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

antioxidants Article Melatonin Targets Metabolism in Head and Neck Cancer Cells by Regulating Mitochondrial Structure and Function Ana Guerra-Librero 1,2,† , Beatriz I. Fernandez-Gil 1,† , Javier Florido 1,2,3, Laura Martinez-Ruiz 1,2,3, César Rodríguez-Santana 1, Ying-Qiang Shen 1, JoséM. García-Verdugo 4, Alba López-Rodríguez 1, Iryna Rusanova 1,2,5 , Alfredo Quiñones-Hinojosa 6, Darío Acuña-Castroviejo 1,2,3 , Jordi Marruecos 7, Tomás De Haro 8and Germaine Escames 1,2,3,*   Citation: Guerra-Librero, A.; Fernandez-Gil, B.I.; Florido, J.; Martinez-Ruiz, L.; Rodríguez-Santana, C.; Shen, Y.-Q.; García-Verdugo, J.M.; López-Rodríguez, A.; Rusanova, I.; Quiñones-Hinojosa, A.; et al. Melatonin Targets Metabolism in Head and Neck Cancer Cells by Regulating Mitochondrial Structure and Function. Antioxidants 2021,10, 603. https://doi.org/10.3390/ antiox10040603 Academic Editors: Helen Galley and Javier González-Gallego Received: 26 February 2021 Accepted: 9 April 2021 Published: 14 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Biomedical Research Center, Health Sciences Technology Park, University of Granada, 18016 Granada, Spain; [email protected] (A.G.-L.); beatrizir[email protected].es (B.I.F.-G.); [email protected] (J.F.); [email protected] (L.M.-R.); [email protected] (C.R.-S.); [email protected] (Y.-Q.S.); [email protected].es (A.L.-R.); [email protected] (I.R.); [email protected] (D.A.-C.) 2CIBERFES, Ibs.Granada, San Cecilio University Hospital, 18016 Granada, Spain 3Department of Physiology, Faculty of Medicine, University of Granada, 18016 Granada, Spain 4Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, 46980 Valencia, Spain; [email protected] 5Department of Biochemistry and Molecular Biology I, Faculty of Science, University of Granada, 18071 Granada, Spain 6Department of Neurologic Surgery, Mayo Clinic, Jacksonville, FL 32224, USA; [email protected] 7Institut Catalàd’Oncologia (ICO), Hospital Universitari Dr. Josep Trueta, 17007 Girona, Spain; [email protected] 8UGC de Laboratorios Clínicos, Hospital Universitario San Cecilio, 18016 Granada, Spain; [email protected] *Correspondence: [email protected]; Tel.: +34-958-241-000 (ext. 20363) † These authors contributed equally to this study. 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 µ M 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: melatonin; head and neck cancer cells; mitochondria; OXPHOS; glycolysis; mitophagy; apoptosis; free radicals Antioxidants 2021,10, 603. https://doi.org/10.3390/antiox10040603 https://www.mdpi.com/journal/antioxidants Antioxidants 2021,10, 603 2 of 20 1. Introduction Head and neck squamous cell carcinoma (HNSCC), one of the most aggressive tumors, accounts for 80–90% of all malignant neoplasms in the oral cavity [ 1 ]. Despite advances in the treatments available, there is a low overall 5-year survival due to high recurrence. New anticancer therapeutic strategies are required to prevent or reverse the development of chemoresistance, which is a major obstacle to HNSCC management. Recent studies highlighted the important role played by tumoral metabolism in HNSCC [ 2 – 4 ]. Increasing evidence indicates that cancer could be considered a mitochondrial metabolic disease in which metabolic reprogramming plays a key role with regard to tumorigenesis and metastasis [ 5 ]. The energy needs of tumor cells are met through aerobic glycolysis, thus, limiting the transformation of glucose into acetyl-coenzyme A (acetyl-CoA). This in turn generates lactate and lowers the pH in the local environment, which play a key role in tumor cell migration and metastasis [ 6 ]. However, the maintenance of glycolysis when oxygen is present requires metabolic reprogramming which involves both epigenetic and genetic changes (the so-called Warburg effect) [ 7 ]. These changes, which are associated with a wide spectrum of metabolic adjustments, increases cell proliferation and self-renewal, as well as poor cell differentiation. Given the differences between cancer and normal cell metabolism, it is vital to precisely elucidate the molecular mechanisms involved in mitochondrial metabolism reprogramming and to provide effective drug repositioning therapeutic strategies to target metabolic reprogramming. Many essential mitochondrial functions play a much more important role in cancer metabolism than previously thought [ 8 ]. Cancer cell mitochondria are novel therapeutic targets given their structural and functional differences from their normal cell counterparts. Increased production of reactive oxygen species (ROS) also generates mutations involving malignant transformation and cancer progression, thus making these organelles promising targets of anticancer therapy. However, ATP production in normal cells might be disrupted by the targeting of mitochondrial ATP production, while mitochondrial ROS-mediated signaling in immune cells might be disrupted when mitochondrial redox status is targeted [ 9 ]. To resolve this problem, cancer cell mitochondria need to be targeted without damaging normal cell mitochondria [5]. Melatonin (N-acetyl-5-methoxytryptamine) is a potent anti-inflammatory and antioxidant molecule. Moreover, melatonin attracts attention as an oncostatic agent. However, the precise mechanism of the action involved in the oncostatic anti-neoplastic impact of melatonin is not fully elucidated. The involvement of mitochondria in the anti-proliferative effect of melatonin on neoplastic cells is of particular relevance for the development of innovative cancer treatments [ 10 – 14 ]. Indeed, we found that melatonin amplifies the cytotoxic effects of chemotherapy and radiotherapy in HNSCC, which are associated with changes in mitochondrial respiratory capacity and ROS balance in these cells [ 10 , 11 ]. Changes in mitochondrial respiration are well-known to contribute to metabolic remodeling, although the mechanism of action involved in melatonin-driven tumoral death remains unclear. Moreover, previous studies showed that melatonin not only enhances the oncostatic effects of radio- or chemotherapy on tumor cells, but also protects normal cells against the adverse effects of these treatments [10,12,14,15]. In this study, we tested the hypothesis that the effects of melatonin on cancer cells can be explained by bioenergetic stress, resulting from the inability of tumor cells to switch on glycolysis, in order to produce ATP, following the inhibition of oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we targeted tumor cell metabolism in HNSCC following melatonin treatment. We confirmed the cytotoxic effect of melatonin on HNSCC and its involvement in a change of metabolism in these cancer cells and also determined the precise mechanism of action involved. We aimed to understand the mechanisms that lead to tumor progression in HNSCC and to provide evidence for melatonin treatment strategies for HNSCC that is aimed at the targeting metabolism. Antioxidants 2021,10, 603 3 of 20 2. Materials and Methods 2.1. Cell Culture and Treatment Head and neck squamous carcinoma cells Cal-27 (ATCC: CRL-2095) and SCC-9 (ATCC: CRL-1629) were obtained from the Cell Bank of the Scientific Instrumentation Centre of the University of Granada. Cal-27 cells were grown, as previously described [ 10 ], in Dulbecco’s Modified Eagles Medium-GlutaMAX (DMEM, high glucose, GlutaMAX ™ Supplement, Fisher Scientific, 41965039, Madrid, Spain) with 10% fetal bovine serum (FBS) (16000044; Fisher Scientific Madrid, Spain), 1 mM sodium pyruvate, and 2% antibiotic/antimycotic solution (15240062, Fisher Scientific, Madrid, Spain). SCC-9 cells were cultured in DMEM-F12 Nutrient Mixture Ham medium (11320033; Fisher Scientific, Madrid, Spain) supplemented with 10% FBS, 2% antibiotic/antimycotic solution, 2 mM L-glutamine (25030081; Fisher Scientific, Madrid, Spain), 0.5 mM sodium pyruvate, and 0.4 µ g/mL hydrocortisone. Both cell lines were maintained in a humidified 37 ◦ C incubator with 5% CO 2 . Cal-27 and SCC-9 cells were negative for mycoplasma, tested using a PCR detection kit, following the manufacturer’s instructions (25235; LiliF Diagnostics, Burlington, MA, USA). Melatonin (33457-24; Fagron Ibérica S.A.U., Terrasa, Spain) stock solution was prepared in 15% propylene glycol (PG; 24414.296; VWR, Radnor, PA, USA) in phosphate buffer solution (PBS) (14190-094, Life Technologies) and filter-sterilized through a 0.2 µ m pore filter (Sartorius Biotech GmbH, Gottingen, Germany). Cells were grown to 60–70% confluence and serum starved for 24 h. They were then treated with different melatonin concentrations (0, 100, 500, and 1500 µ M) for 24, 72, and 120 h (1, 3, and 5 days). Vehicle was added to the control group. 2.2. Metabolomic Analysis The TCA cycle metabolites, citric acid, succinyl-CoA, acetyl-CoA, NADH, and lactic acid, were quantitatively determined by ultraperformance liquid chromatography, in combination with triple quadrupole mass spectrometry (UPLC-MS/MS), at the Center for Omic Sciences (Rovira i Virgili University, Tarragona, Spain). Since metabolic changes are observed in a short period of time, cells were treated with melatonin only for 6 h for metabolomic analysis. After harvesting the cells, the metabolites were extracted by a liquid–liquid extraction with hexane. Samples were analyzed in a 1290 Infinity LC Series chromatograph coupled with a 6495 iFunnel Triple Quad MS/MS (Agilent Technologies, Santa Clara, USA) equipped with an AQUITY UPLC HSS T3 1.8 µ m, 2.1 × 100 mm column (Waters, Milford, USA). The mobile phase used was 20 mM NH4AC and 0.2% acetic acid as solvent A and acetonitrile (ACN) as solvent B. The column flow was 0.5 mL/under the following mobile phase gradient conditions—0–1 min 0% B isocratic, 5 min 50% B, 6–9 min 100% B isocratic, and 10 min 0% B with a post run of 3 min. The volume of injection was 5 µ L. Ionization was performed using a source of electrospray (ESI), with a temperature and flow of Drying Gas (N2) of 290 ◦C and 18 L/min respectively, a nebulization gas (N2) pressure of 20 psi and a sheath gas (N 2 ) temperature and flow of 350 ◦ C and 10 L/min. The fragment or voltage was 380 V, capillary voltage was 3500 V in the positive and 3000 in the negative and the nozzle voltage was 750 V in the positive and 1500 V in the negative mode. Detection was performed in the QqQ detector through acquisition of Multiple Reaction Monitoring (MRM). Quantification of samples was performed through interpolation of the obtained area in the standard curve with the matrix match method. 2.3. Pyruvate Measurement Intracellular pyruvate concentration was measured using a colorimetric assay kit (EPYR-100, BioAssay Systems, Hayward, CA, USA) in a microplate reader spectrophotometer (Power Wave X-1; Bio-Tek Instruments, Inc., Winooski, VT, USA) at 570 nm, according to the manufacturer’s instructions Data were normalized by the protein content measured by Bradford assay. Antioxidants 2021,10, 603 4 of 20 2.4. Mitochondrial Respiration The oxygen consumption rate (OCR) was determined using the Seahorse Extracellular Flux XF-24 analyzer (Seahorse Bioscience, N.Billerica, MA, USA). Cells were seeded in XF 24-well cell culture microplates (Seahorse Bioscience) at a density of 8 × 10 4 cells/well and incubated overnight at 37 ◦ C with 5% CO 2 . Prior to performing the assay, the medium was changed to Base Medium (102353-100, Seahorse Bioscience, N. Billerica, MA, USA) containing 10 mM glucose, 5 mM pyruvate, and 2 mM glutamine, and the cells were equilibrated for 1 h at 37 ◦C without CO2. The OCR was measured as previously described [ 16 ], both at the basal level and in response to modulators of maximal mitochondrial electron transport system (ETS) capacity. A total of 1 µ M oligomycin and 1 µ M trifluorocarbonylcyanide phenylhydrazone (FCCP) (0.5 µ M in two injections) were injected, followed by 1 µ M rotenone/antimycin A. Inhibition of ATP synthase by oligomycin was used to determine the proportion of OCR used for ATP production under basal conditions. Maximal uncoupled respiration was assessed after the addition of FCCP, and the residual nonmitochondrial OCR was measured using rotenone/antimycin A. The average of four readings per cell line was recorded, and it was normalized by the number of cells. Cells were counted in a hemocytometer, and viability was determined by trypan blue staining. 2.5. Measurement of Redox Status ROS production was measured using 100 µ M of 2 0 7 0 dichlorofluorescin diacetate (DCFH-DA) probe (Sigma-Aldrich, Madrid, Spain), according to the protocol described by Shen et al. [ 10 ]. DCFH-DA was transformed into the fluorescent DCF inside the cell. Fluorescence was then measured with the microplate fluorescence reader FLx800 (Bio-Tek Instruments, Inc., Winooski, VT, USA) for 30 min every 5 min, at 485 nm to excitation and 530 nm to emission wavelengths [ 10 ]. Superoxide dismutase (SOD) activity was assayed in terms of its ability to inhibit the auto-oxidation of adrenalin to adrenochrome at pH 10.2. Oxidation of adrenalin was measured at 490 nm for 10 min at 30 ◦ C as Misra et al. described [ 17 ]. SOD activity was expressed as U/mg prot (1 unit = 50% inhibition of epinephrine auto-oxidation). 2.6. Glycolysis Capacity The extracellular acidification rates (ECAR) were measured using the Seahorse Extracellular Flux XF-24 analyzer (Seahorse Bioscience, N. Billerica, MA, USA). Cells were plated in XF24 cell culture microplates at a density of 8 × 10 4 cells/well in DMEM. After 24 h, the culture medium was changed to Base Medium (102353-100, Seahorse Bioscience, N. Billerica, MA, USA) supplemented with 2 mM glucose and was incubated at 37 ◦ C for 1 h in a CO 2 -free incubator. The extracellular acidification rate (ECAR) was measured using the XFp Glycolysis Stress Test Kit (Seahorse Bioscience), according to the manufacturer’s instruction. Accordingly, after baseline measurement, the following injections were made— 10 mM glucose, 1 µ M oligomycin, and 50 mM 2-DG. ECAR values were normalized to the cell number. Cells were counted in a hemocytometer and viability was determined by trypan blue staining. 2.7. Electron Microscopy Analysis Electron microscopy analysis was carried out according to the protocol described by Cebrian et al. [ 18 ]. Cells were fixed in 3% glutaraldehyde in PB buffer 0.1 M pH 7.4 and postfixed in 2% OsO 4 and stained in 2% uranyl acetate in the dark. Finally, cells were dehydrated in ethanol, rinsed with propylene oxide (Lab Baker, Deventry, Holland) and embedded overnight in Araldite (Durcupan, Sigma). Upon polymerization, embedded cultures were detached from the chamber slide and glued (Super glue, Loctite) to Araldite blocks. Semi-thin sections (1.5 µ m) were cut with an ultramicrotome (Ultracut UC-6, Leica, Heidelberg, Germany) mounted onto slides and stained with 1% toluidine blue. Ultrathin Antioxidants 2021,10, 603 5 of 20 sections (50 nm) were obtained with an ultramicrotome using a diamond tipped knife (Ultra 45◦Diatome) and stained with lead citrate (Reynolds Solution). Images were obtained with a transmission electron microscope (FEI Tecnai Spirit BioTwin, Hillsboro, OR, USA) using a digital camera (Morada, Soft Imaging System, Olympus, Tokyo, Japan). At least 10 independent cells were studied for each condition. Among them, 20 mitochondria were analyzed for each cell culture, belonging up to 2 mitochondria to the same cell. The length of the mitochondrial crests per mitochondrial area was quantified using the ImageJ software (La Jolla, California, USA). 2.8. Quantification of Mitochondrial Mass Mitochondrial mass was related to the cardiolipin content. Quantification of cardiolipin was performed using the fluorescent probe Nonyl Acridine Orange (NAO, A1372; Fisher Scientific, Madrid, Spain), according to the protocol described by Shen et al. [ 10 ]. The fluorescence was read using a microplate fluorescence reader FLx800 (Bio-Tek Instruments, Inc., Winooski, VT, USA), at an excitation wavelength of 485 nm and emission of 530 nm. 2.9. Mitochondrial DNA Copy Number Human mitochondrial DNA was quantified by real-time PCR in a Stratagene Mx3005P qPCR System (Agilent Technologies, Inc., Santa Clara, CA, USA), as described by Lopez LC et al. [ 19 ]. We used primers and probe complementary to sequences of the nuclear gene 18 S (TaqMan Gene Expression Assays Hs99999901_s1, Applied Biosystems) and to the sequences of the mtDNA 12 S gene. mtDNA values were normalized to nDNA data (mtDNA/nDNA ratio). 2.10. Cell Proliferation Assay Cell viability was measured using an MTT assay (MTT, V13154; LifeTechnologies, Madrid, Spain), based on the conversion of soluble MTT (yellow) to non-soluble formazan (purple). Cells were seeded in a 96-well culture plate (167008, Thermo Scientific, Madrid, Spain) and MTT assay was performed following the manufacturer’s instructions. 2.11. Cell Cycle Analysis Cellular DNA content was measured by flow cytometry using a propidium iodide (PI)/RNase kit (PI/RNASE, Immunostep, Salamanca, Spain) staining, according to the manufacturer’s instructions. Data were analyzed using flow cytometry in a Becton Dickinson FACSCanto II cytometer (Madrid, Spain). Data were reported as the percentage of cells in each phase of the cell cycle. 2.12. Western Blot Analysis Protein extraction and Western blot analyses were performed as described previously [ 20 ], and Bradford assay was used to determine protein concentration. Adequate amounts of proteins were separated on 7% and 12.5% PhastGel homogeneous gels (GE Healthcare Life Sciences, Barcelona, Spain). Proteins were transferred to Hybond ™ -ECL nitrocellulose membranes (GE Healthcare Life Sciences, Barcelona, Spain) using the Phast- System (GE Healthcare Life Sciences, Barcelona, Spain). Next, the membranes were first incubated in blocking buffer containing 5% BSA in PBS plus 0.1% Tween 20 and were then, incubated with the primary antibodies diluted in blocking buffer overnight at 4 ◦ C. The utilized primary antibodies included OXPHOS Rodent WB Antibody Cocktail (1:1000; ab110413; Abcam, Inc., Cambridge, MA,USA), OPA1 (1:200; sc393296; Santa Cruz Biotechnology, Heidelberg, Germany), MFN2 (1:200; sc5033; Santa Cruz Biotechnology), Drp1 (1:200; sc-32898; Santa Cruz Biotechnology), LETM (1:200; sc-134672; Santa Cruz Biotechnology), Bax (1:200; sc-526; Santa Cruz Biotechnology), Bcl2 (1:200; sc-492; Santa Cruz Biotechnology), NIX (1:1000; N0399; Sigma-Aldrich, Madrid, Spain), LC3 (1:1000; nb100-2220; Novus, Abigdon, UK), ATG12 (1:200; sc-271688; Santa Cruz Biotechnology), MT1 (1:500; TA321736; OriGene Technologies, Rockville, MD, USA), MT2 (1:500; TA314217; OriGene Antioxidants 2021,10, 603 6 of 20 Technologies), ROR α (1:500; TA311450; OriGene Technologies), and GAPDH (1:500; sc- 166574; Santa Cruz Biotechnology). Anti-mouse at dilution 1:1000 (554002; BD Biosciences Pharmigen, San Jose, CA, USA), anti-rabbit at dilution 1:5000 (31460; Thermo Scientific, Madrid, Spain), and anti-goat diluted 1:1000 (sc-2768; Santa Cruz Biotechnology) HRP conjugated secondary antibodies were used according to the manufacturer’s instructions. The images were analyzed using the ECLTM Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Barcelona, Spain), with the Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA), and quantified using 1D Image Analysis software 3.6. Protein band intensities were normalized to GAPDH and data were expressed as the percentage relative to controls. 2.13. Statistical Analysis Data are expressed as the mean ± S.E.M of a minimum of three independent experiments. Statistical analysis was performed with the Prism software (GraphPad, La Jolla, CA, USA), using one-way analysis of variance (ANOVA) with Bonferroni test. Normality test was performed by a Shapiro-Wilk analysis, showing that all date had a normal distribution. Ap-value of < 0.05 was considered to indicate statistical significance. 3. Results 3.1. Melatonin Up-Regulates Key TCA Cycle Metabolites in HNSCC Cells The role of melatonin in regulating cancer cell metabolism remains largely unexplored. To further dissect the effects of melatonin in cancer metabolism, we carried out metabolomic analyses using UPLC-MS/MS, which found clear differences between the control group and the melatonin-treated cells (Figure 1). We found that after 6 h, melatonintreated cells upregulated key TCA cycle metabolites, including acetyl CoA, citric acid, and succinyl-CoA (Figure 1A–C), which led to increased NADH levels (Figure 1D) and reduced levels of pyruvate (Figure 1E). A larger influx of pyruvate into the mitochondria plus the increased NADH levels would lead to enhanced oxidative phosphorylation. These two processes would lead to mitochondrial ROS production. Interestingly, the intracellular lactate increased, compared to the control (Figure 1F). Antioxidants 2021, 10, x FOR PEER REVIEW 7 of 21 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. These findings corroborated the important role played by melatonin to induce a change of head and neck cancer cell metabolism. To independently validate these data, we then assessed the mitochondrial respiration and glycolytic capacity. 3.2. Melatonin Treatment Induces Uncoupling Between Respiration and Phosphorylation in Mitochondria, Correlating with Increased ROS Production in HNSCC Cells To further determine whether the inhibition of metabolic reprogramming in HNSCC cells after melatonin treatment was linked to their mitochondria activity, functional mitochondrial analyses were carried out using the Seahorse XF24 extracellular flux analyzer in Cal-27 cells (Figure 2A–C) and in SCC-9 cells (Figure 2D–F). Consistent with previous findings [10,11], we observed elevated OCR corresponding to increased basal respiration (Figure 2G–I) and maximal respiratory capacity of the electron transport system (ETS), with melatonin treatment of Cal-27 cells in a dose- and time-dependent direct manner (Figure 2J–L). However, at high 500 and 1500 µM doses of melatonin during 3 and 5 days of treatment, melatonin reduced the OCR (Figure 2H,I,K,L), suggesting that the mitochondrial function was defective during long treatments with higher concentrations of melatonin. SCC-9 cells showed no significant differences in basal respiration and ETS capacity (Figure 2G,H), but exhibited a significant decrease in mitochondrial respiration, after 3 and 5 days of melatonin treatment at a dose of 1500 µM (Figure 2H,I,K,L). Thus, confirming the presence of defective mitochondria at high doses of melatonin. No significant changes in ATP turnover were observed in either group tested, following melatonin treatment (Figure 2M–O). 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 µ M. n= 4 per group. Data are presented as mean ±SEM. * p< 0.05, ** p< 0.01, *** p< 0.001 vs. control group. Antioxidants 2021,10, 603 7 of 20 These findings corroborated the important role played by melatonin to induce a change of head and neck cancer cell metabolism. To independently validate these data, we then assessed the mitochondrial respiration and glycolytic capacity. 3.2. Melatonin Treatment Induces Uncoupling between Respiration and Phosphorylation in Mitochondria, Correlating with Increased ROS Production in HNSCC Cells To further determine whether the inhibition of metabolic reprogramming in HNSCC cells after melatonin treatment was linked to their mitochondria activity, functional mitochondrial analyses were carried out using the Seahorse XF24 extracellular flux analyzer in Cal-27 cells (Figure 2A–C) and in SCC-9 cells (Figure 2D–F). Consistent with previous findings [ 10 , 11 ], we observed elevated OCR corresponding to increased basal respiration (Figure 2G–I) and maximal respiratory capacity of the electron transport system (ETS), with melatonin treatment of Cal-27 cells in a dose- and time-dependent direct manner ( Figure 2J–L ). However, at high 500 and 1500 µ M doses of melatonin during 3 and 5 days of treatment, melatonin reduced the OCR (Figure 2H,I,K,L), suggesting that the mitochondrial function was defective during long treatments with higher concentrations of melatonin. SCC-9 cells showed no significant differences in basal respiration and ETS capacity ( Figure 2 G,H), but exhibited a significant decrease in mitochondrial respiration, after 3 and 5 days of melatonin treatment at a dose of 1500 µ M (Figure 2H,I,K,L). Thus, confirming the presence of defective mitochondria at high doses of melatonin. No significant changes in ATP turnover were observed in either group tested, following melatonin treatment (Figure 2M–O). Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 21 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. We then examined OXPHOS protein expression levels by Western blotting (Figure 3). In Cal-27 cells, protein analysis showed that treatment with melatonin led to a significant increase in the expression of complexes I, II, III, and IV, relative to the control cells, especially after 1 and 3 days of treatment (Figure 3A–D). The rate of increase in their expression levels fell after 5 days of treatment (Figure 3E,F), which was consistent with the results for mitochondrial respiratory function. However, no differences in the expression levels of complex V or ATP synthase were observed in any melatonin-treated groups (Figure 3). This correlated with mitochondrial ATP turnover, which remained unchanged (Figure 2M–O). Although SCC-9 cells were more resistant than Cal-27 cells, its values indicated a partial uncoupling between respiration and phosphorylation in mitochondria, which could correlate with an increase in ROS production. Thus, we found that melatonin treatment was able to induce significant levels of ROS production in Cal-27 cells, in a dose- and time-dependent manner (Figure 4A,B). However, ROS production decreased after 5 days of melatonin treatment (Figure 4C), which correlated with the decrease in mitochondrial function. The increase in ROS levels was an indication of the weak scavenging ability of cancer cells. We therefore measured superoxide dismutase (SOD) activity (Figure 4D– F), which constituted the first line of defense against excess ROS. SOD activity was found to significantly decrease following treatment with melatonin, in a dose- and time-depend- ent manner, thus, confirming that melatonin increased oxidative stress in cancer cells. 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 µ M, 500 µ M, and 1500 µ 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< 0.05, ** 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. Antioxidants 2021,10, 603 8 of 20 We then examined OXPHOS protein expression levels by Western blotting (Figure 3). In Cal-27 cells, protein analysis showed that treatment with melatonin led to a significant increase in the expression of complexes I, II, III, and IV, relative to the control cells, especially after 1 and 3 days of treatment (Figure 3A–D). The rate of increase in their expression levels fell after 5 days of treatment (Figure 3E,F), which was consistent with the results for mitochondrial respiratory function. However, no differences in the expression levels of complex V or ATP synthase were observed in any melatonin-treated groups ( Figure 3 ). This correlated with mitochondrial ATP turnover, which remained unchanged ( Figure 2M–O ). Although SCC-9 cells were more resistant than Cal-27 cells, its values indicated a partial uncoupling between respiration and phosphorylation in mitochondria, which could correlate with an increase in ROS production. Thus, we found that melatonin treatment was able to induce significant levels of ROS production in Cal-27 cells, in a dose- and time-dependent manner (Figure 4A,B). However, ROS production decreased after 5 days of melatonin treatment (Figure 4C), which correlated with the decrease in mitochondrial function. The increase in ROS levels was an indication of the weak scavenging ability of cancer cells. We therefore measured superoxide dismutase (SOD) activity (Figure 4D–F), which constituted the first line of defense against excess ROS. SOD activity was found to significantly decrease following treatment with melatonin, in a dose- and time-dependent manner, thus, confirming that melatonin increased oxidative stress in cancer cells. Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 21 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, $$ p < 0.01 vs. aMT 500 µM group. Figure 3. Regulation of OXPHOS protein expression by melatonin in Cal-27 cells. Analysis of OXPHOS protein expression by Western blot after 1 day ( A , B ), 3 days ( C , D ), and 5 days of melatonin treatment ( E , F ). Data for aMT 1500 group are not shown at day 5 because most cells died. n= 6 per group. Data are presented as mean ± SEM. * p< 0.05, ** p< 0.01, *** p< 0.001 vs. control; # p< 0.05, ## p< 0.01, ### p< 0.001 vs. aMT 100 µ M group; $ p< 0.05, $$ p< 0.01 vs. aMT 500 µM group. Antioxidants 2021,10, 603 9 of 20 Antioxidants 2021, 10, x FOR PEER REVIEW 10 of 21 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. 3.3. Effects of Melatonin on Glycolytic Activity in HNSCC Cells Cancer cells are able to use different energy sources. In order to clarify the metabolic flux of HNSCC treated with melatonin, we focused on glycolytic activity, due to its prime role in cancer cell proliferation. Our investigation of the effects of melatonin on glycolytic activity in HNSCC cells showed a decrease in the extracellular acidification rate (ECAR), after 3 and 5 days of treatment with high doses of melatonin (Figure 5A–F). Cellular glycolytic reserve capacity, calculated by subtracting the maximal ECAR from the basal rate, increased significantly, following melatonin treatment, reaching a peak at a dose of 500 µM, after 1 and 3 days of treatment, as compared to the basal control group (Figure 5G– I). 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. 3.3. Effects of Melatonin on Glycolytic Activity in HNSCC Cells Cancer cells are able to use different energy sources. In order to clarify the metabolic flux of HNSCC treated with melatonin, we focused on glycolytic activity, due to its prime role in cancer cell proliferation. Our investigation of the effects of melatonin on glycolytic activity in HNSCC cells showed a decrease in the extracellular acidification rate (ECAR), after 3 and 5 days of treatment with high doses of melatonin (Figure 5A–F). Cellular glycolytic reserve capacity, calculated by subtracting the maximal ECAR from the basal rate, increased significantly, following melatonin treatment, reaching a peak at a dose of 500 µ M, after 1 and 3 days of treatment, as compared to the basal control group (Figure 5G–I). With regard to the effect of melatonin treatment on HNSCC glycolysis, hexokinase II (HK-II) protein levels were determined. Treatment with 500 µ M of melatonin significantly decreased the hexokinase II (HK-II) protein content in Cal-27 cancer cells, after 5 days of treatment (Figure 5J–L). Antioxidants 2021,10, 603 16 of 20 time-dependent manner, which was partly due to decreased mitochondrial SOD activity. High levels of mitochondrial SOD are well-known to greatly stimulate glycolysis through H 2 O 2 production and activation of specific signaling pathways and to strongly inhibit mitochondrial oxidative metabolism [ 41 ]. However, the complex role of ROS in mediating tumor progression remains a subject of controversy. Several studies concluded that ROS, as signaling molecules, facilitate cancer stem cell (CSC) self-renewal and expansion [ 42 ], stimulated cell invasiveness [ 43 ], and potentiated tumor progression. Nonetheless, increased ROS levels were also reported to be detrimental to other cancer cells and to inhibit cancer metastasis [ 42 ]. Furthermore, restoration of OXPHOS activity and increased levels of ROS can sensitize CSCs to chemotherapeutic drugs [44]. Glycolysis is generally used by cancer cells for ATP production rather than mitochondrial respiration, even in the presence of adequate oxygen [37]. In addition, resistant cells can acquire a glycolytic metabolism to survive cytotoxic stress [ 38 ]. Given that glycolytic marker expression in HNSCC is associated with poor survival rates and resistance to chemo and radiotherapy [ 2 ], we observed that melatonin decreases glycolysis. On the whole, these findings indicate that glycolysis inhibition and increased OXPHOS might explain, at least partly, the inhibition of metabolic reprogramming induced by melatonin in HNSCC cells [38]. Mitochondrial metabolic reprogramming is shown to be necessary for tumorigenesis, metastasis, and stemness [ 5 , 30 ]. Guido et al. suggested that remodeling of mitochondrial morphology protects tumor cells against cellular stress with remarkable success, and therefore, that adjustment of mitochondrial morphology could be a potential tool in human cancer treatment [ 45 ]. It was suggested that mitochondrial dynamic changes act as sensors of energy stress and initiators of cancer cell metabolic reprogramming [ 46 ]. However, the effects of melatonin on reprogrammed metabolism mediated by the changes in cancer cell mitochondrial morphology are little understood. Our study showed that melatonin induced mitochondrial elongation and facilitated the formation of cristae in HNSCC cells and thus increased mitochondrial mass. The shape of cristae was also demonstrated to regulate the stability and assembly of respiratory chain supercomplexes, which affected respiratory efficiency [ 27 ]. It was possible to speculate that mitochondrial elongation correlated with a clear metabolic shift from glycolysis to oxidative phosphorylation. Mitochondrial functions were closely associated with the morphodynamics of mitochondria, consisting of both fusion and fission processes. In general, fission, whose pro- or anti-apoptotic function remains a matter of debate, is thought to be pro-tumorigenic [ 25 ]. On the other hand, some authors showed that an increase in activation of mitochondrial fusion in cancer is a metabolic advantage to maintain tumor growth [ 26 ]. In this study we found that melatonin decreased fusion and fission after 5 days of treatment. Our results appear to be congruent with the finding that mitochondrial fusion proteins are responsible for maintaining mitochondrial membrane structures, matrix homogeneity, and mitochondrial genome integrity, which are vital for cell survival [ 47 ]. In addition, the loss of fusion/fission balance is linked to mitochondrial and cellular dysfunctions such as apoptosis. Other studies demonstrated that certain anti-tumor drugs activate autophagy while inducing apoptosis [ 10 , 48 ]. This is in line with our finding that melatonin triggers apoptosis in a dose- and time-dependent manner. Melatonin leads to a decrease in anti-apoptotic factors and to a release of pro-apoptotic factors from the mitochondrial inter-membrane space into the cytosol. This could be partly due to severe impairment of the mitochondrial electron transport chain, which is a major site of ROS production [49]. ROS production triggers mitochondrial autophagy and protein degradation in order to mitigate the cellular stress induced by melatonin. However, autophagy, which plays a dual role, can induce senescence or cell death as a tumor suppressor and can also induce tumorigenesis [ 50 ]. Reprogrammed cancer cells, undergoing glycolysis, display mitochondrial dysfunction and activate autophagy/mitophagy [ 51 ]. Although the specific mechanism of autophagy involved in cancer cells remains largely unknown, Atg5-dependent autophagy was shown to be transiently activated early on the reprogramming process [ 52 ], which Antioxidants 2021,10, 603 17 of 20 was blocked in Agt5 −/− cells in particular. This was consistent with our results which showed that melatonin decreased glycolysis and ATG5 levels after three days of treatment, although melatonin was also found to induce mitophagy and apoptosis. We found that the cytotoxic effects of melatonin in decreasing cell proliferation are dose- and time-dependent. We show that high doses of melatonin, such as those used in this study and elsewhere [ 20 ], with their cytotoxic effect on HNSCC cells, further increased melatonin receptor expression, which might be involved, at least partly, in the indoleamine effect [12]. 5. Conclusions In conclusion, we showed that melatonin increased mitochondrial TCA intermediates and OXPHOS levels, resulting in an inhibition of metabolic reprogramming of aerobic glycolysis. Overall, the study provides new insights into the mechanisms underlying melatonin’s anti-tumor activity in head and neck cancer cells. To the best of our knowledge, this was the first study to demonstrate that the anti-proliferative effects of melatonin in HNSCC are, at least partially, due to an inhibition of metabolic remodeling, resulting in enhanced mitochondrial function. This, in turn, leads to increased oxidative stress and activation of apoptosis and mitophagy in HNSCC cells. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox10040603/s1. Figure S1: Melatonin-induced changes in mitochondrial morphology. Figure S2: Modulation of melatonin receptor expression by melatonin in Cal-27 cells. Figures S3–S8: Full Western blots data. Author Contributions: Conceptualization, G.E.; methodology, G.E., A.G.-L. and B.I.F.-G.; formal analysis, G.E., A.G.-L. and B.I.F.-G.; investigation, A.G.-L., B.I.F.-G., J.F., L.M.-R., C.R.-S., Y.-Q.S., A.L.-R., J.M.G.-V., I.R. and G.E; resources, G.E.; writing-original draft preparation, G.E. and A.G.-L.; writing-review and editing, G.E., A.G.-L., A.Q.-H., D.A.-C., J.M.G.-V., J.M. and T.D.H.; supervision, G.E., D.A.-C., J.M. and T.D.H.; project administration, G.E.; funding acquisition, G.E. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by grants from the Ministerio de Economia, Industria y Competitividad y por el Fondo de Desarrollo Regional FEDER, Spain n ◦ SAF2013-49019, SAF2017-85903-P, and from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P07- CTS- 03135, P10- CTS- 5784, and CTS- 101), Spain. J.F. and L.M. have FPU fellowships from the Ministerio de Educación Cultura y Deporte, Spain. C.R.S. was a schorlarship holder from the Plan Propio de Investigación of the University of Granada. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Acknowledgments: We wish to thank Michael O’Shea for proofreading the paper. Conflicts of Interest: The authors declare no conflict of interest. References 1. Johnson, N.W.; Jayasekara, P.; Amarasinghe, A.A.; Hemantha, K. Squamous cell carcinoma and precursor lesions of the oral cavity: Epidemiology and aetiology. 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