In this study, a no-ozone cold plasma (NCP) device of the dielectric barrier discharge (DBD) type, developed by FEAGLE Corporation located in Yangsan-si, South Korea, was utilized. The plasma source was supplied with argon gas as a buffer, with a flow rate of 2.0 standard liters per minute (slm). By applying a high voltage of 3 kV to the plasma source, an NCP was generated, creating a plasma glow confined within the electrode area without extending to the edge of the electrode. The temperature of the NCP stream was kept below 35 °C for 10 min at a distance of 1 cm from the electrode edge, without any detected ultraviolet (UV) emissions. The distance between the electrodes and the dish was maintained at 1 cm throughout the experiment (Fig. 1A). A real image of the DBD-type plasma handpiece in use is shown in Fig. 1B. When the nozzle of the handpiece is activated, a visible purple plasma jet is emitted, as seen in Fig. 1C. The ozone level in this device was measured to be 0.006 ppm, which is significantly lower than the ozone threshold of 0.05 ppm recommended by the Food and Drug Administration (FDA).
The Squamous Cell Carcinoma cells (SCC-25) and human keratinocyte cells (HaCaT) used in this study were purchased from the American Type Culture Collection (Manassas, Virginia, USA). HaCaT cells, although originally derived from human skin, are widely accepted as a substitute for normal epithelial keratinocytes due to their stable proliferation, differentiation potential, and reproducible epithelial characteristics. In particular, HaCaT cells have been frequently used in studies involving oxidative stress, cytotoxicity, and apoptosis to evaluate the biocompatibility or selectivity of anticancer agents, including plasma-activated media. In this study, they were employed as a reliable control to compare the selective effects of PAM on cancerous and non-cancerous epithelial cells.
The cells were cultured in appropriate culture media, with SCC-25 cells cultured in DMEM-F12 (Dulbecco's Modified Eagle Medium) media and HaCaT cells cultured in DMEM media (Gibco, NY, USA). The culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), along with 100 μg /mL of penicillin and streptomycin. The cells were maintained under controlled conditions at 37 °C in a humidified atmosphere containing 5% CO.
An optical microscope (CX31, Olympus, Tokyo, Japan) was used to observe the differences in cell morphology. Images were captured using an iCM 9.0 digital camera system (IMT I-solution Inc., NY, USA).
We used the sulforhodamine B (SRB) assay to determine the cell survival rates. SCC-25 and HaCaT cells were seeded in 3.5 cm cell culture dishes and cultured for 24 h. Following culture, we treated the cells in three different ways and at three-time intervals in fresh medium.
The three different NCP treatment methods are as follows.
After 24 h, the cells were fixed and stained with sulforhodamine B (SRB) dye. Following staining, the cells were thoroughly dried, and cell images were captured. The stained SRB reagent was then extracted using a 10 mM Tris solution, and the absorbance was measured at 515 nm using a microplate reader (Sunrise Remote Control, Tecan, Austria). The analysis was conducted with three or more repeated experiments.
F-actin staining was conducted to assess the effect of PAM on the intracellular scaffold. Approximately 3 × 10cells were seeded in a cell culture dish and cultured for 24 h. Following the initial incubation, cells were exposed to PAM for 5 min. Subsequently, the treated cells were further cultured for an additional 24 h and then the sample was subjected to washing with Dulbecco's phosphate-buffered saline (DPBS). To fix the cells, a 4% paraformaldehyde solution was used, followed by washing with phosphate-buffered saline (PBS). The cell membranes were permeabilized using 0.1% Triton X-100 for 10 min. Rhodamine phalloidin, a dye, was diluted in 1% BSA and incubated with the cells at 37 °C for 30 min. After washing three times with PBS, the samples were sealed with a solution containing DAPI (4',6-diamidino-2-phenylindole) from Molecular Probes (CA, USA), and subsequent analysis was performed using a confocal laser microscope (LSM 900, Carl Zeiss, Germany). The relative fluorescence intensity was quantified using ImageJ software (NIH -- https://rsb.info.nih.gov/ij/).
Cells were treated with non-treatment (NT) or NCP-PAM for 5 min and cultured for 24 h. Following this, the cells were fixed with a 4% paraformaldehyde solution. They were then blocked at room temperature for 1 h using 0.5% Tween 20/10% BSA solution. Primary antibodies were incubated overnight at 4 °C with cytochrome C and AIF. After washing the cells three times with PBS for 5 min each, they were incubated at room temperature for 1 h with Alexa 488-conjugated goat anti-mouse IgG and Alexa 594-conjugated goat anti-rabbit IgG antibodies (Invitrogen, Carlsbad, CA, USA, 1:200). The expression of each antibody was visualized using a confocal microscope (LSM 900, Zeiss).
The cells were washed with PBS and lysed using a cold lysis buffer (Translab, Korea). After maintaining them at 4 °C for 30 min, the tubes were centrifuged at 4 °C, 12,000 rpm for 20 min. Subsequently, the supernatant was transferred to a new tube. The total protein content of the lysate was assessed by the Bradford Protein Assay method using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). After quantification, lysate samples of 25-35 μg were mixed with 5 × sample buffer and heated at 95 °C for 5 min. Lysate samples were loaded onto SDS/PAGE gels (8-15%). After the transfer process was completed, the protein marker on the membrane was checked and cut to fit the size of each antibody. Experiments were conducted using a cleaved membrane. The membrane was treated with 5% skim milk at room temperature for 1 h. Next, cleaved caspase 3, PARP (1:1000, Cell Signaling, Danvers, MA, USA) Bax and bcl-2 were quantified. β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The MAPKs-related factors, JNK and pJNK (1:1000, Cell Signaling, Danvers, MA, USA), pERK, ERK (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), and p38, pp38 (1:2000, ABclonal, Woburn, MA, USA), were also quantified. Protein bands were detected using ECL™ western blotting detection reagents (Amersham Biosciences, Little Chalfont, UK) and imaged using ImageQuant LAS 4000 (GE, Piscataway, NJ, USA). Protein expression levels were analyzed using ImageJ software. All data were obtained from 1 to 10 h after NCP treatment.
We purchased 5-week-old BALB/c nude mice from Orient-Bio Inc. (Seoul, Korea) and kept them to the Pusan National University School of Medicine animal facility. The mice were placed in cages with controlled environmental conditions, including a room temperature of 23 °C ± 2 °C, humidity maintained at 55% ± 5%, ventilation occurring 10-15 times per hour, and a 12-h light-dark cycle. They were provided with water and food every week.
This animal experiment was conducted in compliance with the guidelines of the Pusan National University Animal Experiment Ethics Committee and was approved by the committee (PNU-2023-0197). Furthermore, the study is reported in accordance with the ARRIVE guidelines to ensure transparent and comprehensive reporting of animal research. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2024-00347292).
We adopted a mouse xenograft model to evaluate anti-OSCC activity in vivo. SCC-25 cells were mixed with Matrigel in a 1:1 ratio at a concentration of 1 × 10⁶ cells/mL in cell media and injected into the dorsal surface of each nude mouse to induce tumor formation. After growing until the tumor size reached a diameter of 0.5 cm, the mice were divided into three groups (CON(-), CON(+), PAM, n = 5).
Subsequently, the CON(-) group received no treatment, whereas the CON( +) group was injected with media without NCP treatment three times a week. The PAM group received injections of NCP-PAM solution, which was generated by treating culture medium with NCP for 5 min, three times per week for 4 weeks. For each injection, a total of 200 µL of PAM was administered, divided equally into four sites around the periphery of the tumor to ensure uniform distribution. Throughout the experimental period, the overall condition of the nude mice was observed, and tumor dimensions were gauged weekly using vernier calipers. Tumor volume was calculated using the following formula: tumor volume = 1/2 × length (longest) × width (shortest). After the final treatment, the tumor tissues were separated, and their weights were measured. Tumor harvesting was conducted in two separate sessions. In our animal experiments, inhalation anesthesia was performed using isoflurane (4-5% for induction and 1-2% for maintenance). Euthanasia was carried out using CO₂ gas. The procedure used 100% CO₂ at a displacement rate of 30% of the chamber volume per minute. CO₂ exposure was continued for at least 5 min. Death was confirmed by the cessation of respiration and heartbeat, and by the presence of fixed, dilated pupils.
Tumor tissues were harvested from sacrificed mice and immediately fixed in 4% paraformaldehyde for 24 h. After fixation, the tissues were embedded in paraffin and sectioned into 5 µm slices. The paraffin sections were deparaffinized using xylene and rehydrated using ethanol. Some deparaffinized sections were stained with hematoxylin and eosin (H&E) following standard clinical pathology protocols and examined under a light microscope for general morphological evaluation. Other sections were stained using the TUNEL assay according to the manufacturer's instructions (TUNEL Assay Kit, Abcam, Cambridge, UK), and observed under the same microscope to assess apoptotic cell death. After TUNEL staining, slides were scanned using a digital slide scanner. Digital image analysis of the scanned slides was performed using QuPath, open-source software for digital pathology image analysis.
The mean ± standard error of the mean is provided for the data, derived from a minimum of three independent experiments. All statistical analyses were performed using Microsoft Excel (Microsoft 2013, Redmond, WA, USA) and IBM SPSS version 26 (IBM, USA). By conducting a two-tailed student's t-test and one-way ANOVA analysis, we identified a significant difference within each group. Duncan's significant difference post hoc test was used for multiple comparisons in the analysis of all data. Statistically significant differences, as determined by one-way ANOVA (p < 0.05), are denoted by different letters (a, b, and c), and the significance of the t-test was set at p < 0.05. SEM was applied to the data.