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 CoQ₁₀ H₂ /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.

Keywords: apoptosis; head and neck cancer cells; melatonin; mitochondria; oxidative damage; reactive oxygen species; reverse electron transport.

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

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

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.

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.

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.

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.