Melatonin modulates cell proliferation and global dna methylation in triple-negative breast cancer cells

Authors

Abstract

AimTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited targeted treatment options. Melatonin has been reported to exhibit anti-cancer and epigenetic regulatory effects. This study aimed to investigate the effects of melatonin on cell proliferation and global DNA methylation in breast cancer cells.
MethodsMDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), while HUVEC cells (ATCC: CRL-1730) were used as a non-cancerous control model. Cells were treated with melatonin at concentrations of 1, 10, 100, and 1000 µM. Cell proliferation was evaluated using the xCELLigence RTCA-DP system and the MTT assay. Global DNA methylation levels were assessed using the MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit.
ResultsMelatonin exhibited a selective anti-proliferative effect on MDA-MB-231 cells, with no significant cytotoxicity observed in HUVEC cells at lower concentrations. The IC50 value for MDA-MB-231 cells was determined as 11.5 µM. A significant reduction in global DNA methylation levels was observed in HUVEC cells at 1000 µM (p < 0.05). In contrast, MDA-MB-231 cells demonstrated a significant decrease in methylation levels at 10 and 100 µM concentrations (p < 0.05).
ConclusionMelatonin exerts selective anti-proliferative effects on breast cancer cells while modulating DNA methylation in a dose-dependent manner. These findings suggest that melatonin may have potential as a supportive therapeutic agent in breast cancer management. Further studies are needed to clarify the underlying molecular mechanisms.

Keywords

Introduction

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression. Representing approximately 10–15% of all breast cancer diagnoses, TNBC is notable for its high proliferative rate, early metastatic spread, and overall poor prognosis. The clinical management of TNBC remains challenging, as the lack of molecular targets precludes endocrine and HER2-directed therapies, leaving chemotherapy as the main systemic option—yet long-term effectiveness is often limited.¹
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone primarily secreted by the pineal gland and is well known for regulating circadian rhythms and the sleep–wake cycle. Beyond its chronobiotic role, melatonin exerts potent anti-inflammatory, antioxidant, immunomodulatory, and anti-cancer effects.^5,6 In multiple cancer models, including breast cancer, melatonin suppresses cellular proliferation, induces apoptosis, inhibits angiogenesis and metastasis, and modulates hormone-dependent signaling pathways. Evidence from both clinical and preclinical studies further suggests that melatonin’s tumor-suppressive actions extend to hormone receptor–negative subtypes such as TNBC.^2,3
Melatonin has also emerged as a promising epigenetic regulator. Growing evidence suggests that it modulates DNA methylation by influencing DNA methyltransferases (DNMTs) or acting through redox-sensitive epigenetic mechanisms. In breast cancer and circadian disruption models, melatonin has been shown to restore global DNA methylation patterns and reactivate tumor suppressor gene expression.^4,5,6
The present study investigates the effects of melatonin on proliferation and global DNA methylation in MDA-MB-231 TNBC cells, using HUVEC cells as a healthy control. We hypothesize that melatonin selectively inhibits TNBC proliferation and modulates global DNA methylation without harming non-cancerous cells. These findings may help position melatonin as a dual-function anti-proliferative and epigenetic modulator in breast cancer, offering potential therapeutic promise for TNBC, where treatment options remain limited.
The aim of this study was to investigate whether melatonin selectively inhibits proliferation and modulates global DNA methylation in triple-negative breast cancer cells compared with non-cancerous cells.

Materials and Methods

Cell Culture and Melatonin TreatmentMDA-MB-231 triple-negative breast cancer cells and human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions (37 °C, 5% CO₂). Melatonin stock solutions were freshly prepared and applied at graded concentrations (1 µM, 10 µM, 100 µM, 1000 µM) to assess dose-dependent effects on proliferation and global DNA methylation. Control groups received no pharmacological treatment.
Real-Time Proliferation Monitoring with xCELLigenceDynamic changes in cell viability and proliferation were quantified using the xCELLigence Real-Time Cell Analysis (RTCA) system. This impedance-based platform enables continuous, label-free monitoring of cell attachment, proliferation, and morphological behavior. Mean cell index values were compared before and after melatonin treatment.
MTT Cell Viability and Proliferation AssayTo further quantify cell metabolic activity and viability, an MTT assay (Abcam, Kit AB2110911000TEST) was conducted following melatonin exposure, which quantifies the NAD(P)H-dependent enzymatic conversion of MTT into insoluble formazan crystals 14. For this assay, cells were seeded at a density of 1×10^4 cells per well in 96-well plates (150 µL/well) and allowed to attach for 24 hours under standard culture conditions (37 °C, 5% CO₂). Following the adhesion period, cells were exposed to increasing concentrations of melatonin (1–1000 µM), and the assay was performed according to the manufacturer’s instructions.
DNA Methylation AnalysisFollowing 72 hours of incubation with or without melatonin, genomic DNA was isolated using the Qiagen DNeasy extraction kit. The purity and concentration of the extracted DNA were assessed with a NanoDrop 2000c spectrophotometer. Global DNA methylation levels, expressed as 5-methylcytosine (5-mC) content, were subsequently quantified using the MethylFlash™ Global DNA Methylation ELISA kit (Epigentek) according to the manufacturer's recommended procedures.
Ethical ApprovalThis study was conducted using established human cell lines and did not require ethical approval according to the relevant institutional and national guidelines.
Statistical AnalysisAll statistical analyses were performed using GraphPad Prism version 9.1.1. Data are presented as mean ± standard deviation (SD). Data sets with normal distribution were analyzed using unpaired t-test or two-way ANOVA, while non-parametric data were analyzed using Mann–Whitney U test or Kruskal–Wallis test, as appropriate. Tukey’s post hoc test was used following ANOVA where applicable. A p value of <0.05 was considered statistically significant.
Reporting GuidelinesNo specific reporting guideline was applicable to this in vitro experimental study.

Results

Melatonin Selectively Inhibits TNBC Cell ProliferationThe combined xCELLigence measurements and MTT data demonstrate that melatonin exerts significant antiproliferative activity in MDA-MB-231 cells at 10 µM (Table 1). The sharp decline in proliferation at this concentration, coupled with the calculated IC₅₀ of 11.5 µM, indicates that TNBC cells are highly responsive to melatonin’s cytostatic effects. Interestingly, higher doses (>10 µM) partially reversed this suppression, producing a rebound increase in impedance. This nonlinear response is consistent with previously reported biphasic melatonin signaling in cancer cells. In contrast, HUVEC cells exhibited enhanced impedance and preserved viability at the same doses, supporting melatonin’s well-established cytoprotective action in normal tissues. In HUVEC cells, increasing melatonin concentrations resulted in a significant enhancement of cell proliferation, with the highest proliferative response observed at the 1000 µM dose (p < 0.05) (Figure 1). The concordance between xCELLigence and MTT findings supports the selective antiproliferative effect of melatonin on malignant cells.
Melatonin Modulates Global DNA Methylation in a Dose- and Cell-Dependent MannerMelatonin reduced global DNA methylation levels in both cell types, with distinct response patterns. MDA-MB-231 cells demonstrated significant sensitivity at lower concentrations (10 and 100 µM), whereas HUVEC cells showed significant reduction only at 1000 µM. These findings suggest a stronger epigenetic responsiveness of malignant cells to melatonin treatment (Figure 2).
Interestingly, we observed that higher melatonin concentrations also reduced methylation levels in HUVEC cells without affecting their proliferation. This finding supports the view that melatonin may function as a methylation normalizer counteracting aberrant hypermethylation in cancer cells while modulating physiological methylation dynamics in normal cells. Melatonin’s capacity to fine-tune epigenetic states may therefore underlie its dual role in proliferation control and epigenomic regulation.

Discussion

The present study demonstrated that melatonin selectively suppresses proliferation of MDA-MB-231 triple-negative breast cancer cells while preserving viability of non-cancerous HUVEC cells. This selective effect is clinically relevant because compounds that inhibit malignant cell growth without harming healthy cells are of particular interest in cancer research.Clinically, this selectivity is particularly advantageous, as melatonin is well-known for its safety profile and lack of toxicity in normal cells, even at pharmacological concentrations.^{6,7,8,9,10,11,12,13,14} In fact, melatonin often acts as a cytoprotectant in healthy tissues under oxidative or inflammatory stress, including endothelial cells.¹⁵
Melatonin’s anticancer activity involves multiple signaling pathways, including ERK, PI3K/Akt, NF-κB, and HIF-1α, which collectively contribute to reduced proliferation and increased apoptosis in tumor cells.^7,16 The decline in global DNA methylation in melatonin-treated MDA-MB-231 cells suggests an epigenetic mechanism potentially mediated through DNMT1 downregulation or redox-sensitive pathways.^11,12 Similar reductions in 5-methylcytosine have been documented across several cancer types following melatonin treatment.^{13,17,18,19,20,21}
Interestingly, we observed that higher melatonin concentrations also reduced methylation levels in HUVEC cells without affecting their proliferation. This finding supports the view that melatonin may function as a methylation normalizer, counteracting aberrant hypermethylation in cancer cells while modulating physiological methylation dynamics in normal cells.¹² Melatonin’s capacity to fine-tune epigenetic states may therefore underlie its dual role in proliferation control and epigenomic regulation.
Further supporting this interpretation, melatonin has been shown to modulate DNA methyltransferase activity and ten-eleven translocation (TET) enzymes, promoting a shift in the methylation–demethylation balance. By enhancing α-ketoglutarate, an essential TET cofactor, melatonin can facilitate active DNA demethylation, while also inhibiting histone deacetylases (HDACs) to increase chromatin accessibility.²⁰ These actions likely contribute to the global hypomethylation observed in both malignant and healthy cells in our study.
The reduction in global DNA methylation observed in MDA-MB-231 cells may indicate modulation of abnormal cancer-associated epigenetic patterns. In contrast, the weaker response in HUVEC cells may reflect greater epigenetic stability in non-malignant cells.
Our findings are also consistent with earlier studies demonstrating that melatonin induces apoptosis and autophagy in TNBC cells. For instance, Önder et al. showed that melatonin treatment upregulates pro-apoptotic markers (Bax, TUNEL) and autophagy-associated proteins (Beclin-1, LC3), while downregulating anti-apoptotic Bcl-2 in MDA-MB-231 cells.¹⁹ Moreover, pharmacological concentrations of melatonin can act as pro-oxidants specifically in tumor cells, generating reactive oxygen species and inducing mitochondrial stress without harming normal cells.^20,21 This cancer-selective oxidative burst triggers caspase activation and DNA damage, contributing to melatonin’s cytotoxic effects against malignant cells.
Given TNBC’s highly aggressive nature and lack of targeted therapies,³ melatonin’s combined anti-proliferative, pro-oxidant, and epigenetic effects address several key hallmarks of the disease. Its potential to synergize with chemotherapies, such as paclitaxel or cisplatin, further strengthens its candidacy as an adjuvant compound.²¹ Recent studies have also linked melatonin to inhibition of TNBC progression through novel mechanisms such as mitophagy regulation.⁵ Our findings contribute to this growing body of evidence by demonstrating that melatonin not only suppresses TNBC proliferation but also induces global DNA methylation changes—providing a two-pronged therapeutic approach.

Limitations

This study has several limitations. First, the experiments were conducted in vitro using a single TNBC cell line. Second, only global DNA methylation was evaluated, whereas gene-specific methylation changes were not assessed. Third, in vivo validation and combination studies with standard therapies were not performed.

Conclusion

Melatonin exerted selective anti-proliferative effects on MDA-MB-231 triple-negative breast cancer cells while preserving viability of non-cancerous cells. In addition, melatonin significantly modulated global DNA methylation in a dose-dependent manner. These findings suggest that melatonin may represent a promising adjunctive candidate with both anti-cancer and epigenetic regulatory potential in TNBC. Further mechanistic and in vivo studies are warranted. Taken together, the combined anti-proliferative and epigenetic effects of melatonin highlight its potential as a safe and multifunctional adjuvant compound in TNBC treatment strategies. Future studies incorporating gene-specific methylation profiling, multiple TNBC models, and in vivo validation will be critical to further elucidate the therapeutic relevance of melatonin-driven epigenetic modulation and to support its translation into clinical applications.

Declarations

Abbreviations

ATCC: American type culture collection
DNMT: DNA methyltransferase
ER: Estrogen receptor
HER2: Human epidermal growth factor receptor 2
HUVEC: Human umbilical vein endothelial cell
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PR: Progesterone receptor
RTCA: Real-time cell analysis
SD: Standard deviation
TET: Ten-eleven translocation
TNBC: Triple-negative breast cancer

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About This Article

Received:

April 1, 2026

Accepted:

June 11, 2026

Published Online:

June 21, 2026