Precise Discovery And Study On Biological Activity Of Anti-triple-negative Breast Cancer Of MRA STAT3-specific Inhibitors In Eupatorium Cannabinum Linn.

ObjectiveMichael reaction acceptors are a class of compounds containing electrophilic conjugated system functional groups formed by conjugation of ethylenic or acetylenic bonds with electron-withdrawing groups.They are important physiologically active molecules,which can exert its biological activity and therapeutic effect by regulating numerous signal transduction pathways in cells via covalent binding with cysteine or arginine residues.STAT3 is an important member of the signal transduction activation protein family.The activation of STAT3 regulates key genes to stimulating tumor cell proliferation,inducing tumor microvascular formation,promoting tumor cell metastasis,invasion and participating in the occurrence and development of tumor.Inhibiting the activation of STAT3,can block the occurrence of tumor and develop multiple pathways associated with.STAT3 Small molecule inhibitors have become one of the hot spots in cancer chemotherapy.STAT3 is an important biomarker for Triple-negative breast cancer(TNBC).The common structure of STAT3 inhibitors is that "α-β unsaturated carbonyl group or sulfone group",which is as the Michael reaction receptor(MRA).MRA is a kind of organic small molecule which has nice application prospect in biomedical field.And the MRA STAT3-specific inhibitor become a hot spot in the research of molecular targeted anti-TNBC drugs.In this paper,glutathione(GSH)was used as a probe,then,using UPLC-MS to rapidly identify the Eupatorium cannabinum Linn.from Eupatoritum L.in Compositae has Michael reaction acceptors.Under the guidance of incubating experiment with GSH as probe,according to the UPLC-MS spectrum,the change of binding peak size and pharmacodynamic activity in vitro,the existence of MRA type compounds in EEECL F4-F10 against triple-negative breast cancer(TNBC)cells was determined.In this study,EEECL F4-F10 was separated and purified by medium pressure column chromatography.The effective fraction of EEECL F4-F10 was obtained by HPLC analysis,then the inhibition of TNBC cells was detected in vitro.To further clarify the chemical structure and biological activity of these compounds,silica gel column chromatography(CC),Medium-Pressure Liquid Chromatography(MPLC),high performance liquid chromatography(HPLC),Ultra Performance Liquid Chromatography(UPLC)and semi-preparative high performance liquid chromatography(Semi-preparative HPLC)were used in this study.The chemical structures of these compounds were identified by 1H NMR,13C NMR,1H-1HCOSY,HSQC,HMBC,NOSEY,HR-ESI-MSI,ESI-MS,IR,the chemical structure of the chemical constituents and the chemical structures of the main compounds in ECL MRAs were elucidated.The inhibitory effect on TNBC cells was systematically evaluated in vitro.Methods1.The methods mainly including silica gel column chromatography(CC),middle pressure column chromatography(MPLC),high performance liquid chromatography(HPLC),ultra performance liquid chromatography(UPLC),preparative liquid chromatography and other modern chromatography technology were used to isolate and purify compounds contained in ECL MRAs.2.The modern spectroscopic techniques including 1H NMR,13C NMR,1H-1HCOSY,HSQC,HMBC,NOSEY,HR-ESI-MSI,ESI-MS,IR were used to analyze chemical structures of the compounds isolated from ECL MRAs.3.Constructing STAT3 luciferase reporter gene assay to screen STAT3 specific inhibitors.4.Microculture tetrazolium(MTT)assay was used to measure the inhibitory effect of the compounds on triple-negative breast cancer(TNBC)cells in vitro.5.Using medium pressure column chromatography to separate and purify EEECL F4-F10.6.The EEECL F4-F10 was analyzed by RP-HPLC,and the effective fraction named as ECL MRAs was obtained by merging the flow fraction.7.Using MTT assay to evaluate the inhibitory effect of ECL MRAs on TNBC cells in vitro.Results1.Using modern chromatography technology to isolate and purify compounds contained in ECL MRAs,we acquired 9 compounds.2.After using modern spectroscopic techniques to identify these compounds structure,we learn that they are all belong to sesquiterpene lactones,3 of them are new compounds.The chemical structure of them are(3aR,4R,6Z,9S,10E,11aR)-9-((2-hydroxy-3-methylbutanoyl)oxy)-6,10-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,8,9,11a-octahydrocyclodeca[b]furan-4-yl(E)-2-(acetoxymethyl)but-2-enoate(1);(3aR,4R,6Z,9S,10E,11aR)-9-hydroxy-6,10-dimethyl-3methylene-2-oxo-2,3,3a,4,5,8,9,11a-octahydrocyclodeca[b]furan-4-yl(E)-2-(acetoxymethyl)but-2-enoate(2);(3aS,4R,6Z,9R,10E,11aR)-9-hydroxy-6,10-dimethyl-3-methylene-2-oxo-2,3,3a,4,5,8,9,11a-octahydrocyclodeca[b]furan-4-yl(E)-2-(acetoxymethyl)but-2-enoate(3),which named as Eucannabnolide A-C;6 known compounds are hiyodorilactone B(4),9β-hydroxy-8β-O-tlgloyl costunohde(5),hiyodorilactone D(6),3β-Hydroxyl-8β-sarracinolyoxycostunolide(7),Eupafor-mosanin(8),Eucannabinolide(9).3.STAT3 reporter gene assay showed that EEECL has STAT3 inhibitory activity and compounds 2-6 and 8-9 also have STAT3 inhibitory activity.4.MTT assay showed that 9 compounds have significant anti-proliferative effect on human breast cancer cells MDA-MB-231 and MDA-MB-468 in vitro.5.EEECL F4-F10 had been separated and purified into flow fractions with MRAs by medium pressure column chromatography.6.The flow fractions with MRAs had been analyzed by RP-HPLC.According to the results of previous incubation experiments with GSH as probe,the effective fraction ECL MRAs was obtained by combining the flow fractions.7.The inhibitory effect of ECL MRAs on human breast cancer cells MDA-MB-231 and MDA-MB-468 had been determined by MTT assay in vitro.The IC50 was 3.297 μM,1.530μM.Conclusion1.The main components of MRA type compounds in Eupatorium cannabinum Linn.are sesquiterpene lactones.Nine sesquiterpene lactones were isolated and their chemical structures were identified.Among them,Eucannabnolide A-C are new compounds.2.The 9 sesquiterpene lactones have significant inhibitory effect on triple-negative breast cancer cells in vitro.3.STAT3 luciferase reporter gene detected that EEECL has inhibitory activity against STAT3,indicating that EEECL is a STAT3 specific inhibitor.Several compounds with STAT3 inhibitory activity were isolated and purified.Compounds 2-6 and 8-9 all have STAT3 inhibitory activity.These compounds are all STAT3 specific inhibitors.4.According to the results of incubation experiment with GSH as probe,the EEECL F4-F10 was separated and purified,the effective fraction is ECL MRAs.5.ECL MRAs was obtained by combining the flow fraction which has inhibitory effect on TNBC cells in vitro.6.To sum up,the main effective substance of MRAs are sesquiterpene lactones,which has anti-tumor effect in vitro.Small anti-tumor lead compounds are found from sesquiterpene lactones,which have a bright application prospect.ECL MRAs as a new anti-tumor drug has potential application values in tumor treatment.