Cysteine Restriction Induces Ferroptosis Depending on the S-adenosylmethionine and Polyamine Biosynthetic Pathways in Hepatic Cancer Cells.

Liver diseases such as hepatocellular carcinoma are known to be affected by nutrition and metabolic activities, but the mechanisms behind them remain unclear. We aimed to reveal the relationship between the concentration of sulfur-containing amino acids and hepatocellular response, and further investigated the mechanism focusing on methionine adenosyltransferase, which plays the central role in methionine metabolism by synthesizing S-adenosylmethionine (SAM). Mouse hepatoma Hepa1 cells were cultured in media with reduced amounts of cysteine, methionine, or both. Cell death was monitored using propidium iodide and annexin V staining followed by flow cytometry. Metabolites were measured by mass spectrometry. Inhibitors of ferroptosis (Fer-1), necroptosis (GSK872), SAM synthesis (cycloleucine), or polyamine synthesis (sardomozide and difluoromethylornithine) were used. Cysteine restriction induced marked cell death, whereas simultaneous restriction of cysteine and methionine fully suppressed the cell death. Cysteine restriction-induced cell death was suppressed with Fer-1 and GSK872, suggesting the involvement of ferroptosis in this process. Cysteine restriction decreased reduced glutathione, which was rescued by simultaneous restriction of cysteine and methionine. Cysteine restriction-induced cell death was also suppressed by knockdown of MAT2A or its inhibitor cycloleucine. Furthermore, inhibitors of several enzymes in the polyamine biosynthetic pathway also suppressed the cell death. In contrast, primary culture of mouse hepatocytes did not show cell death upon cysteine restriction. These results suggest that cysteine-glutathione and SAM-polyamine metabolic pathways are critical modulators of ferroptosis of hepatic cancer cells. Since normal liver cells were more resistant to ferroptosis than cancer cells, cysteine restriction may be exploited in treating hepatic cancer by inducing ferroptosis specifically in cancer cells without affecting normal cells in the liver.