PTEN Promotes Ferroptosis by Inhibiting GCLM Expression
SHANG Kezhuo, WU Yuting, LIU Fangming*
To investigate the role of tumor suppressor Pten in ferroptosis and the mechanism, the Ptendeleted mouse embryonic fibroblasts (Pten–/– MEFs) and its wild-type (Pten+/+) ones were treated with ferroptosisinducing agent (Erastin), with or without ROS scavenging agent, ferroptosis inhibitor, necroptosis inhibitor, caspase inhibitor and autophagy inhibitor. Quantification of cell viability was measured by CCK-8 assay. The levels of lipid hydroperoxide and PI staining analysis were measured by flow cytometry. The level of glutathione was detected by Glutathione Assay Kit. PTEN, P-AKTs473, AKT and GCLM protein expression levels were detected by Western blot. Gclm mRNA expression was measured by RT-qPCR. Gclm-knockdown Pten–/– MEFs and control group were treated with Erastin, and then the quantification of cell viability was measured by CCK-8 assay. The results showed that compared with the control group, Pten–/– MEFs were more resistant to Erastin (P<0.05). Erastin-induced Pten+/+ cell death was reversed by ROS scavenging agent and ferroptosis inhbitor, but not by necroptosis inhibitor, caspase inhibitor and autophagy inhibitor (P<0.05). In addition, lipid ROS level, the marker of ferroptosis, was decreased in Pten–/– cells. These data indicated that Pten-deficiency attenuated the sensitivity of cells to Erastin. Mechanistically, the glutathione levels were increased in Pten–/– cells (P<0.05). mRNA and protein levels of Gclm, which played critical roles in glutathione production, were increased in Pten–/– MEFs. Knocking down GCLM in Pten–/– MEFs increased the lipid hydroperoxide levels and sensitivity to Erastin-induced ferroptosis (P<0.05). Collectively, PTEN promotes ferroptosis by downregulating GCLM-mediated lipid hydroperoxide scavenge.
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