Nitric oxide has diverse effects on head and neck cancer cell proliferation and glycolysis.

Glycolysis is a key energy-providing process and one of the hallmarks of cancer. Nitric oxide (NO), a free radical molecule, regulates glycolysis in various cancers. NO can alter the cell cycle and apoptosis in head and neck squamous cell carcinoma (HNSCC) cells. However, the effect of NO on glycolysis in HNSCC cells remains unresolved. The present study investigated the effects of NO on cell proliferation, glucose transporter (GLUT) gene expression and glycolytic indicators in HNSCC cell lines. Two pairs of isogenic HNSCC cell lines, HN18/HN17 and HN30/HN31, were treated with a NO donor, diethylamine NONOate (DEA-NONOate), for 24, 48 and 72 h. Cell proliferation was assessed using MTT assay and NO concentration was measured using the Griess Reagent System. GLUT1, GLUT2, GLUT3, and GLUT4 gene expression was analyzed using reverse transcription-quantitative PCR. Furthermore, hexokinase (HK) activity and lactate production were measured in NO-treated cells using colorimetric assay. NO exhibited concentration-dependent pro- and anti-proliferative effects on the HNSCC cell lines. Lower NO concentrations (5-200 µM) had pro-proliferative effects, whereas NO >200 µM had an anti-proliferative effect on HNSCC cells. NO (5 µM) promoted proliferation and glycolysis in HN18 cells by upregulating GLUT1 and GLUT2 gene expression and increasing HK activity and lactate levels. At 5-20 µM, NO-induced HN17 and HN30 cells demonstrated enhanced proliferation and GLUT2, GLUT3 and GLUT4 gene expression, whereas the glycolytic pathway was not affected. In conclusion, the present study demonstrated distinct proliferative effects of NO on HNSCC cells. NO may promote cell proliferation by stimulating glucose consumption and the glycolytic rate in HN18 cells. The effects of NO in other cell lines may be mediated by a non-glycolysis mechanism and require further investigation.

High nitric oxide-adapted head and neck cancer cell lines demonstrate altered autophagy and apoptosis.

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. Materials and methods: 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. Results: 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. Conclusion: Our results demonstrate that apoptosis and/or autophagy are increased during HNO adaptation in HNSCC cell lines.