Shikonin promotes ferroptosis though NSUN2-mediated m5C methylation modification of TFRC in acute myelocytic leukemia

Abstract

Shikonin (SHK), extracted from the traditional Chinese herb Lithospermum erythrorhizon, demonstrates a wide range of pharmacological activities. This study aimed to explore the role and underlying mechanisms of the 5-methylcytosine (m⁵C) RNA methyltransferase NOL1/NOP2/SUN domain (NSUN)2 in acute myelocytic leukemia (AML). To assess cell viability and death, we employed Cell Counting Kit-8 and propidium iodide staining. Ferroptosis-related markers were evaluated using commercial kits and Western blot analysis. The m⁵C levels of ferroptosis-associated mRNAs were quantified by methylated RNA immunoprecipitation (MeRIP)-qPCR. The specific m⁵C sites on the transferrin receptor (TFRC) mRNA were identified through a dual-luciferase reporter assay, while the interaction between NSUN2 and TFRC was investigated using RNA immunoprecipitation (RIP). The role of SHK in vivo was explored using a xenografted tumor model. Our findings revealed that SHK significantly reduced cell viability and induced cell death and ferroptosis in HL-60 and NB4 cells. Notably, SHK treatment led to an upregulation of NSUN2 expression. Inhibition of NSUN2 reversed the effects of SHK, restoring cell viability and reducing cell death and ferroptosis. Mechanistically, NSUN2 enhanced TFRC expression via m⁵C-dependent methylation. Overexpression of NSUN2 similarly decreased cell viability and increased cell death and ferroptosis, effects that were mitigated upon silencing of TFRC. In vivo, SHK treatment effectively suppressed tumor growth in xenografted mice. In summary, our study demonstrated that SHK promoted cell death and ferroptosis in AML by modulating NSUN2-mediated m⁵C methylation of TFRC. These findings provided novel insights into potential therapeutic strategies for AML.