Study on Melanoma Suppression by Non-Thermal Atmospheric Pressure Plasma via the PI3K-AKT-ZEB1 Axis

Academic Background

Melanoma is the most aggressive and deadly form of skin cancer, particularly in its advanced stages, making treatment extremely challenging. Although recent advancements in targeted therapy and immunotherapy have improved patient survival rates, overall efficacy remains suboptimal. Therefore, exploring new therapeutic approaches has become a key focus of current research. Non-Thermal Atmospheric Pressure Plasma (NTP), as an emerging physical treatment modality, has shown potential in cancer therapy in recent years. NTP generates Reactive Oxygen and Nitrogen Species (RONS), inducing oxidative stress in cancer cells, thereby exerting anti-tumor effects. However, the molecular mechanisms of NTP, particularly its regulation of microRNAs (miRNAs), have not been fully elucidated.

miRNAs are a class of short non-coding RNAs that regulate gene expression and are involved in various biological processes, including cell proliferation, differentiation, and apoptosis. Their role in cancer is particularly significant, with many miRNAs being identified as tumor suppressors or oncogenes. Therefore, studying the regulatory effects of NTP on miRNAs can help reveal its anti-melanoma mechanisms and provide a theoretical basis for developing new therapeutic strategies.

Source

This study is based on collaborative research by Pradeep Bhartiya, Apurva Jaiswal, Manorma Negi, Neha Kaushik, Eun Ha Choi, and Nagendra Kumar Kaushik. The team primarily consists of researchers from the Plasma Bioscience Research Center at Kwangwoon University, Korea, and the Department of Biotechnology at the University of Suwon, Korea. The paper was published in Journal of Advanced Research, Volume 68, pages 147-161, 2025.

Research Workflow

1. Cell Culture and NTP Treatment

The study initially used three human melanoma cell lines (SK-MEL-2, SK-MEL-31, and G-361) for experiments. Cells were cultured under standard conditions and treated using an NTP device. The NTP device used air as the working gas to generate plasma for cell treatment. To determine the optimal NTP treatment duration, the research team tested different time intervals (0.5, 1, 1.5, 2, and 4 minutes) on SK-MEL-2 cells and assessed cell viability using Alamar Blue dye. Results showed that 2-minute and 4-minute NTP treatments significantly reduced cell viability. Subsequent experiments used 3-minute and 5-minute NTP treatments.

2. Cell Viability and Apoptosis Detection

Cell death was assessed using Propidium Iodide (PI) staining. Results indicated that NTP treatment significantly increased PI uptake, suggesting enhanced cell death. Additionally, real-time quantitative PCR (qPCR) analyzed the expression levels of cell proliferation-related genes (e.g., c-Myc, Ki67, AKT, CDKN2A, p53, and Casp9). NTP treatment significantly reduced the expression of c-Myc and AKT while upregulating CDKN2A, p53, and Casp9, indicating that NTP induced cell cycle arrest and apoptosis to inhibit melanoma cell growth.

3. Clonogenic and Migration Assays

Clonogenic assays demonstrated that NTP treatment significantly reduced the clonogenic ability of SK-MEL-2 cells. Furthermore, scratch wound assays revealed that NTP treatment markedly suppressed cell migration.

4. miRNA Sequencing and Analysis

To further elucidate the molecular mechanisms of NTP, the research team conducted high-throughput miRNA sequencing (miRNA-seq) on NTP-treated SK-MEL-2 cells. Analysis revealed that 82 miRNAs were significantly upregulated and 66 miRNAs were significantly downregulated in the 5-minute NTP treatment group. Notably, miR-200b-3p and miR-215-5p were the most significantly upregulated. Bioinformatics analysis showed that these miRNAs were primarily involved in the PI3K-AKT signaling pathway, cell cycle regulation, and apoptosis.

5. miRNA Functional Validation

To validate the roles of miR-200b-3p and miR-215-5p, the research team used specific inhibitors to suppress these miRNAs and assessed their effects on cell viability and migration. Results showed that inhibiting miR-200b-3p and miR-215-5p significantly attenuated the anti-melanoma effects of NTP, suggesting that these miRNAs play a critical role in NTP-mediated melanoma suppression.

Key Findings

1. NTP Treatment Significantly Inhibits Melanoma Cell Viability and Clonogenic Ability: Experimental data showed that 5-minute NTP treatment significantly reduced the viability of SK-MEL-2, SK-MEL-31, and G-361 cells while inhibiting their clonogenic and migratory capabilities.
   
2. NTP Regulates miRNA Expression: miRNA sequencing revealed that NTP treatment significantly upregulated miR-200b-3p and miR-215-5p while affecting numerous other miRNAs associated with the PI3K-AKT signaling pathway and cell cycle regulation.
   
3. miR-200b-3p and miR-215-5p Play Key Roles in NTP-Mediated Melanoma Suppression: Functional validation experiments demonstrated that inhibiting these miRNAs significantly weakened the anti-melanoma effects of NTP.
   

Conclusions

This study is the first to reveal the molecular mechanisms by which NTP suppresses melanoma through the regulation of miRNAs, particularly miR-200b-3p and miR-215-5p. By upregulating tumor-suppressive miRNAs and downregulating oncogenic miRNAs, NTP induces cell cycle arrest and apoptosis, thereby inhibiting melanoma growth and migration. These findings provide a new theoretical foundation for the application of NTP in melanoma treatment and open new avenues for developing miRNA-based combination therapeutic strategies.

Highlights

1. Innovation: First study to uncover the molecular mechanisms of NTP-mediated melanoma suppression through miRNA regulation.
   
2. Clinical Value: Provides a theoretical basis for the application of NTP in melanoma treatment and proposes miRNA-based combination therapeutic strategies.
   
3. Technical Advancement: Utilizes high-throughput miRNA sequencing and bioinformatics analysis to deeply investigate the molecular mechanisms of NTP.

Significance and Future Directions

This study not only elucidates the mechanisms of NTP in melanoma treatment but also offers important insights for developing novel miRNA-based therapeutic strategies. Future research could further explore the combined application of NTP with other treatment modalities, such as chemotherapy or immunotherapy, to enhance melanoma treatment efficacy. Additionally, miR-200b-3p and miR-215-5p, as potential therapeutic targets, may also play roles in the treatment of other cancer types.