Selvido Diane Isabel, Koontongkaew Sittichai, Kokilakanit Paopanga, Sacharoen Anucha, Korsuwannawong Suwanna, Utispan Kusumawadee
International College of Dentistry, Walailak University, Bangkok, Thailand.
Faculty of Dentistry, Thammasat University (Rangsit Campus), Pathum Thani, Thailand.
J Dent Sci. 2024 Apr;19(2):855-864. doi: 10.1016/j.jds.2023.08.023. Epub 2023 Sep 2.
BACKGROUND/PURPOSE: Autophagy is an intracellular degradative process occurring under stressful conditions. Nitric oxide (NO), a free radical, regulates autophagy and apoptosis in several cancers. However, the effect of head and neck squamous cell carcinoma (HNSCC) cell adaptation to high nitric oxide (HNO) on autophagy remains unknown. We investigated the autophagy and apoptotic changes in the HNO-adapted HNSCC cell lines.
Isogenic primary HNSCC (HN18/HN30) and metastatic (HN17/HN31) cell lines were evaluated. The cells were induced with 1, 2, 3 and 4 mM DEA-NONOate, an NO donor, for 72 h and assessed for cell viability by MTT assay. "HNO-adapted cells" were defined when the cell viability in the treatment group was <10%. The surviving cells were re-treated with HNO to confirm their adaptation. HNO-adapted cells were quantified for apoptosis using flow cytometry. Autophagic structures (autophagosomes) and proteins (LC3A/B and LC3B-II) were investigated using transmission electron and confocal microscopy, respectively.
HNO-adapted concentration for HN18, HN17, HN30 and HN31 cells was 3, 2, 4 and 4 mM, respectively. The HNO-adapted HN18 cells demonstrated a significantly increased apoptotic percentage, whereas no significant apoptotic change was detected in the HNO-adapted HN17, HN30 and HN31 cells compared with the parent cells. Autophagosomes were widely observed across the HNO-adapted cells. Moreover, LC3A/B and LC3B-II proteins were increased in all HNO-adapted cells.
Our results demonstrate that apoptosis and/or autophagy are increased during HNO adaptation in HNSCC cell lines.
背景/目的：自噬是在应激条件下发生的一种细胞内降解过程。一氧化氮（NO）作为一种自由基，在多种癌症中调节自噬和凋亡。然而，头颈部鳞状细胞癌（HNSCC）细胞适应高浓度一氧化氮（HNO）对自噬的影响尚不清楚。我们研究了适应HNO的HNSCC细胞系中的自噬和凋亡变化。
评估了同基因的原发性HNSCC（HN18/HN30）和转移性（HN17/HN31）细胞系。用1、2、3和4 mM的NO供体DEA-NO供体处理细胞72小时，并通过MTT法评估细胞活力。当治疗组细胞活力<10%时，定义为“适应HNO的细胞”。对存活细胞再次用HNO处理以确认其适应性。使用流式细胞术对适应HNO的细胞进行凋亡定量。分别使用透射电子显微镜和共聚焦显微镜研究自噬结构（自噬体）和蛋白质（LC3A/B和LC3B-II）。
HN18、HN17、HN30和HN31细胞适应HNO的浓度分别为3、2、4和4 mM。适应HNO的HN18细胞凋亡百分比显著增加，而与亲代细胞相比，适应HNO的HN17、HN30和HN31细胞未检测到明显的凋亡变化。在适应HNO的细胞中广泛观察到自噬体。此外，所有适应HNO的细胞中LC3A/B和LC3B-II蛋白均增加。
我们的结果表明，在HNSCC细胞系适应HNO的过程中，凋亡和/或自噬增加。
