| Glioma is a primary intracranial tumor,which is one of the most common and highly invasive tumors.The current standardized treatment of glioblastoma is surgical resection in the maximum safety range,followed by radiotherapy alone or chemotherapy combined with temozolomide.But patients with a poor prognosis,prone to relapse.Therefore,it is urgent to find effective treatment strategies for patients with glioma.Ferroptosis is a nonclassical way of cell death reported by Dixon et al.in 2012,with characteristics of iron ion dependence,accumulation of lipid peroxidation products and reactive oxygen species(ROS).It is different from apoptosis,pyroptosis,and necrosis in morphology,biochemistry and mechanism.Clinical and experimental studies have found that inducing ferroptosis could promote the proliferation and development of tumor cells,whereas inhibiting ferroptosis pathway promotes tumor proliferation and malignant transformation.Studies have confirmed that glutathione peroxidase 4(GPX4)protein level in glioma significantly increased and ferroptosis level decreased.Therefore,promoting ferroptosis in glioma cells is one of the strategies for treatment.Erastin,an antitumor drug,inhibits intracellular cysteine(Cys)uptake by inhibiting cystine/glutamate antiporter(System Xc-)on the cell membrane surface,weaken glutathione biosynthesis and destroy the redox balance in cells,resulting in ferroptosis.It is found that ferroptosis was closely related to programmed cell death such as apoptosis,autophagy and pyroptosis.The ferroptosis and apoptosis shared some molecules in their signaling pathways,such as p53,p38,VDAC2 and BAP1.Tumor suppressor p53 was involved in cellular stress-induced cell cycle arrest and apoptosis,and in a study of mice expressing acetylation deficient p533 KR,it was found that p533 KR inhibits a key component of the cystine/glutamate antitransporter expression of SLC7A11,inhibits cystine uptake and sensitizes cells to ferroptosis.The ferroptosis-mediated cleavage of the pro-apoptotic protein Bid promotes the occurrence of apoptosis,and ferroptosis occurred upstream of apoptosis.Autophagy leads to ferroptosis by degrading cellular ferritin in human pancreatic cancer cells.Whether Erastin-induced ferroptosis is accompanied by other types of cell death remains to be further investigated.One recent study found that in the process of ferroptosis,cell releases metabolites through osmotic way,and propagates a "signal" to induce swelling of adjacent cells,ultimately leading to the spread of ferroptosis,while the specific mechanism still unclear.Whether Erastin-induced ferroptotic cell releases certain metabolites to change in the microenvironment,and in turn secondarily leads to other programmed death.Therefore,clarifying whether Erastin can trigger multiple cell death modes and elucidating the relationship between ferroptosis and other cell death and related mechanisms will be crucial for the clinical treatment of glioma and drug development.In this study,C6 cells were treated with different Erastin concentration gradients(0.1 μM,0.5 μM,1 μM,5 μM,10 μM,30 μM,50 μM)to observe the protein expression and cell morphology of ferroptosis,apoptosis,autophagy and pyroptosis.In order to observe ferroptosis,transmission electron microscopy,western blot,flow cytometry,CCK-8 and fluorescence staining were applied to detect the morphological characteristics,intracellular lipid peroxidation level,GPX4 protein expression and mitochondrial reactive oxygen species(ROS)content after Erastin treatment.The level of early apoptosis was detected by flow cytometry(FCM)analysis.The expression changes of cytochrome C(Cyt C)protein,Bcl-2/Bax and cleaved caspase-3 protein were detected to observe the apoptosis phenomenon.Transmission electron microscopy was used to detect cell morphological changes and to detect changes in expression of LC-3 and p62 proteins to observe autophagy.Transmission electron microscopy was used to detect morphological changes in cells and to detect changes in expression of Gasdermin-D(GSDMD),pro caspase-1,and cleaved caspase-1 proteins to observe the phenomenon of pyroptosis in Erastin treatment.Pretreatment of Erastin-injured C6 cells with ferroptosis specific inhibitor Ferrostatin-1(Fer-1),followed by treatment of non-injured C6 cells with conditioned medium containing metabolites.Flow cytometry was applied to detect early apoptosis levels and western blot was used to detect GSDMD,pro caspase-1 and cleaved caspase-1 protein to further investigate the effect of cell metabolites on pyroptosis.Meanwhile,male BALB/c Nude mice were treated with different concentrations(2.74 μg/g,5.47 μg/g,16.41 μg/g)of Erastin by intraperitoneal injection after subcutaneous tumor formation,and the mice were weighed and tumor size changes were measured daily.Changes in the protein content of cleaved caspase-3,GSDMD,pro caspase-1 and cleaved caspase-3 proteins in the tumors of mice,and to investigate the death of glioma cells induced by different doses of Erastin.The results of the study were as follows:1.After treatment of C6 cells with different concentrations of Erastin(5 μM,10 μM,30 μM,50 μM)for 24 hrs,cell viability decreased by 16.8%,41.4%,79.8%,87.1%,92% and 91.9%,respectively,with statistically significant differences compared to the control group(P<0.001).5 μM,10 μM,30 μM and 50 μM Erastin-treated group Trypan blue staining positive cells increased by 68.48%,73.75%,80.68% and 87.7%(P<0.0001)and PI staining positive cells increased 1.04 times,1.29 times,167 times,208.4 times,244.3 times and 266.3 times(P<0.0001).2.After treatment of C6 cells with different concentrations of Erastin,5 μM,10 μM,30 μM and 50 μM groups were no significant change of the nuclei under transmission electron microscope but the mitochondria shrunk and became smaller.Measurements of mitochondrial long axis measurements showed that the mitochondrial length of the 5 μM,10 μM,30 μM and 50 μM Erastin groups decreased by 40.8%,21.0%,17.5% and 18.8%,compared to the DMSO group respectively,with a statistically significant difference(P<0.05,P<0.01).C6 cells treated with different concentrations of Erastin for 24 hours,there was no significant change in the GPX4 protein content in the 0.5 μM Erastin-group compared with the DMSO group(P>0.05).The expression levels of GPX4 protein in the C6 cells after 1 μM、5 μM、10 μM、30 μM and 50 μM Erastin treatment were down regulated by 30.7%,30.6%,31.9%,56.3% and 54.3%,compared to the DMSO group respectively,with statistically significant differences(P<0.05,P<0.001).In addition,the effects of 0.5 μM and 1 μM Erastin had no significant effect on cellular lipid peroxidation(P>0.05),and the levels of cellular lipid peroxidation in the 5 μM,10 μM,30 μM,and 50 μM Erastin-treated groups were significantly increased by 46.8%,64.5%,114.3%,and 54.4%(P<0.001,P<0.0001).10 μM,30 μM and 50 μM Erastin treatment groups the levels of mitochondrial ROS were significantly up-regulated by 53.0%、41.3% and 37.4%(P<0.01).3.C6 cells were treated with different concentrations of Erastin for 24 hrs,the results showed that 5 μM,10 μM,30 μM and 50 μM Erastin treatment groups the levels of early apoptosis were significantly up-regulated by 78.2%,121.3%,177.3% and 237.6%,respectively,compared with the control group(P<0.01,P<0.0001).There was no significant change in the Cyt C protein expression in the 5 μM,10 μM,and 30 μM Erastin-group compared with the DMSO group(P>0.05),and 50 μM Erastin treatment were up-regulated by 148.5%(P<0.001).In contrast,there was no significant change in the protein level of Bcl-2/Bax and cleaved caspase-3 in C6 cells treated with different Erastin treatment(P>0.05).4.C6 cells were treated with different concentrations of Erastin for 24 hrs,5 μM,10 μM,30 μM and 50 μM Erastin-treated groups appeared autophagic vacuolization,which bilayer chambers containing lamellar structures.Measurements of lamellar structures measurements showed that the number of lamellar structures 30 μM and 50 μM Erastin groups increased by 958.5%、812.5%(P<0.01).The expression of autophagy related proteins LC3 Ⅱ in the 5 μM,10 μM and 30 μM drug treatment groups were significantly up-regulated by 428.2%,533.4%,433.1%,respectively,with a statistically significant difference(P<0.01).5.C6 cells were treated with different concentrations of Erastin for 24 hrs,10 μM,30 μM and 50 μM Erastin-treated group appeared the morphological features of pyroptosis,rupture of cell membrane and efflux of contents.There was no significant change in the protein expression level of pro caspase-1 in different Erastin treatment(P>0.05).The level of cleaved caspase-1 protein expression in the 5 μM Erastin-treated group was up-regulated,but it was no statistical significance(P>0.05),and the expression of cleaved caspase-1 protein in the 10 μM and 30 μM drug treatment groups were significantly up-regulated by 43.5% and 44.8%,respectively,with a statistically significant difference(P<0.05).6.After pretreatment of C6 cells with Fer-1 for 30 min,then gave Erastin treatment,followed by treatment of non-injured C6 cells with conditioned medium containing metabolites.Compared with the Erastin alone treated metabolites group,the early apoptosis level of the cells in the 30 μM Erastin-treated metabolites group was increased by 139.3%,respectively,the difference was statistically significant(P<0.05).7.Intraperitoneal injection of Erastin(2.74 μg/g,5.47 μg/g,and 16.41 μg/g)in BALB/c Nude mice after subcutaneous tumor formation,measurements of body weight did not change significantly compared with the control group(P>0.05).Measurements of tumor size showed that tumor volume was down-regulated by 42.2%,48.9% and 49.3% in the 5.47 μg/g Erastin-treated group on days 5-7,respectively,with a statistically significant difference(P<0.05,P<0.01,P<0.001);16.41 μg/g Erastin-treated group was decreased by 53.0%,52.9%,55.1%,54.7% and 56.0% from 3rd to 7th day,respectively,and the differences were statistically significant compared with the control group(P<0.05,P<0.01,P<0.001).After 7days of intraperitoneal injection,the tumor weights of 2.74 μg/g and 5.47 μg/g Erastin treatment groups were decreased,but there was no statistical significance(P>0.05).And the tumor weights of the 16.41 μg/g Erastin-treated group were down-regulated by 34.6%,respectively,with a statistically significant difference(P <0.05).8.Intraperitoneal injection of Erastin in BALB/c Nude mice after subcutaneous tumor formation,2.74 μg/g,5.47 μg/g,and 16.41 μg/g Erastin-treated group under transmission electron microscopy exhibited morphological characteristics of ferroptosis,autophagy and pyroptosis.The above results suggested that ferroptosis in glioma cells increases with the concentration of Erastin increasing,which is accompanied by the occurrence of early apoptosis.Moreover,Erastin induces autophagy and pyroptosis in certain doses.Ferroptosis inhibitor Fer-1 could inhibit the occurrence of ferroptosis,but also inhibit the occurrence of early apoptosis.Erastin induces ferroptosis by secreting metabolites that affect normal C6 cells function and survival.However,which metabolites are released when astroglioma C6 cells undergo ferroptosis,and by which means these metabolites alter the cellular physiological functions or cellular microenvironment to cause other programmed death are largely latent,which still needs to be further investigated.The present study possesses great significance for the in-depth elucidation of the pathogenesis and progression of glioma. |
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