CN101313926B

CN101313926B - Transcription regulating and controlling article composition

Info

Publication number: CN101313926B
Application number: CN 200710109268
Authority: CN
Inventors: 萧培文
Original assignee: Academia Sinica
Current assignee: Academia Sinica
Priority date: 2007-05-30
Filing date: 2007-05-30
Publication date: 2012-12-26
Anticipated expiration: 2027-05-30
Also published as: CN101313926A

Abstract

The invention relates to a method for treating an examinee with a male hormone exciting disease by adding a composition containing an effective dose of IkB kinase subunit alpha activity inhibitors. The invention also provides a method for reducing the risk of prostatic cancer of the examinee who is highly risky in getting the prostatic cancer by adding the composition. Another method relates to a regulator for identifying transcriptional activity regulated by the IkB kinase subunit alpha. Also another method relates to measure the transcriptional activity regulated by the IkB kinase subunit alpha in a nonhuman mammal testing examinee.

Description

Transcription regulating and controlling article composition

Technical field

The present invention relates to a kind of transcription regulating and controlling article composition.

Background technology

Androgen is defined as the major control male characteristic development of broad variety and the steroid hormone of keeping.It brings into play its physiological action via androgen receptor (AR), and this androgen receptor is a member in the steroid hormone nuclear receptor Superfamily.This AR is able to activation through combining androgen (for example testis sterone) or relevant ligand.Activated AR priming signal transduction path, it drives the increase of transcribing of numerous ＂ androgen response ＂ target genes (for example PSA (PSA)).It should be noted that the signal transduction step in this path is not illustrated as yet fully.

By androgen activation AR, then increase transcribing of androgen response target gene, can excite numerous diseases, for example carcinoma of prostate.Therefore, the treatment carcinoma of prostate generally includes and throws and the AR antagonist.Unfortunately, owing to often become androgen independence by the AR that cancerous cell show, meaning is composition active (for example owing to suddenling change), so its effectiveness often is short-term.Therefore, even still driving the target gene of high-caliber support persistency cancer growth in the presence of the AR antagonist, ligand dependent/non-dependent AR transcribes.

Therefore, need in the signal transduction step in AR activation downstream, regulate and control androgen and respond the compositions that target gene is transcribed, and the method that is used for its identification and therapeutic use.

Summary of the invention

The present invention's part finds that based on accident promptly I kappa b kinase subunit α (IKK α) transcribes very crucial with AR interaction and its activity for the androgen responsive genes.

Therefore, one aspect of the present invention relates to through the person under inspection being thrown the method for treating the disease of androgen stimulation with the compositions that contains the IKK alpha active inhibitor of effective dose.Preferably, before throwing and said composition, this person under inspection suffers from the disease that this androgen stimulates after diagnosing.Any health anomalies disease of the disease that androgen stimulates for causing or worsen, for example carcinoma of prostate, benign prostatauxe, breast carcinoma, male's alopecia, propionibacterium acnes, PCO disease, Autosome dominance POLYCYSTIC KIDNEY DISEASE or the too much disease of androgen by the active institute of AR.Compositions can contain the extract from Herbia Wedeliae (Wedelia chinensis), Eclipta prostrata (Eclipta prostrata) or Herba Ecliptae (Eclipta alba).IKK alpha active inhibitor can be leaf and contains 1,8,9-trihydroxy-3-methoxy-benzo[4,5 (wedelolactone), leaf flavone (luteolin), Cortex querci dentatae ketone (quercetin), foreign first Sui flavin (apigenin), 15-deoxidation-Δ ^12,14-prostaglandin J ₂Or PGA1.This method is also included within measuring from the PSA content in this person under inspection's the Biosample before or after person under inspection's throwing and the above-mentioned composition.Before dispensing, measure PSA content and be applicable to that the diagnosis person under inspection suffers from the disease (for example carcinoma of prostate) that androgen stimulates.The PSA content measured is used to treat the reliable index of treatment of method of the disease of androgen stimulation for response is above-mentioned after dispensing.

The present invention relates to through the excessive risk person under inspection being thrown the method that reduces this person under inspection's carcinoma of prostate risk with the IKK alpha active inhibitor that contains effective dose on the other hand.

The present invention relates to the method that is used to discern modulators (meaning is the inhibitor or the stimulant of the transcriptional activity of IKK α adjusting) on the other hand.The transcriptional activity inhibitor is applicable to the disease that the androgen of (for example) treatment such as carcinoma of prostate etc. stimulates.This method comprises that (1) provides mammalian cell; It expresses IKK α and AR; And contain the isolating nucleic acid that the androgen that is connected on the coding report body peptide sequence is responded the promoter sequence with comprising operability; Thereby make this cell also express this report body polypeptide, (2) contact this cell with test composition, and the activity of (3) calibrating expression (meaning is mRNA or protein expression amount) or report body polypeptide.If the activity of the expression that this test composition increase or reduction are relevant with the negative control treatment or the report body polypeptide of coding thinks that then it is the modulators by the transcriptional activity of IKK α adjusting.AR through expressing can be the composition active mutant.If it is active that AR is non-composition, then this method comprises cell is contacted with the AR agonist.This method also can comprise judge test composition to the work of IKK alpha active in order to further characterization mechanism, test composition is through this mechanism regulating transcriptional activity of regulating of kinases thus.

The present invention relates on the other hand and is used for measuring the method for non-human mammal test person under inspection by the transcriptional activity of IKK α adjusting.This method is applicable to that (for example) judge the in vivo effect of candidate IKK alpha inhibitor compositions.This method comprises that (1) introduce numerous cells; Its performance IKK α and androgen receptor; And contain the androgen that is connected on the coding report body peptide sequence with having operability and respond the isolating nucleic acid of promoter sequence, thereby make this cell also express this report body polypeptide; And the activity by the expressed report body polypeptide of these cells is detected in (2).The transcriptional activity of regulating by IKK α among the detection indication test person under inspection of report body active (for example in vivo).This method also can comprise the IKK alpha active degree in the Biosample of measuring the self-test person under inspection.

Further feature of the present invention or advantage will also reach claim obviously because of following embodiment.

Embodiment

Hereinafter is described and is used to discern for example androgen response target gene and the IKK α adjustment type of transcribing at the method for compositions of (IKK α RT) of specific regulation and control.Hereinafter is also described the method for treating the high risk person under inspection of disease tool who suffers from disease that androgen stimulates or stimulate for androgen with the affiliated compositions of effective dose through throwing.

IKK α RT regulating and controlling article composition (meaning promptly reduces or increase the compositions of IKK α RT) is surveyed through the health check-up of androgen response report and is discerned.

Androgen response report body structure merges through the nucleic acid that androgen is responded promoter and coding report body polypeptide and produces.It is can be by the activatory promoter in AR dependent signals transduction path that androgen is responded promoter.Body structure is reported in this androgen response to be introduced in host cell to produce the somatic cell of androgen response report, this androgen response promoter driving report body polypeptide expression in this androgen response report somatic cell.For being evaluated at the ability of test composition regulation and control IKK α RT in the report somatic cell, can be through having or not having under the situation of test composition affiliated cell contacted with androgen (for example testis sterone or 5 α-dihydro-testosterone sterone) to increase and report somatic IKK alpha active.With cell and androgen add or do not have contact under the situation of test composition after, measure that the report body surface reaches or active.If in the presence of test composition, compare with no test composition, the expression of the report body that the response androgen stimulates or activity increase or reduce, and think that then said composition is an IKK α RT modulators.

Test composition can be (for example) compositions, plant extract, protein, peptide or micromolecule (for example being selected from the chemical compound in the synthetic storehouse of combination) from biogenic purification or synthetic natural generation.

Suitable androgen response report somatic cell can be derived from mammalian tissues or cell strain (for example derived from the mankind, monkey, mice, rat or hamster).Host cell endogenous or heterologous ground performance IKK α (for example Gen Bank is NP_001269 number) and AR (for example Gen Bank is AAA51729 number).For example, derive from American type culture collection (American Type CultureCo1lection) ( Preserving number: CRL-2505 ^TM) human PCa22Rv1 cell strain can be used for producing androgen and respond report somatic cell strain.

In the report body measurement, can avoid androgen to stimulate through using the become second nature report somatic cell of active A R of performance group, people (1995) such as Marce1li for example, Endocrinology the 136th volume, the 3rd is interim said.

The method that is used for producing the report body structure, is introduced into cell and measures multiple report body polypeptide active is in (for example) Current Protocols in MolecularBiology; John Wiley & Sons; N.Y. (2005) specify among 3.16-3.17 and the 9.1-9.14 respectively.

Androgen is responded promoter should comprise at least a in the following androgen response element:

AGAACAGCAAGTGCT(SEQ?ID?NO：1)

GGATCAGGGAGTCTC(SEQ?ID?NO：2)

GGAACATATTGTATC(SEQ?ID?NO：3)

Referring to people such as Cleutjens (1997), Mol.Endocrinol.11 (9): 1256-65.

For example, androgen is responded promoter and can be PSA promoter, and it contains the listed androgen response element of whole three kinds of preceding text, and has following sequence:

AGCTTCTAGTTTTCTTTTCCCGGTGACATCGTGGAAAGCACTAGCATCTC

TAAGCAATGATCTGTGACAATATTCACAGTGTATTGCCATCCAGGGAACT

CAACTGAGCCTTGATGTCCAGAGATTTTTGTGTTTTTTTCTGAGACTGAG

TCTCGCTCTGTGCCCAGGCTGGAGTGCAGTGGTGCAACCTTGGCTCACTG

CAAGCTCCGCCTCCTGGGTTCACGCCATTCTCCTGCCTCAGCCTCCTGAG

TAGCTGGGACTACAGGCACCCGCCACCACGCCTGGCTAATTTTTTTGTAT

TTTTAGTAGAGATGGGGTTTCACTGTGTTAGCCAGGATGGTCTCAGTCTC

CTGACCTCGTGATCTGCCCACCTTGGCCTCCCAAAGTGCTGGGATTACAG

GCGTGAGCCACTGCGCCTGGCCGATATCCAGAGATTTTTTGGGGGGCTCC

ATCACACAGACATGTTGACTGTCTTCATGGTTGACTTTTAGTATCCAGCC

CCTCTAGAAATCTAGCTGATATAGTGTGGCTCAAAACCTTCAGCACAAAT

CACACCGTTAGACTATCTGGTGTGGCCCAAACCTTCAGGTGAACAAAGGC

ACTCTAAACTGGCAGGATATTCCAAAGCATTAGAGATGACCTCTTGCAAA

GAAAAAGAAATGGAAAAGAAAAAGAAAGAAAGGAAAAAAAAAAAAAAAAA

GAGATGACCTCTCAGGCTCTGAGGGGAAACGCCTGAGGTCTTTGAGCAAG

GTCAGTCCTCTGTTGCACAGTCTCCCTCACAGGGTCATTGTGACGATCAA

ATGTGGTCACGTGTATGAGGCACCAGCACATGCCTGGCTCTGGGGAGTGC

CGTGTAAGTGTATGCTTGCACTGCTGAATGGCTGGGATGTGTCAGGGATT

ATCTTCAGCACTTACAGATGCTCATCTCATCCTCACAGCATCACTATGGG

ATGGGTATTACTGGCCTCATTTGATGGAGAAACTGGCTGTGGCTCAGAAA

GGGGGGACCACTAGACCAGGGACACTCTGGATGCTGGGGACTCCAGAGAC

CATGACCACTCACCAACTGCAGAGAAATTAATTGTGGCCTGATGTCCCTG

TCCTGGAGAGGGTGGAGGTGGACCTTCACTAACCTCCTACCTTGACCCTC

TCTTTTAGGGCTCTTTCTGACCTCCACCATGATACTAGGACCCCATTGTA

TTCTGTACCCTCTTGACTCTATGACCCCCACTGCCCACTGCATCCAGCTG

GGTCCCCTCCTATCTCTATTCCCAGCTGGCCAGTGCAGTCTCAGTGCCCA

CCTGTTTGTCAGTAACTCTGAAGGGGCTGACATTTTACTGACTTGCAAAC

AAATAAGCTAACTTTCCAGAGTTTTGTGAATGCTGGCAGAGTCCATGAGA

CTCCTGAGTCAGAGGCAAAGGCTTTTACTGCTCACAGCTTAGCAGACAGC

ATGAGGTTCATGTTCACATTAGTACACCTTGCCCCCCCCAAATCTTGTAG

GGTGACCAGAGCAGTCTAGGTGGATGCTGTGCACACGGGGTTTGTGCCAC

TGGTGAGAAACCTGAGATTAGGAATCCTCAATCTTATACTGGGACAACTT

GCAAACCTGCTCAGCCTTTGTCTCTGATGAAGATATTATCTTCATGATCT

TGGATTGAAAACAGACCTACTCTGGAGGAACATATTGTATCGATTGTCCT

TGACAGTAAACAAATCTGTTGTAAGAGACATTATCTTTATTATCTAGGAC

AGTAAGCAAGCCTGGATCTGAGAGAGATATCATCTTGCAAGGATGCCTGC

TTTACAAACATCCTTGAAACAACAATCCAGAAAAAAAAAGGTGTTGCTGT

CTTTGCTCAGAAGACACACAGATACGTGACAGAACCATGGAGAATTGCCT

CCCAACACTGTTCAGCCAGAGCCTTCCACCCTTGTCTGCAGGACAGTCTC

AACGTTCCACCATTAAATACTTCTTCTGTCACATCCTGCTTATTTATGCC

TAACCAAGGTTCTAGGTCCCGATCGACTGTGTCTGGCAGCACTCCACTGC

CAAACCCAGAATAAGGCAGCGCTCAGGATCCCGAAGGGGCATGGCTGGGG

ATCAGAACTTCTGGGTTTGAGTGAGGAGTGGGTCCACCCTCTTGAATTTC

AAAGGAGGAAGAGGCTGGATGTGAAGGAACTGGGGGAGGGAAAGTGTCAG

TTCCGAACTCTTAGGTCAATGAGGGAGGAGACTGGTAAGGTCCCAGCTCC

CGAGGTACTGATGTGGGAATGGCCTAAGAATCTCATATCCTCAGGAAGAA

GGTGCTGGAATCCTGAGGGGTAGAGTTCTGGGTATATTTGTGGCTTAAGG

CTCTTTGGCCCCTGAAGGGCAGAGGCTGGAACCATTAGGTCCAGGGTTTG

GGGTGATAGTAATGGGATCTCTTGATTCCTCAAGAGTCTGAGGATCGAGG

GTTGCCCATTCTTCCATCTTGCCACCTAATCCTTACTCCACTTGAGGGTA

TCACCAGCCCTTCTAGCTCCATGAAGGTGCCCCTGGGCAAGCACAATCTG

AGCATGAAAGATGCCCCAGAGGCCTTGGGTGTCATCCACTCATCATCCAG

CATCCACACTCTGAGGGTGTGGCCAGCACCATGACGTCATGTTGCTGTGA

CTATCCCTGCAGCGTGCCTCTCCAGCCACCTGCCAACCGTAGAGCTGCCG

ACATCCTCCTCTGGTGGGAGTGGCCTGCATGGTGCCAGGCTGAGGCCTAG

TGTCAGACAGGGAGCCTGGAATCATAGGGATCCAGGACTCAAAAGTGCTA

GAGAATGGCCATATGTCACCATCCATGAAATCTCAAGGGCTTCTGGGTGG

AGGGCACAGGGACCTGAACTTATGGGTTTTCCCCAAGTCTATTGCTCTCC

CAAGTGAGTCTCCCAGATACGAGGCACTGTGCCAGCATCAGCCTTATCTC

CACCACATCTTGTAAAAGGGACTACCCAGGGCCCTGATGAACACCATGGT

GTGTACAGGAGTAGGGGGTGGAGGCACGGACTCCTGTGAGGTCACAGCCA

AGGGAGCATCATCATGGGTGGGGAGGAGGCAATGGACAGGCTTGAGAACG

GGGATGTGGTTGTATTTGGTTTTCTTTGGTTAGATAAAGTGCTGGGTATA

GGATTGAGAGTGGAGTATGAAGACCAGTTAGGATGGAGGATCAGATTGGA

GTTGGGTTAGAGATGGGGTAAAATTGTGCTTCGGATGAGTTTGGGATTGA

CACTGTGGAGGTGGTTTGGGATGGCATGGCTTTGGGATGGAAATAGATTT

GTTTTGATGTTGGCTCAGACATCCTTGGGGATTGAACTGGGGATGAAGCT

GGGTTTGATTTTGGAGGTAGAAGACGTGGAAGTAGCTGTCAGATTTGACA

GTGGCCATGAGTTTTGTTTGATGGGGAATCAAACAATGGGGGAAGACATA

AGGGTTGGCTTGTTAGGTTAAGTTGCGTTGGGTTGATGGGGTCGGGGCTG

TGTATAATGCAGTTGGATTGGTTTGTATTAAATTGGGTTGGGTCAGGTTT

TGGTTGAGGATGAGTTGAGGATATGCTTGGGGACACCGGATCCATGAGGT

TCTCACTGGAGTGGAGACAAACTTCCTTTCCAGGATGAATCCGGGGAAGC

CTTAATTCACGTGTAGGGGAGGTCAGGCCACTGGCTAAGTATATCCTTCC

ACTCCAGCTCTAAGATGGTCTTAAATTGTGATTATCTATATCCACCTCTG

TCTCCCTCACTGTGCTTGGAGTTTACCTGATCACTCAACTAGAAACAGGG

GAAGATTTTATCAAATTCTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAG

TCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCAGTCTCGGCTCACT

GCAACCTCTGCCTCCCAGGTTCAAGTGATTCTCCTGCCTCAGCCTCCTGA

GTTGCTGGGATTACAGGCATGCAGCACCATGCCCAGCTAATTTTTGTATT

TTTAGTAGAGATGGGGTTTCACCAATGTTTGCCAGGCTGGCCTCGAACTC

CTGACCTGGTGATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAG

GCGTCAGCCACCGCGCCCAGCCACTTTTGTCAAATTCTTGAGACACAGCT

CGGGCTGGATCAAGTGAGCTACTCTGGTTTTATTGAACAGCTGAAATAAC

CAACTTTTTGGAAATTGATGAAATCTTACGGAGTTAACAGTGGAGGTACC

AGGGCTCTTAAGAGTTCCCGATTCTCTTCTGAGACTACAAATTGTGATTT

TGCATGCCACCTTAATCTTTTTTTTTTTTTTTTAAATCGAGGTTTCAGT

CTCATTCTATTTCCCAGGCTGGAGTTCAATGGCGTGATCACAGCTCACTG

TAGCCTTGAACTCCTGGCCTTAAGAGATTCTCCTGCTTCGGTCTCCCAAT

AGCTAAGACTACAGTAGTCCACCACCATATCCAGATAATTTTTAAATTTT

TTGGGGGGCCGGGCACAGTGGCTCACGCCTGTAATCCCAACACCATGGGA

GGCTGAGATGGGTGGATCACGAGGTCAGGAGTTTGAGACCAGCCTGACCA

ACATGGTGAAACTCTGTCTCTACTAAAAAAAAAAAAAATAGAAAAATTAG

CCGGGCGTGGTGGCACACGGCACCTGTAATCCCAGCTACTGAGGAGGCTG

AGGCAGGAGAATCACTTGAACCCAGAAGGCAGAGGTTGCAATGAGCCGAG

ATTGCGCCACTGCACTCCAGCCTGGGTGACAGAGTGAGACTCTGTCTCAA

AAAAAAAAAATTTTTTTTTTTTTTTTGTAGAGATGGATCTTGCTTTGTTT

CTCTGGTTGGCCTTGAACTCCTGGCTTCAAGTGATCCTCCTACCTTGGCC

TCGGAAAGTGTTGGGATTACAGGCGTGAGCCACCATGACTGACCTGTCGT

TTAATCTTGAGGTACATAAACCTGGCTCCTAAAGGCTAAATATTTTGTTG

GAGAAGGGGCATTGGATTTTGCATGAGGATGATTCTGACCTGGGAGGGCA

GGTCAGCAGGCATCTCTGTT.GCACAGATAGAGTGCACAGGTCTGGAGAAC

AAGGAGTGGGGGGTTATTGGAATTCCACATTGTTTGCTGCACGTTGGATT

TTGAAATGCTAGGGAACTTTGGGAGACTCATATTTCTGGGCTAGAGGATC

TGTGGACCACAAGATCTTTTTATGATGACAGTAGCAATGTATCTGTGGAG

CTGGATTCTGGGTTGGGAGTGCAAGGAAAAGAATGTACTAAATGCCAAGA

CATCTATTTCAGGAGCATGAGGAATAAAAGTTCTAGTTTCTGGTCTCAGA

GTGGTGCAGGGATCAGGGAGTCTCACAATCTCCTGAGTGCTGGTGTCTTA

GGGCACACTGGGTCTTGGAGTGCAAAGGATCTAGGCACGTGAGGCTTTGT

ATGAAGAATCGGGGATCGTACCCACCCCCTGTTTCTGTTTCATCCTGGGC

GTGTCTCCTCTGCCTTTGTCCCCTAGATGAAGTCTCCATGAGCTACAGGG

CCTGGTGCATCCAGGGTGATCTAGTAATTGCAGAACAGCAAGTGCTAGCT

CTCCCTCCCCTTCCACAGCTCTGGGTGTGGGAGGGGGTTGTCCAGCCTCC

AGCAGCATGGGGAGGGCCTTGGTCAGCCTCTGGGTGCCAGCAGGGCAGGG

GCGGAGTCCTGGGGAATGAAGGTTTTATAGGGCTCCTGGGGGAGGCTCCC

CAGCCCCAAGCTT(SEQ?ID?NO：4)

Those skilled in the art will think that other promoter that structurally reaches equivalence on the function can be used in the said just now promoter report health check-up survey; The promoter that for example has at least 75% homogeneity (that is any homogeneity percent between 75% and 100%) with SEQ ID NO:4.These promoter should comprise at least one duplicate of androgen response element, for example any among the SEQ ID NOs:1-3.

The instance of equivalence promoter comprises those and leaves out or adds the active equivalent promoter of promoter up to 40 kinds of nucleotide and maintenance at least 70% SEQ ID NO:4.The purposes of these promoter is also in category of the present invention.

The promoter activity can be expressed (for example according to the fluorescent hybridization technology) through characteristic (for example enzymatic activity or fluorescence), report body expression of polypeptides (for example according to the ELISA calibrating) or the report body mRNA that measures report body polypeptide and come quantitatively.Be exposed to AR agonist (for example testis sterone or 5 α-dihydro-testosterone sterone) and test composition first group and be exposed to that relatively PSA promoter is active between second group of independent AR agonist.If with respect to this second group, the report body is active or express and increase or reduce in this first group, thinks that then this test composition is an IKK α RT modulators.

Suitable report body polypeptide comprises (for example) LUC Photinus pyralis LUC Photinus pyralis FL, jellyfish luciferases (Renilla luciferase), fluorescence protein (for example enhanced type GFP egfp), beta galactosidase, β-Nei vinegar amine enzyme, alkali phosphatase and horseradish peroxidase.

For example, uciferase activity can detect through the suitably luminous matter (for example be used for the fire fly luminescence of LUC Photinus pyralis LUC Photinus pyralis FL plain or be used for the coelenterazine (coelenterazine) of jellyfish luciferases) that receives is provided.Uciferase activity can be come quantitatively by for example cold light photometry numerous standard techniques such as (luminometry) in the presence of the matter suitably receiving.

Can make report body live vol mark standard change into the protein concentration in the cell lysates.Protein concentration can use the Ku Masi (Coomassie) of commercial reagent to measure by (for example).Perhaps, the second report body structure (meaning is ＂ standardization report body structure ＂) can be used for active measurement of standardization report body.Standardization report body polypeptide by standardization report body structure coding has the activity that is different from above-mentioned androgen response report body polypeptide usually.In addition, standardization report body structure generally includes weak composition promoter, for example drives the herpetic thymidine kinase promoter of report body expression of polypeptides.This standardization report body structure can separate with the identical nucleic acid that comprises the first report body structure or be the part of this nucleic acid the part of plastid (for example as).The promoter activity can be come quantitatively through the ratio of measuring androgen response report body polypeptide active and standardization report body polypeptide active.For example, in the health check-up of two luciferase report was surveyed, LUC Photinus pyralis LUC Photinus pyralis FL can be used as indication androgen response promoter active report body polypeptide and jellyfish luciferases can be used as standardization report body polypeptide.The detailed description that comprises two luciferase detection methods of high-yield method is disclosed in United States Patent (USP) the 5th, 744, in No. 320.

The report body surface shows or activity can or be examined and determine in living cells in acellular calibrating (for example cell lysates), and this is decided by matter according to report body polypeptide that is selected for detection or report body enzyme.Acellular detection can be carried out in any suitable vessel (for example microtiter plates, test tube, color comparison tube and microcentrifugal tube).Living cells calibrating can be carried out in the container (for example micro-cell culture dish, porous disc, Tissue Culture Dish and Tissue Culture Flask) that mammalian cell cultivates in any being suitable for.The porous cell culture plate is applicable to the direct cold light photometry or the fluorometry (fluorimetry) of cell in the hole of this dish or cell lysates.Uciferase activity can be in living cells through suitable luciferase being received the upright light emission that connects (meaning does not promptly have dissolving step) in the cultured cell that is added in the cell culture medium and measure directly measure from intact cell.Can be with Viviren ^TMReceive matter (Promega, WI) or other suitable cell permeability luciferase be added in the cell to measure uciferase activity by upright connecing.

Fluorescent polypeptide (for example EGFP) can be in living cells thus in the technology known numerous detection methods (for example fluorometry or fluorescence microscopy) detect and quantitatively.In living cells, using the report health check-up of fluorescent polypeptide decide the detailed description (comprising high-yield method) of screening can be in discovery in No. the 6th, 875,578, (for example) United States Patent (USP).

Report body mRNA and protein performance amount can be measured by numerous prior art method.For example; Can be from the report body mRNA of treated and undressed cell performance by standard rna ink dot analytic process monitoring or can be by PCR; Especially for known similar techniques in quantitative PCR (qPCR) or this technology assist (referring to, Current Protocols in Molecular Biology for example, John Wiley & Sons; N.Y. (2005), 15.5-15.7).Immunoassays also can be used for detecting or monitoring report body content of peptides.For numerous report body polypeptide (for example EGFP, luciferase or beta galactosidase), many strains of specificity or monoclonal antibody are commercially available and can be used in any standard immunoassay calibrating form.Be applicable to that the algoscopy that measures report body content of peptides comprises that competitiveness and noncompetitive algoscopy, radioimmunoassay, bioluminescence and chemiluminescence assay, fluorimetry, sandwich assay, the some stain method of the use of ink and water, enzyme join algoscopy (comprising ELISA), microtiter plates and band that applies through antibody or the gauge rod that is used for fast monitored blood.For each method, confirm the scope, sensitivity, precision, reliability, specificity and the repeatability that detect.The instance of some immunoassay is described in detail in (for example) Current Protocols inMolecular Biology, John Wiley & Sons, and N.Y. (2005) is among the 11.1-11.3.

The IKK alpha kinase level of activity of regulating through AR according to circumstances, can be measured in the report somatic cell that is exposed to test composition or negative control compositions (for example cell culture medium).For example; After having or not having the androgen irritation cell of IKK α RT-modulators test composition; The IKK alpha kinase content that can prepare cell lysates and AR misfit can be by using the AR monoclonal antibody (for example from Santa Cruz Biotechnology; Inc. the immunoprecipitation of (Santa Cruz, AR-441 CA)) is then by using histone H 3 as measured by the in vitro protein kinase algoscopy of matter.The IKK alpha active degree that the degree indication of histone H 3 phosphorylation stimulates through AR.Perhaps, the IKK alpha active can in vitro be measured in the presence of test composition.For example, can at first use to derive from (for example) Santa Cruz Biotechnology, (SantaCruz, the polyclonal antibody of the anti-IKK α of many strains CA) is with whole IKK α immunoprecipitation in cell lysates for Inc..Then detecting kinases through immunoprecipitation is having or is not having that it makes the ability of histone H 3 phosphorylation under the situation of test composition.

Above-mentioned androgen response report somatic cell (for example androgen response report somatic cell strain) is applicable to that also the IKK α RT that measures (for example in mice, rat, hamster, guinea pig, rabbit, Canis familiaris L., cat or the monkey) among the non-human mammal test person under inspection is active.Animal model (for example nude mouse or nude rat) to xenograft tool toleration is especially suitable.At first will report somatic cell and inject the test person under inspection.In Biosample that the self-test person under inspection obtains (for example the living tissue sample of blood sample, skin sample, prostata tissue etc.), detect report body activity subsequently.Other or in addition, can be directly (meaning promptly in vivo) to detect report body active in the test person under inspection.Luciferase is the report body polypeptide that especially is suitable for that is used in vivo detecting.For example, the somatic test of the report that contains expressing luciferase person under inspection is thrown and the suitable luminous matter (for example fluorescein) that receives.Then (for example) detected the photo emissions by in vivo uciferase activity produced in vivo by the optical tomography art described in No. the 6th, 596,257, (for example) United States Patent (USP).The charge coupled device camera system that is applicable in vivo imaging is available from (for example) Xenogen (Alameda, IVIS CA)

system.Come the IKK alpha active degree in self-test person under inspection's the Biosample also can measure by said method.The Biosample of IKK alpha active to be determined can comprise only reports somatic cell, report somatic cell and blastema or blastema only.

The measurement of report body and IKK alpha active is applicable to that (for example) is evaluated at the IKK α RT modulators effectiveness of being discerned in the above-mentioned test composition report health check-up survey in vivo in the test person under inspection.

The health check-up of above-mentioned promoter report is surveyed and can be used for discerning the compositions that suppresses IKK α RT.Affiliated compositions comprises (for example) plant extract from Herbia Wedeliae, Eclipta prostrata or Herba Ecliptae, and suppresses the pure compound of IKK alpha active, and for example leaf contains 1,8,9-trihydroxy-3-methoxy-benzo[4,5, leaf flavone, Cortex querci dentatae ketone, foreign first Sui flavin, 15-deoxidation-Δ ^12,14-prostaglandin J ₂And PGA1.

IKK α RT inhibitor can be used for treating the disease that androgen stimulates.For example; Can throw the disease that the compositions (for example containing the compositions that Eclipta prostrata extract or leaf contain 1,8,9-trihydroxy-3-methoxy-benzo[4,5) with the IKK α RT inhibitor that contains effective dose stimulates with the treatment androgen, for example carcinoma of prostate, benign prostatauxe, breast carcinoma, male's alopecia, propionibacterium acnes, PCO disease, Autosome dominance POLYCYSTIC KIDNEY DISEASE or the too much disease of androgen to the person under inspection of needs.Before throwing and inhibitor combination, this person under inspection is diagnosed as the disease of suffering from the androgen stimulation or suffers from this sick excessive risk.For example, under the situation of carcinoma of prostate, in from this person under inspection's Biosample (for example blood sample), measure PSA content.The detection that is used for PSA can be carried out described in 088 like (for example) United States Patent (USP) the 6th, 300.The measurement of PSA content also can be carried out in different time points after throwing and the inhibitor combination as the treatment indicator that responds this treatment.

In some cases, the person under inspection can have the excessive risk of suffering from carcinoma of prostate, and is for example indicated by too high PSA content.Therefore, can reduce this person under inspection's carcinoma of prostate risk through this person under inspection prophylactically being thrown with the material that contains the IKK α RT inhibitor of effective dose.

Above-mentioned for example comprise from the plant extract of Herbia Wedeliae, Eclipta prostrata or Herba Ecliptae and for example leaf contain 1,8,9-trihydroxy-3-methoxy-benzo[4,5, leaf flavone, Cortex querci dentatae ketone, foreign first Sui flavin, 15-deoxidation-Δ ^12,14-prostaglandin J ₂Or the inhibitor combination of the pure compound of PGA1 can be incorporated in the medical composition for prevention or treatment use.For example, medical composition can comprise that the leaf of effective dose contains 1,8,9-trihydroxy-3-methoxy-benzo[4,5 and pharmaceutically acceptable supporting agent.Term ＂ effective dose ＂ refers to that the person under inspection to treatment grants the amount that produces the required active compound of prevention or therapeutical effect.Those skilled in the art considered that effective dose can and handle the probability of sharing with other prevention or treatment according to the disease type of being treated, disease seriousness, person under inspection's holistic health and/or age, previous treatment, dosing way, excipient usage and change.

Be the method for embodiment of the present invention, can be non-throw and active compound through intestinal, per os, per nasal, per rectum, part or through the approach of buccal.Among this paper used term ＂ non-through intestinal ＂ refer in subcutaneous, Intradermal, intravenous, intramuscular, intraarticular, intra-arterial, synovial membrane, in the breastbone, in the sheath or intralesional and any suitable injection technique.

The sterile injectable compositions can be nontoxic non-solution or suspension in intestinal acceptable diluent or solvent, such as the solution in the 1,3 butylene glycol.Spendable accept mediator and solvent be mannitol, water, ringer's solution (Ringer ' s solution) and etc. open sodium chloride solution.In addition, known fixed oil is as solvent or suspension media (for example synthetic monoglyceride or two glyceride).Such as oleic fatty acid and glyceride ester derivatives thereof is to be applicable to prepare the injectable thing of going up acceptable oil like natural medicaments, such as olive oil or Oleum Ricini, specifically is the ethylating form of its polyoxy.Said oil solution or suspension also can contain long-chain alcohol diluent or dispersant, carboxymethyl cellulose or similar dispersant.From the allotment purpose, also can use other interfacial agent commonly used (such as soil temperature (Tween) or span (Span) or other similar emulsifying agent) or the biological usability reinforcing agent that are usually used in making pharmaceutically acceptable solid, liquid or other dosage form.

The compositions that is used for oral administration medicine supplying can be any per os acceptable forms, comprises capsule, lozenge, emulsion and waterborne suspension, dispersion liquid and solution.Under the situation of lozenge, supporting agent commonly used comprises lactose and corn starch.Also add such as lubricants such as magnesium stearate usually.For the oral administration medicine supplying of capsule form, suitable diluents comprises lactose and dried corn starch.When oral administration throwing and waterborne suspension or emulsion, can or be dissolved in the oil phase that makes up with emulsifying agent or suspending agent the active ingredient suspension.If need, then add some sweeting agent, flavoring agent or coloring agent.

The technology preparation that nasal spray or composition for inhalation are known in can the technology according to pharmaceutical formulation.For example, known other solubilizing agent or dispersant in absorption enhancer, fluorocarbon and/or this technology of use benzylalcohol or other suitable antiseptic, enhancing biological usability can be formed in the solution in the normal saline solution with the said composition preparation.

The suppository form that also can be used for rectal administration is thrown and active compound.

Supporting agent in medical composition compatibility (and preferably, can make active ingredient stable) and with regard to the harmless aspect of person under inspection to be treated, be necessary for the acceptable ＂ of ＂ with regard to the active ingredient of itself and said composition.One or more solubilizing agent can be used as the medical excipient that transmits reactive compound.The instance of other supporting agent comprises colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate and D&C Yellow#10.

Following instantiation is only explained as exemplary, and is limited the application's remainder in no case.Not having under the situation about further carefully stating, believe that knowing this operator can farthest utilize the present invention based on the explanation among this paper.The full text of all documents of being quoted among this paper is incorporated herein by reference.

The generation and the characteristic of instance 1 carcinoma of prostate report somatic cell strain

The 22Rv1 cell strain system is available from ATCC.Circular plastid (p5.7kb-PSA-Luc) comes construction through 5.7kb PSA promoter is inserted in the pGL3 plastid (Promega).The 22RvPSA36-103 cell strain obtains in p5.7kb-PSA-Luc and pGK-puro stable transfection to the 22Rv1 cell and described fast little element is selected (Fryer and Archer, 1998) through making.

Detect 8 * 10 for uciferase activity ⁴Individual cell derived from 22Rv1 is grown in 48 hole porous discs.Cell culture medium is the RPMI that contains the glucosan absorption hyclone (Hyclone) that 5% charcoal encapsulates.After cell was grown one day, change its growth medium comprising experiment or the reference composition that is described below in culture, and the cell growth was continued 20 hours again.Make cytolysis through adding passive dissolution buffer (Promega), then use luciferase detection system (Promega) and VICTOR ²Many mark calculating instruments (PerkinElmer) carry out luciferase and detect, and are standardized into the solubilising protein that is measured as by Ku Masi (Bradford) protein detection cover group (Pierce).

AR signal transduction path promotes the carcinoma of prostate development.Therefore, the inventor sets up based on the model of cell and responds the promoter activity with the androgen in the observation human benign prostatic cancerous cell.The inventor is with responding report somatic cell strain to produce the 22RvPSA36-103 androgen in PSA-luciferase report body stable transfection to the 22Rv1 cell.The 22RvPSA36-103 cell was cultivated 20 hours in the presence of androgen.Stimulate PSA-uciferase activity up to about 100 times androgen, testis sterone or 5 α-dihydro-testosterone sterone (10nM) with the dose dependent mode through surpassing baseline.Because steroid hormone 17-and progesterone fail to induce the PSA-luciferase expression; Therefore inducing the PSA-luciferase expression in the 22RvPSA36-103 cell is specific to androgen; Be similar to like people such as Shao; (2003), Prostate, the ketosteroid specificity of the in vivo CWR22 xenograft among the 57:1-7.Active the reaching by androgen in low basis proves that to effectively inducing of PSA-luciferase expression the 22RvPSA36-103 cell can be used for discerning androgen and responds the active modulators of promoter.

Instance 2 androgens induce AR and IKK α to interact

The inventor studies IKK α or whether IKK β can interact with activated AR in the 22Rv/PSA36-103 cell after androgen stimulates.After the 5 α dihydro-testosterone sterone that cell are exposed to 10nM last five minutes, the coimmunoprecipitation of AR reaction in the misfit thing of detection and IKK α.The IKK alpha content relevant with AR accumulated in 10 to 15 minutes and reaches peak value, then after 30 minutes, descended, and after 60 minutes, reduced fully.On the contrary, at all time points, detect IKK β and in AR misfit thing, only be in utmost point low content.Such as by the indirect IF staining institute mensuration of using AR and IKK Alpha antibodies, during AR and IKK α are positioned to examine altogether.It follows the time-histories of being measured by said proteinic coimmunoprecipitation reaction for being similar to after location altogether.

The result proves jointly in the AR activation inducing cell nuclear that the formation of misfit thing is transcribed with activation androgen response target gene between the AR and IKK α.

Instance 3 IKK α suppress to reduce androgen and respond the promoter activity

Use above-mentioned luciferase to detect and tried IKK inhibitor 15-deoxidation-Δ ^12,14-prostaglandin J ₂, PGA1 and leaf contain 1,8,9-trihydroxy-3-methoxy-benzo[4,5 and suppress androgen and respond the active ability of promoter.Said chemical compound suppresses 5 α-inductive luciferase expression of dihydro-testosterone sterone with the dose dependent mode.On the contrary, IKK-β-specific inhibitor SC-514 does not play a role to androgen response promoter activity under any concentration of being tested.This result hints that AR signal transduction path is via IKK alpha specific generation effect.

For confirming this conclusion, the 22RvPSA36-103 cell strain that inventor's construction can induce the RNAi-of IKK α or IKK β to knock out.

The employed bobby pin RNAi of inventor expression system is the modification of the derivable expression system reported of other places (people such as vandeWetering, 2003).Use following hair clip to knock out sequence:

IKK α (sense strand 5 '-GCAGGCUCUUUCAGGGACA-3 ')

IKK β (sense strand 5 '-GGUGAAGAGGUGGUGGUGAGC-3 ')

Two sequences are reported in people such as Takaesu, in 2003.The H1-bobby pin rna expression frame that construction tet operon is regulated and be inserted into that (Carlsbad is in pcDNA6tTRTM plastid CA) from Invitrogen.Make each cell strain in plastid transfection to the 22RvPSA36-103 cell that knocks out with the generation stable transfection.IKK α or IKK β's knocks out through western blot determination in the daughter cell strain.Show that two cell strains that significantly knock out are used for further research.For inducing knocking out of RNAi adjusting, make comparisons with the mediator matched group, cell was grown 3 days in the culture medium that contains 1 μ g/ml doxycycline.Carry out subsequently processing and detection according to above-mentioned luciferase validation program.

Consistent with above-mentioned IKK inhibitor result, the RNAi of IKK α knocks out and makes the inductive PSA-luciferase expression of androgen reduce 50% to 60%.On the contrary, the demonstration that knocks out of IKK β does not have effect to the inductive luciferase of androgen, and this hints IKK α but not IKK β is required for androgen response promoter activity.

Instance 4 IKK alpha inhibitors are to the effect of prostate gland cancer cell growth

The activation of supposing AR signal transduction path stimulates prostatic cell proliferation and inventor to find to relate in this path IKK α, and the inventor manages to confirm that IKK α suppresses whether can reduce the propagation of prostate gland cancer cell.For this purpose, having or do not having in the presence of the androgen of IKK inhibitor, the colony of 22Rv1 cell and 22RvPSA36-103 cell forms efficient system through making 2 * 10 ⁴Individual cell is grown in 12 hole porous discs and is measured over 6 days.Changed growth medium in per three days.When growth cycle finished, 0.1% crystal violet that is used for the phosphate-buffered normal saline solution dyeed cell colony, dry and photography.The cell retentivity of crystal violet is through 20% acetic acid extraction in the water and at OD ₅₉₅Measure down to form the quantitative target of efficient as colony.

Under same concentrations, being used for above-mentioned experiment responds the active leaf of promoter to contain the 1,8,9-trihydroxy-3-methoxy-benzo[4,5 proof effective to the colony growing height of the 22Rv1 that suppresses androgen with the dose dependent mode and stimulate and 22RvPSA36-103 cell to suppress androgen.On the contrary, IKK β-specific inhibitor SC-514 fails to suppress the colony growth.The RNAi of IKK α knocks out the colony growth that reduces the androgen stimulation also effective.On the other hand, the RNAi of IKK β knocks out and fails to reduce the colony growth.

These results confirm that when IKK α came across in the carcinoma of prostate, its cell proliferation to the androgen stimulation was very crucial, and this cancer can be effectively treated in the inhibition of IKK α.

Other embodiment

All characteristics that disclosed in this description can be combined with any combination.Each characteristic that is disclosed in this description can be substituted by the alternative characteristics as identical, equivalence or similar applications.For example, the ability of candidate IKK α RT regulating and controlling article composition inhibition IKK alpha active can be examined and determine in IKK α-express cell of responding except that androgen the report somatic cell.Certainly, said mensuration is also in the present invention's category.

Describe through preceding text, be familiar with this operator and can be easy to definite basic feature of the present invention, and can make multiple variation of the present invention and modification under spirit of the present invention and the category so that it adapts to multiple usage and condition not breaking away from.Therefore, other embodiment is also contained in the present invention.

The present invention also provides following technical proposals:

1. the purposes of the compositions of an I kappa b kinase subunit alpha inhibitor that contains effective dose; It is used to prepare such medicament; Said medicament is used for treating the disease that person under inspection's androgen of needs treatment stimulates; The disease that this androgen stimulates is a carcinoma of prostate, and wherein this I kappa b kinase subunit alpha inhibitor is that leaf contains 1,8,9-trihydroxy-3-methoxy-benzo[4,5 (wedelolactone).

2. like the purposes of item 1, wherein before throwing and this medicament, further diagnose this person under inspection to suffer from the disease of carcinoma of prostate.

3. like the purposes of item 1, wherein said composition is a plant extract.

4. like the purposes of item 3, wherein this plant is Herbia Wedeliae (Wedelia chinensis).

5. as 1 purposes, wherein throw with this medicament before or after further measure from the prostatic special antigen content in this person under inspection's the Biosample.

6. the purposes of the compositions of an I kappa b kinase subunit alpha active inhibitor that contains effective dose; It is used to prepare such medicament; Said medicament is used to reduce the carcinoma of prostate risk for the person under inspection of the high ill risk of carcinoma of prostate tool, and wherein this I kappa b kinase subunit alpha inhibitor is that leaf contains 1,8,9-trihydroxy-3-methoxy-benzo[4,5 (wedelolactone).

7. method that is used to discern the modulators of the transcriptional activity of regulating by I kappa b kinase subunit α, it comprises:

Mammalian cell is provided; It expresses I kappa b kinase subunit α and androgen receptor; And contain separated nucleic acid; Said nucleic acid is connected in the androgen of coding report body peptide sequence with comprising operability and responds the promoter sequence, so that this cellular expression this report body polypeptide, wherein this androgen response promoter sequence comprises SEQ ID NO:4;

This cell is contacted with test composition; And

Detect the activity of expression or expressed report body polypeptide; Wherein compare the transcriptional activity that these test composition regulation and control of active difference indication of this expression or this report body polypeptide are regulated by I kappa b kinase subunit α in the presence of this test composition with the situation of not having this test composition.

8. like the method for item 7, wherein this androgen response promoter comprises at least one duplicate among the SEQ ID NOs:1,2 or 3.

9. like the method for item 8, wherein this androgen response promoter comprises at least one duplicate among the SEQ ID NOs:1,2 and 3.

10. as 7 method, it further comprises this cell is contacted with the agonist of this androgen receptor.

11. like the method for item 7, wherein this androgen receptor is the composition active mutant of this androgen receptor.

12. like the method for item 7, it further is included in after this contact procedure, measures the level of activity of this I kappa b kinase subunit α.

Sequence table

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Claims

2. purposes as claimed in claim 1 wherein before throwing and this medicament, further diagnoses this person under inspection to suffer from the disease of carcinoma of prostate.

3. purposes as claimed in claim 1, wherein said composition is a plant extract.

4. purposes as claimed in claim 3, wherein this plant is Herbia Wedeliae (Wedelia chinensis).

5. purposes as claimed in claim 1 was wherein further measured from the prostatic special antigen content in this person under inspection's the Biosample before or after throwing and this medicament.

This cell is contacted with test composition; And

8. method as claimed in claim 7, wherein this androgen response promoter comprises at least one duplicate among the SEQ ID NOs:1,2 or 3.

9. method as claimed in claim 8, wherein this androgen response promoter comprises at least one duplicate among the SEQ ID NOs:1,2 and 3.

10. method as claimed in claim 7, it further comprises this cell is contacted with the agonist of this androgen receptor.

11. method as claimed in claim 7, wherein this androgen receptor is the composition active mutant of this androgen receptor.

12. method as claimed in claim 7, it further is included in after this contact procedure, measures the level of activity of this I kappa b kinase subunit α.