Melatonin Targets Metabolism in Head and Neck Cancer Cells by Regulating Mitochondrial Structure and Function

Abstract

Metabolic reprogramming, which is characteristic of cancer cells that rapidly adapt to the hypoxic microenvironment and is crucial for tumor growth and metastasis, is recognized as one of the major mechanisms underlying therapeutic resistance. Mitochondria, which are directly involved in metabolic reprogramming, are used to design novel mitochondria-targeted anticancer agents. Despite being targeted by melatonin, the functional role of mitochondria in melatonin's oncostatic activity remains unclear. In this study, we aim to investigate the role of melatonin in mitochondrial metabolism and its functional consequences in head and neck cancer. We analyzed the effects of melatonin on head and neck squamous cell carcinoma (HNSCC) cell lines (Cal-27 and SCC-9), which were treated with 100, 500, and 1500 µM of melatonin for 1, 3, and 5 days, and found a connection between a change of metabolism following melatonin treatment and its effects on mitochondria. Our results demonstrate that melatonin induces a shift to an aerobic mitochondrial metabolism that is associated with changes in mitochondrial morphology, function, fusion, and fission in HNSCC. We found that melatonin increases oxidative phosphorylation (OXPHOS) and inhibits glycolysis in HNSCC, resulting in increased ROS production, apoptosis, and mitophagy, and decreased cell proliferation. Our findings highlight new molecular pathways involved in melatonin's oncostatic activity, suggesting that it could act as an adjuvant agent in a potential therapy for cancer patients. We also found that high doses of melatonin, such as those used in this study for its cytotoxic impact on HNSCC cells, might lead to additional effects through melatonin receptors.

Keywords: OXPHOS; apoptosis; free radicals; glycolysis; head and neck cancer cells; melatonin; mitochondria; mitophagy.

Figure 1

Upregulation by melatonin of key TCA cycle metabolites in HNSCC cell line Cal-27. Metabolomic study of intracellular levels of acetyl-CoA (A), citric acid (B), succinyl-CoA (C), NADH (D), and lactate (F). Pyruvate levels were measured using a colorimetric test (E). Treatment groups included vehicle (control) and melatonin (aMT) at a concentration of 500 µM. n = 4 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group.

Figure 2

Effect of melatonin on mitochondrial respiration in HNSCC cell lines Cal-27 (in blue) and SCC-9 (in black and grey). Oxygen consumption rate (OCR) after 1 day (A,D), 3 days (B,E), and 5 days (C,F) of melatonin treatment, basal respiration (G–I), maximal respiratory capacity (ETS) (J–L) and ATP turnover (M–O). Treatment groups include vehicle (control) and melatonin (aMT) at concentrations of 100 µM, 500 µM, and 1500 µM. Data for aMT 1500 group are not shown at day 5 because most cells died. n = 6 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. aMT 100 µM group; $$ p < 0.01, $$$ p < 0.001 vs. aMT 500 µM group.

Figure 3

Regulation of OXPHOS protein expression by melatonin in Cal-27 cells. Analysis of OXPHOS protein expression by Western blot after 1 day (A,B), 3 days (C,D), and 5 days of melatonin treatment (E,F). Data for aMT 1500 group are not shown at day 5 because most cells died. n = 6 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. aMT 100 µM group; $ p < 0.05, $$ p < 0.01 vs. aMT 500 µM group.

Figure 4

Melatonin-induced oxidative stress in Cal-27 cells. Measurements of intracellular ROS levels by fluorometry after staining with the DCF fluorescent probe (A–C) and SOD activity (D–F). Data for aMT 1500 group are not shown at day 5 because most cells died. n = 6 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ## p < 0.01, ### p < 0.001 vs. aMT 100 µM group.

Figure 5

Effect of melatonin on glycolysis in Cal-27 cells. Extracellular acidification rate (ECAR) (A–C), glycolytic capacity (D–F), glycolytic reserve (G–I) analyzed by Seahorse, and hexokinase II protein expression analyzed by Western blot (J–L). Data for the aMT 1500 group are not shown at day 5 because most cells died; n = 6 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. aMT 100 µM group; $$ p < 0.01 vs. aMT 500 µM group.

Figure 6

Melatonin-induced changes in mitochondrial morphology. Alterations in mitochondrial morphology analyzed by EM in Cal-27 cells (A), cristae length analyzed using the Image J software (B–D) in Cal-27 (in blue) and SCC-9 cells (in black and grey). In the control cells, the typical morphology of the Cal-27 mitochondria could be observed, with a round shape and few mitochondrial cristae (asterisks). No changes were detected over time. With aMT 100 concentration, mitochondria tend to get smaller and adopt elongated shapes, especially after 5 days of treatment (arrows). At dose of aMT 500, mitochondria do not show much change between day 1 and day 3, but at 5 days, mitochondria can be seen with a marked increase in cristae number (asterisks). At a concentration of 1500, at one day of treatment, mitochondria are similar to those in the control cells, although heterogeneous dense bodies are occasionally appreciated (arrows). After 3 days of treatment, the mitochondria maintain their shape and appearance, with abundant cristae. After 5 days, dense bodies continue to be seen (arrows) and it is difficult to detect cristae in some mitochondria. n = 6 per group. Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001 vs. control; ### p < 0.001 vs. aMT 100 µM group; $$$ p < 0.001 vs. aMT 500 µM group. Scale bar = 1 µm.

Figure 7

Melatonin-altered mitochondrial dynamics in Cal-27 cells. Western blot analysis of OPA-1 (A–C,M), MFN2 (D–F,M), Drp1 (G–I,M), and LETM1 (J–L,M). Data for aMT 1500 group are not shown at day 5 because most cells died. n = 6 per group. Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. aMT 100 µM group; $$ p < 0.01 vs. aMT 500 µM group.

Figure 8

Increases in apoptosis and autophagy caused by melatonin in Cal-27 cells. Western blot analysis of Bax (A–C,J), Bcl-2 (D–F,J), Bax/Bcl-2 ratio (G–I), NIX (K–M,T), LC3 (N–P,T), and ATG12-ATG5 (Q–T). Data for aMT 1500 group are not shown at day 5 because most cells died. n = 6 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p <0.01, ### p < 0.001 vs. aMT 100 µM group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. aMT 500 µM group.

Figure 9

Decreased proliferation of Cal-27 and SCC-9 cells caused by melatonin. Morphological alterations (A), proliferation rate (B–D), percentage of cells in each cell cycle phase (E–G), and representative plots showing cell redistribution (H). Data for aMT 1500 group are not shown at day 5 because most cells died. n = 6 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ## p < 0.01, ### p < 0.001 vs. aMT 100 µM group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. aMT 500 µM group.