m6A methylation dependent autophagy activation is required for anti-tumor effects of artesunate through ferroptosis in hepatocellular carcinoma.

BACKGROUND & AIMS: Artesunate (Art), a semisynthetic derivative of Artemisinin isolated from the traditional Chinese medicinal plant Artemisia annua L., has recently emerged as a candidate agent for hepatocellular carcinoma (HCC) therapy. Although accumulating preclinical evidence suggests its potent antitumor efficacy against HCC, the precise molecular mechanisms underlying its therapeutic effects remain poorly characterized. This investigation employs a multimodal approach to delineate the pharmacodynamic mechanism of Art in HCC pathogenesis, while establishing a mechanistic foundation for developing targeted combination therapies against this malignancy.

METHODS

In vitro metastatic potential was quantified through standardized wound-healing assays and Matrigel-based transwell invasion systems. Autophagic flux dynamics and ferroptosis biomarkers were systematically profiled using commercial assay kits coupled with live-cell confocal imaging and Western blot. Mechanistic interrogation employed RIP-seq analysis and dual-luciferase reporter systems with in situ mutation controls. In vivo therapeutic efficacy was validated using orthotopic HCC xenograft models in immunocompromised BALB/c nude mice.

RESULTS

Art demonstrated antitumor efficacy, significantly attenuating HCC cell motility and suppressing orthotopic xenograft growth. Mechanistic profiling revealed Art-induced ferroptosis through autophagy, mediated by m6A-dependent post-transcriptional regulation. Specifically, Art enhanced WTAP-mediated deposition of m6A modifications at the RRACH motif of ATG5 mRNA, facilitating YTHDC2-dependent translation. This epitranscriptomic reprogramming increased ATG5 protein synthesis, triggering autophagy dependent ferritin degradation and subsequent ferroptosis.

CONCLUSION

Art emerges as a clinically potential HCC therapeutic that elicits its oncosuppressive activity through WTAP/YTHDC2-mediated m6A methylation of ATG5 transcripts, thereby causes ferroptosis of HCC mediated by autophagy.