Targeting Cancer Metabolism: Modulation of Metformin Antitumor Effects by Caffeine with Involvement of p53 Signaling

Background: Cancer remains a major global health challenge, with treatment efficacy limited 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 cell lines, with the lowest IC₅₀ values in HeLa and A549 cells after 48 h (2.28 and 3.30 mM; p < 0.05). Caffeine showed moderate antiproliferative activity, with strongest effects at 2.03 mM in HeLa and 2.01 mM in HT29 cells (p < 0.05). Combined treatment demonstrated variable effects depending on the cell line and treatment duration, with limited synergistic interaction observed only under specific conditions, while predominantly antagonistic effects were detected overall. Increased apoptosis and elevated p53 expression suggest activation of tumor-suppressive pathways. Conclusions: Metformin exhibits significant anticancer activity in vitro, supporting metformin 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.