| Cisplatin is the first-line drug for the treatment of various malignant tumors including ovarian cancer.However,most patients with advanced ovarian cancer develop resistance to platinum drugs.Therefore,how to increase the sensitivity of cisplatin has become a clinical problem.Autophagy is an important degradation pathway in cells that degrades damaged proteins and organelles by forming autophagolysomes.This complete process is also known as autophagic flux.Although people believed autophagy is a double-edged sword in cancer,current studies have confirmed that autophagy can help tumor cells antagonize the damage caused by chemotherapy which is involved in drug resistance.However,it is difficult to judge the level of autophagy in tumor tissues of ovarian cancer patients.p62 is one of the receptors of autophagy.Previous studies have confirmed that p62 is highly expressed in a variety of malignancies including ovarian cancer.Our current study suggests that the expression of p62 was higher in cisplatin-resistant SKOV3/DDP ovarian cancer cells which have higher levels of autophagy.Exploring further mechanism,we found highly expressed p62 activated NF-κB pro-survival signaling pathway through ZZ domain which mediated cisplatin resistance of ovarian cancer cells.However,autophagy inhibitor Chloroquine or Bafilomycin can increase the sensitivity of cells to cisplatin.And the expression of p62 was also significantly increased(inducible p62).These results suggest that p62 plays a pro-survival role when cells are at high levels of autophagy;conversely,when autophagic flux is blocked,p62 may play a role in promoting death.It is indicated that the expression levels can’t be used to judge the role of p62 in ovarian cancer.The level of autophagy(autophagic flux)may determine whether p62 promotes cell survival or death.Therefore,it is necessary to further explore the regulatory relationship between p62 and autophagy.p62 has abundant protein-binding sequences,including UBA(ubiquitinatedprotein domain),LIR(autophagy membrane LC3 binding sequence),and ZZ(zinc finger domain)functional domains,which help multifunctional protein p62 also function as a "signal hub" through interacting with different proteins to that regulate cell survival and apoptosis.Caspase 8 is a classical extrinsic apoptotic signal promoter and is proved low expressed in malignant ovarian cancer cells.Recent study found inhibition of Caspase 8 expression in ovarian cancer cells can reduce the apoptosis sensitivity of chemotherapy.And autophagy also participate in Caspase 8 activation and degradation in addition to the proteasome system.We believe exploring the mechanism of p62 activated Caspase 8-mediated apoptosis pathway may be useful to explain autophagy inhibition increased sensitivity to chemotherapy of ovarian cancer.This study investigate the role of accumulated p62 caused by autophagy suppression in cisplatin induced Caspase 8 activation through changing functional domain of p62 in vitro and in vivo.And provide new clues for increasing the sensitivity of cisplatin by targeting autophagy in ovarian cancer cells.Methods:1.Xenograft experiment.Mice were treated with 35 mg/kg chloroquine,3 mg/kg cisplatin or the combination treatment.And the expression of p62 was detected by immunohistochemical staining.The enzyme activity of Caspase 8 was detected by Caspase activity assay and the apoptosis rate was detected by TUNEL staining.2.SKOV3 cells were transfected with wild-type p62 and UBA/ LIR domain mutations,and the following experiments were carried out:1).SKOV3 cells were transfected with wild-type p62 and UBA domain mutants.MTT assay was used to detect the sensitivity of each group to cisplatin.2).Cells were treated with 6μg/ml cisplatin for 24 h.Apoptosis was detected by AnnexinV/PI staining,Hoechst staining and Caspase 3/7 activity assy.3).Cells were treated with 6μg/ml cisplatin for 12 h,and the activation of Caspase 8 was detected by enzyme activity assy and Western Blot.4).Autophagic flux evaluation.Cells were treated with 6μg/ml cisplatin cells after transfected with wild type p62 and UBA /LIR domain mutations.The expression of insoluble ubiquitinated proteins were exanined by 1% Triton-SDS isolation;m-Cherry-GFP report asssy was used to detecte autophagic flux;the proteinsdegradation through autophagy-lysosome were detected by DQ-BSA assy.5).The SKOV3 cells were transfected with wild-type p62 and UBA/ LIR domain mutations.After cisplatin treatment,the binding of p62,LC3 and Caspase 8 were detected by immunoprecipitation and immunofluorescence.The GST fusion proteins of wild-type p62,UBA and LIR mutants were purified to explore their combination in xenograft mice tissues.3.The expression of p62 and Caspase 8 in 160 cases of ovarian cancer patients were detected by immunohistochemical staining.The correlation between protein expression and survival time,TNM stage and risk of recurrence were analyzed.Results:1.Immunohistochemical staining showed that the combination of chloroquine and cisplatin promoted the expression of p62 in tumor tissues.At the same time,the enzyme activity of Caspase 8 and positive TUNEL staining was increased with the combination of chloroquine and cisplatin.2.MTT results showed that SKOV3 cells were less sensitive to cisplatin with transfection of UBA domain mutant p62 compared with overexpressing wild-type p62.3.The results of apoptosis experiment showed that the positive rate of Annexin V/PI staining decreased after UBA domain mutant p62 transfection;Hoechst staining revealed that compared with wt-p62,the nuclear fragmentation induced by cisplatin treatment was significantly reduced;the enzymatic activity of Caspase 3/7 and Caspase 8 were decreased.4.Evaluation of autophagy flux revealed that overexpression of wild-type p62 led to an increased amount of insoluble ubiquitinated protein accumulation,which was similar to the accumulation observed with autophagy inhibition caused by chloroquine treatment.In contrast,the main part of ubiquitinated proteins were in the soluble fractions with p62 mutants;compared with wild-type p62,there were higher numbers of mature autophagosomes in SKOV3 cells transfected with the p62 mutants;at the same time,expression of p62 mutants lacking the LIR and UBA domains resulted in increased proteins degraded through autophagy-lysosome pathway.These results indicated that UBA or LIR domain mutations increased the autophagic flux of SKOV3 cells.5.The results of immunofluorescence and co-immunoprecipitation showed that UBA and LIR domain mutations significantly inhibited the binding of Caspase 8 and LC3 compared with overexpression of wild-type p62;GST-pull down experiments showed that these mutations significantly inhibited the binding of p62 to Caspase 8and LC3.6.Spearman correlation analysis confirmed that p62 expression was positively correlated with Caspase 8(pho=0.499,P<0.01);among 160 patients with ovarian cancer,53 of them both highly expressed p62 and Caspase 8;Kaplan–Meier analysis suggested that patients with both p62 and Caspase 8 high expression had a significantly longer survival time than those with high p62 but low Caspase 8expression.Furthermore,expression of Caspase 8 was negatively correlated with tumor-node-metastasis(TNM)stages and less relapse risks in tissues with high p62 expression.Conclusion:1.Experiments in vitro and in vivo confirmed that autophagy inhibition promoted the activation of Caspase 8 induced by cisplatin.The activated Caspase 8 may be involved in the mechanism that autophagy suppression increase the cisplatin sensitivity of ovarian cancer cells.2.Cells transfected with UBA or LIR mutants inhibit the cisplatin-induced Caspase 8activation and increase the autophagy flux of ovarian cancer cells.These results suggest that autophagic flux may regulate p62 mediated Caspase 8 activation and is related to UBA or LIR domains of p62.3.Co-localization of p62,Caspase 8 and LC3 in ovarian cancer cells was found by immunoprecipitation,immunofluorescence and GST-pull Down assay.These results suggest that autophagosome membrane may provide a "molecular platform" for Caspase8 activation mediated by p62.And autophagic flux is the key factor determining whether p62 activates Caspase 8.Different from primary highly expressed p62,autophagy inhibition induced p62 accumulation may promote sensitivity of cisplatin by increasing the activation of Caspase 8 in ovarian cancer cells.4.The results of tissue microarray show the ovarian cancer patients with high expression of Caspase 8 and p62 have longer survival time,lower malignancy andless relapse risks,which suggest that Caspase 8 may serve as an auxiliary molecule of p62 to increase the accuracy of ovarian cancer prognosis.In summary,this study find that Caspase 8 not only mediates apoptosis signal,but participates in autophagy level evaluation combined with p62 in ovarian cancer cell.Exploring these mechanisms may be helpful to clarify why autophagy inhibition increases sensitivity of chemotherapy,which will provide new clues for prognosis and chemotherapeutic drugs sensitivity improvement of ovarian cancer. |
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