Gene Expression Alteration by Non-thermal Plasma- Activated Media Treatment in Radioresistant Head and Neck Squamous Cell Carcinoma
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Objectives. Head and neck squamous cell carcinoma (HNSCC) exhibits high recurrence rates, particularly in cases of ra- dioresistant HNSCC (RR-HNSCC). Non-thermal plasma (NTP) therapy effectively suppresses the progression of HNSCC. However, the therapeutic mechanisms of NTP therapy in treating RR-HNSCC are not well understood. In this study, we explored the regulatory role of NTP in the RR-HNSCC signaling pathway and identified its signature genes. Methods. After constructing two RR-HNSCC cell lines, we prepared cell lysates from cells treated or not treated with NTP- activated media (NTPAM) and performed RNA sequencing to determine their mRNA expression profiles. Based on the RNA sequencing results, we identified differentially expressed genes (DEGs), followed by a bioinformatics analy- sis to identify candidate molecules potentially associated with NTPAM therapy for RR-HNSCC. Results. NTPAM reduced RR-HNSCC cell viability in vitro. RNA sequencing results indicated that NTPAM treatment acti- vated the reactive oxygen species (ROS) pathway and induced ferroptosis in RR-HNSCC cell lines. Among the 1,924 genes correlated with radiation treatment, eight showed statistical significance in both the cell lines and The Cancer Genome Atlas (TCGA) cohort. Only five genes—ABCC3, DUSP16, PDGFB, RAF1, and THBS1—showed consis- tent results between the NTPAM data sequencing and TCGA data. LASSO regression analysis revealed that five genes were associated with cancer prognosis, with a hazard ratio of 2.26. In RR-HNSCC cells, NTPAM affected DUSP16, PDGFB, and THBS1 as activated markers within 6 hours, and this effect persisted for 12 hours. Furthermore, enrich- ment analysis indicated that these three DEGs were associated with the extracellular matrix, transforming growth factor-beta, phosphoinositide 3-kinase/protein kinase B, and mesenchymal-epithelial transition factor pathways. Conclusion. NTPAM therapy exerts cytotoxic effects in RR-HNSCC cell lines by inducing specific ROS-mediated ferropto- sis. DUSP16, PDGFB, and THBS1 were identified as crucial targets for reversing the radiation resistance induced by NTPAM therapy, providing insights into the mechanisms and clinical applications of NTPAM treatment in RR-HN- SCC.
Abstract
Objectives. Head and neck squamous cell carcinoma (HNSCC) exhibits high recurrence rates, particularly in cases of ra- dioresistant HNSCC (RR-HNSCC). Non-thermal plasma (NTP) therapy effectively suppresses the progression of HNSCC. However, the therapeutic mechanisms of NTP therapy in treating RR-HNSCC are not well understood. In this study, we explored the regulatory role of NTP in the RR-HNSCC signaling pathway and identified its signature genes. Methods. After constructing two RR-HNSCC cell lines, we prepared cell lysates from cells treated or not treated with NTP- activated media (NTPAM) and performed RNA sequencing to determine their mRNA expression profiles. Based on the RNA sequencing results, we identified differentially expressed genes (DEGs), followed by a bioinformatics analy- sis to identify candidate molecules potentially associated with NTPAM therapy for RR-HNSCC. Results. NTPAM reduced RR-HNSCC cell viability in vitro. RNA sequencing results indicated that NTPAM treatment acti- vated the reactive oxygen species (ROS) pathway and induced ferroptosis in RR-HNSCC cell lines. Among the 1,924 genes correlated with radiation treatment, eight showed statistical significance in both the cell lines and The Cancer Genome Atlas (TCGA) cohort. Only five genes—ABCC3, DUSP16, PDGFB, RAF1, and THBS1—showed consis- tent results between the NTPAM data sequencing and TCGA data. LASSO regression analysis revealed that five genes were associated with cancer prognosis, with a hazard ratio of 2.26. In RR-HNSCC cells, NTPAM affected DUSP16, PDGFB, and THBS1 as activated markers within 6 hours, and this effect persisted for 12 hours. Furthermore, enrich- ment analysis indicated that these three DEGs were associated with the extracellular matrix, transforming growth factor-beta, phosphoinositide 3-kinase/protein kinase B, and mesenchymal-epithelial transition factor pathways. Conclusion. NTPAM therapy exerts cytotoxic effects in RR-HNSCC cell lines by inducing specific ROS-mediated ferropto- sis. DUSP16, PDGFB, and THBS1 were identified as crucial targets for reversing the radiation resistance induced by NTPAM therapy, providing insights into the mechanisms and clinical applications of NTPAM treatment in RR-HN- SCC.