Activated SIRT1 Contributes To Deoxypodophyllotoxin--Induced Glioma Cell Parthanatos

Background and Objective:Parthanatos is a programmatic cell death pathway that relies on the hyperactivation of poly(ADP-ribose)polymerase 1(PARP1).The excessive activation of PARP1 leads to the depletion of nicotinamide adenine dinucleotide(NAD+)and the generation of poly(ADP-ribose)(PAR)polymers.These PAR polymers can induce the release of apoptosis-inducing factor(AIF)from mitochondria,for ming a complex with macrophage migration inhibitory factor(MIF)that enters the cell nucleus.MIF,functioning as a nucleotidase,can degrade DNA,thereby resulting in cell death.Parthanatos plays a significant role in various tumor cell death processes and has emerged as a promising research field in malignant tumor therapy.Silent information regulator 1(SIRT1)is a highly conserved deacetylase primarily located in the cell nucleus.Literature reports suggested that activation of SIRT1 could promote programmed cell death processes such as autophagic cell death,apoptosis,and necroptosis.However,in the context of parthanatos,SIRT1 was often believed to inhibit the occurrence of parthanatos due to its deacetylation effect on PARP1.Nevertheless,the activation of SIRT1 depleted cellular NAD+,reducing its levels.Although NAD+ could enhance SIRT1 activation,the enzymatic activity of SIRT1 did not solely rely on NAD+.Some studies indicated that NAD+ depletion could contribute to the accumulation of reactive oxygen species(ROS)and DNA double-strand breaks(DSBs),which is one of the classical pathways for PARP1 activation.Thus,the deacetylation role of SIRT1 and its ability to induce DNA double-strand breaks through NAD+ depletion appear to present a certain contradiction regarding PARP1 activation.Therefore,further research is needed to investigate the role of SIRT1 in parthanatos and its underlying mechanisms.Deoxypodophyllotoxin(DPT)is a lignan compound with biological properties such as anti-inflammatory and anti-tumor effects.Our previous studies have demonstrated that DPT exerts cytotoxic effects on glioblastoma cells,including inducing excessive activation of PARP1,generation of PAR polymers,and triggering parthanatos.To further investigate the mechanism of action of SIRT1 in glioma cell parthanatos,we conducted in-depth studies using U87,U251,U118 glioma cell lines,and a subcutaneous xenograft model in nude mice.Methods: 1.The lactate dehydrogenase release assay was employed to measure the cell death rate induced by DPT and FK866 in glioma cells.2.The expression levels of SIRT1,NOX2,NAT10,and JNK were knocked down using si RNA.3.The neutral comet assay was employed to analyze DNA double-strand breaks induced by DPT.4.The NADPH oxidase activity assay kit was utilized to measure changes in intracellular NADPH oxidase activity after DPT treatment.5.The NAD+/NADH assay kit was used to measure changes in intracellular NAD+ levels after DPT and FK866 treatment.6.The DCFH-DA probe was used to detect the impact of DPT and FK866 treatment on intracellular ROS levels.7.Immunofluorescence staining was performed to observe the changes in γ-H2 AX and NAT10 in the cell nucleus after DPT treatment using confocal microscopy.8.Co-immunoprecipitation(Co-IP)analysis was conducted to exa mine the interaction between NAT10 and PARP1 and the acetylation level of PARP1.9.Western blotting was employed to measure the expression levels of SIRT1,pSIRT1(Ser27),PARP1,PAR,γ-H2 AX,p-ATM,NOX2,NAT10,ASK1/JNK,and other proteins in glioma cells after DPT treatment.Combine Co-IP analysis to study protein-protein interactions.10.U87 cells were subcutaneously implanted into BALB/c nude mice to establish an in vivo model.DPT was ad ministered via intraperitoneal injection for therapeutic purposes.The effects of DPT on glioma tumor growth were observed,and hydrogen peroxide levels,NAD+ levels,expression levels of various proteins,and their interactions in glioma tissues were measured.Results: 1.The previous research conducted by our research group had confirmed that DPT induced excessive activation of PARP1 and parthanatos in glioma cells.Subsequent investigations revealed that DPT induced a time-dependent upregulation of SIRT1 and p-SIRT1(Ser27)within the cell nucleus.Inhibition of SIRT1 with EX527 or SIRT1 si RNA not only suppressed DPT-induced cell death in glioma cells but also inhibited the upregulation of SIRT1,p-SIRT1(Ser27),PARP1,and PAR accumulation.Conversely,treatment with the SIRT1 activator SRT2183 had the opposite effect.These findings suggest that DPT can induce the upregulation of SIRT1 and phosphorylation at the Ser27 site in glioma cells,thereby promoting the activation of PARP1 and the occurrence of parthanatos.2.EX527 inhibited the comet tail formation induced by DPT and the upregulation of p-ATM and γ-H2 AX,while the use of SRT2183 showed the opposite effect.This indicates that SIRT1 can exacerbate DPT-induced DNA double-strand breaks.Furthermore,after DPT treatment,the acetylation level of PARP1 showed a timedependent increase.EX527 inhibited the DPT-induced increase in PARP1 acetylation level,while SRT2183 further enhanced the acetylation level of PARP1.This suggests that SIRT1 enhances the DPT-induced acetylation of PARP1.3.DPT induced an increase in intracellular ROS levels and enhanced NADPH oxidase activity.Additionally,DPT also promoted an upregulation of NOX2 expression in the cytoplasm.The inhibition of NOX2 using GSK2795039 or NOX2 si RNA not only suppressed cell death and ROS accumulation induced by DPT but also inhibited the upregulation of p-ATM,γ-H2 AX,activation of PARP1,and increase in PAR levels.This suggests that the upregulation and activation of NOX2 in DPT-induced parthanatos in glioma cells can exacerbate DNA double-strand breaks by promoting ROS generation,thereby promoting the activation of PARP1.Furthermore,EX527 inhibited the accumulation of ROS and the increase in NADPH oxidase activity induced by DPT.It also inhibited the upregulation of NOX2 expression and downregulation of p-AKT caused by DPT,while SRT2183 showed the opposite effect.This indicates that SIRT1 promotes the upregulation and activation of NOX2.4.DPT induced an increase in NAT10 expression in the nucleus of glioblastoma cells.The inhibition of NAT10 using remodelin or NAT10 si RNA suppressed cell death and the activation of PARP1 induced by DPT.This suggests that the upregulation and activation of NAT10 promote the activation of PARP1 induced by DPT.Furthermore,under the influence of remodelin,the interaction between NAT10 and PARP1 as well as the acetylation level of PARP1 induced by DPT were inhibited.This indicates that the activation of NAT10 enhances the acetylation of PARP1 induced by DPT.Further investigations revealed that EX527 inhibited the upregulation of NAT10 and p-CREB expression induced by DPT,while SRT2183 had the opposite effect.This suggests that SIRT1 can enhance the expression of NAT10 by promoting the phosphorylation of CREB.5.DPT treatment resulted in intracellular NAD+ depletion.FK866 exacerbated the NAD+ depletion and cell death induced by DPT.It also enhanced the expression and acetylation level of PARP1,while exogenous NAD+ had the opposite effect.This suggests that NAD+ depletion promotes the activation of PARP1 induced by DPT.Furthermore,FK866 also promoted ROS accumulation and increased NADPH oxidase activity induced by DPT.It further enhanced the downregulation of p-AKT and the upregulation of NOX2,NAT10,and p-CREB expression induced by DPT.It also increased the interaction between NAT10 and PARP1.On the other hand,exogenous NAD+ exhibited the opposite effects compared to FK866.Additionally,the activation of SIRT1 was found to cause NAD+ depletion.This suggests that SIRT1 promotes the activation and upregulation of NOX2 and NAT10 induced by DPT through NAD+ depletion.Further simulation experiments confirmed that NAD+ depletion can enhance the upregulation and activation of NOX2 and NAT10.6.DPT treatment caused an upregulation of p-JNK in the cells.The JNK inhibitor SP600125 or JNK si RNA could inhibit cell death,activation of PARP1,and increase of PAR induced by DPT.This indicates that JNK promotes the activation of PARP1 and parthanatos induced by DPT.Additionally,SP600125 and JNK si RNA also suppressed the upregulation of SIRT1,p-SIRT1(Ser27),NOX2,and NAT10,as well as the downregulation of acetyl-p53 induced by DPT.They also inhibited the NAD+ depletion induced by DPT.This suggests that the activation of JNK promotes the upregulation and phosphorylation at Ser27 of SIRT1,thereby increasing its activity,in the process of parthanatos in glioma cells.7.DPT treatment resulted in an upregulation of ASK1,p-ASK1(T845),and pJNK in the cytoplasm,while GSK2795039 and NOX2 si RNA inhibited the expression of these proteins.This indicates that NOX2 promotes the activation of the ASK1/JNK pathway induced by DPT.Additionally,exogenous NAD+ and EX527 were able to attenuate the upregulation of ASK1,p-ASK1(T845),and p-JNK caused by DPT,while FK866 and SRT2183 had the opposite effect.This suggests that NAD+ depletion,dependent on SIRT1,enhances the activation of the ASK1/JNK signaling pathway by DPT through NOX2.8.DPT effectively inhibits the growth of glioma tissue in vivo while inducing an increase in NADPH oxidase activity,elevated H2O2 levels,and NAD+ depletion within the tissue.The expression of various proteins in glioma tissue and their interactions observed after DPT treatment are consistent with the results obtained from in vitro experiments.Conclusion: 1.In glioma cells,DPT induces the upregulation of SIRT1 expression and phosphorylation at the Ser27 site,leading to the activation of SIRT1.This activation further promotes the activation of PARP1 and the occurrence of parthanatos.2.Activation of SIRT1 leads to NAD+ depletion,and NAD+ depletion exacerbates DPT-induced DNA double-strand breaks and PARP1 acetylation,thereby promoting PARP1 activation.3.NAD+ depletion plays two roles in DPT-induced glioma cell parthanatos:(1)NAD+ depletion upregulates and activates NOX2,promoting ROS generation and DNA double-strand breaks;(2)NAD+ depletion upregulates and activates NAT10,enhancing PARP1 acetylation levels.4.During DPT-induced glioma cell parthanatos,the activation of JNK promotes the upregulation of SIRT1 expression and phosphorylation at the Ser27 site,increasing its activity.SIRT1-dependent NAD+ depletion upregulates and activates NOX2,and the ROS produced by NOX2 can activate the ASK1/JNK signaling pathway,for ming a positive feedback loop between SIRT1 and JNK.