The Mechanistic Study On The Inhibition Of Glycolysis In Glioma By Niditine Chloride

BackgroundGliomas are the most common primary malignant tumor in the nervous system,accounting for approximately 80%of intracranial malignancies.Gliomas are highly proliferative,aggressive and drug-resistant.Currently,patients with glioma are mostly treated with a treatment strategy of surgical resection plus radiotherapy.However,the survival rate of glioma patients has not improved significantly.In particular,the median survival of patients with aggressive glioma is only 14.4 months.The recurrence and drug resistance are of vital importance reasons for the poor prognosis of glioma.Altered forms of energy metabolism are often considered to be a major marker of cellular malignancy.The change is mainly reflected in the preferential reliance of tumor cells on the glycolytic pathway for energy production under both aerobic and anaerobic conditions.This approach allows tumor cells to increase glucose uptake and accelerate the rate of production of adenosine-triphosphate(ATP),ultimately providing a stable source of energy for tumor cells.It has been shown that aerobic glycolysis plays an important role in the onset of drug resistance and malignant progression of tumors over layers.The development of novel effective glycolysis inhibitors will be significant.Nitidine chloride(NC),a natural product extract with antimalarial activity,has been shown to have anticancer effects in a variety of malignancies,including colon,breast,and ovarian cancers.However,its role in the study of glioma is still superficial.Pre-experimental studies by our group revealed that NC is a novel and effective glycolysis inhibitor,but its role in inhibiting malignant progression of glioma and glycolysis and the specific regulatory network mechanism are still unclear.Therefore,an in-depth investigation of the molecular mechanism of the anticancer role of NC in glioma is beneficial to improve the therapeutic ideas and enhance the therapeutic effects of glioma.Objectives1.To elucidate the inhibitory effect of nitidine chloride on glycolysis of glioma.2.To investigate the molecular mechanisms underlying the specific role of nitidine chloride in the inhibition of glycolysis in glioma.3.To identify potential targets of action that can effectively inhibit glycolysis in glioma.Methods1.The appropriate concentration of drug action was found by CCK-8 assay.The changes of glucose and lactate in the internal and external environment of glioma cells after NC treatment were detected by using glucose kit and lactate detector kit.The changes of pH in the supernatant of NC-treated cell culture medium were detected by using pH meter.2.GBM#P3 cells treated with PBS and NC for 24 hours,respectively,were collected for transcriptome sequencing and analyzed for changes in glycolysis-related genes.Kegg analysis was used to explore the molecular mechanisms.3.Western blot was used to detect changes in the key enzymes of glycolysis and related molecular mechanisms in the control and drug-treated groups.4.Analyze sequencing data to find the most relevant signaling pathway regulatory molecule,extracellular matrix glycoprotein-C(Tenascin-C,TNC).Analysis of differential expression of TNC in glioma and normal brain tissue,survival analysis associated with differential TNC expression using the GEPIA public database.Download TCGA database,classify TNC-related genes by correlation analysis,limma package,etc.and further perform Kegg analysis,GO analysis.5.The TNC gene fragment was knocked down in glioma cells with small interfering RNA,and the changes in pathway activity and expression of key enzymes of glycolysis were analyzed after knockdown.Results1.NC has an inhibitory effect on glycolysis in glioma cells.2.Whole-transcriptome sequencing of the GBM#P3 cell line showed significant changes in key glycolysis-related genes,and positive correlation was found between changes in the activity of key glycolytic enzymes and changes in the activity of the PI3K/AKT/mTOR pathway.3.Upstream molecular analysis of the pathway showed that TNC is a regulatory molecule of the PI3K/AKT/mTOR signaling pathway and is significantly overexpressed as an oncogene in glioma,with expression significantly downregulated in glioma cells after NC action.4.In vitro experiments showed that when TNC was knocked down in glioma cells,PI3K/AKT/mTOR pathway activity was also decreased in glioma cells,while glycolysis was also inhibited.Conclusions1.Nitidine chloride inhibits glycolysis in glioma cells.2.In glioma cells,the PI3K/AKT/mTOR signaling pathway has a regulatory role in glycolysis,and the relationship between the two is positively correlated.3.Tenascin-C is highly expressed in glioma cells and high expression of TNC in gliomas is associated with poor survival prognosis of patients.4.TNC could be a potential target to inhibit glycolysis in glioma cells.