Abstract Cancer cell metabolism represents a critical therapeutic target, particularly under conditions of metabolic stress induced by glycolysis inhibition. Nitroglycerin (glyceryl trinitrate, GTN), a nitric oxide donor, and 2-deoxy-D-glucose (2-DG), a glycolysis inhibitor, have individually demonstrated anticancer potential through modulation of cellular metabolism and redox balance. In this study, we investigated the cytotoxic and combined effects of GTN and caffeine under 2-DG-induced metabolic stress in human cancer cell lines ( HeLa, A549, HT29, and MRC-5). Cell viability was assessed using the sulforhodamine B assay after 24 and 48 h treatments, while drug interactions were evaluated using the Chou–Talalay method and combination index (CI) values. 2-DG alone reduced cell viability in a concentration- and time-dependent manner, with IC 50 values ranging from 2.01 to 7.05 mM depending on the cell line and exposure period. The combined treatment further enhanced cytotoxicity, particularly in A549 cells, where viability decreased to approximately 63% after 48 h and the calculated IC 50 value for GTN in the presence of caffeine reached 0.143 μM. CI analysis demonstrated synergistic interactions in HeLa and A549 cells (CI 1). However, strong synergistic effects were also observed in MRC-5 fibroblasts, indicating limited selectivity toward cancer cells. Molecular docking suggested favorable in silico binding of GTN to aldehyde dehydrogenase 2 (ALDH2) and caffeine to the adenosine A2A receptor. Nevertheless, these findings should be considered exploratory and hypothesis-generating because target expression, enzymatic activity, and pathway activation were not experimentally validated. Overall, the results suggest that GTN enhances caffeine-induced cytotoxicity under metabolically stressed conditions through combined metabolic and redox perturbation, although the magnitude of the response depends on cellular context and warrants further mechanistic investigation. 2. Results The cytotoxic effects of nitroglycerin, 2-deoxy-D-glucose (2-DG), and caffeine were evaluated in HeLa, A549, and HT29 cancer cell lines, as well as in normal lung fibroblasts (MRC-5), using the SRB assay. Cells were treated with individual agents and their combinations for 24 h and 48 h. 2.1. Effects of Nitroglycerin Nitroglycerin (GTN) is an organic nitrate that acts as a prodrug and requires enzymatic bioactivation to exert its biological effects. The primary pathway of GTN bioactivation involves mitochondrial aldehyde dehydrogenase (ALDH2), which catalyzes the conversion of GTN into nitric oxide (NO) or related nitrogen reactive species. These molecules regulate multiple cellular processes, including vasodilation, mitochondrial respiration, oxidative stress, apoptosis, and autophagy. Although both normal and malignant cells possess the enzymatic machinery necessary for GTN bioactivation, cancer cells frequently exhibit altered redox balance, mitochondrial dysfunction, and dysregulated NO signaling, which may influence their sensitivity to NO-mediated cytotoxic effects. Consequently, GTN has attracted interest as a potential anticancer agent, particularly in combination with therapies targeting tumor metabolism and oxidative stress. GTN alone exerted minimal cytotoxic effects in HeLa and A549 cells across the tested concentration range, with cell viability remaining above 85% after both 24 h and 48 h treatments. These findings suggest that GTN, as a single agent, does not significantly impair cell survival under the tested conditions, which may be related to its prodrug nature and the requirement for enzymatic bioactivation. In contrast, HT29 cells showed a moderate response, particularly after prolonged exposure, indicating cell-type-dependent sensitivity. Normal MRC-5 fibroblasts exhibited negligible sensitivity, confirming the low intrinsic cytotoxicity of GTN under these conditions. 2.2. Combined Treatment with 2-Deoxy-D-Glucose The addition of 2-DG significantly enhanced cytotoxicity in all cancer cell lines, with the most pronounced effects observed after 48 h. As shown in Table 1, 2-DG alone exhibited concentration- and time-dependent cytotoxic effects, with IC 50 values ranging from 2.01 to 7.05 mM depending on the cell line and incubation period. In HeLa cells, combination treatment reduced viability to approximately 59–61%, compared to >85% observed with single agents. A549 cells demonstrated a similar trend, with viability decreasing to approximately 60% at higher GTN concentrations. HT29 cells displayed a weaker response, indicating partial resistance to metabolic stress-induced cytotoxicity. Notably, MRC-5 fibroblasts also showed a substantial reduction in viability (~35%), highlighting the limited selectivity of the combination treatment. These findings suggest that glycolytic inhibition may sensitize cancer cells to GTN-mediated cytotoxicity under metabolic stress conditions, although the pronounced effects observed in normal fibroblasts indicate an important limitation for therapeutic application ( Figure 1). Treatment with 2-deoxy-D-glucose induced pronounced morphological alterations characteristic of metabolic stress and cell death, including cellular shrinkage, loss of normal morphology, and reduced cell density, supporting its antiproliferative and cytotoxic activity ( Figure S1). The effects of the combined treatment of NTG and 1 mM 2DG on cell viability are shown in Figure 2. 2.3. Combined Treatment with Caffeine The addition of caffeine further modulated cytotoxic responses. While limited effects were observed after 24 h, prolonged exposure (48 h) resulted in enhanced cytotoxicity, particularly in A549 cells, where cell viability decreased below 50% at higher concentrations. This finding suggests that caffeine may potentiate GTN-induced cytotoxicity, possibly through modulation of cell cycle regulation and DNA damage response pathways. A marked reduction in viability was also observed in MRC-5 fibroblasts, further confirming the limited selectivity of the combination treatment. HT29 cells were the least sensitive to the combined treatment ( Table S2). Cell viability remained above 60% after 24 h and above 75% after 48 h even at the highest tested concentration (10 μM GTN + 1 mM caffeine), indicating that the IC 50 value exceeded the investigated concentration range. These results support the hypothesis that caffeine enhances the cytotoxic response of cancer cells under metabolically stressed conditions, although additional studies are needed to clarify the underlying mechanisms. 2.4. Docking Experiments Proteins 1O01 and 1O02, representing ALDH2 structures, were selected for molecular docking with nitroglycerin (GTN) based on the previously reported involvement of ALDH2 in GTN bioactivation and nitric oxide (NO)-related signaling [ 4, 11]. Therefore, ALDH2 was considered a literature-supported target for the theoretical evaluation of GTN binding. However, it should be emphasized that the present docking analysis does not demonstrate ALDH2 expression, enzymatic activity, NO release, or pathway activation in the investigated cell lines. Accordingly, the interaction between GTN and ALDH2 should be interpreted as a theoretical and hypothesis-generating observation rather than direct experimental evidence of ALDH2-mediated bioactivation in HeLa, A549, HT29, or MRC-5 cells. The molecular docking results obtained using CB-Dock2 ( Table 2) showed that the predicted binding energies of GTN and caffeine with the selected target proteins ranged from −6.2 to −6.5 kcal/mol. Negative binding energy values indicate energetically favorable in silico ligand–protein interactions, with lower values generally corresponding to more stable predicted complexes. Nevertheless, docking scores represent theoretical estimates of binding affinity and should not be interpreted as experimental evidence of target engagement, receptor activation or inhibition, or functional pathway involvement in the tested cell lines. Docking scores represent predicted in silico binding affinities and should be interpreted only as theoretical estimates. They do not provide direct evidence of target expression, target engagement, pathway activation, or functional involvement in the tested cell lines. 2.5. Chou–Talalay Analyses CI–Fa analysis revealed distinct interaction patterns across the examined cell lines. HeLa cells consistently showed synergistic effects (CI 1 indicates antagonism. Figure 3. Combination index (CI) versus fraction affected (Fa) plots for the interaction between nitroglycerin (GTN) and 2-deoxy-D-glucose (2-DG) after 48 h treatment. ( a) HeLa and A549 cells showing predominantly synergistic interactions (CI 1 indicates antagonism.
