The Regulating Effects Of Propofol On Invasion Of Gliomas And ADAR2-CPARs-system X_c~- Pathway

Objective: Glioma,a devastating glial-derived brain tumor,has a poor prognosis and represents a significant health burden.Although various therapies are available,surgery is still the chief approach for managing gliomas.Because brain tumor patients will be exposed to perioperative sedatives and anesthetics,the influence of anesthetics on tumor recurrence is of interest to scientists,and selection of optimal anesthetics is required to reduce any potential patient risk.Ca2+ permeability of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA)receptors is considered to be one of the essential factors for the invasion of glioma.In neurons and astrocytes,AMPA receptors,which are Ca2 + impermeable,are composed of Glu R1-Glu R4 subunits.While the lacking of Glu R2 subunit is crucial for Ca2 + permeability and this form of AMPA receptors are named Ca2 + permeable AMPA receptors(CPARs).Studies indicate that glioma cells express CPARs which can bind to Glu and induce Ca2 + influx,promote cell viability and glioma invasiveness.Adenosine deaminase acting on RNA2(ADAR2)determines the Ca2+ permeability of AMPA receptors through editing pre-m RNA of Glu R2 and changing the construction of AMPA receptors.The other key factor,which effects the Ca2+ influx of CPARs,is glutamate(Glu).Glu can be binding with CPARs and cause Ca2+ influx through CPARs.The cystine/glutamate antiporter system(system xc-)is an important regulator of extracellular Glu level.System xc-imports cystine(Cys-Cys)in exchange for Glu release at 1:1 ratio.In addition,Glu release from gliomas occurs predominantly via system xc-which is highly expressed in glioma cells.Propofol(2,6-diisopropylphenol)is a commonly used intravenous sedative hypnotic.Numerous previous studies indicate that propofol had an anti-tumor effect on several tumor types.Our previous work suggested that propofol was neuroprotective by regulating the ADAR2-AMPA receptors pathway in the ischemia-reperfulion injury mode.Thus,we investigated the effect of propofol on system xc-and discussed any potential relationship between CPARs and system xc-,which are pivotal aspects of invasiveness of glioma cells.Methods: The experiments were divided into two parts.The first part was used to explore the effect of propofol on C6 glioma cells.C6 glioma cells were cultured and divided into 4 groups,control group(group C)was treated normally,propofol groups(P1-P3)were treated with propofol in defferent concentrations(1.2 μg/ml,4 μg/ml,12.4 μg/ml).The cell viability,invasiveness,and migration were measured with MTT assay,transwell invasion assay and scratch assay respectively.The expression of Glu R2 and x CT were analyzed by Western bolt and immunofluorescent staining.In the second part,the role of ADAR2-CPARs-system xc-pathway in the inhibition of propofol on C6 glioma cells was investigated.The N-acetylcysteine(NAC,100 μM),an agonist of system xc-,1-naphthyl acetyl spermine(NAS,100 μM),a specific inhibitor of CPARs,and(R,S)-AMPA(100 μM),an activator of AMPARs were used to explore the effect of propofol on ADAR2-CPARs-system xc-and discuss the relationship between CPARs and system xc-.To illustrate the relationship between ADAR2 and Glu R2,we transfected C6 glioma cells with ADAR2-si RNA which can silence ADAR2 genes.Except for MTT assay,transwell invasion assay,scratch assay,Western blot,glutamate level and reactive oxygen species(ROS)level were also measured.Results: the first part,compared with group C,the cell viability,invasion and migraion were statistically decreased in all propofol groups,the expression of ADAR2 and Glu R2 were upregulated,while the expression of x CT was downregulated(P<0.05).The second part,the inhibition of propofol on glioma could be reversed by the application of an agonist of system xc-NAC and an activator of AMPA receptors(R,S)-AMPA.The transfection with ADAR2-si RNA decreased the editing of Q/R site of Glu R2 pre-m RNA in C6 glioma cells,and the cell viability,invasion and migraion of C6 glioma cells were decreased statistically;meanwhile,the effects of transfection can be reversed by propofol.Excess extracellular glutamate increase of cell viability,invasiveness,and migration of C6 glioma cells,meanwhile propofol could inhibit the promotion via adding excess exogenous glutamate.Conclusion: Our present results suggest that propofol can decrease cell viability,invasiveness,and migration of C6 glioma cells and these anti-glioma effects are related to upregulation of the AMPARs Glu R2 subunit,namely inhibiting CPARs,and downregulating system xc-.We show that propofol can inhibit Glu release via the system xc-and prevent the activation of CPARs as well as confirmed that activity of CPARs could regulate system xc-as an upstream regulator.