| Epstein-Barr virus（EBV）is the first tumor-associated virus to be discovered.The occurrence and progression of nasopharyngeal carcinoma（NPC）is closely related to EBV infection,and recurrence and metastasis are the main reasons for treatment failure of advanced NPC.Therapeutic strategies that target EBV-positive tumor cells by activating EBV lytic replication have important clinical value in advanced refractory NPC.Heat shock protein 90（Hsp90）is highly expressed in many kinds of tumors,including nasopharyngeal carcinoma（NPC）.Hsp90 inhibitors can activate unfolded protein response（unfolded protein response,UPR）to affect the growth and survival of tumor cells.Curcumin（CUR）is a widely bioactive compound extracted from the rhizome of Curcuma,which has significant anti-tumor activity and has been proved to inhibit Hsp90.Curcuminoid C210 synthesized by our research group has a stronger affinity for Hsp90.In this study,we will explore the action characteristics of C210 on UPR signal pathway and whether C210 affects EBV lytic replication through UPR signal pathway,laying a foundation for the development of a safe and efficient novel inducer.Objective:To explore whether curcuminoid C210 activates EBV lytic replication by affecting UPR signal pathway,which provides an important theoretical basis for targeted therapy of EBV-related tumors.Method:1.The inhibitory effect of C210 on the proliferation of human nasopharyngeal carcinoma cell line C666-1 and CNE2 was detected by MTT assay;2.The effects of C210 on the expression of EBV lytic genes mRNA（Zta,BMRF1,gp350,gp110）,EBV DNA replication and UPR main signal mRNA（CHOP,Trb3,BiP,XBP1s）in human nasopharyngeal carcinoma cell line C666-1 was detected by qPCR;3.The effects of C210 on the expression of EBV lytic viral proteins（Zbra,EA-D）and UPR signal proteins（p-PERK,p-eIF2α,ATF4,CHOP,ATF6,BiP,p-IRE1α,XBP1s）in human nasopharyngeal carcinoma cell line C666-1 was detected by Western Blot.4.Lentivirus was used to knock down PERK and XBP1 in human nasopharyngeal carcinoma cell line C666-1 respectively.qPCR was used to detect the expression of EBV lytic genes Zta and gp350 mRNA in knock-down group under the action of C210,and Western Blot was used to detect the expression of EBV lytic protein Zebra in knock-down group under the action of C210.Result:1.C210 inhibited the proliferation of human nasopharyngeal carcinoma cell line C666-1 and CNE2,and the inhibitory effect of C210 on C666-1 and CNE2 cells was dose-dependent and time-dependent.The IC50values of C666-1 cells at 24h and 48h were（2.94±0.38）μg/mL、（1.14±0.20）μg/mL.The IC50 values of CNE2 cells at24h and 48h were（3.32±0.68）μg/mL、（2.53±0.69）μg/mL.2.C210 could activate EBV lytic replication.（1）EBV lytic genes Zta,BMRF1,gp350 and gp110 mRNA were up-regulated with the increase of concentration of C210,while Zta,BMRF1 and gp350 mRNA were up-regulated with the prolongation of treatment time at low concentration of C210,while low concentration of C210 had no significant effect on gp110 mRNA.（2）The copy number of EBV DNA increased.（3）The expression of Zebra（Zta）and EA-D（BMRF1）of EBV lytic protein increased.3.C210 can activate three branches of UPR signal pathway.（1）PERK signal pathway:C210 can up-regulate downstream signal CHOP,Trb3 mRNA and major signal proteins p-PERK,p-eIF2α,ATF4,CHOP;（2）ATF6 signal pathway;C210 can up-regulate downstream signal BiP mRNA and major signal proteins ATF6,BiP;（3）IRE1 signal pathway;C210 can up-regulate downstream signal XBP1s mRNA and main signal proteins p-IRE1α,XBP1s.4.C210 initiates the EBV lytic replication cycle by activating XBP1 signal.Knocking down XBP1 attenuated the effect of C210 on the expression of Zta and gp350mRNA and Zebra protein,while knocking down PERK had no significant effect on the expression of Zta and gp350 mRNA but Zebra protein expression was increased which activated by C210.Conclusion:Curcuminoid C210 can activate UPR signal pathway,and the activation of PERK signal and XBP1 signal can initiate EBV lytic replication cycle. |
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