Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma

Abstract

Sorafenib is the first-line medication for advanced hepatocellular carcinoma (HCC), but it can only extend limited survival. It is imperative to find a combination strategy to increase sorafenib efficacy. Artesunate is such a preferred candidate, because artesunate is clinically well-tolerated and more importantly both drugs can induce ferroptosis through different mechanisms. In this study we investigated the combined effect of sorafenib and artesunate in inducing ferroptosis of HCC and elucidated the involved molecular mechanisms. We showed that artesunate greatly enhanced the anticancer effects of low dose of sorafenib against Huh7, SNU-449, and SNU-182 HCC cell lines in vitro and against Huh7 cell xenograft model in Balb/c nude mice. The combination index method confirmed that the combined effect of sorafenib and artesunate was synergistic. Compared with the treatment with artesunate or sorafenib alone, combined treatment induced significantly exacerbated lipid peroxidation and ferroptosis, which was blocked by N-acetyl cysteine and ferroptosis inhibitors liproxstatin-1 and deferoxamine mesylate, but not by inhibitors of other types of cell death (z-VAD, necrostatin-1 and belnacasan). In Huh7 cells, we demonstrated that the combined treatment induced oxidative stress and lysosome-mediated ferritinophagy, two essential aspects of ferroptosis. Sorafenib at low dose mainly caused oxidative stress through mitochondrial impairments and SLC7A11-invovled glutathione depletion. Artesunate-induced lysosome activation synergized with sorafenib-mediated pro-oxidative effects by promoting sequential reactions including lysosomal cathepsin B/L activation, ferritin degradation, lipid peroxidation, and consequent ferroptosis. Taken together, artesunate could be repurposed to sensitize sorafenib in HCC treatment. The combined treatment can be easily translated into clinical applications.

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Fig. 1: Artesunate sensitized HCC cells to sorafenib-induced death.
Fig. 2: The combined treatment inhibited xenograft tumors in vivo through extensive cell death.
Fig. 3: The combined treatment-induced lipid peroxidation and ferroptosis.
Fig. 4: The combined treatment impaired mitochondrial functions.
Fig. 5: The combined treatment-induced lysosomal activation and ferritin degradation.
Fig. 6: The combined treatments caused consistent changes in FTL degradation and MDA production in xenograft nodules in vivo.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81672370 and 81972291), the Guangxi Natural Science Foundation Key Grant (2018GXNSFDA050006), the Hundred-Talent Program (2016), the Guangxi Medical University Training Program for Distinguished Young Scholars (2017), the OpenGrant from Key Laboratory of High-Incidence Tumor Prevention & Treatment (Guangxi Medical University, GKE2018-02) (all awarded to GDL); the Guangxi Natural Science Foundation Key Grant (2017GXNSFDA198020) awarded to JZ, and the Guangxi Specially Appointed Professor Grant awarded to JW.

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ZJL, XWH, and HQD equally performed the majority of experiments and analyzed the data; ZJL, XWH, and YJL performed the nude mouse xenograft experiment; HQD and JF conducted the Seahorse metabolic experiments and flow cytometry analysis; YW prepared the figures; XMY, ZXW, and JHD provided technical support; PHC and HS performed the literature review; JGW and GDL designed the research and summarized the data; JZ and GDL wrote the paper.

Corresponding authors

Correspondence to Ji-gang Wang or Jing Zhou or Guo-dong Lu.

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The authors declare no competing interests.

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Li, Zj., Dai, Hq., Huang, Xw. et al. Artesunate synergizes with sorafenib to induce ferroptosis in hepatocellular carcinoma. Acta Pharmacol Sin (2020). https://doi.org/10.1038/s41401-020-0478-3

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Keywords

  • Sorafenib
  • Artesunate
  • HCC
  • Ferroptosis
  • Mitochondria
  • Lysosome

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