Therapeutic potential of no-ozone cold plasma-activated saline in osteosarcoma through mitochondrial and MAPK pathway modulation

Abstract

Plasma has the unique ability to trigger potent chemical reactions, making it a promising tool in biomedical applications. Recent advancements focus on low-temperature plasmas, particularly no-ozone cold plasma (NCP), which produces RONS at body-compatible temperatures. NCP has shown notable potential in inducing apoptosis in various cancer cells. This study investigated the efficacy of no-ozone cold plasma-activated saline in human osteosarcoma cell lines, and elucidated the underlying molecular mechanisms. Cells were indirectly exposed to plasma-activated media (PAM) and PAS. Cell viability was assessed using the SRB assay, while wound healing and oxidative stress were evaluated through migration, H₂O₂, and ROS assays. Immunofluorescence and Western blotting were used to analyze apoptotic markers and signaling pathways. PAS treatment significantly reduced osteosarcoma cell viability in a cell selective manner, impaired MG-63 cell migration and increased ROS levels in a time-dependent manner. Apoptotic indicators such as cytochrome c and AIF translocation, caspase-3 and PARP cleavage, Bax upregulation, and Bcl-2 downregulation were observed. Moreover, PAS modulated MAPK signaling by enhancing p38 phosphorylation and reducing ERK activity. These findings highlight PAS as a selective and noninvasive potential therapy for osteosarcoma as its selectively targets osteosarcoma cells while sparing normal cells, and triggers apoptosis through both mitochondrial dysfunction and MAPK pathway modulation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-23282-7.

Introduction

Osteosarcoma is one of the most common and highly aggressive primary malignant bone tumors and affects mostly children and adolescents, with a high incidence rate in women^1,2. It generally metastasizes to the lungs and is undetectable by diagnostic tools. The most common site includes long bones (near metaphyseal growth plates)³. Despite its low frequency relative to other solid tumors, osteosarcoma is one of the leading causes of cancer mortality in children, with a 5-year survival rate of under 30% in metastatic cases and 20% in recurring tumors^4,5.

Current treatment modalities include a combination of surgery, chemotherapy, and, in select cases, radiotherapy. Moreover, gene targeted therapies such as cryosurgery, laser ablation, and thermal coagulation have gained popularity. However, these approaches are often associated with substantial adverse effects, including systemic toxicity and impaired bone remodeling. Moreover, resistance to standard therapies and the lack of effective options for treating metastatic or recurrent disease necessitate the development of novel, minimally invasive therapeutic strategies capable of inducing tumor-selective apoptosis⁶.

Plasma, often referred to as the fourth state of matter, is a highly reactive medium composed of electrons, ions, radicals, and excited molecules. In previous years, plasma generation required extreme temperatures of more than 3000 °C or a vacuum, but recent advancements have enabled its production at room temperature and atmospheric pressure^7–9.

Cold atmospheric plasma (CAP) or Non Thermal Plasma (NTP), created by electrical discharge in a gas (helium or argon), generates reactive species, free radicals, UV radiation, and electromagnetic fields, resulting in biological effects such as antimicrobial activity, blood coagulation, and wound healing¹⁰. CAP has also demonstrated selective cytotoxicity against cancer cells in more than 20 cancer types while sparing healthy tissues^7,11,12.

A promising application of CAP is plasma-activated solutions, particularly plasma-activated saline (PAS) and plasma-activated media (PAM), which retain reactive species and allow indirect application to biological systems. These reactive species, such as hydrogen peroxide and nitric oxide, can penetrate cancer cells and induce oxidative stress, DNA damage, and mitochondrial dysfunction, ultimately leading to apoptosis.^1,8,13,14.

In this study, a plasma device capable of generating No-ozone cold plasma (NCP) was developed. This device operates below 30 °C and has an ozone concentration of only 0.006 ppm (below FDA safety thresholds).^8,15.

Apoptosis, a form of programmed cell death mechanism essential for proper organism development by removing abnormal cells. Two primary pathways regulate apoptosis: the intrinsic (mitochondrial) and extrinsic (death receptor-mediated) pathways. Both ultimately activate caspases 3, 6, and 7, leading to the breakdown of apoptotic bodies by phagocytes¹⁶. Mitochondria play a pivotal role in initiating apoptosis, with a decline in the mitochondrial membrane potential triggering the release of apoptotic factors. The balance between proapoptotic and antiapoptotic proteins within the Bcl-2 family regulates cytochrome C release into the cytoplasm, which in turn activates caspases and promotes apoptotic body formation. Caspase-3, an executioner caspase, becomes active when its precursor, pro-caspase-3, is cleaved into cleaved caspase-3. This active form then cleaves pro-PARP into cleaved PARP, ultimately leading to apoptosis¹⁷.

Moreover, the mitogen-activated protein kinase (MAPK) family, comprising extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), plays important roles in cell proliferation, differentiation, apoptosis and the stress response¹⁸. Phosphorylated ERK promotes cell survival, whereas JNK and p38 MAPK activate apoptosis. Studies have shown that the accumulation of reactive oxygen species (ROS) can activate the MAPK pathway, leading to apoptotic cell death^19,20.

Given the extensive research currently being conducted on osteosarcoma and its treatment strategies, much of the focus has been directed toward gene therapy, therapeutic agents, and direct plasma irradiation^21,22 However, the clinical utility of direct plasma irradiation is limited by the shallow penetration depth of the plasma. This limitation highlights a critical research gap in developing plasma-based therapies that are both selective and clinically applicable. Thus, we explored an indirect approach utilizing no-ozone cold plasma (NCP) in the form of plasma-activated media (PAM) and plasma-activated saline (PAS). It offers a promising solution, as it can be generated under no-ozone conditions, retains stable reactive species, and allows for minimally invasive administration to deeper lesions. In this study, we aimed to investigate the efficacy of PAS in inducing apoptosis in human osteosarcoma cell lines (MG-63 and Saos-2), with a focus on the mitochondrial and MAPK signaling pathways. By elucidating these mechanisms, we aim to provide a foundation for future translational and in vivo studies.

Materials and methods

No-ozone cold plasma device

In this study, a No-Ozone Cold Plasma (NCP) device based on the dielectric barrier discharge (DBD) technology with a coaxial geometry, consisting of a stainless-steel inner electrode and an outer electrode encasing the ceramic nozzle developed by the FEAGLE Corporation located in Yangsan-si, Gyeonsangnam-do, Republic of Korea (as shown in Fig. 1) was used. The NCP device utilized argon gas as a buffer gas with a flow rate of 2.0 standard liters per minute (slm). Plasma was ignited by applying a sinusoidal high-voltage power supply at 20 kHz with an amplitude of 3 kV (peak-to-peak)^23,24.

Fig. 1.

(A) Schematic diagram of Plasma torch. (B) Image of NCP Device and the plasma jet with Argon.

To ensure safety and control, the temperature of the NCP flow was meticulously maintained below 35 °C, specifically at a distance of 1 cm from the electrode orifice, for a duration of 10 min. Notably, the measured ozone concentration was exceptionally low at 0.006 parts per million (ppm), which is significantly below the ozone level recommended by the Food and Drug Administration (FDA), which is set at less than 0.05 ppm. Notably, no ultraviolet (UV) radiation was detected under these experimental conditions. To maintain experimental consistency, the distance between the cell culture dishes and the electrodes was carefully maintained at 1 cm throughout the study.

Cell lines and culture conditions

Human osteosarcoma cells (Saos-2 and MG-63) and Nontumorigenic epidermal keratinocyte HaCaT cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and American Type Culture Collection (Manassas, NJ) respectively. They were cultured (Saos-2, MG-63 & HaCaT—passages 5–10) in suitable media (Saos-2, McCoy’s 5A media; MG-63, MEM media; HaCaT, DMEM media) supplemented with 10% of fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in a humidified 5% CO₂ atmosphere.

HaCaT cells are a reliable, well-established human cell line that is widely utilized in plasma medicine research to examine redox biology, so they were selected as a non-tumorigenic control. Since epithelial cells are among the first tissues to be exposed to plasma-activated solutions in clinical settings, while being obtained from the skin, they offer a physiologically relevant comparison for assessing selective cytotoxicity.

Cell seeding and NCP application

The cells were seeded in 35 mm dishes at 1.3–1.8 × 10⁵ cells/ml. and incubated for 24 h in a CO₂ incubator. The experimental groups were divided into CON (Non- Treat), PAM (Plasma activated media) and PAS (Plasma activated saline). For preparation, 2 mL of either culture medium (for PAM) or PBS supplemented with 10% FBS and 1% penicillin/streptomycin (for PAS) was placed in a 35 mm Petri dish. The plasma jet nozzle was positioned vertically at a fixed distance of 1 cm above the liquid surface, and the solution was exposed to NCP for 5 min under the above-described discharge conditions. Importantly, the cells themselves were not directly exposed to NCP; instead, freshly prepared PAM or PAS was immediately applied to the cells to enable indirect plasma treatment. Control (non-treated) was handled under identical conditions without plasma exposure. After incubation for 24 h after plasma treatment, the experiments were performed.

Preliminary experiments were conducted at durations of 1, 3, and 5 min. Among these, the 5-min treatment consistently showed the most pronounced and reproducible effects on osteosarcoma cells. Therefore, subsequent experiments focused on the 5-min condition, which was selected as the representative treatment duration for detailed analyses.

SRB cell viability assay

For these experiments, the cells were seeded in 35 mm dishes and incubated for 24 h in a CO₂ incubator. One day later, the fresh media (media and saline respectively) were subjected to NCP for 5 min and were transferred to the cell dishes indirectly after removing the old media (Fig. 2). After 24 h of NCP treatment, cells were fixed with 4% PFA for 1 h and then washed thrice for 5 min. After drying, the cells were stained with sulforhodamine B (SRB) dye for 1 h. After 1 h, the cells were washed with 1% acetic acid for 4 times. When the cell dishes dried completely, photomicrograph was taken.

Fig. 2.

Application of NCP in two different solutions: (A) NCP application to the media. (B) NCP application to the saline. In both cases, only the liquid (medium or saline) was directly exposed to NCP for 5 min; the cells themselves were never exposed to the plasma jet. The plasma-treated solutions were then immediately transferred to the cells for indirect treatment.

Later, the cells were treated with 1 ml of 10 mM Tris solution to elute the stained SRB dye, and 150 μl of each solution were transferred into 96-well plates. Absorbance was detected using a microplate reader at a wavelength of 515 nm. All assays were performed in triplicate.

Wound healing assay

MG63 cells were inoculated into 60 mm cell dishes at a density of 1 × 10⁵ cells/ml for 24 h. A 1 ml sterile pipette tip and a ruler were used to make a scratch. A single scratch was made at the start of the assay, and no additional mechanical manipulation was performed during the observation period. After which the cells were washed thrice with PBS. Finally, the cells were indirectly treated with PAS. The microscope (Nikon ECLIPSE Ts2) was used to capture images of the cells in the same field of view at 0, 24, and 48 h. Scratch width and cell migration were observed and recorded. Image J software was used to measure cell migration and % of open wounds.

Hydrogen peroxide (H₂O₂) assay

A hydrogen peroxidase assay kit was used to determine the effect of PAS on the oxidation of Amplex Red. The cells were seeded in a 35 mm dish at a density of 1.2 × 10⁵ cells/mL and incubated for 24 h. Following treatment with PAS for CON (non-treat), and 5 min, the media samples were collected at 0, 0.5, 2, and 4 h post treatment. The level of hydrogen peroxide was quantified using the Hydrogen Peroxidase Assay Kit (Cat. No. A22188, Invitrogen, CA, USA), following the manufacturer’s guidelines. The same procedure was applied to the experimental samples without cells. Each sample was treated with 50 µL of Amplex Red reagent/HRP working solution for 30 min, and absorbance was measured at 560 nm via a microplate reader (AMR-100, ALLSHENG, China).

Intracellular ROS assay

The cell-permeable, fluorogenic probe, DCFH-DA was used to detect reactive oxygen species (ROS) and oxidative stress in cells. The cells were seeded in a black 96-well microplate at a density of 1 × 10⁴ cells per well and incubated for 24 h in an incubator. The samples were then exposed to 5-µM of 2′,7′ dichlorodihydrofluorescein diacetate (H2DCFDA) solution (Cat. No. D399, Invitrogen, CA, USA), 150 µl of which was added to each well of a plate, and incubated at 37 °C for 30 min in the dark. After the cells were washed with PBS three times, and then treated with 100 µl of NCP-PAS for 0, 0.5, 2 and 4 h. Intracellular ROS levels were measured using the fluorescence microscopy and was quantifed by a fluorescence multiwell plate reader (BioTek, Highland Park, VT, USA) (excitation:490 nm; emission: 535 nm).

Immunofluorescence analysis

For the immunofluorescence assay, MG-63 and HaCaT cells were fixed in a 4% paraformaldehyde solution at 4 °C and then permeabilized for 1 h at 4 °C. Thereafter, the cells were incubated with primary antibodies at 4 °C overnight. The following day, cells were washed with PBS buffer + 0.01% Tween 20 and then incubated with secondary antibody for 1 h at room temperature. Then, the nuclei were counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) for 10 min. A negative control was obtained by omitting the primary antibody. The fluorescence signal was examined with a fluorescence microscope (Olympus IX50) with appropriate filters. At 40X magnification, images of positive representative fields were captured.

Western blot analysis

The cells were washed with phosphate-buffered saline (PBS) and lysed in ice-cold lysis buffer containing nonionic detergent, protease inhibitors, and phosphatase inhibitors (TLP-121CETi, TransLab, South Korea). After incubation at 4 °C for 30 min, the samples were centrifuged at 13,000 rpm at 4 °C for 15 min. The supernatant was collected and transferred to fresh tubes. The total protein content in the lysate was quantified using the Bradford Protein Assay with the Pierce BCA Protein Assay kit (Cat No- 23,225, Thermo Scientific, USA). Lysate samples (30–35 µg) were mixed with 5X sample buffer and heated at 95 °C for 5 min. Proteins were separated using SDS-PAGE (10–15% gel) and transferred onto PVDF membranes (Merck, NJ, USA). Protein expression levels were analyzed using the ImageJ software (https://imagej.net/ij/) without additional plug-ins.

Following transfer, protein markers on the membrane were identified and cut according to the molecular weight of each target antibody. The membrane sections were blocked with 3% BSA for 1 h at room temperature and then incubated with primary antibodies against cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), Bax, Bcl-2 and MAPK factors JNK, ERK, and p38. The protein bands were detected using advanced ECL™ western blotting detection reagents (Amersham Biosciences, Little Chalfont, UK). β-actin and a-tubulin were used as loading controls. Images were captured using the ImageQuant LAS 4000 system (GE, Piscataway, NJ, USA).

Statistical analysis

All experiments were conducted with a minimum of three independent biological replicates, and each biological replicate included three technical replicates. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using Microsoft Excel and SPSS (ver. 16.0, SPSS Inc., Chicago, IL, USA). Differences between two groups were assessed using Student’s t-test, while comparisons among multiple groups were evaluated by one-way or two-way ANOVA followed by Tukey’s post-hoc test. A p-value of less than 0.05 was considered statistically significant.

Results

NCP selectively impaired the viability of osteosarcoma cell lines

The SRB assay was performed to assess the impact of NCP treatment on osteosarcoma cell viability in vitro. First, human osteosarcoma cells (MG-63 and Saos-2) and human keratinocyte cells (HaCaT) were cultured and seeded. The cells were subjected to PAM (plasma activated media) and PAS (plasma activated saline) treatment using the indirect treatment method for 5 min duration—with the NCP device, while an untreated control group (CON) was also included. The MG-63 and Saos-2 cells exhibited a significant and time-dependent decline in viability following NCP treatment. However, the HaCaT cells retained their viability post-treatment (Fig. 3A).

Fig. 3.

(A) Effects of NCP (PAM & PAS) on the viability of MG63, Saos-2 and HaCaT cells. (Scale bar: 50 µm) (B) Comparison of the effects of NCP on the viability of MG63, Saos-2 and HaCaT cells.*p < 0.05; **p < 0.01

Additionally, statistical significance analysis was carried out for both cell lines. After 5 min of PAS treatment, MG-63 cell viability decreased to 15.2% ± 3.1 of control levels (p < 0.001), whereas Saos-2 cells retained 35.7% ± 4.5 viability (p < 0.01). In contrast, HaCaT cells maintained 78.4% ± 5.2 viability, showing no statistically significant difference compared to untreated controls (p = 0.21). PAM treatment was less effective, reducing viability to 48.9% ± 6.0 in MG-63 and 55.1% ± 5.4 in Saos-2 cells (p < 0.05), while HaCaT cells remained at 85.6% ± 4.7.

Compared with those of the Saos-2 and HaCaT cells in the PAS treatment group, an evident reduction in MG-63 cell viability was noted after 5 min of PAS treatment. A statistically significant difference was confirmed between the PAM and PAS groups for the 5-min treatment. Based on these findings, subsequent experiments employed the 5-min PAS treatment method, and MG-63 cells were selected to proceed further with the experiments. Two-way ANOVA with post-hoc test was conducted to determine statistical significance across different groups (Fig. 3B).

PAS suppressed migratory ability of MG-63 cells

Malignant cancer cells are defined by their ability to spread and grow uncontrollably. In this study, compared with the control treatment, PAS treatment significantly reduced the ability of MG-63 cells to form colonies. There was a significant increase in the migrating surface of MG-63 cells following NCP treatment, and this increase was time dependent. Quantitative analysis revealed that PAS inhibited MG-63 cell migration by 62.5% ± 4.3 at 24 h and 74.1% ± 3.9 at 48 h compared to controls (p < 0.001 at both time points), indicating that PAS effectively inhibits the proliferation and migration of MG-63 cells over time. (Fig. 4).

Fig. 4.

PAS inhibited the migration of MG-63 cells (A) Images of the wound healing assay. (B). Quantification of the wound healing area. The data is presented as the mean of three independent experiments.

H₂O₂ concentration shift in the media following PAS exposure

To investigate the mechanism of apoptosis in MG-63 cells after NCP-PAS treatment, the H₂O₂ concentration in the media was assessed using the Amplex Red assay. Media samples were prepared on dishes with and without MG-63 cell seeding and treated with NCP-PAS for varying durations (CON [Non-treat] and 5 min) for 0 h, 0.5 h, 2 h and 4 h. Immediately following NCP-PAS exposure, H₂O₂ levels in the media increased progressively with increasing treatment time.

However, in media containing MG-63 cells, the H₂O₂ concentration significantly declined within 0.5 h post-treatment and continued to decrease at 2 and 4 h, approaching levels similar to the CON group. In contrast, in media without MG-63 cells, H₂O₂ levels remained stable for up to 4 h, with concentrations increasing proportionally to the NCP-PAS treatment durations of 5 min. (Fig. 5) These findings suggest active ROS consumption or neutralization by MG-63 cells.

Fig. 5.

The variation in H₂O₂ concentration in response to PAS treatment, with and without MG-63 cells. In the presence and absence of MG-63 cells, PAS was applied to plasma non-treated samples and those treated for 5 min, followed by culturing for 0 h (immediately after treatment), 0.5 h, 2 h, and 4 h and H₂O₂ concentration was then measured. * p < 0.05, ** p < 0.01.

PAS treatment led to a transient increase in intracellular ROS levels in MG-63 cells

To assess the impact of NCP-PAS treatment on osteosarcoma cell apoptosis, intracellular oxidative stress levels were evaluated. The intracellular ROS levels were measured using the DCFH-DA assay. MG-63 and HaCaT cells were treated with either CON (Non-treated) or PAS for 5 min, and ROS level changes were monitored immediately after treatment and at 0.5, 2, and 4 h. A one-way ANOVA with Tukey’s post-hoc test was conducted to compare the groups.

In MG-63 cells, ROS levels increased approximately 1.5 times, 30 min after PAS treatment compared to immediately after treatment in the 0.5-h group, relative to NT. No significant differences were observed between the cells at 0.5 h. However, at 2 h post-treatment, MG-63 cells exhibited a gradual increase in ROS levels compared to NT cells. In contrast, HaCaT cells showed no significant differences in ROS levels between treatment groups, except in the PAS 5-min group, where a significant difference was noted between the two cell types. Additionally, significant differences were observed between MG-63 CON and 5 min group, as well as between MG-63 5 min and HaCaT-CON at 2 h.

At 4 h post-treatment, ROS levels in MG-63 cells significantly increased compared to NT in the 5-min group. No ROS level differences were observed among HaCaT cell groups, but a significant difference was found between the two cell types. By 4 h after NCP-PAS treatment, ROS levels in MG-63 cells had decreased compared to immediately after treatment. (Fig. 6).

Fig. 6.

Variations in intracellular ROS levels in response to PAS treatment in MG-63 and HaCaT cells. The cells were subjected to PAS treatment for CON and for 5 min, followed by incubation for 0 h (immediately after treatment), 0.5 h, 2 h, and 4 h, after which the intracellular ROS levels were measured. * indicates p < 0.05, ** indicates p < 0.01.

PAS treatment induced mitochondrial-mediated apoptosis

An additional apoptotic parameter, which indicates changes in mitochondrial outer membrane permeability, was implemented. Immunofluorescence analysis enabled tracking the cellular distribution of cytochrome c and AIF. In untreated MG-63 cells, cytochrome c and apoptosis-inducing factor (AIF) were localized within the mitochondria, exhibiting punctate cytoplasmic staining.

Upon PAS exposure, both proteins redistributed—cytochrome c diffused into the cytosol and AIF translocated to the nucleus, as confirmed by DAPI counterstaining (Fig. 7). These findings support mitochondrial outer membrane permeabilization and activation of caspase-independent apoptosis pathways.

Fig. 7.

Immunofluorescence images showing cytochrome c release and AIF nuclear translocation in PAS-treated MG-63 cells.

PAS activated the intrinsic apoptotic pathway via MAPK signaling

To verify the mechanism of apoptosis induced by PAS treatment, apoptosis-related factors and protein expression were analyzed using Western blot. The proteolytic activation of cleaved caspase-3 was detected immediately after 2 h of PAS treatment and showed a steady increase over time. Similarly, cleaved PARP was detected at 4 h after the treatment and expressed most at 10 h. Bax levels gradually increased, whereas Bcl-2 levels decreased over time.

To further elucidate the mechanism of MAPK pathway, additional Western blot experiments were performed. While the ratio of phosphorylated JNK (pJNK/JNK) remained unchanged, the phosphorylated p38 (pp38/p38) ratio increased in a time-dependent manner. In contrast, the phosphorylated ERK (pERK/ERK) ratio gradually decreased over time. The protein expression was quantified using β-actin and a-tubulin as a loading control. (Fig. 8) These data suggest that PAS-mediated apoptosis is regulated through ROS-induced activation of the p38 MAPK pathway, with concurrent inhibition of ERK signaling.

Fig. 8.

Western Blot analysis of apoptotic and MAPK pathway proteins in MG-63 cells following PAS treatment. Western blot analysis of cleaved caspase-3, cleaved PARP, Bax, Bcl-2, and MAPK pathway proteins (ERK, JNK, p38) in MG-63 cells following PAS treatment (5 min). Cells were harvested at 0, 2, 4, 6, 8 and 10 h post-treatment. β-actin and α-tubulin were used as loading controls. Data represent mean ± SD from three independent biological replicates. Quantification of band intensities was performed using ImageJ. Statistical analysis was performed by one-way ANOVA. *p < 0.05, **p < 0.01.

Discussion

Osteosarcoma (OS) is a primary malignant bone tumor associated with a low survival rate, ranging from 5 to 60%, depending on the stage at diagnosis. This highlights the urgent need for alternative therapeutic approaches beyond conventional surgery and chemotherapy as metastasis and invasion continue to be major challenges contributing to treatment failure¹

Recent studies have shown that No-ozone Cold Plasma (NCP) is effective in biomedical applications. Moreover, they provide clinicians with a minimally invasive method that can be safely applied to living tissues^25,26 The development of plasma devices especially Dielectric Barrier Discharge (DBD) has made them increasingly suitable for in vitro and in vivo experiments. The protocol for NCP treatment has been outlined in previous studies^15,27–29 Since its introduction for cancer therapy in the 2000s, it has been effective in treating several cancer types. Previous studies have examined the direct impact of No-ozone cold plasma (NCP) on OS cell lines, and demonstrated the reduced proliferation or apoptosis of OS cells, whereas normal cells remain unharmed^2,8,12,14,30.

The MAPK signaling pathway plays a significant role in the development of drug resistance across various cancer types. It can be activated by external stimuli or intracellular signals, including physical stress and inflammatory responses. The MAPK family primarily consists of ERK1/2, JNK, and p38, each with distinct functions. ERK1/2 is essential for cell growth, differentiation, and proliferation, while JNK is involved in apoptosis, stress response, and cell differentiation. p38 regulates cytokine production, transcription, and apoptosis. As such, its components are considered key targets for therapeutic intervention and may also serve as valuable indicators for predicting patient outcomes^16,20,31 Additionally, the MAPK pathway has been implicated in driving the aggressive nature of osteosarcoma (OS). However, its potential as a major therapeutic target in OS has been relatively underexplored. These observations led us to investigate the expression levels of MAPK pathway components after treating OS cells with NCP.

In this study, the cytotoxicity and cell death mechanisms induced by NCP were assessed in osteosarcoma cell line (Saos-2 and MG-63) and one non-malignant human keratinocyte cell line (HaCaT) by SRB cell viability assay. The findings revealed that the NCP exposure selectively reduced the viability of OS cells, while non-malignant cells maintained viability levels at or above 75%. Specifically, cell viability decreased to about 50–55% in both Saos-2 and MG-63 cells after PAM treatment, and further dropped to approximately 35% in Saos-2 and 15% in MG-63 cells following a 5-min PAS treatment. (Fig. 3) These results align with earlier studies on other cancer types, which similarly showed that indirect treatment using NCP could induce DNA damage and apoptosis primarily in cancer cells, while sparing normal cells from such effects^2,32 This selectivity offers substantial clinical potential by minimizing the adverse side effects often associated with current therapies such as chemotherapy. Considering that direct NCP application to bone tissue would necessitate surgical access, we explored the potential of plasma-conditioned liquids as a minimally invasive alternative. In one of the experiments of this study, PAM (Plasma activated media) & PAS (Plasma activated saline)—generated by treating standard culture medium and saline with NCP—were applied to the cells. Both the treatments yielded cytotoxic and selective effects comparable to those of direct plasma exposure, but PAS was more effective. Therefore, in subsequent experiments, the PAS treatment method was selected due to its greater apoptotic effect, and the MG63 cell line was chosen among the osteosarcoma (OS) cell lines, as it exhibited a more pronounced reduction following treatment.

The prognosis for osteosarcoma (OS) patients remains poor because of the presence of metastases, particularly hematogenous dissemination. The progression of the disease is often driven by the ability of malignant tumor cells to migrate and invade surrounding tissues or enter the bloodstream. These processes are complex and involve multiple steps^33–35 To assess the effect of PAS on MG-63 cell migration and invasion, a wound healing assay was conducted. The findings revealed that PAS significantly suppressed the migratory and invasive capabilities of MG-63 cells. This reflects cytotoxic effects of PAS on cells located at the wound boundary, leading to apoptosis and detachment rather than migration into the wound area. It shows the strong inhibitory effect of PAS on osteosarcoma cell motility and viability, distinguishing it from the typical wound closure observed in untreated controls. (Fig. 4).

Research has shown that NCP triggers intracellular reactive oxygen species (ROS) production, induces cell cycle arrest, and activates both intrinsic and extrinsic apoptotic pathways in cancer cells³⁶ To evaluate the effects of different concentrations of reactive oxygen species (ROS) generated by PAS, both the Amplex Red and DCFH-DA assays were performed. A significant increase in hydrogen peroxide (H₂O₂) levels was observed in the culture medium immediately following PAS treatment, with the concentration continuing to maintain the same level over time. However, 0.5 h post-treatment, H₂O₂ levels in the medium containing MG-63 cells declined gradually. (Fig. 5) In particular, 30 min after treatment, the intracellular ROS levels in the MG-63 cells were approximately 1.5 times higher than those in the untreated controls, although the difference between the 0-min and 30-min groups was not statistically significant. (Fig. 6) These findings suggest that the rise in intracellular ROS is likely due to the diffusion of NCP-derived ROS across the cell membrane. This led to a decrease in osteosarcoma cell viability following PAS exposure. Like this study, other studies revealed that H₂O₂ and ROS cooperatively mediate cell death^37–40 Although this study quantified hydrogen peroxide as a representative ROS, other long-lived RONS such as nitrite (NO₂⁻) and nitrate (NO₃⁻) are also known to accumulate in plasma-activated solutions and contribute to biological activity. Previous studies have reported micromolar to millimolar levels of NO₂⁻ and NO₃⁻ in PAS depending on plasma conditions. Future studies will incorporate quantitative analyses of these species to provide a more comprehensive understanding of PAS chemistry in relation to its biological effects^40,41.

Apoptosis induction in tumor cells is widely recognized as a key and effective strategy in cancer therapy. In this study (Fig. 7), we observed that cytochrome C and apoptosis-inducing factor (AIF), which are typically confined to the mitochondria under normal physiological conditions, translocated to the cytoplasm or nucleus exclusively in MG-63 osteosarcoma cells following treatment with PAS. These findings indicate that PAS selectively triggers apoptotic pathways in cancer cells. In support of these findings, previous studies have shown that indirect plasma treatment can lead to mitochondrial network disruption and apoptosis specifically in tumor cells, while sparing normal cells. These effects have been linked to the accumulation of mitochondrial reactive oxygen species (ROS), particularly hydrogen peroxide (H₂O₂), with cancer cells exhibiting greater vulnerability to mitochondrial oxidative stress than their normal counterparts⁴².

Apoptosis is a fundamental phenomenon in cancer pharmacotherapy, and serves as a primary mechanism for eliminating tumor cells. The apoptotic signaling pathway is categorized into intrinsic (mitochondria-mediated), triggered by intracellular stress signals, and the extrinsic pathway, which are activated through death receptors on the cell surface. Both pathways converge on the activation of caspases—cysteine proteases that are typically synthesized as inactive precursors (procaspases) and require activation to initiate the apoptotic cascade. They also play a major role in recognizing and protecting damaged cells by degrading PARP within the cell. Caspase-9 functions as a key initiator caspase in the intrinsic pathway, amplifying apoptotic signaling downstream of mitochondrial dysfunction. On the other hand, caspase-8 serves as a central initiator in the extrinsic pathway. During apoptosis, different caspases are sequentially activated, with caspase-3 acting as a crucial executioner that leads to the degradation of cellular components.

Bax facilitates the opening of the mitochondrial voltage-dependent anion channel (VDAC), resulting in a loss of mitochondrial membrane potential and the release of cytochrome C into the cytosol. In contrast, Bcl-2 functions as an anti-apoptotic regulator by preventing cytochrome C release. Thus, an increase in Bax expression alongside a decrease in Bcl-2 is indicative of apoptotic progression. Exposure to No-Ozone Cold Plasma (NCP) activates caspase-9 and caspase-3, leading to the cleavage of poly (ADP-ribose) polymerase (PARP), a hallmark of apoptosis^43,44 Furthermore, NCP downregulates Bcl-2 expression while upregulating Bax, promoting its translocation to the mitochondria. This sequence of events initiates the intrinsic apoptotic pathway and facilitates the mitochondrial release of cytochrome C^45,46.

To identify the mechanism of the intrinsic apoptotic pathway involved in PAS induced cell death, we examined the activation levels of caspase-3, PARP, Bax and Bcl-2. Notably, cleaved caspase-3 and cleaved PARP were significantly increased. Similarly, Bax gradually increased over time, whereas Bcl-2 decreased. These findings suggest that PAS predominantly induces apoptosis via the intrinsic mitochondrial pathway. (Fig. 8).

To further investigate the mechanism underlying the activation of the intrinsic apoptotic pathway, we focused on the role of the MAPK signaling cascade. Previous studies have highlighted the critical involvement of MAPKs in converting various extracellular stimuli—particularly reactive oxygen species (ROS)—into specific cellular responses. These pathways regulate a broad range of cellular functions, including proliferation, differentiation, and apoptosis⁴⁷ The MAPK family is primarily composed of three subfamilies: ERK, JNK, and p38 MAP kinases⁴⁸ ERK is typically activated by mitogens and growth factors, promoting cell proliferation and survival. In contrast, JNK and p38 are predominantly activated in response to proinflammatory cytokines and oxidative stress, such as ROS, and are associated with the induction of apoptotic processes⁴⁹.

To explore the connection between PAS treatment and the activation of the intrinsic apoptotic pathway, we performed additional Western blot analyses. Our results revealed that p38 MAPK was phosphorylated and activated following PAS exposure, whereas ERK—known to support cell survival—was notably underphosphorylated. No significant changes were observed in the phosphorylation levels of JNK. These findings suggest that PAS-induced apoptosis in cancer cells is mediated through the intrinsic pathway, likely triggered by ROS-induced activation of the MAPK signaling cascade, which specifically involves p38. (Fig. 9).

Fig. 9.

A schematic diagram illustrating NCP-induced apoptosis via the mitochondrial and MAPK pathways in MG-63 cells.

Our findings are consistent with previous studies demonstrating the selective cytotoxicity of plasma-conditioned liquids in cancer cells while sparing normal cells. For example, Hamouda et al⁶ reported that plasma-treated media selectively impaired the viability of bone cancer cell lines, while Canal et al⁶ showed plasma-induced selectivity in bone cancer cell death. Similarly, Tanaka et al¹⁴ highlighted the therapeutic potential of plasma-treated solutions (PTS) in various cancer models. In line with these reports, our study confirms that plasma-activated saline (PAS) reduces osteosarcoma cell viability with minimal impact on non-tumorigenic HaCaT cells. Importantly, however, our work extends beyond prior studies by demonstrating that PAS generated under no-ozone conditions is more potent than plasma-activated media (PAM) and acts through defined molecular mechanisms. Specifically, we show for the first time in osteosarcoma that PAS induces apoptosis via mitochondrial dysfunction, including cytochrome c and AIF redistribution, and through modulation of MAPK signaling, characterized by p38 activation and ERK inhibition. These findings not only tell of the selective anti-cancer potential of plasma-treated solutions but also provide novel mechanistic insights that differentiate PAS from earlier plasma-based approaches.

Nevertheless, further investigation is required to identify other reactive species and bioactive components produced during NCP exposure. To fully elucidate the anti-cancer mechanisms of NCP—especially in different osteosarcoma cell lines—additional experimental approaches and in vivo studies are essential. The development of standardized or next-generation NCP devices and optimized treatment protocols is critical for enhancing the clinical applicability and therapeutic efficacy of these devices.

These findings underscore the potential of no-ozone cold plasma-activated saline (PAS) as a promising therapeutic approach for osteosarcoma treatment. PAS demonstrated selective cytotoxicity by significantly reducing the viability of osteosarcoma cells and inducing apoptosis, without harming normal cells—a therapeutic advantage over plasma-activated medium (PAM), which has lower efficacy. Additionally, PAS treatment led to a time-dependent change in hydrogen peroxide (H₂O₂) levels following a 5-min exposure, with concentrations peaking shortly after treatment and subsequently declining in the presence of cells. These temporal dynamics contributed to oxidative stress and the generation of reactive oxygen species (ROS), ultimately leading to apoptosis through redox imbalance. Furthermore, PAS activated both the mitochondrial and MAPK signaling pathways, indicating its ability to initiate apoptosis via both intrinsic and extrinsic mechanisms.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, D.H., and M.J; Methodology: M.J.; Formal analysis, M.J.; Investigation, M.J.; Data Curation, M.J.; Writing—Original draft preparation, M.J.; Writing—Review and Editing, M.J., Y.J. and D.H.; Supervision, M.J., Y.J.; Project administration and Funding acquisition, D.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT), Grant Number- RS-2024–00347292. None of the funding bodies had any influence on the study design; data collection, analysis, or interpretation; or the writing of the manuscript.

Data availability

All data generated or analyzed during this study are included in this article (and its Supplementary Information files).

Declarations

Competing interests

The authors declare no competing interests.