Proteasome Inhibitor Epox Promotes P53 Mitochondrial Translocation And Increases Cisplatin Sensitivity In Ovarian Cancer

Background: Surgical resection and cisplatin-based chemotherapy are still the main methods for the treatment of ovarian cancer.However,platinum drugs such as cisplatin still face many problems such as toxic side effects and poor therapeutic effect.Researchers are looking forward to increasing the effect of clinical treatment of ovarian cancer by combining inhibitors of different carcinogenic pathways and improving the anti-tumor activity of chemotherapy drugs such as cisplatin.However,compared with the mechanism of action of single anti-tumor drugs,the mechanism of combination therapy for tumors is still poorly understood.Further researches and exploration of the mechanism of drug combination therapy can provide new clues for the clinical treatment of ovarian cancer.Cellular protein homeostasis is regulated by 2 highly conserved degradation pathways,the ubiquitin-proteasome system(UPS)and macroautophagy/autophagy.By regulating the degradation of proteins,proteasomes play a key role in maintaining protein homeostasis and promoting cell survival.Proteasome inhibitors such as MG132 have been found to reduce the degradation of tumor suppressor protein p53 and thus induce apoptosis.Cisplatin can also induce the expression of p53 protein by damaging cellular DNA,suggesting that the combination of proteasome inhibitors and cisplatin may produce more effective anti-tumor effects.Recent studies have also found that inhibition of proteasomes not only causes protein toxicity,but also causes abnormal protein accumulation leading to mitochondrial dysfunction.taking mitochondrial dysfunction as an entry point may further elucidate the role of p53 in inhibiting proteasome-induced apoptosis.The UPS or autophagy system degrades the aggregation of unfolded or misfolded proteins through ubiquitin chains,and there is a mutual compensatory interaction between the two systems.When the proteasome is inhibited,if the autophagy flux is smooth,it can play a compensatory role to promote cell survival,and when the autophagy flux is blocked,it will aggravate the abnormal protein aggregation of cells,resulting in cell death.The autophagy-related protein p62 with ubiquitin-associated domain(UBA)combines with polyubiquitinated proteins to form protein aggregates through its UBA domain and is considered an important communication node between UPS and autophagy.Our previous research found that p62,which was accumulated by inhibiting autophagy,can form a death platform to promote tumor cell apoptosis by recruiting Caspase-8.Researchers have found that mitochondrial translocation of p53 can lead to cell apoptosis,and targeting mitochondrial p53 is considered to be one of the more effective modes of action in inhibiting tumor cells.Therefore,it is speculated that the proteasome inhibitor Epox in combination with cisplatin may affect mitochondrial p53 translocation through p62,resulting in mitochondrial dysfunction and apoptosis of ovarian cancer cells.The crosstalk between the nucleus and the mitochondria is key to determining the fate of cells.Recent studies have found that ATF5,as a transcription factor,mediates the mitochondrial unfolded protein response(UPRmt)by participating in the regulation of the expression of HSP10,HSP60,HSP70 and CLPP,YMEL1,LONP1 proteases in mitochondrial matrix and anti-apoptotic proteins to coordinate the crosstalk between the nucleus and mitochondria to promote cell survival.Therefore,the role of ATF5 in tumor development and drug therapy has received attention.Although it has been reported that inhibition of proteasomes can induce UPRmt,the relationship between ATF5-mediated UPRmt and cisplatin sensitivity in tumor cells is unclear.The above research results suggest that by exploring the mechanism of targeting ATF5-mediated UPRmt to inhibit the survival of ovarian cancer cells,it may provide a theoretical basis for elucidating the mechanism of proteasome inhibitors combined with chemotherapy drugs to induce apoptosis of ovarian cancer cells.In this study,based on the results of bioinformatics analysis,taking the epithelial ovarian cancer cell line as the research object,we first explored the effect of the selective proteasome inhibitor Epox on the sensitivity of ovarian cancer cells to cisplatin and the expression of mitochondrial p53;Next,we used p62 functional domain mutation technology to determine the effect of p62 UBA and LIR domain mutations on the expression of mitochondrial p53;Finally,we analyzed the changes in expression of nuclear-encoded proteins and the staining results of histopathological tissues in patients with clinical ovarian cancer and elucidated the mechanism of Epox combined with cisplatin to interfere with mitochondrial function through nucleus-mitochondrion crosstalk and induce apoptosis in ovarian cancer cells from the perspective of ATF5-mediated UPRmt,aiming to provide theoretical and experimental basis for drug combination therapy in clinical ovarian cancer.Methods: 1.Analysis of differential gene expression in ovarian cancer tissue chip GSE9891 chip in the GEO database were used to analyze differentially expressed genes in different ovarian cancer tissues:.(1)GO enrichment analysis of differentially expressed genes in different ovarian cancer tissues was performed.(2)KEGG analysis of differentially expressed genes in different ovarian cancer tissues was performed.2.Effect of cisplatin on p53 protein expression and effect of Epox combined with cisplatin on cell viability and mitochondrial function(1)Effect of cisplatin on the expression of p53 protein in ovarian cancer cells and the effect of Epox on the sensitivity of ovarian cancer cells to cisplatin:(1)A2780 cells and SKOV3 cells were treated with different doses of cisplatin for 24 h and cell viability were detected by MTT assay.The two cell lines were treated with 2 μg/m L cisplatin for 3h,6h,12 h and 24 h and the expression of p53 was detected by Western blot to clarify the difference in the expression of p53 in ovarian cancer cells with different cisplatin sensitivity.(2)A2780 cells and SKOV3 cells were treated with different doses of cisplatin alone or in combination with Epox for 24 h,and the cell viability was detected by MTT assay.(2)The effect of Epox combined with cisplatin on mitochondrial function and expression of p53 and OXPHOS complexes subunits:(1)A2780 cells were treated with100 n M Epox and 2μg/m L cisplatin alone or both for 12 h,JC-1 staining and flow cytometry were performed to detect mitochondrial membrane potential;ATP levels were detected by a ATP detection kit;mt DNA copy numbers were detected by RT-q PCR.(2)Mitochondrial proteins were isolated and the expression of POLRMT and OXPHOS complexes subunits proteins were detected by Western blot;m RNA levels of the OXPHOS subunits encoded by mt DNA were detected by RT-q PCR.(3)A2780 cells were transfected with wt-p53 plasmid and the m RNA expression level of the OXPHOS complexes subunits encoded by mt DNA were detected by RTq PCR.3.p62 affects p53 mitochondrial translocation through the UBA domain leading to mitochondrial dysfunction(1)Effects of Epox and cisplatin alone or in combination on the subcellular localization of p62 and p53:(1)A2780 cells were treated with 100 n M Epox and 2μg/m L cisplatin alone or in combination for 12 h,the nuclear and cytoplasmic proteins were isolated,and the expressions of p62 and p53 were detected by Western blot.(2)A2780 cells were treated with Epox and cisplatin alone or in combination for 12 h,the mitochondrial and cytoplasmic proteins were isolated,and the expressions of p62 and p53 were detected by Western blot.(3)Co-localization of p53 and p62 with mitochondria in cells detected by immunofluorescence.(2)Effects of Epox or cisplatin alone or in combination on autophagy flux and aggregation status of p62 and p53 proteins:(1)A2780 cells were treated with 100 n M Epox and 2μg/m L cisplatin alone or in combination for 12 h,autophagy-specific inhibitor chloroquine was added,and changes of autophagy protein LC3,p62 and ubiquitin protein accumulation were detected by Western blot to evaluate whether the autophagy flux was smooth.(2)A2780 cells were treated with Epox and cisplatin alone or in combination for 12 h,the protein was isolated by 1% Triton-SDS,and the expression of p62 and p53 in the soluble components of the protein and the insoluble components were detected by Western blot to confirm the protein aggregation status of p62 and p53.(3)Effects of UBA domian and LIR domain of p62 protein on the expression of p53 mitochondria and OXPHOS complexes and the sensitivity to cisplatin:(1)Wild-type p62(wt-p62),UBA domain truncated mutant p62(ΔUBA)and LIR domain truncated mutant p62(ΔLIR)were constructed and transfected into A2780 cells.After treatment with 2 μg/m L cisplatin for 12 h,mitochondrial proteins were isolated and the expression of mitochondrial p53 were detected by Western blot.(2)A2780 cells were transfected with wt-p62,p62(ΔUBA)and p62(ΔLIR)and then treated with 2μg/m L cisplatin for 12 h.m RNA expression changes of 13 OXPHOS complexes subunits encoded by mt DNA were detected by RT-q PCR.(3)A2780 cells were transfected with wt-p62 and p62(ΔUBA)and treated with 2 μg/m L cisplatin for 24 h and cell viability was detected by MTT assay;A2780 cells transfected with p62(ΔUBA)were treated with 100 n M Epox and 2 μg/m L cisplatin alone or in combination for 12 h,and mitochondiral p53 was detected by Western blot 4.Mechanism of Epox combined with cisplatin inhibiting ATF5-mediated UPRmtinduced apoptosis of A2780 cells(1)Effects of Epox combined with cisplatin on the expression of mitochondrial ATF5 and apoptosis-related proteins in A2780 cells: A2780 cells were treated with 100 n M Epox or 2μg/m L cisplatin alone or in combination for 12 h,mitochondrial and cytosolic proteins were isolated,and the expression of ATF5 and its downstream apoptosis-related proteins BCL-2,MCL-1,BAX were detected by Western blot.(2)Effect of Epox combined with cisplatin on transcriptional activity of ATF5:(1)The nuclear and cytoplasmic proteins were isolated,and the expression of ATF5 in the nucleus was detected by Western blot.(2)The m RNA levels of LONP1,HSP10,HSP60 and mt HSP70 which are downstream of UPRmt mediated by ATF5 were detected by RT-q PCR,and the effect of Epox combined with cisplatin on the transcriptional activity of ATF5 was evaluated.(3)Detection of COXIV,p53,HSP60,YME1L1,ATF5 expression in human ovarian cancer tissue: Mitochondrial content,ATF5,p53,HSP60,YME1L1 protein expression in ovarian cancer tissues were detected by HE staining,immunohistochemistry and immunofluorescence staining in order to verify the relationship between mitochondria with its related regulatory proteins and tumor malignant potential.Results: 1.Analysis of differential gene expression in ovarian cancer tissue chip(1)GO enrichment analysis showed that the differentially expressed genes mainly focus on the biological processes such as the binding activity of ubiquitin like protein ligase,the regulation of DNA replication,the activity of ubiquitin protein transferase.(2)KEGG signaling pathway analysis showed that the differentially expressed genes are mainly enriched in protein digestion and degradation pathways,p53 signaling pathways,PI3K-Akt signaling pathways,Foxo signaling pathways,and cell cycle signaling pathways.2.Effect of cisplatin on p53 protein expression and effect of Epox combined with cisplatin on cell viability and mitochondrial function(1)Effect of cisplatin on the expression of p53 protein in ovarian cancer cells and the effect of Epox on the sensitivity of ovarian cancer cells to cisplatin:(1)MTT assay results showed that A2780 cells were more sensitive to cisplatin than SKOV3 cells.Western blot results showed that under the same dose of cisplatin,the expression of p53 in A2780 cells gradually increased with the extension of cisplatin action time,while the expression level of p53 protein in SKOV3 cells did not change significantly,indicating that the cisplatin sensitivity of ovarian cancer cells was related to the expression of p53.(2)MTT assay showed that compared with SKOV3 cells,Epox combined with cisplatin significantly reduced the cell viability of A2780 cells,suggesting that Epox can significantly increase the cisplatin sensitivity of A2780 cells.(2)The effect of Epox combined with cisplatin on mitochondrial function and expression of p53 and OXPHOS complexes subunits:(1)Mitochondrial function test showed that epox combined with cisplatin led to a significant decrease in mitochondrial membrane potential,a decrease in ATP level and a decrease in mt DNA copy numbers in A2780 cells,which suggests that miitochondrial dysfunction is associated with increased cisplatin sensitivity by inhibiting the proteasome.During Epox treatment,ATP levels decreased,and the expression of OXPHOS complexes subunits m RNA encoded by mt DNA increased.It is suggested that OXPHOS biosynthesis may be one of the compensatory mechanisms for ovarian cancer cell survival.(2)Epox combined with cisplatin resulted in decreased protein levels of the mitochondrial RNA polymerase POLRMT,protein levels of the OXPHOS complexes subunits,and m RNA levels of the OXPHOS complexes subunits encoded by mt DNA.(3)After overexpression of wild-type p53 in A2780 cells,RT-q PCR results showed a significant decrease in m RNA expression of most of the OXPHOS complexes subunits encoded by mt DNA.It is suggested that epox in combination with cisplatin causes mitochondrial dysfunction by inhibiting OXPHOS biosynthesis in A2780 cells,which may be related to p53 3.p62 affects p53 mitochondrial translocation through the UBA domain leading to mitochondrial dysfunction(1)Effects of Epox and cisplatin alone or in combination on the subcellular localization of p62 and p53:(1)Western blot results showed that compared with the Epox group or cisplatin group,the nuclear expression of p53 in the epox combined cisplatin group did not change significantly,suggesting that the nuclear expression of p53 may not be the reason for the combination of Epox and cisplatin to induce apoptosis in ovarian cancer cells.(2)Western blot results showed that compared with the Epox group or cisplatin group,the nuclear expression of p53 in the Epox combined cisplatin group was significantly increased,suggesting that p53 expressed in mitochondria may be the main reason for the combination of Epox and cisplatin to induce apoptosis in ovarian cancer cells(3)Immunofluorescence results showed that Epox combined with cisplatin can increase the co-localization of p53 and p62 with mitochondria respectively.These above results suggest that Epox combined with cisplatin increases the localization of p53 and p62 in mitochondria.(2)Effects of Epox or cisplatin alone or in combination on autophagy flux and aggregation status of p62 and p53 proteins:(1)The addition of chloroquine to the Epox group inhibited autophagy,and the results showed that the expression of LC3,p62 and ubiquitinated proteins increased significantly.It was suggested that Epox could increase the level of autophagy in A2780 cells,and the autophagy flux was smooth.When chloroquine was added in the epox combined with cisplatin group,the expression of LC3 and p62 was down-regulated,suggesting that Epox combined with cisplatin may cause the autophagy flux of A2780 cells to be blocked.(2)Western blot results showed that Epox increased the expression of p53 and p62 proteins in insoluble proteins,while the overall level of ubiquitinated proteins increased,suggesting that Epox may affect subcellular localization of p53 by causing abnormal protein accumulation.(3)Effects of UBA domain and LIR domain of p62 protein on the expression of p53 mitochondria and OXPHOS complex and the sensitivity to cisplatin::(1)Western blot results showed that compared with wild-type p62,the p62 protein UBA and LIR domain mutant group of A2780 cells treated with cisplatin could significantly down-regulate the mitochondrial expression of p53.It is suggested that the UBA domain and LIR domain of p62 may be involved in regulating the expression of p53 in mitochondria.(2)RT-q PCR results showed that the UBA domain mutation of p62 protein upregulated the levels of OXPHOS complexes subunits m RNA,while the mutation of p62 protein LIR domain did not.These results suggest that the UBA domain of p62 may be involved in regulating the expression of the OXPHOS complexes subunits m RNA encoded by mt DNA in ovarian cancer A2780 cells,while the LIR domain is weaker.(3)MTT results showed that mutations in the p62 protein UBA domain led to a decrease in the sensitivity of cisplatin in A2780 cells.Western blot results showed that there was no significant difference in the expression level of p53 between Epox group and Epox combined cisplatin group.After transfection with UBA domain truncated mutants,the combination of Epox and cisplatin did not result in significantly higher mitochondrial expression of p53 than in the Epox group,which further proved that the UBA domain of p62 may be involved in the regulation of p53 mitochondrial translocation.4.Mechanism of Epox combined with cisplatin inhibiting ATF5-mediated UPRmt-induced apoptosis of A2780 cells(1)Effects of Epox combined with cisplatin on the expression of mitochondrial ATF5 and apoptosis-related proteins in A2780 cells: Western blot results showed that Epox and cisplatin combined treatment inhibited mitochondrial expression of ATF5 and anti-apoptotic proteins BCL-2 and MCL-1 in A2780 cells,and increased the BAX/BCL-2 ratio.It is suggested that the apoptosis of ovarian cancer cells induced by Epox combined with cisplatin may be related to the decrease of mitochondrial ATF5 expression.(2)Effect of Epox combined with cisplatin on transcriptional activity of ATF5:(1)Western blot results showed that compared with the Epox group,the expression of ATF5 in the nucleus of A2780 cells in the Epox combined cisplatin group was downregulated to a certain extent.(2)RT-q PCR results showed that the m RNA expression of UPRmt-related genes LONP1,HSP10,HSP60 and mt HPS70 as downstream of ATF5 in the Epox group was up-regulated.Compared with the Epox group,the m RNA expression of LONP1,HSP10,HSP60 and mt HSP70 in the Epox combined with cisplatin group was down-regulated.The above results suggest that Epox combined with cisplatin interferes with mitochondria-nucleus crosstalk by interfering with ATF5-mediated UPRmt,thereby interfering with mitochondrial quality control,inhibiting OXPHOS biosynthesis,inhibiting UPRmt-mediated pro-survival effects and leading to mitochondrial dysfunction,(3)Detection of COXIV,p53,HSP60,YME1L1,ATF5 expression in human ovarian cancer tissue: Analysis of mitochondrial content in ovarian cancer tissues showed that the mitochondrial content of cancerous tissues was higher than that of adjacent tissues,suggesting that ovarian cancer cells need to increase mitochondrial content to maintain their own metabolism and proliferation;The results of immunohistochemical staining and immunofluorescence staining showed that the expression of ATF5,p53,HSP60 and YME1L1 in ovarian cancer tissues was significantly higher than that in paracancerours tissues,and was mainly located in mitochondria.These results indicates that ATF5-mediated UPRmt is related to the malignant potential of ovarian cancer.Conclusions: 1.The cisplatin sensitivity of ovarian cancer is related to the expression of p53 protein,and Epox can significantly increase the cisplatin sensitivity of ovarian cancer A2780 cells.2.p62-mediated p53 mitochondrial translocation leads to mitochondrial dysfunction by affecting the expression of the mitochondrial RNA polymerase POLRMT and mitochondrial mt DNA encoding the OXPHOS complexes subunit,which may be the main reason for Epox’s increased cisplatin sensitivity.3.Eopx combined with cisplatin induces protein aggregation between the nucleus and mitochondrial p62 protein,and the stacked p62 protein promotes protein aggregation through the UBA domain,which plays a key "bridge" role in inducing apoptosis of ovarian cancer cells.4.Epox combined with cisplatin interferes with ATF5-mediated mitochondrial unfolded protein reaction(UPRmt),resulting in abnormal protein aggregation and aggravating mitochondrial dysfunction.