The Methods And Molecular Mechanisms Of Enhancing Sorafenib Sensitization In Liver Cancer

Background and objective:Liver cancer is one of the most common malignant tumors.In addition to radiotherapy,surgery and interventional therapy,chemotherapy is also an important means of treatment for liver cancer,but it is currently very difficult to cure liver cancer using available chemotherapy drugs.It produces severe drug resistance and therapeutic resistance.Therefore,it is particularly important to enhance the therapeutic effect of chemotherapy drugs and adopt new drug combination programs.Sorafenib,the first FDA-approved molecular drug targeted for liver cancer,is widely recognized for its effectiveness and safety,and has a significant effect on prolonging the progression of disease and survival.However,a considerable number of patients receiving sorafenib not only had no definite curative effect,but also increased the side effects and heavy financial burden.Therefore,how to inhibit the occurrence of sorafenib resistance in liver cancer cells and improve their therapeutic effects has gradually become a hot spot in recent years.The autophagy-related 4B(ATG4B)is a key protein in the regulation of autophagy in mammalian cells.The activity of ATG4 B is closely related to the occurrence and development of liver cancer and chemotherapy resistance.Acetylation/deacetylation modification is an important way to regulate protein activity,but whether it is involved in the regulation of ATG4 B activity has not been reported.Alteration of ATG4 B acetylation may modulate autophagy by affecting ATG4 B activity.Since the elevation of protective autophagy is an important mechanism leading to chemo-resistance,we investigated whether the signal axis of ATG4 B acetylation/ATG4 B activity/autophagy regulates the sorafenib resistance in liver cancer cells.Based on this,the methods to enhance the sensitivity of sorafenib in liver cancer cells were designed,and so provide a new strategy and basis for overcoming the resistance of sorafenib.The resistance of sorafenib is closely related to the hypoxia microenvironment of tumor cells.Dichloroacetate(DCA)is an inhibitor of pyruvate dehydrogenase(PDH)kinase and an important anti-metabolic drug.Studies have shown that DCA can inhibit the proliferation of tumors and enhance the anti-tumor effect of multi-class chemotherapy drugs.Because DCA has the effect of reversing tumor glycolysis and changing the acidic microenvironment around tumor cells,it has become an attractive tumor chemotherapeutic agent.Therefore,to explore whether DCA can enhance the effect of sorafenib and associated molecular mechanism,we checked the effect of DCA combined sorafenib on cell growth,apoptosis and autophagy of liver cancer cells.Contents and methods:Part ? Inhibition of SIRT2-ATG4B(Acetylation)-autophagy pathway enhances the effect of sorafenib on liver cancer1.CCK-8 experiments were performed to confirm the effect of sorafenib on the survival of hepatoma cells and the induction of autophagy.2.Immunoprecipitation and immunoprecipitation combined with mass spectrometry were used to identify whether ATG4 B is acetylated and its possible modification sites.3.Immunoprecipitation and CCK-8 experiments were used to demonstrate the role of ATG4 B acetylation on sorafenib-induced protective autophagy.4.Immunoprecipitation,Western blot,gene silencing technology and deacetylase inhibitors were used to screen the deacetylase or acetylase which mediates ATG4 B acetylation.5.Immunoprecipitation,Western blot and AU4 S system were used to study the role of SIRT2 on ATG4 B acetylation and ATG4 B functional activity.6.Specific site mutations,Western blot,immunoprecipitation and CCK-8 assays were used to identify the acetylation sites of ATG4 B mediated by SIRT2 and their biological significance.7.Gene silencing technology was used to analyze the effect of inhibiting SIRT2 on autophagy and proliferation inhibition in sorafenib-treated liver cancer cells.8.Confirming the role of the SIRT2-ATG4B(Acetylation)-autophagy axis on enhancing sorafenib sensitization in liver cancer in vivo.Part ? The effect of dichloroacetate combined with sorafenib on liver cancer1.CCK-8 assay was used to assess the sensitizing effect of dichloroacetate on sorafenib-treated liver cancer cells.2.Flow cytometry and Western blot were used to explore the effect of DCA combined sorafenib on the apoptosis of liver cancer cells.3.Western blot and gene over-expression technology were used to detect the effect of the combination treatment on anti-apoptotic protein levels in liver cancer cells.4.Protein synthesis and degradation were inhibited at the transcriptional level and proteasome level respectively,and the mechanisms of anti-apoptotic protein levels changes upon the combination treatment were analyzed.5.Flow cytometry was used to detect the ROS level after the combination treatment in liver cancer cells.6.Western blot was used to detect the effect of the combination treatment on the levels of JNK activation.7.Western blot and CCK-8 experiments were used to explore the effect of the combination treatment on autophagy.8.Confirming the effect of DCA on enhancing sorafenib sensitization in liver cancer in vivo.Results:Part ? Inhibition of SIRT2-ATG4B(Acetylation)-autophagy pathway enhances the effect of sorafenib on liver cancer1.Sorafenib induces protective autophagy,which plays an important role on the process of liver cancer cells resisting to sorafenib.2.ATG4 B can be acetylated.3.The level of acetylation of ATG4 B is related to the ATG4 B activity and the level of autophagy after treatment by sorafenib.4.SIRT2 is a key deacetylase that regulates the acetylation of ATG4 B.5.SIRT2 regulates the activity and function of ATG4 B by deacetylating lysine at 39 and 259 sites.6.Inhibition of SIRT2-ATG4B(Acetylation)-autophagy pathway can enhance the effect of sorafenib on liver cancer cells.7.Suppression of the SIRT2-ATG4B(Acetylation)-autophagy axis enhanced sorafenib sensitization in liver cancer in vivo.Part ? The effect of dichloroacetate combined with sorafenib on liver cancer1.Dichloroacetate can significantly enhance the killing effect of sorafenib on liver cancer cells.2.The combination treatment can significantly enhance the apoptosis rate of liver cancer cells.3.The combination treatment can significantly reduce the levels of anti-apoptotic proteins Mcl-1 and Bcl-2 by promoting their degradation.4.The combination treatment significantly enhanced the ROS level and JNK activity in liver cancer cells.5.The combination treatment significantly activated the ROS/JNK pathway and promoted apoptosis of liver cancer cells.6.The combination treatment induces protective autophagy in liver cancer cells.7.DCA significantly enhanced sorafenib sensitization in liver cancer in vivo.Conclusion:1.In liver cancer cells,sorafenib activates the SIRT2-ATG4B(Acetylation)-autophagy pathway,resulting in a significant decrease on ATG4 B acetylation at 39 and 259 sites,enhancing the functional activity of ATG4 B and increasing autophagy.The enhanced autophagy protects the cells from apoptosis,resulting in the resistance of liver cancer cells to sorafenib treatment.Targeting SIRT2 or ATG4 B acetylation modification can enhance the killing effect of sorafenib on liver cancer.2.Dichloroacetate significantly enhanced the killing effect of sorafenib on liver cancer cells.The related mechanism was closely related to the activation of ROS/JNK pathway and the decrease of anti-apoptotic proteins Mcl-1 and Bcl-2.Dichloroacetate combined with sorafenib can significantly promote the increase of ROS level and JNK activation,and decrease the levels of Mcl-1 and Bcl-2.At the same time,the decrease of Mcl-1 also enhances the level of protective autophagy of liver cancer cells.The results of these experiments provide a new idea and basis for the clinical use of sorafenib in liver cancer.