Molecular Insights into the Interaction Between Metformin and Caffeine: Time-Dependent Antagonism and Modulation of p53 Signaling in Cancer Cells

Background:Cancer remains a major global health challenge, with treatment efficacy li-mited by drug resistance and adverse effects. Drug repurposing offers opportunities for novel anticancer strategies. This study evaluated the cytotoxic, antiproliferative, and pro-apoptotic effects of metformin and caffeine, alone and in combination, in human cancer cell lines, and their potentialinteraction mechanisms. Methods:Human cervical carcinoma (HeLa), lung adenocarcinoma (A549), and colorectal carcinoma (HT29) cell lines were treated with metformin (0.05–50 mM) and caffeine (0.5–5 mM), alone or combined, for 24 and 48 h. Cell viability and proliferation were assessed using Trypan Blue and sulforhodamine B (SRB) assays. Apoptosis was analyzed by Annexin V/propidium iodide flow cytometry, and p53 expression in HeLa cells was determined by ELISA. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. Results:Metformin induced dose- and time-dependent cytotoxicity in all tested cell lines, with the lowest IC₅₀ values observed in HeLa and A549 cells after 48 h (2.28 and 3.30 mM, respectively; p < 0.05). Caffeine showed moderate antiproliferative activity, with the strongest effects at 2.03 mM in HeLa and 2.01 mM in HT29 cells (p < 0.05). The combined treatment produced effects that varied depending on both the cell line and exposure time. At earlier time points, transient synergistic effects were observed in certain cell lines, particularly HeLa; however, these effects were not sustained over time. With prolonged exposure, the interaction shifted toward predominantly antagonistic effects, indicating a reduced overall efficacy of the combination compared to expected additive outcomes.Increased apoptosis and elevated p53 expression further support the activation of tumor-suppressive pathways. Conclusions:Metformin exhibits significant anticancer activity in vitro, supporting met-formin repurposing in oncology. However,the addition of caffeine does not uniformly enhance its efficacy and appears to exert context-dependent effects.Further in vivo studies are required to confirm its clinical relevance. Keywords:AMPK;Antitumor activity; Apoptosis; Caffeine; Cancer cell lines;Chou–Talalay method; Drug repurposing;Docking;Metfomin;Molecular docking; p53;