Ferroptosis Induced By RSL3 Can Promote The Chemotherapeutic Effect Of TMZ On Glioblastoma

Glioblastoma（GBM）is the most malignant glioma with 5-year survival rate less than 5%and patient average survival time about 14 months.Standard therapeutic strategy for GBM is surgical resection in combination with radiotherapy and chemotherapy with alkylating agents（such as temozolomide）.However,GBM is difficult to cure because of its highly invasive growth and drug resistance.Clinically,about 90%of the patients are diagnosed as IDH1/2wild type by molecular typing.Most of these patients have no history of glioma and belonged to primary GBM.In addition,about 10%of the patients harbor IDH1/2 mutations in molecular typing,most of which were associated with a history of low-grade gliomas and are classified into secondary GBM.GBM patients with IDH1/2 mutations are sensitive to temozolomide treatment,and the prognosis is significantly better than that of IDH1/2 wild type patients.The genetic and transcriptomic maps of GBM have been published in TCGA database,and GBM is divided into four molecular subtypes:classical,mesenchymal,neural and proneural subtypes.These different molecular subtypes correspond to different survival time and different therapeutic responses in the clinical treatment and prognosis of GBM.In view of the difficulty of clinical treatment of GBM,revealing new and effective treatment is an urgent clinical problem to be solved.Ferroptosis is a newly discovered form of cell death,characterized by the accumulation of lipid peroxides caused by iron overload,which is different from apoptosis or necrosis.Current studies have shown that it is triggered by blocking systerm Xc^-or inhibiting glutathione-dependent antioxidant enzyme GPX4.Systerm Xc^-is a transmembrane dimer complex composed of light chain SLC7A11 and heavy chain SLC3A2.Its function is to transport cystine into cells and reduce cystine to cysteine for the synthesis of GSH.Then GSH is absorbed and utilized by GPX4 to reduce lipid peroxides to lipids.Other studies suggest that ferroptosis may be caused by the destruction of the antioxidant system of GSH-GPX4.Iron has two states:oxidized state and reduced state:Fe²⁺or Fe³⁺.Normal iron balance can protect cells from ferroptosis,which is a normal metabolic process for the human body.The destruction of iron redox cycle may affect the sensitivity of cells to ferroptosis.In cells,Fe³⁺binds to transferrin in serum and is absorbed by the transferrin receptors（TFRC and TFR2）on the cell membrane.At this time,cytoplasmic iron is mainly stored in ferritin,which is composed of ferritin light chain（FTL）and ferritin heavy chain 1（FTH1）.At present,there are four known ferroptosis inducers（FINs）,which induce ferroptosis through different mechanisms.Class I（such as Erastin and sorafenib）mainly inhibits the activity of systerm Xc^-and consumes GSH.Erastin reduces glutathione synthesis by inhibiting cystine uptake by SLC7A11,which leads to cell death caused by the accumulation of reactive oxygen species（ROS）.Class II ferroptosis inducers（such as RSL3）directly inhibit the activity of GPX4,resulting in the decrease of antioxidant capacity and the increase of lipid peroxides,resulting in ferroptosis.The class III drugs（such as FIN56 or statins）can induce the degradation of GPX4 and disturb the production of mevalonate pathway intermediates,which directly inhibit the production of coenzyme Q10（Co Q10）,thus deplete GPX4 and cause rapid accumulation of lipid peroxides.Class IV ferroptosis inducers（such as FINO2）stimulates lipid peroxidation and the loss of GPX4 activity triggers ferroptosis.To sum up,GPX4 is undoubtedly the core regulatory target of ferroptosis.We focused on RSL3,an inhibitor of GPX4,to explore the important role of ferroptosis in tumorigenesis and drug resistance.Then,we elaborate the topic from six aspects:1.RSL3 could inhibit the proliferation and migration of GBM cells.Firstly,we evaluated the effect of RSL3 on GBM cell line.Through wound healing experiment,proliferation and Transwell chamber invasion experiments,we found that RSL3could significantly inhibit the proliferation,migration and invasion of GBM.The results of western blot assay also confirmed that RSL3 did not affect the expression of apoptosis marker cleaved caspase 3 protein.In order to study the downstream gene network,we analyzed the control group and RSL3-treated LN18 and primary GBM1 cells by transcriptomic RNA sequencing.By analyzing the sequencing data,it was found that down-regulated genes more than 2 times were significantly involved in the interaction between cell cycle and extracellular matrix,while up-regulated genes were concentrated in inflammation and hypoxia-related genes.GSEA（geneset enrichment analysis）showed that RSL3 treatment significantly enriched the genesets related to ferroptosis.In addition,the down-regulated genes significantly enriched genesets related to cell mobility and cell proliferation,including epithelial-mesenchymal transformation（EMT）,invasive growth and metastasis.EMT was related to cell mobility,so we detected several EMT markers in LN18,LN229 and GBM1 cell lines.The data confirmed that the expression of key transcription factors related to N-Cadherin,Vimentin and EMT decreased after RSL3 treatment,suggesting that RSL3inhibited the proliferation and migration of GBM cells.2.Combination of RSL3 and TMZ could inhibit the viability and migration ability of GBM cells.Recently,it has been found that ferroptosis may be involved in the drug resistance of GBM cells to TMZ,so we began to study the effect of RSL3 and TMZ on GBM cells.We found that RSL3 can promote the sensitivity of GBM cells to TMZ.Invasion experiments showed that the combination of RSL3 and TMZ could inhibit the invasion of GBM cells more obviously than RSL3 or TMZ alone.At the same time,in order to accurately detect ferroptosis,we also used transmission electron microscope to observe the changes of cells after treatment with drugs.Electron microscope experiments found that cells treated with TMZ alone did not damage mitochondria,but cells treated with RSL3 could cause mitochondrial damage.It is known that mitochondrial damage is a typical effect of ferroptosis.The combination of RSL3 and TMZ can significantly affect mitochondria and lysosomes,which confirms that TMZ can enhance the effect of ferroptosis induced by RSL3.3.Animal experiments showed that the combined effect of RSL3 and TMZ significantly inhibited the growth of GBM cells.We want to observe the effect of RSL3 and TMZ in vivo through experiments,so we build an animal model by nude mice.We found that TMZ or RSL3 alone slightly inhibited the growth of GBM tumor in nude mice,but it was obvious that the tumor inhibition effect was stronger when RSL3 and TMZ were used at the same time.We collected the transplanted tumor and made immunohistochemical staining of the tumor.The results showed that the number of Ki67 positive cells in the tumor cells treated with RSL3 and TMZ at the same time was less than that in the other single drug group and the control group.The expression of GPX4 protein in the transplanted tumor was decreased in RLS3 with TMZ simultaneous treatment group and RLS3 monotherapy group.In addition,transmission electron microscope experiments also showed that the mitochondrial damage in RSL3 and TMZ simultaneous treatment group was more serious.Therefore,the combined application of RSL3 and TMZ could have a stronger anti-tumor effect than each inhibitor alone.4.IDH1 mutant status did not affect the combined action of RSL3 and TMZ.IDH1 mutation is the most common molecular phenotype in gliomas.Clinical experiments have confirmed that the prognosis of GBM patients with IDH1-R132H mutation is better than that of patients without IDH1 mutation.The main purpose of this section is to verify that RSL3 has an impact on the state of IDH1.Through the cell viability test,we detected the sensitivity of cell lines with different IDH1 states to drugs,and found that the cell lines with IDH1 mutations were more sensitive to RSL3.However,the combination of two drugs made IDH1-wild-type（insensitive to drug response）tumor cells sensitive to chemical inhibiton.We then sequenced the cells with different molecular types by RNA sequencing.the results showed that the number of significantly altered genes in IDH1 mutant tumors was much more than that in wild type tumors.The sequencing results of IDH1-wild type and mutant type showed that there was a large part of gene overlap between them,which indicated that the genesets affected by IDH1-mutant type glioma after RSL3 treatment were larger than those of IDH1-wild type glioma.In other words,IDH1-mutant type group was more sensitive to RSL3 than IDH1-wild type group.Although RSL3 had no significant effect on the state of IDH1,the inhibitory effect of TMZ on the invasion of mutant GBM cells was significantly stronger than that of wild type GBM.On the contrary,TMZ alone might not effectively inhibit IDH1wild-type GBM,only the combination of RSL3 and TMZ completely counteracted the effect of IDH1 on drug sensitivity.Thus,the combination of two drugs might effectively inhibit IDH1 wild-type GBM with poor therapeutic effect.Therefore,TMZ alone could not significantly inhibit the GBM cells of some refractory IDH1-wild type,while the addition of RSL3 promoted the inhibitory effect of TMZ on refractory GBM.5.FTH1 and FTL were tightly related to TMZ resistance.We analyzed the correlation between ferroptosis and GPX4,SLC7A11,FTH1,FTL,TF,TFRC,TFR2,NQO1 and ATL1genes.Through the analysis of glioma data in TCGA database,we found that there was a tight correlation among FTH1,FTL and GPX4,and we speculated that there was a relationship between gene FTH1,FTL and drug RSL3.Through literature review,we found that MGMT played a decisive role in temozolomide resistance,and thus we analyzed the correlation between key iron death regulatory factors and gene MGMT in TCGA glioma database.The results showed that the expression of FTH1 and FTL was related to MGMT gene expression,which proved that ferroptosis was related to temozolomide resistance.Then we analyzed the sequencing data of temozolomide resistant LN229 cell line from GSE113510 and found that the high expression of FTH1 and FTL could also enrich the gene set related to drug resistance.Therefore,the expression of FTH1 and FTL genes leaded to insensitivity of GBM cells to temozolomide.6.FTH1 and FTL were correlated with prognosis and progression of GBM.Immunohistochemical analysis of GBM samples using tissue microarray showed that the expression of FTH1 and FTL genes was related to the malignant degree of GBM.The results of survival analysis showed that the prognosis of patients with low expression of FTH1 or FTL was significantly better than that of patients with high expression.We arranged and combined the expression status of these two genes and found that patients with high expression of FTH1 and FTL had a shorter survival time than patients with other expression combinations.In addition,it was mentioned earlier that there are four molecular types of GBM:mesenchymal type,classical type,anterior nerve type and nerve type.Database analysis showed that the expression of FTH1 and FTL in mesenchymal GBM with the worst prognosis was significantly higher than that in the other three subtypes.Therefore,we believe that the expression of FTH1 and FTL could predict the poor prognosis of patients with GBM.In summary,our study confirmed that RSL3 inhibited the growth and invasion of GBM and provided evidence that the combination of RSL3 and TMZ effectively inhibited both IDH1 wild-type and mutant.In addition,our study revealed that FTH1 and FTL could be used as biomarkers to predict the poor prognosis of GBM patients,and played an important role in drug resistance towards TMZ.