Ponicidin triggered ferroptosis in esophageal squamous cell carcinoma by suppressing the SLC7A11/Glutathione/GPX4 signalling axis

Abstract

Background: Ponicidin, a diterpenoid derived from Rabdosia rubescens, exhibits potent antitumor activity. However, its mechanisms against esophageal squamous cell carcinoma (ESCC) remain obscure. This study aims to explore the effects of ponicidin against ESCC and reveal its molecular mechanisms.

Methods: The anti-ESCC effects of ponicidin were evaluated using CCK-8 assay, colony formation and transwell invasion assays. Cell cycle progression and mitochondrial membrane potential were analyzed using flow cytometry. Proteomics was applied to explore ponicidin's mechanisms. Ferroptosis induction was evaluated by quantifying reactive oxygen species, Fe²⁺, malondialdehyde, glutathione, and lipid peroxidation levels. Docking and molecular dynamics simulations were conducted to identify the targets. siRNA was employed to validate target. The efficacy of ponicidin on tumorigenicity was explored in tumor xenograft mouse models, and its biosafety was evaluated via hemolysis assays, plasma ALT, AST, BUN, and CRE levels, as well as histopathological examinations. Western blot was used to analyze protein expression levels.

Results: Ponicidin inhibited ESCC cell proliferation, arrested cells in the G₂/M phase, reduced mitochondrial membrane potential, and suppressed tumor growth without evident toxicity. Proteomics identified that ponicidin-induced ferroptosis is the predominant mechanism against ESCC. Ponicidin increased reactive oxygen species, malondialdehyde, Fe²⁺, lipid peroxidation and glutathione depletion. Ferrostatin-1 pretreatment reduced lipid peroxidation, rescued PON induced inhibition of cell viability, and reversed the decreased expression of SLC7A11, GPX4 and GSR. Molecular docking revealed strong binding affinity of PON to GPX4 (-7.31±0.55 kcal/mol) and SLC7A11 (-8.19±0.37 kcal/mol). Molecular dynamics simulations confirmed stabilized complexes with total interaction energies of -23.43 ± 2.13 kcal/mol (GPX4-PON) and -31.42 ± 0.84 kcal/mol (SLC7A11-PON). siRNA-mediated knockdown of GPX4 and SLC7A11 reduced ESCC sensitivity to ponicidin-induced ferroptosis.

Conclusion: This study provides the first evidence that ponicidin triggers ferroptosis in ESCC cells via suppression of the SLC7A11/glutathione/GPX4 signalling axis, offering actionable targets for ferroptosis-enhancing combination therapies.

Keywords: Esophageal squamous cell carcinoma; Ferroptosis; Ponicidin; SLC7A11/Glutathione/GPX4 signalling axis.