Autophagy Activated By Silibinin Contributes To Glioma Cell Death Via Induction Of Oxidative Stress Mediated BNIP3-dependent Nuclear Translocation Of AIF

Background:Glioma is an aggressive malignant brain tumor and constitutes the major causes leading to death in both pediatric and adult populations[1].The median survival time of the patients with newly diagnosed glioma is only 14.6 months,despite these patients are treated with surgical removal of tumor followed by radiotherapy and chemotherapy with temozolomide(TMZ)[2].As an alkylating agent,TMZ is widely used for treating primary and recurrent high-grade gliomas,but its efficacy is often limited by development of resistance[3].Therefore,new medicines are needed for glioma treatment.Autophagy that is characterized morphologically with formation of autophagosomes or autolysosomes in cytoplasm is a degradation pathway by which intracellular materials or impaired organelles are delivered to lysosomes for clearance[4].Autophagy plays dual roles in regulation of cell destiny.On the one hand,it protects cells against detrimental stresses.On the other hand,it contributes to cell death,which is designated as autophagic death[4].Autophagy was reported to abrogate cisplatin-induced death in hepatocellular carcinoma cells via inhibiting accumulation of reactive oxygen species(ROS)by clearing damaged mitochondria[5].It was also found that autophagy protected neuroblastoma cells by removing misfolded proteins which were generated during the process of oxidative stress[6].Thus,the protection of autophagy against cell damage is closely associated with suppression of oxidative stress.In the case of autophagic death,autophagy is also accompanied with accumulation of intracellular ROS and generally thought to be activated by ROS[7,8].However,it remains elusive whether autophagy could promote cell death via improving intracellular ROS or inducing mitochondria damage.Silibinin is the most biologically active component of silymarin that is a polyphenolic extract from milk thistle(Silybum marianum).It has been widely used to treat and prevent many types of hepatobiliary disorders such as alcoholic liver disease,nonalcoholic fatty liver disease and mushroom poisoning[9,10].Moreover,silibinin not only exert potent inhibitory effect on various types of cancer cells such as breast cancer cells,prostate cancer cells and glioma cells,but also sensitizes glioma cells to TMZ,TRAIL or arsenic trioxide treatment and prevents hepatocyte injury induced by chemotherapy agent cisplatin.Although silibinin could induce autophagic death in cancer cells,the biochemical events downstream silibinin-induced autophagy remains unclear.Therefore,in this study,we used glioma cell lines and nude mice with xenografted glioma to investigate whether autophagy plays a role in promoting mitochondria damage and its potential mechanism.Objective:In this study,we use multiple glioma cell lines and nude mice with xenografted glioma to investigate whether BNIP3 related autophagy contributes to silibinin-induced glioma cell death.Methods:1.MTT assay was used to examine the viability of glioma cells.2.LDH release assay was used to detect the morbidity of glioma cells.3.Western-blotting was used to detect the expression level of related proteins in glioma cells,as well as xenograft glioma tissue.4.Combined with JC-1 probe staining,flow cytometry was used to analyze the changes of mitochondrial membrane potential.5.The morphologial changes were observed under fluorescence microscope loaded with JC-1?DCFH-DA?Mitosox or Mitotracker green probe.6.Immunofluorescence staining was used to detect the distribution of related proteins in glioma cells through laser scanning confocal microscope.7.The oxidative level of glioma cells were tested by DCFH-DA probe,and H2O2 assay.8.The glioma cells were infected by Stub RFP-Sens GFP-LC3 B lentivirus to detected the condition of autophagy.9.Intracellular cysteine and GSH was measured to reflected the content of reducing substances in glioma cells,as well as xenograft glioma tissue.10.Knockdown of BNIP3,AIF or ATG5 with small interfering RNA was used to investigate their functions in silibinin-induced nuclear translocation of AIF and excessive mitophagy.11.C6 cells were xenografted in the athymic BALB/c nude mice to establish the mice model of xenograft glioma,and silibinin was injected to investigate the effect of silibinin in vivo.Results:1.MTT assay showed that silibinin inhibited the viabilities of U87,U251,SHG-44 and C6 glioma cells in a dosage-dependent manner.Western blotting analysis showed that silibinin induced time-dependent upregulation of ATG5 and LC3-II,but downregulation of autophagy substrate p62(SQSTM1).Representative images of the cells tranfected with Stub RFP-Sens GFP-LC3 B lentivirus under confocal microscope revealed that silibinin induced formation of autophagosomes(yellow punta)and autolysosomes(violet puncta).Western blotting analysis showed that 3MA abrogated silibinin-induced upregulation of LC3-II and reduction of p62.Bafilomycin A1 reversed silibinin-induced p62 reduction,but it further improved LC3-II levels.LDH release assay proved that silibinin-induced glioma cell death was prevented in the presence of 3MA or bafilomycin A1.Western blotting demonstrated that knockdown of ATG5 with Si RNA inhibited silibinin-induced upregulation of LC3-II and reduction of p62.LDH release assay showed that silibinin-induced glioma cell death was abrogated when ATG5 was knocked down with Si RNA.2.Representative images of the cells stained with JC-1 under fluorescence microscope showed that silibinin treatment resulted in obvious reduction of red fluorescence in U87 and U251 cells.Flow cytometry analysis combined with JC-1staining confirmed that silibinin induced time-dependent dissipation of mitochondria membrane potentials.Representative images of the cells stained with Mitosox red under fluorescence microscope showed that the red fluorescence exhibited by Mitosox red was apparently stronger in silibinin-treated cells than that in control group.Statistical analysis of the red fluorescence intensity exhibited by Mitosox red proved that silibinin triggered mitochondrial accumulation of superoxide in a time-dependent manner.Western blotting analysis revealed that silibinin induced AIF translocation from mitochondria to nuclei in a time-dependent manner.Representative images acquired by confocal microscopy combined with immunochemical staining showed that silibinin induced accumulation of AIF in the nucleus of U87 cell.Knockdown of AIF with Si RNA suppressed silibinin-induced accumulation of AIF in nuclear fraction.LDH release assay proved that knockdown of AIF prevented silibinin-induced glioma cell death.Statistical analysis of the red fluorescence intensity exhibited by Mitosox red showed that silibinin-induced mitochondrial accumulation of superoxide was significantly inhibited in the presence of 3MA or bafilomycin A1.Western blotting proved that knockdown of ATG5 with Si RNA prevented silibinin-induced AIF translocation from mitochondria to nuclei.Western blotting revealed that silibinin-induced nuclear translocation of AIF was suppressed when ATG5 was knocked down with Si RNA.Knockdown of ATG5 with Si RNA abrogated the improvement of mitochondrial superoxide induced by silibinin.3.Western blotting analysis showed that silibinin induced BNIP3 upregulation and promoted BNIP3 accumulation on mitochondria in a time-dependent manner.Representative.images.acquired.by.confocal.microscopy.combined.with.immunocytoc hemical staining showed that the upregulated BNIP3 induced by silibinin were colocalized with mitochondria.Knockdown of BNIP3 with Si RNA inhibited silibinin-induced BNIP3 upregulation and accumulation on mitochondria.LDH release assay showed that silibinin-induced glioma cell death was prevented when BNIP3 was knocked down with Si RNA.Statistical analysis of the intensity of the red fluorescence exhibited by the cells incubated with Mitosox red proved that knockdown of BNIP3 with Si RNA prevented silibinin-induced accumulation of mitochondrial superoxide.Flow cytometry analysis combined with JC-1 staining showed that knockdown of BNIP3 alleviated silibinin-induced mitochondria depolarization.Western blotting analysis revealed that silibinin-induced AIF translocation from mitochondria to nuclei was suppressed when BNIP3 was knocked down with Si RNA.Western blotting proved that silibinin-induced BNIP3 upregulation and accumulation on mitochondria were both inhibited in the presence of 3MA or bafilomycin A1.4.Western blotting proved that silibinin induced downregulation of HIF-1?,but upregulation of GPX4 in a time-dependent manner.H2O2 assay showed that intracellular H2O2 was improved time-dependently in silibinin-treated glioma cells.GSH assay proved that silibinin treatment resulted in time-dependent depletion of GSH.H2O2 assay demonstrated that supplement of exogenous GSH prevented silibinin-induced increase of H2O2.LDH release assay showed that silibinin-induced glioma cell death was prevented when GSH was supplemented.Western blotting revealed that pretreatment with GSH prevented silibinin-induced BNIP3over-expression and accumulation on mitochondria.Western blotting showed that H2O2 not only induced BNIP3 over-expression,but also improved BNIP3 levels in mitochondrial fractions.GSH assay showed that silibinin-induced GSH depletion was inhibited in the presence of 3MA or bafilomycin A1.H2O2 assay showed silibinin induced improvement of H2O2 was reversed in the cells pretreated with 3MA or bafilomycin A1.5.Cysteine assay showed that silibinin induced time-dependent depletion of cysteine.Western blotting analysis proved that silibinin downregulated x CT,but upregulated p53 and phospho-p53 in a time-dependent manner.These were all prevented in the presence of 3MA or bafilomycin A1.Cysteine assay demonstrated that silibinin-induced depletion of cysteine was prevented in the presence of 3MA or bafilomycin A1.Western blotting demonstrated that knockdown of ATG5 with Si RNA prevented silibinin-induced downregulation of CT and upregulation of p53 and phospho-p53.Cysteine assay proved that silibinin-induced depletion of cysteine was inhibited when ATG5 was knocked down with Si RNA.6.Silibinin depleted ATP,glucose-6-phosph and pyruvate in a time-dependent manner.Western blotting revealed that silibinin induced time-dependent downregulation of HK-II,PFKP and PKM2.Pretreatment with GSH prevented silibinin-induced depletion of ATP,glucose-6-phosph and pyruvate.Western blotting proved that the downregulation of HK-II,PFKP,PKM2 and p62 and the upregulation of ATG5 and LC3-II induced by silibinin were all inhibited in the presence of GSH.7.The mice model of C6 xenograft glioma revealed that the volume of xenograft gliomas became smaller in the silibinin group.Western blotting analysis revealed that silibinin upregulated ATG5 and LC3-II,but downregulated p62.Silibinin treatment resulted in depletion of GSH and cysteine,but improved H2O2.Western blotting analysis showed that silibinin downregulated x CT,but upregulated p53 and phospho-p53.Moreover,silibinin triggered BNIP3 upregulation and accumulation on mitochondria and promoted AIF translocation from mitochondria to nuclei.Silibinin induced depletion of ATP,glucose-6-phosph and pyruvate.Silibinin treatment resulted in downregulation of HK.Conclusion:1.Silibinin inhibited the viabilities of glioma cells in vitro and in vivo.2.Silibinin induced autophagic death in glioma cells,autophagy contributed to mitochondria damage.3.Autophagy promoted silibinin-induced BNIP3 expression and accumulation onmitochondria.4.Autophagy promoted silibinin-induced increase of H2O2 via causing GSH depletion.5.Autophagy.promoted.silibinin-induced.cystine.depletion.and.p53.phosphorylati on.6.Silibinin suppressed glycolysis in glioma cells.