Gramine suppresses triple-negative breast cancer by inducing ferroptosis via CUL3-mediated ubiquitination of MTDH

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer lacking effective targeted therapies and frequently exhibiting chemotherapy resistance. Natural compounds are valued for their multi-target therapeutic potential and lower toxicity profiles. This study explores gramine (GM) as a potential candidate for TNBC treatment and investigates its mechanism of action. We screened 27 indole alkaloids via CCK-8 assays. LIP-MS, molecular docking, CETSA, and DARTS assays validated direct binding to candidate targets. Expression of MTDH, SLC3A2, GPX4 was assessed by Western blot. Ferroptosis markers and mitochondrial morphology were measured. Ferroptosis rescue and MTDH knockdown were used to confirm mechanism. In vivo efficacy was assessed by 4T1 and MDA-MB-231 xenograft mouse models. GM selectively inhibited TNBC cell growth (IC₅₀ ∼ 22–28 µM). Proteomic analysis revealed the involvement of ferroptosis pathways and identified MTDH as a key effector. GM directly bound to CUL3, reducing its E3 ubiquitin ligase activity toward MTDH, thereby stabilizing MTDH, which downregulated ferroptosis inhibitors (SLC3A2, GPX4) and upregulated ferroptosis markers (ROS, Fe²⁺, and MDA), along with decreased GSH and mitochondrial morphological changes. Ferroptosis rescue or MTDH knockdown significantly reversed GM’s anti-TNBC effects both in vivo and in vitro . Furthermore, GM markedly suppressed tumor growth in vivo without obvious systemic toxicity. GM inhibits TNBC growth by targeting the CUL3–MTDH axis to trigger ferroptosis. This study uncovers a novel regulatory pathway in ferroptosis, highlighting the significant potential of GM as an effective therapeutic candidate for TNBC.