DPhA-EtOBz-TSC targets cystathionine γ-lyase (CSE) to trigger ferroptosis and inhibit colorectal cancer growth in vitro and in vivo.

Colorectal cancer (CRC) remains one of the most prevalent malignancies globally, with limited therapeutic options for advanced-stage patients due to acquired drug resistance and systemic toxicity. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a promising therapeutic target for CRC, while cystathionine γ-lyase (CSE)-a key enzyme in hydrogen sulfide (H2S) biosynthesis-plays a critical role in maintaining redox homeostasis and suppressing ferroptosis in cancer cells. Here, we synthesized a novel small-molecule compound, DPhA-EtOBz-TSC, and systematically evaluated its anti-CRC efficacy and underlying molecular mechanism. The chemical structure of DPhA-EtOBz-TSC was confirmed by 1H nuclear magnetic resonance (1H NMR), 13C NMR, and high-resolution mass spectrometry (HRMS). In vitro studies using human CRC cell lines (HCT8 and SW480) demonstrated that DPhA-EtOBz-TSC specifically targeted and inhibited CSE: molecular docking and cellular thermal shift assay (CETSA) confirmed direct binding between DPhA-EtOBz-TSC and CSE (binding affinity ΔG = -29.07 ± 3.85 kcal/mol), while Western blot analysis revealed a concentration-dependent reduction in CSE protein levels in SW480 and HCT8 cells treated with DPhA-EtOBz-TSC. Mechanistically, DPhA-EtOBz-TSC-induced CSE downregulation disrupted the CSE-H2S-GPX4 axis, leading to reduced glutathione (GSH) levels, decreased glutathione peroxidase 4 (GPX4) activity, and accumulation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA)-hallmarks of ferroptosis. Additionally, Electron microscopy and JC-1 staining revealed that DPhA-EtOBz-TSC induced significant alterations in mitochondrial morphology and a marked reduction in mitochondrial membrane potential (ΔΨm). Functionally, DPhA-EtOBz-TSC exhibited potent in vitro anti-CRC activity with IC50 values of 25.15 μM (HCT8) and 26.11 μM (SW480) respectively, and suppressed migration and invasion. In vivo, DPhA-EtOBz-TSC (10 mg/kg/day, i.p.) significantly suppressed the growth of CT26 cell-derived xenograft tumors in BALB/c mice without causing obvious histopathological abnormalities in major organs (heart, liver, spleen, lung, kidney). Immunohistochemical staining of tumor tissues confirmed increased lipid peroxidation and decreased CSE/GPX4 expression in DPhA-EtOBz-TSC-treated mice, consistent with in vitro findings. In conclusion, DPhA-EtOBz-TSC is a novel CSE-targeting compound that induces ferroptosis in CRC cells and inhibits tumor growth in vivo. Our findings identify DPhA-EtOBz-TSC as a potential lead compound for CRC therapy and highlight the CSE-H2S-GPX4 axis as a viable therapeutic target for ferroptosis-based anticancer strategies.