Cisplatin Induces Autophagy To Enhance Hepatitis B Virus Replication Via Activation Of ROS/JNK And Inhibition Of The Akt/mTOR Pathway

ObjectiveHepatitis B virus(HBV)infection has become a serious global health concern,with approximately 350 million chronic HBV carriers worldwide.Chronic HBV infection is a major global risk factor for hepatocellular carcinoma(HCC).HBV reactivation generally occur less in these people with chronic hepatitis B virus and carriers asymptomatic,when these patients receving chemotherapeutic agents and/or immunosuppression,some of these patients will occur the phenomenon of HBV reactivation,a large number of instantaneous activation in patients with HBV may cause serious clinical problems such as acute hepatitis,acute severe hepatitis,and even liver failure.Many clinical data indicate that HBV reactivation occurs undergoing chemotherapeutic drugs.However,The mechanism underlying the enhanced viral replication during cytotoxic chemotherapy remains unclear.Thus,the underlying mechanisms require further investigation.Autophagy is a catabolic process for the removal of damaged organelles and long-lived proteins for the maintenance of cellular homeostasis through lysosome-dependent machinery.Autophagy plays vital roles in numerous physiological and pathophysiological processes.Autophagy is a doubleedged swordduring viral replication.On the one hand,autophagy can respond to cellular stress such as nutrient deprivation,unfolded protein response(UPR),risk-related molecular patterns(DAMP),hypoxia,redox stress and mitochondrial damage.The virus can trigger these stimulations to induce autophagy at different stages of the virus replication cycle,which may act as an antiviral pathway.On the other hand,autophagy plays a beneficial role in the viral replication life cycle,and viruses can evade antiviral innate immunity to enhance virus replication.Previous studies have indicated that the relationship is complicated between viruses and autophagy.There are three main outcomes:(1)Viruses hijack autophagy as a scaffold to promote viral replication.(2)The viruses destroy or inhibit the autophagy process to avoid limiting its replication.(3)Autophagy limits viral replication.Cisplatin is an effective chemotherapeutic agent widely used to treat various types of cancers,including lymphomas,sarcomas,ovarian cancer,cervical cancer,small cell lung cancer,and bladder cancer.However,several clinical studies have reported that HBV reactivation is one of the serious complications in cancer patients receiving chemotherapy containing cisplatin.Host immunity impairment upon viral replication is considered the initial factor contributing to HBV reactivation associated with chemotherapy.But HBV reactivation reportedly sometimes occurs in the early stage of chemotherapy,indicating that chemotherapeutic agents may directly stimulate HBV reactivation besides disturbing the balance between immunity and viral replication.Thus,HBV reactivation seems to be a complex process relativing chemotherapeutic agents and/or immunosuppression,and the underlying mechanisms require further investigation,which can provide new ideas and theoretical basis for the prevention and treatment of HBV reactivation.Our previous dates suggested that cisplatin can enhance HBV replication and synthesis of HBV viral proteins both in an HBV-replicating cell model and in an HBV-transgenic mouse model.Thus,our study explores the molecular mechanism of HBV reactivation induced by cisplatin.Methods:1.The levels of HBV replication of treatment with cisplatin were measured by ELISA,Real-Time PCR,Southern blot and Western blot in HepAD38 and HepG2-HBV1.3 cells.2.The levels of autophagy proteins and HBV viral proteins were measured by Western blot and confocal immunofluorescence microscopy in HepAD38 and HepG2-HBV1.3 cells.The levels of HBV replication were detected underlying treatment with cisplatin and autophagy inhibitors of Chloroquine diphosphate salt(CQ),3-methyl adenine(3-MA),and siATG5.The relationship among the cisplatin,autophagy and HBV replication was analyzed.3.The levels of ROS were detected by confocal immunofluorescence microscopy underlying treatment with cisplatin.Cells were treated with cisplatin alone or cisplatin combined with oxidative stress inhibitor N-acetylL-cysteine(NAC).The levels of autophagy proteins and HBV viral proteins were measured by ELISA,Real-Time PCR,Southern blot and Western blot.4.Through western blot and reviewing literatures to research MAPKs pathway-associated signaling molecules.HepAD38 Cells were treated with cisplatin alone or cisplatin combined with shJNK,NAC,SC79 and MK2206 to explore the molecular mechanism of HBV reactivation.5.The effect of cisplatin promoted HBV replication was detected in in an HBV-transgenic mouse model.Mice were administered 40 mM NAC in the drinking water or were intraperitoneally injected with chloroquine(30 mg/kg body weight).Forty-eight hours later,the mice were intraperitoneally injected with cisplatin(5 mg/kg body weight)or PBS(control)daily for five consecutive days.At the sixth day post cisplatin injection,mouse serum and liver tissue specimens were harvested for ELISA,Real-Time PCR,confocal immunofluorescence microscopy,Western blot,Southern blot and IHC analysis.Results:1.HepAD38 cells and HepG2-HBV1.3 cells transiently transfected with HBV were treated with cisplatin,compared with PBS control,Cisplatin significantly increased HBsAg and HBeAg levels in the culture supernatants,the HBV 3.5-kb RNA levels,HBV DNA levels,HBV replicative intermediates and intracellular HBsAg and HBcAg expression.It showed that cisplatin enhances HBV reactivation in vitro.2.The expression of LC3 in HepAD38 cells and HepG2-HBV1.3 cells underlying treatment with cisplatin were detected by western blot and confocal immunofluorescence microscopy.Treatment with cisplatin significantly upregulated LC3-II expression and suppressed p62 expression by western blot.The confocal immunofluorescence microscopy experiment revealed that Cisplatin significantly increased RFP-LC3 puncta and GFPLC3 puncta formation,but RFP-LC3 puncta is more pronounced than GFPLC3 puncta.Taken together,these data indicated that cisplatin induces complete autophagic flux in virto.Moreover,HBV-expressing hepatoma cells were treated with cisplatin alone or cisplatin combined with CQ,3-MA and siATG5,the level of autophagy and the level of HBV replication decreased by western blot.3.The levels of ROS were detected in cisplatin-treated cells by confocal immunofluorescence microscopy.The results of confocal immunofluorescence microscopy showed that compared with PBS group,the levels of ROS increases in the cisplatin group.Cells were treated cisplatin alone or cisplatin combined with NAC,the experiment revealed that compared with cisplatin group,the cisplatin+NAC group,the levels of LC3,HBcAg and HBsAg proteins,HBeAg and HBsAg levels in the culture supernatants,the HBV 3.5-kb RNA levels,HBV DNA levels and HBV replicative intermediates were significantly increased.Taken together,these data indicated that cisplatin induces HBV replication by inducing ROS generation and subsequent autophagy activation.Inhibitor of ROS reversed partly cisplatin-induced stimulation of HBV replication.4.MAPKs pathway-associated signaling molecules were detected by western blot.The results of western blot showed that Cisplatin increased JNK phosphorylation,but did not affect p38 phosphorylation in HepAD38 cells.Moreover,Akt phosphorylation and mTOR phosphorylation were significantly downregulated in cisplatin-treated cells.Additionaly,pretreatment with NAC drastically reversed JNK phosphorylation in HepAD38 cells,but had little effect on the levels of p-AKT and p-mTOR.Cells treated with cisplatin alone or cisplatin combined with shJNK(siRNAmediated knockdown of JNK),SC79(Akt activator)or MK2206(Akt inhibitor).Both JNK knockdown or SC79 pretreatment significantly attenuated cisplatin-induced autophagy,HBV replication,and viral proteins production.In contrast,MK2206 observably increased the expression of the autophagy-related proteins,HBsAg and HBcAg in the presence of cisplatin.Moreover,JNK knockdown,SC79 or MK2206 did not affect cisplatinmediated ROS induction and JNK activation.Taken together,these results suggested that cisplatin activates ROS/JNK and inhibits Akt/mTOR signaling,which may be associated with cisplatin-induced autophagy and viral-replication promotion.5.Cisplatin administration significantly stimulated HBV replication and biosynthesis and induced inflammation in the mouse model of HBV infection.Concomitantly,significant increases in ROS generation,MDA levels and LC3 expression in liver tissues were observed in cisplatinadministrated mice.Pretreatment with the ROS inhibitor NAC or the autophagy inhibitor CQ sharply reversed cisplatin-mediated autophagy activation and enhanced HBV replication,whereas CQ administration did not suppress ROS induction by cisplatin.Conclusion:In this study,we investigated that cisplatin stimulated HBV replication both in an HBV-replicating cell model and in an HBV mouse model.Further,the potential involvement of ROS/JNK and Akt/mTOR signaling in cisplatin-induced autophagy were investigated with the aim to elucidate the underlying mechanisms.We expected our findings to contribute to the development of new therapeutic strategies against chemotherapy-mediated HBV reactivation.