CN101008009A - Cell model for quick screening of histone deacetylase inhibitor - Google Patents
Cell model for quick screening of histone deacetylase inhibitor Download PDFInfo
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- CN101008009A CN101008009A CNA2006100112727A CN200610011272A CN101008009A CN 101008009 A CN101008009 A CN 101008009A CN A2006100112727 A CNA2006100112727 A CN A2006100112727A CN 200610011272 A CN200610011272 A CN 200610011272A CN 101008009 A CN101008009 A CN 101008009A
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Abstract
The invention discloses a cell sifting moudle for fast sifting of antineoplastic- histone precursor histone deacetylase inhibitor compound from compound storage during new drug development. The moudle makes use of speciality that the precursor histone deacetylase inhibitor can activate some starting subsequence, constructs te oexpression vector containing special starting factor and firefly luciferase reporting gene, and builds cos- 7 clone that stably expresses said vector. The cell contains starting subsequence with different degree of activity for luciferase, the starting factor can be acitvated by histone precursor histone deacetylase inhibitor and then the expression of luciferase reporting gene is intensified, and so the detection for histone precursor histone deacetylase inhibitor activity in sample through detection of luciferase activity change can be finished in a short time. The cell sifting moudle can be used for fast and high- flux sifting of new antineoplastic and histone precursor histone deacetylase inhibitor compound from compound storage during new drug development.
Description
Technical field
The invention belongs to employedly in the novel compounds medicament research and development a kind ofly can carry out fast compound library, high flux screening, to obtain the cell model of NSC 630176 lead compound.
Background technology
Histone is the important component part that constitutes human body chromatin basal component group corpusculum, twines different eight aggressiveness of histone by the dna double chain and constitutes the group corpusculum.Studies show that the Methionin afterbody at histone exists multiple modification, comprise phosphorylation, methylate, acetylize and ubiquitinization etc. that these modifications have important regulation for gene transcription and expression.Exist two kinds of enzymes to participate in the adjusting of acetylation of histone in the known cell, promptly can strengthen acetylizad histone acetyltransferase, and can suppress acetylizad histon deacetylase (HDAC) (HDAC).Under normal circumstances, the acetylize of histone and the adjusting of deacetylation state have been kept in the running balance between these two kinds of enzymic activitys.When histone was high acetylize state, the space conformation of group corpusculum was comparatively open, is beneficial to various transcription factors near DNA, promotes genetic transcription.The deacetylation of histone then makes between DNA and the histone and is intimate-association state, is unfavorable for genetic transcription, causes some expression of gene to be closed.Present studies show that histone is the deacetylation state in tumor tissues, and this state mediated gene is transcribed inhibition, promotes the generation of tumour.And adopt hdac inhibitor to strengthen the acetylize of histone, and then can activate the expression of corresponding gene, suppress tumor proliferation, the blocking-up cell cycle, promote cytodifferentiation or apoptosis.Confirmed that hdac inhibitor all has the activity of good tumor-killing and inhibition metastases in kinds of tumor cells system and animal-transplanted tumor model, and it is lower to normal cytotoxicity, the effect characteristics of this high-efficiency low-toxicity make it become rather ideal anti-cancer agent, present several hdac inhibitors, it is clinical to enter the II phase in the U.S. as FK228, SAHA, MS275 etc.In view of such classes of compounds more, and mostly by extracting in plant and the microbe metabolite, and our country has abundant Chinese herbal medicine resource, by setting up the reliable and stable system of screening fast, can be expected to from existing monomeric compound storehouse or known Chinese herbal medicine effective ingredients, seek novel hdac inhibitor.
Summary of the invention
The purpose of this invention is to provide 2 kinds of cell models that can be used for anti-cancer agent histon deacetylase (HDAC) (HDAC) inhibitor rapid screening.It is characterized in that: contain in the cell can specificity by hdac inhibitor activatory exogenous promoter sequence:
The sequence that model 1 contains is: GCCGCCCCG ACTGCATCTG CGTGTTCGAA
TTCGCCAATG ACAAGACGCT GGGCGGGGTT TGTGTCATCA
TAGAACTAAA GACATGCAAA TATATTTCTT CCGGGGACAC
CGCCAGCAAA CGCGAGCAAC GGGCCACGGG GATGAAGCAG；
The sequence that model 2 contains is: TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT
AGCCCATATA TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC
TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG
TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG
TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC
AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT
ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC
GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA
TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA
TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA
AAATGTCGTA ACAACTCCG；
The establishment method of above-mentioned cell model is the upstream structure carrier for expression of eukaryon that above-mentioned promoter sequence is inserted the Photinus pyralis LUC reporter gene, method through the plasmalogen mediation imports the COS7 cell respectively with above-mentioned carrier, obtains the model cell of stable expression of exogenous gene after G418 drug screening.When adopting hdac inhibitor transaction module cell, exogenous promoter in the cell model can be activated and be strengthened the expression of luciferase, measure by fluorescein and luciferase noclilucence system, can detect the variation of cell uciferase activity before and after drug treating extremely sensitive, efficiently, judge the active state of promotor whereby.Wherein the characteristics of model 1 are low, the high specificities of detection background, can effectively get rid of false-positive screening sample.The effect characteristics of model 2 are highly sensitive, can effectively avoid because of the active low omission that occurs of sample.By the screening of uniting of above-mentioned 2 kinds of cell models, can discern and have the active compound of hdac inhibitor.This cell model has certain application value for lead compound and the follow-up medicament research and development of finding novel hdac inhibitor provides the important techniques platform.
Embodiment
The present invention is described further below in conjunction with adopting different types of hdac inhibitor Depsipeptide (FK228), suberoylanilidehydroxamic acid (SAHA), Trichostatin A (TSA) and other 4 kinds of medicine 5-aza-2 '-deoxycytidine (DAC), vitamin A acid, taxol and 17-AAG with different genera of anti-tumor activity to handle the experiment of cell model respectively.
1. experimental technique
In containing the commercial carrier of luciferase reporter gene, insert above-mentioned promoter sequence fragment respectively 1.1 adopt the method for molecular cloning, behind the plasmid amplification purifying, adopt the method rotaring redyeing COS 7 cell of plasmalogen mediation, G418 (1mg/ml) pressurization screening is after 21 days, obtain the cell resistance clone of stable expression of exogenous gene, this resistant cell can be used for follow-up drug screening as model.
1.2HDAC inhibitor FK228 and TSA are provided by David doctor Schrump of NIH institute of oncology, hdac inhibitor SAHA is provided by Britain's AstraZeneca (AstrZeneca) company.Other anti-tumor agents are respectively available from Sigma company.The working concentration of FK228 is respectively 12.5,25 and 50ng/ml, and the working concentration of SAHA is respectively 2.5,5 and 10 μ M, and the working concentration of TSA is respectively 300,600 and 900nM.
1.3 model cell is inoculated 24 orifice plates with appropriate to the occasion ratio, adopt the drug treating of different concns next day, after the drug treating 24 hours, the fluorescein detection kit that adopts Promaga company to provide, carry out lysis by the test kit specification sheets, and on the TD20/20 photometer that U.S. Turner Designs company produces, adopt single pipe method to carry out enzyme assay.Also can inoculate 96 orifice plates adopts and to read the plate photometer and carry out enzyme assay.
2. experimental result
2.1 three kinds of hdac inhibitors and 4 kinds of other antitumor drugs to the influence of the plain enzymic activity of model 1 cell fluorescence by shown in Figure 1,3 kinds of hdac inhibitors all can be the activity that dose-dependently strengthens the plain enzyme of cell fluorescence, increased activity 2.3-5.8 doubly after the medication, its minimal effective concentration is respectively FK22812.5ng/ml, SAHA2.5 μ M and TSA300nM, be lower than the effective concentration that the medicine pair cell kills and wounds, indicating system has hypersensitivity to the reaction of medicine, and other antitumor drugs all do not show the increase of uciferase activity in effective cell casualty-producing concentrations scope.
2.2 three kinds of hdac inhibitors and 4 kinds of other antitumor drugs are to the influence of the plain enzymic activity of model 2 cell fluorescences as shown in Figure 2,3 kinds of hdac inhibitors all can be the activity that dose-dependently strengthens the plain enzyme of cell fluorescence, increased activity 2.1-8.5 doubly after the medication, other antitumor drugs are handled the back uciferase activity and are also slightly strengthened, but the enhancing amplitude is no more than 2 times, and is not the dose-dependently increase.
Description of drawings:
Fig. 1 is that 3 kinds of hdac inhibitors and 4 kinds of antitumor drugs are in model 1 cell activity detected result; Show among the figure that 3 kinds of hdac inhibitors are dose-dependently and strengthen the plain enzymic activity of cell fluorescence, and other antitumor drugs do not show the uciferase activity increase.
Fig. 2 is that 3 kinds of hdac inhibitors and 4 kinds of antitumor drugs are in model 2 cell activity detected results; Show among the figure that 3 kinds of hdac inhibitors all can be the activity that dose-dependently strengthens the plain enzyme of cell fluorescence, maximum value can strengthen 8.5 times.Other antitumor drugs are handled also slightly increase of uciferase activity in the cell of back, but the enhancing amplitude is no more than 2 times, and are not the dose-dependently increase.
Claims (2)
- Two kinds can be by the specific, activated promoter sequence of NSC 630176:Sequence 1:GCCGCCCCG ACTGCATCTG CGTGTTCGAA TTCGCCAATGACAAGACGCT?GGGCGGGGTT?TGTGTCATCA?TAGAACTAAAGACATGCAAA?TATATTTCTT?CCGGGGACAC?CGCCAGCAAACGCGAGCAAC?GGGCCACGGG?GATGAAGCAGSequence 2:TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCATAGCCCATATA?TGGAGTTCCG?CGTTACATAA?CTTACGGTAAATGGCCCGCC?TGGCTGACCG?CCCAACGACC?CCCGCCCATTGACGTCAATA?ATGACGTATG?TTCCCATAGT?AACGCCAATAGGGACTTTCC?ATTGACGTCA?ATGGGTGGAG?TATTTACGGTAAACTGCCCA?CTTGGCAGTA?CATCAAGTGT?ATCATATGCCAAGTACGCCC?CCTATTGACG?TCAATGACGG?TAAATGGCCCGCCTGGCATT?ATGCCCAGTA?CATGACCTTA?TGGGACTTTCCTACTTGGCA?GTACATCTAC?GTATTAGTCA?TCGCTATTACCATGGTGATG?CGGTTTTGGC?AGTACATCAA?TGGGCGTGGATAGCGGTTTG?ACTCACGGGG?ATTTCCAAGT?CTCCACCCCATTGACGTCAA?TGGGAGTTTG?TTTTGGCACC?AAAATCAACGGGACTTTCCA?AAATGTCGTA?ACAACTCCG；
- 2. contain the application of eukaryotic cell in screening NSC 630176 lead compound of above-mentioned promoter sequence and Photinus pyralis LUC reporter gene expression carrier.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979090A (en) * | 2010-09-25 | 2011-02-23 | 北京师范大学 | Medicament for treating tumors |
| CN103293315A (en) * | 2012-06-14 | 2013-09-11 | 中国食品药品检定研究院 | RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog |
| CN104388538A (en) * | 2014-11-18 | 2015-03-04 | 中国人民解放军第三军医大学 | Method for screening histone deacetylase inhibitor |
| CN104714024A (en) * | 2014-11-13 | 2015-06-17 | 贵阳医学院 | Fluorescent activity detection method for human-derived silent information regulator 5 |
| CN102140494B (en) * | 2010-01-29 | 2015-07-08 | 杭州景杰生物科技有限公司 | Method for screening and testing activity of lysine propionylation removal enzyme and butyrylation removal enzyme |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001149081A (en) * | 1999-11-29 | 2001-06-05 | Cyclex Co Ltd | Method for assaying activities of deacetylases, and method for screening inhibitor or activator of these enzymes |
| WO2005059167A1 (en) * | 2003-12-18 | 2005-06-30 | Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa | Method for identifying histone deacetylase inhibitors |
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2006
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140494B (en) * | 2010-01-29 | 2015-07-08 | 杭州景杰生物科技有限公司 | Method for screening and testing activity of lysine propionylation removal enzyme and butyrylation removal enzyme |
| CN101979090A (en) * | 2010-09-25 | 2011-02-23 | 北京师范大学 | Medicament for treating tumors |
| CN103293315A (en) * | 2012-06-14 | 2013-09-11 | 中国食品药品检定研究院 | RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog |
| CN103293315B (en) * | 2012-06-14 | 2016-05-25 | 中国食品药品检定研究院 | RGA method detects insulin secretion accelerating peptide fusion BA |
| CN104714024A (en) * | 2014-11-13 | 2015-06-17 | 贵阳医学院 | Fluorescent activity detection method for human-derived silent information regulator 5 |
| CN104388538A (en) * | 2014-11-18 | 2015-03-04 | 中国人民解放军第三军医大学 | Method for screening histone deacetylase inhibitor |
| CN104388538B (en) * | 2014-11-18 | 2017-01-18 | 中国人民解放军第三军医大学 | Method for screening histone deacetylase inhibitor |
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