Plasma-activated medium induces apoptosis of oral squamous cell carcinoma through the MAPKs pathway

No-ozone cold plasma (NCP) is actively being researched as an alternative to address various side effects of cancer treatment. Compared to traditional direct procedures, the indirect method using plasma-activated medium (PAM) has advantages like greater tissue penetration and convenience. The purpose of this study is to assess the efficacy of plasma-activated medium on oral squamous cell carcinoma both in vitro and in vivo. Squamous Cell Carcinoma cells (SCC-25) and human keratinocyte cells (HaCaT) were used in this experiment. The evaluation of cell viability was conducted through sulforhodamine B (SRB) analysis. The effect of PAM was observed on the cytoskeletal structure through F-actin staining. To analyze cell selectivity, immunofluorescence staining was conducted to monitor alterations in the intracellular localization of cytochrome C and AIF. Western blotting was carried out to investigate the mechanism of apoptosis induced by PAM. Mitochondrial intrinsic pathway-related factors, cleaved caspase 3, PARP, Bax and bcl-2 were quantified. The MAPKs-related factors, JNK and pJNK, as well as pERK, ERK, and p38, pp38, were also quantified. Animal experiments were conducted to validate factors verified in vitro for their reproducibility in vivo. To induce tumor formation, SCC-25 cells were injected into nude mice xenograft model and then divided into three groups (CON(-)/CON(+)/PAM) for a 4-week experiment, and the treatment was performed three times per week. Tumor volume and mouse weight were measured, and at the end of the 4-week experiment, tumors were excised and weighed. Tumor tissues were processed for histological analysis, including H&E staining for morphology and TUNEL staining for apoptosis. PAM significantly decreased the viability of SCC-25 cells. F-actin staining showed that PAM disrupted the fibrous structure of SCC-25 cells and reduced the number of cells. Immunofluorescence staining revealed that cytochrome C and AIF, normally present in the mitochondria of normal cells, translocated to the cell nucleus or cytoplasm after PAM treatment. Western blot analysis showed cleaved forms of PARP and caspase 3, factors associated with the intrinsic pathway of apoptosis, and an increase in Bax over time, while bcl-2 decreased. The MAPKs-related factors, JNK and p38, showed increased phosphorylation, whereas ERK phosphorylation decreased. In animal experiments, tumor volume decreased by 32% compared to the control group when treated with PAM, and tumor weight also decreased by 25%. No significant changes were observed in the overall weight of the mice. H&E staining revealed significant morphological changes in tumor tissues following PAM treatment, accompanied by extensive apoptosis, with TUNEL-positive cells markedly increased to 75% compared to 30-42% in controls. 1. PAM can overcome the shortcomings of direct treatment (DT), such as limited tissue penetration and inconveniences of storage and use. 2. PAM exhibits selective cytotoxicity toward oral squamous cell carcinoma cells while preserving viabilityof human keratinocyte cells, suggesting partial cell selectivity. 3. PAM is presumed to induce apoptosis in oral squamous cell carcinoma through the mitochondrial-related intrinsic pathway via MAPK signaling. However, further validation using pathway inhibition studies is needed to confirm this mechanism.