Metformin regulates ferroptosis in Skin cutaneous melanoma via ATF3/NRF2 axis

Skin Cutaneous Melanoma (SKCM) remains a highly aggressive malignancy characterized by rapid proliferation and significant resistance to conventional therapeutic interventions. Prior research has shown that metabolic reprogramming plays a central role in the survival and progression of these malignant melanocytes within the tumor microenvironment. The induction of non-apoptotic cell death pathways has emerged as a promising alternative for overcoming drug resistance in advanced skin cancers that fail to respond to standard chemotherapy. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, represents a potential vulnerability in melanoma cells that can be exploited for therapeutic gain. While metformin is widely recognized for its anti-diabetic properties and metabolic effects, its specific influence on iron-mediated death pathways in melanoma remains poorly understood. This gap motivated the current investigation into how this biguanide compound modulates intracellular oxidative stress and iron metabolism to inhibit tumor growth.

This investigation evaluates the impact of metformin on the proliferative capacity and ferroptotic signaling of skin cutaneous melanoma cells to identify new treatment strategies. The researchers sought to determine whether metformin could trigger iron-dependent cell death by altering the balance of intracellular antioxidants and pro-oxidants in malignant cells. A primary objective involved identifying the specific molecular mediators that govern the response of melanoma cells to metformin exposure across different concentrations. The study aimed to map the transcriptomic changes induced by this treatment to uncover relevant biological functions and signaling pathways associated with cell death. Investigators focused on the potential involvement of the Activating Transcription Factor 3 (ATF3) and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) signaling axis as a regulatory mechanism for ferroptosis induction. This absence of evidence motivated the development of a theoretical foundation for utilizing metformin as a sensitizing agent in melanoma therapy.

The research team employed the Cell Counting Kit-8 (CCK-8) assay to quantify the inhibitory effects of metformin on melanoma cell proliferation over specified time intervals. Intracellular oxidative status was assessed using specialized kits to measure Glutathione (GSH) content, Reactive Oxygen Species (ROS), and Lipid Peroxide (LPO) levels in treated versus control groups. Malondialdehyde (MDA) concentrations were also determined to serve as a definitive marker for lipid peroxidation and ferroptotic activity within the cellular membrane. High-throughput RNA-seq sequencing was performed to identify differentially expressed genes and characterize the global transcriptomic response to metformin treatment in skin cutaneous melanoma. Western blot analysis allowed for the precise detection of Activating Transcription Factor 3 (ATF3) and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) protein levels to map signaling interactions. Statistical correlations were calculated to link ATF3 mRNA expression with metformin concentration and clinical tissue samples obtained from melanoma patients.

Metformin treatment significantly suppressed the proliferation of skin cutaneous melanoma cells in a dose-dependent manner, confirming its potent anti-tumor activity. Treated cells exhibited a marked reduction in Glutathione (GSH) levels alongside a substantial accumulation of Reactive Oxygen Species (ROS) and Lipid Peroxide (LPO) within the cytoplasm. Elevated Malondialdehyde (MDA) levels confirmed the induction of ferroptosis following exposure to the biguanide compound, indicating a shift in the cellular redox balance. Transcriptomic profiling revealed 2068 differentially expressed genes, including 897 up-regulated and 1171 down-regulated transcripts that participate in various metabolic processes. Pathway analysis indicated that iron metabolism disorders and ferroptosis-related signaling were specifically activated by the treatment, highlighting the molecular impact of the drug. Molecular analysis showed that metformin increased ATF3 mRNA and protein expression while simultaneously decreasing NRF2 protein levels to facilitate cell death.

The findings suggest that metformin serves as a potent inducer of ferroptosis in skin cutaneous melanoma through the modulation of the ATF3/NRF2 axis. Upregulation of ATF3 appears to suppress NRF2, thereby compromising the antioxidant defense system and promoting iron-dependent cell death in malignant cells. This mechanism offers a novel strategy for improving the therapeutic outcomes of patients diagnosed with aggressive skin cancers that are resistant to apoptosis. Integrating metformin into existing treatment protocols may enhance the efficacy of drugs that target oxidative stress pathways or iron metabolism in tumors. Future research should focus on validating these molecular interactions in vivo to confirm the clinical utility of the ATF3/NRF2 regulatory pathway in human subjects. These results establish a clear link between biguanide treatment and the activation of iron-mediated death in malignant melanocytes, providing a basis for clinical trials.