SIRT1 Sensitizes Glioma Cells To Ferroptosis Via Induction Of NAD+ Depletion-dependent Activation Of ATF3

Background: Ferroptosis is a form of programmed cell death caused by iron-dependent lipid peroxidation accumulation.Its main biological characteristic is the oxidation of polyunsaturated fatty acids on the cell membrane by reactive oxygen species(ROS)in the presence of iron ions,forming toxic lipid peroxides,destroying the cell membrane,and causing cell death.Inducing ferroptosis to kill tumor cells is a novel cancer treatment strategy.Activating Transcription Factor 3(ATF3)as a member of the ATF/CREB transcription factor family can participate in the occurrence of ferroptosis by inhibiting the transcription of SLC7A11 and GPX4,and ATF3 can be activated by the endoplasmic reticulum stress pathway,but it is not yet clear whether other factors are involved in the activation of ATF3.Sirtuin 1(SIRT1)is a deacetylase that removes acetyl amino acid residues from target proteins such as P53,PARP1 by consuming NAD+.It plays a variety of important roles in cell biology,such as inflammation,metabolism,oxidative stress,and apoptosis.Activated SIRT1 protects non-tumorous cells against various ferroptosis-inducing factors,such as inhibiting heat shock-induced ferroptosis in lung epithelial cells,and ischemia/hypoxia-induced ferroptosis in neuronal cells.Mechanistically,SIRT1-mediated deacetylation inhibits ferroptosis through two pathways: one is deacetylation of P53 to inhibit P53-dependent downregulation of SLC7A11,and the other is deacetylation of NRF2 to drive NRF2-mediated upregulation of GPX4.However,activated SIRT1 adversely affects tumor cells through multiple pathways.Literature reports that SIRT1 promotes pyroptosis in breast cancer cells induced by metformin through enhancing NF-κB nuclear translocation;SIRT1 aggravates autophagic cell death in lung cancer cells induced by quercetin through activating AMPK;SIRT1 also promotes PARP1-dependent death in glioma cells induced by deoxypodophyllotoxin through upregulating the acetyltransferase NAT10.Therefore,the role and mechanism of SIRT1 in glioma cell ferroptosis require further investigation.As a classic ferroptosis inducer,RSL3 triggers ferroptosis by specifically inhibiting Glutathione Peroxidase 4(GPX4).RSL3,as a compound that can specifically induce ferroptosis,has potential value in the research and development of new cancer treatment drugs.Our group’s previous research found that RSL3 can cause glioma cell death by activating lethal autophagy,showing certain killing effects on glioma cells both in vivo and in vitro.Therefore,further exploring the role and mechanism of RSL3-induced ferroptosis in glioma cells not only provides references for the study of the molecular mechanism of ferroptosis but also offers a theoretical basis for the clinical application of RSL3.Objective: Using U87,U251,and U118 human glioma cell lines and a subcutaneous xenograft tumor model in nude mice,the role and mechanism of SIRT1 in the ferroptosis of glioma cells induced by RSL3 were investigated.Methods: 1.Detection of changes in the levels of ferrous ions in glioma cells induced by RSL3 and the effect of inhibitors such as DFO,FAC,SRT2183,EX527 on these changes using the Ferro-Orange fluorescence probe;2.Determination of the degree of lipid peroxidation in glioma cells induced by RSL3 and the effect of various inhibitors on this process using malondialdehyde content assay kits;3.Measurement of the extent of glioma cell death induced by RSL3 and the effect of inhibitors like Fer-1,NAD+,SRT2183 on this process through lactate dehydrogenase release assays;4.Examination of changes in the levels of proteins such as SLC7A11,GPX4,transferrin,transferrin receptor,SIRT1,ATF3,AROS in glioma cells treated with RSL3 and the impact of various inhibitors on these changes via Western Blotting;5.Studying the effect of knocking down the expression levels of SIRT1,ATF3,and AROS with siRNA on the changes in ferroptosis-related indicators of glioma cells induced by RSL3;6.Observing the impact of RSL3 on the localization and expression levels of SIRT1,ATF3,and AROS proteins in glioma cells under a laser confocal microscope after immunofluorescence staining;7.Detecting changes in reactive oxygen species levels in glioma cells treated with RSL3 and the effect of the inhibitor NAC on these changes after DCFH-DA probe staining;8.Measuring the degree of cysteine consumption in glioma cells induced by RSL3,SRT2183 or FK866 and the effect of various inhibitors on this process;9.Assessing the degree of glutathione depletion in glioma cells induced by RSL3,SRT2183 or FK866 and the effect of various inhibitors on this process;10.Evaluating the extent of NAD+ depletion in glioma cells induced by RSL3 or SRT2183 and the effect of various inhibitors on this process;11.Establishing a subcutaneous xenograft tumor model in nude mice by subcutaneous injection of U87 human glioma cells,intraperitoneal injection of RSL3 to observe the effect of RSL3 on tumor cell growth,and assessing changes in related indicators within tumor tissues using western blotting,malondialdehyde content assay,NAD+ content assay,divalent iron ion content assay,hydrogen peroxide content determination,cysteine content measurement,and glutathione content measurement.Results: 1.RSL3 induces ferroptosis in glioma cells;(1)RSL3 induces an upregulation of ferrous ions and the lipid peroxidation product MDA in glioma cells;(2)Pre-treatment of cells with iron chelators and lipid peroxidation inhibitors can alleviate glioma cell death caused by RSL3,transferrin and transferrin receptor upregulated after RSL3 treatment in glioma cells,suggesting RSL3 induces ferroptosis in glioma cells.2.SIRT1 promotes RSL3-induced ferroptosis in glioma cells;(1)SIRT1 expression is upregulated in glioma cells treated with RSL3.Pre-treatment with the SIRT1 activator SRT2183 significantly enhances the expression of SIRT1,transferrin,transferrin receptor increased by RSL3 and the cell death caused by RSL3,indicating SIRT1 promotes ferroptosis in glioma cells by regulating ferrous ion uptake;(2)Knocking down SIRT1 expression with siRNA or inhibiting SIRT1 activity with EX527 significantly inhibits the expression increase of SIRT1,transferrin,transferrin receptor induced by RSL3 and glioma cell death caused by RSL3,further proving SIRT1 promotes ferroptosis in glioma cells induced by RSL3.3.SIRT1 promotes the downregulation of GPX4 and SLC7A11 expression levels induced by RSL3;(1)RSL3 induces a decrease in the expression levels of GPX4 and SLC7A11 in glioma cells;(2)Pre-treatment of cells with the SIRT1 activator SRT2183 significantly enhances the downregulation of GPX4 and SLC7A11 expression levels induced by RSL3,while pre-treatment with the SIRT1 inhibitor EX527 or knocking down SIRT1 expression with siRNA significantly inhibits this downregulation,suggesting SIRT1 promotes RSL3-induced ferroptosis in glioma cells by suppressing GPX4 and SLC7A11 expression levels;(3)Treatment of glioma cells with a cell death-inducing dose of SRT2183(40μmol/L)leads to the upregulation of SIRT1 expression and the downregulation of GPX4,SLC7A11,and Acetyl-P53 expression,as P53 becomes inactivated after deacetylation by SIRT1,further proving that activated SIRT1 can downregulate GPX4 and SLC7A11 protein levels,not via the P53 pathway.4.SIRT1 induces the downregulation of GPX4 and SLC7A11 through NAD+ depletion;(1)SRT2183 exacerbates the decline in NAD+ levels caused by RSL3,while EX527 inhibits this decline,indicating RSL3 reduces NAD+ levels by activating SIRT1;(2)FK866 exacerbates cell death and NAD+ level reduction after RSL3 treatment,while NAD+ supplementation inhibits both,suggesting RSL3 induces glioma cell death by lowering NAD+;(3)Western blotting shows FK866 exacerbates the downregulation of GPX4 and SLC7A11 induced by RSL3,while NAD+ inhibits it.Exogenous NAD+ supplementation can inhibit the downregulation of GPX4 and SLC7A11 induced by SRT2183 and the reduction in cysteine and glutathione levels,further confirming that activated SIRT1 suppresses GPX4 and SLC7A11 expression through NAD+ depletion.5.NAD+ depletion dependent on SIRT1 is the cause of ATF3 activation;(1)RSL3 induces the downregulation of GPX4 and SLC7A11 expression through ATF3 activation;(2)FK866 enhances the upregulation of ATF3 induced by RSL3,while exogenous NAD+ inhibits this upregulation.Prolonging the action time of FK866 to further reduce the intracellular NAD+ level could induce the up-regulation of ATF3 expression,which is significantly inhibited by pre-treatment with exogenous NAD+,indicating NAD+ depletion is the upstream signal for ATF3 activation;(3)SRT2183 promotes the upregulation of ATF3 expression induced by RSL3,while EX527 inhibits it.SRT2183 treatment significantly increases ATF3 expression,but this increase is significantly inhibited by co-incubation with EX527 or exogenous NAD+,proving SIRT1 promotes ATF3 expression through NAD+ depletion.6.AROS is involved in SIRT1 activation induced by RSL3;(1)AROS expression is upregulated in glioma cells treated with RSL3;(2)The upregulation of SIRT1 induced by RSL3,along with the downregulation of its target protein acetylated P53 and its substrate NAD+,can be alleviated by knocking down AROS with siRNA,suggesting RSL3 induces SIRT1 activation by upregulating AROS.7.Reactive oxygen species are involved in AROS-dependent SIRT1 activation;(1)Pre-treating glioma cells with the antioxidant NAC not only inhibits the increase in intracellular reactive oxygen species(ROS)levels caused by RSL3 but also suppresses the upregulation of AROS、SIRT1 and the downregulation of acetylated P53 expression,suggesting that RSL3 upregulates AROS to activate SIRT1 by increasing ROS levels;(2)Pre-treating glioma cells with the antioxidant NAC not only inhibits the increase in intracellular reactive oxygen species(ROS)levels caused by hydrogen peroxide but also suppresses the upregulation of AROS 、 SIRT1 and the downregulation of acetylated P53 expression,indicating that the increase in reactive oxygen species levels induces SIRT1 activation dependent on AROS.(3)SRT2183 exacerbates cell death,SIRT1 upregulation,and acetylated P53 downregulation caused by hydrogen peroxide,while EX527 inhibits these effects.indicating that SIRT1 is activated by hydrogen peroxide.Knocking down AROS with siRNA similarly inhibits cell death,SIRT1 upregulation,and acetylated P53 downregulation caused by hydrogen peroxide,further verifying that hydrogen peroxide-induced SIRT1 activation depends on AROS up-regulation.8.RSL3 can inhibit the growth of subcutaneously transplanted gliomas in nude mice.In the subcutaneous tumor tissues of nude mice treated with RSL3,the levels of related proteins,ferrous ions,lipid peroxidation product malondialdehyde,cysteine,GSH,hydrogen peroxide content,and NAD+ showed changes consistent with in vitro experiments compared to the control group.Conclusion 1.RSL3 can cause the upregulation of AROS expression by increasing the level of reactive oxygen species(ROS)inside glioma cells,with the upregulated AROS further activating SIRT1.2.RSL3 induces SIRT1 activation leading to NAD+ depletion,which promoting ferroptosis in glioma cells.3.NAD+ depletion is the upstream signal for ATF3 activation,with SIRT1-dependent NAD+ depletion inducing ATF3 nuclear translocation,which further inhibits the activity of SLC7A11 and GPX4.4.During the process of ferroptosis in glioma cells,an increase in ROS levels can cause the upregulation of AROS expression.High levels of AROS activate SIRT1,leading to NAD+ depletion and further activation of ATF3,which suppresses the expression levels of SLC7A11 and GPX4,making glioma cells more sensitive to ferroptosis.