Corilagin Induces Apoptosis And Autophagy On U251 Cells Through Inhibiting PI3K/AKT/mTOR Signaling Pathway

[Objective] Glioma is the most common malignant primary brain tumor,and its pathogenesis is not clear.At present,the effect of routine treatment for glioma is not satisfactory,and it is easy to appear Radiotherapy and chemotherapy resistance,which leads to poor prognosis of glioma.Therefore,exploring and developing new and effective anti-glioma drugs is an urgent problem to be solved in current basic and clinical research.Corilagin(Corilagin,cor)is a kind of polyphenol tannic acid compounds extracted from natural plants such as leaf bead,matsumura leafflower herb,longan and so on.At present,Corilagin has many biological activities,such as anti-inflammation,anti-virus,anti-oxidation and anti-tumor,but there are few reports about the anti-glioma effect of Corilagin.The aim of this study was to explore the effect of Corilagin on apoptosis and autophagy of glioma U251 cells and to provide a new strategy for clinical treatment of glioma.[Methods] U251 cells were cultured and treated with different concentrations of Corilagin(0,25,50,100 ?g/ml).The morphological changes of U251 cells were observed under optical microscope.The cytotoxicity and proliferation characteristics of U251 cells were detected by cck-8 assay.Annexin V-FITC/PI staining and hoechst 33342 staining were used to detect apoptosis,PI single staining was used to detect cell cycle,cell scratch test and transwell cell migration assay were used to detect the ability of cell migration and invasion.The characteristics of autophagy in U251 cells treated with Corilagin were observed by transmission electron microscope,and the changes of autophagy level in U251 cells infected with lc3 double-labeled adenovirus were observed.Rt-pcr and western blot were used to detect the expression levels of PI3K/AKT/m TOR signaling pathway and downstream related molecules and autophagy-associated genes m RNA and protein.First,the cytotoxicity of Corilagin on human glioma cell U251 and human normal hepatocyte LO2 and its inhibitory effect on U251 proliferation were detected by CCK-8 assay.Secondly,the effects of Corilagin on apoptosis and cell cycle of U251 cells were detected.The morphological changes of U251 cells were observed by phase contrast inversion microscope,the apoptotic morphology of U251 cells was observed by hoeschst 33342 staining,the apoptosis rate of U251 cells was detected by Annexin V-FITC/PI double staining and flow cytometry.Pi single staining and flow cytometry were used to detect the cell cycle changes.Thirdly,the migration ability of U251 cells was detected by cell scratch assay,and the invasive ability of U251 cells was detected by Transwell cell migration assay.Then,the morphological characteristics of autophagy bodies in U251 cells were observed by transmission electron microscope,and the U251 cells were transfected with LC3 double-labeled autophagic adenoviruses and the expression level of autophagy was observed by fluorescence microscope.Finally,RT-PCR and Western Blot were used to detect the expression of PI3K/AKT/m TOR signaling pathway,downstream related molecules and autophagy-associated genes m RNA and protein.[Results] 1.When the concentration of Corilagin was less than 100 ? g/ml,it had no obvious cytotoxicity to human normal hepatocyte LO2,but could significantly inhibit the proliferation of U251 cells,and Corilagin inhibited the proliferation of U251 cells in a concentration-and time-dependent manner;2.The normal U251 cell morphology is full and grows well.The cell morphology become wrinkled and deformed and the density of adherent cells decreased with the increase of the concentration of Corilagin,and more suspended cell fragme appeared gradually in the medium;3.When U251 cells were treated with different concentrations of Corilagin,after hoechst 33342 staining,the nuclei of U251 cells were stained bright and the chromatin shrank,and the typical apoptotic bodies could be observed under fluorescence microscope.Annexin V-FITC/PI staining and flow cytometry showed that Corilagin could induce apoptosis of U251 cells;4.U251 cells were treated with different concentrations of Corilagin and PI was singly stained.The results of flow cytometry showed that Corilagin could induce U251 cells to stagnate in the S phase;5.Cell scarification test showed that the wound healing ability of U251 cells treated with high or medium concentration of Corilagin was lower than the control group after 48 h treatment with different concentrations of Corilagin.Transwell cell invasion test showed that the ability to penetrate the membrane of U251 cells was decreased in the medium and high concentration of Corilagin treatment group;6.When U251 cells were treated with 100 ?g/ml Corilagin,autophagy vesicles was increased obviously under transmission electron microscope,which showed the morphology of autophagy bodies with typical bilayer membrane structure;7.The optimal MOI value of LC3 double labeled autophagic adenovirus infected U251 cells was 40.A large number of autophages and autophagy lysosomes were found in U251 cells treated with 100 ? g/ml Corilagin,which indicated that Corilagin could induce autophagy in U251 cells;8.The results of RT-PCR and western blot showed that Corilagin could significantly inhibit the activation of PI3K/AKT/m TOR signaling pathway,affect the expression of downstream related genes m RNA and protein,and then promote cell apoptosis,inhibit cell migration and invasion,and induce autophagy.[Conclusion] 1.In the presence of no obvious cytotoxicity to human normal hepatocytes,Corilagin could significantly inhibit the proliferation of glioma U251 cells.The anti-glioma activity of Corilagin was associated with its promotion of apoptosis and the induction of cell S-phase arrest and and inhibit cell migration and invasion.The anti-glioma biological activity of Corilagin may be due to the inhibition of PI3K/AKT/m TOR signaling pathway and the expression of downstream related molecules;2.The anti-glioma activity of Corilagin may also be related to the induction of autophagy,which leads to autophagic death.Corilagin may induce autophagy by promoting the expression of beclin-1 and autophage-associated gene ATG9 B.