Redox modulation and induction of ferroptosis by dimethyl fumarate in cervical carcinoma

Cervical cancer (CC) is the fourth most common cause of women death. The papillomavirus (HPV) persistent infection is the lead- ing cause for the development of CC that progresses through multistep transformation. Drug resistance and relapse are fre- quently observed in CC after conventional chemotherapy and radiotherapy. Therefore, there is a need to find new drugs against CC. Dimethyl fumarate (DMF) is an FDA-approved anti-inflam- matory drug and emerging studies suggest that DMF also exert an anti-tumor activity in some cancers. We were interested to fer- roptosis, a type of cell death caused by iron-dependent lipid per- oxidation. Using cell viability and colony assay, we tested the effectiveness of DMF alone in comparison to other well-known inducers of ferroptosis in SiHa and C4I cells. We performed fer- roptosis related-genes expression analysis, lipid peroxidation and malondialdehyde assays demonstrating that DMF induces ferrop- tosis in a concentration-dependent manner in both cell lines. Next, we investigated if the combination of DMF with sub-cyto- toxic ferroptotic-drugs was able to ameliorate ferroptosis. We found that co-treatments of DMF/sulfasalazine (SAS) were asso- ciated with enhanced cell death. To elucidate molecular mecha- nisms underlying these effects we analyzed the NRF2 antioxidant pathway. Real-time PCR and western blot assays showed an induction of NRF2 protein and NRF2-dependent genes, such as SLC7A11 involved in GSH synthesis. In contrast with the observed SLC7A11 increase, we detected a strong reduction of glutathione (GSH) under DMF/SAS treatments. These results indicate that SAS cooperates in GSH depletion favoring ferrop- tosis. Since DMF has been found to influence NF-kB, STAT3 signaling we also tested IL-6 levels, a NF-kB target, and phos- phorylation of STAT3 in DMF/SAS treatments. Our results demonstrate that both these pathways are reduced in presence of SAS, implicating that DMF/SAS exhibited a strong killing-effect than either DMF or SAS alone.