Study On The Mechanism Of Gambogenic Acid Inducing Autophagy In Glioma U-87 Cells Based On PI3K/Akt/m-TOR Signaling Pathway

Gliomas are the most common type of primary malignant brain tumor,accounting for around 54% of adult malignant brain tumors and an annual incidence of 100000 population.Malignant gliomas arise in a multistep process involving sequential and cumulative genetic alterations resulting from intrinsic and environmental factors.Significant advances have been made in anti-cancer treatment over the past 50 years,but little has been done in the treatment of gliomas therapy.Rapid proliferation and uncontrolled invasion have been obstacles to clearing glioma.Owing to itsspecial growth mode and location of the occurrence,most of them have no obvious boundary with the surrounding normal brain tissue,especially in the brain functional area tumor,the operation is difficult to completely remove,and the prognosis is very poor.Effective chemoradiotherapy regimens are constantly being explored,But most are limited.Therefore,finding safe and effective chemotherapy drugs is one of the hot issues in glioma research.GNA is one of the major active compounds extracted from a traditional Chinese medicinal herb named Garcinia hanburyi.Recent studies showed that GNA has broad spectrum and significant anti-tumour effects.The mechanism is related to the different cell death patterns and the inhibition of the proliferation of tumor cells.On the basis of previous studies,this experiment further explored whether GNA is related to the effect of inducing autophagy in U-87 cells of glioma and inhibiting tumor cell proliferation from the perspective of autophagy Objective: Study on the mechanism of GNA inducing autophagy in glioma U-87 cells based on PI3K/Akt/m-TOR signaling pathway.Methods: Effect of GNA on cell viability by MTT assay.An inverted optical microscope was used to observe the morphological changes.PI staining assay detects the effect on cell proliferation;MDC staining inverted fluorescence microscope to observe the formation of autophagic vacuoles.AO staining flow cytometry was used to quantitative analysis of acidic vesicular organelles.Observation of autophagosome formation by transmission electron microscopy.The expression of tumor Autophagy factor LC3 m RNA and Beclin-1 m RNA was detected by q RT-PCR.Western blot was used to measure the expression of a Autophagy-related proteins LC3-Ⅱ /LC3-Ⅰ、beclin-1 and p62.Western blot was used to measure the expression of a Autophagyrelated proteins p-PI3K、p-Akt、p-m-TOR、PI3K、Akt、m-TOR.Western blot analyses were the expression of PI3K/AKT/m-TOR pathway-related proteins after PI3K/m-TOR inhibitor(LY24002)intervention.Results:(1)MTT assay showed that GNA inhibited the proliferation of U-87 cells in the concentration range of 1～16 μmol·L-1.The morphology of U-87 cells was observed by inverted microscope.It was found that U-87 cells were treated with GNA and compared with normal cells、cell gap was widened、cell antennae contracted、wrinkled and rounded,intracellular particles increased and refractive index decline.PI staining flow analysis showed that the GNA had a significant killing effect on tumor cell U-87 in a certain concentration range.(2)MDC staining inverted fluorescence microscopy showed that GNA promoted the formation of autophagic vacuoles in U-87 cells;AO staining flow cytometry showed that GNA increased the number of acidic vesicle organelles in U-87 cells;transmission electron microscopy The results showed that the autophagosomes in U-87 cells without drug treatment were less,and the autophagosomes in the cells treated with new gambogic acid were significantly increased.The results of q RT-PCR showed that GNA acts on U-87 cells.Up-regulation of m RNA expression of LC3 and Beclin-1 Western blotting illustrated the ratio of LC3-II/LC3-I were increased,the expression of beclin-1were increased and p62 were reduced.The experimental results suggest that GNA can enhance autophagy after U-87 cells.(3)Western blotting assay revealed that compared with the control group,GNA inhibited PI3 K phosphorylation,and dramaticly decreased the levels of phospho-Akt,leading to the down-regulation of downstream phospho-m-TOR in a dose-dependent manner.The expression of PI3 K,Akt and m-TOR in each groups had no substantial changes.The ratio of PI3K/PI3 K,p-Akt/Akt,p-m-TOR/m-TOR was decreased compared with the control group;after treatment with the specific PI3 K inhibitor LY294002 and GNA,the results showed that the expression of p-Akt,p-m-TOR was lower than that of the GNA group alone.The experimental results suggest that the PI3K/Akt/m-TOR pathway is involved in GNA-induced autophagy.Conclusion: Studies have shown that GNA can inhibit the proliferation of tumor cells by regulating PI3K/Akt/m-TOR induced autophagy in glioma U-87 cells.