Based On The Interaction Between C-FLIP And Endoplasmic Reticulum Stress Pathway To Explore The Mechanism Of Jiedu Fang Improving The Sorafenib Resistance

Background:Sorafenib(Sora)is one of molecular targeted therapy recommended for the advanced stage treatment of hepatocellular carcinoma(HCC),but in the treatment process there will be a variety of side effects.More importantly,a number of patients become resistant to sorafenib.Based on the researches at present,the mechanism of sorafenib resistance is complex,involving many signal pathways and biological molecules.However,the current researches cannot fully reveal the mechanism of sorafenib resistance.It has been reported that sorafenib can activate CD95/Fas to induce apoptosis,and caspase-8 is the important initiation protein in the exogenous apoptosis pathway.Caspase-8 activation can lead to a series of cascade reactions.C-FLIP is an important inhibitor of caspase-8,which is highly expressed in many tumors,and is closely related to the inhibition of apoptosis.At the same time,it is located in the membrane of endoplasmic reticulum(ER),with the ability of regulating endoplasmic reticulum stress(ERS),and ERS is closely related to the occurrence and development of cancers,involved in drug resistance.Thus,we assume that whether the c-FLIP and ERS signaling pathway are involving in the complex process of resistance and is one of the mechanisms of sorafenib resistance.At the same time,the members of the project group in the prevention and treatment of liver cancer with traditional Chinese medicine has certain research foundation and rich clinical experience,and summarize the experience and medication in long-term clinical studies.We summed up the small compound “Jiedu Fang”.In a multi-center clinical study based on the “Jiedu Fang”,we found that it could prolong the survival of patients with advanced hepatocellular carcinoma,especially in patients with sorafenib resistantance or side effects.Traditional Chinese medicine has a unique advantage in reversing the drug resistance of cancer chemotherapy.So whether the Chinese small compound “Jiedu Fang” can improve the resistance of sorafenib,and whether the mechanism is involved in the regulation of c-FLIP/ERS?Objective: 1.To construct sorafenib resistant human hepatocellular carcinoma cell strains and observe the effects of sorafenib on the proliferation and apoptosis of sensitive/resistant hepatocellular carcinoma cells.2.To observe the effect of sorafenib on ERS and downstream apoptosis and autophagy pathway in the sensitive/resistant hepatocellular carcinoma cell lines.And to study the effect of c-FLIP on the apoptosis pathway and its possible mechanism.3.To investigate whether “Jiedu Fang” in vitro can improve sorafenib resistance by regulating c-FLIP and ERS related pathway.4.To further demonstrate the inhibitory effect of Jiedu Fang combined with sorafenib on subcutaneous transplantation tumor of drug-resistant hepatocellular carcinoma cells in vivo.Methods: 1.The concentrations of sorafenib on HepG2 and Huh7 were screened by MTT assays.The sustained sorafenib intervention was used to induce HepG2-R and Huh7-R with the concentrations lower than IC50.Proliferation and apoptosis were detected by flow cytometry and MTT assays to verify the resistance.2.To observe the effect of ERS pathway in the resistance of sorafenib,and the ERS inhibitor 4-Phenylbutyric acid(4-PBA)was used for inverse validation.Further compare the sensitive with the resistant in terms of apoptosis and autophagy and the expression of related protein was detected by Western Blot.Observation of autophagosome was constructed under electron microscope.To study the role of ERS in drug resistance of sorafenib.3.According to the effect of ERS on the apoptosis pathway,we observed the effect of siRNA for an inhibitor of apoptosis protein cFLIP on the sorafenib resistance.4.To observe the inhibitory effect of Jiedu Fang combined with sorafenib on drug resistant hepatocellular carcinoma cell line in vitro,and to explore the effect of combined therapy on the expression of c-FLIP and ERS.5.Nude mice models were established with sorafenib resistant human hepatocellular carcinoma cell strains and in vivo to observe the effect and mechanism of Jiedu Fang on improving sorafenib resistance.Results: 1.The sorafenib resistant human hepatocellular carcinoma cell line HepG2-R and Huh7-R were successfully constructed,and the concentration of sorafenib was about 6μM,and 9μM,respectively.After the treatment of sorafenib,the IC50 of HepG2-R,HepG2,were 18.17μM and 9.26μM for 48 h,12.74μM and 6.48μM for 72 h respectively,HepG2-R,HepG2,IC50,23.36μM and 10.13μM for 48 h,16.88μM and 5.84μM for72 h,respectively.And the differences between the groups were statistically significant(p<0.05).The results of flow cytometric detection of resistant and sensitive strains were consistent with the results of MTT,and the apoptotic rate of 48 h was significantly lower than that of the sensitive strains(p<0.05).Under the same concentration of sorafenib,compared with the proteins in the resistant,PARP,caspase-8,caspase-3 and were activated in the sensitive.2.Study on the effect of ERS on resistance in the process.By comparing the sensitive/resistant cell ERS related protein,the activation degree of IRE1 and PERK signal pathway was higher,the expression level of ERS protein was higher,the use of 4-PBA(ERS inhibitor)could increase the resistance strain sensitivity to sorafenib,and decrease the level of PERK and Bip,increase Chop level,and apoptosis protein such as aspase-8,caspase-9,caspase-3 PARP were activated.However,the effect of ERS on apoptosis related caspase-12 was not obvious.The further study of ERS related to autophagy found that compared with sensitive strains,the level of autophagy in resistant cells was higher,the expression of P62 decreased,the expression of LC3 II increased obviously,ERS inhibitor 4-PBA combined with sorafenib could increase P62,LC3 I to II conversion decreased,electron microscopy observed similar results,drug resistant strain and sorafenib group could have more autophagosomes.However,after 4-PBA treatment,the autophagosomes were decreased.After treated with autophagy inhibitor 3-Methyladenine(3-MA)and Chloroquine(CQ)treatment,resistant strains of sorafenib on proliferation inhibition rate and apoptosis rate increased significantly.3.Through the comparison of sensitive strains and resistant strains of apoptosis protein caspase-8,9,12,we found that caspase-8 of sensitive cells with sorafenib treatment was significantly activated while in resistant strains it was not easy to be activated.So we further observe the inhibition of caspase-8 protein c-FLIP in sorafenib resistance mechanism.After Sorafenib treatment,the expression of c-FLIP in resistant strains was higher than that in sensitive cells.By siRNA interference,c-FLIP,both the sensitivity of resistant Huh7-R or HepG2-R to sorafenib increased significantly,and compared with NC group,the c-FLIP siRNA group cell proliferation inhibition rate and apoptosis rate increased(p<0.05).To observe the c-FLIP si RNA interference effect on ERS related protein and caspase-8,after sorafenib treatment,PERK and Bip level decreased,Chop level increased,cleaved-caspase-8 increased significantly in HepG2-R.It showed that c-FLIP played an important role in the ERS pathway in regulation of resistant cells and inhibition of caspase-8 activation.Overexpression of c-FLIP can reduce the inhibitory effect of Jiedu Fang combined with sorafenib on resistant cells,and further confirms that Jiedu Fang can improve sorafenib resistance by reducing c-FLIP.4.Jiedu Fang can improve sorafenib resistance in vitro.The different concentrations of Jiedu Fang(JDF0.1mg/ml,JDF0.2mg/ml,JDF0.4mg/ml),sorafenib(Sora2.5μM,Sora5μM,Sora7.5μM)and Jiedu Fang(JDF0.2mg/ml,JDF0.4mg/ml,JDF0.5mg/ml),sorafenib(Sora5 M,Sora10 M,Sora15 M)were exposed to HepG2-R and Huh7-R for 48 h.We found the proliferation and survival rate were greater than 80%(p>0.05).The different concentration of JDF combined with sorafenib,it can significantly increase the inhibition rate of sorafenib resistant strains with significant difference(p<0.05)in a concentration dependent manner.By calculating the combined index(CI),we found that CIHep G2-R=0.58(JDF0.4mg/ml+Sora5μM)and CIHuh7-R=0.33(JDF0.4mg/ml+Sora5μM).And the results showed that it had a synergistic effect in JDF and sorafenib.By Western blot,with the combined effects of JDF and sorafenib,c-FLIP expression decreased in a dose-dependent manner,PERK and IRE1 pathway activation and Chop and Bip increased.5.The results in vivo showed that JDF combined with sorafenib could significantly inhibit the growth of subcutaneous tumor cells.The construction of Huh7,Huh7-R subcutaneous xenograft tumor model in nude mice,the mice of two groups were treated with low dose of sorafenib for 4 weeks.The subcutaneous transplantation tumor only appeared in nude mice of Huh7-R group,while had no obvious subcutaneous tumor was found in Huh7 group.In vivo it showed that the resistance model was constructed successfully.Vehicle,Sorafenib,JDF+Sorafenib,JDF and were administrated respectively after 15 days,the tumor weight of JDF group compared with those of Vehicle group,the difference was not statistically significant(p>0.05),and the tumor weight of the JDF+Sorafenib group is lower than that in the Vehicle group,and the difference was statistically significant(p<0.05).Through the detection of protein of tumor,JDF and sorafenib could activate IRE1 and PERK pathways,upregulate of the pro-apoptotic chop protein level at the same time,inhibite of cFLIP expression and activate caspase-8 consistent results in vitro.In addition,the pathological results of liver and kidney tissues in nude mice showed that JDF and sorafenib had no toxicity in vivo.Conclusion: 1.Sorafenib resistance is related with inhibition of caspase-8 activation induced by high expression of c-FLIP and autophagy activation and apoptosis inhibition through ERS.2.Jiedu Fang improve the resistance of sorafenib with the mechanism of regulation of c-FLIP/ERS.