Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport

Abstract

The oncostatic effects of melatonin correlate with increased reactive oxygen species (ROS) levels, but how melatonin induces this ROS generation is unknown. In the present study, we aimed to elucidate the two seemingly opposing actions of melatonin regarding its relationship with free radicals. We analyzed the effects of melatonin on head and neck squamous cell carcinoma cell lines (Cal‐27 and SCC‐9), which were treated with 0.5 or 1 mM melatonin. We further examined the potential effects of melatonin to induce ROS and apoptosis in Cal‐27 xenograft mice. Here we report that melatonin mediates apoptosis in head and neck cancer by driving mitochondrial reverse electron transport (RET) to induce ROS production. Melatonin‐induced changes in tumoral metabolism led to increased mitochondrial activity, which, in turn, induced ROS‐dependent mitochondrial uncoupling. Interestingly, mitochondrial complex inhibitors, including rotenone, abolished the ROS elevation indicating that melatonin increased ROS generation via RET. Melatonin also increased membrane potential and CoQ₁₀H₂/CoQ₁₀ ratio to elevate mitochondrial ROS production, which are essential conditions for RET. We found that genetic manipulation of cancer cells with alternative oxidase, which transfers electrons from QH₂ to oxygen, inhibited melatonin‐induced ROS generation, and apoptosis. RET restored the melatonin‐induced oncostatic effect, highlighting the importance of RET as the site of ROS production. These results illustrate that RET and ROS production are crucial factors in melatonin's effects in cancer cells and establish the dual effect of melatonin in protecting normal cells and inducing apoptosis in cancer cells.

1. INTRODUCTION

Cases of cancer, which is the second‐leading cause of mortality worldwide, are expected to increase by 2025, ¹ and head and neck squamous cell carcinoma (HNSCC) is responsible for 500 000 new cases each year. Despite ongoing advances in surgery, radiotherapy, and chemotherapy, 5‐year survival rates remain under 50%. ² , ³ Recurrent HNSCC contributes to significant morbidity and portends overall poor survival, ⁴ and research into new therapeutic targets and treatments is quite urgent.

Because of its oncostatic influence and lack of association with adverse effects, melatonin (N‐acetyl‐5‐methoxytryptamine) is of particular relevance to the development of innovative cancer treatments. ⁵ , ⁶ , ⁷ Several groups have reported that high concentrations of melatonin can promote reactive oxygen species (ROS) generation in a variety of cancers. ⁶ , ⁷ , ⁸ , ⁹

Melatonin and its derivatives, however, are potent free radical scavengers and broad‐spectrum antioxidants, which are evolutionarily conserved properties. ¹⁰ , ¹¹ Thus, how ROS production is involved in melatonin's antineoplastic impact remains unclear, and elucidating this mechanism is important for possible clinical applications.

Melatonin targets mitochondria to enhance their function, maintaining mitochondrial integrity and leading to reduced electron leakage and mitochondrial ROS (mtROS) generation in nontumor cells. ¹ , ¹² , ¹³ The respiratory chain is the major source of ROS, and complexes I and III are generally regarded as the main ROS sources, whereas the contribution of intact complex II seems to be negligible. ¹⁴ , ¹⁵ Recent studies have highlighted an important role for reverse electron transport (RET) in producing mtROS. ¹⁵ , ¹⁶ , ¹⁷ RET occurs when the pool of coenzyme Q (CoQ) is over‐reduced with electrons from respiratory complex II. In the presence of a high proton motive force (Δp), complex I reduces NAD⁺ to NADH with electrons received from the ubiquinol pool, generating a high level of mtROS. All of these cellular alterations may lead to apoptosis. ¹⁸

Finally, complex I produces ROS in either the forward or reverse direction depending on the substrates used to feed the respiratory chain, suggesting that a change in cell metabolism could induce RET. ¹⁸ In this context, we have previously demonstrated that melatonin reverses metabolic reprogramming in HNSCC cells. ⁶ Taking these previous results together with known effects of melatonin in modifying tumor metabolism, we hypothesized that melatonin might increase mtROS by RET.

2. MATERIALS AND METHODS

2.1. Cell culture and treatment

The head and neck squamous carcinoma (HNSCC) cell lines 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. The Cal‐27 cell line was grown as previously described. ⁵ Previously, we performed a dose‐response study, and the maximal effect was obtained at 500 and 1000 µM. ⁵ , ⁶ , ⁷ Therefore, cells were treated with melatonin 500 and 1000 µM for 48 h. The vehicle was added to the control group. Details are available in the Supporting Information.

2.2. Measurement of ROS production and mitochondrial mass

Mitochondrial superoxide was detected using the fluorescent MitoSox Red probe and mitochondrial mass with the fluorescent MitoTracker Green probe.

ROS production was measured using 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) as previously described. ⁶ Details are available in the Supporting Information.

2.3. Apoptosis

Apoptotic cells were detected using an annexin V/propidium iodide (PI) kit (ANXVKF‐100T; Inmunostep). Once cells were treated as described above, annexin V/PI staining was performed according to the manufacturer's instructions. HNSCC cells were analyzed using a flow cytometer (Becton Dickinson FACSCanto II cytometer).

2.4. Calcium retention capacity in digitonin‐permeabilized cells

The calcium retention capacity was measured in digitonin‐permeabilized cells by adding sequential Ca²⁺ pulses until mitochondrial permeability transition pore (mPTP) was opened. Measurements of extramitochondrial Ca²⁺ were carried out fluorometrically. ¹⁹ , ²⁰ Details are available in the Supporting Information.

2.5. Western blot analysis

Protein extraction and western blot analyses were performed as previously described ⁶ and the Bradford assay was used to determine protein concentration. Details are available in the Supporting Information.

2.6. Electron transport chain (ETC) complex activity assays

Mitochondrial respiratory chain activities were measured for each complex using kits from Abcam according to the manufacturer's instructions. Details are available in the Supporting Information.

2.7. Measurement of mitochondrial respiration

Oxygen consumption rate (OCR) was determined using the Seahorse Extracellular Flux (XF‐24) analyzer as previously described. ⁶ Details are available in the Supporting Information.

2.8. Measurement of oxygen consumption in intact cells

Oxygen consumption was measured into a thermostatically controlled oxygraph vessel equipped with a Clark oxygen electrode at 37°C (Strathkelvin MS200A system). ²¹ Details are available in the Supporting Information.

2.9. Measurement of oxygen consumption in digitonin‐permeabilized cells

Oxygen consumption was measured in digitonin‐permeabilized cells in a thermostatically controlled oxygraph vessel equipped with a Clark oxygen electrode at 37°C. ²¹ Details are available in the Supporting Information.

2.10. Determination of adenosine triphosphate (ATP) levels

ATP concentration was determined using the ATP assay kit from Abcam according to the manufacturer's instructions. Details are available in the Supporting Information.

2.11. Determination of CoQ₁₀ and CoQ₁₀H₂ by liquid chromatography and mass spectrometry (UPLC‐MS/MS)

CoQ₁₀ pools were extracted as previously described ²² and analyzed by ultraperformance liquid chromatography (UPLC) with tandem mass spectrometry (MS/MS). The UHPLC system was coupled online with a Q Exactive Focus mass spectrometer (Thermo) as previously described. ²³ Data were analyzed using Tracefinder 4.1 software (Thermo). Details are available in the Supporting Information.

2.12. Mitochondrial membrane potential analysis

Mitochondrial membrane potential was measured by fluorometric analysis using tetramethylrhodamine ethyl ester (TMRE) with the microplate fluorescence reader FL × 800 (Bio‐Tek Instruments) at 549 nm to excitation and 575 nm to emission. ²⁴ Details are available in the Supporting Information.

2.13. Mitochondrial mass analysis by flow cytometry

Cell suspensions were incubated in the presence of 50 nM of Mitotracker GreenFM for mitochondrial mass measurement for 15 min in a 5% CO₂ humidified atmosphere at 37°C and protected from light. They then were immediately analyzed by fluorescence‐activated cell sorting at 488 nm to excitation and 530 nm to emission. ²⁵

2.14. Determination of tricarboxylic acid (TCA) cycle intermediates by UPLC‐MS/MS

TCA intermediates were extracted as previously described. ²⁶ Samples were then analyzed by UPLC‐MS/MS. Data were acquired with MassLynx 4.0 software and calibrated and quantified by QuanLynx software. Details are available in the Supporting Information.

2.15. NAD/NADH quantification

NAD+ levels were measured using a oxidized and reduced nicotinamide adenine dinucleotide (NAD/NADH) assay kit (ab65348; Abcam) according to the manufacturer's instructions. NAD+ and NADH levels were measured with a microplate reader spectrophotometer (Power Wave X‐1) at 450 nm. Values for NAD+ and NADH levels were determined from a standard calibration curve. NAD+ and NADH concentrations were normalized by protein content as measured using the Bradford assay.

2.16. Evaluation of supercomplex (SC) formation by blue native gel electrophoresis (BNGE)

BNGE was performed on crude mitochondrial fractions as previously described. ²⁷ Details are available in the Supporting Information.

2.17. Production of alternative oxidase (AOX) stable lines

Lentiviral vectors carrying the cDNA encoding an AOX (sequence from Ciona intestinalis genome, DM193474.1, 1110 pb) were used to generate a Cal‐27 cell line expressing AOX. The AOX stable cell line was established according to the manufacturer's instructions. Details are available in the Supporting Information.

2.18. Animal models

All animal experiments were performed following a protocol approved by the Institutional Animal Care and Use Committee of the University of Granada (procedures 11‐CEEA‐OH‐2013), developed in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (CETS # 123) and Spanish law (R.D. 53/2013). Details are available in Supporting Information.

2.19. Histology

Apoptosis was evaluated using the DeadEnd Fluorometric terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (G3250; Promega) system in tumor tissue sections following the manufacturer's indications. Hoechst (33342; ThermoFisher Scientific) was used to stain the nuclei. Images were acquired under fluorescence microscopy (Nikon Eclipse Ni‐U microscope), with quantification of TUNEL+ per field using five random fields per slide. Images were analyzed with ImageJ software. Details are available in Supporting Information.

2.20. Data and statistical analysis

All statistical analyses were performed using Prism 8 scientific software (GraphPad Software Inc.). Unpaired Student's t‐tests were used to compare differences between experimental groups and their respective untreated controls. Data were expressed as the mean ± standard error of the mean of a minimum of three independent experiments. A p < .05 was considered statistically significant.

3. RESULTS

3.1. Melatonin induces apoptosis via ROS‐dependent production

As the direct anticancer effects of melatonin correlate with increased ROS levels and increased apoptosis in HNSCC, ⁶ we first analyzed ROS levels by MitoSox‐Red and MitoTracker green fluorescent staining (Figure 1A) and fluorometric techniques (Figure 1B). Consistent with previous findings, ⁵ , ⁶ , ⁷ melatonin treatment induced an increase in ROS production in Cal‐27 cells, especially after 48 h at 500 or 1000 µM (Figure 1A,B), which correlated with a marked increase in apoptosis (Figure 1C). Similar results were obtained in SCC‐9 cells (Supporting Information: Figure S1A,B). Of note, pretreating cancer cells with N‐acetylcysteine (NAC) at 5 mM to scavenge ROS (Figure 1D) inhibited melatonin‐induced apoptosis (Figure 1E).

Figure 1.

mtROS production drives melatonin‐induced proapoptotic effects in Cal‐27. (A) ROS (red) and mitochondria (green) detected by fluorescence microscopy after incubation with MitoSox‐Red (5 µM) and MitoTracker (50 nM) for 20 min in Cal‐27 cells treated with melatonin (aMT) for 48 h. Scale bar = 50 µm. (B) Measurements of intracellular ROS levels by fluorimetry after staining with the DCFA fluorescent probe after 24 and 48 h of melatonin treatment. (C) Apoptosis level (AV+/PI+ and AV+/PI−) analyzed by flow cytometry after 24 and 48 h of melatonin treatment. For (A–C), treatment groups included vehicle (control) and melatonin (aMT) at a concentration of 500 or 1000 µM for 24 h (gray range) or 48 h (blue range) in Cal‐27 cells. (D) Measurements of intracellular ROS levels by fluorimetry after staining with the DCF fluorescent probe in Cal‐27 cells treated with melatonin at 1000 µM alone and pretreated with NAC 5 mM. (E) Apoptosis level (AV+/PI+ and AV+/PI−) analyzed by flow cytometry in Cal‐27 cells treated with melatonin at 1000 µM alone and pretreated with NAC 5 mM. (F) Calcium retention capacity detected by fluorometric assay in Cal‐27 cells treated with melatonin at 500 or 1000 µM for 48 h. (G) Measurements of intracellular ROS levels by fluorimetry after staining with the DCF fluorescent probe in Cal‐27 cells treated with melatonin at 500 or 1000 µM for 48 h and pretreated with CsA at 1 µM. (H) Apoptosis level (AV+/PI+and AV+/PI−) analyzed by flow cytometry in Cal‐27 cells treated with melatonin at 500 or 1000 µM for 48 h and pretreated with CsA at 1 µM. (I) Measurements of intracellular ROS levels by fluorimetry after staining with the DCF fluorescent probe in Cal‐27 cells treated with melatonin at 500 or 1000 µM for 48 h and pretreated with 3 nM rotenone, 10 mM malonate, 50 µM TTFA, 2 µM ant A, 200 µM KCN, or 200 nM FCCP. (J) Apoptosis level (AV+/PI+ and AV+/PI−) analyzed by flow cytometry in Cal‐27 cells treated with melatonin at 500 or 1000 µM for 48 h and pretreated with 3 nM rotenone, 10 mM malonate, 50 µM TTFA, 2 µM antimycin A, 200 µM KCN, or 200 nM FCCP. Data are presented as mean ± standard error of the mean (n =  4–10 for each group; one‐tailed unpaired t‐test; *p < .05; **p < .01; ***p < .001 vs. control; ^δ p < .05, ^δδ p < .01 vs. aMT 1000 µM). ant A, antimycin A; AV, annexin V; DCF, 2′,7′‐Dichlorodihydrofluorescein; DCFA, dichlorodihydrofluorescein diacetate; FCCP, carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone; KCN, potassium cyanide; mtROS, mitochondrial ROS; NAC, N‐acetylcysteine; PI, propidium iodide; ROS, reactive oxygen species; TTFA, thenoyltrifluoroacetone

ROS production may trigger mPTP with further stimulation of ROS. Therefore, we measured the calcium retention capacity in Cal‐27 cells treated with melatonin at 500 or 1000 µM after 48 h. Melatonin treatment decreased calcium retention capacity for both cell lines (Figure 1F and Supporting Information: Figure S1C), indicating that it increased the mPTP opening state in Cal‐27 and SCC‐9 cells. To corroborate whether the mPTP opening state induced apoptosis, cells were pretreated with an mPTP opening inhibitor, cyclosporin A (CsA) at 1 µM in both cell lines. As shown in Figure 1G and Supporting Information: Figure S1D, CsA decreased melatonin‐induced ROS production. Moreover, consistent with the above results, the reduced ROS production led to decreased apoptosis (Figure 1H and Supporting Information: Figure S1E). Overall, these findings suggest that the apoptotic effect of melatonin arises from ROS‐induced mPTP opening. These data further support that melatonin‐induced apoptosis requires sustained ROS production.

To investigate whether the activity of ETC complexes is essential for ROS production, we applied different inhibitors of mitochondrial respiration. Of interest, pretreatment of Cal‐27 and SCC‐9 cells with rotenone, malonate, thenoyltrifluoroacetone, antimycin A, potassium cyanide, or carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP) inhibited melatonin‐induced ROS production (Figure 1I and Supporting Information: Figure S1F). As expected, these results correlated with decreased melatonin‐induced apoptosis (Figure 1J and Supporting Information: Figure S1G). These findings suggest that melatonin acts through high mitochondrial complex activities and high membrane potential to elevate mitochondrial ROS production and that all mitochondrial complex inhibitors, including rotenone, can abolish the ROS elevation (Figure 1I and Supporting Information: Figure S1F). Rotenone inhibits complex I RET, ¹⁸ , ²⁸ so it is possible that melatonin alters mitochondrial respiration via RET.

3.2. Melatonin induces mitochondrial uncoupling in HNSCC cells

Mitochondrial ROS burst is a hallmark of complex I RET, and the activity of ETC complexes and the mitochondrial proton gradient is essential for RET. ¹⁵ For these reasons, to elucidate the mechanism underlying the melatonin‐induced ROS increase, we analyzed mitochondrial function in HNSCC cells. We evaluated OXPHOS protein levels by western blot analysis and OXPHOS activity by spectrophotometric analysis (Figure 2A–K). In agreement with the effects of melatonin in nontumor tissues, ¹² , ²⁹ , ³⁰ , ³¹ melatonin increased the levels of complexes I and IV relative to control cells at 1000 μM after 48 h of treatment (Figure 2A,D). This increase was reflected primarily in increased complex I and IV activities after 24 h of treatment (Figure 2G,J). To our surprise and in contrast to results with the mitochondria of nontumor cells, ³⁰ melatonin at 48 h highly increased levels of complex II, and at 24 h increased its activity, at 1000 µM (Figure 2B,H). Complex III and complex V were unaffected (Figure 2C,E,I,K). Furthermore, we found a decrease in complex II and complex V activity after 48h of melatonin treatment at 1000 µM (Figure 2H,K), suggesting that the high concentration of melatonin during longer treatments decreased mitochondrial function.

Figure 2.

Effect of melatonin on mitochondrial function in the HNSCC Cal‐27 cell line. (A–F) Analysis of OXPHOS protein expression by western blot (WB). (G–K) Analysis of mitochondria complex (CI–CV) activity by spectrophotometric analysis. (L–N) OCR corresponding to (M) basal respiration and (N) ETS capacity, as analyzed by SeaHorse. (O) OCR in digitonin‐permeabilized Cal‐27 cells with glutamate/malate without ADP (state 2), in the presence of 500 µM ADP (state 3) or 0.16 µg/ml oligomycin A (state 4), and RCR calculation (state 3/state 4). (P) OCR in digitonin‐permeabilized Cal‐27 cells with succinate without ADP (state 2), in the presence of 500 µM ADP (state 3) or 0.16 µg/ml oligomycin A (state 4), and RCR calculation (state 3/state 4). (Q) Ratio of ATP levels measured by fluorometric test. (R,S) Ratio of PAMPK and AMPK expression analyzed by western blot. Treatment groups included vehicle (control) and melatonin (aMT) at 500 or 1000 µM for 24 h (gray range) or 48 h (blue range) in Cal‐27 cells. Data are presented as mean ± standard error of the mean (n = 3–8 for each group; one‐tailed unpaired t‐test; *p < .05; **p < .01 vs. control). ADP, adenosine diphosphate; ETS, electron transport system; HNSCC, head and neck squamous cell carcinoma; OCR, oxygen consumption rate; OD, optical density; AMPK, adenosine monophosphate protein kinase; PAMPK, phosphorylated adenosine monophosphate protein kinase; RCR, respiratory control ratio; WB, western blot

Next, using the Seahorse XF24 extracellular flux analyzer, we examined whether the change in mitochondrial complexes correlated with increased mitochondrial respiration. Again, to our surprise and in contrast to results observed for complex activities, melatonin did not modify the OCR corresponding to basal respiration (Figure 2L,M). However, melatonin induced increased electron transport system (ETS) within 24 h in Cal‐27 cells treated with 1000 μM melatonin, without any change at 48 h (Figure 2L,N). These results were confirmed using an oxygraph vessel equipped with a Clark oxygen electrode (Supporting Information: Figure S2A,B), indicating that melatonin treatment did not modify basal OCR after 48 h of treatment.

To further confirm these results, we conducted experiments using oxygraph respiration in permeabilized Cal‐27 cells. OCR was measured in the presence of glutamate/malate (G/M) (Figure 2O) or succinate as substrates (state 2) (Figure 2P), after the addition of adenosine diphosphate (state 3) and followed by the addition of oligomycin (state 4). Consistent with the results obtained with intact cells (Supporting Information: Figure S2A), melatonin at any dose did not modify the respiratory control ratio (RCR: state 3/state 4) with G/M, related to complex I‐dependent respiration (Figure 2O). However, melatonin decreased the RCR with succinate, which is related to complex II‐dependent respiration (Figure 2P). These data suggest that melatonin induced an increase in some components of the ETC (complexes I, II, and IV) without increasing mitochondrial respiration, suggesting uncoupling between the ETC and phosphorylation in the mitochondria of Cal‐27 cells.

To clarify the partial uncoupling between respiration and oxidative phosphorylation in mitochondria induced by melatonin, we analyzed steady‐state levels of ATP by fluorometric assay (Figure 2Q) and adenosine monophosphate‐activated protein kinase (AMPK) activation, which depends on the AMP/ATP ratio, by western blot analysis (Figure 2R,S). Melatonin reduced ATP levels after 48 h of treatment and activated AMPK (Figure 2Q–S), confirming that melatonin induced a mitochondrial uncoupling in Cal‐27 cells, increasing mtROS production.

3.3. Melatonin increases RET‐ROS by modification of the CoQ redox state and mitochondrial membrane potential (Δψm)

Given that RET occurs when the pool of CoQ is over‐reduced with electrons from respiratory complex II ¹⁷ , ³² and that melatonin notably increased complex II activity and expression (Figure 2B,H), we analyzed the CoQ redox state by UPLC‐MS/MS (Figure 3A,B). Consistent with our prediction, melatonin 1000 µM enhanced the CoQ₁₀H₂/CoQ₁₀ ratio after 24 h of treatment (Figure 3A), without increasing total CoQ (Figure 3B). We obtained similar results with melatonin after 48 h of treatment (Figure 3A,B).

Figure 3.

Increased RET‐ROS by modification of CoQ redox and mitochondrial membrane potential in Cal‐27 cells treated with melatonin. (A, B) CoQ10 and CoQ10H₂/CoQ10 ratio analyzed by UPLC‐MS/MS. (C, D) (C) Mitochondrial membrane potential and (D) mitochondrial mass analyses by fluorimetry after staining with TMRE or MTG fluorescent probe, respectively. (E, F) Western blot analysis of UCP‐1, UCP‐2, and UCP‐3. Treatment groups included vehicle (control) and melatonin (aMT) at 500 or 1000 µM for 24 h (gray range) or 48 h (blue range) in Cal‐27 cells. Data are presented as mean ± standard error of the mean (n =  3–8 for each group; one‐tailed unpaired t‐test; *p < .05; **p < .01; ***p < .001 vs. control). CoQ, coenzyme Q; MS, mass spectrometry; MTG, mitotracker green; RET, reverse electron transport; ROS, reactive oxygen species; TMRE, tetramethylrhodamine ethyl ester; UCP, uncoupling protein; UPLC, ultraperformance liquid chromatography.

The production of mitochondrial ROS via complex I RET tightly depends on high Δψm, and even a slight decrease in Δψm induced by FCCP reduces ROS generation via complex I RET. ³³ As we showed above, pretreatment of Cal‐27 cells with FCCP abolished the melatonin‐induced mitochondrial ROS burst (Figure 1I). Moreover, cell incubation with melatonin for 24 h significantly increased Δψm in Cal‐27 cells (Figure 3C) without altering the mitochondrial mass, as measured by MitoTracker Green FM fluorescence (Figure 3D), further supporting melatonin‐induced ROS generation via complex I RET. However, we observed a drop in Δψm after 48 h of melatonin treatment (Figure 3C), which could be explained by the fact that melatonin‐induced sustained ROS production after 48 h may trigger mPTP opening (Figure 1F), inducing Δψm decline. Because uncoupling proteins (UCPs) are the direct downstream mediators of ROS‐dependent mitochondrial production ³⁴ related to a drop in Δψm, we analyzed UCP protein levels by western blot analysis. As shown in Figure 3F,E, melatonin significantly increased UCP2 levels in a dose‐dependent manner, explaining the steep reduction in Δψm.

3.4. Melatonin increases RET‐ROS by modification of metabolism

To examine how melatonin induces a modification of metabolism, we first carried out metabolomic analyses using UPLC‐MS/MS and found that melatonin upregulated key TCA cycle metabolites in Cal‐27 cells (Figure 4A–D), especially succinic acid, the principal substrate of RET (Figure 4A). This upregulation led to an increased NADH/NAD ratio (Figure 4E), further supporting that melatonin induced ROS generation via complex I RET.

Figure 4.

Melatonin‐induced change in Cal‐27 cell metabolism by upregulation of fatty acid oxidation and TCA metabolites. (A–D) Metabolomic study of intracellular levels of (A) succinic acid, (B) fumaric acid, (C) malic acid, and (D) ketoglutaric acid. (E) NADH and NAD+ levels measured using a colorimetric test and expressed as a ratio. (F–I) Western blot analysis of (F) MDAC, (G) EHHADH, and (H) PACC/ACC ratio. (J–M) Evaluation of SC formation by BNGE. Treatment groups included vehicle (control) and melatonin (aMT) at 500 or 1000 µM for 24 h (gray range) or 48 h (blue range) in Cal‐27 cells. Data are presented as mean ± standard error of the mean (n = 3–7 for each group; one‐tailed unpaired t‐test; *p < .05; **p < .01; ***p < .001 vs. control). BNGE, blue native gel electrophoresis; EHHADH, enoyl‐CoA hydratase and 3‐hydroxyacyl CoA dehydrogenase; PACC/ACC, phosphorylated/no‐ phosphorylated acetyl‐CoA carboxylase; SC, supercomplex; TCA, tricarboxylic acid.

When fatty acids are oxidized, NADH increases, resulting in an over‐reduction of the CoQ pool and an increase in RET‐ROS. ³⁵ We analyzed medium‐chain acyl‐CoA dehydrogenase (MCAD) and enoyl‐CoA hydratase and 3‐hydroxyacyl CoA dehydrogenase (EHHADH), which regulate fatty acid oxidation, and acetyl‐CoA carboxylase (ACC), which is involved in lipid biosynthesis. ³⁶ In a dose‐ and time‐dependent manner, melatonin triggered increased levels of medium‐chain acyl‐CoA dehydrogenase (Figure 4F,I), with a tendency for EHHADH at 48 h (Figure 4G,I), and an increased phosphorylated/No‐ phosphorylated acetyl‐CoA carboxylase ratio (Figure 4H,I), most likely due to the activated phosphorylated adenosine monophosphate protein kinase. Collectively, these results suggest that melatonin activated fatty acid oxidation as well as inhibiting lipid biosynthesis.

The high CoQ₁₀H₂/CoQ₁₀ ratio during β‐oxidation leads to RET‐produced ROS and localized RET leads to changes in SC formation. ³⁵ As expected, melatonin reduced the formation of SCs involving complex I (Figure 4J,M) but increased the formation of SCs involving complex III within 48 h in a dose‐dependent manner (Figure 4K–M). These results indicate that melatonin rearranges the formation of SCs degrading CI and stimulating the formation of complexes III + IV SCs to favor the oxidation of fatty acids. These findings support the important role of melatonin in inducing changes in HNSCC cell metabolism, indicating that melatonin could be exerting its oncostatic effect via RET‐ROS.

3.5. AOX expression reduces RET and inhibits melatonin's oncostatic effects in vitro and in vivo

To confirm whether RET underlies melatonin‐induced ROS production in HNSCC cells, we used the AOX, a cyanide‐insensitive oxidase that transfers electrons from QH₂ to oxygen, decreasing the CoQH₂/CoQ ratio. ¹⁶ For this reason, we expressed AOX in Cal‐27 cells to examine whether reducing CoQ₁₀H₂ accumulation decreased ROS production and then apoptosis. AOX expression restored a normal CoQ₁₀H₂/CoQ₁₀ ratio (Figure 5B) without inducing significant changes in the total CoQ₁₀ amount (Figure 5A). Moreover, the effects of melatonin in inducing ROS production and apoptosis were abolished in cells expressing AOX (Figure 5C,D). This result indicated that complex I RET induced by melatonin is also required for melatonin‐induced sustained ROS production. Furthermore, to ensure AOX functionality, cells were treated with complex III inhibitor antimycin A. AOX cell line showed an increase in Cal‐27 cell proliferation and mitochondrial respiration compared to WT in presence of antimycin A, indicating AOX functionality (Supporting Information: Figure S5).

Figure 5.

Melatonin‐induced oncostatic effects are inhibited in Cal‐27 expressing AOX in vitro and in vivo. (A,B) (A) Levels of total CoQ10 and (B) CoQ10H2/CoQ10 ratio in Cal‐27 cells expressing AOX in vitro, analyzed by UPLC‐MS/MS. Groups included vehicle (control) and melatonin (aMT) at 1000 µM for 24 h (treatment with the greatest difference vs. wild‐type Cal‐27). (C) Measurements in vitro of intracellular ROS levels by fluorimetry after staining with the DCF fluorescent probe in Cal‐27 cells expressing AOX. Groups included vehicle (control) and melatonin (aMT) at 500 µM for 48 h (treatment with the greatest difference vs. wild‐type Cal‐27). (D) Apoptosis level (AV+/PI+ and AV+/PI−) analyzed in vitro by flow cytometry in Cal‐27 cells expressing AOX. Groups included vehicle (control) and melatonin (aMT) at 500 µM for 48 h (treatment with the greatest difference vs. wild‐type Cal‐27). (E) Tumor volume difference analyzed in vivo with a caliper vernier. (F) Western blot analysis of carbonyl protein. (G,H) (H) TUNEL+ nuclei (apoptotic nuclei, green) and DAPI‐stained nuclei (total nuclei, blue). Scale bar = 100 µm. (G) The percentage of apoptotic cells was designated as the apoptotic index. For (E–G), treatment groups included WT Cal‐27 xenograft treated intratumorally with vehicle (control WT) and melatonin at 3% (aMT 3% WT) or Cal‐27–expressing AOX xenograft treated intratumorally with vehicle (control AOX) and melatonin at 3% (aMT 3% AOX). Data are presented as mean ± standard error of the mean (n = 3–8 for each group; one‐tailed unpaired t‐test; *p < .05; **p < .01 vs. control). AOX, alternative oxidase; CoQ, coenzyme Q; DAPI, 4′,6‐diamidino‐2‐phenylindole; DCF, 2′,7′‐dichlorodihydrofluorescein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild type.

We further confirmed these results in vivo, using mice injected with wild‐type Cal‐27 cells or cells expressing AOX. Considering that our in vivo previous results suggested that melatonin administered intraperitoneally did not reach the mitochondria at a sufficient concentration, ⁵ both groups were treated intratumorally with vehicle or with melatonin, which did not reach the plasma increasing melatonin level into the tumor. After 35 days of treatment, melatonin, which reduced greatly the tumor volume in wild‐type Cal‐27 cells, did not have any effect on volume in tumors containing cells expressing AOX (aMT 3% AOX) compared with its controls (Figure 5E). To investigate whether melatonin‐induced complex I RET led to apoptosis, we analyzed carbonyl protein level and apoptosis using the TUNEL assay. As shown in Figure 5F–H, the effects of melatonin in increasing ROS production and apoptosis disappeared in tumors expressing AOX (aMT AOX) compared with controls. Overall, these findings support that melatonin induces ROS generation via complex I RET.

4. DISCUSSION

Melatonin has two seemingly opposing actions regarding its relationship with free radicals. Numerous studies have shown that it plays an effective role in maintaining mitochondrial homeostasis, ¹² , ²⁹ , ³⁰ , ³¹ , ³⁷ but the mechanism by which melatonin induces ROS production in cancer cells is poorly understood. In this study, we provided evidence that melatonin mediates apoptosis in HNSCC cells by targeting mitochondria to induce ROS by complex I RET.

The complex role of ROS in mediating tumor progression remains a subject of controversy. Cancer cells often exhibit altered mitochondrial function, including elevated ROS generation, ³⁸ , ³⁹ which is involved in the initiation and progression of cancer. ⁴⁰ , ⁴¹ However, toxic levels of ROS production in cancers are antitumorigenic. ⁵ , ⁶ , ⁷ , ⁴² The current findings show that melatonin‐induced apoptosis was abolished by pretreatment of Cal‐27 cells with the antioxidant NAC, suggesting that ROS levels are increased by melatonin above a toxicity threshold that results in oxidative damage and cell death.

Moreover, an amplified ROS signal may cause perceptible mitochondrial and cellular injury. ⁴³ It seems that the main sites of mtROS generation in vivo are respiratory complexes I and III. ¹⁸ , ²⁸ However, under specific conditions such as hypoxia, complex II subunits can lead to increased ROS generation via RET. ¹⁸ , ²⁸  One way to differentiate RET‐ROS production is by using rotenone, which increases ROS production in the forward direction, but decreases it via RET. ⁴⁴ Our current findings indicate that ROS production induced by melatonin was reduced under complex I inhibition with rotenone. Moreover, melatonin increased the expression and activities of complexes I, II, and IV, as well as ETS capacity, and especially surprising to us was the significant increase in complex II with no effect on complex III. Complex II can adapt to different roles as a producer or modulator of mtROS depending on the activity of other complexes such as complex III. ¹⁵ In contrast, we observed a lack of effect of melatonin on complex V with a decrease in ATP production, which, in part, supports an earlier observation of melatonin‐induced mitochondrial uncoupling. ⁴⁵ These data further support that melatonin‐induced uncoupling depends on complex II respiration, as melatonin‐induced ROS production involved both complexes I and II. When complex II‐linked substrates, such as succinate, are used, a portion of the electrons flows through complex I in the reverse direction. ROS produced under these conditions depend on the redox state of CoQ and Δψm. ¹⁶ , ³² Furthermore, our results showed that melatonin increased the CoQ₁₀H₂/CoQ₁₀ ratio and mitochondrial membrane potential (Figure 6). These results appear to be congruent with the finding that ROS production via RET requires some essential mitochondrial features in the cell, such as the certain activity of mitochondrial complexes to maintain a high Δψm, especially the high activity of complex II to over‐reduce the CoQ pool. ¹⁵ Furthermore, our data suggest that there is a correlation between the cell content of melatonin and ROS production, thus supporting the notion that high concentrations of melatonin in cancer cells are required to its the cytotoxic effect. Previously, we demonstrated that melatonin markedly decreases cell viability in the irradiated cells in a dose‐dependent manner, especially at doses of 500 and 1500 µM. Surprisingly, 100 µM melatonin did not significantly reduce viability. ⁷ Consistently, other authors found that melatonin at high concentrations (10–1000 µM) was able to promote ROS generation and induced apoptosis in human leukemic Jurkat cells. When tested at concentrations of <10 µM, melatonin did not induce significant ROS generation in these cancer cells. ⁸

Figure 6.

Melatonin mediates apoptosis in head and neck cancer by driving RET to induce ROS production. (A) Melatonin increases succinate levels, complex I, complex IV, and highly complex II activity and expression inducing an increase in CoQH₂/CoQ ratio and Δψm. (B) As a consequence, melatonin induces RET‐ROS. (C), which leads to mitochondrial transition pore opening and exacerbated ROS production. (D) Finally, a high amount of ROS produces cancer cell apoptosis. Image created with BioRender.com. CoQ, coenzyme Q; RET, reverse electron transport; ROS, reactive oxygen species.

Moreover, we recently demonstrated that high doses of melatonin, such as those used in this study and elsewhere, ⁴⁶ significantly increased MT1 gene expression and led to a marked decrease in MT2 and RORα levels, ⁶ suggesting that melatonin receptor expression may be involved, at least partly, in the indoleamine effect. ³¹

Finally, using AOX, a cyanide‐insensitive oxidase that transfers electrons from QH₂ to oxygen and thus inhibits RET‐ROS production, we confirmed the mechanism of action of melatonin in inducing ROS production in cancer cells via complex I RET. In vitro results in the current work demonstrated that complex I RET mediated the proapoptotic effects of melatonin via ROS production (Figure 6). Consistent with this finding, in vivo expression of AOX in a Cal‐27 xenograft model abolished the therapeutic effects of melatonin

The question that arises is why melatonin produces large amounts of ROS by RET at complex I in HNSCC cells. In response to hypoxia, hypoxia‐inducible factor (HIF) maintains ROS production at a physiologically low level and sustains integrity by decreasing respiratory activity. ⁴⁷ However, melatonin can destabilize HIF‐1α. ⁴⁸ Previously, we have shown that melatonin inhibits tumor cells by reversing aerobic glycolysis. ⁶ Therefore, a key step in melatonin‐based suppression of aerobic glycolysis is to destabilize HIF‐1α. ⁴⁹ These results align with the current observation that melatonin increased Krebs cycle intermediates, including succinate and respiratory capacity (Figure 6).

Our data show for the first time that melatonin induces ROS complex I RET under anaerobic metabolism and leads to apoptosis from excessive ROS production, only at high doses. This pathway likely represents a new mechanism underlying the oncostatic actions of melatonin in HNSCC cells.

AUTHOR CONTRIBUTIONS

Conceptualization: Germaine Escames. Methodology: Germaine Escames, Javier Florido, Laura Martinez‐Ruiz, and César Rodriguez‐Santana. Formal analysis: Germaine Escames, Javier Florido, and Laura Martinez‐Ruiz. Investigation: Javier Florido, Laura Martinez‐Ruiz, César Rodriguez‐Santana, Alba López‐Rodríguez, Agustín Hidalgo‐Gutiérrez, Cécile Cottet‐Rousselle, Frédéric Lamarche, Ana Guerra‐Librero, Paula Aranda‐Martínez, and Luis C. López. Resources: Germaine Escames and Uwe Schlattner. Writing‐original draft preparation: Germaine Escames and Javier Florido. Writing‐review and editing: Germaine Escames, Javier Florido, Uwe Schlattner, Darío Acuña‐Castroviejo, and Luis C. López. Supervision: Germaine Escames, Uwe Schlattner, Darío Acuña‐Castroviejo, and Luis C. López. Project administration: Germaine Escames. Funding acquisition: Germaine Escames

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

Supplementary information.

Florido J, Martinez‐Ruiz L, Rodriguez‐Santana C, et al. Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport. J Pineal Res. 2022;73:e12824. 10.1111/jpi.12824

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.