| Background and Objective:Gliomas are the most common type of malignant brain tumors with higher mortality.The median survival of the patients with high-grade glioma is not longer than 14.6 months,even if they accept surgery and then are treated with chemotherapy and radiotherapy.It has been found that glioma cells are resistant to the therapy based on induction of apoptosis.Thus,new medicines that induce non-apoptotic cell death might be useful to eliminate glioma.Ferroptosis is a newly established type of programmed necrosis and implicated in multiple pathological processes such as neurodegenerative diseases,carcinogenesis,ischemia-reperfusion,brain trauma and cerebral bleeding.Morphologically,it is featured by presence of mitochondria with condensed mitochondrial membrane densities and decreased size.Biochemically,ferroptosis is characterized with intracellular accumulation of iron which results from imbalance between iron uptake,storage and export.Intracellular iron level is mainly regulated by transferrin receptor which accounts for transporting extracellular iron-transferrin complex into cells via clathrin-mediated endocytosis,ferritin composed of light chain and heavy chain and responsible for storing iron,and ferroportin in charge of iron exportation.The increased iron disrupts membrane integrity by peroxidizing polyunsaturated fatty acid(PUFA)chains of membrane phospholipids via two pathways.One is to serve as a cofactor for nonheme iron‐containing lipoxygenase to enzymatically catalyze PUFA peroxidation[6].The other is to react with H2O2 via fenton reaction to generate toxic hydroxyl radicals,which have potent capacity to peroxidize PUFA.Induction of ferroptosis could effectively eliminate various types of malignant tumor cells from prostate cancer,colorectal cancer,hepatocellular carcinoma and even cisplatin-resistant lung cancer cells and glioma stem cells.Thus,ferroptosis inducers might be potential medicine for glioma treatment.Activating transcription factor 3(ATF3)belongs to the ATF/CREB family of basic-leucine zipper transcription factors.Investigation by using removed glioma tissue revealed that ATF3 is over-expressed in human gliomas and its expression level is positively associated with tumor malignancy.ATF3 plays dual roles in regulation of cancer cell demise.On one hand,it inhibited tumorigenesis and progression of hepatocellular carcinoma cells,repressed epithelial-mesenchymal transition of cholangiocarcinoma cells and limited invasion and migration of human colorectal cancer cells.On the other hand,it could protect breast cancer cells against radiotherapy by improving Akt phosphorylation.ATF3 is upregulated during the process of ferroptosis,as well as in apoptosis and necrosis.It was found that ATF3 contributed to erastin-induced ferroptosis in fibrosarcoma cells or retinal pigment epithelial cells via inhibiting x CT expression.However,it remains elusive whether ATF3 is involved in glioma cell ferroptosis or regulates ferroptosis via other pathways.Brucine(Fig.1A)is a weak alkaline indole alkaloid extracted from the seeds of Strychnos nux-vomica and usually used to relieve arthritis and traumatic pain.Recent studies reveal that brucine has potent anti-tumor activity in various types of cancers,including colon adenocarcinoma,hepatocellular carcinoma,multiple myeloma,breast cancer and glioma.Its anti-tumor effects are found to be associated with induction of apoptosis and cycle arrest,activation of JNK,inhibition of angiogenesis and epithelial mesenchymal transition and repression of tumor cell migration and metastasis.However,it remains elusive whether ferroptosis and ATF3 are both involved in brucine-induced cancer cell death.Therefore,in this study,we investigated the roles of ferroptosis and ATF3 in brucine-induced glioma cell death.Methods:1.Detect the inhibition of brucine on the proliferation of human glioma cell lines by MTT assay;Detect cell death ratio of gliomas induced by brucine and the effects of inhibitor on brucine-induced cell death by LDH release assay.2.Measurement of ferrous ion concentration: The ferrous ion detection kit was used to induce changes in iron ion concentration in human glioma cells at different concentrations of brucine;and the inhibitors were tested for effects on changes in iron concentrations in human glioma cells induced by brucine.3.Western Blotting was used to detect the relative protein expression caused by brucine on glioma cells at different times,and the changes of relative protein content in samples of brucine on glioma cells after ATF3 and NOX4 gene silence.4.To determine the degree of lipid oxidation induced by brucine in human glioma cells and the effect by inhibitors such as DFO,Fer-1,Lip-1,GSH and 4-PBA on lipid oxidation in brucine-treated human glioma cells.5.Detect the concentration of H2O2 in brucine-treated human glioma cells and effect by inhibitors include DFO.6.Detect the activation of NADPH oxidase in brucine-treated human glioma cells and the effect of inhibitors such as DFO on the anti-oxidative stress activity of brucine-induced human glioma.7.Test the effect of silencing ATF3 and NOX4 by siRNA on brucine-induced ferroptosis process in glioma cells.8.Human U87 glioma was xenografted in BALB / c nude mice and brucine were injected intraperitoneally to observe the effect on U87 glioma.Changes of iron concentration and relative protein expression in tumor tissues were detected.Results:1.Brucine induced glioma cell death.2.Brucine induced ferroptosis in glioma cells.3.ATF3 contributed to brucine-induced glioma cell ferroptosis.4.ATF3 contributed to brucine-induced iron increase by improving H2O2.5.ATF3 improved H2O2 by upregulating NOX4 and down-regulating x CT.6.Brucine induced a positive feedback between ER stress and H2O2 generation.7.Brucine inhibited tumor proliferation in vivo.Conclusion:1.Brucine inhibited glioma proliferation and induced glioma cell death both in vivo and in vitro.2.Brucine induced glioma cell death accompanied by increased intracellular ferrous iron and improved lipid peroxidation3.Brucine induced ferroptosis in glioma cells through ATF3.4.Brucine regulated intracellular ferrous iron and hydrogen peroxide through activation of ATF3 in glioma cells.5.Brucine induced endoplasmic reticulum stress in glioma cells by improved hydrogen peroxide levels,and positive feedback further promoted hydrogen peroxide accumulation. |
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