Artesunate directly targeting MMP9 to reduce angiogenesis and increase CD8 + T cell infiltration inhibits hepatocellular carcinoma

BACKGROUND: Artesunate (ART) is one of the semi-synthetic derivatives of artemisinin. It is currently mainly used in the clinic for the rescue of cerebral malaria and various critical malaria. Recent studies have shown that it has a wide range of anti-tumor effects. However, its treatment for hepatocellular carcinoma (HCC) and its specific mechanism remain unclear. PURPOSE: This study aims to investigate ART therapeutic effects on HCC and elucidate its molecular mechanisms. METHODS: This study explored the inhibitory effect of ART on HCC through in vivo and in vitro experiments. Cellular experiments evaluated the effects of ART on HCC cell lines (HepG2, H22, Hepa1-6) using CCK-8, Annexin/PI staining, wound healing, and Immunofluorescence. In vivo, ART was tested in HepG2 xenografts and H-ras12V transgenic mice, with tumor growth monitored by Doppler ultrasound. Multi-omics (transcriptomics, network pharmacology, single-cell sequencing) identified MMP9 as a key target. Mechanisms were explored via molecular docking/dynamics, thermal shift assays, and Western blot analysis. Flow cytometry and immunofluorescence confirm CD8 + T cell infiltration. RESULTS: ART inhibited the proliferation of HCC cell lines and promoted its apoptosis. In vivo experiments showed that it had a certain inhibitory effect on the development of immune-deficient mice transplanted tumors. Continuous monitoring of tumor growth by Doppler ultrasound found that its effect was more obvious in the immune-competent spontaneous H-ras12V mouse model of liver cancer, especially for the development of small tumors. Mechanistically, ART can inhibit tumor growth by inhibiting the classic PI3K-AKT signaling pathway, which is consistent with the results in other tumor studies. At the same time, ART can directly target MMP9 and promote its degradation, thereby inhibiting tumor's angiogenesis. To explain why its effect was more pronounced in immune-competent mice, we found ART can significantly increase the tumor infiltration of CD8 + T cells and its effect is also achieved by degrading MMP9. CONCLUSION: T cell infiltration. This study provides a mechanistic basis for the potential clinical application of ART in HCC therapy and identifies MMP9 as a promising therapeutic target.