The Mechanism Of Ferroptosis Induced By Xanthohumol In SK-N-SH Human Neuroblastoma Cells

Background and significance:Hops are perennial herbs in the genus Humulus of the mulberry family.The female inflorescences of the plants give beer its unique flavour and aroma,and in recent years have also attracted attention for their health functions as medicinal and food plants.Xanthohumol is the main isoprenoid flavonoid contained in hops and has a variety of physiological activities including anti-tumour,anti-inflammatory and anti-bacterial.Neuroblastoma is a common and highly prevalent tumour in children,which is highly heterogeneous,and high-risk malignant neuroblastoma is prone to metastasis,for which there is no ideal intervention strategy.In this study,we investigated the in vitro anti-tumour activity of XN using human neuroblastoma SK-N-SH cells cultured in vitro,and explored the molecular mechanism of XN killing SK-N-SH cells in terms of redox homeostasis and iron death,with a view to providing a scientific basis for the anti-tumour application of XN.Methods:（1）Microscopic observation of XN on SK-N-SH cell morphology.（2）Proliferation inhibition and mortality of XN-treated cells were detected by SRB method and Taipan Blue rejection method.（3）The extent of LDH release from the cells was detected using a kit.（4）Fluorescent probes DCFH-DA,BODIPY^TM581/591 C11,Ferro Orange staining,fluorescence microscopy and flow cytometry were used to detect total reactive oxygen species and lipid reactive oxygen species,and fluorescence microscopy combined with enzyme marker was used to detect intracellular Fe²⁺levels.（5）The levels of lipid peroxidation product MDA,glucose hexakisphosphate dehydrogenase G6PDH,reduced nicotinamide adenine dinucleotide phosphate NADPH and reduced glutathione GSH were detected by using kits.（6）Western blot technique was applied to detect the expression of iron death-related proteins（GPX4,FTH1）.Results:（1）XN inhibited SK-N-SH cell proliferation and induced cell death in a time（24,48,96 h）and concentration（0-80μmol/L）dependent;（2）cell membrane damage,cellular reactive oxygen species and lipid peroxidation stress levels were significantly increased after 10 and 20μmol/L XN treatment for 24 h;（3）after 20μmol/L XN treatment for 24 h G6PDH activity was significantly reduced,NADPH and GSH levels were also reduced,and GPX4 protein expression was significantly decreased,indicating that cellular antioxidant defense was weakened and intracellular redox homeostasis was dysregulated;（4）intracellular Fe²⁺overload and FTH1 protein expression were reduced after 24 h treatment with 10 and 20μmol/L XN,suggesting the occurrence of iron death;（5）iron death specific inhibitor（Fer-1）had a protective effect on XN-induced cell proliferation inhibition and damage,and significantly attenuated XN-induced oxidative stress levels and Fe²⁺overload.Conclusion:This study demonstrates that XN can induce iron death in human neuroblastoma（SK-N-SH）by triggering dysregulation of redox homeostasis and Fe²⁺overload.