| Objective:Endocrine therapy plays an important role in the treatment of breast cancer.Tamoxifen,which targets the estrogen receptor(ER),has changed the history of female endocrine therapy.However,tamoxifen can excite some estrogen receptors and cause adverse reactions such as endometrial cancer.Fulvestrant,a pure inhibitor of estrogen receptor,is approved for advanced breast cancer,which not only competes with the estrogen receptor but also protects the ER receptor by binding to ER.But its biological activity and utilization in vivo is limited.HSP90 is a member of chaperone family involved in the folding,transport and degradation of various proteins.HSP90 is closely related to the occurrence,development and resistance of breast cancer.Firstly,ER / PR,which is related to the proliferation of estrogen receptor positive breast cancer cells,is the chaperone of HSP90.Second,the AKT signaling pathway which associated with breast cancer drug resistance,is a HSP90 chaperone protein;at the same time,HER2,Raf is also the chaperones of HSP90.Therefore,the role of HSP90 in breast cancer is particularly worth studying.XH formula has been used for the treatment of breast cancer since 1740,it was founded by the well-known doctor Wang Wei De of the Qing Dynasty and recorded in the "Wai Ke Zheng Zhi Quan Sheng Ji",then many of the ancient books mentioned its role in the treatment of breast cancer.XH formula consists of olibanum,myrrh,calculus bovis,musk.Modern pharmacological studies have found that XH formula have a therapeutic effect in breast cancer,lung cancer,colon cancer and lymph node metastasis.However,its mechanism has not been further studied.In order to understand the multi-target and multi-component prescriptions of XH formula for the treatment of breast cancer,this study predicted the main active components and targets of XH formula against breast cancer using network pharmacology and molecular docking.And the predicted results were verified by in vitro and in vivo studies.Methods1.To detect the proliferation of ER-positive cell line,HER2-positive cell line andtriple negative breast cancer cell line,Type-blue staining method and CCK8 cytotoxic activity assay were used.The nude mouse breast cancer cell lines(T47D,SKBr3)xenografts were used to detect the inhibitory effect of XH and its single agents in vivo.The effect of XH and its single agents on the proliferation of xenografts was also detected by PNCA immunohistochemistry.2.Flow cytometry,Annexin V-FITC / PI apoptosis detection kit and PI reagent were used to detect the effect of XH and its single active ingredients on cell cycle and apoptosis of ER positive cells,HER2 positive cells,and triple negative breast cancer.TUNEL kit was used to detect the apoptosis of xenografted tumor in nude mice.3.Wound-healing experiment and Transwell chamber assay were used to detect the migration of XH and the single agents on the migration of ER positive cell line,HER2 positive cell line and triple negative breast cancer cell line.4.Apply the TTD database to find the existing drug targets of breast cancer,and refer to the relevant literature to build molecular targets of breast cancer.5.Apply TCMSP database to find out the drug components of XH,and use PUBCHEM to search the 3D structure of drug components,and then use Chemmaper to search the predicted target of drug components.6.Using cytoscape to construct and analyze the target of XH and the molecular target of breast cancer,draw the single component-target map,and predict the main active ingredients of XH exerting effects on breast cancer and their targets.7.Using systemsDock to conduct molecular docking of the main active ingredient and target of XH,analyze its binding site and binding affinity with the target.8.The results of the experiments were validated by Western blot to verify the target protein expression,immunofluorescence to verify the characteristics of intracellular expression,co-immunoprecipitation was used to detect protein interactions,immunohistochemical was used to detect the protein expression of xenograft tumor.The mRNA expression after the XH treatment was detected by RNA-seq assay and RT-PCR.The ITC assay(Isothermal Titration Calorimetry)wasused to verify the binding of the major active ingredient to the estrogen receptor.9.The cytotoxic activity was compared after XH treatment on different breast cancer cell lines using CCK8.Results:1.The inhibitory concentration(IC50)of XH on ER positive cell lines MCF-7,T47 D were 49.39 μg/mL and 43.78 μg/mL,respectively.The inhibitory effect(IC50)of XH on HER2 positive breast cancer cell lines and triple negative Breast cancer cell lines,were 105.50 μg/mL,138.70 μg/mL.2.In XH formula,the ratio of olibanum,myrrh,calculus bovis,musk is 30: 30:4.5: 1,but the IC50 of calculus bovis,musk in all four breast cancer cell lines were >200 μg/mL.Therefore,the directed anti-proliferation of calculus bovis,musk in XH formula is weak.3.XH and its single agent also have inhibitory effect on the breast cancer cell xenografts.The inhibitory rate on ER-positive breast cancer cells T47 D was 72.54%and the tumor inhibition rate on HER2-positive cell line SKBr3 was 56.05%.4.XH and its main components of olibanum,myrrh were able to block the cell cycle,induce apoptosis and inhibit cell migration.XH and its main components of olibanum,myrrh also inhibited the transplanted tumor in vivo.5.XH has the characteristics of multi-component and multi-target.According to the prediction of network pharmacology,there are 120,264,16,19 active components respectively in olibanum,myrrh,calculus bovis,musk and 641,645,369,621 predicted targets,respectively;among which there were 30,34,21,33 breast cancer-related targets.6.The main active ingredient of XH against breast cancer is20α-hydroxy-4-pregnen-3-one(20αHP),Guggulsterone,Guggulsterol V,Guggulsterone M,Guggulsterol-I,Olibanumol I,Acetyl-β-boswellic acid,Acetyl-α-boswellic acid,β-boswellic acid.The main targets of XH for the breast cancer treatment are ER,HSP90,VEGF,EGFR and Bcl-2.7.Experimental studies had confirmed that XH could block the binding of ERαand HSP90 binding;promote the degradation of ERα through the proteasome pathway,and hinder ERα into the nucleus.XH does not affect mRNA expression of ERα nor the protein and mRNA expression of HSP90.However,XH can reduce theexpression of other HSP90 chaperones,such as EGFR,HER2,ErbB3,Raf,Erk and Akt.8.ITC experiments(Isothermal Titration Calorimetry)studies have confirmed that Guggulsterone,the main active ingredient of XH,could bind to ERα and The values of Ka,ΔH and ΔS were 348 ± 24.4/M,-2037 ± 24.4cal/mol,5.06 cal/mol/deg,respectively.The energetics of estradiol compound binding to ERα was measured using ITC.The binding reaction was exothermic and exhibited slope of the ITC curve indicating binding reaction.The values of Ka,ΔH and ΔS were 652 ± 100/M,-1770 ±16.23 cal/mol,7.17 cal/mol/deg,respectively.9.Guggulsterone and 20αHP,the main active ingredients of XH,which only can bind to ERα,inhibited the activity of ER-positive cell lines,but can not inhibit the activity of HER-2-positive cell lines and triple negative breast cancer.Acetyl-β-boswellic acid,Acetyl-α-boswellic acid and β-boswellic acid,which can bind to HSP90,have significant inhibitory effect on ER positive cell line,HER2 positive cell line and triple negative breast cancer.ConclusionIn conclusion,we found that XH,which has been used since 1740,has antiestrogenic effects in ER+ breast cancer via causing the degradation of ERα,antagonizing its effect and disrupting nuclear localization,similar to fulvestrant.More importantly,XH also caused the dissociation of ERα and other oncoproteins via binding to HSP90.The major active ingredients are 20α-hydroxy-4-pregnen-3-one(20αHP),Guggulsterone,Guggulsterol V,Guggulsterone M,Guggulsterol-I;Olibanumol I,Acetyl-β-boswellic acid,β-boswellic acid. |
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