CN102058580A - New application of 15-oxospiramilactone to inhibition of Wnt signal path - Google Patents
New application of 15-oxospiramilactone to inhibition of Wnt signal path Download PDFInfo
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Abstract
The invention discloses 15-oxospiramilactone capable of inhibiting a classic Wnt signal path. Therefore, the 15-oxospiramilactone can be applied to preparing medicaments for adjusting the classic Wnt signal path and can also be applied to researching the classic Wnt signal path. The 15-oxospiramilactone provides a new thought of screening the medicaments for adjusting a Wnt signal transduction path or medicaments for preventing and/or treating diseases associated with the Wnt signal path.
Description
Technical field
The present invention relates to drug world, in particular, the present invention relates to of the application of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone as novel Wnt signal pathway inhibitor.
Background technology
The Wnt signal pathway is at the conservative signal transduction pathway of organism evolutionary process camber, regulates the numerous vital movement processes of control.In the animal body early development, a series of critical events such as the formation of Wnt signal deciding dorsoventral axis, germinal layer foundation; And directly control propagation, differentiation, apoptosis and the isocellular destiny of anti-apoptosis.
The Wnt signaling molecule is a class secreting type glycoprotein family, its tens members by with cell membrane on different receptor acting participate in the unlike signal approach.These approach mainly are divided into classical Wnt signal pathway (Wnt/ beta-catenin) that depends on beta-catenin/TCF transcription complex and non-classical Wnt signal pathway (Wnt/JNK approach) and the Wnt/Ca2+ signal pathway that does not rely on beta-catenin/TCF transcription complex.Classical Wnt signal research is comparatively thorough, and its abnormal activation is that the Wnt signal pathway participates in tumor, for example colon cancer, breast carcinoma and pulmonary carcinoma, and the main mode of the generation of some familial heredopathias such as Alzheimer or multicystic kidney disease, development.The classical Wnt signal transduction path also plays an important role in the regulation and control bone development.
When in the classical Wnt approach, not having the Wnt signal, APC, GSK3 β, CK I and axle albumen (Axin) albumen form a degraded complex, APC and axle albumen as the scaffolding protein in the complex make CKI specifically in the phosphorylation endochylema Ser-45 of beta-catenin then GSK3 β again its Thr-41, Ser-37 and Ser-33 are carried out phosphorylation [1-3].The beta-catenin of phosphorylation form is discerned by F-box albumen β-TrCP after ubiquitinization, finally by proteasome degradation pathway be degraded [4-6].The C of beta-catenin end includes a transcriptional activation domain [7], but itself is not in conjunction with DNA but be attached on the transcription factor TCF/LEF.Other protein can be regulated their transcriptional activity on specific promoter by mutually combining with this transcription complex.The transcriptional activation ability increases [8] on the c-MYC site going out thereby the lys-49 that acetyltransferase CBP can the acetylation beta-catenin causes them.CBP is raised specific promoter and acetylated histones makes that thereby the loose transcription complex of chromatin can be better in conjunction with promoter sequence [9].The activity of beta-catenin/TCF in the nuclear has been subjected to legless (Lgs) [10] and pygopus (pygo) [10-13] regulates.Wherein legless is as the bridge that connects beta-catenin and pygopus, and pygopus thinks that then its NHD may regulate the activation that albumen promotes beta-catenin in conjunction with certain.TCF must be in conjunction with beta-catenin competence exertion transcriptional activation [7,14].When not having the Wnt signal, TCF/LEF is last in conjunction with the Profilin Groucho[15 of wide expression, 16], thus it can cause chromatic agglutination to suppress the transcribing of target gene [17] of TCF by raising histon deacetylase (HDAC).When Wnt albumen is attached to its seven transmembrane receptor Frizzle and co-receptor LRP5, [18-24] when LRP6 is last simultaneously, axle albumen can move near the cell membrane and interact and become thereupon unstable with LRP5, simultaneously thereby Wnt is attached to and can causes the excessive phosphorylation of Dsh to suppress the activity of GSK3 β on the Frizzle and beta-catenin is escaped the destiny that is degraded and accumulated, enter transcribe [25,26] that nucleus and TCF/LEF open the downstream target gene subsequently.
The target gene such as the c-myc in classical Wnt signal downstream, cyclin D1, MMP-7 etc. participate in physiological process such as cell growth, propagation, angiogenesis, and the abnormal activation and the cancer of Wnt/ beta-catenin approach have important relation.Have now found that colon cancer, breast carcinoma, tens kinds of frequently-occurring cancers such as pulmonary carcinoma all have relation with Wnt/ beta-catenin abnormal activation.Colon cancer (CRC) patient body inner cell above 90% all contains a sudden change that can activate the classical Wnt signal, finally causes beta-catenin stable and accumulation in nucleus.Beta-catenin is the sign that the classical Wnt signal is activated in the nuclear, and the slight abnormality that the sudden change of Wnt signaling molecule causes also can be seen the obvious accumulation of examining interior beta-catenin.Because the sudden change of classical Wnt signal often appears on the CRC, so CRC becomes research Wnt/ beta-catenin abnormal signal activation and cancer relation than proper model.[27]
The Wnt abnormal activation can produce carcinogenesis, and in animal model and tissue lines, Wnt and neoplastic transformation have direct relation.Studies show that at present the unusual generation with human tumor of Wnt gene and regulatory factor thereof may be relevant.For example, relevant [28] may take place, develop with colon cancer with Wnt-5a in Wnt-2.DKK-1 inactivation in colon cancer progress, also may play an important role [29].Wnt11 expresses in stomach cancer cell line MKN45 and cervical cancer cell strain SKG-IIIa all rise, and in a lot of human primary tumors such as adenoma of colon, the level of mRNA all has rise [30] in the renal cell carcinoma.Wnt2 in gastric cancer because its mRNA level of interaction of tumor and substrate has rise, and at the colon cancer polyp, colon cancer A-C phase and colon cancer also frequent up-regulated in the transfer process of liver, it has become the label [31] of gastric cancer and colon cancer.
The relation that apc gene and colon cancer take place is very close, can cause beta-catenin stable and accumulation in nucleus after its sudden change, causes the colon epithelial cell hyperplasia, forms polyp, finally forms tumor.Can detect apc gene deletion mutation or inactivation in the about 80% sporadic colon cancer, and the disappearance of apc gene or inactivation are acknowledged as the early stage incident that colon cancer takes place.
Axle albumen is the important member of APC/ axle albumen/GSK degraded complex, APC or beta-catenin sudden change do not take place in some colons and the rectum cancer cell system, but sudden change has taken place the axle protein gene.These sudden changes cause the disappearance of Dsh or GSK binding site, cause the nuclear accumulation [32] of beta-catenin.In the HCC (hepatocarcinoma) that does not have the CTNNB1 sudden change, also find to have the proteic sudden change of axle equally.
The gene of beta-catenin (CTNNB1) sudden change itself also is the reason that causes it to accumulate unusually in kytoplasm.The normal localization and expression of beta-catenin cell membrane reduces in the colon of 70-84% and rectal cancer, and kytoplasm and nuclear beta-Lian abnormal protein are expressed and increased in 66-79% colon and rectum cancer tissue.
Tcf family comprises 4 different albumen Tcf-1, Tcf-2, Tcf-3, Tcf-4/Lef.Tcf-4 gene in the generation of colon cancer, play an important role [33].All can cause beta-catenin behind a variety of causes excessive activation Wnt signal pathway and accumulate in kytoplasm, free beta-catenin enters in the nuclear and combines the formation complex with Tcf-4, promotes tumor to take place and cell cycle progression thereby start transcribing unusually of downstream target gene.These target bases prisoner is how relevant with apoptosis, cell growth, angiogenesis and tumor invasion transfer, as c-myc, cyclin D1, MMP-7 etc.
Because Wnt/ beta-catenin signal pathway is relevant with numerous cancers, thus Recent study its in cancer takes place effect and become the domestic and international research focus at its therapeutic strategy.Existing therapeutic strategy comprises that the multiple non-cell toxicity medicine that utilizes regulates and control this approach, and non-steroidal anti-inflammatory drug for example is as indometacin (indomethacin) and aspirin [34,35] and sulindac (sulindac) [36]; Plant compounds, for example one of composition of curcumin (curcumin) [37], LVHUA Broccoli (broccoli) Sulforaphane (sulforaphane) [38] and β-lapachol [39]; And at the molecular targeted property treatment of this approach signal of interest molecule, for example Antybody therapy; Micromolecular inhibitor, as, tyrosine kinase inhibitor imatinib mesylate (Glivec/Gleevec) [40], blood vessel endothelium chalone (endostatin) [41,42]; And gene therapy, be the gene therapy method of target spot for example with the beta-catenin, with Tcf-4 gene therapy method [43,44] of target spot etc.
In sum, the target for modulation and the means of downward modulation Wnt/ beta-catenin signal pathway mainly contain three kinds: 1. cross by Wnt antibody, sFRP and DKK on extracellular and cell membrane level and be expressed in extracellular blocking-up Wnt signal path; 2. be by mistake expression APC, axle albumen on the endochylema level, siRNA blocking-up beta-catenin level suppresses the Wnt signal path; 3. the transcribing of inhibition Wnt downstream genes of interest such as activity of blocking-up downstream beta-catenin/TCF transcription complex in nucleus or antisense interference [45,46] by micromolecular compound or virus.Because sudden change has all taken place beta-catenin in a lot of cancers,, do not have actual application value and cross inhibitive factor such as expression APC so it is little that the signal of beta-catenin upstream is suppressed effect.What therefore feasibility and extensive use are arranged most is a kind of at last.Molecular targeted therapeutic strategy at this signal pathway is expected to for tumor treatment provides new effective way, and multiple non-cell toxicity medicines such as non-steroidal anti-inflammatory drug and plant compounds also will play important role for the prevention and the Comprehensive Treatment of tumor.
Summary of the invention
First aspect present invention relates to the application of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone in the preparation medicine, and described medicine is used to prevent or treat disease or the imbalance that is caused by Wnt/ beta-catenin signal pathway abnormal activation or need specificity inhibition Wnt/ beta-catenin signal pathway.In a preferred embodiment, described disease is selected from Alzheimer, multicystic kidney disease or cancer, is more preferably colon cancer.
The present invention relate on the other hand a kind of be used to prevent or treat cause by Wnt/ beta-catenin signal pathway abnormal activation or need specificity to suppress the disease of Wnt/ beta-catenin signal pathway or the pharmaceutical composition of imbalance, it is characterized in that, described pharmaceutical composition comprises the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone for the treatment of effective dose, and pharmaceutically acceptable excipient.In a preferred embodiment, described disease is selected from Alzheimer, multicystic kidney disease or cancer.In a more preferred embodiment, described cancer is selected from colon cancer.
A further aspect of the invention relates to 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone is used for suppressing the active material of Wnt/ beta-catenin signal pathway reporter gene Top-flash in preparation application.
A further aspect of the invention relates to 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone is used for regulating and control the material of bone development in preparation application.
Other advantages of the present invention and benefit can be known from hereinafter detailed description and embodiment.
Description of drawings
Fig. 1 .15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the active of Wnt signal pathway reporter gene Top-flash and not influence the protein level and the distribution of cell caryoplasm of beta-catenin.A. dye the Top-flash reporter gene adds specific concentrations after 17 hours 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and solvent control DMSO at the HEK293T transit cell.The LiCl that adds Wnt conditioned medium or 10mM after 1 hour.Receive the cell detection reporter gene activity after 6 hours.B.HEK293T adds the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and the solvent control of specific concentrations, adds control compound and Wnt chemical compound after 1 hour.Divide Cytoplasm and nucleus to detect the beta-catenin protein level after 6 hours, 'beta '-tubulin is as the Cytoplasm label, and SP1 is as the nucleus label.
Fig. 2 .15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the activity of the Top-flash in the Wnt approach abnormal activation tumor cell and not influence the protein level and the distribution of cell caryoplasm of beta-catenin.A. left side figure be that SW480 cell transfecting Top-flash/Fop-flash reporter gene adds and to contain serum DMEM and stop transfection and add specific concentrations 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and 3 hours reporter gene activity result of solvent control (is 1 with the Top-flash of DMSO and the ratio of Fop-flash) simultaneously, and right figure is adding specific concentrations 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and 24 hours reporter gene activity result of solvent control during for Caco-2 cell termination transfection.The B.SW480 cell adds the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and the solvent control of specific concentrations and divides Cytoplasm and nucleus after 3 hours, detects the protein level of beta-catenin, and 'beta '-tubulin is as the Cytoplasm label, and SP1 is as the nucleus label.1 is DMSO, and 2 is the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of 1.25 μ g/ml, and 3 is the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of 2.5 μ g/ml, and 4 is the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of 5 μ g/ml.
Fig. 3 .15-oxo Ramulus et Folium Spiraeae Salicifolia lactone specificity suppresses the activity of Top-flash and protein or the mRNA level that dose dependent ground suppresses Wnt target base prisoner.A.HEK293T cell transfecting report base prisoner is after 17 hours, added the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of 2.5 μ g/ml and solvent control 1 hour, add corresponding stimulation Wnt chemical compound then, ionomycin (Ionomycin) 1 μ g/ml, Forskolin (Forskolin) 10 μ M examining report gene activity after 6 hours.The 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of B.SW480 cell adding specific concentrations and solvent control were received cell and are made the level that RT-PCR detects axle albumen 2mRNA after 3 hours.Cultivate the fixed time after the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of C.SW480 cell adding specific concentrations and the solvent control and receive sample, make the Western trace and detect.
Fig. 4 .15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can influence the interaction of beta-catenin and TCF4.The A.SW480 cell adds the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and the solvent control of specific concentrations, special time is received cell and is made endogenous co-immunoprecipitation experiment (1:DMSO, 2:1.25 the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of μ g/ml, the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of 3:5 μ g/ml).The 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of B.SW480 cell adding prescribed concentration and solvent control 2 hours are carried out the ChIP experiment then, and the method for reuse PCR in real time detects its relative amount.
Fig. 5: 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can influence the energy for growth of colon cancer cell.A, C: vertical coordinate is represented the relative value of the MTT reading of cell, with 0 hour be 1, abscissa is the time.B, D: vertical coordinate is the growth inhibition ratio of variable concentrations, and deducting its read-around ratio of ordering with respect to DMSO by 1 is worth coming, and abscissa is different concentration.Experiment is three independent experiment meansigma methodss (meansigma methods ± SE).
Fig. 6: 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the expression of Cdc2, Cdc25c and cause the tumor cell cell cycle arrest in the G2/M phase.Receiving cell Western trace behind the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone of A.SW480 cell adding specific concentrations and the solvent control effect special time detects.The B.SW480 cell adds the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and the solvent control of specific concentrations and receives cell fixation and PI dyeing after 36 hours, detects by FACS.The cartogram of C.B figure.Experiment is twice independent experiment meansigma methods.
Fig. 7 .15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the non-anchorage dependence energy for growth of tumor cell.The 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and the solvent control that add specific concentrations when A. coil soft agar experiment shop, 18 days purple dyeing counting statistics of post crystallization.The cartogram of B.A figure (meansigma methods ± SE).
Fig. 8 .15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the growth of SW480 transplanted tumor in the nude mice.A. the cartogram of two groups of tumor weights (P=0.03＜0.05 of meansigma methods ± SE).B. gross tumor volume-injection natural law cartogram (meansigma methods ± SE).C. nude mice body weight cartogram (meansigma methods ± SE).D. representational tumor picture in processed group and the matched group.
The specific embodiment
The inventor finds first that through further investigation 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the classical Wnt signal pathway.Because classical Wnt signal pathway and multiple disease, for example tumor and and some familial heredopathias, relevant as Alzheimer or multicystic kidney disease and regulation and control bone development, find that therefore 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the classical Wnt signal pathway and treat these diseases new thinking is provided for developing new medicine.
Specifically, the inventor finds that by mammalian cell reporter gene screening system 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the activity of classical Wnt signal pathway reporter gene Top-flash, finds that by the Western trace protein level and cell caryoplasm that it does not influence beta-catenin distribute.The inventor finds that also 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress Top-flash active in the tumor cell of Wnt approach abnormal activation and not influence the protein expression level and the distribution in the cell caryoplasm thereof of beta-catenin.15-oxo Ramulus et Folium Spiraeae Salicifolia lactone specificity suppresses the activity of Wnt signal pathway reporter gene Top-flash and albumen or the mRNA level that dose dependent ground suppresses the Wnt target gene.In addition, 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can influence the interaction of beta-catenin and TCF4.Based on above-mentioned discovery, the inventor has finished the present invention.
The present invention relates to the application of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone in the preparation medicine, described medicine is used to prevent or treat disease or the imbalance that is caused by Wnt/ beta-catenin signal pathway abnormal activation or need specificity inhibition Wnt/ beta-catenin signal pathway.
" disease or imbalance " herein comprises any symptom that can benefit from processing of the present invention.It is including, but not limited to various chronic and acute imbalances or disease.In a preferable embodiment, described disease or imbalance are familial heredopathia such as Alzheimer or multicystic kidney disease.In another preferable embodiment, described disease or imbalance are cancers, as pulmonary carcinoma, breast carcinoma, colorectal cancer, melanoma, colon cancer, mesothelioma, ovarian cancer, gastric cancer, renal carcinoma, bladder cancer, carcinoma of prostate, uterus carcinoma, thyroid carcinoma, cancer of pancreas, cervical cancer, esophageal carcinoma, head and neck cancer, hepatocarcinoma, the cerebral tumor, cancer of vagina, carcinoma of testis, sarcoma, leukemia, lymphoma, glioma and glioblastoma.In preferred scheme, described cancer is a colon cancer.
The present invention also provide a kind of be used to prevent or treat cause by Wnt/ beta-catenin signal pathway abnormal activation or need specificity to suppress the disease of Wnt/ beta-catenin signal pathway or the pharmaceutical composition of imbalance, it is characterized in that, described pharmaceutical composition comprises the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone for the treatment of effective dose, and pharmaceutically acceptable excipient.Term " comprise " refer to " containing " and " by ... constitute ", the compositions that for example " comprises " X can be made of X fully, perhaps can contain the material outside the X, for example X+Y.Term used herein " treatment effective dose " refers to the therapeutic agent treatment, alleviates or prevent the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.This effect for example can detect by chemical labeling or antigen levels.Therapeutic effect also comprises alleviating of physiological symptom.Depend on the nature and extent of the build of this object and health status, disease and the therapeutic agent selecting to give and/or the combination of therapeutic agent for the accurate effective dose of a certain object.Therefore, specifying accurately in advance, effective dose is useless.Yet, for certain given symptom, can determine this effective dose with normal experiment, the clinicist can judge.
Described pharmaceutically acceptable excipient (or carrier) refers to be used for the treatment of the excipient of agent administration, and itself and consumption thereof should not induce the individuality of accepting said composition to produce deleterious antibody, and do not have undue toxicity after the administration.Pharmaceutically acceptable excipient generally includes the preparation adminicle of nontoxic solid, semisolid or liquid filling agent, diluent, lapping or any general type.Suitable excipient includes but are not limited to water, glucose, glycerol, saline, ethanol and combination thereof.Described excipient also can contain other reagent such as wetting agent or emulsifying agent, pH buffer agent or strengthen the adjuvant that preparation is renderd a service.Other materials such as antioxidant, wetting agent, viscosity stabiliser and similar reagents can be added as required.Liposome is also included within the definition of pharmaceutically acceptable excipient.
In a preferred embodiment, pharmaceutical composition of the present invention also can comprise other one or more active components, other Wnt signal pathway inhibitor/antagonist for example known in the art is as excretory Frizzled associated protein (sFRP) family, WNT-inhibitive factor-1 (WIF-1), Dickkopf (DKK) family etc.; Non-steroidal anti-inflammatory drug is as indometacin (indomethacin) and aspirin [34,35] and sulindac (sulindac); Plant compounds, for example one of composition of curcumin (curcumin) [37], LVHUA Broccoli (broccoli) Sulforaphane (sulforaphane) and β-lapachol; And at the molecular targeted property treatment of this approach signal of interest molecule, for example Antybody therapy; Micromolecular inhibitor is as tyrosine kinase inhibitor imatinib mesylate (Glivec/Gleevec), blood vessel endothelium chalone (endostatin) etc.The content of described other active component can be determined with routine test as the case may be by those skilled in the art.
Usually, pharmaceutical composition can be made injectable agent, for example liquid solution or suspension; Also can be made into before injection, be fit to allocate in solution or the suspension, the solid form of liquid-carrier.
In addition, the invention still further relates to 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and be used for suppressing the application of the active material of Wnt/ beta-catenin signal pathway reporter gene Top-flash, and 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone is used for regulating and control the application of the material of bone development in preparation in preparation.
The present invention also provides a kind of method of screening the inhibitor that suppresses the classical Wnt signal pathway, the activity of described inhibitor blocking-up beta-catenin/TCF transcription complex, but the protein level and the cell caryoplasm that do not influence beta-catenin distribute, and described method comprises:
1). with reporter gene Top-flash transfectional cell;
2). with the cell after the Wnt conditioned medium incubation transfection that contains LiCl;
3). measure reporter gene activity, comprising:
A). the SCREENED COMPOUND for the treatment of with prescribed concentration acts on the cell fixed time;
B). collecting cell;
C). cell lysis;
D). measure the proteic fluorescence of GFP in the cell pyrolysis liquid, as the interior mark of cellular expression amount;
E). in this lysate, add the substrate of luciferase;
F). measure uciferase activity;
G). make it standardization with GFP intensity.
4). pair cell carries out endogenous co-immunoprecipitation to detect the protein level of beta-catenin;
5). the Cytoplasm of isolated cell and nucleus, the Western trace detects the caryoplasm distribution situation of beta-catenin;
6). carry out the ChIP test and whether be affected with the interaction that detects endogenous beta-catenin and TCF4.
Detecting interacts between albumen and the albumen can adopt technology well known to those skilled in the art.In the specific embodiment of the present invention, adopt the immunoprecipitation technology.Its principle is: keeping under the condition of protein-protein interaction, and results and cell lysis, immunoprecipitation destination protein specifically from cell extract is then by method separating immune precipitate such as electrophoresis.Proteic co-precipitation with known features can adopt the Western trace to detect.
The present invention adopts ChIP experiment (chromatin immunoprecipitation analysis) to come researching DNA and protein interactions, it is based on the method that the body inner analysis grows up, can reflect the modulin that is combined on the DNA sequence truely and completely, be to determine at present and bonded genome area of specific protein or the definite and bonded proteinic best method in specific gene group zone.
The mechanism that the present invention illustrates 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone inhibition tumor first is to suppress the classical Wnt signal pathway, thereby, and provide direction for looking for the chemical compound that can suppress this signal pathway and then can treat classical Wnt signal pathway relevant disease for further research Wnt signal pathway provides favourable instrument.
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are for the present invention is described but not limit the scope of the invention.Concrete experimental technique that embodiment adopted and experiment condition are the method and the conditions of this area routine, or the manufacturer method and the condition of advising.Unless otherwise defined, identical with known to scientific words and those skilled in the art of used specialty in the literary composition.
Embodiment
1. experiment material
1.1 reagent
Protease inhibitor (adequate proteins enzyme inhibitor mixing tablet) and NP-40 are available from (the Roche of Roche Holding Ag in Indianapolis, state of Indiana city, Indianapolis, IN), the conditioning culture medium that contains Wnt-3a prepares with the cell strain (available from ATCC) that can secrete Wnt-3a, protein A/G agarose is available from (the Santa Cruz Biotechnology of Santa Cruz Bioisystech Co., Ltd in San Diego, California city, Inc, San Diego, CA).15-oxo Ramulus et Folium Spiraeae Salicifolia lactone provides available from the Hao Xiaojiang researcher of Kunming plant research institute of the Chinese Academy of Sciences.
1.2 antibody
The 'beta '-tubulin monoclonal antibody is that laboratory is provided for oneself, fluorescence two anti-mices of the link coupled affinity purification of anti-IRDye 800CW or tame rabbit igg are purchased in (the Rockland of Roc Lande Co., Ltd. in gilbert Si Weier city, guest's sunset method Buddhist nun state, Inc, Gilbertsville, PA).Cdc2, Cdc25C, cyclin D1, Bcl 2 antibody are available from Santa Cruz biotech company.
1.3 plasmid
PTop-flash, pFop-flash, pNFAT-luc are provided by professor Wu Dianqing of Yale University.
1.4 primer
2. experimental technique
2.1 cell culture
HEK293T (human embryonic kidney cell), SW480, LS174T, HCT116 derive from ATCC, cultivation is at the DMEM culture fluid that contains 10% hyclone (but the Ji Bu company) ((Gibco of Ji Buke company in Carlsbad, California city, Carlsbad, CA)) in.Incubator remains on 37 ℃, and contains 5% carbon dioxide.
2.2 cell transfecting
Transient transfection reagent is liposome Lipofectamine reagent and Plus reagent (hero company (Invitrogen Inc), Carlsbad city, California).Top-flash, Fop-flash, NF-AT luc and pTAL-CRE luc reporter gene are surveyed the experiment of living and are generally used 24 porose discs: add 250ng plasmid/hole in the HEK293T cell, comprising 20ngTop-flash and pTAL-CRE luc or 40ng NF-AT is luc, 25ng GFP, all the other mend 250ng with Lac Z; In SW480, HCT116, LS174T cell, add 500ng plasmid/hole, comprising the 100ng reporter gene, 100ng GFP, all the other mend 500ng with Lac Z.To mix plasmid and plus reagent mix 15 minutes,, change the cell of serum-free medium then into, add said mixture again with lipofectamine reagent mix 15 minutes, transfection change into after 3 hours contain 10% hyclone culture fluid to stop transfection.
2.3 surveying, reporter gene lives
2.4 endogenous co-immunoprecipitation
SW480 cell (10cm dish) two dishes of dosing being handled the fixed time discard culture medium and add PBS, and place on ice.With cell scrape accept cell after, add 3ml hypotonic buffer liquid (1mM pyrophosphoric acid, 2mM Na
3VO
4, 10mM NaF and protease inhibitor, 10mM HEPES, pH 7.9,1.5mM MgCl
2, 10mM KCl), evenly placed 10 minutes on ice the back with micropipettor piping and druming, then the 7ml homogenizer of pausing with U.S.'s favour (WHEATON, USA) pull is 10 times, 4 ℃ 1, centrifugal 10 minutes of 000rpm.Abandon supernatant, precipitation 1ml lysate (1mM pyrophosphoric acid, 2mM Na
3VO
4, 10mM NaF and protease inhibitor, 50mM Tris-HCl pH7.4,150mM NaCl, 1%Triton X-100,10mM EDTA) and cell lysis.4 ℃ of rotation mixed pyrolysis are after 15 minutes, and 4 ℃ 13, centrifugal 15 minutes collecting cell lysate supernatants of 000rpm are in another clean EP pipe.Getting 50 μ l gives over to supernatant and mixes with the SDS sample buffer.Add corresponding antibody in all the other lysate supernatants, after 4 ℃ of rotation mixing are spent the night, add protein A/G resin (50 μ l/ pipe), 4 ℃ are continued mixing and use lysis buffer washing resin 5 times after 5 hours, at last add SDS sample buffer 50 μ l in resin, 100 ℃ are boiled.Sample carries out doing the immunoblotting detection behind the SDS-PAGE electrophoresis.
2.5 caryoplasm separates
Cultivation is removed culture fluid after the 293T cell of 60-mm dish adds suitable stimulation, place on ice, the PBS 0.75ml that adds pre-cooling, scrape cell with cell, collect in the 1.5ml ampoul tube (Ep pipe), in dish, add the 0.75mlPBS washing again, scrape with cell equally and collect with in the pipe ampoul tube, 4 ℃ of 2.5krpm * 10 minute receipts cells remove PBS, add 300 μ l hypotonic buffer liquid (10mM HEPES in cell precipitation, pH 7.9,1.5mM MgCl
2, 10mM KCl is with before adding protease inhibitor and 1mM pyrophosphoric acid), with putting 10 minutes on ice behind the micropipettor piping and druming mixing, with the insulin needle pull to broken back 4 ℃ of 2.5krpm * 10 of microscopy cell membrane minute, 4 ℃ 100 of supernatant, centrifugal 1 hour of 000G, getting super is the cytoplasm component from supernatant.Add the piping and druming washing of 300 μ l hypotonic buffer liquid in the precipitation, 4 ℃ of 2.5krpm * 10 minute add 60 μ l high osmotic buffer (20mM HEPES pH 7.9,420mM NaCl, 0.2mM EDTA, 25% glycerol, 1mM pyrophosphoric acid, 2mM Na in precipitation
3VO
4, 10mM NaF and protease inhibitor), placed on ice 30 minutes, last 4 ℃ 100, centrifugal 1 hour of 000G, getting supernatant is nuclear consitution.
2.6FACS flow cytometer is surveyed cell cycle
After the cell process prescribed concentration drug treating of 60mm dish, culture fluid is transferred in the Ep pipe, used the PBS washed cell, the PBS of washing also forwards to in the pipe, use trypsin digestion cell, the culture fluid that adds firm receipts stops digestion, and the piping and druming cell receives that 800rpm received cell in centrifugal 10 minutes with in the pipe Ep pipe, after twice of the PBS rinsing, add 300 μ l PBS rotation and mix with complete suspension cell, add the 700ul dehydrated alcohol while rotate then to mix in cell, 4 ℃ are fixedly spent the night.Centrifugal 5 minutes of 800rpm removes supernatant, with the PBS washed cell once, add 500 μ l PBS (containing 50 μ g/mlPI (propidium iodide), 200 μ g/ml RNase, 0.1%Triton X-100) suspension cell, 37 ℃ of lucifuge incubations 1 hour carry out flow cytometry analysis.
2.7ChIP
With adding in the SW480 cell of 100-mm dish among the post-stimulatory culture fluid 4.5ml, add final concentration 1% formaldehyde, 37 ℃ after crosslinked 10 minutes, the glycine that adds final concentration 125mM stops, room temperature was placed 0.5 hour, sop up after going to add 5ml pre-cooling PBS washed cell surface behind the clean culture fluid, add the PBS of an amount of pre-cooling again, scrape collection with cell, 4 ℃ were removed supernatant after (13krpm) in centrifugal 5 minutes, the Mg-NI buffer of reuse 1ml pre-cooling (15mM Tris-HCl, pH7.4,5mM MgCl
2, 60mM KCl, 0.5mM DTT, 15mM NaCl, 300mM sucrose) and washing precipitation, 4 ℃ centrifugal 2 minutes (13krpm) back supernatant discarded, the Mg-NI-NP40 buffer of reuse 1ml pre-cooling (15mMTris-HCl, pH7.4,5mM MgCl
2, 60mM KCl, 0.5mM DTT, 15mM NaCl, 300mM sucrose, 1%NP40) washing precipitation was placed 10 minutes on ice, 4 ℃ of centrifugal 13krpm * 2 minute, supernatant discarded is with Ca-NI buffer (15mM Tris-HCl, pH7.4, the l mM CaCl of 1ml pre-cooling
2, 60mM KCl, 0.5m M DTT, 15mM NaCl, 300mM sucrose) washing precipitation, 4 ℃ of centrifugal 13krpm * 2 minute, remove supernatant, use 250 μ l lysis buffer (50mM Tris-HCl, 1%SDS at last, 10mM EDTA) cracking precipitation, the reuse ultrasound wave interrupts DNA, power 280W, 10 seconds working times, had a rest 30 seconds, and circulated 6 times.4 ℃ of centrifugal 13krpm * 10 minute, get supernatant 10 μ l and give over to the supernatant sample to-20 ℃, get supernatant 100 μ l again, be added to 0.9ml dilution buffer liquid (16.7mMTris-HCl, pH8.0,0.01%SDS, 1.1%Triton X-100,1.2mM EDTA, 167mM NaCl) in, add various antibody 4 μ g, 4 ℃ of rotations mix spends the night.Add 50 μ l protein A/G agar, 4 ℃ of rotations mixed 2 hours, 4 ℃ of centrifugal 5krpm * 1 minute, remove supernatant, with 0.5ml SB140 buffer (50mM HEPES, pH7.9,1mM EDTA, 1%Triton X-100,0.1%SDS, 140mM NaCl, 0.1% dexycholate) washing precipitation twice, each room temperature rotation mixed 5 minutes, reuse 0.5ml SB500 buffer (50mM HEPES, pH7.9,1mM EDTA, 1%Triton X-100,0.1%SDS, 500mM NaCl, 0.1% dexycholate) washing precipitation twice, use 0.5ml LiCl buffer (20mM HEPES then, pH8.0,1mM EDTA, 250mM LiCl, 0.5% dexycholate, 0.5%NP40) washing precipitation is twice, use 0.5ml TE (10mM Tris-HCl, pH8.0,1mM EDTA) washed twice at last, precipitation adds 110 μ l elution buffer (50mM Tris-HCl, 1%SDS, 10mM EDTA), place 10 minutes eluting for 65 ℃, get supernatant 100 μ l after centrifugal to clean Ep pipe, add 150ul and contain 0.67% the further eluting of TE in precipitation again, placed 10 minutes for 65 ℃, centrifugal back supernatant is incorporated into in the pipe eluent, 250 μ l altogether, get μ l supernatant sample on the previous day 10 simultaneously, add the TE that 240 μ l contain 1%SDS, together put separate to 65 ℃ crosslinked more than 6 hours.Every pipe is added 50 μ l E.C. 3.4.21.64 buffer (TE that contains 20 μ g E.C. 3.4.21.64s and 20 μ g glycogens), and 56 ℃ were reacted 2 hours, phenol/chloroform extracting DNA, and ethanol precipitation spends the night.Dry after the washing precipitation, the supernatant sample is with 60 μ lTE, and elution samples is with 20 μ l TE solution precipitations.Respectively getting 2.5 μ l draws PCR to detect the combination in target gene promoters district as template.
2.8MTT method detects cell viability
With suitable cell density cell is assigned in 96 orifice plates, selected appropriate drug concentration and drug treating time.To treat that then the culture fluid in the gaging hole siphons away, change fresh medium into, and add MTT incubation 4 hours in 37 ℃ of incubators.Remove the culture fluid for the treatment of to contain in the gaging hole MTT, add DMSO dissolving MTT.The room temperature placement allowed it fully dissolve in 5 minutes or directly blew and beat mixing with micropipettor, read absorption value at 570nM at last.
2.9 the soft agar colony forms
Prepare aseptic 1.2% and 0.6% agar solution, melt postcooling to 40 ℃ with microwave before using.Get and spread into 6 porose discs cooled and solidified with the 1.5ml/ hole after isopyknic 1.2% agar solution adds 2 * DMEM (containing additives such as serum) mixing.Get isopyknic 0.6% agar solution again and add 2 * DMEM (containing additives such as serum) mixing, coil with shop, 1.5ml/ hole after adding the cell mixing of suitable concentration then.Put into incubator after the cooled and solidified and cultivate about 2 weeks, with the counting of taking pictures after the violet staining.
2.10 transplanted tumor in nude mice experiment
Get the 3-4 male nude mouse in age in week, every group of five nude mices.With SW480 cell tryptase enzymic digestion and resuspended, with 2 * 10
6Cell/side injection is to both sides, nude mice back.The tumor mean size is 50mm after 14 days etc.
3The time with concentration intraperitoneal injection of drugs or the solvent control of 90ug/kg, measure the major diameter and the minor axis of mouse body weight, tumor simultaneously, according to formula: tumor size=A * B
2/ 2 (A represents the major diameter of tumor, and B represents the minor axis of tumor) are calculated the size of tumor.Measured once in per two days.Put to death nude mice after 17 days, strip tumor, carry out statistical analysis after weighing.
3. experimental result
3.1 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the active of Wnt signal pathway reporter gene Top-flash and not influence the protein level and the distribution of cell caryoplasm of beta-catenin
The chemical constitution of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone is
It is that diterpene alkaloid (being mainly spiradine A, B, C and D) by the compound group of pink blossom Ramulus et Folium Spiraeae Salicifolia is under the highly basic effect, be converted into diterpene-kind compound Ramulus et Folium Spiraeae Salicifolia lactone (spiramilactone) and Ramulus et Folium Spiraeae Salicifolia lactone C (spiramilactone C) through hydrolysis and intramolecularly dismutation reaction, (in the accompanying drawing name be called 043) that the Ramulus et Folium Spiraeae Salicifolia lactone obtains through oxidation again.Anti-tumor activity mechanism for 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone is not appeared in the newspapers.
The inventor finds that by mammalian cell reporter gene screening system 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the Wnt conditioned medium in dose dependent ground, and LiCl stimulates the Top-flash reporter gene activity (Figure 1A) that produces.The mechanism of action of LiCl is the kinase activity [47] that suppresses GSK3 β in the beta-catenin degraded complex, thereby causes beta-catenin to be stablized accumulation by phosphorylation.The activation effect that therefore can suppress LiCl illustrates that the target spot of its effect should be on the beta-catenin/TCF transcription complex level in degraded complex downstream.Because beta-catenin can also not relied on the active degradation pathway of GSK3 beta kinase degrade [48-50] by siah, RXR, PPAR γ etc., the inventor has separated HEK293T at post-stimulatory Cytoplasm of Wnt and nucleus, and the Western trace detects finds the distribution not obviously influence (Fig. 1 β) of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone to the protein level and the cell caryoplasm of beta-catenin.
3.2 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the activity of the Top-flash in the Wnt approach abnormal activation tumor cell and not influence the protein expression level and the distribution in the cell caryoplasm thereof of beta-catenin
Thereby colon cancer cell SW480 and Caco-2 cause beta-catenin to be degraded owing to the APC of degraded in the complex undergos mutation stablize accumulation and can to enter transcribing of nucleus startup downstream target gene.The inventor finds that 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can dose dependent ground suppresses the Top-flash reporter gene activity (Fig. 2 A) in SW480 and the Caco-2 cell, and does not influence the protein level of the beta-catenin in the SW480 cell and caryoplasm distribute (Fig. 2 B) too.
3.3 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone specificity suppresses the activity of Wnt signal pathway reporter gene Top-flash and albumen or the mRNA level that dose dependent ground suppresses the Wnt target gene
In order to verify whether 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone suppresses the Wnt signal pathway specifically, the inventor in transfection in the HEK293T cell of report base prisoner pTAL-CRE luc of the reporter gene NF-AT luc of Wnt signal pathway reporter gene Top-flash, NF-AT and CREB, add the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone or the solvent control of specific concentrations simultaneously.Find that 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone specificity has produced the activity of Top-flash reporter gene and suppress and both do not suppress effect (Fig. 3 A) to all the other.Axle albumen 2 and cyclin D1 all be forefathers empirical tests the target gene of Wnt signal path, the inventor has detected the mRNA level of axle albumen 2 and the protein level that Western trace method has detected cyclin D1 by real-time PCR method, finding 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can its level of dose dependent ground inhibition, (Fig. 3 B C) and to the protein level of Bcl2 and 'beta '-tubulin does not have any influence.
3.4 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can influence the interaction of beta-catenin and TCF4
But 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone does not influence that the protein level of beta-catenin and cell caryoplasm distribute the expression that can influence downstream target base prisoner again, illustrates that it may regulate the activity of beta-catenin and TCF4 transcription complex.This exists several probabilities: the one, regulated both interactions, and the 2nd, the binding ability of having regulated TCF4 and DNA, the 3rd, regulated the expression of TCF4, the 4th, regulated the activation capability of beta-catenin.At first the inventor has detected the interaction of endogenous beta-catenin and TCF4, finds that 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can weaken both interactions (Fig. 4 A).By ChIP experiment, further find 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can the appreciable impact beta-catenin in the gathering (Fig. 4 B) of the promoter region of axle albumen 2.
3.5 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can influence the energy for growth of colon cancer cell
APC sudden change has caused the beta-catenin/TCF4 transcriptional activity of abnormal activation among colon cancer cell SW480 and the Caco-2, the energy for growth that beta-catenin/TCF4 transcription complex inhibitor that forefathers find can suppress colon cancer cell with become the tumor ability.In order to verify whether 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone has equal effect, the inventor utilizes the MTT method to detect the influence of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone to SW480 and Caco-2 energy for growth, find the growth of two kinds of cells of inhibition that it can dose dependent, and also can cause apoptosis (Fig. 5) under the high concentration, long duration of action.By calculating 72 hours 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone to the IC of SW480
50=0.567 μ g/ml (1.7 μ M), the IC of Caco-2
50=0.313 μ g/ml (0.9 μ M).
3.6 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the expression of Cdc2, Cdc25c and cause the tumor cell cell cycle arrest in the G2/M phase
The inventor finds in test to add that cellular morphology changes behind the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone certain hour, nucleus becomes not obvious.Detect the discovery cell by PI dyeing FACS and be stuck in the G2/M phase (Fig. 6 B), and detect Cdc25C, the expression of Cdc2 finds also to be subjected to obvious inhibition (Fig. 6 A).
3.7 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the non-anchorage dependence energy for growth of tumor cell
The non-anchorage dependence energy for growth of tumor cell is its important indicator that becomes the tumor ability, in order to detect the influence of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone for the non-anchorage dependence energy for growth of SW480 cell, the inventor chooses 1/8,1/4,1/2 of least concentration 156ng/ml in the MTT experiment and finishes this experiment.In the soft agar experiment, in the dish of shop, add the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone and the solvent control of specific concentrations, count with violet staining after 18 days.Find that 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can obviously suppress the non-anchorage dependence energy for growth (Fig. 7) of tumor cell.
3.8 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone can suppress the growth of SW480 transplanted tumor in the nude mice
In order to verify that can 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone suppress the growth of in-vivo tumour, the inventor has transplanted the SW480 cell on nude mice, waits tumor average volume long to 50mm
3The time lumbar injection 90 μ g/kg 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone, injection in 24 hours is once.Measure nude mice body weight and gross tumor volume every other day, inject after 17 days and to put to death mouse and claim tumor weight.Statistics finds that the gross tumor volume of 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone group and 50%, two group of mouse body weight that weight average is about matched group do not have notable difference.The dosage of injection is very low and can have so strong inhibition effect proof 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone for colon cancer good therapeutic effect (Fig. 8) may be arranged.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
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Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉15-oxo Ramulus et Folium Spiraeae Salicifolia lactone suppresses the new application of Wnt signal pathway
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Claims (8)
1.15-the application of oxo Ramulus et Folium Spiraeae Salicifolia lactone in the preparation medicine, described medicine are used to prevent or treat disease or the imbalance that is caused by Wnt/ beta-catenin signal pathway abnormal activation or need specificity inhibition Wnt/ beta-catenin signal pathway.
2. application as claimed in claim 1 is characterized in that described disease is selected from Alzheimer, multicystic kidney disease or cancer.
3. application as claimed in claim 2 is characterized in that described cancer is selected from colon cancer.
One kind be used to prevent or treat cause by Wnt/ beta-catenin signal pathway abnormal activation or need specificity to suppress the disease of Wnt/ beta-catenin signal pathway or the pharmaceutical composition of imbalance, it is characterized in that, described pharmaceutical composition comprises the 15-oxo Ramulus et Folium Spiraeae Salicifolia lactone for the treatment of effective dose, and pharmaceutically acceptable excipient.
5. pharmaceutical composition as claimed in claim 4 is characterized in that described disease is selected from Alzheimer, multicystic kidney disease or cancer.
6. pharmaceutical composition as claimed in claim 5 is characterized in that described cancer is selected from colon cancer.
7.15-oxo Ramulus et Folium Spiraeae Salicifolia lactone is used for suppressing the application of the active material of Wnt/ beta-catenin signal pathway reporter gene Top-flash in preparation.
8.15-oxo Ramulus et Folium Spiraeae Salicifolia lactone is used for regulating and control the application of the material of bone development in preparation.
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| CN200910198665A CN102058580B (en) | 2009-11-12 | 2009-11-12 | New application of 15-oxospiramilactone for inhibition of Wnt signal path |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103193754A (en) * | 2013-04-19 | 2013-07-10 | 贵州省中国科学院天然产物化学重点实验室 | 7-acyl-15-oxo-spiraea lactone derivative as well as preparation method and applications thereof |
| WO2014169711A1 (en) * | 2013-04-19 | 2014-10-23 | 中国科学院昆明植物研究所 | 15-oxospiramilactone derivatives, preparation method therefor, and uses thereof |
| CN107595839A (en) * | 2016-07-12 | 2018-01-19 | 中国科学院上海生命科学研究院 | The action target spot CARF of 15 oxo meadow sweet lactones discovery and its function in classical Wnt signal path |
| CN107596386A (en) * | 2016-07-12 | 2018-01-19 | 中国科学院上海生命科学研究院 | Functions of the CARF in classical Wnt signal path |
| CN109164260A (en) * | 2018-08-20 | 2019-01-08 | 广州伯信生物科技有限公司 | A kind of co-immunoprecipitation method that can exclude antibody signal interference |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101239964B (en) * | 2008-03-07 | 2012-01-04 | 中国科学院昆明植物研究所 | 15-oxospiramilactone and its medicament composition, preparation method and application |
-
2009
- 2009-11-12 CN CN200910198665A patent/CN102058580B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103193754A (en) * | 2013-04-19 | 2013-07-10 | 贵州省中国科学院天然产物化学重点实验室 | 7-acyl-15-oxo-spiraea lactone derivative as well as preparation method and applications thereof |
| WO2014169711A1 (en) * | 2013-04-19 | 2014-10-23 | 中国科学院昆明植物研究所 | 15-oxospiramilactone derivatives, preparation method therefor, and uses thereof |
| US10112918B2 (en) | 2013-04-19 | 2018-10-30 | Kunming Institute Of Botany, The Chinese Academy Of Sciences | 15-oxospiramilactone derivatives, preparation method and uses thereof |
| CN107595839A (en) * | 2016-07-12 | 2018-01-19 | 中国科学院上海生命科学研究院 | The action target spot CARF of 15 oxo meadow sweet lactones discovery and its function in classical Wnt signal path |
| CN107596386A (en) * | 2016-07-12 | 2018-01-19 | 中国科学院上海生命科学研究院 | Functions of the CARF in classical Wnt signal path |
| CN107596386B (en) * | 2016-07-12 | 2020-11-20 | 中国科学院分子细胞科学卓越创新中心 | Function of CARF in canonical Wnt signaling pathway |
| CN109164260A (en) * | 2018-08-20 | 2019-01-08 | 广州伯信生物科技有限公司 | A kind of co-immunoprecipitation method that can exclude antibody signal interference |
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| CN102058580B (en) | 2012-09-12 |
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