P65-PKM2 Mediated Fenofbrate-Induced Inhibition Of Glucose Metabolism In Human Glioblastoma Cells

Research background: Cancer cells use aerobic glycolysis preferentially for energy provision even in the presence of oxygen and this metabolic change is important for tumour growth.NF-?B(p65)-dependent PKM2 upregulation is required for glycolysis and tumorigenesis.Fenofibrate,a fibric acid derivative,is known to possess lipid-lowering effects.In recent years,related studies have shown that fenofibrate has been shown to exert anticancer effects.Recent studies have reported that fenofibrate has exerted strong anti-proliferative,anti-metastatic,and pro-apoptotic effects in various tumors of neuroectodermal origin,including melanoma,medulloblastoma,and glioblastoma.Although fenofibrate treat patients with high cholesterol mainly depending on activation of PPAR?,the anti-tumor of mechanism is still not clear.Yamasaki et al reported that fenofibrate suppresses growth of the human hepatocellular carcinoma cell via PPAR?-independent mechanisms.So far,researches on fenofibrate-induced inhibition of tumor largely confined to the anti-proliferation,invasion and apoptosis.In this study we explored that p65-PKM2 mediated fenofbrate-induced inhibition of glucose metabolism in human glioblastoma cells depending on activation of PPAR?.Methods: 1.CCK8 assay,cell flow cytometry were used to access the proliferation ability and cell cycle in glioma cells treated with fenofibrate.ECAR was also measured by The Seahorse XF Analyzer.2.The expression of p65 was down-regulated by sh-p65 plasmid in human glioma cell line U87 and U251 cells.The effects of proliferation were evaluated by CCK8 assay and cell flow cytometry.3.The expression of p65 was up-regulated by p65 overexpression plasmid in human glioma cell line U87 and U251 cells.ECAR was measured by The Seahorse XF Analyzer.Then Western blot was used to detect the PKM1,PKM2 and Hn RNPs protein level.4.Luciferase reporter assay and Western blot was used to analysis whether PKM is the target of p65.5.Co-immunoprecipitation assays were used to explore the interaction between different types of proteins.6.To test the effects of fenofibrate/p65-PKM2 signaling,we employed a resilience experiment.Results:1.Fenofibrate inhibited cell growth and glucose metabolism in gliomas in PPAR?-dependent manner.2.Abrogating expression of p65 significantly inhibited the growth of glioma cells,and delayed cell cycle at G1 phrase for both U87 and U25 l cells.The concentration of lactate and ECAR was decreased.3.The over-expression of p65 increased the concentration of lactate and ECAR for both U87 and U25 l cells,and the western blot analysis revealed that the expression level of PKM2 was remarkably increased whereas PKM1 was slightly increased.On the other hand,the Hn RNPs protein level was not significantly affected by p65 over-expression.4.PKM2 is positively regulated by p65 at the transcriptional level.The over-expression of p65 enhanced the activity of PKM in the presence of HIF1?.5.The treatment with fenofibrate significantly promotes the interaction of PPAR? and p65 in the cytoplasm.Fenofibrate-mediated activation of PPAR? not only notably suppressed p65 transcriptional activity but also promoted the separation of p65 and HIF1?.Overexpression-p65 significantly reversed fenofibrate-induced inhibition of PKM2 and glucose metabolism.Conclusions:1.Fenofibrate inhibited cell growth and glycolysis depending on activation of PPAR?2.P65-PKM2 mediated fenofibrate-induced inhibition of glucose metabolism in human glioblastoma cells.