and then treated with nsPEFs at electric fields of 0, 10, 20, 30 and 50 kV/cm. After incubation for 24, 48, and 72 h, 20 μL MTT (5 mg/ml) was added to each well, and cells were further incubated at 37 °C for 4 h. The medium was then removed and 200 μL of DMSO was added to dissolve the reduced formazan product. MTT dye intensity was then read on a micro plate reader (BioRad) at 492 nm. The survival rates under different field strengths were calculated according to the following formula [24]: Survival rate = A value of tested well/A value of control well × 100%. Using this same method, we investigated the anti-
Scheme 1. A schematic diagram of the experimental arrangement, including logarithmic growth phase cells preparation, cells collecting, nsPEFs treatment, cell culture, radiotherapy of Tca8113 cells and data detection.
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
37
2.6. Cell-cycle distribution
Fig. 2. Tca8113 cell survival after treatment with nsPEFs. Cells were treated with nsPEFs at 10, 20, 30 and 50 kV/cm, 20 pulses, respectively. After incubation for 24 h, 48 h and 72 h, cell survival rates were determined by MTT assay. Results are presented as the percentage of the decreased values from the control cells.
proliferative effects of nsPEFs combined with radiation. In the treatment of sensitizing cells with nsPEFs before radiation treatment, cells were detached and treated with nsPEFs, then dispensed into 90-mm plates and further incubated at 37 °C for 24 h. After that, Cells were treated with different doses of radiation (2, 4, 6 and 8 Gy) and cell survival rates were analyzed after 24, 48, and 72 h by MTT assay. A schematic diagram of the experimental arrangement is shown in Scheme 1.
2.5. Clonogenic survival assay In radiation biology, clonogenic assay is the gold standard assay for determining reproductive cell death after radiation treatment [25]. In this study, the survival fraction (SF) of Tca8113 cells in each group under different conditions was determined by clonogenic assay. After irradiation, Tca8113 cells were immediately trypsinized and suspended. The cells were then seeded in triplicate in 60 mm petri dishes at a different cell densities (control 200 cells, nsPEFs 200 cells, 2 Gy 800 cells, 4 Gy 1600 cells, 6 Gy 3200 cells and 8 Gy 6400 cells). After incubation for 10 days, the cells were washed twice with phosphate-buffered saline, fixed in methanol, and then stained with Giemsa stain. Colonies (≥50 cells) were counted for computing percent growth inhibition. Plating efficiency (PE) and the survival fraction (SF) were calculated according to the following formula: PE = (clone count/cell count) × 100%, SF = (PE in observed group/PE in 0 Gy group) × 100%. Then, a single-hit multi−D N
target (SHMT) radiobiological model {S ¼ 1−ð1−e D0 Þ } was applied to delineate survival curves [26]. The mean lethal dose (D0), quasithreshold dose (Dq), survival fraction at irradiation dose of 2 Gy (SF2) and sensitization enhancement ratio (SER) were calculated based on cell survival curves of single-hit multi-target model, respectively. Cell survival curves (Fig. 4B) were fitted with Sigmaplot™ 10.0 software (Systat Software, Inc., Chicago, IL, USA).
The cell cycle was determined with a flow cytometer and the cellcycle phases were analyzed by measuring the DNA fragments stained with propidium iodide (PI; Sigma-Aldrich). Tca8113 cells at log phase were inoculated in 6-well plate at 5.0 × 105 cells in each well. Four groups were set as the control group, nsPEFs group, radiotherapy group and combination group, respectively. Tca8113 cells were harvested and centrifuged at 24 h after different treatments. Then, the cells were washed twice with pre-cooled phosphate-buffered saline. After this the cells were fixed and permeabilized overnight by adding 1 mL of 70% pre-cooled ethanol to each tube at 4 °C. After centrifugation, the fixatives were decanted and the cell pellets were resuspended in 0.5 mL of staining solution containing 200 μL each of DNAse-free RNAse (Sigma-Aldrich) and PI and incubated for 30 min at room temperature in the dark [27]. The distribution of the Tca8113 cells in the cell cycle phases was analyzed from the DNA histogram using a FACSCaliber™ flow cytometer and CellQuest™ software (BD Biosciences, San Jose, CA, USA). 2.7. Cell apoptosis An Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharmingen) was used for assessing apoptosis induced by nsPEFs combined with radiation [28]. Annexin-V-FITC and PI were used to evaluate normal cells (no staining), early apoptotic cells (Annexin-V-FITC positive, PI negative) and necrotic cells (Annexin-V-FITC and PI positive). The Tca8113 cells were harvested and centrifuged at 24 h after different treatments. Then cells stained with Annexin-V-FITC in a dark at room temperature for 15 min, and cells were stained with PI on ice for 30 min. Samples were assessed by FACSauto flow cytometry (Becton Dickinson, USA). 2.8. JC-1 Mitochondrial potential assay JC-1 is cationic dye that changes fluorescence with mitochondrial membrane potential. Mitochondrial membrane potential (ΔΨm) was evaluated by fluorescence microscope using 5,5, 6,6′-tetrachloro-1, 1′, 3,3′ -tetraethylbenzimidazole-carbocyanide iodine (JC-1; Beyotime, China) staining [29]. After nsPEF treatment, cells were stained with JC1 at 37 °C for 20 min. Images were taken with a fluorescence microscope. When mitochondrial membrane potential drops, the fluorescent emission changes from red to green. The intensity of red and green emission was analyzed to represent mitochondrial membrane potential changes. 2.9. Measurement of nitric oxide (NO) Total NO concentration in the nsPEF treated buffer system (without Tca8113 cells) was determined by measuring the concentration of
Fig. 3. Cell survival rates of Tca8113 cells were determined after nsPEFs plus radiation combination treatment for 24 h, 48 h and 72 h. Results are presented of the decreased values from the control group. The statistical significance among groups of radiation and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t-test. *p b 0.05.
38
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
Fig. 4. Clonogenic survival for Tca8113 cells after nsPEFs, radiation alone and combined treatment. a.) Photographic difference in colony formation in treated groups. b.) Dose-survival curves fitted using a single-hit multi-target model showing the radiosensitizing effects of nsPEFs plus radiation in Tca8113 cells. Data points are mean values (plus standard deviations) from three independent determinations.
nitrate and nitrite, a stable metabolite of NO, according to the Griess assay with the Total Nitric Oxide Assay Kit (Beyotime Institute of Biotechnology, China). 2.10. Statistics All data were processed by Origin Professional 8.0 software. The statistical significance between the control, nsPEF, radiation and combination treatment groups was calculated by ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with a Student-t-test and significance was considered when p b 0.05. 3. Results and discussion Survival rates of Tca8113 cells treated with nsPEFs are shown in Fig. 2. When a high field of strength 50 kV/cm and 20 pulses were applied, Tca8113 cell viability (43.87 ± 1.21) was reduced to 50% of control group at 72 h. After 20 kV/cm, 20 pulses treatment, the growth survival rates were 86.13 ± 1.02 (24 h), 81.96 ± 3.41 (48 h) and 73.42 ± 0.21 (72 h), respectively. Thus, high fields strengths (over 20 kV/cm) showed that nsPEFs reduced Tca8113 cell survival in a dose (field strength & incubation time) dependent manner. This result is consistent with our previous report of the nsPEF synergistic effect of gemcitabine on TSCC cancer cell line Cal-27 [15,16]. However, Tca8113 cell inhibition effects caused by low field strength 10 kV/cm, 20 pulses were not statistically significant compared with control samples (P N 0.05). The results showed a different effect on Tca8113 cells proliferation and these results are in accordance with the previous studies. Bo Su et al. suggested that low field intensity could significantly improve the pre-growth of Haloxylon ammodendron [13]. Kun Zhang et al. demonstrated that exposing chondrocytes to nsPEFs at low field intensity led to enhanced proliferation and de-differentiation [30]. In all, both academic analyses and medical experiment results showed that a low field intensity can induce cell proliferation [13,30] while a high field intensity induce cell inhibition [15,16]. These results agree with previous findings that nsPEFs showed a window effect on different biological cells [31]. Based on this consideration, we chose 20 kV/ cm, 20 pulses as a moderate field strength for combination treatment with radiation. Fig. 3 shows cell survival rates after nsPEF (20 kV/cm, 20 pulses) and radiation (2, 4, 6 and 8 Gy) alone and in combination by MTT assay.
When radiation was used alone, there was a dose-dependent increase in cell death. Combination treatment groups showed more significant inhibition effects on tumor cells growth compared with that in the other two groups (radiation alone, nsPEFs alone), and the differences were significant (P b 0.05). Survival rate of the 20 KV/cm plus 2 Gy combination group (83.8 ± 1.5%, 24 h) is lower than the survival rate of 4 Gy group (86.6 ± 1.81%, 24 h). Meanwhile, the 20 kV/cm plus 4 Gy combination group, survival rate (75.6 ± 3.25%, 24 h) is lower than the 6 Gy radiation alone group survival rate (81.2 ± 2.11%, 24 h), while the 20 kV/cm plus 6 Gy combination group survival rate (71.14 ± 3.25%, 24 h) is lower than the 8 Gy radiation alone survival rate (73.47 ± 2.11%, 24 h). More importantly, these results show that the combination treatments have a dose and time-dependent effect on Tca8113 cells. Taking a different viewpoint, for similar treatment outcomes on Tca8113 cells, we speculated that nsPEFs could significantly reduce the radiation dose in TSCC cancer treatment. For example, the 20 kV/cm plus 4 Gy combination group had a similar treatment effect compared with the 6 Gy radiation alone group. More specifically, nsPEFs reduced radiation dose exposure to Tca8113 cancer cells by 33%. Furthermore, through the above data analysis we can easily see that a potential radiosensitizing effect of nsPEFs on Tca8113 cells. The mean lethal dose (D0) is defined as that amount of radiation required to reduce the survival by 67% from any point on the linear portion of the curve. Quasi-threshold dose (Dq) is a measure of how much damage occurs before Tcsa8113 cells is lethal. SF2 is a survival fraction at irradiation dose of 2 Gy. In this study, a single-hit multi-target model was used to plot survival curves and calculate overall radiosensitivity parameters (D0, Dq, SF2, SER) [25,26]. Tca8113 cell viability was assessed by clonogenic assay and the combination treatment decreased the ability of Tca8113 cells to repair radiation-induced sub-lethal injury (Fig. 4). From the results in Fig. 4A, we can see there was a dose dependent increase in growth inhibition with radiation treatment. This result from clonogenic assays was consistent with the MTT data shown in Fig. 3. Dose–survival curves fitted using a single-hit multi-target model are shown in Fig. 4B. SER = 1.453 ± 0.038 N 1, which shows that nsPEFs has a significant radiosensitizing effect on Tca-8113 cells. The radiosensitivity parameters D0, Dq and SF2 were significantly reduced in irradiated cells pretreated with nsPEFs. After combination treatment, mean lethal dose of radiation
Table 2 Impact of nsPEFs plus radiation on G2/M (mitotic) phase arrest in Tca8113 cell. Table 1 The radiosensitivity parameters of Tca8113 cell survival curves after radiation and combination treatment. Group
D0
Dq
SF2
Radiation nsPEFs + Radiation
3.40 2.34
1.37 0.7
0.8 0.45
Group
G1 Phase(%)
S Phase(%)
G2/M Phase(%)
Control 4 Gy 20 kV/cm 20 kv/cm + 4Gy
52.93 41.12 49.85 40.43
33.73 26.26 32.07 19.84
13.34 32.62 18.08 39.73
± 16.84 ± 1.46⁎ ± 8.23 ± 1.47⁎
± 9.31 ± 3.21⁎ ± 3.27 ± 2.41⁎⁎##
± 2.61 ± 1.96⁎⁎ ± 3.06* ± 0.48⁎⁎##
VS control group ⁎P b 0.05, ⁎⁎P b 0.01; VS radiation group #P b 0.05, ##P b 0.01.
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
39
Fig. 5. Tca8113 cell apoptosis under different conditions (control group, nsPEFs group, radiotherapy group and combination group). a.) Cells were assessed by FACSauto flow cytometry. b.) The early apoptosis induced by different groups. c.) The late apoptosis induced by different groups. The statistical significance among groups of nsPEFs, radiotherapy and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t-test. (*P b 0.05).
(D0, Gy) value decreased from 3.40 to 2.34, indicating an enhancement of radiation sensitivity of nsPEFs on Tca8113 cells. The value of the quasithreshold dose (Dq, Gy) also decreased from 1.37 to 0.7, suggesting nsPEFs treatment led to the inhibition of cellular capacity to repair potentially lethal radiation damage. Thus, combined with the proliferation results in Fig. 3 we concluded that radiosensitization of Tca8113 cells by
Fig. 6. JC-1 mitochondrial potential assay of Tca8113 cells treated with nsPEFs and radiation. Data expressed as mean of 590 nm/520 nm emission spectrum ± SEM, n = 4. The statistical significance among groups of nsPEFs, radiotherapy and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t-test. (*P b 0.05).
nsPEFs was also field strength-dependent. So, we can change nsPEFs parameters to achieve complete treatment sensitization of different cancer cells. Cell cycle arrest was the major reason for death induced by radiosensitization drugs [32]. It is well known that there is different radiation sensitivity at different phases of the cell cycle with more radiation sensitivity at G2/M phase and cell cycle arrest [33]. Furthermore, the mechanism of classical chemical sensitization in the radiotherapy always induces G2/M phase arrest [34,35]. In order to make sure the radiosensitivity of nsPEFs was due to cell cycle arrest, the influence of nsPEFs plus radiation treatment on cell cycle distribution was observed. As shown in Table 2, there was a significant difference between the number of cells found in G2/M phase in the control group (13.34 ± 2.61) and the nsPEFs group (18.08 ± 3.06), P b 0.05. The number of cells in G2/M phase was found to be increased in the 4 Gy radiation group, 20 kV/cm nsPEFs group and the 4 Gy plus 20 kV/cm combination group. Especially in the combination treatment group, the G2/M phase cells (39.73 ± 0.48) were clearly increased compared with the control group (13.34 ± 2.61) and the radiation group (32.62 ± 1.96), P b 0.01. Importantly, recent studies of nsPEFs on the cell cycle suggested that the nsPEF effects can be shown to be cell cycle specific and could induce G2/M arrest [19,20,36]. However, the exact mechanism which occurs in the cell cycle process is still not clear. Currently, it is well known that exposure to nsPEFs can cause DNA damage [14,36,37]. Thus, we speculate that nsPEFs at 20 kV/cm induced
Fig. 7. Total NO concentration in the nsPEF treated buffer system (without Tca8113 cells). Bars indicate average expression (± SD) of three replicates. Statistically significant comparisons are indicated by a star (Student's t-test, *P b 0.05).
40
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
DNA damage and evoked a cell cycle checkpoint (such as Cyclin B) in Tca8113 cells. The cell cycle checkpoint (such as Cyclin B) can then prevent or delay Tca8113 cell progression through the cell cycle to repair DNA damage before re-entering the cell cycle and finally resulting in a G2/M phase arrest. Generally, for fast-dividing mammalian cells (Tca8113), the length of the cell cycle is approximately 24 h [38]. So, we choose a 24 h of delay between the electric field treatment and radiotherapy (Fig. 2). Interestingly, EH H et al. found that nsPEFs effects on the nucleus and cytoskeleton may provide synergistic therapeutic actions with other agents, such as ionizing radiation or chemotherapy agents [39]. Based on these results, we conclude that nsPEF induced G2/M phase arrest in Tca8113 cells is one of the mechanism for radiosensitization. (See Table 1.) Apoptosis was the major reason for cell death induced by radiosensitization drugs [40]. The numbers of apoptotic and necrotic cells were determined by Annexin- V-FITC and PI double staining. As shown in Fig. 5, there was a significant difference in the early apoptosis rate between the combination group (26.72 ± 3.74) and radiation group (13.21 ± 2.31), P b 0.05. Greater numbers of Tca8113 cells showing early apoptosis were observed when nsPEFs were combined with 4 Gy. Therefore, apoptosis induction has a close relationship to the improved radiosensitivity of TSCC due to nsPEFs. One early characteristic of apoptosis is the loss of mitochondrial membrane potential [41]. In this study, we examined the mitochondrial membrane potential (ΔΨm) by the JC-1 staining. The ratio of red to green fluorescence serves as a readout of mitochondrial membrane potential. JC-1 staining (Fig. 6A) and relative fluorescence intensity (Fig. 6B, C) shows that combination treatment with nsPEFs and radiotherapy can make green fluorescence intensity higher than with other. This indicates that the mitochondrial membrane potential of Tca-8113 cells is significantly decreased after combination treatment. This result is in accordance with the cell apoptosis assay in Fig. 5. Thus, it can be seen that the mechanism of nsPEFs sensitization is similar to that of chemical sensitization agents, both of which were caused by apoptosis induction. Our previous study reported that nsPEFs at 20 kV/cm can induce exogenous NO and endogenous. NO production [13]. Furthermore, NO was almost as efficient as oxygen in improving the radiosensitivity of different cancer cells [42,43]. In order to determine whether nsPEFs induced radiosensitization is related to NO, total NO concentration was also measured in this study. We tested the total NO concentration of each sample group without Tca8113 cells. As shown in Fig. 7, the total NO concentrations in the buffer solution (RPMI-1640 medium) of controls (10.01 ± 1.93 μmol/L) are significantly lower than those of nsPEF treated groups (p b 0.05). Significantly, nsPEFs increased NO production in treated buffer solution. The NO concentration detected in nsPEF treatment groups were 15.29 ± 2.09, 20.64 ± 2.57, 22.36 ± 0.64 and 29.71 ± 3.12 μmol/L for 10 to 50 kV/cm, 20 pulses. The existence of exogenous NO in buffer solution may depend on the arc discharge during the pulse electric field and other physical process [44]. According to arc discharge theory, it is possible that trace amounts of N2 and O2 from dissolved air in the buffer solution react with nsPEFs and generate the exogenous NO. These results suggest that NO may be a factor in the enhancement of radiosensitivity in TSCC by nsPEF treatment.
4. Conclusion In summary, nsPEFs exhibited potent radiosensitivity against the human oral tongue cancer cell line Tca8113 in vitro. This radiosensitivity was variously related to cell cycle arrest at G2/M phase, cell apoptosis induction and NO production in nsPEF treated system. Our study provides an experimental basis for the clinical application of nsPEFs combined with radiotherapy in TSCC cancer treatment. As an alternative strategy, nsPEFs could provide a potentially feasible way for radiosensitization of oral tongue squamous cell carcinoma.
Acknowledgement This project was supported by the Fundamental Research Funds for the Central Universities (Grant Number: lzujbky-2014-159), National Natural Science Foundation of China (Grant Number: 81372893), Natural Science Foundation of Gansu Province (Grant Number: 1208RJZA193) and the Peking University Biomed-X Foundation.
References [1] L.M. Brown, D.P. Check, S.S. Devesa, Oral cavity and pharynx cancer incidence trends by subsite in the United States: changing gender patterns, Journal of Oncology 4 (2012). [2] S. Cpatel, W. Rcarpenter, S. Tyree, M. Ecouch, M. Cweissler, T. Hackman, D. Neilhayes, C. Gshores, B. Schera, Increasing incidence of oral tongue squamous cell carcinoma in young white women, age 18 to 44 years, J. Clin. Oncol. 29 (2011) 1488–1494. [3] P. Erikpetersen, Oral cancer prevention and control – the approach of the World Health Organization, Oral Oncol. 45 (2009) 454–460. [4] K. Omura, Current status of oral cancer treatment strategies: surgical treatments for oral squamous cell carcinoma, Int. J. Clin. Oncol. 19 (2014) 423–430. [5] S. Krishnamurthi, V. Shanta, M. Krishnannair, Studies of chemical sensitization in the radiotherapy of oral and cervical carcinomas, Cancer 20 (1967) 822–825. [6] R. Sealy, A. Williams, S. Cridland, M. Stratford, A. Minchinton, C. Hallet, A report on misonidazole in a randomized trial in locally advanced head and neck cancer, Int. J. Radiat. Oncol. Biol. Phys. 8 (1982) 339–342. [7] L.L. Herscher, J. Cook, Taxanes as radiosensitizers for head and neck cancer, Curr. Opin. Oncol. 11 (1999) 183–186. [8] E.T. Shinohara, A. Maity, B.S.N. Jha, R.A. Lustig, Sirolimus as a potential radiosensitizer in squamous cell cancer of the head and neck, Head Neck 31 (2009) 406–411. [9] K.H. Schoenbach, S.J. Beebe, E.S. Buescher, Intracellular effect of ultrashort electrical pulses, Bioelectromagnetics 22 (2001) 440–448. [10] S.J. Beebe, P.M. Fox, L.J. Rec, K. Somers, R.H. Stark, K.H. Schoenbach, Nanosecond pulsed electric field (nspef) effects on cells and tissues: apoptosis induction and tumor growth inhibition, IEEE Transactions on Plasma Science. 30 (2002) 286–292. [11] B. Hargreaves, F. Li, Nanosecond pulse electric field activated-platelet rich plasma enhances the return of blood flow to large and ischemic wounds in a rabbit model, Phys. Rep. 3 (2015). [12] A. Guionet, F. David, C. Zaepffel, M. Coustets, K. Helmi, C. Cheype, D. Packan, J. Garnier, V. Blanckaert, J. Teissie, E. coli electroeradication on a closed loop circuit by using milli-, micro- and nanosecond pulsed electric fields: Comparison between energy costs, Bioelectrochemistry (2015) 65–73. [13] B. Su, J. Guo, W. Nian, H. Feng, K. Wang, J. Zhang, J. Fang, Early growth effects of nanosecond pulsed electric field (nsPEFs) exposure on Haloxylon ammodendron, Plasma Process. Polym. 12 (2015) 372–379. [14] K.H. Schoenbach, R.P. Joshi, J.F. Kolb, N. Chen, M. Stacey, P.F. Blackmore, Ultrashort electrical pulses open a new gateway into biological cells, Proc. IEEE 92 (2004) 1122–1137. [15] J. Wang, J. Guo, S. Wu, H. Feng, S. Sun, J. Pan, J. Zhang, S.J. Beebe, Synergistic effects of nanosecond pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro, PLoS One 7 (2012). [16] W. Qi, J. Guo, S. Wu, B. Su, L. Zhang, J. Pan, J. Zhang, Synergistic effect of nanosecond pulsed electric field combined with low-dose of pingyangmycin on salivary adenoid cystic carcinoma, Oncol. Rep. 31 (2014) 2220–2228. [17] R. Nuccitelli, R. Wood, M. Kreis, B. Athos, J. Huynh, K. Lui, P. Nuccitelli, E. Hepstein, First-in-human trial of nanoelectroablation therapy for basal cell carcinoma: proof of method, Exp. Dermatol. 23 (2014) 135–137. [18] S. Wu, Y. Wang, J. Guo, Q. Chen, J. Zhang, J. Fang, Nanosecond pulsed electric fields as a novel drug free therapy for breast cancer: an in vivo study, Cancer Lett. 343 (2014) 268–274. [19] M. Stacey, J. Stickley, P. Fox, V. Statler, K.H. Schoenbach, S.J. Beebe, S. Buescher, Differential effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA damage, and cell cycle analysis, Mutation Research/fundamental & Molecular Mechanisms of Mutagenesis. 542 (2003) 65–75. [20] L. Estlack, B.L. Ibey, Effects of nano-second electrical pulses (nspefs) on cell cycle progression and susceptibility at various phases, Proceedings of SPIE - The International Society for Optical Engineering. 8585 (2013). [21] J.F. Kolb, K. Susumu, K.H. Schoenbach, Nanosecond pulsed electric field generators for the study of subcellular effects, Bioelectromagnetics 27 (2006) 172–187. [22] S. Romeo, L. Zeni, M. Sarti, A. Sannino, P.T. Vernier, O. Zeni, DNA electrophoretic migration patterns change after exposure of jurkat cells to a single intense nanosecond electric pulse, PLoS One 6 (2011), e28419. [23] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity, Cancer Res. 47 (1987) 936–942. [24] X.D. Jiang, Y. Qiao, P. Dai, Q. Chen, J. Wu, D.A. Song, Enhancement of recombinant human endostatin on the radiosensitivity of human pulmonary adenocarcinoma a549 cells and its mechanism, J. Bioenerg. Biomembr. 301931 (2012). [25] G.S. Tiwana, R. Prevo, F.M. Buffa, S. Yu, D.V. Ebner, A. Howarth, Identification of vitamin b1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen, Oncotarget 6 (2015) 5978–5989.
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41 [26] A. Hercbergs, P.J. Davis, F.B. Davis, M.J. Ciesielski, J.T. Leith, Radiosensitization of gl261 glioma cells by tetraiodothyroacetic acid (tetrac), Cell Cycle 8 (2009) 2586–2591. [27] S.M. Huang, J.M. Bock, P.M. Harari, Epidermal growth factor receptor blockade with c225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of the head and neck, Cancer Res. 59 (1999) 1935–1940. [28] Y.F. Ho, S.A. Karsani, W.K. Yong, S.N. Abd Malek, Induction of apoptosis and cell cycle blockade by helichrysetin in a549 human lung adenocarcinoma cells, Evid. Based Complement. Alternat. Med. 221-229 (2013). [29] A. Cossarizza, M. Baccaranicontri, G. Kalashnikova, C. Franceschi, A new method for the cytofluorometric analysis of mitochondrial membrane potential using the j-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolcarbocyanine iodide (jc-1), Biochem. Biophys. Res. Commun. 197 (1993) 40–45. [30] K. Zhang, J. Guo, Z. Ge, J. Zhang, Nanosecond pulsed electric fields (nspefs) regulate phenotypes of chondrocytes through wnt/β-catenin signaling pathway, Sci. Report. 4 (2014) 5836. [31] C. Yao, X. Hu, Y. Mi, C. Li, C. Sun, Window effect of pulsed electric field on biological cells, Dielectrics & Electrical Insulation IEEE Transactions on. 16 (2009) 1259–1266. [32] C.J. Mcginn, E.M. Miller, M.J. Lindstrom, The role of cell cycle redistribution in radiosensitization: implications regarding the mechanism of fluorodeoxyuridine radiosensitization, International Journal of Radiation Oncologybiologyphysics. 30 (1994) 851–859. [33] S.J. Knox, W. Sutherland, M.L. Goris, Correlation of tumor sensitivity to low-doserate irradiation with g2/m-phase block and other radiobiological parameters, Radiat. Res. 135 (1993) 24–31. [34] A.F. Wahl, K.L. Donaldson, C. Fairchild, F.Y. Lee, S.A. Foster, G.W. Demers, Loss of normal p53 function confers sensitization to taxol by increasing g2/m arrest and apoptosis, Nat. Med. 2 (1996) 72–79.
41
[35] S.Q. He, H. Rehman, M.G. Gong, Y.Z. Zhao, Z.Y. Huang, C.H. Li, Inhibiting survivin expression enhances trail-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest, Cancer Biology & Therapy. 6 (2007) 1258–1268. [36] M.A. Mahlke, C. Navara, B.L. Ibey, Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and jurkat cells, Proceedings of SPIE - The International Society for Optical Engineering. 8941 (2014). [37] E.H. Hall, K.H. Schoenbach, S.J. Beebe, Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null hct116 colon carcinoma cells, Apoptosis 12 (2007) 1721–1731. [38] H.A. Cameron, R.D. Mckay, Restoring production of hippocampal neurons in old age, Nat. Neurosci. 2 (1999) 894–897. [39] E.H. Hall, K.H. Schoenbach, S.J. Beebe, Nanosecond pulsed electric fields have differential effects on cells in the s-phase, DNA Cell Biol. 26 (2007) 160–171. [40] R.J. Muschel, W.G. Mckenna, E.J. Bernhard, Radiosensitization and apoptosis, Oncogene 17 (1998) 3359–3363. [41] A. Mcgill, A. Frank, N. Emmett, D.M. Turnbull, M.A. Birchmachin, N.J. Reynolds, The anti-psoriatic drug anthralin accumulates in keratinocyte mitochondria, dissipates mitochondrial membrane potential, and induces apoptosis through a pathway dependent on respiratory competent mitochondria, FASEB J. 19 (2005) 1012–1014. [42] B.F. Jordan, P. Sonveaux, O. Feron, V. Grégoire, N. Beghein, C. Dessy, Nitric oxide as a radiosensitizer: evidence for an intrinsic role in addition to its effect on oxygen delivery and consumption, Int. J. Cancer 109 (2004) 768–773. [43] J.F. Jeannin, L. Leon, M. Cortier, N. Sassi, C. Paul, A. Bettaieb, Nitric oxide-induced resistance or sensitization to death in tumor cells, Nitric Oxide 19 (2008) 158–163. [44] M.S. Stark, J.T.H. Harrison, C. Anastasi, Formation of nitrogen oxides by electrical discharges and implications for atmospheric lightning, J. Geophys. Res. 101 (1996) 6963–6969.
Contents lists available at ScienceDirect
Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Radiosensitization of oral tongue squamous cell carcinoma by nanosecond pulsed electric fields (nsPEFs) Jinsong Guo a, Yu Wang b, Jing Wang c,⁎, Jue Zhang a,b,⁎⁎, Jing Fang a,b a b c
College of Engineering, Peking University, Beijing, 100871, China Academy of Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China Department of Oral Medicine, School of Stomatology, Lanzhou University, Lanzhou, Gansu, China
a r t i c l e
i n f o
Article history: Received 10 November 2015 Received in revised form 16 September 2016 Accepted 20 September 2016 Available online 22 September 2016 Keywords: Radiosensitization nsPEFs TSCC G2/M arrest Nitric oxide Apoptosis
a b s t r a c t Nanosecond pulsed electric fields (nsPEFs) are a non-thermal and non-toxic technology that induce a myriad of biological effects. They have been proven to be effective in tumor shrinkage, but few studies focus on its radiosensitization in oral tongue squamous cell carcinoma. The purpose of this research was to study the radiosensitization effect of nsPEFs on a human oral tongue cancer cell line Tca8113 and to investigate the potential antitumor mechanism. A Tca8113 cell line was tested respectively by MTT assay, clonogenic assay, flow cytometry assay, annexin V-FITC/PI assay, mitochondrial potential assay and total nitric oxide assay. Our results showed that nsPEFs had a time and field strength dependent inhibition effect on Tca8113 cells. The sensitization enhancement ratio (SER) of nsPEFs was 1.453 ± 0.038. Furthermore, radiation induced G2/M arrest was augmented by treatment with nsPEFs. We observed many more Tca8113 cells showing early apoptosis after nsPEFs combined with radiotherapy. Additionally, the NO concentration was significantly increased after nsPEFs treatment. These findings indicate that combination of nsPEFs with radiotherapy can enhance the radiosensitivity of Tca8113 cells and nsPEFs could be a potential radiosensitizer for oral tongue squamous cell carcinoma. © 2016 Published by Elsevier B.V.
1. Introduction Oral tongue squamous cell carcinoma (TSCC) is one of the most common malignancies of the head and neck region. Recent survey data suggests that the incidence of TSCC is actually increasing in young and middle age populations [1,2]. Despite advances in cancer treatment, the 5-year survival rate has remained at about 50% over the past several decades [3]. Usually, operation and irradiation are the mainstay treatments for TSCC patients. However, surgical treatment for patients often produces a significant adverse effect on speech function, chewing and swallowing ability. Further, there is a high recurrence rate after surgery [4]. Thus, how to further improve the sensitivity of oral tongue cancer cells to radiation has become a research hotspot. Recently, looking for new, safe and effective radiosensitizers has become a novel strategy in oral tongue cancer treatment. Since the 1960s chemical sensitization in radiotherapy has already been studied [5]. Up until now, we have obtained some stable and high radiosensitizing effect radiosensitizers, such as misonidazole (MISO), taxanes and sirolimus. With advances in new radiosensitizers, the survival of TSCC patients have improved significantly [6–8]. ⁎ Corresponding author. ⁎⁎ Correspondence to: J. Zhang, College of Engineering, Peking University, Beijing 100871, China. E-mail addresses: [email protected] (J. Wang), [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.bioelechem.2016.09.002 1567-5394/© 2016 Published by Elsevier B.V.
However, it is difficult to avoid the toxicity effects induced by the chemical radiosensitizers, especially for the advanced cancer patients and those with TSCC recurrence after radiation therapy. Overall, we need to find an effective sensitization technology for radiotherapy in TSCC treatment without toxic side effects. Typically, the effects of pulsed electric fields on biological cells have been investigated since the late 1950s. More recently, the duration of the electric fields has been shortened to nanoseconds [9]. Many previous studies have found that nanosecond pulsed electric fields (nsPEFs) are able to induce a series of medical and biological effects. The pertinent areas include tumor shrinkage, wound healing, sterilization, and plant growth promoting [10–13]. Meanwhile, the present work describes in detail the anticancer activity of nsPEFs, including cell apoptosis, cytochrome C release, calcium release and DNA damage [14]. Additionally, some studies have also showed that nsPEFs have synergistic effects on low concentration chemotherapy drugs in oral cancer cell lines [15,16]. In particular, Nuccitelli R completed the first-in-human safety trials of nsPEFs in basal cell carcinoma (BCC). The results indicated that nsPEFs are safe and may offer a fast and scarless alternative to cancer treatment [17]. Also, Shan Wu, et al. found that nsPEFs treatment did not create permanent damage to the skin or normal tissues and only left shallow marks on the skin that faded completely in two weeks [18]. Moreover, we concluded that nsPEFs can effectively induce cell cycle arrest at G2/M phase [19,20]. Moreover, we inferred that NO was positively involved in the early growth effects of Haloxylon ammodendron seed
36
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
Fig. 1. a.) The photo of 60 ns nsPEFs generator used in this experiment. b.) Typical oscillogram of 60 ns pulse generator. c.) Circuit diagram of the basic Blumlein pulse forming system. Two cable length of 12 m and one cable length of 6 m were chosen in order to generate 60 ns pulses.
after nsPEFs exposure [13]. However, so far, there is no study focusing on the effect of nsPEFs for radiosensitization in oral tongue squamous cell carcinoma. The present study aimed to investigate the proliferation, apoptosis and cell cycle effects of nsPEFs combined with radiation in a human tongue squamous cell carcinoma Tac8113 cell line, providing a new theoretical basis of nsPEFs for radiosensitization in oral tongue squamous cell carcinoma. 2. Materials and methods 2.1. Cell line and cell culture Human squamous cell carcinoma Tca8113 cells were cultured in RPMI-1640 medium (GIBCO BRL, Rockville, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). No antibiotics were used in the preparation of the medium. Cells were grown under 5% CO2 in a humidified atmosphere at 37 °C. Cells in the logarithmic growth phase were used to perform the experiments described follows. 2.2. nsPEFs treatment In this study, the nsPEF generator was applied as previously described [21,22]. A Blumlein line pulse generator (Fig. 1A.) produced nearly rectangular 60 ns pulses that were delivered to the Tca8113 cells by a 2 mm cuvette. The electric field strength varied from 10 kV/cm to 50 kV/cm. A digital phosphor oscilloscope (DPO4054, Tektronix) with a probe (P6015A, Tektronix) was utilized to monitor the voltage waveform (Fig. 1B.). Tca8113 cells were counted with a hemocytometer, and 2.0 × 106 cells suspended in 500 μL culture medium were added to 2 mm gap cuvettes (Biosmith, San Diego, CA). Then the experimental
cuvettes were exposed to 20 pulses of nsPEFs of 60 ns duration at an electric field strength of 10, 20, 30 and 50 kV/cm, respectively. Cuvettes that did not undergo nsPEF treatment served as the control group. The time between each pulse was about 1 s. 2.3. Radiation platform To simulate clinical conditions, Tca8113 cells were irradiated with Xrays at 6 MV at room temperature using a linear accelerator (Elekta Precise, Stockholm, Sweden) under the source-to-skin distance (the distance from the radiation source to the central surface of the plate – 100 cm), and the dose rate was 2.0 Gy/min. 2.4. Cell proliferation assay The effects of nsPEFs on Tca8113 cells were determined with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The MTT assay uses a MTT dye which acts as a substrate for viable cell reductase enzyme. This enzyme reduces MTT yellow dye to purple colour formazan which is in proportional to viable cell eventually [23]. When cells began to grow exponentially, they were harvested and resuspended at a concentration of 2.0 × 106 cells/ml. A 500 μl cell suspension (1.0 × 106 cells) was placed in a 2 mm gap cuvette (Biosmith) and then treated with nsPEFs at electric fields of 0, 10, 20, 30 and 50 kV/cm. After incubation for 24, 48, and 72 h, 20 μL MTT (5 mg/ml) was added to each well, and cells were further incubated at 37 °C for 4 h. The medium was then removed and 200 μL of DMSO was added to dissolve the reduced formazan product. MTT dye intensity was then read on a micro plate reader (BioRad) at 492 nm. The survival rates under different field strengths were calculated according to the following formula [24]: Survival rate = A value of tested well/A value of control well × 100%. Using this same method, we investigated the anti-
Scheme 1. A schematic diagram of the experimental arrangement, including logarithmic growth phase cells preparation, cells collecting, nsPEFs treatment, cell culture, radiotherapy of Tca8113 cells and data detection.
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
37
2.6. Cell-cycle distribution
Fig. 2. Tca8113 cell survival after treatment with nsPEFs. Cells were treated with nsPEFs at 10, 20, 30 and 50 kV/cm, 20 pulses, respectively. After incubation for 24 h, 48 h and 72 h, cell survival rates were determined by MTT assay. Results are presented as the percentage of the decreased values from the control cells.
proliferative effects of nsPEFs combined with radiation. In the treatment of sensitizing cells with nsPEFs before radiation treatment, cells were detached and treated with nsPEFs, then dispensed into 90-mm plates and further incubated at 37 °C for 24 h. After that, Cells were treated with different doses of radiation (2, 4, 6 and 8 Gy) and cell survival rates were analyzed after 24, 48, and 72 h by MTT assay. A schematic diagram of the experimental arrangement is shown in Scheme 1.
2.5. Clonogenic survival assay In radiation biology, clonogenic assay is the gold standard assay for determining reproductive cell death after radiation treatment [25]. In this study, the survival fraction (SF) of Tca8113 cells in each group under different conditions was determined by clonogenic assay. After irradiation, Tca8113 cells were immediately trypsinized and suspended. The cells were then seeded in triplicate in 60 mm petri dishes at a different cell densities (control 200 cells, nsPEFs 200 cells, 2 Gy 800 cells, 4 Gy 1600 cells, 6 Gy 3200 cells and 8 Gy 6400 cells). After incubation for 10 days, the cells were washed twice with phosphate-buffered saline, fixed in methanol, and then stained with Giemsa stain. Colonies (≥50 cells) were counted for computing percent growth inhibition. Plating efficiency (PE) and the survival fraction (SF) were calculated according to the following formula: PE = (clone count/cell count) × 100%, SF = (PE in observed group/PE in 0 Gy group) × 100%. Then, a single-hit multi−D N
target (SHMT) radiobiological model {S ¼ 1−ð1−e D0 Þ } was applied to delineate survival curves [26]. The mean lethal dose (D0), quasithreshold dose (Dq), survival fraction at irradiation dose of 2 Gy (SF2) and sensitization enhancement ratio (SER) were calculated based on cell survival curves of single-hit multi-target model, respectively. Cell survival curves (Fig. 4B) were fitted with Sigmaplot™ 10.0 software (Systat Software, Inc., Chicago, IL, USA).
The cell cycle was determined with a flow cytometer and the cellcycle phases were analyzed by measuring the DNA fragments stained with propidium iodide (PI; Sigma-Aldrich). Tca8113 cells at log phase were inoculated in 6-well plate at 5.0 × 105 cells in each well. Four groups were set as the control group, nsPEFs group, radiotherapy group and combination group, respectively. Tca8113 cells were harvested and centrifuged at 24 h after different treatments. Then, the cells were washed twice with pre-cooled phosphate-buffered saline. After this the cells were fixed and permeabilized overnight by adding 1 mL of 70% pre-cooled ethanol to each tube at 4 °C. After centrifugation, the fixatives were decanted and the cell pellets were resuspended in 0.5 mL of staining solution containing 200 μL each of DNAse-free RNAse (Sigma-Aldrich) and PI and incubated for 30 min at room temperature in the dark [27]. The distribution of the Tca8113 cells in the cell cycle phases was analyzed from the DNA histogram using a FACSCaliber™ flow cytometer and CellQuest™ software (BD Biosciences, San Jose, CA, USA). 2.7. Cell apoptosis An Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharmingen) was used for assessing apoptosis induced by nsPEFs combined with radiation [28]. Annexin-V-FITC and PI were used to evaluate normal cells (no staining), early apoptotic cells (Annexin-V-FITC positive, PI negative) and necrotic cells (Annexin-V-FITC and PI positive). The Tca8113 cells were harvested and centrifuged at 24 h after different treatments. Then cells stained with Annexin-V-FITC in a dark at room temperature for 15 min, and cells were stained with PI on ice for 30 min. Samples were assessed by FACSauto flow cytometry (Becton Dickinson, USA). 2.8. JC-1 Mitochondrial potential assay JC-1 is cationic dye that changes fluorescence with mitochondrial membrane potential. Mitochondrial membrane potential (ΔΨm) was evaluated by fluorescence microscope using 5,5, 6,6′-tetrachloro-1, 1′, 3,3′ -tetraethylbenzimidazole-carbocyanide iodine (JC-1; Beyotime, China) staining [29]. After nsPEF treatment, cells were stained with JC1 at 37 °C for 20 min. Images were taken with a fluorescence microscope. When mitochondrial membrane potential drops, the fluorescent emission changes from red to green. The intensity of red and green emission was analyzed to represent mitochondrial membrane potential changes. 2.9. Measurement of nitric oxide (NO) Total NO concentration in the nsPEF treated buffer system (without Tca8113 cells) was determined by measuring the concentration of
Fig. 3. Cell survival rates of Tca8113 cells were determined after nsPEFs plus radiation combination treatment for 24 h, 48 h and 72 h. Results are presented of the decreased values from the control group. The statistical significance among groups of radiation and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t-test. *p b 0.05.
38
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
Fig. 4. Clonogenic survival for Tca8113 cells after nsPEFs, radiation alone and combined treatment. a.) Photographic difference in colony formation in treated groups. b.) Dose-survival curves fitted using a single-hit multi-target model showing the radiosensitizing effects of nsPEFs plus radiation in Tca8113 cells. Data points are mean values (plus standard deviations) from three independent determinations.
nitrate and nitrite, a stable metabolite of NO, according to the Griess assay with the Total Nitric Oxide Assay Kit (Beyotime Institute of Biotechnology, China). 2.10. Statistics All data were processed by Origin Professional 8.0 software. The statistical significance between the control, nsPEF, radiation and combination treatment groups was calculated by ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with a Student-t-test and significance was considered when p b 0.05. 3. Results and discussion Survival rates of Tca8113 cells treated with nsPEFs are shown in Fig. 2. When a high field of strength 50 kV/cm and 20 pulses were applied, Tca8113 cell viability (43.87 ± 1.21) was reduced to 50% of control group at 72 h. After 20 kV/cm, 20 pulses treatment, the growth survival rates were 86.13 ± 1.02 (24 h), 81.96 ± 3.41 (48 h) and 73.42 ± 0.21 (72 h), respectively. Thus, high fields strengths (over 20 kV/cm) showed that nsPEFs reduced Tca8113 cell survival in a dose (field strength & incubation time) dependent manner. This result is consistent with our previous report of the nsPEF synergistic effect of gemcitabine on TSCC cancer cell line Cal-27 [15,16]. However, Tca8113 cell inhibition effects caused by low field strength 10 kV/cm, 20 pulses were not statistically significant compared with control samples (P N 0.05). The results showed a different effect on Tca8113 cells proliferation and these results are in accordance with the previous studies. Bo Su et al. suggested that low field intensity could significantly improve the pre-growth of Haloxylon ammodendron [13]. Kun Zhang et al. demonstrated that exposing chondrocytes to nsPEFs at low field intensity led to enhanced proliferation and de-differentiation [30]. In all, both academic analyses and medical experiment results showed that a low field intensity can induce cell proliferation [13,30] while a high field intensity induce cell inhibition [15,16]. These results agree with previous findings that nsPEFs showed a window effect on different biological cells [31]. Based on this consideration, we chose 20 kV/ cm, 20 pulses as a moderate field strength for combination treatment with radiation. Fig. 3 shows cell survival rates after nsPEF (20 kV/cm, 20 pulses) and radiation (2, 4, 6 and 8 Gy) alone and in combination by MTT assay.
When radiation was used alone, there was a dose-dependent increase in cell death. Combination treatment groups showed more significant inhibition effects on tumor cells growth compared with that in the other two groups (radiation alone, nsPEFs alone), and the differences were significant (P b 0.05). Survival rate of the 20 KV/cm plus 2 Gy combination group (83.8 ± 1.5%, 24 h) is lower than the survival rate of 4 Gy group (86.6 ± 1.81%, 24 h). Meanwhile, the 20 kV/cm plus 4 Gy combination group, survival rate (75.6 ± 3.25%, 24 h) is lower than the 6 Gy radiation alone group survival rate (81.2 ± 2.11%, 24 h), while the 20 kV/cm plus 6 Gy combination group survival rate (71.14 ± 3.25%, 24 h) is lower than the 8 Gy radiation alone survival rate (73.47 ± 2.11%, 24 h). More importantly, these results show that the combination treatments have a dose and time-dependent effect on Tca8113 cells. Taking a different viewpoint, for similar treatment outcomes on Tca8113 cells, we speculated that nsPEFs could significantly reduce the radiation dose in TSCC cancer treatment. For example, the 20 kV/cm plus 4 Gy combination group had a similar treatment effect compared with the 6 Gy radiation alone group. More specifically, nsPEFs reduced radiation dose exposure to Tca8113 cancer cells by 33%. Furthermore, through the above data analysis we can easily see that a potential radiosensitizing effect of nsPEFs on Tca8113 cells. The mean lethal dose (D0) is defined as that amount of radiation required to reduce the survival by 67% from any point on the linear portion of the curve. Quasi-threshold dose (Dq) is a measure of how much damage occurs before Tcsa8113 cells is lethal. SF2 is a survival fraction at irradiation dose of 2 Gy. In this study, a single-hit multi-target model was used to plot survival curves and calculate overall radiosensitivity parameters (D0, Dq, SF2, SER) [25,26]. Tca8113 cell viability was assessed by clonogenic assay and the combination treatment decreased the ability of Tca8113 cells to repair radiation-induced sub-lethal injury (Fig. 4). From the results in Fig. 4A, we can see there was a dose dependent increase in growth inhibition with radiation treatment. This result from clonogenic assays was consistent with the MTT data shown in Fig. 3. Dose–survival curves fitted using a single-hit multi-target model are shown in Fig. 4B. SER = 1.453 ± 0.038 N 1, which shows that nsPEFs has a significant radiosensitizing effect on Tca-8113 cells. The radiosensitivity parameters D0, Dq and SF2 were significantly reduced in irradiated cells pretreated with nsPEFs. After combination treatment, mean lethal dose of radiation
Table 2 Impact of nsPEFs plus radiation on G2/M (mitotic) phase arrest in Tca8113 cell. Table 1 The radiosensitivity parameters of Tca8113 cell survival curves after radiation and combination treatment. Group
D0
Dq
SF2
Radiation nsPEFs + Radiation
3.40 2.34
1.37 0.7
0.8 0.45
Group
G1 Phase(%)
S Phase(%)
G2/M Phase(%)
Control 4 Gy 20 kV/cm 20 kv/cm + 4Gy
52.93 41.12 49.85 40.43
33.73 26.26 32.07 19.84
13.34 32.62 18.08 39.73
± 16.84 ± 1.46⁎ ± 8.23 ± 1.47⁎
± 9.31 ± 3.21⁎ ± 3.27 ± 2.41⁎⁎##
± 2.61 ± 1.96⁎⁎ ± 3.06* ± 0.48⁎⁎##
VS control group ⁎P b 0.05, ⁎⁎P b 0.01; VS radiation group #P b 0.05, ##P b 0.01.
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
39
Fig. 5. Tca8113 cell apoptosis under different conditions (control group, nsPEFs group, radiotherapy group and combination group). a.) Cells were assessed by FACSauto flow cytometry. b.) The early apoptosis induced by different groups. c.) The late apoptosis induced by different groups. The statistical significance among groups of nsPEFs, radiotherapy and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t-test. (*P b 0.05).
(D0, Gy) value decreased from 3.40 to 2.34, indicating an enhancement of radiation sensitivity of nsPEFs on Tca8113 cells. The value of the quasithreshold dose (Dq, Gy) also decreased from 1.37 to 0.7, suggesting nsPEFs treatment led to the inhibition of cellular capacity to repair potentially lethal radiation damage. Thus, combined with the proliferation results in Fig. 3 we concluded that radiosensitization of Tca8113 cells by
Fig. 6. JC-1 mitochondrial potential assay of Tca8113 cells treated with nsPEFs and radiation. Data expressed as mean of 590 nm/520 nm emission spectrum ± SEM, n = 4. The statistical significance among groups of nsPEFs, radiotherapy and combination treatment was calculated with ANOVA analysis in SPSS. The significance between two groups (two orders of treatments) was calculated with Student-t-test. (*P b 0.05).
nsPEFs was also field strength-dependent. So, we can change nsPEFs parameters to achieve complete treatment sensitization of different cancer cells. Cell cycle arrest was the major reason for death induced by radiosensitization drugs [32]. It is well known that there is different radiation sensitivity at different phases of the cell cycle with more radiation sensitivity at G2/M phase and cell cycle arrest [33]. Furthermore, the mechanism of classical chemical sensitization in the radiotherapy always induces G2/M phase arrest [34,35]. In order to make sure the radiosensitivity of nsPEFs was due to cell cycle arrest, the influence of nsPEFs plus radiation treatment on cell cycle distribution was observed. As shown in Table 2, there was a significant difference between the number of cells found in G2/M phase in the control group (13.34 ± 2.61) and the nsPEFs group (18.08 ± 3.06), P b 0.05. The number of cells in G2/M phase was found to be increased in the 4 Gy radiation group, 20 kV/cm nsPEFs group and the 4 Gy plus 20 kV/cm combination group. Especially in the combination treatment group, the G2/M phase cells (39.73 ± 0.48) were clearly increased compared with the control group (13.34 ± 2.61) and the radiation group (32.62 ± 1.96), P b 0.01. Importantly, recent studies of nsPEFs on the cell cycle suggested that the nsPEF effects can be shown to be cell cycle specific and could induce G2/M arrest [19,20,36]. However, the exact mechanism which occurs in the cell cycle process is still not clear. Currently, it is well known that exposure to nsPEFs can cause DNA damage [14,36,37]. Thus, we speculate that nsPEFs at 20 kV/cm induced
Fig. 7. Total NO concentration in the nsPEF treated buffer system (without Tca8113 cells). Bars indicate average expression (± SD) of three replicates. Statistically significant comparisons are indicated by a star (Student's t-test, *P b 0.05).
40
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41
DNA damage and evoked a cell cycle checkpoint (such as Cyclin B) in Tca8113 cells. The cell cycle checkpoint (such as Cyclin B) can then prevent or delay Tca8113 cell progression through the cell cycle to repair DNA damage before re-entering the cell cycle and finally resulting in a G2/M phase arrest. Generally, for fast-dividing mammalian cells (Tca8113), the length of the cell cycle is approximately 24 h [38]. So, we choose a 24 h of delay between the electric field treatment and radiotherapy (Fig. 2). Interestingly, EH H et al. found that nsPEFs effects on the nucleus and cytoskeleton may provide synergistic therapeutic actions with other agents, such as ionizing radiation or chemotherapy agents [39]. Based on these results, we conclude that nsPEF induced G2/M phase arrest in Tca8113 cells is one of the mechanism for radiosensitization. (See Table 1.) Apoptosis was the major reason for cell death induced by radiosensitization drugs [40]. The numbers of apoptotic and necrotic cells were determined by Annexin- V-FITC and PI double staining. As shown in Fig. 5, there was a significant difference in the early apoptosis rate between the combination group (26.72 ± 3.74) and radiation group (13.21 ± 2.31), P b 0.05. Greater numbers of Tca8113 cells showing early apoptosis were observed when nsPEFs were combined with 4 Gy. Therefore, apoptosis induction has a close relationship to the improved radiosensitivity of TSCC due to nsPEFs. One early characteristic of apoptosis is the loss of mitochondrial membrane potential [41]. In this study, we examined the mitochondrial membrane potential (ΔΨm) by the JC-1 staining. The ratio of red to green fluorescence serves as a readout of mitochondrial membrane potential. JC-1 staining (Fig. 6A) and relative fluorescence intensity (Fig. 6B, C) shows that combination treatment with nsPEFs and radiotherapy can make green fluorescence intensity higher than with other. This indicates that the mitochondrial membrane potential of Tca-8113 cells is significantly decreased after combination treatment. This result is in accordance with the cell apoptosis assay in Fig. 5. Thus, it can be seen that the mechanism of nsPEFs sensitization is similar to that of chemical sensitization agents, both of which were caused by apoptosis induction. Our previous study reported that nsPEFs at 20 kV/cm can induce exogenous NO and endogenous. NO production [13]. Furthermore, NO was almost as efficient as oxygen in improving the radiosensitivity of different cancer cells [42,43]. In order to determine whether nsPEFs induced radiosensitization is related to NO, total NO concentration was also measured in this study. We tested the total NO concentration of each sample group without Tca8113 cells. As shown in Fig. 7, the total NO concentrations in the buffer solution (RPMI-1640 medium) of controls (10.01 ± 1.93 μmol/L) are significantly lower than those of nsPEF treated groups (p b 0.05). Significantly, nsPEFs increased NO production in treated buffer solution. The NO concentration detected in nsPEF treatment groups were 15.29 ± 2.09, 20.64 ± 2.57, 22.36 ± 0.64 and 29.71 ± 3.12 μmol/L for 10 to 50 kV/cm, 20 pulses. The existence of exogenous NO in buffer solution may depend on the arc discharge during the pulse electric field and other physical process [44]. According to arc discharge theory, it is possible that trace amounts of N2 and O2 from dissolved air in the buffer solution react with nsPEFs and generate the exogenous NO. These results suggest that NO may be a factor in the enhancement of radiosensitivity in TSCC by nsPEF treatment.
4. Conclusion In summary, nsPEFs exhibited potent radiosensitivity against the human oral tongue cancer cell line Tca8113 in vitro. This radiosensitivity was variously related to cell cycle arrest at G2/M phase, cell apoptosis induction and NO production in nsPEF treated system. Our study provides an experimental basis for the clinical application of nsPEFs combined with radiotherapy in TSCC cancer treatment. As an alternative strategy, nsPEFs could provide a potentially feasible way for radiosensitization of oral tongue squamous cell carcinoma.
Acknowledgement This project was supported by the Fundamental Research Funds for the Central Universities (Grant Number: lzujbky-2014-159), National Natural Science Foundation of China (Grant Number: 81372893), Natural Science Foundation of Gansu Province (Grant Number: 1208RJZA193) and the Peking University Biomed-X Foundation.
References [1] L.M. Brown, D.P. Check, S.S. Devesa, Oral cavity and pharynx cancer incidence trends by subsite in the United States: changing gender patterns, Journal of Oncology 4 (2012). [2] S. Cpatel, W. Rcarpenter, S. Tyree, M. Ecouch, M. Cweissler, T. Hackman, D. Neilhayes, C. Gshores, B. Schera, Increasing incidence of oral tongue squamous cell carcinoma in young white women, age 18 to 44 years, J. Clin. Oncol. 29 (2011) 1488–1494. [3] P. Erikpetersen, Oral cancer prevention and control – the approach of the World Health Organization, Oral Oncol. 45 (2009) 454–460. [4] K. Omura, Current status of oral cancer treatment strategies: surgical treatments for oral squamous cell carcinoma, Int. J. Clin. Oncol. 19 (2014) 423–430. [5] S. Krishnamurthi, V. Shanta, M. Krishnannair, Studies of chemical sensitization in the radiotherapy of oral and cervical carcinomas, Cancer 20 (1967) 822–825. [6] R. Sealy, A. Williams, S. Cridland, M. Stratford, A. Minchinton, C. Hallet, A report on misonidazole in a randomized trial in locally advanced head and neck cancer, Int. J. Radiat. Oncol. Biol. Phys. 8 (1982) 339–342. [7] L.L. Herscher, J. Cook, Taxanes as radiosensitizers for head and neck cancer, Curr. Opin. Oncol. 11 (1999) 183–186. [8] E.T. Shinohara, A. Maity, B.S.N. Jha, R.A. Lustig, Sirolimus as a potential radiosensitizer in squamous cell cancer of the head and neck, Head Neck 31 (2009) 406–411. [9] K.H. Schoenbach, S.J. Beebe, E.S. Buescher, Intracellular effect of ultrashort electrical pulses, Bioelectromagnetics 22 (2001) 440–448. [10] S.J. Beebe, P.M. Fox, L.J. Rec, K. Somers, R.H. Stark, K.H. Schoenbach, Nanosecond pulsed electric field (nspef) effects on cells and tissues: apoptosis induction and tumor growth inhibition, IEEE Transactions on Plasma Science. 30 (2002) 286–292. [11] B. Hargreaves, F. Li, Nanosecond pulse electric field activated-platelet rich plasma enhances the return of blood flow to large and ischemic wounds in a rabbit model, Phys. Rep. 3 (2015). [12] A. Guionet, F. David, C. Zaepffel, M. Coustets, K. Helmi, C. Cheype, D. Packan, J. Garnier, V. Blanckaert, J. Teissie, E. coli electroeradication on a closed loop circuit by using milli-, micro- and nanosecond pulsed electric fields: Comparison between energy costs, Bioelectrochemistry (2015) 65–73. [13] B. Su, J. Guo, W. Nian, H. Feng, K. Wang, J. Zhang, J. Fang, Early growth effects of nanosecond pulsed electric field (nsPEFs) exposure on Haloxylon ammodendron, Plasma Process. Polym. 12 (2015) 372–379. [14] K.H. Schoenbach, R.P. Joshi, J.F. Kolb, N. Chen, M. Stacey, P.F. Blackmore, Ultrashort electrical pulses open a new gateway into biological cells, Proc. IEEE 92 (2004) 1122–1137. [15] J. Wang, J. Guo, S. Wu, H. Feng, S. Sun, J. Pan, J. Zhang, S.J. Beebe, Synergistic effects of nanosecond pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro, PLoS One 7 (2012). [16] W. Qi, J. Guo, S. Wu, B. Su, L. Zhang, J. Pan, J. Zhang, Synergistic effect of nanosecond pulsed electric field combined with low-dose of pingyangmycin on salivary adenoid cystic carcinoma, Oncol. Rep. 31 (2014) 2220–2228. [17] R. Nuccitelli, R. Wood, M. Kreis, B. Athos, J. Huynh, K. Lui, P. Nuccitelli, E. Hepstein, First-in-human trial of nanoelectroablation therapy for basal cell carcinoma: proof of method, Exp. Dermatol. 23 (2014) 135–137. [18] S. Wu, Y. Wang, J. Guo, Q. Chen, J. Zhang, J. Fang, Nanosecond pulsed electric fields as a novel drug free therapy for breast cancer: an in vivo study, Cancer Lett. 343 (2014) 268–274. [19] M. Stacey, J. Stickley, P. Fox, V. Statler, K.H. Schoenbach, S.J. Beebe, S. Buescher, Differential effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA damage, and cell cycle analysis, Mutation Research/fundamental & Molecular Mechanisms of Mutagenesis. 542 (2003) 65–75. [20] L. Estlack, B.L. Ibey, Effects of nano-second electrical pulses (nspefs) on cell cycle progression and susceptibility at various phases, Proceedings of SPIE - The International Society for Optical Engineering. 8585 (2013). [21] J.F. Kolb, K. Susumu, K.H. Schoenbach, Nanosecond pulsed electric field generators for the study of subcellular effects, Bioelectromagnetics 27 (2006) 172–187. [22] S. Romeo, L. Zeni, M. Sarti, A. Sannino, P.T. Vernier, O. Zeni, DNA electrophoretic migration patterns change after exposure of jurkat cells to a single intense nanosecond electric pulse, PLoS One 6 (2011), e28419. [23] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity, Cancer Res. 47 (1987) 936–942. [24] X.D. Jiang, Y. Qiao, P. Dai, Q. Chen, J. Wu, D.A. Song, Enhancement of recombinant human endostatin on the radiosensitivity of human pulmonary adenocarcinoma a549 cells and its mechanism, J. Bioenerg. Biomembr. 301931 (2012). [25] G.S. Tiwana, R. Prevo, F.M. Buffa, S. Yu, D.V. Ebner, A. Howarth, Identification of vitamin b1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen, Oncotarget 6 (2015) 5978–5989.
J. Guo et al. / Bioelectrochemistry 113 (2017) 35–41 [26] A. Hercbergs, P.J. Davis, F.B. Davis, M.J. Ciesielski, J.T. Leith, Radiosensitization of gl261 glioma cells by tetraiodothyroacetic acid (tetrac), Cell Cycle 8 (2009) 2586–2591. [27] S.M. Huang, J.M. Bock, P.M. Harari, Epidermal growth factor receptor blockade with c225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of the head and neck, Cancer Res. 59 (1999) 1935–1940. [28] Y.F. Ho, S.A. Karsani, W.K. Yong, S.N. Abd Malek, Induction of apoptosis and cell cycle blockade by helichrysetin in a549 human lung adenocarcinoma cells, Evid. Based Complement. Alternat. Med. 221-229 (2013). [29] A. Cossarizza, M. Baccaranicontri, G. Kalashnikova, C. Franceschi, A new method for the cytofluorometric analysis of mitochondrial membrane potential using the j-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolcarbocyanine iodide (jc-1), Biochem. Biophys. Res. Commun. 197 (1993) 40–45. [30] K. Zhang, J. Guo, Z. Ge, J. Zhang, Nanosecond pulsed electric fields (nspefs) regulate phenotypes of chondrocytes through wnt/β-catenin signaling pathway, Sci. Report. 4 (2014) 5836. [31] C. Yao, X. Hu, Y. Mi, C. Li, C. Sun, Window effect of pulsed electric field on biological cells, Dielectrics & Electrical Insulation IEEE Transactions on. 16 (2009) 1259–1266. [32] C.J. Mcginn, E.M. Miller, M.J. Lindstrom, The role of cell cycle redistribution in radiosensitization: implications regarding the mechanism of fluorodeoxyuridine radiosensitization, International Journal of Radiation Oncologybiologyphysics. 30 (1994) 851–859. [33] S.J. Knox, W. Sutherland, M.L. Goris, Correlation of tumor sensitivity to low-doserate irradiation with g2/m-phase block and other radiobiological parameters, Radiat. Res. 135 (1993) 24–31. [34] A.F. Wahl, K.L. Donaldson, C. Fairchild, F.Y. Lee, S.A. Foster, G.W. Demers, Loss of normal p53 function confers sensitization to taxol by increasing g2/m arrest and apoptosis, Nat. Med. 2 (1996) 72–79.
41
[35] S.Q. He, H. Rehman, M.G. Gong, Y.Z. Zhao, Z.Y. Huang, C.H. Li, Inhibiting survivin expression enhances trail-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest, Cancer Biology & Therapy. 6 (2007) 1258–1268. [36] M.A. Mahlke, C. Navara, B.L. Ibey, Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and jurkat cells, Proceedings of SPIE - The International Society for Optical Engineering. 8941 (2014). [37] E.H. Hall, K.H. Schoenbach, S.J. Beebe, Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null hct116 colon carcinoma cells, Apoptosis 12 (2007) 1721–1731. [38] H.A. Cameron, R.D. Mckay, Restoring production of hippocampal neurons in old age, Nat. Neurosci. 2 (1999) 894–897. [39] E.H. Hall, K.H. Schoenbach, S.J. Beebe, Nanosecond pulsed electric fields have differential effects on cells in the s-phase, DNA Cell Biol. 26 (2007) 160–171. [40] R.J. Muschel, W.G. Mckenna, E.J. Bernhard, Radiosensitization and apoptosis, Oncogene 17 (1998) 3359–3363. [41] A. Mcgill, A. Frank, N. Emmett, D.M. Turnbull, M.A. Birchmachin, N.J. Reynolds, The anti-psoriatic drug anthralin accumulates in keratinocyte mitochondria, dissipates mitochondrial membrane potential, and induces apoptosis through a pathway dependent on respiratory competent mitochondria, FASEB J. 19 (2005) 1012–1014. [42] B.F. Jordan, P. Sonveaux, O. Feron, V. Grégoire, N. Beghein, C. Dessy, Nitric oxide as a radiosensitizer: evidence for an intrinsic role in addition to its effect on oxygen delivery and consumption, Int. J. Cancer 109 (2004) 768–773. [43] J.F. Jeannin, L. Leon, M. Cortier, N. Sassi, C. Paul, A. Bettaieb, Nitric oxide-induced resistance or sensitization to death in tumor cells, Nitric Oxide 19 (2008) 158–163. [44] M.S. Stark, J.T.H. Harrison, C. Anastasi, Formation of nitrogen oxides by electrical discharges and implications for atmospheric lightning, J. Geophys. Res. 101 (1996) 6963–6969.