Research On The Identification Of Human Targets Of (20S)G-Rh2 And Molecular Basis For Its Ant-Cancer Activity

(20S)G-Rh2 is a secondary saponin generated by the process from ginseng to red ginseng,which present superior anti-cancer activity.Recent researches reveal that(20S)G-Rh2 induces apoptosis in multiple cancer cells including leukemia cells,cervical cancer cells,ovarian cancer cells,liver cancer cells and breast cancer cells,and both mitochondria-dependent pathway and membrane-death-receptor-dependent pathway are simultaneously activated under(20S)G-Rh2 treatment.Researches on molecular mechanism demonstrates that(20S)G-Rh2 induces cell cycle arrest at G1 phase via the inhibition of JNK,Akt,MAPK and ?-catenin signaling pathway in cancer cells,which inhibits the growth of cancer cells.In breast cancer cells,(20S)G-Rh2 inhibits the multiple drug resistance induced by microRNAs.In polymorphic malignant glioma cells,(20S)G-Rh2 down-regulates VEGF-A,inhibits EGFR/PI3k/Akt/mTor signaling pathway and finally narrow the invasiveness of cancer cells.Moreover,animal studies prove that(20S)G-Rh2 inhibits the growth of various implantation tumors in mouse xenograft model.In the article,interacting proteome of(20S)G-Rh2 was studied,aiming at revealing the cellular targets of(20S)G-Rh2,which finally explained the molecular basis for the anti-cancer activity of(20S)G-Rh2.In order to explore the interacting proteins of(20S)G-Rh2,we performed an affinity pull-down analysis with(20S)G-Rh2-PEGA resin and(20R)G-Rh2-PEGA resin respectively in HepG2 lysates,and finally acquired 381 proteins by liquid chromatography tandem mass spectrometry.113 proteins within were collected with high binding capability to(20S)G-Rh2 after abundance of each protein attached to(20S)G-Rh2-PEGA resin and(20R)G-Rh2-PEGA resin was compared and analyzed.We finally identified 13 proteins out of 113(20S)G-Rh2 interacting proteins were over-expressed in hepatocellular carcinoma with the help of gene expression database.Taking the advantage of GO analysis of(20S)G-Rh2 interactome,we finally assigned Annexin A2 to further function study.Annexin A2 is a multi-function proteins involved in cellular processes as cell proliferation,cell migration and cell adhesion.Annexin A2 constructs protein complexes with S100 family members in a membrane-dependent manner,promoting the recruitment of proteins onto the surface of the membrane,which enhances cell proliferation and cell migration.None-membrane-attached Annexin A2 promotes the activation of transcription factors including STAT3,STAT6 and NF-?B,up-regulating the expression of inflammatory cytokines and anti-apoptosis genes,which enhances cell survival.Annexin A2 is excessively expressed in multiple cancers,promoting survival,growth and migration of cancer cells as well as the resistance to chemicals.In order to determine the interaction between Annexin A2 and(20S)G-Rh2,we performed isothermal titration calorimetry,molecular docking studies,competitive pull-down analysis and thermal shift assay,and finally identified that Annexin A2 interacted with(20S)G-Rh2,demonstrating Annexin A2 was a cellular target of(20S)G-Rh2.NF-?B functions as an accelerant in carcinogenesis and development of hepatocellular carcinoma.Liver injury at the early stage activates NF-?B,which up-regulates the expression of pro-survival genes and prevent the death of injured cells.The constitutive activation of NF-?B simultaneously results in the expression and secretion of inflammatory cytokines,promoting cancerization of normal cells nearby,which contributes to the carcinogenesis.NF-?B also enhances the expression of anti-apoptosis genes after a tumor takes shape,facilitating the generation of chemical resistance.The over-expression of cytokines and chemokines regulated by NF-?B rebuilds surrounding environment,promoting cancerization of normal cell for another time as well as tube formation.Cellular studies reveals Annexin A2 binds to NF-?B p50 subunit,enhance their nuclear co-localization,promote NF-?B activation,and up-regulates the expression of anti-apoptosis genes,which contributes to the generation of chemical resistance and promotes cancer development.In the present study,(20S)G-Rh2 interacted with Annexin A2,prevented its interaction and nuclear co-localization with NF-?B p50 subunit,inhibited the activation of NF-?B,and down-regulated the expression of anti-apoptosis genes,which enhanced apoptosis in Hep G2 cells and suppressed proliferation of HepG2 cells.In the article,the interaction of Annexin A2 and other ginsenosides was determined by molecular docking study and thermal shift assay,and G-Rg5,G-Rk1,and C-K were proven able to bind to Annexin A2.All of three ginsenosides interacted with Annexin A2,prevented its interaction and nuclear co-localization with NF-?B p50 subunit,inhibited the activation of NF-?B,and down-regulated the expression of anti-apoptosis genes,which enhanced apoptosis in HepG2 cells and suppressed proliferation of HepG2 cells.The main achievements:1.We constructed the study method of(20S)G-Rh2-PEGA mediated pull-down attached to liquid chromatography tandem mass spectrometry to identify(20S)G-Rh2 interacting proteome,and finally acquired(20S)G-Rh2 interactome as well as its GO characteristics and relative expression level in hepatocellular carcinoma.2.We identified the interaction between Annexin A2 and(20S)G-Rh2 via isothermal titration calorimetry,molecular docking studies,competitive pull-down analysis and thermal shift assay,and constructed Annexin A2-K302 A mutant with binding deficiency to(20S)G-Rh2.3.We reveal the molecular basis for the anti-cancer activity of(20S)G-Rh2.(20S)G-Rh2 interacted with Annexin A2,prevented its interaction and nuclear co-localization with NF-?B p50 subunit,inhibited the activation of NF-?B,and down-regulated the expression of X-IAP,c-IAP1,c-IAP2 and Surivin,promoting the activation of Caspase 9,resulting in the apoptosis in HepG2 cells.4.We screened for three nature compound antagonists for Annexin A2 including G-Rg5,G-Rk1 and C-K,and reveal their molecular mechanisms of anti-cancer activity.G-Rg5,G-Rk1 and C-K interacted with Annexin A2,prevented its interaction and nuclear co-localization with NF-?B p50 subunit,inhibited the activation of NF-?B,and down-regulated the expression of X-IAP,c-IAP1,c-IAP2 and Surivin,promoting the activation of Caspase 9,resulting in the apoptosis in HepG2 cells.