The Effects And Mechanisms Of NOX4-mediated ROS On Honokiol-induced Pyroptosis In Glioma Cells

Background:Glioma is the most common primary intracranial tumor which is prone to metastasis and has a poor prognosis.Clinical data have shown that the average survival of glioblastoma patients was less than one year^[21].Although in recent years,the diagnosis,surgery,radiotherapy and chemotherapy of glioma have improved,the final therapeutic effect is still not satisfactory^[22].Glioma is not sensitive to radiotherapy and temozolomide chemotherapy because of the resistance to apoptosis^[23].Therefore,inducing programmed cell death such as pyroptosis in glioma cells may be an effective therapeutic strategy.Pyroptosis is an inflammatory programmed cell death characterized by membrane perforation,cell swelling,cell lysis and release of inflammatory cytokines^[24].Apoptosis is mainly regulated by caspase and gasdermin family.In canonical pyroptosis,NOD-like receptors(NLR)activated by inflammatory stimuli can bind and activate pro-caspase-1.The activated caspase-1 further cleaves GSDMD and the cleaved GSDMD aggregates on cell membrane to induce membrane perforation and the release of inflammatory factors,eventually leading to cell death^[24].NLRP3 is a well-studied NLR,which can be activated by a variety of stimuli such as viral RNA,potassium efflux,ROS and Ca²⁺to induce pyroptosis^[25].In non-small cell lung cancer,simvastatin activates NLRP3/caspase-1-dependent cell pyroptosis and has tumor-suppressive effects^[10].However,during the NSCLC pyroptosis caused by Polyphyllin VI,the intracellular ROS and oxidative stress play a key role in the activation of NLRP3/caspase-1 pathway^[26].NOX4 belongs to the NADPH oxidase family,and its main function is to catalyze the reduction of oxygen to superoxide anions,which is one source of intracellular ROS^[27].Coxsackievirus B3 increases intracellular ROS level through activation of NOX4 and ultimately induces apoptosis of cardiomyocytes^[28].NOX-induced ROS accumulation plays an important role in the regulation of programmed cell death.Recently,it has also been reported that NOX4 plays an important role in inducing NLRP3/caspase-1-dependent pyroptosis^[29,30].Honokiol,a natural compound isolated from Magnolia officinalis,has shown anti-tumor properties in a variety of human tumor cells^[31].Honokiol is able to cross the blood-brain barrier,contributing to its application in nervous system disease^[12].Honokiol has been reported to inhibit proliferation and invasion of glioma cells^[32].However,the specific mechanism of the glioma cell death caused by honokiol remains to be further explored.Objectives:U87,U251 and xenograft nude mouse models were used to investigate whether honokiol can induce cell pyroptosis and its mechanism.Besides,the role of NOX4 and ROS in honokiol-induced cell death was further explored.Methods:1.MTT and LDH assays were used to examine the viability and mortality of cells.2.DCFH-DA probe was used to evaluate the level of intracellular ROS.3.NADPH oxidase activity detection kit was used to evaluate NADPH oxidase activity in glioma cells.4.GKT137831,NAC,MCC950,VX765 were used to investigate the role of NOX4,ROS,NLRP3,caspase-1 in honokiol-induced glioma pyroptosis.5.siRNA of NOX4,NLRP3 and caspase-1 were used to investigate the role of NOX4,NLRP3 and caspase-1 in honokiol-induced glioma pyroptosis.6.The mice model of U87 xenograft glioma was used to investigate the effect of honokiol on glioma cells in vivo.7.Western Blot was used to detect the expression level of NOX4,NLRP3,caspase-1,GSDMD,IL-1? and IL-18 in glioma cells and xenografts.8.ELISA was used to detect the release of IL-1? and IL-18 from glioma cells.Results;1.Honokiol resulted in decreased survival and increased mortality in glioma cells,with little effect on normal liver cells.Honokiol resulted in NADPH oxidase and ROS increasing in glioma cells,and the increase of IL-1? and IL-18 in culture medium.Western Blot proved that honokiol activated NOX4,NLRP3,caspase-1,GSDMD,IL-? and IL-18 in glioma cells.2.VX765 and siRNA of caspase-1 decreased cell death induced by honokiol,suppressing GSDMD and IL-1?.ELISA proved that VX765 decreased the release of IL-1? and IL-18 induced by honokiol.3.MCC950 and siRNA of NLRP3 decreased cell death induced by honokiol,suppressing caspase-1,GSDMD and IL-1?.4.NAC decreased cellular ROS and cell death induced by honokiol,suppressing NLRP3,caspase-1,GSDMD and IL-1?.NAC decreased cellular ROS and cell death induced by hydrogen peroxide,suppressing NLRP3,caspase-1,GSDMD and IL-1?.5.GKT137831 and siRNA of NOX4 decreased NADPH oxidase activity,ROS and cell death induced by honokiol,suppressing NLRP3,caspase-1 and IL-1?.6.Honokiol increased NADPH oxidase activity and suppressed the growth of xenograft.Western Blot proved that NOX4,NLRP3,caspase-1,GSDMD,IL-1? and IL-18 were activated by honokiol in xenograft.Conclusions:1.Honokiol induced glioma pyroptosis in vivo and vitro.2.Honokiol can activate NOX4 to increase the content of ROS,and eventually lead to glioma cell death.3.Pyroptosis induced by honokiol can help to avoid the problem of drug resistance caused by apoptosis tolerance,which has clinical application value.