Inhibition Of Metabotropic Glutamate Receptor 5 Affects The Viability Of Hypoxia-Induced Glioma Cell

Gliomas,as the most frequent primary brain tumor,have rapid progression and dismal prognosis of less than 1-year median survival.Hypoxia is prevalent in the microenvironment of gliomas with changed metabolism.However,it impedes radiotherapy,as oxygen is required to generate free radicals to destroy tumor cell,and chemotherapy,as it reduced drug uptake,stability and penetration into tumors.Therefore,hypoxia in the glioma microenvironment needs to be addressed for better treatment.At present,there are many molecular targeted drugs for glioma clinical research,such as imatinib,gefitinib and bevacizumab.However,signal transduction pathways are complicated,and there are interactions among different pathways.Single targeted drug therapy has limited effect on malignant glioma.The combination of complementary targeted drugs,targeted drugs and cytotoxic drugs such as temozolomide,and radiotherapy will be the key to improve the efficacy.Accumulating evidences indicate that,aside from neuro-modulation and maintenance of cellular homeostasis of the central nervous system,mGluRs are expressed in other types of tissues and play other functional roles,such as cancer.mGluRs are G-protein-coupled receptors mostly expressed in the central nervous system,which function in neuronal excitability and synaptic plasticity,as well as modulate the feedback inhibition of neurotransmitter release.mGluR family has three groups according to their sequence homology,pharmacology and associated second messenger signaling pathways.Group I mGluR signaling is mediated by GTP-binding proteins(G-proteins)and downstream second messengers such as cyclic adenosine monophosphate,diacylglycerol and inositol 1,4,5-triphosphate.On the other hand,signaling pathways of group II and group III mGluRs are mostly mediated by Gi/oproteins,which can liberate the G?? subunits to modulate activity of adenylyl cyclase,ion channel and other downstream signaling molecules.The aim of this study was to observe the effects of metabolic glutamate receptor type I(mGluR 1 and mGluR 5)on the death/survival of human glioma cells in different environments and to explore possible mechanisms.In the first part of this study,we observed the effects of antagonistic/ agonistic metabolic glutamate receptor type I(mGluR 1 and mGluR 5)on the survival rate of human glioma cells under normal and hypoxic conditions.In the second part of this study,we further clarified the reason why inhibiting metabolic glutamate receptor 5(mGluR5)reduces the survival rate of human glioma cells under hypoxia,and further explored the related signaling pathways and their effects on cell survival rate and mitochondrial oxidative function.In the third part of this study,we collected some clinical cases and carried out related studies to clarify the expression level of metabolic glutamate receptor 5(mGluR5)in human glioma cells.Part one The effect of group I mGluR on human glioma cell viability under normoxia or hypoxia conditionObjective: To observe the effects of antagonistic/agonistic metabolic glutamate receptor type I(mGluR 1 and mGluR 5)on human glioma cell viability under normal and hypoxic conditions.Methods: We probed their functions using a potent,selective and systemically active mGlu5 antagonist MPEP,mGluR1-specific antagonist CPCCOEt,and group I mGluR(mGluR1 and mGluR5)agonist DHPG in four human glioma cell lines: LNT-229,LNT-308,LNT-428 and G55 under normoxia and hypoxia conditions,respectively for 24 h.We used two assays to evaluate cell viability upon pharmaceutical treatments with and without hypoxia: lactate dehydrogenase(LDH)release assay using the Cytotoxicity Detection Kit(Roche),and using propidium iodide(PI)uptake staining followed by BD Canto II flow cytometry.Results: We found that under normoxia,treatment with MPEP,CPCCOEt or DHPG had no effect on the viability of LNT-229,LNT-308,LNT-428 or G55 cells.Under hypoxia,treatment with MPEP induced significantly more LDH release than control,CPCCOEt or DHPG,as evidenced by decreased cell viability of LNT-229,LNT-308,LNT-428 and G55 cells.Under normoxia,treatment with MPEP,CPCCOEt or DHPG had no effect on the PI staining of LNT-229,LNT-308,LNT-428 and G55 cells.Under hypoxia,treatment with MPEP induced significantly more PI staining than control,CPCCOEt or DHPG,indicated by decreased cell viability of LNT-229,LNT-308,LNT-428 and G55 cells.Summary: These results indicated that selective antagonizing mGluR5 facilitated glioma cell death under hypoxia but not normoxia,whereas antagonizing mGluR1 or stimulating mGluR1 and mGLuR5 didn't affect the glioma cell viability.Part Two The reduced viability of hypoxia-induced glioma cell under inhibition of metabotropic glutamate receptor 5 relates AktObjective: To understand why inhibition of mG luR5 under hypoxia increased cell death,we tried to explore the related signaling pathways involved and their effects on glioma cell viability and mitochondrial oxidative function.Methods: We used RT-PCR to evaluate expression of genes related to mitochondrial oxidative function in LNT-229 cells after 8h MPEP or CPCCOEt treatment at normoxia condition or hypoxia condition.We examined expression of peroxisome proliferator-activated receptor gamma coactivator(PGC-1a),peroxisome proliferator-activated receptor beta coactivator(PGC-1b),cytochrome b(MT-CYB),oestrogenic-related receptor-a(ERR-a),cytochrome c oxidase subunit 1(MT-CO1)and Cytochrome c oxidase subunit 2(MT-CO2),synthesis of cytochrome c oxidase 2(SCO2),ATP synthase Fo subunit C(ATP5G1)in LNT-229 cells by real-time PCR using the CFX96 Real-Time PCR Detection System(Bio-Rad)and SYBR Premix Ex Taq II(2 ×)(Tli RNaseH Plus,TaKaRa).Housekeeping gene succinate-dehydrogenase complex subunit A(SDHA)was used as a loading control.We normalized expression of mRNAs to that of the control using the comparative threshold cycle(2-? CT)method.We assessed the activation of these two pathways by measuring ERK and Akt phosphorylation in hypoxic LNT-229 cells with 8h treatment of MPEP,CPCCOEt and DHPG.We lysed the harvested LNT-229 cells treated for 8 hours with MPEP,DHPG,CPCCOEt or control under hypoxia condition with radioimmunoprecipitation buffer containing a phosphatase and protease inhibitor cocktail.We boiled the lysates in sodium dodecylsulfate(SDS)loading buffer,used SDS-PAGE to separate proteins,and then transferred it to nitrocellulose membrane for blocking with 5% nonfat milk and Tween-20 in phosphate-buffered saline at room temperature for 1 h.We incubated the membrane with primary antibodies as a loading control.We used secondary antibodies(1:10,000)from Bio-Rad and detected the antigen–antibody complexes using enhanced chemiluminescence.We measured cell viability and mitochondrial oxidative function with MPEP and Akt agonist SC79 after 24 h.Results: We found that under hypoxia,treatment with MPEP increased expression of PGC-1a,PGC-1b,ERR-a,SCO2 and ATP5G1 compared tocontrol and CPCCOEt treatment,whereas expression of MT-CO1,MT-CYB and MT-CO2 were not affected.We found that in hypoxic LNT-229 cells,MPEP,CPCCOEt or DHPG treatment didn't affect the ERK phosphorylation ratio,while MPEP treatment significantly decreased Akt phosphorylation compared with control,CPCCOEt or DHPG.We found that in hypoxic LNT-229 cells,MPEP-induced elevation in LDH release and PI staining were suppressed by SC79 co-application.We also observed that Akt activation suppressed the MPEP-enhanced expression of mitochondrial oxidative function related genes.Summary: These results indicated antagonizing mG luR5 promoted expression of some mitochondrial oxidative genes.Antagonizing mG luR 5 depressed Akt phosphorylation.mGluR5 inhibition promoted hypoxia-inducedglioma cell death via deactivating Akt.Part Three Expression of Metabolic Glutamate Receptor 5 in Human Glioma CellsObjective: In order to clarify the expression level of metabolic glutamate receptor 5 in human glioma cells,we collected some clinical cases and carried out related studies.Methods: We collected 27 patients with direct diagnosis and treatment from June 2018 to December 2018 in Neurosurgery of the Second Hospital of Hebei Medical University.All primary gliomas and paracancerous tissues were collected from surgical specimens of Neurosurgery of the Second Hospital of Hebei Medical University.No radiotherapy or chemotherapy was received before operation.All specimens of glioma were confirmed by pathological diagnosis.The content of this study was examined and approved by the Ethics Committee of the Second Hospital of Hebei Medical University.All the selected patients signed the informed consent form for biological specimens.The expression of metabolic glutamate receptor 5 in cancer tissues and adjacent tissues(1 cm away from the edge of the tumor)was observed by Westem blot.Results: The expression of metabolic glutamate receptor 5 in cancer tissues was higher than that in adjacent tissues.Western blot was used to detect the expression of metabolic glutamate receptor 5 in cancer tissues and adjacent tissues.We found that the expression of metabotropic glutamate receptor 5 in cancer tissues of three patients was higher than that in adjacent tissues(P <0.05).Summary: Metabolic glutamate receptor 5 has been detected in human glioma cells and is highly expressed compared with adjacent tissues,making it a potential suitable biomarker for glioma under certain types or specific conditions.Conclusion: 1.When we inhibited mGluR5 using its antagonist MPEP,we found thatcell viability under hypoxia was reduced.2.mGluR5 inhibition was correlated with increased expression of genes involved in mitochondrial respiratory function,such as PGC-1a,PGC-1b,ERR-a,SCO2 and ATP5G1.Our data indicated inhibition of mGluR5 didn't affect ERK protein level or phosphorylation,but promoted Akt phosphorylation/activation in the hypoxic glioma cells.We further tested the effect of Akt activation by its agonist SC79 on mGluR5 inhibition and glioma cell viability as well as mitochondrial gene expression.The results showed that co-application of SC79 reversed the MPEP inhibition on mGluR5-induced elevation of LDH release,PI staining and expression of mitochondrial genes.Therefore it seems that Akt phosphorylation functions downstream of mGluR5 inhibition to regulate cell viability under hypoxia.3.mGluR5 was also found in vitro in our current study that it was detected at high expression level,in glioma cells,making it a potentially suitable biomarker for gliomas of certain type or in certain condition.