| Background: At present,the standard glioma treatment is surgery,followed by chemotherapy and radiotherapy.Temozolomide(TMZ)is the first-line chemotherapy drug for gliomas.However,even with TMZ management,the overall survival of patients with GBM has been reported to only be prolonged by about 3-5 months.Thus,improving the therapeutic effect of TMZ is a major issue that needs to be addressed.Several studies have indicated that autophagy participates in the regulation of the sensitivity to TMZ.According to context,autophagy could either play a pro-survival,or pro-death role under treatment with TMZ,but the underlying mechanism has not been fully understood,making it difficult to distinguish the specific role autophagy might play,and thus target it for increased TMZ efficacy.Many autophagy-related proteins have been shown to be involved in the regulation of apoptosis,so the autophagic flux condition has been considered as a key factor determining the role of autophagy.Hence,there is a pressing need to better understand the role of autophagy in apoptosis,and the regulation of the sensitivity to TMZ under different autophagic flux backgrounds.Autophagy is a highly dynamic,multi-step biological process,including initiation,formation of autophagosome membrane nucleus,extension,maturation and closure,fusion with lysosomes,formation of autophagolysosomes,and then lysosomal degradation.Several studies have indicated that interfering with different autophagic flux stages might result in different cell fate under treatment with TMZ,suggesting that autophagic flux conditions under treatment with TMZ might lead to different cell fate,and the accumulation state of autophagy substrates might be the key to determining the role of autophagy.Sequestosome-1(SQSTM1),also known as the ubiquitin-binding protein p62,is a specific substrate for selective autophagy and an important part of the autophagosomal membrane.The degradation of p62 is mainly regulated by the autophagic flux [14].An earlier study of our research group suggested that basic high expression of p62 and fluent autophagy flux could endow tumor cells with higher resistance to chemotherapeutics [15,16].When autophagic flux was blocked with CQ or bafilomycin A1(Baf A1),apoptotic sensitivity was significantly increased.Meanwhile,Wen et al.,showed that knocking out autophagy related 4C cysteine peptidase(ATG4C)in U87 MG cells,could significantly inhibit autophagy flux,leading to accumulation of p62 and LC3-phosphatidylethanolamine conjugate(LC3-II),thus significantly improving the cytotoxicity of TMZ [18].These findings suggested that p62 might be the key factor in regulating autophagy under treatment with TMZ,but expression of p62 could not exactly reflect the conditions of autophagic flux and distinguish the role of autophagy in TMZ-induced cell death.Thus,uncovering the mechanisms of p62 in TMZ-induced cell death under different autophagic flux conditions remains an urgent issue that needs to be solved.Caspase-8 is known as a key protein in the exogenous apoptotic pathway.Studies have shown that although Caspase-8 is highly expressed in GBM,but its apoptotic function is not activated [20].Activating the apoptotic function of Caspase-8 might be an optional strategy in improving the efficacy of TMZ.Several studies had shown that p62 could determine cell fate through mediating the activation and degradation of Caspase-8.Besides,a recent study discovered that autophagosomal membrane could serve as platform for intracellular death-inducing signaling complex(i DISC),leading to activation of Caspase-8 and apoptosis independent of activation of death receptors.Thus,studying the interaction of Caspase-8 and p62 might help reveal the precise mechanism of regulation of autophagy flux on apoptosis,and provide a reference for accurately judging the role of autophagy under treatment with TMZ.Therefore,whether autophagy flux could modulate the therapeutic effect of TMZ by regulating the p62-mediated activation of Caspase-8 was examined through cell models with different autophagic flux and xenograft in this study.Methods:(1)Bioinformatics analysis and glioma tissue microarray were used to analyze the correlations between autophagy-related genes or Caspase-8 and glioma patient prognosis.(2)MTT assay was used to exam temozolomide and CQ induced cell viability inhibition.(3)p62 and LC3 expressions evaluated by Western Blotting and m RFP-GFP-LC3 system were used to detect autophagic flux.(4)Apoptosis was evaluated by Caspase-8 and Caspase-3 activity,TUNEL,Annexin-V/PI double staining and Western Blotting.(5)Immunofluorescence and co-immunoprecipitation were used to estimate the interaction between p62 and Caspase-8 and the translocation of Caspase-8 to autophagosomal membrane.(6)p62 knockdown or overexpression of p62-wild type or UBA or LIR truncated mutants were applied,and Caspase-8 activity was analyzed by kit and Western Blotting to evaluate the effects of p62 expression and its UBA and LIR domain on Caspase-8 activation.(7)U87MG glioma cells were xenografted in Balb/c nude mice to build animal model,and the animal model was used to evaluate the effect of temozolomide and chloroquine on xenograft growth in vivo.Results:(1)Bioinformatic analyses and glioma tissue microarray results showed that autophagy and lysosomal-related genes were highly expressed in gliomas,and were positively correlated with tumor grade and significantly correlated with poor prognosis.And in p62 high expression subgroup,survival of patients with high Caspase-8 expression was longer.(2)SHG44 with low basal autophagic flux and U87 MG with high low basal autophagic flux were screened out.And temozolomide(TMZ)could increase autophagy induction and autophagic flux in both glioma cell lines.Chloroquine(CQ)blocked autophagic flux resulting in p62 accumulation,and then increasing TMZ-induced Caspase-8 activation and apoptosis.(3)CQ could increase the interaction of p62 and Caspase-8,and the translocation of Caspase-8 to autophagosomal membrane.p62 knockdown could decrease Caspase-8 activation induced by TMZ and CQ.Besides,p62 overexpression increased TMZ-induced Caspase-8 activation and apoptosis.And compared with p62,UBA and LIR truncated mutant significantly reduced Caspase-8 activation.(4)In xenograft model,CQ could effectively enhance TMZ-induced tumor grouth inhibition.CQ could increase p62 accumulation and Caspase-8 activation induced by TMZ in vivo.Conclusions:(1)Autophagic flux was higher in high grade gliomas than low grade gliomas,and higher autophagic flux indicated worse prognosis.(2)When autophagic flux was blocked or the initiation of autophagy is greater than autophagy-associated lysosome degradation capacity,it would result in p62 accumulation,which in turn led to Caspase-8 activation.Autophagic flux was involved in temozolomide sensitivity regulation through regulating the formation or degradation of i DISC,which resulted in Caspase-8 activation.(3)i DISC-mediated Caspase-8 activation was dependent on UBA and LIR domain of p62.(4)Caspase-8 may act as a cofactor helping p62 to evaluate autophagic flux in gliomas,p62/Caspase-8 expression pattern may be a more accurate prognosis marker. |
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