Disulfiram/Copper Induce Ferroptosis in Triple-Negative Breast Cancer Cell Line MDA-MB-231

Background: The complex formed by disulfiram (DSF) and copper (Cu) is safe and effective for the prevention and treatment of triple-negative breast cancer (TNBC). Although previous studies have shown that DSF/Cu induces ferroptosis, the mechanism remains unclear. Methods: The mitochondrial morphology of TNBC treated with DSF/Cu was observed by transmission microscopy, and intracellular levels of iron, lipid reactive oxygen species (ROS), malondialdehyde, and glutathione were evaluated to detect the presence of ferroptosis. Target genes for the DSF/Cu-activated ferroptosis signaling pathway were examined by transcriptome sequencing analysis. Expression of the target gene, HOMX1, was detected by qRT-PCR, immunofluorescence and western blot. Results: The mitochondria of TNBC cells were significantly atrophied following treatment with DSF/Cu for 24 h. Addition of DSF/Cu supplement resulted in significant up-regulation of intracellular iron, lipid ROS and malondialdehyde levels, and significant down-regulation of glutathione levels, all of which are important markers of ferroptosis. Transcriptome analysis confirmed that DSF/Cu activated the ferroptosis signaling pathway and up-regulated several ferroptosis target genes associated with redox regulation, especially heme oxygenase-1 (HMOX-1). Inhibition of ferroptosis by addition of the ROS scavenger N-acetyl-L-cysteine (NAC) significantly increased the viability of DSF/Cu-treated TNBC cells. Conclusions: These results show that DSF/Cu increases lipid peroxidation and causes a sharp increase in HMOX1 activity, thereby inducing TNBC cell death through ferroptosis. DSF/Cu is a promising therapeutic drug for TNBC and could lead to ferroptosis-mediated therapeutic strategies for human cancer.