This study investigated how metformin, a common anti-diabetic drug with anti-cancer properties, affects selective autophagy pathways - mitophagy (targeting damaged mitochondria) and lipophagy (targeting lipid molecules) - in breast cancer cells. We aimed to determine if metformin's effects are specific to these pathways and if they vary by breast cancer subtype.
We used two breast cancer cell lines: aggressive triple-negative MDA-MB-231 and estrogen receptor-positive luminal MCF-7 cells. Both were treated with a non-toxic, clinically relevant dose (85 μM) and high (10 mM) metformin concentrations for 24 hours. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was used as a positive control for mitochondrial impairment. Mitochondrial dysfunction was assessed using fluorescent microscopy and flow cytometry, quantifying mitochondrial reactive oxygen species (ROS) with MitoSOX and mitochondrial membrane potential with tetramethylrhodamine ethyl ester (TMRE). Lipid droplets were evaluated by flow cytometry to quantify lipid droplets and microscopic visualization of intracellular lipid distribution using BODIPY fluorescent dye.
Metformin treatment induced mitochondrial stress in both cell lines, as demonstrated by increased mitochondrial ROS and decreased membrane potential. These effects were more pronounced in the aggressive MDA-MB-231 cells compared to the luminal MCF-7 cells. Alterations in lipid droplet abundance and spatial organization were also observed; however, these findings require further validation to determine whether lipophagy is specifically involved.
Overall, our results show that metformin promotes mitochondrial dysfunction and alters lipid homeostasis in breast cancer cells, with a stronger response in triple-negative MDA-MB-231 cells than in MCF-7 cells. These data improve our understanding of metformin’s cellular effects and suggest that breast cancer subtype may influence treatment response.