| Background:Parthanatos,also known as poly（ADP-ribose）polymerase 1（PARP1）-dependent cell death,is a type of non-apoptotic programmed cell death initiated by PARP1overactivation and characterized by apoptosis inducing factor（AIF）-dependent DNA degradation.After being activated upon occurrence of DNA double strand breaks（DSBs）,nuclear PARP1 utilizes nicotinamide adenine dinucleotide（NAD+）as substrate to synthesize cytotoxic PAR polymers,which then translocate into cytoplasm and target mitochondria to induce AIF translocation from mitochondrial to nuclei.Within nuclei,AIF and microphage migration inhibitory factor（MIF）cause irreversible DNA degradation and cell death after being recruited to the location of DSBs.Thus,nuclear translocation of AIF is a key step to complete the final stage of parthanatos.Although PAR polymers are regarded as the executioner to cause nuclear translocation of AIF during the process of parthanatos,it remains unclear whether activated PARP1 induces AIF translocation from mitochondria to nuclei via regulation of other signals.Mitochondrial respiratory chain complex Ⅰ is a protein complex responsible for acquiring electrons by oxidizing NADH to NAD+and transferring these electrons to Co Q.It is also a primary site where electrons leak into mitochondrial matrix and bind with molecular oxygen to form superoxide.Given that mitochondrial superoxide is a crucial factor to cause nuclear translocation of AIF,activated complex Ⅰ might play a role in promoting AIF translocation from mitochondria to nuclei.TAX1BP1（Tax1 binding protein 1）is a protein highly and specifically expressed in brain and has dual roles in modulating the destinies of cancer cells.On one hand,it inhibits the genesis and development of hepatocarcinoma,and reduces the drug resistance of pancreatic cancer cell.On the other hand,it contributes to breast and gastric cancer cells’resistance to chemotherapeutic agent.However,the role of TAX1BP1 in glioma remains unclear.As a protein mainly located in cytoplasm,TAX1BP1 was found to localize at mitochondria with decreased membrane potentials and accumulated superoxide,but it is unclear of its role in regulating mitochondrial respiratory complex Ⅰ activity.Deoxypodophyllotoxin（DPT）is not only a natural lignan with multiple pharmacological effects such as anti-inflammation,antivirus and anti-allergy,but also could induce parthanatos in glioma cells.However,the underlying mechanism accounting for its induction of parthanatos is still needed to be clarified.The study of the regulatory mechanism of parthanatos induced by DPT can not only provide reference for the molecular mechanism of parthanatos,but also provide theoretical basis for the clinical application of DPT.Objective:Human U251,U87 MG and U118 MG glioma cells as well as xenograft nude mouse models were used to investigate the role of TAX1BP1 in DPT-induced glioma cell parthanatos and its underlying mechanism.Methods:1.Lactate dehydrogenase release assay was used to determine the degree of glioma cell death induced by DPT and the effects of various inhibitors on DPT-induced cell death.2.Western blotting was used to detect the effects of DPT on the levels of TAX1BP1,AIF,PARP1,PAR,mitochondrial respiratory chain complex Ⅰ subunits and DSBs-related protein levels as well as the effects of various inhibitors on the above changes.3.Laser confocal microscopy was used to observe the effects of DPT on the localization and expression levels of TAX1BP1,AIF and ND1 in glioma cells after immunofluorescence staining.4.Small interfering RNA（si RNA）was used to knock down the expression levels of TAX1BP1,PARP1 and ND1 in order to examine their effects on DPT-induced biochemical changes in glioma cells.5.Flow cytometry combined with JC-1 staining was used to detect the changes of mitochondrial membrane potential in cells treated with DPT and the effects of various inhibitors on DPT-induced mitochondrial membrane potential changes.6.Fluorescence microscopy was used to observe the changes of mitochondrial superoxide levels in cells treated with DPT and the effects of various inhibitors on DPT-induced changes after Mito SOX probe staining.The fluorescence intensity values of each group were measured by microplate reader.7.Fluorescence microscopy was used to observe the changes of ROS levels in cells treated with DPT and the effects of various inhibitors on DPT-induced changes after DCFH-DA probe staining.The fluorescence intensity values of each group were measured by microplate reader.8.The mitochondrial respiratory chain complex Ⅰ activity assay kit was used to detect the changes of NADH oxidation rate in glioma cells treated with DPT and the effects of various inhibitors on DPT-induced changes.9.Co-immunoprecipitation was performed to detect DPT-induced changes in the interaction between ND1 and ND2,NDUFS2 as well as NDUFS4.10.Seahorse XF-24 energy metabolism analyzer was used to detect the change of mitochondrial oxygen consumption rate in order to reflect the oxidative phosphorylation ability of mitochondria in DPT-treated cells.11.RT-q PCR was used to detect TAX1BP1 m RNA levels in cells treated with DPT or transfected with TAX1BP1 si RNA.12.The NAD+assay kit was used to detect intracellular NAD+level changes in DPT-treated cells,and the effects of various inhibitors on DPT-induced changes.13.Human U87 MG glioma was xenografted in BALB/c nude mice to build subcutaneous tumor model,and DPT was injected intraperitoneally to observe its effects on the changes of subcutaneous tumor volume.Western blotting,NAD+assay kit and complex Ⅰ activity assay kit were used to detect the changes of parthanatos-related indexes in tumor tissues,and H2O2 assay kit was used to detect the ROS levels in tumor tissues.Results:1.TAX1BP1 contributed to DPT-induced nuclear translocation of AIF（1）DPT triggered expressional upregulation of PARP1 and PAR,mitochondrial depolarization as well as AIF nuclear translocation,indicating that DPT triggered parthanatos in glioma cells.（2）DPT increased transcription of TAX1BP1,and boosted TAX1BP1translocation from cytoplasm to mitochondria.Knockdown of TAX1BP1 with si RNA inhibited DPT-induced mitochondrial depolarization,AIF nuclear translocation and glioma cell death,indicating that TAX1BP1 contributed to DPT-induced glioma cell parthanatos by regulating AIF nuclear translocation.2.TAX1BP1 contributed to AIF nuclear translocation by boosting mitochondrial superoxide accumulation.（1）DPT triggered mitochondrial superoxide accumulation in glioma cells.Mn TBAP,which was a mitochondrial superoxide inhibitor,could inhibit DPT-induced mitochondrial superoxide accumulation,mitochondrial depolarization,AIF nuclear translocation and cell death,indicating that superoxide contributed to DPT-induced AIF nuclear translocation.（2）Knockdown of TAX1BP1 with si RNA inhibited DPT-induced mitochondrial superoxide accumulation,indicating that TAX1BP1 contributed to AIF nuclear translocation by promoting mitochondrial superoxide accumulation.3.TAX1BP1 boosted mitochondrial superoxide accumulation by activating mitochondrial respiratory chain complex Ⅰ.（1）DPT enhanced mitochondrial respiratory chain complex Ⅰ activity,triggered expressional upregulation of ND1,ND2 and promoted complex Ⅰ assembly in glioma cells.Inhibition of complex Ⅰ activity by rotenone or ND1 si RNA inhibited DPT-induced complex Ⅰ activity enhancement,mitochondrial superoxide accumulation and AIF nuclear translocation,indicating that the increased complex Ⅰ activity could promote AIF nuclear translocation by causing superoxide accumulation.（2）Knockdown of TAX1BP1 with si RNA inhibited the increased complex Ⅰ activity in oxidation of NADH,expressional upregulation of ND1,ND2 and increased complex Ⅰ assembly caused by DPT,indicating that TAX1BP1 promoted superoxide accumulation by activating complex Ⅰ.（3）Knockdown of TAX1BP1 with si RNA mitigated DPT-induced expressional downregulation of mitochondrial catalase and GPX4,indicating that TAX1BP1promoted superoxide accumulation by downregulation of catalase and GPX4.4.PARP1 promoted DPT-induced TAX1BP1 translocation to mitochondria in glioma cells.（1）Inhibition of PARP1 by 3-AB or PARP1 si RNA not only suppressed DPT-induced expressional upregulation of PARP1 and PAR,but also prevented DPT-induced TAX1BP1 mitochondrial translocation,indicating that PARP1 activation triggered mitochondrial translocation of TAX1BP1.（2）Inhibition of PARP1 by 3-AB or PARP1 si RNA inhibited DPT-induced expressional upregulation of ND1 and ND2,complex Ⅰ activity enhancement and mitochondrial superoxide accumulation,indicating that PARP1 was an upstream signal of TAX1BP1 and could regulate TAX1BP1-induced complex Ⅰ activation and superoxide accumulation.5.The NAD+depletion induced by PARP1 activation promoted DPT-induced mitochondrial translocation of TAX1BP1 in glioma cells.（1）DPT triggered NAD+depletion in glioma cells,which could be prevented by3AB,indicating that DPT caused PARP1-dependent NAD+depletion in glioma cells.（2）The NAD+depletion,TAX1BP1 mitochondrial translocation,expressional upregulation of ND1 and ND2,complex Ⅰ activity enhancement,mitochondrial superoxide accumulation caused by DPT were inhibited by exogenous supplement of NAD+,but exacerbated when NAD+regeneration was suppressed by FK866.6.Mitochondrial superoxide reversly reinforced DPT-induced PARP1 activation by inducing ROS-dependent DNA double strand breaks.（1）DPT triggered ROS accumulation,which was mitigated by Mn TBAP,indicating that mitochondrial superoxide promoted DPT-induced ROS accumulation.（2）DPT triggered expressional upregulation of p-ATM,p-H2AX,PARP1 and PAR,which could be mitigated by Mn TBAP,indicating that mitochondrial superoxide promoted PARP1 activation by causing ROS-dependent DNA double strand breaks.（3）Inhibition of complex Ⅰ activity by rotenone or knockdown of ND1 with si RNA could inhibit DPT-induced ROS accumulation,DNA double strand breaks as well as PARP1 activation,indicating that the mitochondrial superoxide accumulation triggered by complex Ⅰ could promote PARP1 activation by causing ROS-dependent DNA double strand breaks.7.DPT inhibited the growth of tumor volume in subcutaneous tumor-bearing nude mice,and the changes of NAD+level,complex Ⅰ activity,ROS level,TAX1BP1and AIF levels in tumor tissues were all consistent with in vitro experiments.Conclusion1.The overactivated PARP1 induced by DPT causes NAD+depletion in glioma cells,which boosts TAX1BP1 translocation from cytoplasm to mitochondria.2.TAX1BP1 contributes to DPT-induced AIF nuclear translocation via inducing mitochondrial superoxide accumulation by activation of mitochondrial respiratory chain complex Ⅰ activity.3.The increased mitochondrial superoxide triggered by TAX1BP1 reversely reinforced PARP1 activation by inducing ROS-dependent DNA double strand breaks.4.TAX1BP1 acts as a downstream signal of activated PARP1 to trigger nuclear translocation of AIF by activation of mitochondrial respiratory chain complex Ⅰ. |
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