Psudolaric Acid B Triggers Ferroptosis In Glioma Cells Via Activation Of NOX4 And Inhibition Of XCT

Background:Glioma is the most common and aggressive form of intrinsic brain tumor[1].Clinical therapy generally consists of surgical resection followed by radiotherapy in conjunction with alkylating agent temozolomide.Gross Total Resection(GTR)can reduce the recurrence of gliomas[28,29].However,the prognosis is still very poor,with a median survival of about 14.6 months[2].Thus,novel therapeutic strategy is needed urgently.Iron plays a key role in the metabolism of all cells and is also found to regulate DNA replication,cell proliferation and oxidative stress[3].Cancer cells exhibit an enhanced dependence on iron relative to their normal counterparts,which is termed iron addiction[3,4].Although chelation of iron could sensitize glioma cells to temozolomide or radiotherapy[5],the role of excessive intracellular iron in regulation of glioma cell remains elusive.Ferroptosis is a newly-established form of regulated cell death,which is coined after the requirement for free ferrous iron[6].Biochemically,the process of ferroptosis is characterized by accumulation of lipid peroxidation products and lethal reactive oxygen species(ROS)derived from iron metabolism.The excessive production of reactive oxygen species in the organism leads to changes in the microenvironment and affects the normal biological functions of the cells[30].Morphologically,the cells undergoing ferroptosis present normal-sized nuclei free of chromatin condensation and shrinking mitochondria with increased membrane density[7].Ferroptosis is involved in multiple pathological processes including ischemia/reperfusion injury,acute renal failure and neurodegeneration[6].Therefore,triggering ferroptosis is emerging to be an effective approach to eliminate cancer cells.Pseudolaric acid B(PAB),a diterpene acid isolated from the root and trunk bark of Cortex pseudolaricis,has various bioactivities such as antimicrobial activity,antiangiogenic activity and antivirus activity[17,18].Several reports have shown that PAB treatment could lead to death in human cancer cells from prostate cancer,cervical cancer and breast cancer [19-21].Although previous study has shown that PAB could induce apoptosis and autophagic death in cancer cells,accumulating evidences have demonstrated that ferroptosis could occur simultaneously with apoptosis and autophagy[26,27].In this study,we thus used rat and human glioma cell lines and nude mice model of xenograft glioma to investigate whether PAB could induce ferroptosis and its underlying mechanism.Objective:Rat C6 glioma cells and human SHG-44,U87 MG,U251 glioma cells and xenograft nude mouse models were established to investigate whether PAB could induce the death of ferroptosis and its mechanism.Methods:1.MTT assay: detecting the inhibitory rate of PAB to glioma cells and HL-7702 liver cells;LDH release assay: detecting the toxicity of PAB to tumor cells and the killing effect of PAB after added inhibitor and promoter.2.Iron detection: Iron colorimetric kit detects the iron concentration change of high/low concentration and in the long/short term of glioma cells induced by PAB;The changes of iron content in PAB-induced glioma cells were detected by DFO pre-treatment for 1h.3.Western Blotting assay: detecting protein expression levels of PAB-induced time gradient glioma cells sample,PAB-induced glioma cells sample pretreated by inhibitor and promoter for 1h,PAB-induced glioma cells sample pretreated by silencing NOX4 and GPX4 gene.4.Light microscope observe morphological changes of PAB-induced glioma cells pretreated by inhibitor for 1h;transmission electron microscope observe mitochondrial changes of PAB-induced glioma cells.5.JC-1 probe staining: mitochondrial morphology and fluorescence changes after PAB treatment to glioma cells were observed by fluorescence microscope;mitochondrial membrane potential changes after PAB induction of glioma cells were detected by flow cytometry.fluorescence changes after PAB treatment to glioma cells were observed by microplate reader.6.MDA detection: detecting lipid peroxidation products expression levels ofPAB-induced glioma cells sample.Detecting lipid peroxidation products expression levels of PAB-induced glioma cells pretreated by inhibitor and promoter for 1h sample.7.DCFH-DA probe fluorescence intensity,DHE probe fluorescence intensity,H2O2 content detection: oxidative stress index change of high/low concentration and in the long/short term of glioma cells induced by PAB;Oxidative stress index change of PAB-induced glioma cells pretreated by inhibitor and promoter for 1h sample;fluorescence microscope observation fluorescence changes of PAB-induced glioma cells,loading DCFH-DA probe,DHE probe.8.NADPH oxidase detection: detecting NOX expression levels of PAB-induced glioma cells sample.Detecting NOX expression levels of PAB-induced glioma cells pretreated by inhibitor and promoter for 1h sample.9.Glutathione and cysteine detection: detecting glutathione and cysteine expression levels of PAB-induced glioma cells sample.Detecting glutathione and cysteine levels of PAB-induced glioma cells pretreated by inhibitor and promoter for1 h sample.10.si RNA technology: After silencing of GPX4 gene and NOX4 gene,detecting cell death ratio and protein expression level of PAB-induced glioma cells.11.C6 glioma cells were xenografted in Balb /c nude mice and PAB was injected intraperitoneally to observe the inhibitory effect on tumor.Detecting tissue sample changes of ferrous iron content,MDA,H2O2,glutathione and cysteine.Results:1.PAB inhibited the proliferation of glioma cells and induces its death,with little effect on liver cells.2.Iron regulated PAB-induced glioma cell death.3.Iron contributed to PAB-induced glioma cells occurring lipid peroxidation.4.Iron modulated PAB-induced over-production of superoxide and H2O2.5.Iron contributed to PAB-induced activation of NOX4.6.Iron promoted PAB-induced down-regulation of x CT leading to glutathione depletion.7.PAB inhibited the growth of tumor tissue in vivo and showed dose-related effect.Conclusion:1.PAB inhibited the viabilities of glioma cells in vitro and in vivo.2.The increase of intracellular iron caused by PAB was related to upregulated transferrin receptor.3.PAB induced lipid peroxidation via activation of Nox4 in an iron-dependent manner.4.The depletion of GSH caused by PAB was via p53-regualted inhibition of x CT.5.The glioma cell death triggered by PAB was decided by intracellular iron and lipid peroxidation.