Natural Saponin Formosanin C‐induced Ferroptosis in Human Hepatocellular Carcinoma Cells Involved Ferritinophagy

Formosanin C (FC) is an anti‐tumor diosgenin saponin isolated from Paris formosana Hayata (Liliaceae) which is a perennial herb grown in the humid and dark forests of Taiwan at about the level of 1000 m. The plat has been used as a folk remedy for snake‐bite inflammation and tumors. FC is also a component of “Yunnan Bai Yao”, a traditional Chinese hemostatic medicine. Ferroptosis is a new form of iron‐dependent cell death, which occurs via an increased labile iron pool and lipid reactive oxygen species (ROS). Ferritinophagy is a ferritin (an iron storage protein) degradation pathway, depending on a selective autophagic cargo receptor nuclear coactivator 4 (NCOA4). Increased labile iron pool by ferritinophagy has been recognized as an upstream regulator of ferroptosis. Thus, ferroptosis has been reported to be a form of autophagic cell death. Although iron is an essential nutrient that facilitates cell proliferation and growth, overexpression of ferritin and down‐regulation of ferroportin (an iron export protein) has recently been observed in human hepatocellular carcinoma (HCC), indicating the increased dependence of iron in the cancer cells. In the present study, FC caused a significant dosage‐related population growth inhibition in HCC HepG2 and Hep3B cells using SRB assay, which can be partially blocked by co‐administration of ferroptosis inhibitor ferrostatin‐1 (a scavenger of lipid ROS). FC induced elevation in lipid ROS formation can also be partially reversed by addition of either ferrostatin‐1, or the presence of antioxidant vitamin C and/or vitamin E using flow cytometry, confirming FC‐induced ferroptosis. Flow cytometric analysis also revealed that FC elevated the percentage of acidic vesicular organelles‐positive cells, representing the formation of autolysosomes and induction of autophagic flux in both cell lines. The FC‐induced autophagy was confirmed using Western blotting, characterized by the production of LC3‐II and a further elevation of LC3‐II expression by addition of autophagy inhibitor bafilomycin A1 to prevent the fusion of autophagosomes and lysosomes for cargo degradation. In the present of bafilomycin A1, accumulation of NCOA4 and ferritin was observed in both FC‐treated cell lines using Western blotting. Concurrently, increase in co‐localization of LC3 puncta with NCOA4 or ferritin was exhibited using confocal microscopy, suggesting FC‐induced ferritinophagy. It is noteworthy that HepG2 cells with a higher NCOA4 expression were more sensitive to FC‐induced ferroptosis and autophagy compared to Hep3B cells with a higher ferritin expression. Overall, our data suggest chemotherapeutic potential of phytochemical FC for treatment of HCC with higher expression of NCOA4 via ferritinophagy and ferroptosis. Support or Funding Information Supported in part by the Ministry of Science and Technology, Taiwan, No. MOST 106‐2320‐B‐003‐006‐MY3 and 107‐2320‐B‐003‐002‐MY2