Additive Anticancer and Antioxidant Effects of Metformin and Luteolin in Breast and Colorectal Cancer Cell Lines.

Background/Objectives: Metformin (Met) is a potent antidiabetic drug that also exhibits anticancer, antioxidant, and organ-protective properties. Luteolin (Lut), a naturally occurring flavonoid found in many plant species, possesses anticancer, antioxidant, and anti-inflammatory effects. Since both compounds affect cellular metabolism and oxidative balance, the analysis of metabolites produced in living cells provides insight into biochemical alterations occurring in cancer cells and enables monitoring of treatment response. Methods: In this study, Met (1-20 mM) and Lut (1-100 µM) were tested in vitro, both individually and in combination, to evaluate their effects on cell viability, free radical levels, and metabolite profile alterations in cancer and normal cell lines (MDA-MB-231, SW620, and V79). Cell viability was assessed using the MTT assay at two time points (24 h and 48 h), while reactive oxygen species (ROS) levels were measured after hydrogen peroxide stimulation (100 µM H2O2) using the DCF-DA assay. Metabolomic changes induced by Met and Lut were analyzed by 1H NMR spectroscopy. Results: The analysis showed that Lut reduced the viability of MDA-MB-231 cells at both time points, whereas Met decreased viability only after prolonged incubation. Met did not inhibit the proliferation of SW620 colorectal cancer cells, while Lut reduced viability at higher concentrations (100 µM after 24 h, and 50-100 µM after 48 h). Conclusions: The combination of metformin and luteolin demonstrated additive effects in reducing cell viability and oxidative stress compared with single-compound treatments. Normal V79 fibroblasts responded to both Met and Lut, individually and in combination. Both compounds exhibited moderate antioxidant properties in cells exposed to 100 µM H2O2. Lut (25 µM) reduced free radical levels in MDA-MB-231 cells, whereas Met (2.5 mM) did so in SW620 cells. The combination of both compounds increased ROS levels in SW620 cells subjected to oxidative stress. Overall, co-treatment with metformin and luteolin altered metabolic pathways and induced changes in intra- and extracellular metabolite levels across all tested cell lines. The observed additive effects suggest that the combined use of metformin and luteolin may enhance anticancer and antioxidant responses, warranting further in vivo studies to confirm these interactions.