Jonathan H. Sherman, MD is a current member of the PLOS One Editorial Board. His co-authorship on this manuscript does not alter the authors' adherence to the PLOS ONE Editorial policies and criteria.
Conceived and designed the experiments: AS OV XC SK KB FM MK JS. Performed the experiments: AS OV XC SK. Analyzed the data: AS OV XC SK MK JS. Contributed reagents/materials/analysis tools: KB FM MK JS. Wrote the paper: AS OV XC SK KB FM MK JS.
Current address: Department of Radiation Oncology, Stanford School of Medicine, Stanford, CA, United States of America
Cold atmospheric plasma (CAP) has recently been shown to selectively target cancer cells with minimal effects on normal cells. We systematically assessed the effects of CAP in the treatment of glioblastoma.
Three glioma cell lines, normal astrocytes, and endothelial cell lines were treated with CAP. The effects of CAP were then characterized for viability, cytotoxicity/apoptosis, and cell cycle effects. Statistical significance was determined with student's t-test.
CAP treatment decreases viability of glioma cells in a dose dependent manner, with the ID50 between 90-120 seconds for all glioma cell lines. Treatment with CAP for more than 120 seconds resulted in viability less than 35% at 24-hours posttreatment, with a steady decline to less than 20% at 72-hours. In contrast, the effect of CAP on the viability of NHA and HUVEC was minimal, and importantly not significant at 90 to 120 seconds, with up to 85% of the cells remained viable at 72-hours post-treatment. CAP treatment produces both cytotoxic and apoptotic effects with some variability between cell lines. CAP treatment resulted in a G2/M-phase cell cycle pause in all three cell lines.
This preliminary study determined a multi-focal effect of CAP on glioma cells in vitro, which was not observed in the non-tumor cell lines. The decreased viability depended on the treatment duration and cell line, but overall was explained by the induction of cytotoxicity, apoptosis, and G2/M pause. Future studies will aim at further characterization with more complex pre-clinical models.
Cold atmospheric plasma (CAP) is a partially ionized gas that can focally deliver low levels of ultraviolet (UV) radiation and ionized particles to synergistically generate reactive oxygen species (ROS). This technology has demonstrated efficacy in several biomedical applications ranging from anti-bacterial decontamination to promotion of wound healing [
Glioblastoma is a highly malignant primary central nervous system neoplasm associated with poor survival and is invariably fatal. It is considered one of the most aggressive forms of human cancers, characterized largely by its rapid growth, extensive angiogenesis, and invariable resistance to all current therapies. Consequently, treatments remain largely palliative despite recent advances with the integration of multi-modal therapies. The current standard of care, known as the Stupp protocol, improves median survival up to 14.6 months [
Recurrence of glioblastoma is typically local, due in large part to poor treatment distribution, limited therapeutic penetration, and development of drug resistance to chemotherapeutic agents [
Glioma cell lines (U87, U373, A172) and human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection (Manassas, VA). Human normal astrocytes E6/E7 were generously donated by Dr. Andrew Parsa at The University of California San Francisco. All the cell lines were maintained at 37°C, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) plus 1% penicillin/streptomycin mixture. At ~70% confluence, the cells were re-plated onto (1) 96-well plates for the MTT assay and ApoTox-Glo™ Triplex assay, or (2) 12-well plates for cell cycle determination through propidium iodide staining followed by flow cytometry. Experiments were undertaken 48 hours post-plating, at ~40% confluence.
The CAP jet is the dielectric barrier discharge in helium that is described elsewhere [
(A) Cold atmospheric plasma (CAP) jet, with which can be mounted for treatment directly over culture plate containing various glioma cell lines (U87, U373, A172) for various durations. (B) The parameters of the CAP were an output voltage between 4.5–5 kV, a frequency of 13 kHz and a helium flow rate at 10 L/min. The distance between the jet and the base of the culture plate was maintained at 20 mm, yielding an average jet dose of 0.8 J/sec/cm2.
The ionized nitrogen species and highly reactive oxygen radicals are present in the CAP spectrum, which was stable over time, and consisted largely reactive nitrogen and oxygen species (
Spectra of reactive species generated by CAP on media (DMEM, 10% FBS, 1% abx), which produces a stable proportion of reactive species over the duration of treatment (30 to 180 seconds).
The MTT assay was used to quantify viability of each cell line after treatment with CAP for 60 to 180 seconds. The cells were incubated with 1.7 mM of Thiazolyl Blue Tetrazolium Bromide (Sigma, cat#M2128) for 3 hours at 37°C. This tetrazolium salt is metabolically reduced by the mitochondria of viable cells to yield a blue insoluble Formosan product that is detectable spectrophotometrically. The percentage of surviving treated cells compared to control cells was calculated from the average OD570 values obtained in each experiment, and plotted over time. The MTT assay was performed at 24, 48, and 72 hours post-CAP treatment.
Untreated controls were maintained throughout the experiment as a negative control, also measured at 570 nm. The viability of the treated cells were calculated as a percentage this negative control.
The ApoTox-Glo Triplex Assay was purchased from Promega Corp (G6320; Madison, WI). This assay combines three distinct chemistries to assess viability, cytotoxicity, and caspase 3/7 activity within a single well format, as per protocol. Briefly, the simultaneous measurement of two proteases was used as markers of viability and cytotoxicity through glycylphenylalanyl-aminofluorocoumarin (GF-AFC) and bis-alanylalanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110), respectively. GF-AFC is cell permeable and thus enters an intact cell where it is cleaved by a live-cell protease to generate a fluorescent signal proportional to the number of living cells. Conversely, this live-cell protease becomes inactive upon loss of cell membrane integrity and leakage into the surrounding culture medium. Bis-AAF-R110 is used to measure dead-cell protease activity, which is released with loss of cell membrane integrity. As bis-AAF-R110 is not cell-permeable, no signal is generated from intact cells. The simultaneous determinaton of both products was made possible due to different excitation/emission spectra, as measured by the BioTek Synergy H1 Hybrid Microplate Reader. The viability and cytotoxicity were measured at 4, 24, and 48 hours post-CAP treatment.
The second component of the assay utilizes a caspase-Glo™ 3/7 reagent in lysed cells, which produces luminescent signal by luciferase that is measured by a BioTek Synergy H1 Hybrid Microplate Reader. The caspase 3/7 activity was measured at 4, 24, and 48 hours post-CAP treatment.
Each of the glioma cells were plated in 24-well plates. After 48 hours, at ~ 40% confluence, the cells were treated for either 90 or 120 s CAP. After treatment with CAP, the DNA content stained with propidium iodide/RNase staining buffer (BD Pharmingen, cat# 550825) in 0.1% Triton X-100. Cells were trypsinized with 0.25% Trypsin EDTA (Invitrogen, Gibco, cat# 25200), washed with PBS prior to fixation in 70% ethyl alcohol, stored at -20°C for 24 hours and then stained.
DNA content was measured using flow cytometry on a FACSCalibur DxP8 at the GWU Flow Cytometry Core Facility (manufactured by BD Biosciences and upgraded by Cytek Development). The analyzer is equipped with three lasers (providing excitation wavelengths of 488, 637 and 407 nm) and eight detectors for fluorescence. The DNA content was measured at 24 and 48 hours post-CAP treatment.
Results are expressed as mean ± SEM. A one-way analysis of variance was performed for multiple comparisons, and if there was a significant variation between the treatment groups, the mean values for a treated group were compared with those of the control by Student’s
The
Results of MTT assay from (A) A172, (B) U373, (C) U87, (D) NHA and (E) HUVEC treated with varying durations of cold atmospheric plasma (CAP). After treatment, the MTT assay was performed at 24, 48, and 72 hours. Viability was calculated relative to control. All three glioma cell lines exhibited a dose-dependent decline in viability with CAP treatments ranging from 60 to 180 seconds. The effects of CAP were sustained up to 72 hours post-treatment. Compared to the other two cancer cell lines, U87 seemed to be more resistant to CAP treatment for 90 seconds. No significant response was seen with NHA, and only transient effects observed on HUVEC cells. * denotes statistical significance compared to control (P <0.05).
MTT Assay Post-Treatment (hours) | Duration of CAP Treatment (seconds) | Glioma Cell Line % Viability (SEM) | ||
---|---|---|---|---|
A172, (n = 22) | U373, (n = 22) | U87 (n = 22) | ||
24 | 60 | 86 (0.09) | 97 (0.04) | 90 (0.05) |
90 | 69 (0.03) |
64 (0.06) |
76 (0.04) |
|
120 | 17 (0.04) |
28 (0.07) |
32 (0.09) |
|
180 | 4 (0.04) |
6 (0.01) |
7 (0.01) |
|
48 | 60 | 88 (0.2) | 67 (0.05) |
81 (0.04) |
90 | 45 (0.1) |
42 (0.06) |
56 (0.04) |
|
120 | 14 (0.03) |
28 (0.08) |
31 (0.03) |
|
180 | 13 (0.04) |
28 (0.09) |
7 (0.02) |
|
72 | 60 | 114 (0.14) | 59 (0.05) |
74 (0.04) |
90 | 33 (0.04) |
31 (0.06) |
55 (0.02) |
|
120 | 9 (0.02) |
18 (0.05) |
13 (0.03) |
|
180 | 12 (0.05) |
10 (0.03) |
6 (0.02) |
Results of MTT assay from three glioma cell lines, A172, U373, and U87 treated with varying durations of cold atmospheric plasma (CAP). After treatment, the MTT assay was performed at 24, 48, and 72 hours. Viability was calculated relative to control.
*Denotes significance to p< 0.05 relative to control.
From this data, the duration of treatment necessary to decrease the viability by 50% (ID50) was calculated for each cell line at 24, 48 and 72 hours post-treatment (
Duration of CAP treatment necessary to decrease viability by 50%, termed the inhibitory duration (ID
In contrast to the glioma cells, CAP treatment did not significantly decrease the viability on NHA and HUVEC cells as assessed by the MTT assay. At the maximal treatment duration of 180 seconds, the viability of the NHA remained 78% at 72 hours post-treatment. No significant decline in viability was observed for treatment times between 60 to 120 seconds in NHA cells (
A transient but significant decline to >85% viability was observed at 24 hours with CAP treatments of 60 to 120 seconds, followed by reconstitution to greater than 95% viability by 72 hours (
Treatment with CAP for 60 seconds resulted in significant cytotoxicity for A172 at 24 and 48 h, and for U373 at 4 and 48 hours (
Briefly, cells were treated with CAP for 60, 90 or 120 seconds, incubated with bis-AAF-R110 at 4, 24 and 48 hours post-treatment for 30 minutes for 30 minutes, and emission quantified, as per protocol. bis-AAF-R110 cleavage calculated and plotted relative to untreated control cells. (A) Cytotoxicity was greatest for A172 24 hours post-treatment. (B) Cytotoxicity was greatest for U373 at 3 and 48 hours post-treatment. (C) CAP treatment did not produce a significant amount of cytotoxicity, as measured by bis-AAF-R110 cleavage.* denotes statistical significance compared to control (P <0.05).
Apoptosis, as measured by functional caspase 3/7 activity, was also variable across the glioma cell lines (
Briefly, cells were treated with CAP for 60, 90 or 120 seconds, incubated with DEVD at 4, 24 and 48 hours post-treatment for 30 minutes, and emission quantified, as per protocol. DEVD cleavage calculated and plotted relative to untreated control cells. (A) Treatment with the lowest dosage of CAP, 60s, induced apoptosis in A172. Higher dosages induced cytotoxicity instead of apoptosis. (B) Apoptosis was significantly increased at 48 hours post-CAP at 120 seconds. At 4 and 24 hours post-CAP, a significant decline in caspase 3/7 activity was detected, likely representing a decline in total population due to the cytotoxic effects observed in
Consistent with our previous findings [
Briefly, each of the glioma cells were treated with 90s CAP, and DNA content was measured at 24, 48, and 72 hours post-CAP treatment. In A172, CAP treatment for 90s resulted in a significant G2/M arrest at (B) 48 and (C) 72 hours, but not at (A) 24 hours. In U373, 90s CAP treatment resulted in a significant increase in the S-phase and G2/M population at (D) 24 hours and sustained at (E) 48 and (F) 72 hours. In U87, CAP treatment for 90s resulted in a significant increase in the G2/M population at (G) 24 and (H) 48 hours that diminished by (I) 72 hours. * denotes statistical significance compared to control (P <0.05).
The unique chemical and physical properties of cold atmospheric plasmas have permitted broad applications in biomedicine, and the recent emergence of the field of plasma healthcare. The most widely established use of plasma is for the sterilization of fragile materials sensitive to thermal damage. Subsequent studies have identified several clinical uses ranging from dental care to various skin diseases such as wound healing, foot fungus, and general hand sterilization without disturbing the skin barrier [
Investigations into the mechanism of injury have pointed to a synergy of several major biologically active components: (1) reactive oxygen species, (2) charge particles, (electric field) (3) UV (primarily UV-C) radiation and (4) ionizing radiation to generate reactive species to cause further damage [
While prior research displayed a dose dependent effect of CAP on such tumors as colon cancer, our data indicate a dose-dependent effect of CAP on glioma cells, and suggests an optimal treatment duration between 90 to 120 seconds based on the ID50 [
Among the three glioma cell lines, some variability in sensitivity to CAP treatment is seen based on the MTT assay, with the greatest albeit non-significant resilience seen in the U87 cell line. Examination into the cytotoxic and apoptotic effects also suggests that U87 glioma cells were more resistant to CAP treatment. However, the ID50 for U87 remained between 90 and 120 seconds. With regards to the U373 and A172, treatment with CAP resulted in varying but significant degrees of cytotoxicity and caspase 3/7 activation that account for the decreased viability seen with the MTT assay. In specific, U373 exhibited early and delayed cytotoxic effects from CAP with a significant increase in apoptosis observed 48 hours post-treatment. Coupled with the observation that the cell cycle of U373 cells are exquisitely sensitive to CAP treatment, it is likely that the decreased viability in U373 cells are due to early direct effects and also delayed indirect effects induced by CAP. With regards to the A172 cell line, the predominant mechanism of CAP-mediated injury was cytotoxicity, with a significant increase in apoptosis at the lower CAP treatment duration. The results also indicate that varying the duration of CAP treatment can favor either cytotoxicity resulting in necrosis or apoptosis.
Gene expression studies have indicated that glioblastoma are composed of distinct subtypes [
Although more rigorous studies are necessary, CAP may be a promising option to address the limitations of the current treatment algorithm. Glioblastoma is a highly infiltrative tumor that extends beyond the margins of a clinical target volume. Whether it is "micro-infiltration" or en masse infiltration, current therapies can benefit from the addition of a treatment modality specifically directed towards the resection cavity as a great majority of the recurrences are local [
Synergistic effects of UV and ionizing radiation have been previously described [
Ultimately, the efficacy of CAP will depend on the ability to optimize the chemical and physical properties of plasmas to maximize therapeutic efficacy with minimal side-effects. This study identifies the optimal treatment duration between 90–120 seconds with our CAP settings, which largely are seen to be effective in decreasing the viability in glioma cells but not in NHA or HUVEC cell lines.
Treatment of malignant cells with CAP