背景:急性单核细胞白血病-M5(AML-M5)由于其高发病率和不良预后,仍然是一种具有挑战性的疾病。除了前面提到的证据,多项研究表明,程序性细胞死亡(PCD)在AML-M5的治疗中起着至关重要的作用。然而,铁凋亡和坏死性凋亡在AML-M5中的作用和关系尚不清楚.
方法:THP-1细胞主要用Erastin和IMP-366处理。CCK-8、westernblot检测铁凋亡和坏死水平的变化,实时定量PCR,和电子显微镜。应用流式细胞术检测ROS和脂质ROS水平。MDA,4-HNE,通过ELISA试剂盒评估GSH和GSSG。通过免疫荧光染色和蛋白质印迹研究FSP1的细胞内分布。
结果:将肉豆蔻酰化抑制剂IMP-366添加到经过erastin处理的急性单核细胞白血病细胞系THP-1细胞中,不仅导致对以脂质过氧化为特征的铁凋亡的敏感性更高,谷胱甘肽(GSH)耗竭和线粒体收缩,由于膜上的FSP1位置被抑制,但也增加了p-RIPK1和p-MLKL蛋白的表达,以及caspase-8表达的减少,并引发了特征性的坏死现象,包括细胞质半透明,线粒体肿胀,FSP1通过结合输入蛋白α2迁移到细胞核中的膜性骨折。有趣的是注意到铁凋亡抑制剂fer-1逆转了坏死。
结论:我们证明了IMP-366对肉豆蔻酰化的抑制作用能够改变THP-1细胞中的铁凋亡和铁凋亡依赖性坏死。在这些发现中,FSP1介导的铁凋亡和坏死被描述为THP-1细胞PCD的替代机制。为AML-M5提供潜在的治疗策略和靶点。
BACKGROUND: Acute monocytic leukemia-M5 (AML-M5) remains a challenging disease due to its high morbidity and poor prognosis. In addition to the evidence mentioned earlier, several studies have shown that programmed cell death (PCD) serves a critical function in treatment of AML-M5. However, the role and relationship between ferroptosis and necroptosis in AML-M5 remains unclear.
METHODS: THP-1 cells were mainly treated with Erastin and IMP-366. The changes of ferroptosis and necroptosis levels were detected by CCK-8, western blot, quantitative real-time PCR, and electron microscopy. Flow cytometry was applied to detect the ROS and lipid ROS levels. MDA, 4-HNE, GSH and GSSG were assessed by ELISA kits. Intracellular distribution of
FSP1 was studied by immunofluorescent staining and western blot.
RESULTS: The addition of the myristoylation inhibitor IMP-366 to erastin-treated acute monocytic leukemia cell line THP-1 cell not only resulted in greater susceptibility to ferroptosis characterized by lipid peroxidation, glutathione (GSH) depletion and mitochondrial shrinkage, as the
FSP1 position on membrane was inhibited, but also increased p-RIPK1 and p-MLKL protein expression, as well as a decrease in caspase-8 expression, and triggered the characteristic necroptosis phenomena, including cytoplasmic translucency, mitochondrial swelling, membranous fractures by
FSP1 migration into the nucleus via binding importin α2. It is interesting to note that ferroptosis inhibitor fer-1 reversed necroptosis.
CONCLUSIONS: We demonstrated that inhibition of myristoylation by IMP-366 is capable of switching ferroptosis and ferroptosis-dependent necroptosis in THP-1 cells. In these findings,
FSP1-mediated ferroptosis and necroptosis are described as alternative mechanisms of PCD of THP-1 cells, providing potential therapeutic strategies and targets for AML-M5.