Yonsei Med J.  2014 Nov;55(6):1640-1647. 10.3349/ymj.2014.55.6.1640.

Nonthermal Plasma Induces Apoptosis in ATC Cells: Involvement of JNK and p38 MAPK-Dependent ROS

• Lee SY
• Kang SU
• Kim KI
• Kang S
• Shin YS
• Chang JW
• Yang SS
• Lee K
• Lee JS
• Moon E
• Kim CH

• KMID: 2070214
• DOI: http://doi.org/10.3349/ymj.2014.55.6.1640

Abstract

PURPOSE
To determine the effects of nonthermal plasma (NTP) induced by helium (He) alone or He plus oxygen (O2) on the generation of reactive oxygen species (ROS) and cell death in anaplastic thyroid cancer cells.
MATERIALS AND METHODS
NTP was generated in He alone or He plus O2 blowing through a nozzle by applying a high alternating current voltage to the discharge electrodes. Optical emission spectroscopy was used to identify various excited plasma species. The apoptotic effect of NTP on the anaplastic thyroid cancer cell lines, such as HTH83, U-HTH 7, and SW1763, was verified with annexin V/propidium staining and TUNEL assay. ROS formation after NTP treatment was identified with fluorescence-activated cell sorting with DCFDA staining. The mitogen-activated protein kinase pathways and caspase cascade were investigated to evaluate the molecular mechanism involved and cellular targets of plasma.
RESULTS
NTP induced significant apoptosis in all three cancer cell lines. The plasma using He and O2 generated more O2-related species, and increased apoptosis and intracellular ROS formation compared with the plasma using He alone. NTP treatment of SW1763 increased the expression of phosphor-JNK, phosphor-p38, and caspase-3, but not phosphor-ERK. Apoptosis of SW1763 as well as expressions of elevated phosphor-JNK, phosphor-p38, and caspase-3 induced by NTP were effectively inhibited by intracellular ROS scavengers.
CONCLUSION
NTP using He plus O2 induced significant apoptosis in anaplastic cancer cell lines through intracellular ROS formation. This may represent a new promising treatment modality for this highly lethal disease.

Keyword

Nonthermal plasma; ROS; anaplastic thyroid cancer; apoptosis; helium; oxygen

MeSH Terms

Apoptosis/*drug effects
Caspase 3/*metabolism
Flow Cytometry
Humans
Male
Plasma Gases/*pharmacology
Reactive Oxygen Species/*metabolism
Spectrometry, X-Ray Emission
Thyroid Carcinoma, Anaplastic
p38 Mitogen-Activated Protein Kinases/*metabolism
Caspase 3
Plasma Gases
Reactive Oxygen Species
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 (A) Schematic diagram of the plasma torch. (B) Image of the plasma jet with helium (He) and oxygen (O2). (C) Plasma emission spectra with different gases, comparing He versus He+O2 at 2 kV.

• 

  Fig. 2 Cell viability assay on ATC cells. (A) SW1736. (B) HTH83. (C) U-HTH 7. Cells were treated with gas only or each type of plasma jets at 2 kV for 1 s. At 24 hours after plasma treatment, cell viability was measured by a MTT assay. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 with Student's t-test compared with the control. ATC, anaplastic thyroid cancer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

• 

  Fig. 3 Analysis of the death of ATC cells by annexin V/PI staining. (A) SW1736. (B) HTH83. (C) U-HTH 7. Cells were treated with gas only or plasma jets at 2 kV for 1 s and then incubated for 24 hours, and stained with annexin V-FITC and propidium iodide (PI). Apoptosis was detected using a FACS system. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 with Student's t-test compared with the control. FITC, fluorescein isothiocyanate; ATC, anaplastic thyroid cancer; FACS, fluorescence activated cell sorter.

• 

  Fig. 4 Analysis of ATC cell death using TUNEL staining. (A) SW1736. (B) HTH83. (C) U-HTH 7. The TUNEL analysis was performed for DNA fragmentation using an in situ cell death detection kit under a light microscope; the digital images of apoptotic cells were selected randomly. Scale bars denote 50 µm. ATC, anaplastic thyroid cancer.

• 

  Fig. 5 Measurement of ROS production by nonthermal plasma. After plasma treatment, the SW1736 cells were treated with carboxy-H2DCFDA dye and then analyzed by flow cytometry. The FACS data from three independent experiments were quantified. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01 with Student's t-test compared with the control. NS, not significant. NAC, N-acetyl cysteine; ROS, reactive oxygen species; FITC, fluorescein isothiocyanate; FACS, fluorescence activated cell sorter.

• 

  Fig. 6 Evaluation of cell death due to ROS generated by nonthermal plasma using annexin V/PI staining. The SW1736 cells were treated with gas only or plasma jets at 2 kV for 1 s and then incubated for 24 hours, and stained with annexin V-FITC and propidium iodide (PI). Apoptosis was detected using a FACS Canto system. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 compared with the control using Student's t-test. NS, not significant; ROS, reactive oxygen species; FITC, fluorescein isothiocyanate; FACS, fluorescence activated cell sorter.

• 

  Fig. 7 Western blot analysis. Cells were suspended in RIPA buffer supplemented with PhosSTOP and cOmplete Mini EDTA-free. The proteins from SW1736 cells were electrotransferred to Immobilon-P membranes. Specific proteins were detected with an ECL Western Blotting Kit. NAC, N-acetyl cysteine; EDTA, ethylenediaminetetraacetic acid; ECL-enhanced chemiluminescent.

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