The Nrf2/PGC1? Pathway Regulates Anti-Oxidant And Proteasomal Activity To Alter Cisplatin Sensitivity In Ovarian Cancer

Objective:Ovarian cancer is the most deadly gynecological malignancy,and drug resistance has become a major challenge in its treatment in recent years.Resistance to chemotherapy in tumor cells is related to multiple factors,including the repair of DNA damage,blockade of drug-induced apoptosis,reactive oxygen species(ROS)-mediated redox state,and protein degradation.Current research suggests that exploring the interaction between different signals can shed light on the mechanisms of tumor drug resistance.Proteasomal activity plays important roles in tumorigenesis and chemotherapy resistance,and abnormally elevated proteasome levels are found in a variety of cancers.The proteasome can eliminate oxidative and damaged proteins to reduce oxidative stress damage,and enhanced anti-oxidant capacity can promote the expression of 20S and 19S proteasome subunits.These findings suggest the existence of an interaction between proteasomal activity and redox function,and exploring this relationship will be more helpful in solving the problem of chemotherapy resistance in ovarian cancer.The ubiquitin-proteasome system(UPS)maintains cell homeostasis by regulating proteins involved in signal transduction and cell cycle pathways Inhibition of the proteasome can cause the accumulation of pro-apoptotic proteins and induce tumor cell apoptosis.However,recent clinical studies found that some tumors are not sensitive to proteasome inhibitors.Proteasome inhibitor-resistant tumors generally have higher expression levels of anti-oxidant genes.The nuclear factor E2-related factor 2(Nrf2,gene name NEF212)-mediated anti-oxidant stress pathway was identified as a resistance mechanism in the proteasome inhibitor-resistant phenotype.Nrf2 can regulate the transcriptional activity of proteasome mature protein,which can promote resistance to proteasome inhibitors.This suggests that Nrf2-mediated anti-oxidant activity may be related to proteasome-mediated tumor resistance.Nrf2 is an important element of the anti-oxidant response element(ARE)transcription complex,and it can regulate the expression of various protective genes.Nrf2 and anti-oxidant-related gene expression is elevated in drug-resistant tumor cells,and this mainly occurs through the dissociation of Nrf2 and Kelch-like ECH-associated protein 1(Keapl),so that Nrf2 enters the nuclear regulatory resistance Transcription of the oxidase gene.In addition,the decreased activity of glycogen synthase kinase 3?(GSK3?)can also lead to the dissociation of Nrf2 from?-transducin repeat-containing protein(?-TrCP),thereby reducing Nrf2 degradation via the ?-TrCP-mediated proteasome pathway.Peroxisome proliferator-activated receptor-? coactivator la(PGCla,gene name PPARGC1A)can also coordinate the expression of multiple anti-oxidant genes to protect cells against oxidative stress damage.PGCla can coactivate Nrf2 to induce anti-oxidant gene expression in an aging disease model,and studies have confirmed the protein-protein interaction between them.Therefore,the mechanism by which PGC1? and Nrf2 interact may play a synergistic role in anti-oxidation,but it is unclear whether their interaction is related to drug resistance.In addition,the maintenance of mitochondrial redox homeostasis plays an important role in proteasome activity.Mitochondria are the main sources of ROS,its impaired function can lead to excessive ROS production can increase the burden on the UPS?Nrf2 can inhibit mitochondrial ROS production by upregulating Heme Oxidase(HO-1)and Superoxide Dismutase 2(SOD2),and can interact with a variety of mitochondrial proteins to regulate its protein stability and structural integrity,including PGC1? and PGC1?.These results suggest that the homeostasis of mitochondrial function mediated by Nrf2 and PGCla also regulates the redox homeostasis and thus maintains proteasome activity.In this study,the "UPS-Anti-oxidation Axis" was taken as the entry point to explore the roles of PGCla and Nrf2 in the regulation of anti-oxidant and mitochondrial functions in maintaining proteasome activity.The findings provide new ideas for reversing drug resistance in ovarian cancer.Methods:1.To investigate the effect of proteasome inhibition on the sensitivity of A2780 and SKOV3 cells to cisplatinCells were treated with different doses of the proteasome inhibitor Epox or 100 nM Epox combined with different doses of cisplatin for 24 h,the cell viability was measured by MTT assay2?To investigate the effect of proteasome inhibition on nuclear expression of Nrf2 in A2780 and SKOV3 cellsCells were treated with 100nM Epox for 24 h.The expression and nuclear expression of Nrf2 were measured by Western blot and immunofluorescence assay;the colocalization of Keapl and Nrf2 was detected by immunoprecipitation and immunofluorescence3?To investigate the effect of proteasome inhibition on the redox fuction and apoptosis activity of A2780 and SKOV3 cells? Cells were treated with 100 nM Epox for 12 h.RT-qPCR was used to detect the mRNA expression of anti-oxidant related genes and PPARGC1A;DCFH-DA staining flow cytometry was used to detect the total intracellular ROS level? Cells were treated with 100 nM Epox for 24 h.AnnexinV/PI staining flow cytometry was to detecte the apoptosis;Western blot was used to detecte the protein expression of PGC1?4?To explore the interaction between Nrf2 and PGCla in SKOV3 cells?Cells were treated with 100 nM Epox for 12 h or transfected with overexpressing Nrf2 plasmid.The co-localization of Nrf2 and PGCla in nucleus was determined by staining and observed by fluorescence microscope;the promoter activity of PGCla was determined by dual-luciferase reporter assays;the mRNA expression of NEF212 was determined by RT-qPCR? Cells were treated with ZLN005(a PGC1? transcription activator)for 24 h or transfected with PGCla-shRNA plasmid.Western blot was used to detect the expression of Nrf2 in the nucleus and the expression of p38?GSK3? and phosphorylation levels of p38 and GSK3?;the colocalization of Keap1 and Nrf2 was detected by immunoprecipitation;the expression of NEF212 mRNA was determined by RT-qPCR.5?To investigate the effect of proteasome inhibition or overexpression of Nrf2 onmitochondrial function of SKOV3 cellsSKOV3 cells were treated with 100 nM Epox for 12 h or transfected with overexpressing Nrf2 plasmid.JC-1 or Mitotracker Red staining flow cytometry was used to detect the mitochondrial membrane potential or the number of mitochondrial;the oxygen consumption(OCR)and ATP level were detected by corresponding assay kits;RT-qPCR was used to detect the mitochondrial gene copy numbers;Western blot was used to measure the expression of mitochondrial repiratory chain protein6?To investigate the effect of PGC1? knockdown combined with Epox on thesensitivity of SKOV3 cells to apoptosisSKOV3 cells were transfected with Scr-shRNA and PGCla-shRNA plasmid,and/or treated with 100nM Epox for 12 h or 24 h.JC-1 staining flow cytometry was used to detect the mitochondrial membrane potential;AnnexinV/PI staining was used to evaluate the apoptosis fraction and apoptosis proteins were analyzed by western blotResults:1?MTT resluts showed that,compared with A2780 cells,SKOV3 cells were less sensitive to cisplatin and the proteasome inhibitor Epox.Epox can significantly enhance the sensitivity of A2780 cells to cisplatin,but not SKOV3 cells2?The immunoprecipitation and immunofluorescence results showed decreased colocalization of Keapl and Nrf2 after Epox treatment,which promoted Nrf2 localization to the nucleus3?Evaluation of cell redox levels revealed that,Epox significantly reduced the mRNA expression of anti-oxidant genes and PPARGC1A in A2780 cells,increased ROS production and promoted apoptosis,while in SKOV3 cells showed no significant changes4?Detection of the interaction between PGCla and Nrf2 revealed that Epox enhanced colocalization of Nrf2 and PGCla in the nucleus of SKOV3 cells.Epox or overexpression of Nrf2 markedly upregulated the transcriptional activity of PGC1?After ZLN005 treatment for 24 h,nuclear Nrf2 expression was increased through the reduced colocalization of Keap1 and Nrf2,and the increased phosphorylation of p38 and GSK3?.Furthermore,the gene expression of NEF212 was also increased after ZLN005 treatment.However,transfection with PGCla-shRNA plasmids had the opposite effect5?Detection of mitochondrial function-related indicators and found that,Epox treatment or Nrf2 overexpression enhanced the mitochondrial membrane potential(MMP,??m),the number of mitochondria,the ATP levels,mitochondrial gene copy numbers,the oxygen consumption rate(OCR)and the protein expression of mitochondrial respiratory chain in SKOV3 cells6?Apoptosis results showed that transfection with PGCla-shRNA plasmid reduced the mitochondrial membrane potential,increased the rate of apoptosis and the expression of apoptosis-related proteins of SKOV3 cells.The above resluts were further exacerbated when combined with EpoxConclusions:1.Epox cannot enhance the sensitivity of SKOV3 cells to cisplatin.It is suggested that SKOV3 cells may develop multidrug resistance including proteasome inhibitors2.The increased nuclear expression of Nrf2 induced by Epox treatment enhanced anti-oxidant levels and PGCla gene expression in SKOV3 cells.This suggested that the Nrf2/PGC1? pathway may be involved in the regulation of anti-oxidant signaling pathway induced by proteasome inhibition3.Epox treatment or Nrf2 overexpression can enhance the mitochondrial function,while inhibition of PGCla can promote Epox-induced SKOV3 cells apoptosis.This suggested that the Nrf2/PGCla pathway also maintains redox homeostasis by regulating mitochondrial function,and thus participate in drug resistance regulation of SKOV3 cells.