Molecular Signaling Mechanism Of Dcf1 Inhibition Of Autophagy And Apoptosis In Human Glioblastoma Cells

Glioblastoma is the most common and lethal primary malignant brain tumor with a high recurrence rate and extreme lethality that threat seriously to human health and life.Currently,there is no effective treatment,and the postoperative survival is about 14 months on average.the main reason is that the pathology and molecular biology responsible for their generation and development and progression are not fully elucidated.In this study,a total of 24 WHO grade IV human glioblastoma tissues and 6 normal trauma tissues were collected,and a total of 11 human glioblastoma cells were cultured successfully by tissue isolation.We identified that the dcf1(dendritic factor 1,a single transmembrane protein)expression were decreased in glioblastomas tissue without mutation,and overexpression of dcf1 in vitro could inhibit the proliferation,migration,invasion,and wound healing of glioblastoma cells,and promote the adsorption of glioblastomas cells to type I.Collagenase and the apoptosis of glioblastoma cells,but had no effect on the cell cycle of glioblastoma cells.We applied the method of isobaric tags for relative and absolute quantitation(iTRAQ)to explore the molecular mechanism of dcf1 inducing apoptosis in glioblastoma cells thoroughly on glioblastoma tissue,normal trauma tissues and GBM cells that transfected with dcf1.We identified that 234 proteins in glioblastoma tissues were significantly changed on expression level and dcf1 overexpression induced significant differences in 176 proteins in GBM cells.Through proteomic analysis,it was found that dcf1 induces a reverse of protein expression in tissues and subcellular localization differences of dcf1 in lysosomes and endosomes.,which affected the balance of organelles interconnection,these results suggested that dcf1 mediates molecular changes in protein expression and possible functional changes of organelles in glioblastoma cells.Mechanistically,dcf1 was first found to inhibit the expression of HistoneH2 A isomers and reduce the level of histoneh2 a isomers ubiquitization and nucleosome-associated protein,impair nucleosome structure and genetic material stability,and lead to DNA damage.dcf1 overexpression decreased the copy of mtDNA,reduced the number of mitochondria through SIRT1/3-PGC-1?-NRF1-TFAM,the mitochondrial membrane potential,increased mitochondrial membrane permeability,decreased the expression and activity of IDH1 and inhibited ATP production and thus leads to the reduction of energy supply of cells,and eventually leads to mitochondrial damage.dcf1 inhibited the expression of PARL and increased the ratio of BECN1/Bcl-2,but did not affect the localization of the complex,indicating the apoptosis of glioblastoma cells after autophagy.dcf1 enhanced the expression of LC3-II/LC3-I and increased autophagy structure,indicating dcf1 induces autophagy;GFP-LC3 puncta decreased but mCherry-LC3 puncta increased suggested that dcf1 promoted the fusion of autophagocytic vacuole with lysosome.dcf1 overexpression leads to decreased number of lysosomes,increased membrane permeability,decreased membrane function proteins V-ATPase,TFEB,increased expression of ACCP,CB,CD and,formation of damaged lysosomes.In addition,the pH of glioma cells was slightly alkaline,the activity of acidic phosphatase decreased and the contents of lysosome changed.At the same time,dcf1 upregulates PS1 to promote the fusion of lysosome and autophagic vacuoles,but the expression of LAMP1 that regulates the fusion of autophagic vesicles and lysosomes is decreased,making the fusion of autophagic vacuoles and lysosomes form defective autophagosomes,which hinders the autophagy process.dcf1 regulates autophagy through the PI3K/AKT/MAPK/mTOR/Ulk1 signaling pathway.dcf1 induces release of CB and CD in lysosomes to cytoplasm,cuts and activates BID,down-regulates Bcl-2 expression and localizes to mitochondria to bind VDAC1,and induces mitochondrial release of pro-apoptotic factors.Finally,dcf1 overexpression induced apoptosis-related protein TRAIL casp3/7 /GZMB expression in glioma cells,and induced apoptosis by increasing activation of TRAIL casp8/10-casp3/7 /GZMB exogenous death receptor pathway.In general,we for the first time found that dcf1 the expression is correlated with glioma malignant degree.Overexpression of dcf1 can inhibit the survival of human glioma cells by inducing autophagy and apoptotic signaling pathways.It reveals a key role of dcf1 in the process of the occurrence and development human glioma which provides a new idea for the treatment of glioma,and also lays a foundation for further research.