Research On The Anti-tumor Effect Of Rare Ginsenosides Targeting And Inhibiting The HSP90A-Cdc37 System

Ginsenosides are bioactive ginseng metabolites,which have curative efficacy in the treatment of inflammation,cancer,diabetes,and hyperlipidemia.The major ginsenosides Rb1,Rb2,Rg1,Re,Rf,Rc,and Rd are in high abundance,accounting for 80–90%of the total ginsenoside content.However,the major ginsenosides have lower pharmacological activity and bioavailability due to their large glycosyl groups and molecular weight.Red ginseng is a processed product obtained from ginseng roots after steaming and drying,and it is rich in a variety of rare ginsenosides,which are derived from the deglycosylation of major ginsenosides during ginseng processing.For example,the diol-type ginsenosides Rb1,Rd,and Rc are deglycosylated on carbon-20during heat treatment and gradually transformed into the rare ginsenosides(20S)G-Rg3,(20R)G-Rg3,(20S)G-Rh2,(20R)G-Rh2,G-Rg5,and G-Rk1.Numerous studies have shown that rare ginsenosides have higher bioactivity and oral bioavailability,so they are more suitable for drug development.In recent years,people's interest in rare ginsenosides has focused on their anti-tumor mechanisms.Our previous studies have shown that(20S)G-Rh2 can initiate both exogenous and endogenous apoptotic pathways,thus inducing apoptosis in human cervical cancer He La cells,non-small cell lung cancer A549cells,liver cancer Hep G2 cells,and breast cancer MCF-7 cells.In addition,rare ginsenosides such as C-K,G-Rk1,and G-Rg5 also have significant inhibitory effects on various cancer cells.We selected(20S)G-Rh2 as our research subject,aiming to investigate the anti-tumor mechanism of(20S)G-Rh2 by screening and studying its intracellular targets of liver cancer cells.PEGA resin covalently conjugated with(20S)G-Rh2 or(20R)G-Rh2 was used to enrich the proteins in Hep G2 cell lysate.Then,the proteins eluted from PEGA resin were analyzed by liquid chromatography tandem mass spectrometry,and we found 355 proteins bound to both(20S)G-Rh2 and(20R)G-Rh2 to a certain extent.Based on the enrichment difference in these two ginsenosides,we obtained 214 proteins that preferred to bind(20S)G-Rh2(Ratio R/S<1).Combined with the investigation of liver cancer-related genes in Gene Cards database,25 targets of(20S)G-Rh2 were further screened(including STAT3,HSP90AA1,TRIP13,HSPB1,SFN,ACTB,KRT18,NPM1,HSPA5,ITGB1,KRT8,HSPA9,MSH2,HSPA4,PHB,HSP90B1,ANXA2,IQGAP1,COX5A,CD44,FASN,MSH6,BSG,LRRC59,and PC).We inferred that HSP90A(encoded by HSP90AA1)may be the critical target for(20S)G-Rh2 to inhibit the proliferation of liver cancer cells through network pharmacology analysis and enrichment analysis of these 25potential targets.Investigation of the TCGA hepatocellular carcinoma dataset revealed that the HSP90A level was positively correlated with the grade of tumor malignancy and negatively correlated with the overall survival of patients,indicating that HSP90A indeed plays a key role in the development of liver cancer.The Hsp90 family includes HSP90A,HSP90B,Grp94,and Trap1.Their major function is to help the proper folding of nascent peptides and promote the maturation of hundreds of proteins.Hsp90 is highly expressed in a variety of tumor tissues,and the high expression of Hsp90 is considered to be a marker of poor prognosis in various cancers,including bladder cancer,esophageal cancer,and lung cancer.Hsp90 can regulate the growth,adhesion,invasion,metastasis,and angiogenesis of tumor cells by stabilizing and activating oncoproteins.To verify the interaction between HSP90A and(20S)G-Rh2,we performed molecular docking analysis and thermal shift assay.The results demonstrated that(20S)G-Rh2 can bind to HSP90A,and may occupy both the ATP binding site and the Cdc37 binding site on HSP90A.This suggested that(20S)G-Rh2 may disrupt the interaction of HSP90A with Cdc37.As one of the most well-studied Hsp90 co-chaperones,Cdc37 takes part in the regulation of various kinases.First,activated Cdc37 selectively recognizes and binds unfolded clients,recruiting them to the Hsp90 homodimer.Then,Cdc37 is dephosphorylated and dissociated from the complex,and the Hsp90 dimer undergoes a conformational change and hydrolyzes ATP to complete the correct folding of client proteins.Based on the above molecular mechanism,we performed co-immunoprecipitation experiments and confirmed that(20S)G-Rh2 can indeed inhibit the interaction of HSP90A and Cdc37,indicating that the maturation of their CDKs clients would be affected.To verify this hypothesis,we analyzed the levels of cell cycle-related proteins by immunoblotting.The results showed that the levels of CDK1,CDK2,CDK4,and CDK6 decreased significantly under treatment with(20S)G-Rh2.In addition,the levels of Cyclin A,Cyclin B,and Cyclin E were significantly down-regulated,while the levels of Cyclin D and p21 were up-regulated.Cyclin D-CDK4/6,a cell cycle G₀-G₁ phase promoter,can phosphorylate Rb to relieve its inhibition on E2F transcription factors.As we expected,the phosphorylation level of Rb decreased significantly,and the cell cycle was arrested at G₀-G₁ phase in treatment of(20S)G-Rh2.Cyclin E is the regulator of G₁-S phase,and Cyclin A is the regulator of S phase.They are also the downstream targets of E2F.Meanwhile,the results showed that the levels of Cyclin E,Cyclin A,and Cyclin B were also decreased in a dose-dependent manner of(20S)G-Rh2.In the same conditions,the Cyclin D and p21 level was significantly up-regulated,and we suggested that the decrease in CDK4 and CDK6 led to the compensatory upregulation of Cyclin D.The results of cell viability assay showed that(20S)G-Rh2 can inhibit the proliferation of Hep G2 cells and SK-HEP-1 cells in a dose-dependent manner with IC₅₀ values of 5.02?M and 5.25?M,respectively.The results of quantitative real-time PCR demonstrated that the m RNA levels of CDK2,CDK4,and CDK6 showed an increment instead of a decrement under treatment with(20S)G-Rh2,indicating that the decrease in CDKs protein level was due to protein degradation.We then investigated the degradation pathway of CDKs,and found that(20S)G-Rh2-induced CDKs degradation was mediated by the proteasome pathway rather than the autophagy-lysosome pathway.Taken together,we have elucidated the anti-tumor mechanism of(20S)G-Rh2 in liver cancer cells.(20S)G-Rh2 can inhibit the HSP90A-Cdc37 system by targeting HSP90A,thus disrupting the maturation of CDKs and leading to a G₀-G₁ phase arrest of liver cancer cells.Based on the structural similarity of ginsenosides,we calculated the probability of HSP90A interacting with other ginsenosides using molecular docking,and selected G-Rk1 and G-Rg5 for further study.We then successively elucidated the anti-tumor mechanism of G-Rk1 and G-Rg5 in Hep G2 cells according to the previous(20S)G-Rh2 study.G-Rk1 and G-Rg5 can both inhibit the interaction of HSP90A and Cdc37 by targeting HSP90A,thus affecting the proper folding of CDKs and inducing a G₂-M phase arrest of liver cancer cells.Ginsenosides,as natural products from ginseng,are different from other synthetic compounds.They can target multiple proteins to modulate the disease-related signaling pathways,which reduces the probability of drug resistance.For example,we found that(20S)G-Rh2 can promote the apoptosis of tumor cells by targeting and inhibiting Annexin A2 in our previous study.Moreover,ginsenosides have moderate affinities with target proteins,and tend to have less side effects,which make ginsenosides a promising anti-tumor adjuvant drug in cancer therapy.