MitoQ Induces Ferroptosis By Regulating NCOA4-FTH Signaling Pathway

Background:Ferroptosis,a non apoptotic form of cell death observed in cancer cells with oncogenic Ras mutations,is morphologically,biochemically and genetically distinct from other forms of cell death.It is typically characterized by massive accumulation of free iron and unrestricted lipid peroxidation,which can be specifically rescued by iron death inhibitors or iron chelators.Since the discovery of ferroptosis,the molecular mechanism and its potential applications have become a hot research topic.Although the major pathway of ferroptosis is well established,the specific molecular mechanisms underlying this regulatory network remain unknown.Ferroptosis generally results from inhibition of the cystine/glutamate retromer(System XC~-)or loss of glutathione(GSH)peroxidase 4(GP X4)activity.System XC~-is thus critical for cystine import,sustaining de novo synthesis of glutathione,an important nonenzymatic antioxidant in the cell.Inducing ferroptosis in cancer cells is a potential anticancer therapeutic strategy.Therefore,mechan isms that trigger ferroptosis,such as inhibition of system XC~--GSH-GPX4 system,may provide new therapeutic avenues to treat cancer or reverse cancer drug resistance.As a widely used mitochondria targeted antioxidant,mitoquinone(Mito Q)largely accumulates in mitochondria and can slow down oxidative stress damage to mitochondria and activate cellular energy.Previous studies by our group also demonstrated that Mito Q ameliorates CISD3 related ferroptosis by regulating mitophagy and mitochondrial dynamics processes.However,Mito Q may also have negative effects,and the mechanisms underlying its negative roles in tumor development and ferroptosis remain unclear.Purpose:This topic is mainly to investigate the related specific mechanism of ferroptosis induced by Mito Q,aiming to investigate the possible negative effect of Mito Q as an antioxidant in mitochondria,and to investigate the molecular mechanism by which ferroptosis occurs and provide a theoretical basis for the clinical application of Mito Q.Methods:Cell survival was assessed using CCK-8 Kit;The cell proliferation ability was detected by the flat cloning assay;The levels of intracellular lipid peroxides,cellular ROS,intracellular free iron,mitochondrial morphology,intramitochondrial ROS,and mitochondrial membrane potential were assessed by staining with C11-BODIPY,DCF-DA,RPA,mito Tracker Red,mito SOX Red,and TMRM probes,respectively;Autophagic flux was next confirmed using m RFP-GFP-LC3 adenovirus;Immunoblotting experiments were performed to detect the protein expression levels in the cells.Results:Cell proliferation assay showed that Mito Q induced cell death and inhibited cell proliferation in a conc entration dependent manner;At the same time,using confocal fluorescence microscopy with fluorescent probes,we found that Mito Q promoted the accumulation of intracellular lipid peroxides,cellular reactive oxygen species,as well as free iron.Meanwhile,both ferroptosis chelators and autophagy inhibitors(DFO and Ba FA1)could rescue this phenomenon.In addition,increasing doses of Mito Q resulted in severely impaired mitochondrial morphology,highly accumulated mitochondrial reactive oxygen species,and depolarized mitochondrial membrane potential.On the other hand,Mito Q was also found to downregulate the m TOR/P70S6K signaling pathway by activating the phosphorylation of AMPK to induce autophagy,which was confirmed by m RFP-GFP-LC3 adenovirus.Finally,the present study confirmed that Mito Q induced iron death possibly through regulation of the NCOA4-FTH signaling pathway by knockdown of NCOA4and overexpression of FTH.Conclusion:Mito Q may induce the occurrence of ferroptosis by regulating t he NCOA4-FTH signaling pathway,so Mito Q as a mitochondria targeted antioxidant must be used with caution in the clinic.