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Glyceryl Trinitrate Enhances Caffeine Cytotoxicity Under Metabolic Stress in Cancer Cells

Vesna Zeljković1, Mirjana Bogavac2, Tanja V Soldatović3

  • 1Department of Biomedical Sciences, State University of Novi Pazar, Vuka Karadžića 9, 36300 Novi Pazar, Serbia.

Molecules (Basel, Switzerland)
|June 12, 2026
PubMed

Insights

Nitroglycerin (GTN) and caffeine show enhanced cancer cell killing when combined with 2-deoxy-D-glucose (2-DG) metabolic stress. Synergistic effects were observed in some cancer cells, but also in normal fibroblasts, suggesting further investigation is needed.

Area of Science:

  • Biochemistry
  • Pharmacology
  • Oncology

Background:

  • Cancer cell metabolism is a key therapeutic target, especially under metabolic stress.
  • Glycolysis inhibitors like 2-deoxy-D-glucose (2-DG) and nitric oxide donors like nitroglycerin (GTN) have shown anticancer potential.
  • Investigating drug combinations under metabolic stress can reveal novel therapeutic strategies.

Purpose of the Study:

  • To evaluate the combined cytotoxic effects of nitroglycerin (GTN) and caffeine on human cancer cell lines under 2-DG-induced metabolic stress.
  • To determine the drug interaction patterns (synergistic, additive, or antagonistic) of GTN and caffeine in combination with 2-DG.
  • To explore potential molecular targets using in silico methods.

Main Methods:

  • Human cancer cell lines (HeLa, A549, HT29) and fibroblasts (MRC-5) were treated with 2-DG, GTN, and caffeine.
  • Cell viability was assessed using the sulforhodamine B assay at 24 and 48 hours.
  • Drug interactions were quantified using the Chou-Talalay method to calculate combination index (CI) values.
  • Molecular docking was performed to predict binding interactions of GTN and caffeine with potential targets.

Main Results:

  • 2-DG alone reduced cell viability in a dose- and time-dependent manner across cell lines.
  • Combined treatment with GTN and caffeine under 2-DG stress enhanced cytotoxicity, particularly in A549 cells.
  • Synergistic interactions (CI < 1) were observed in HeLa and A549 cells, while HT29 cells showed predominantly antagonistic responses (CI > 1).
  • Strong synergistic effects were also noted in MRC-5 fibroblasts, indicating a lack of cancer cell selectivity.
  • Molecular docking suggested favorable binding of GTN to ALDH2 and caffeine to the adenosine A2A receptor.

Conclusions:

  • GTN enhances caffeine-induced cytotoxicity under 2-DG-induced metabolic stress via combined metabolic and redox perturbation.
  • The effectiveness and selectivity of the GTN-caffeine combination are context-dependent and require further mechanistic validation.
  • Findings suggest potential for combination therapies but highlight the need for experimental validation of molecular targets and pathway activation.
Keywords:
2-deoxy-D-glucoseALDH2Chou–Talalay methodcaffeinecancer metabolismcytotoxicityglyceryl trinitratemolecular dockingnitroglycerinsynergistic effect

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