CN102517373A - Antineoplastic drug screening cell model utilizing STAT3 as target and creation and application thereof - Google Patents
Antineoplastic drug screening cell model utilizing STAT3 as target and creation and application thereof Download PDFInfo
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Abstract
The invention provides an antineoplastic drug screening cell model utilizing STAT3 as target, belonging to the field of biomedicine. The model is created by a reporting system carrier containing an STAT3 specific binding sequence on the basis of active cells with constitutive STAT3, and the model is used for screening treatment medicine of carcinomatous changes and tumors caused by STAT3 constitutive activation. The active cells with the constitutive STAT3 are utilized, so the reporter gene in the model cell is situated at an overactive state, and no additional stimulant is necessary; besides, the obtained stable cell model can be directly used for screening the compounds, thereby not only greatly reducing the screening cost, but also simplifying the whole screening process from cell cultivation, STAT3 activation, compound feed and detection to cell cultivation, compound feed and detection, shortening the screening period, reducing the operation steps, and improving the stability, efficiency and usability of the system, so that the model is more suitable for high throughput medicine screening.
Description
Technical field
The invention belongs to biomedicine field, relating to a kind of is the screening anti-tumor medicine cell model of target spot with STAT3, relates in particular to a kind of to have the screening anti-tumor medicine cell model that composing type STAT3 active cells is the basis; The present invention also relates to the construction process and the application of this screening anti-tumor medicine cell model simultaneously.
Background technology
Signal transduction and activating transcription factor 3 (STAT3) are being brought into play important effect as important transcription factor in the generation of cancer and development, be acknowledged as the cancer GAP-associated protein GAP.In many tumour cells; In colorectal carcinoma, mammary cancer, neuroglial cytoma, lung cancer, Head and Neck cancer or the like cell, STAT3 usually exists with the form of continuous activation, and STAT3 receives that the stream signal cytokine stimulates or under the state of some Tyrosylprotein kinase continuous activation; 705 tyrosine of its C end are formed dimer by phosphorylation; And then go into the nuclear adjusting and transcribe, start the regulation and control of series of genes, promoting cell transformation, shifting, prevent to bring into play multiple action aspect the apoptosis.Usually born of the same parents regulate outward STAT3 cytokine IL-6 arranged, LIF, OSM, EGF, PDGF, G-CSF etc., the factor of keeping the STAT3 continuous activity in the born of the same parents comes from continuous activation or self sudden change of upper reaches acceptor or nonreceptor tyrosine kinase.Research shows, suppresses the STAT3 activity and will reduce growth, existence and the transfer of kinds of tumor cells greatly, thereby reduce their grade malignancies (people: Cancer Res 2000 such as Ni greatly; People such as Leong: Proc Natl Acad Sci 2003; People such as Barton: Mol Cancer Ther 2004; People such as Kortylewski: Nat Med 2005; People such as Xin: Cancer Res 2009).Research shows in addition; In many tumour cells, STAT3 can promote and keep the activity of NF-κ B, and NF-κ B then is another important factor of facilitating that tumour takes place and shifts; It is active in this type tumour, to suppress STAT3; The tumor-related gene that has not only suppressed STAT3 regulation and control itself is also reduced the activity of NF-κ B to a certain extent, reaches purpose (people: the Cancer Res 2005 such as Yang J of any two target; People: Genes & Dev 2007 such as Yang J; People: Nat Cell Biol 2008 such as Torres J; People: Cancer Cell 2009 such as Lee H; People such as Yu H: Nat Rev Cancer 2009).
Be that the medicament sifting motion system of target spot has shortcomings such as inefficiency, complex steps usually at present based on STAT3; For example sieve medicine at every turn and all need use the foreign cell factor for example interleukin 6 (IL-6) and the interior STAT3 of leukocyte inhibitory factor (LIF) activating cells; Extra step will make the stability of sieve medicine system reduce greatly, and cost also will increase because of the use of IL-6 or LIF greatly.
Summary of the invention
The objective of the invention is to the problem that exists in the prior art, providing a kind of is the screening anti-tumor medicine cell model of target spot with STAT3.
It is the construction process of the screening anti-tumor medicine cell model of target spot with STAT3 that another object of the present invention provides a kind of.
A further object of the invention, just providing a kind of is the application of screening anti-tumor medicine cell model in screening anti-tumor medicine of target spot with STAT3.
(1) with STAT3 is the screening anti-tumor medicine cell model of target spot
The present invention is the screening anti-tumor medicine cell model of target spot with STAT3; Be to utilize the reporting system carrier that contains STAT3 specificity binding sequence; Be the basis to have composing type STAT3 active cells, being built into stable is target spot high-throughput screening anti-tumor medicine cell model with the STAT3 signal.
The specificity binding sequence of said STAT3 is 5 '-TT (C/A) CNNNAA-3 ', and N is a base.
Said reporting system carrier is in containing the report carrier MCS of luciferase reporter gene, to insert to include the response sequence of STAT3 specificity binding sequence as the reporting system carrier, the reporting system carrier that is built into.
Said have composing type STAT3 active cells and be: DU145 prostate cancer cell, H1299 human lung carcinoma cell, A549 human lung carcinoma cell, HCT119 human colon cancer cell, Hela human cervical carcinoma cell, MCF-7 human breast cancer cell, MDA-MB-468 human breast cancer cell or MDA-MB-231 human breast cancer cell or the like.
(2) with STAT3 be the structure of the screening anti-tumor medicine cell model of target spot.
The present invention is the structure of the drug screening cell model of target spot with STAT3, comprises following process step:
(1) reporting system vector construction: utilize molecular biology method in containing the report carrier MCS of luciferase reporter gene, to insert to include STAT3 specificity binding sequence and 3 ' end to contain the DNA fragment of TATA box sequence; As the response sequence of reporting system carrier, be built into the reporting system carrier.Insertion sequence is positioned at the upper reaches of luciferase reporter gene, when the STAT3 activation, just can strengthen transcribing and the luciferase translation of luciferase reporter gene, thereby strengthen the activity of luciferase, can detect stronger fluorescence when adding the luciferase substrate; When suppressing the STAT3 activity, the level of translating with luciferase of transcribing of luciferase reporter gene all reduces, and also just suppresses the activity of luciferase in the cell, and fluorescence intensity also just weakens when adding the luciferase substrate.
(2) structure of cell model: after the transfection of reporting system carrier had composing type STAT3 active cells; Carry out the positive colony screening with tetracycline; Choose the agent of known STAT3 signal suppressing (like PD-180970; Etc. STAT3 stream signal suppressor factor in the known specific cells) clone of response is arranged, being with STAT3 is the drug screening cell model of target spot.
In having the active cell of composing type STAT3; The reporting system carrier can be activated well; Fluorescence is significantly suppressed, and the cell model of structure also just need not under exogenous stimulation, to be used for delicately high-flux medicaments sifting.
(3) with STAT3 be the application of screening anti-tumor medicine cell model in screening anti-tumor medicine of target spot.
The present invention is that the screening anti-tumor medicine cell model of target spot is used to screen canceration and the tumor treatment medicine that is caused by the activation of STAT3 composing type with STAT3.The method of concrete screening antineoplastic drugs comprises following process step:
(1) with said screening anti-tumor medicine cell model with 100 μ l culture medium inoculateds in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40%;
(2) SCREENED COMPOUND 0.1 μ l ~ 2 μ l are treated in adding in substratum, continue to cultivate 24 hours;
(3) in above-mentioned 96 orifice plates, add 50 μ l stable form luciferase substrates, use fluorescence/chemiluminescent analyzer to measure fluorescence intensity and analysis of fluorescence inhibiting rate, obtain the primary dcreening operation product more than 40% when the fluorescence inhibiting rate reaches;
(4) with the coup injury of compound cytotoxicity experiment elimination compound pair cell model, the fluorescence inhibiting rate reaches still and is obtaining multiple sieve product more than 40%, and being with STAT3 is the inhibition medicine of target spot.
The present invention has the following advantages with respect to prior art: with the STAT3 signal path is target spot; Employing is strategy based on the clone with composing type STAT3 active (it is active promptly to continue STAT3); Make reporting system this is in the height active state in cell; The stabilized cell model that obtains need not to use to add stimulation, can directly be used for the screening of compound; Not only greatly reduce the cost of screening; Also make simultaneously whole screening process be reduced to " cell cultures → add compound → detection " from " cell cultures → activation STAT3 → add compound → detection "; Shortened the cycle of screening; Reduce operation steps, promoted stability, high efficiency and the ease for use of system, more be applicable to high-flux medicaments sifting.
Description of drawings
Fig. 1 detects the active result of STAT3 in the cell involved in the present invention for using the Western-Blot method.
Fig. 2 prevents test (EMSA) to detect the active result of STAT3 in the cell involved in the present invention for using gel electrophoresis.
Fig. 3 is used report carrier basic framework.
Fig. 4 is the sequencing result figure of constructed reporting system carrier S IE-luc insertion sequence.
Fig. 5 is for utilizing 293T cell detection reporting system carrier S IE-luc responding ability.
Fig. 6 detects the STAT3 activity change of A549 cell after known suppressor factor PD180970 handles for using the Western-Blot method.
Fig. 7 is that screening anti-tumor medicine cell model A549R is to the active response that reduces or raise of STAT3.
3412 pairs of STAT3 activatory inhibition of compound ability that Fig. 8 is sieved for using the Western-Blot method to detect.
Embodiment
Be example with the A549 cell below, the structure of screening anti-tumor medicine cell model of the present invention, construction process and application are described further.
The instrument and the reagent that are adopted in the experiment are following:
Instrument: PerkinElmer Victor3 fluorescence/chemiluminescent analyzer.
Reagent: property restriction endonuclease, T4 dna ligase are available from Fermentas company; Synthetic and the order-checking of primer, Shanghai is given birth to the worker and is provided; DMEM (high sugar) and foetal calf serum are available from Hyclone (Thermo company), and transfection reagent GenEscort II is biological available from the intelligent base in Nanjing; Photinus pyralis LUC is measured test kit available from Promega company; IL-6 is available from Peprotech company; PD180970 and Puromycin are available from Sigma-Aldrich company; 96 orifice plates that are used to sieve medicine are available from Corning company; Compound to be screened is from national micromolecular compound resource center; Various antibody are available from Cell Signaling Technology company.
1, reporting system vector construction
Dna fragmentation with 16 SIE that comprise (being STAT3 specificity binding sequence) of synthetic and the general auxiliary preface 5 ' of TATA box-TATATAA-3 '; Restriction enzyme site with KpnI and HindIII is inserted into general luciferase reporting carrier pBR322-luc-puro, the reporting system carrier called after SIE-luc that obtains.Above-mentioned STAT3 specificity binding sequence comprises 85 '-TTCCCGTAA-3 ' (M67 sequences; Hyperactive mutated form of SIE); 85 '-TTCCTGTAA-3 ' (from the Ly6E gene) alternately connect, and two kinds of elements all have the avidity of high special to STAT3.Luciferase reporting carrier pBR322-luc-puro is by inserting North America Photinus pyralis LUC (Firefly Luciferase between pBR322 carrier Hind III and the Sal I; Referring to people such as J R de Wet: reporter gene and tetracycline gene (Puromycin Mol. Cell. Biol. 1987); Referring to people: Gene.1989 such as Lacalle RA; GenBank:M25346.1) gained; And luciferase reporter gene gets into sequence (IRES) with the tetracycline screening-gene through internal ribosome and links to each other, and it is poly A tailing signal that there is the AATAAA sequence in tetracycline gene terminator codon downstream.The pBR322-luc-puro MCS is between former EcoR I and Hind III (referring to Fig. 3).
So far, the complete insertion sequence on pBR322-luc-puro luciferase reporting carrier (sequencing result is seen Fig. 4) as follows:
2, reporting system carrier property test
Because the 293T cell is relatively more responsive to the stimulation of the various foreign cell factors, be convenient to add the activation situation that detects the corresponding signal path when cytokine stimulates outside, so use this cell as representing examining report system carrier whether to respond normally.(see figure 5) shows as a result, in the carrier response of operation report system of corresponding signal path activation time institute normally, stimulates the back and stimulates the ratio of preceding uciferase activity to be about 18.7:1, and wherein the 293T cell uses IL-6 to stimulate activation.The 293T cell is grown to 50% ~ 70% density in 12 orifice plates, transfection SIE-luc (2 μ g/ hole), the usage ratio of carrier and transfection reagent is 1:2 (i.e. 2 μ g plasmids; 4 μ l transfection reagents respectively are dissolved in 50 μ l DMEM serum free mediums, are mixed into 100 μ l premixed liquids; Room temperature leaves standstill 20 min, adds 150 μ l serum-free DMEM again, adds behind the mixing in each hole of 12 orifice plates) change perfect medium (the DMEM substratum that promptly contains 10% foetal calf serum) into after 4 hours; After 12 hours, handled 24 hours, inhale and remove substratum with IL-6 (250 ng/ml); Add 200 μ l lysate lysing cell, jog 10min gets 150 μ l in 96 orifice plates; Add the common Photinus pyralis LUC substrate of 20 μ l, use fluorescence/chemiluminescent analyzer to measure the activity of (in the 1min) luciferase immediately, contrast and each 3 repetition of processing; And independent revision test 3 times (* * *, p 0.001).
3, the medicaments sifting model cell is selected
The activity of preventing STAT3 in the experimental analysis cell through Western-Blot and EMSA gel; Obtain multiple tumor cell line (Fig. 1 that is suitable for the STAT3 active high (need not cytokine irritation cells such as extra use IL-6) of this report system carrier; Fig. 2), Fig. 1 detects the active result of STAT3 in the cell involved in the present invention for using the Western-Blot method.The result shows that a series of tumor cell ratio normal cells that comprise A549 have higher STAT3 activity; Phosphorylation with the 705th tyrosine of STAT3 (Tyr705) characterizes; Be labeled as " pTyr705-STAT3 ", phosphorylation level is high more, and activity is high more.Fig. 2 prevents test (EMSA) to detect the active result of STAT3 in the cell involved in the present invention for using gel electrophoresis.Show that used several kinds of tumour cell activatory STAT3 have higher DNA binding ability than normal cell HME1 among Fig. 1.Wherein, HME1, people's mammary gland epidermic cell (normal control cell); DU145, prostate cancer cell; HepG2, human liver cancer cell; H1299 and A549 are human lung carcinoma cell, HCT119, human colon cancer cell; Hela, human cervical carcinoma cell; MCF-7, MDA-MB-468 and MDA-MB-231 are human breast cancer cell.
4, medicaments sifting model makes up and performance test
With SIE-luc transfection A549 cell (10cm petridish) after 48 hours, 1 passes 3, and adds puromycin and carry out the positive colony screening, and starting point concentration is 5 μ g/ml, reduces to 2.5 μ g/ml after 2 weeks.Behind the clone to be formed (altogether about 3 weeks), in 96 orifice plates, change 48 orifice plates with the trysinization mono-clonal after having grown again over to, 6 orifice plates are until 10 cm culturing bottles and frozen, during the use final concentration be 2.5 μ g/ml puromycin continue kept for 2 weeks.Whether first-selection is chosen luciferase male clone, detect these clones again and respond normal; Though be the composing type activation, cell generally can also externally add cytokine and react to some extent, so the uciferase activity of selected positive colony should increase when adding the IL-6 stimulation to some extent.Through checking, add and to make about 2.5 times of the active liftings of this cell model STAT3 when IL-6 (250 ng/ml) stimulates, and, use the suppressor factor of STAT3 signal path that the activity of luciferase is reduced owing to be constitutive activation.PD180970 is the suppressor factor of Tyrosylprotein kinase Src, and the STAT3 of bibliographical information A549 cell continuous activation is responsive to PD180970; The PD180970 that is selected for use (250 nM) concentration confirms that through cell growth inhibition test the A549 cell is not had growth-inhibiting (24 hours); The activity of using PD180970 under the concentration that does not influence the cell growth, all significantly to suppress STAT3 (is analyzed referring to Fig. 6 Western-Blot; The A549 passage is in 10 cm petridish; During to 60% ~ 70% degree of converging; Add 250 nM PD180970 and handle 24h, add DMSO simultaneously as contrast), thus the expression that has suppressed luciferase reaches 52.2%.Select the clone No. 18 through screening is final, be labeled as A549R, referring to Fig. 7 (* * *, p < 0.001).Cell inoculation is in 96 orifice plates (10; 000/>hole; 100 μ l) growth is 12 hours, changes the new nutrient solution that contains above-mentioned IL-6 (250 ng/ml) or suppressor factor PD180970 (250 nM), continues to cultivate after 24 hours; Add 50 μ l stable form luciferase substrates (Steady-Glo), lucifuge uses fluorescence/chemiluminescent analyzer to measure the activity of luciferase after reacting 10 min.
5, model is used
With the drug screening cell model with 100 μ l culture medium inoculateds in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40%; Add and treat SCREENED COMPOUND 0.25 μ l (25 μ M), continue to cultivate 24 h, in corresponding 96 orifice plates, add 50 μ l stable form luciferase substrates, use fluorescence/chemiluminescent analyzer to measure fluorescence intensity and analysis of fluorescence inhibiting rate.Obtain 8 kinds and inhibition and inhibiting rate are arranged at the compound more than 40%; The screening that reduces by half once more of these 8 kinds of compound concentrations; And eliminate the coup injury of compound pair cell model here with compound cytotoxicity experiment (testing with MTT); Obtaining still drug screening cell model uciferase activity inhibiting rate being had a kind (inhibiting rate reaches 70%) at the compound more than 40%, be the multiple sieve product that the ability specificity suppresses STAT3, is 3412 compound for numbering.
This is numbered 3412 compound except can suppressing the STAT3 reporting system is produced among the A549R fluorescence through the Western-Blot analysis verification; STAT3 to Human Prostate Cancer Cells DU145 continuous activation; And the activation of STAT3 has extraordinary inhibition effect (referring to Fig. 8) among the IL-6 inductive 293T; Prove that this medicaments sifting model not only has good feasibility and safety, good versatility (compound that is sieved can suppress the activity of STAT3 under multiple situation) is arranged simultaneously.Wherein, the DU145 passage is in 10 cm petridish, and during to 60% degree of converging, adding is numbered 3412 compound (10 μ M) processing 2h, simultaneously to add DMSO as contrast; The 293T passage is in 10 cm petridish, and during to 60% degree of converging, adding is numbered 3412 compound (10 μ M) and pre-treatment 30 min, adds IL-6 (250 ng/ml) again and handles 2h.
Above-mentioned experiment showed, through being that the screening anti-tumor medicine cell model of target spot sieves the compound that obtains again really for being the inhibition medicine of target spot with STAT3 with STAT3, thus the validity of this drug screening cell model proved.
A large amount of experiments show to have in the active cell of composing type STAT3 multiple, and that the present invention with STAT3 is that the drug screening cell model of target spot can both be obtained is convenient, screening effect fast and efficiently.
Claims (7)
1. with STAT3 the screening anti-tumor medicine cell model of target spot; It is characterized in that: utilize the reporting system carrier that contains STAT3 specificity binding sequence; Be the basis to have composing type STAT3 active cells, being built into stable is target spot high-throughput screening anti-tumor medicine cell model with the STAT3 signal.
2. be the screening anti-tumor medicine cell model of target spot with STAT3 according to claim 1, it is characterized in that: the specificity binding sequence of said STAT3 is 5 '-TT (C/A) CNNNAA-3 ', and N is a base.
3. be the screening anti-tumor medicine cell model of target spot according to claim 1 with STAT3; It is characterized in that: said reporting system carrier is in containing the report carrier MCS of luciferase reporter gene, to insert to include the response sequence of STAT3 specificity binding sequence as the reporting system carrier, the reporting system carrier that is built into.
4. be the screening anti-tumor medicine cell model of target spot with STAT3 according to claim 1, it is characterized in that: said to have composing type STAT3 active cells be DU145 prostate cancer cell, H1299 human lung carcinoma cell, A549 human lung carcinoma cell, HCT119 human colon cancer cell, Hela human cervical carcinoma cell, MCF-7 human breast cancer cell, MDA-MB-468 human breast cancer cell or MDA-MB-231 human breast cancer cell.
5. be the construction process of the screening anti-tumor medicine cell model of target spot with STAT3 according to claim 1, comprise following process step:
(1) reporting system vector construction: utilize molecular biology method in containing the report carrier MCS of luciferase reporter gene, to insert to include STAT3 specificity binding sequence and 3 ' end to contain the DNA fragment of TATA box sequence; As the response sequence of reporting system carrier, be built into the reporting system carrier;
(2) structure of cell model: after the transfection of reporting system carrier had composing type STAT3 active cells; Carry out the positive colony screening with tetracycline; Choosing to STAT3 signal suppressing agent has the clone of response, and being with STAT3 is the drug screening cell model of target spot.
6. the screening anti-tumor medicine cell model that with STAT3 is target spot according to claim 1 is used to screen canceration and the tumor treatment medicine that is caused by the activation of STAT3 composing type.
7. of claim 6 is that the screening anti-tumor medicine cell model of target spot is used to screen canceration and the tumor treatment medicine that is caused by the activation of STAT3 composing type with STAT3, and it is characterized in that: the method for said screening antineoplastic drugs comprises following process step:
(1) with said screening anti-tumor medicine cell model with 100 μ l culture medium inoculateds in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40%;
(2) SCREENED COMPOUND 0.1 μ l ~ 2 μ l are treated in adding in substratum, continue to cultivate 24 hours;
(3) in above-mentioned 96 orifice plates, add 50 μ l stable form luciferase substrates, use fluorescence/chemiluminescent analyzer to measure fluorescence intensity and analysis of fluorescence inhibiting rate, obtain the primary dcreening operation product more than 40% when the fluorescence inhibiting rate reaches;
(4) with the coup injury of compound cytotoxicity experiment elimination compound pair cell model, the fluorescence inhibiting rate reaches still and is obtaining multiple sieve product more than 40%, and being with STAT3 is the inhibition medicine of target spot.
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