The Study On The Mechanism Of BmK CT Inhibitis Glioma Cell Proliferation

Cancer cell proliferation and survival are associated with metabolic remodeling.As a hallmark of cancer,metabolic remodeling has been central to the study following Otto Warburg's pioneering work on aerobic glycolysis.Glioma is one of the most common intracranial tumors associated with signifivant morbidity and mortality.It has been regarded as a difficult problem in clinical treatment.A chlorotoxin-like toxin in the venom of the scorpion Buthus martensii Kirsch(BmK CT)exhibits a significant anti-glioma effect,but the underlying mechanism remains unclear.In this study,we used metabonomics to explore molecular mechanism of BmK CT inhibits glioma proliferation.First,the recombinant protein GST-BmK CT was expressed and purified,then the secondary structure was analyzed using CD spectra.The data showed that the proteins had a-helix structure and thermal stability.MTT assays were used to examine the proliferation of glioma cells.We identified 63 metabolites using GC-MS,and the metabolic profiling analysis confirmed that GST-BmK CT significantly down-regulated the level of pyruvate acid and up-regulated the levels of glutamic acid and glutamine in glioma compared with GST-treated cells.Pyruvate kinase M2(PKM2),a key enzyme regulating aerobic glycolysis,is overexpressed in many human cancers.Here,we showed that BmK CT inhibited expression of PKM2.Our results indicated that BmK CT inhibited the proliferation of glioma cells by down-regulating PKM2-mediated aerobic glycolysis.But according to the glucose consumption,aerobic glycolysis can't decrease ATP level by 87.66%.Our results indicated that GST-BmK CT binded to MMP-2 and ?v?3 complex on the surface of glioma cells,inhibited PI3K/AKT signaling pathway,and then induced downstream cascade reactions including up-regulation of SIRT4 and down-regulation of HIF-1?.Thereby GST-BmK CT inhibited the transcriptional expression and enzyme activity of GLUD1,a key enzyme regulating glutamic acid into a-ketoglutarate.At the same time,BmK CT also reduced the transcriptional of y-GCS,which is a rate-limiting enzyme for glutathione synthesis.BmK CT promoted apoptosis by releasing cytochrome C and up-regulating expression of caspase-9/caspase-3.The main results are as follows:1.The fusion protein GST-BmK CT was expressed and purified based on the results of previous laboratory experiments.The secondary structure and thermal stability of GST-BmK CT were determined by CD spectra at pH 6.5 and pH 7.4,respectively.MTT assays confirmed that GST-BmK CT inhibited the proliferation of glioma cells in a concentration-dependent manner and time-dependent manner.2.Typical GC-MS total ion chromatograms(TIC)of the U251 cells treated with PBS,GST,GST-BmK CT,respectively.A total of 63 metabolites were analyzed.PCA and HCA analyses confirmed that BmK CT increased the level of glutamic acid and glutamine and decreased the content of pyruvate in glioma cells.In addition,the pathway impact and enrichment analyses revealed that glioma was linked to glutamate metabolism.Meanwhile,HPLC showed that BmK.CT reduced the content of glutamate.3.The expression of PKM2 and HIF-1? was detected by qPCR and Western blot.The results showed that BmK CT decreased the expression of PKM2 by decreasing HIF-1?,and inhibited the aerobic glycolysis of glioma.4.Immunocytochemical analysis revealed that MMP-2,integrin-?v?3 and GST-BmK CT could interact on glioma membrane(U251,U87 and A172).The levels of PI3K/AKT-associated proteins including PI3K,AKT and AKT phosphorylation were investigated using western blot analysis.The results demonstrated that in GST-BmK CT treated glioma,PI3K and phosphorylation of AKT were signifcantly decreased.5.Using qPCR and enzyme activity detection,it was found that BmK CT decreased transcription level and catalytic activity of GLUD1.Previous studies have confirmed that the transcriptional level of GLUD1 is positively correlated with HIF-1?,and the activity of GLUD1 is negatively correlated with SIRT4.We validated this conclusion by western blot and qPCR,respectively.6.q-PCR results showed that BmK CT inhibited the transcription of y-GCS and increased the ROS levels in cells.Flow cytometry results showed that BmK CT promoted apoptosis of glioma.However,GST-BmK CT and NAC-treated(a ROS scavenger)glioma reversed cell apoptosis.The levels of apoptosis-associated proteins were investigated using western blot analysis.The results demonstrated that the levels of cytochrome C,cleaved caspase-9 and cleaved caspase-3 were increased in GST-BmK CT-treated glioma compared with GST-treated group.