Cisplatin Enhances Hepatitis B Virus Replication And PGC-1α Expression Through ER Stress

Introduction:There are about 350 million people infected with hepatitis B virus(HBV)around the world.Those people were settled in the Western Pacific and the African region.Besides,about 0.8 million people die of complications related to hepatitis B virus infection every year.HBV reactivation generally occur less in these people caused by chronic hepatitis B virus and carriers asymptomatic.Due to the use of different chemotherapeutic and immune inhibitors.HBV reactivation can discontinue chemotherapy and induce hepatic injury or severe liver failure.Therefore,this study aims to enlarge the knowledge on biological mechanism underlying persistent HBV infection and HBV reactivation,which will lay a foundation in order to obtain the corresponding clinical solution.The endoplasmic reticulum(ER)normally responsible for lipid metabolism,protein processing,Calcium storing,to maintenance of cells’ internal and external balance.While the organism responds to those situations of hypoxia,virus infection,hypoglycemia,the physiological function of the cell endoplasmic reticulum disorder.ER stress can initiate unfolded protein response(UPR)via three different sensors:inositol-requiring enzyme 1(IRE1),activating transcription factor 6(ATF6),and protein kinase R-like endoplasmic reticulum kinase(PERK),on the one hand which can degradation the unfolded protein or misfolded protein to maintenance of cells’ internal and external balance,on the other hand,which play an important role in the process of viruses infected human.For example,HBs Ag protein can induces ER stress;HBx protein increase the expression of CREB1 by activated ER stress,resulted to the occurrence of hepatitis,liver cirrhosis and liver cancer.Recent studies have shown that ER stress is involved in the process of cisplatin mediated tumor cell death,thus ER stress may play an essential role in drug resistance in tumor cells.The precise mechanisms of HBV reactivation were related with the impairment of host immune system and the virus mutation.However,Recent research have showed that nearly half of HBV reactivation occurred in the early stage of chemotherapeutic regimen,which indicates that mechanisms other than rely on the perturbation of the immunity balance,but direct stimulatory effects of anticancer drugs,may be responsible for HBV reactivation.More and more data suggested that cytotoxic chemotherapy,including doxorubicin,etoposide,or vincristine,may directly stimulate HBsAg secretion and HBV DNA replication in a dose-dependent manner.However,cisplatin is a broad-spectrum cytotoxic anti-caner drugs,which has been used extensively in the clinical application and has destruct effect on liver cancer cell.The molecular mechanisms involving chemotherapeutic agent or immunosuppression--related HBV reactivation needs to explore.Our preliminary data has indicated that cisplatin directly upregulated PGC-1α and HNF-4α,thus promoting HBV replication in vivo and in vitroexperiment.In order to explore the molecular mechanism of HBV reactivation induced by cisplatin,we suspect that ER stress is also correlated with cisplatin induced HBV replication.The aim of this study includes: 1)the impact on endoplasmic reticulum stress related signaling pathways induced by Cisplatin.2)the endoplasmic reticulum stress has effect on HBV replication.3)the expression of transcription factor PGC-1α aroused by ERS.4)Cisplatin promotes HBV replication by active ERS-PGC-1 α signaling pathways,which will be identified in transgenic mice.Methods:1.HepG2.2.15 and HepAD38 cells were transfected with plasmid ERSE,and then incubated with cisplatin,Tg alone or cisplatin combined with 4μ8C or cisplatin combined with PBA,respectively.At 72 h after treatment,cells were collected to detect ER stress induced by Cisplatin were analyzed by Western blot,real-time PCR and luciferase assay.2.HepG2.2.15 and Hep AD38 cells were treated with cisplatin alone or cisplatin combined with ER stress inhibitor PBA or 4μ8C for 96 h,respectively.Then ER stress-related HBV reactivation were analyzed by Western blot,real-time PCR,ELISA and Southern blot,etc.3.HepAD38 cells were transfected with plasmid PGC-1ɑ,followed with cisplatin,Tg alone or cisplatin combined with ER stress inhibitor PBA or 4μ8C for 96 h,respectively.Then cells were collected to detect PGC-1ɑ induced by Cisplatin-related ER stress,which were detected by ChIP,real-time PCR,Western blot,etc.4.HBV transgenic mice were randomly received PBA or 4μ8C for 1day,followed by treatment with PBA or 4μ8C combined with cisplatin daily for 5 days.On the sixth day,the mice were sacrificed and serum and tissue samples were detected by Western-Blot,real-Time PCR and Southern blot.Result:1.Compared with PBS control,Cisplatin or Tg treatment improved pERSE luciferase,Meanwhile pERSE luciferase was markedly reduced by PBA and 4μ8C administration(p<0.05).2.Compared with PBS control,The experiment result showed that cisplatin induce ERS signaling pathways to promoter HBV replication,especial the activation of XBP1-IRE1,but ER stress inhibitor PBA or4μ8C can reduce the expression of ER stress markers and reverse cisplatin-induced HBV replication(p<0.05).3.The experimental results showed that cisplatin administration induced PGC-1α promoter activity,and Chromatin immunoprecipitation assay(CHIP)revealed that cisplatin treatment enhanced recruitment of CREB to the PCG-1α promoter binding site,compared with PBS control,and ER stress inhibition significantly decreased CREB recruitment to the PCG-1α promoter.Western blot showed that ER stress markers,PCG-1αHBs Ag and HBcAg were greatly increased after cisplatin intervention but were reduced after PBA or 4μ8C administration(p<0.05).4.The result of liver samples and serum in HBV-Tg mouse model revealed that ER stress markers,PGC-1α and HBV protein were remarkably reduced when ER stress inhibitor was co-administered,and cisplatin remarkably reversed ER stress inhibitor induced the expression ofERS markers,PGC-1α and HBV protein detected by western blot,real-time PCR and southern blot,etc.Conclusions:1.Cisplatin promotes HBV replication by inducing endoplasmic reticulum stress signal pathway,the XBP1-IRE1 activation particularly in HBV-expressing cell line,and the transcription were blocked by ERS inhibitor PBA or IRE1 inhibitor 4μ8C.2.Cisplatin promoter HBV replication by induced ER stress signal pathway,but ER stress inhibitor PBA or 4μ8C reverse cisplatin-induced HBV replication.3.Cisplatin induced ERS may mediate PGC-1α upregulation in a CREB dependent manner,but Cisplatin-induced activation of PGC-1α was markedly reduced and reversed HBV reactivation by PBA and 4μ8C.the expriment showed that cisplatin-induced PGC-1α upregulation by CREB phosphorylation is ER stress dependent.4.Cisplatin promoter HBV replication by induced ERS-PGC-1αsignal pathway,which were blocked by PBA and 4μ8C in HBV-Tg mice model.Collectively,chemotherapy drugs cisplatin stimulates the HBV reactivation directly.Therefore,novel potential therapeutic schemes should target ER stress,or PGC-1α for the treatment of chemotherapy-related HBV reactivation.