The Ketogenic Microenvironment Enhances The Effect Of Temozolomide Against Glioma And Its Related Mechanism

Background: High expression of MGMT plays an important role on glioma’s resistance to temozolomide(TMZ).In recent years,studies have shown that Wnt/β-catenin and NF-κB pathway are related to MGMT-dependent TMZ resistance of glioma.The ketogenic diet has been shown to have a strong anti-glioma effect in experimental and clinical studies,but its effect on enhancing TMZ’s potency against glioma has been relatively less studied.Previous studies have been shown that the ketogenic diet could regulate NF-κB and Akt pathways,but its effect on MGMT is less studied.Objective: To explore the effect of the ketogenic microenvironment on glioma cells’ viability,and under this basis,to further investigate whether chemo-resistance to TMZ of glioma cells would be improved under the ketogenic microenvironment and its underlying mechanism.Methods: CCK8 assay was used to detect the effect of different concentrations of β-hydroxybutyric acid(BHB)on the viability of glioma cells and human umbilical vein endothelial cells(HUEVCs)in high and low glucose environments.The effects of the ketogenic microenvironment on glioma cell viability were detected by CCK8 assay and colony formation assay.The effects of the ketogenic microenvironment on the cell cycle distribution and apoptosis rate of glioma cells were detected by PI/RNase and Annexin V-FITC/PI double staining.Western blot was used to detect the effects of the ketogenic microenvironment on cell cycle related proteins and apoptosis-related proteins.CCK8 assay and colony formation assay were used to detect the effect of TMZ combined with the ketogenic microenvironment compared with TMZ alone on cell activity.The changes of apoptosis ratio and mitochondrial membrane potential of glioma cells were detected by Annexin V-FITC/PI and JC10 Kit.The expression levels of key protein in the pathways associated with TMZ resistance in glioma were detected by western blot.The changes of migration ability of glioma cells treated by the ketogenic microenvironment combined with TMZ versus TMZ alone were investigated by Wound Healing Assays and Western blot.Results: 1.High concentration of BHB inhibited the viability of glioma cells in both low and high glucose environments.In the high glucose environment,low concentration of BHB had no significant effect on the viability of glioma cells,while in the low glucose environment,low concentration of BHB slightly promoted the activity of glioma cells LN18 and T98 G,but had no significant effect on glioma cells A172 and U118.At the same BHB concentration,the activity of glioma cells was significantly inhibited in the low glucose environment compared with the high glucose environment.HUVECs were used to evaluate the effect of the ketogenic microenvironment on normal cells.The viability of HUVECs was not significantly inhibited in the low glucose environment,which was equivalent to the high glucose environment.2.CCK8 assay and colony formation assay demonstrated that the ketogenic microenvironment could inhibit the activity of glioma cells.The ketogenic microenvironment led to the cell cycle arrest of glioma cells in G0/G1 phase,and the expression levels of G1-related proteins Cyclin D1,CDK4 and CDK6 were significantly decreased.The ketogenic microenvironment induced apoptosis of glioma cells,and the apoptosis rate of glioma cells and the apoptotic related proteins cleaved PARP-1 and cleaved Caspase-3 increased with the prolonging of treatment time,while it had no significant effect on the apoptosis ratio of HUVECs cells.3.CCK8 assay and colony formation assay demonstrated that the ketogenic microenvironment combined with TMZ enhanced the inhibition of glioma cell viability by TMZ,and the IC50 value of TMZ significantly decreased in all glioma cell lines.Compared with TMZ monotherapy,the apoptosis rate of glioma cells was significantly increased,the mitochondrial membrane potential was significantly decreased,and the expression levels of apoptotic related proteins cleaved PARP-1,cleaved Caspase-3,and Bax/Bcl-2 were significantly up-regulated.4.TMZ monotherapy up-regulated the expression of MGMT protein in glioma cells,also increased the expression of phosphorylated Akt,phosphorylated GSK3β,β-catenin and phosphorylated p65 protein.The ketogenic microenvironment combined with TMZ administration inhibited the expression of MGMT protein in glioma cells,and the expression of phosphorylated Akt,phosphorylated GSK3β,β-catenin,phosphorylated p65 protein and Bcl-2 were also down-regulated.5.The ketogenic microenvironment combined with TMZ significantly inhibited the migration ability of glioma cells,and western blot results showed that the expression levels of MMP9 and Vimentin were further down-regulated by the combined therapy compared with TMZ alone.Conclusion: 1.The ketogenic microenvironment inhibited the activity of glioma cells,but did not significantly inhibit the activity of human umbilical vein endothelial cells.The ketogenic microenvironment induced G0/G1 cell cycle arrest in glioma cells.The ketogenic microenvironment induced the apoptosis of glioma cells in a time-dependent manner,but had no significant pro-apoptotic effect on human umbilical vein endothelial cells.2.The ketogenic microenvironment combined with temozolomide enhanced the inhibition of glioma cell viability by temozolomide,promoted the induction of glioma cell apoptosis by temozolomide,and further weakened the migration ability of glioma cells.3.The drug resistance of gliomas to temozolomide may be related to the activation of Wnt/β-catenin and NF-κB pathways,and the ketogenic microenvironment may improve the drug resistance of gliomas to temozolomide by inhibiting the Wnt/β-catenin and NF-κB pathways as well as the expression of the downstream MGMT and anti-apoptotic protein Bcl-2.