Zheng Sicong, Piao Yudan, Jung Seung-Nam, Oh Chan, Lim Mi Ae, Nguyen QuocKhanh, Shen Shan, Park Se-Hee, Cui Shengzhe, Piao Shuyu, Kim Young Il, Kim Ji Won, Won Ho-Ryun, Chang Jae Won, Shan Yujuan, Liu Lihua, Koo Bon Seok
Department of Medical Science, Chungnam National University College of Medicine, Daejeon, Korea.
Dental Department, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Clin Exp Otorhinolaryngol. 2025 Feb;18(1):73-87. doi: 10.21053/ceo.2024.00238. Epub 2025 Jan 6.
Head and neck squamous cell carcinoma (HNSCC) exhibits high recurrence rates, particularly in cases of radioresistant 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.
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 analysis to identify candidate molecules potentially associated with NTPAM therapy for RR-HNSCC.
NTPAM reduced RR-HNSCC cell viability in vitro. RNA sequencing results indicated that NTPAM treatment activated 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 consistent 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, enrichment 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.
NTPAM therapy exerts cytotoxic effects in RR-HNSCC cell lines by inducing specific ROS-mediated ferroptosis. 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-HNSCC.
头颈部鳞状细胞癌（HNSCC）具有较高的复发率，尤其是在耐放射性HNSCC（RR-HNSCC）病例中。非热等离子体（NTP）疗法可有效抑制HNSCC的进展。然而，NTP疗法治疗RR-HNSCC的机制尚不清楚。在本研究中，我们探讨了NTP在RR-HNSCC信号通路中的调节作用，并确定了其特征基因。
构建两种RR-HNSCC细胞系后，我们从经NTP激活培养基（NTPAM）处理或未处理的细胞中制备细胞裂解物，并进行RNA测序以确定其mRNA表达谱。基于RNA测序结果，我们鉴定了差异表达基因（DEG），随后进行生物信息学分析，以识别可能与RR-HNSCC的NTPAM疗法相关的候选分子。
NTPAM在体外降低了RR-HNSCC细胞的活力。RNA测序结果表明，NTPAM处理激活了活性氧（ROS）途径，并在RR-HNSCC细胞系中诱导了铁死亡。在与放射治疗相关的1924个基因中，有8个在细胞系和癌症基因组图谱（TCGA）队列中均显示出统计学意义。在NTPAM数据测序和TCGA数据之间，只有5个基因——ABCC3、DUSP16、PDGFB、RAF1和THBS1——显示出一致的结果。LASSO回归分析显示，5个基因与癌症预后相关，风险比为2.26。在RR-HNSCC细胞中，NTPAM在6小时内影响DUSP16、PDGFB和THBS1作为激活标记，且这种影响持续12小时。此外，富集分析表明，这三个DEG与细胞外基质、转化生长因子-β、磷酸肌醇3-激酶/蛋白激酶B和间充质-上皮转化因子途径相关。
NTPAM疗法通过诱导特定的ROS介导的铁死亡在RR-HNSCC细胞系中发挥细胞毒性作用。DUSP16、PDGFB和THBS被确定为逆转NTPAM疗法诱导的放射抗性的关键靶点，为NTPAM治疗RR-HNSCC的机制和临床应用提供了见解。
