Sorafenib Induces Programmed Cell Death In Hepatic Stellate Cell By Coordinating The Cross-talk Between Apoptosis And Autophagy Via The Akt And JNK Signaling Pathway

Hepatic fibrosis is a wound-healing response characterized by theaccumulation of extracellular matrix (ECM) proteins as results of chronicliver injury. Activation of hepatic stellate cells (HSCs), the main source ofECM proteins, represents a crucial event in the pathogenic sequence offibrosis and thus, provides an important framework to define potentialstrategies for anti-fibrotic therapy. Beside the inhibition of fibrogeniccytokines, the induction of HSCs death is now considered an effectivestrategy for the resolution of hepatic fibrosis. The ideal cell death strategyshould selectively target activated HSCs while maintaining quiescent HSCsbut not alert the hepatic inflammatory response. In contrast to theunorganized way of death “necrosis” that triggers the inflammatory response,apoptosis and autophagy proceed with the participation of organized andcontrolled cellular processes, which achieve a cleaner cellular execution.Death by apoptosis commonly signals through the activation of initiatorand executioner caspases, resulting in the formation of apoptotic bodies thatare removed by phagocytes. The autophagic pathway involves the formationof a membrane around a targeted region of the cell, resulting in theautophagosome formation, which fuses with a lysosome to form anautophagolysosome where the contents are degraded via acidic lysosomalhydrolases. Although apoptosis is the primary mechanism ofchemotherapy-induced cell death, an alternative mode of cell death, termedautophagic cell death, has emerged recently as an important mechanism ofcell death. However, at present, manipulation of autophagy in favor of celldeath is still confusing and controversial due to the paradoxical roles ofautophagy in cell survival and cell death. In addition, the latest research found that there is complex interaction between autophagy and apoptosis,because some moleculars which play an important role in autophagy alsoparticipate in apoptosis, such as beclin1and Atg5; meanwhile the caspaseand Bcl-2family which are involved in apoptosis regulate autophagy bycleaving or binding Beclin1. Most recently, autophagy has been implicatedin the release of lipid droplets from HSCs that further promoted fibrogenesisof activated HSCs. Paradoxically, Rickmann et al. have reported thattocotrienols counteracted pancreatic fibrogenesis through the induction ofapoptosis and autophagy in activated pancreatic stellate cells. Therefore,autophagy is a double-edged sword which is a friend as well as an emeny forcell death depending on the different stages of the disease, cellular nichesand different treatment. There are a large number of studies showing thatvarious anti-cancer drugs play a role in anti-tumor effect by inducing tumorcells to undergo autophagic cell death, such as bufalin, cannabidiol,cucurbitacin and so on.Sorafenib is a multiple receptor tyrosine kinase inhibitor targeting theRaf/ERK signaling pathways. It is reported that: sorafenib could inducekinds of tumor cell to undergo autophagy due to its effect on regulatingmultiple autophagy-related signaling pathways, such as: MAPK/ERK, Akt/mTOR/p70S6K and JNK/c-Jun signaling pathway.. There has been a recentinterest regarding the potential efficacy of sorafenib in the treatment of themost common and progressive forms of fibrosis. Previous studies indicatedthat sorafenib induced a significant decrease in portal pressure andangiogenesis in rats with liver fibrosis. Our previous study showed thatsorafenib inhibited proliferation and induced apoptosis in activated HSCs.However, the molecular mechanisms involved in sorafenib anti-fibroticeffects have not been completely characterized.Thus, we hypothesize that sorafenib has a therapeutic effect on liverfibrosis by inhibiting proliferation meanwhile inducing apoptosis, or byinducing HSC autophagic cell death in HSC.The aim of this project is toexplore sorafenib’s effect on inducing autophagic cell death in human HSC-LX2cell lines and primary rat HSCs as well as the relationshipbetween autophagy and apoptosis and potential molecular mechanism. Theexperiment consists of the following three parts:Part1: Sorafenib induces the apoptosis and ACD of HSCsObjective: To study the effect of sorafenib on apoptosis and ACDMethods:To obtained primary HSCs, rat liver tissues sequentialdigestion with pronase and collagenase, followed by single step densitygradient centrifugation with Nycodenz. The identification of HSC was madeby using fluorescent microscope and alpha-SMA immunocytochemicalstaining. Different concentration of Sorafenib (2.5μmol/L,5.0μmol/L,10.0μmol/L) treated HSC-LX2cell lines at the time point of6h,12h or24h,then use MTT assay to detect cell viability, Annexin-V/Propidium iodide (PI)double-labeled the apoptosis of HSCs rate by flow cytometry. HSC-LX2celllines were preincubated by apoptosis inhibitor Z-VAD-FEK (20μmol/L) orthe necrosis inhibitor IM-54(10μmol/L) for1hour, then co-incubated with5μmol/L sorafenib for another11h. The cell viability was also determinedby MTT assay, while the changes of the percentage of Annexin V+cellnumber and PI+cells was dectected by flow cytometry. After primary HSCor HSC-LX2cell lines were treated by sorafenib (5μmol/L) for12h,transmission electron microscopy and acridine orange staining were used toabserve the autophagy activation of HSCs, laser confocal microscope wasused to track the changes of autophagy marker protein LC3; RT-PCR methodwas explored to detect autophagy-related gene Beclin1, Atg7and Atg5aftersorafenib treatment. Western Blot was used to study the effect of sorafeniband autophagy inhibitor’s on the expression of autophagy-related proteinLC3, Beclin1, Atg7, Atg5, and p62/SQSTM1.Results:①The average havest rate of HSC cells was1.2to2.0×107perrat and the cell purity and survival rate were more than90%. At the verybeginning the extracted HSCs were round and cytoplasm contained a greatdeal of lipid droplets, emitting blue ray under the320nm fluorescence microscope. After the HSCs were cultured for10days, the rested cells wereactivated and present a shape of fusiform or star, and the lipid droplets rich invitamin A disappeard.②The identification of the characteristic of primaryrat HSC. Inactivated HSCs had no expression of alpha-SMA; when the cellswere cultured for14days, fully activated HSCs did have alpha-SMAexpression in cytoplasmic.③sorafenib inhibited HSC-LX2cell survivaland promoted apoptosis. The MTT assay and flow cytometry results showedthat sorafenib inhibited HSCs survival and promoted cell apoptosis in atime-and concentration-dependent manner,5μmol/L sorafenib treatment for12h made the cell viability reduced to50.19±0.19%(P <0.001) comparedwith100%of the control group, and the apoptosis rate (41.22±2.425%)was significantly higher than the control group (7.73±1.34%)(P <0.01).④sorafenib induced the cell non-apoptotic cell death in HSC. The Z-VAD-FEKPre-incubation group had no difference in cell survival rate with sorafenibgroup, but less than the control group (P <0.01).However, the PI+rate(54.19±4.03%) was significantly higher than Annexin V+rate (10.22±2.03%)(P <0.01). The cell viability (50.36±4.73%) of IM-54Pre-incubation group was still less than the control group (P <0.01),both PI+rate and Annexin V+rate had no significantly differences with sorafenibtreated group.⑤The observation of HSCs morphological changes byacridine orange staining and transmission electron microscopy. After5μmol/L sorafenib treated HSCs for12h, the electron microscopy showedthe characteristics of autophagy: a large number of vesicles withdouble-membran which contained cytoplasmic substances, sometimes itshowed wrapped organelles such as mitochondria. Besides, lysosomal wasactivated which was fused with bilayer membrane vesicle; Under thefluorescence microscopy, large numbers of particles with orange flurescencewere dispersed in the cytoplasm around the nucleus.⑥"Autophagy tide"was induced by sorafenib in HSCs. The HSCs were pre-incubated withlysosomal inhibitors E64d and pepstatin A for1h, after then co-incubatedwith5μmol/L sorafenib for another11h. Western blot displayed the LC3II/I exrepssion is further increased in the E64d and pepstatin A pre-incubationgroup compared with only sorafenib treated group; Under the confocalmicroscope the primary HSC of both E64d and pepstatin A pre-incubationgroup showed increase LC3green spots.⑦RT-PCR detected theexpression of autophagy-related gene Beclin1, Atg7and Atg5geneincreased.⑧Western blot results showed that autophagy-related proteinBeclin1, Atg5and LC3II/I were significantly increased, while p62decreased significantly after sorafenib treatment which could be reversed byadding3-MA.Conclusion: Sorafenib could promote death of HSCs by inducingapoptosis and autophagic cell death. It is speculated that the HSCs couldphagocyte, digestion and eliminate cytoplasm substances and organelles byactivating autophagic death pathway.Part II: Sorafenib induces programmed cell death in hepatic stellate cellby coordinating the cross-talk between apoptosis and autophagyObjective: To probe the cross-talk between apoptosis and autophagyinduced by sorafenib.Methods:5μmol/L sorafenib treated HSCs at different time points (0h,3h,6h,12h,24h) or different concentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) treated HSCs for12h. To study the HSCs’ autophagyand apoptosis, the expression of blot LC3II/I and Cleaved-PARP wasdetected by Western blot. After HSCs were treated by differentconcentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) for12h,acridine orange staining or PI/Annexin V double staining were used to detectthe AVOs and changes of the apoptotic cell number; Western blot methodwas explored to detect the expression of autophagy-related protein Beclin1and apoptosis-related proteins cleaved-caspase-3,-8. HSCs werepre-incubated with Z-VAD-FEK (20μmol/L) for1h then incubated with10μmol/L sorafenib for another11h. Western blot was used to detect theautophagy-related protein LC3II/I and Beclin1change. And after autophagy inhibitor3-MA (5mmol/L) pre-incubated for1h and5μmol/L sorafenibincubated for another11h, apoptosis rate of HSCs was detected by flowcytometry, and the expression of autophagy-related protein Beclin1andapoptosis-related proteins cleaved-caspase-3,-8were detected by Westernblot. In addition, Atg5-siRNA technology is applied to interfere LX2, after5μmol/L sorafenib treated for12h, the autophagy and apoptosis-relatedprotein changes were checked by Western blot.Results:①Autophagy induced by sorafenib is prior to apoptosis. Thewestern blot results showed that: after the HSCs were treated with sorafenibfor24h, Cleaved-PARP expression was significantly increased, while LC3II/I expression was significantly increased at the time point of6h, andreached the peak at12h. when treatment time extended to24h, the LC3II/Iexpression returned to baseline levels.②Different concentrations ofsorafenib had different effect on autophagy and apoptosis in HSCs. Westernblot showed that in sorafenib (5μmol/L) group LC3II/I expression wassignificantly increased (P <0.01); In sorafenib group (10μmol/L), LC3II/Iexpression was decreased, but Cleaved-PARP expression was significantincreased (P <0.01). Flow cytometry results showed that in sorafenib group(5μmol/L), the AVOS cell percentage (62.19±6.11%) was significantlyhigher than the percentage of apoptotic cells (38.28±4.91%)(P <0.01),while in sorafenib (10μmol/L) group, the apoptosis rate (95.90±3.33%)was significantly higher than in sorafenib (5μmol/L) group (P <0.01),butthere was no significant change of the autophagy rate(73.18±5.04%).③The activated caspases cleaved Beclin1. The Western Blot resultsdemonstrated that in sorafenib (10μmol/L) group,the expression of thecleaved-caspase-3,-8enhanced, but Beclin1decreased. Z-VAD-FMK canreverse this situation, and increase the LC3II/I expression.④Inhibition ofautophagy can promote sorafenib-induced apoptosis. After pre-incubationwith3-MA, HSCs were detected by flow cytometry. The results showed thatAnnexin V-positive cells (81.94±5.34%) were much higher the sorafenibtreated group (42.34±3.60%)(P<0.01). Western blot showed that the expression of the apoptosis-related proteins, such as cleaved-caspase-3,-8and cleaved-PARP could significantly increase after pre-incubated with3-MA. At the mean time, Atg5-siRNA could inhibit sorafenib-inducedincrease of LC3II/I, meanwhile increase expression of the cleaved-caspase-3,-8and cleaved-PARP.Conclusion: The HSCs treated with sorafenib undergo autophagic celldeath pior to apoptosis, autophagic cell death was inhibited when theapoptosis was started, which may due to the activated caspases whichinvolves in cleavaging Beclin1. After blocking the autophagy induced bysorafenib, the apoptosis increased in a compensatory manner, which is analternative way as a kind of programmed cell death.Part III: The signal transduction mechanism of sorafenib inducingprogrammed cell death in hepatic stellate cellsObjective: To investigate the regulatory function of sorafenib onAkt/mTOR/p70S6K and JNK/c-Jun signal transduction pathway in HSCcells.Methods: HSC-LX2cell line was treated with5μmol/L sorafenib atdifferent time porints (0h,0.5h,1.5h,3h,6h,12h,24h), expression ofAkt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K, JNK and p-JNK,weredetected by Western blot. Laser scanning confocal microscopy was exploredto track pc-Jun nuclear translocation. After HSCs was treated by differentconcentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) for12h,Acridine orange staining or Annexin-V/Propidium iodide (PI) double-labeledstaining by flow cytometry and changes of the apoptotic cellss number; afterHSCs were treated with5μmol/L sorafenib and10μmol/L SP600125for12h, Western blot was used to detect JNK, p-JNK, c-Jun, p-c-Jun, Beclin1andLC3II/I expression.Results:①Sorafenib inhibited phosphorylation of Akt/mTOR/p70S6Ksignaling pathway in HSC-LX2cells. The ratio of p-Akt/Akt,p-mTOR/mTOR and p-p70S6K/p70S6K were reduced (P<0.01) respectively compared to control group.②Sorafenib induced cell apoptosis byAkt/mTOR/p70S6K signal pathway. Flow cytometry revealed that the rate ofapoptosis in LY294002+sorafenib group (69.44±6.71%) increased by about53%compared to the sorafenib group (38.22±4.66%) while LY294002hadno effect on the AVOs cell percentage.③Sorafenib initiated autophagythrough JNK/c-Jun signaling pathway. Sorafenib activated phosphorylationof JNK/c-Jun signaling pathway in HSC-LX2cells. The ratio of p-JNK/JNKsignificantly increased compared to the control group by sorafenibintervention, a significant increase of p-c-Jun and its translocation to thenucleus were proved by confocal macrophage. SP600125inhibited thephosphorylation level of JNK/c-Jun and also inhibited the sorafenib-inducedLC3II/I and Beclin1increasing.Conclusion: Sorafenib inhibited Akt/mTOR/p70S6k signaling pathwayand activated JNK/c-Jun signaling pathway, in which Akt/mTOR/p70S6kpathway leaded to apoptosis, and JNK/c-Jun signal pathway participated insorafenib-induced autophagy directly.