Huachansu Injection induces ferroptosis in multiple myeloma through NRF2/HO-1 signaling pathway.

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Multiple myeloma (MM) is a common hematologic malignancy. Huachansu (HCS) is extracted from the skin of Bufo bufo gargarizans, known for its well-established and multi-target anti-tumor effect. It has been reported to be effective in treating patients with multiple myeloma but its underlying mechanism remains unclear. This study aims to investigate the cellular and molecular mechanisms in which HCS induces cell death of MM. Cell viability was assessed using the CCK-8 assay. The effect of HCS on the gene expression of MM were screened by transcriptome sequencing and validated by quantitative real-time PCR, western blot, and immunofluorescence. The ferroptosis phenotype were evaluated by measuring iron ion concentration, lipid peroxidation degree in terms of malondialdehyde (MDA), and reduced glutathione (GSH) level. Flow cytometry was adopted to measure intracellular ROS and PGSK levels. The ability of ferroptosis inhibitors to reverse these effects was also assessed. The treatment effect and ferroptosis induction of HCS on MM in vivo were explored on a xenograft nude mice model, with mitochondrial damage observed by transmission electron microscopy. HCS modulated the NRF2/HO-1 pathway, upregulating PRP and ZIP8, leading to Fe2+ accumulation and PGSK elevation, while increasing ROS and MDA levels and reducing GSH content. These effects were significantly reversed by the ferroptosis inhibitor Ferrostin-1. HCS induced MM cell ferroptosis through the NRF2/HO-1 pathway in vivo, inhibiting MM progression similarly to the positive control drug bortezomib. These results indicate that HCS can induce ferroptosis in MM cells via the NRF2/HO-1 pathway, thereby controlling MM progression. Our study provides a solid theoretical basis for the clinical use of HCS in treating MM. Additionally, it suggests an innovative treatment alternative based on natural medicine, proposing the combined use of HCS and chemotherapy drugs as a new therapeutic avenue for MM.