CN104056267A - Method for improving therapeutic effect of breast cancer endocrine drugs by tissue factor antibody - Google Patents
Method for improving therapeutic effect of breast cancer endocrine drugs by tissue factor antibody Download PDFInfo
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- CN104056267A CN104056267A CN201310084936.2A CN201310084936A CN104056267A CN 104056267 A CN104056267 A CN 104056267A CN 201310084936 A CN201310084936 A CN 201310084936A CN 104056267 A CN104056267 A CN 104056267A
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Abstract
A method for improving therapeutic effect of breast cancer endocrine drugs by a tissue factor antagonist. The tissue factor antagonist improves or reestablishes the expression of estrogen receptors in breast cancer cells by intracellular signal transduction pathway, and thus plays a role of enhancement and assistance of the therapeutic effect of estrogen-blocking breast cancer endocrine drugs.
Description
Technical field
The present invention relates to using-system factor antagonist for breast carcinoma or after performing the operation, prevent that cancer from expanding the effect of calculating, promote the expression of estrogen receptor in breast cancer cell by tissue factor antagonist, thereby improve the therapeutic effect with the endocrine medicine of estrogen-receptor inhibitor class.Described tissue factor antagonist, as the antibody for tissue factor, comprises for the albumen of at least one tissue factor or the described antibody of its fragments specific or its variant.
Background technology
The sickness rate of breast carcinoma is number two in whole cancer, be the modal malignant tumor of China women, and sickness rate is ascendant trend year by year.At present comprise operative treatment, radiotherapy, chemotherapy, endocrine therapy and molecular targeted therapy for the treatment means of breast carcinoma.At present still taking excision as main, under the modern breast cancer treatment new concept of science and humane combination instructs, the treatment trend of breast carcinoma comprises reservation breast and the better targeted drug treatment of the Minimally Invasive Surgery of axillary fossa, more accurate stereotactic radiotherapy and selectivity.Molecular targeted therapy is the part that breast cancer treatment research field is enlivened the most in recent years, and likely becomes the main direction of breast cancer medicines research and development from now on.Simultaneously according to the different typings of breast carcinoma, body inner estrogen acceptor (Estrogen receptor, ER), progesterone receptor (Progesterone receptor, PR) and ErbB-2 (HER2) all present negative patient, endocrine therapy and targeting medication are all subject to great restriction, can only be taking chemotherapy as main.Therefore, explore the signal transduction pathway that works for tumor proliferation, invasion and attack and transfer, for new molecular targeted agents with improve endocrine medication and provide fundamental basis, be extremely important for improving breast carcinoma clinical therapeutic efficacy.
Tissue factor (tissue factor, TF) is a cross-film strand glycoprotein being made up of 263 amino acid residues, and molecular weight is about 47kDa.Wherein 219 amino acid residues of aminoterminal are positioned at outside cell membrane, and 23 amino acid residues are following closely through cell membrane, and all the other 21 amino acid residues are positioned at Cytoplasm.Under normal circumstances, tissue factor is positioned at blood vessel wall adventitial cell, hold the fibroblast of blood vessel, the fibrous capsule of the organs such as liver,spleen,kidney, and in the epidermis cell of skin outer layer, glomerular epithelium cell, brain cortex, myocardial cell, pulmonary alveolar macrophage, gastrointestinal tract wall, part genitourinary tract and endometrial stromal cell, and very rare at tunica media or theca interna.Therefore tissue factor is not present in circulation or not and contacts with blood circulation under normal circumstances, only has TF in the time that the integrity of blood vessel wall is destroyed to be just exposed to blood circulation, by activating coagulation cascade reaction performance anastalsis.Tissue factor is by being combined and starting blood coagulation cascade reaction with factor VII/VIIa.And TF relies on performance " anchor " effect of combining closely of itself and cell membrane, make physiological coagulation process be confined to damage location, and do not send out to distant place from the initial position of blood coagulation.In the time that the TF of cell surface expression is exposed to plasma protein, TF will have with it factor VII (factor VII, F VII) phase adhesion of high-affinity.And TF2 VIIa complex can further activate free F VII.The activation of catalytic factor X rapidly of TF2 VIIa complex, these processes finally cause thrombin generation.Thrombin and then catalysis fibre proteinogen are transformed into fibrin and aggregate into fibrin blood clot, in thrombosis, play an important role.TF is except the function showing under normal physiological state, and increasing evidence shows that TF plays an important role in the evolution of tumor, the overexpression of TF often with being associated of tumor.
Estrogen receptor (Estrogen receptor, ER) be positioned at nucleus, mediate estrogenic genotype effect, regulate effect by regulating transcribing of specific target gene to bring into play " genotype ", ER plays an important role in the generation of breast carcinoma and treatment.If ER signal path imbalance can cause related gene expression unbalance or change protein function in kytoplasm, impel mammary glandular cell hyper-proliferative or apoptosis in mammary gland to be obstructed, thereby cause the generation of breast carcinoma; The current endocrine therapy for breast carcinoma on the other hand, greatly mainly with estrogen receptor targeting, approximately having clinically 70% patient with breast cancer is estrogen receptor positive, and the effective percentage that estrogen receptor positive person applies endocrine therapy is 50%～60%, and receptor negative person effective percentage is lower than 10%.The trial demethylation medicines such as Giacinti can make the ER of breast cancer cell recover to express, and improve the sensitivity of tumor to antiestrogen, thereby improve therapeutic effect.Plescia etc. are the molecular chaperoneses of many key proteins in breast cancer disease process and resistance mechanism according to HSP90, affect the stable of ER and the biological function in conjunction with hormone, and attempt carrying out breast cancer treatment with the inhibitor shepherdin of HSP90.
In W094/05328, disclose using-system factor antibody and suppressed outbreak and the development of the transfer of tumor, thereby realized inhibition transfer by eliminating the long-term adhesion of transitional cell in microvasculature.But and unexposed whether by and endocrine Drug combination, and reach by the expression of regulation and control estrogen receptor the endocrinotherapy effect that promotes estrogen-receptor inhibitor class, thereby reach the transfer outbreak that suppresses breast carcinoma.
The method that suppresses tumor growth with anti-tissue factor antibodies is disclosed in patent 1829531, mainly suppress tumor vessel by tissue factor antibodies and form, thereby after mice, detect the impact of tissue factor antibodies for the growth of tumor for the tumour transplatation such as breast carcinoma, cancer of pancreas.But and unexposed tissue factor antibodies is for the machine-processed research of tumor growth impact, also and not
Examination research for the impact of estrogen receptor expression for tissue factor antibodies, and unexposed tissue factor antibodies and estrogen receptor antagon coupling are for the inhibitory action of growth and the transfer of the tumor cell of estrogen receptor negative.
Summary of the invention
Object of the present invention: research organization's factor antagonist is for the situation that affects of the expression of estrogen receptor alpha, the drug action of the endocrine medicine of lift pins to ER α.
Antibody of the present invention is a kind of Fab antibody, can be the human monoclonal antibodies of Mus source, Ren Yuan and inserted type or their fragment.Described antibody possesses and prevents that tissue factor from passing through the characteristic of the signal conduction that its cell intracellular domain produces.This antibody-like should be known in the art, and can be in method of the present invention.
Tissue factor inhibitor of the present invention has promoted the expression of the estrogen receptor in breast cancer cell.
Tissue factor inhibitor of the present invention can with breast carcinoma hormone Drug combination, include but not limited to the medicines such as tamoxifen (tamoxifen), toremifene (fareston), fulvestrant (fulvestrant), raloxifene, for the patient with breast cancer of estrogen receptor negative and estrogen receptor positive, can promote therapeutic effect.
Brief description of the drawings
Below in conjunction with accompanying drawing and specific embodiment, the present invention will be further described in detail.
Fig. 1 illustrates ERa is subject to the situation that affects of TF and antibody thereof in different breast cancer cells.
Fig. 2 illustrates affecting tumor cell proliferation and invading profit situation of in different breast cancer cells TF and antibody thereof.
Fig. 3 illustrates the tumor growth rate (stereomutation) of the human breast cancer cell of subcutaneous implantation mice, from the 0th day to using weekly TF antibody one time, and the situation of negative control PBS.
Detailed description of the invention
Below in conjunction with specific embodiment, such scheme is described further, should be understood that these embodiment are not limited to limit the scope of the invention for the present invention is described.The implementation condition adopting in embodiment can be done further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in normal experiment.
Specific embodiments one, experiment in vitro
1, (TF is negative to express breast carcinoma cell strain MC F-7 cells, ER positive expression) and MDA-MB-231 (TF positive expression, ER is negative to express) purchased from ATC C, cultivate in DMEM culture fluid at 5%CO2 respectively, MCF-7 cell transfecting wild type TF (WTF), excised cytoplasm domain part TF fragment TF Δ CT (purchased from Rockville company of the U.S.).MCF-7 cell after transfection, by PCR, ELISA and Western blotting technology for detection, the m RNA of ER α and the expression of albumen.MCF-7 cell after transfection adds TF inhibitor MAB-5G9 antibody 1mg/ml, after 48 hours, uses equally ELISA, and RT-PCR and Western blotting detect the expression of ER α.
ELISA: get 1 × 10
6mCF-7 cell, broken rear centrifugal 1200rpm 8 minutes, gets supernatant as testing sample.ER α antigen 3ug/ml, 100ul/ hole, 4 DEG C of coated spending the night, PBS washes plate 3 times, 5 minutes/time; Within second day, add the PBS of 2%BSA, 37 DEG C are sealed 1.5 hours; Wash 37 DEG C of testing samples that add the every hole of 100ul after plate, hatch 1h; Again wash the anti-humanER that adds the HRP labelling in 100ul/ hole after plate, 37 DEG C, hatch 1h; Wash plate, every hole adds 100ul nitrite ion, and within static 5 minutes, after developing the color, every hole adds 2M H2SO4 color development stopping, OD280mn reading Analysis.
RT-PCR: get 2 × 10
6/ ml MCF-7 cell, is put in 1.5ml EP pipe, adds 0.2ml chloroform, acutely shakes 30s, room temperature 3min.12000 × g, 4 DEG C centrifugal, 15min.Suct the colourless water of layer, move into (about 0.5ml) in another EP pipe.Add equal-volume isopropyl alcohol ,-20 DEG C, 30min.12000 × g, 4 DEG C centrifugal, 10min.The visible micro-RNA precipitation in pipe bottom.Abandon supernatant, add 75% ethanol 1ml, vibration 7500 × g, 4 DEG C are centrifugal, 10min.Abandon supernatant, carefully draw residual liquid, drying at room temperature 5-10min with filter paper.Precipitation is dissolved in 20 μ l DEPC water, gets 1 μ l and adds 79 μ l DEPC water to survey OD260/OD280 calculating concentration and purity.
Reverse transcription synthesizes cDNA
Reaction system is as table 1:
Table 1
Mix once centrifugal fast
Reaction condition is as table 2
Table 2
PCR reacts as table 3
Table 3
Mix once centrifugal fast
Reaction condition is as table 4
Table 4
Get PCR product 8 μ l and add 5 × Loading Buffer, 2 μ l2% agarose gel electrophoresis 120V 100mA 30min ethidium bromide stainings, gel imaging instrument imaging saving result
Western blotting: collect MCF-7 cell 2 × 10
6/ ml, boils through freezing and heat, smudge cells, and ultrasonic rear centrifugal 12000rp, 6 minutes, gets supernatant.Preparation SDS-PAGE glue, is directly loaded to protein sample in SDS-PAGE glue well, uses BIO-RAD standard electrophoretic apparatus, low pressure 80v, high pressure 120v, 90 minutes.Transferring film is selected nitrocellulose filter (NC film), uses the standard wet type membrane-transferring device of Bio-Rad, and setting transferring film electric current is 300-400mA, and the transferring film time is 60 minutes.After transferring film, immediately albuminous coat is placed in preprepared Western cleaning mixture, rinsing 1-2 minute, to wash away the transferring film liquid on film.Add Western confining liquid, slowly shake on shaking table, 4 DEG C of sealings are spent the night.With mini desktop vacuum pump exhaustion confining liquid, add immediately the primary antibodie of having diluted, 4 DEG C are slowly shaken and hatch one hour on side-sway shaking table.Reclaim primary antibodie.Add Western cleaning mixture, on side-sway shaking table, slowly 5-10 minute is washed in shake.Exhaust after cleaning mixture, then add cleaning mixture, washing 5-10 minute.Wash altogether 3 times.Two anti-diluted horseradish peroxidase (HRP) labellings two anti-, exhausts cleaning mixture with mini desktop vacuum pump, adds immediately dilute two anti-, 4 DEG C on side-sway shaking table slow shake hatch one hour.Add Western cleaning mixture, on side-sway shaking table, slowly shake is washed 10 minutes.Exhaust after cleaning mixture, then add cleaning mixture, wash 10 minutes.Wash altogether 3 times, use BeyoECL, the ECL class reagent such as Western luciferase assay reagent detect albumen.With developing fixing test kit, preparing developer liquid and fixative solution carry out craft and develop a film voluntarily.
2, MDA-MB-231 cell adds TF antibody MAB-5G9, and adds tamoxifen (Tamoxifen) estrogen receptor antagon, detect cell propagation, invade profit ability.
1.) cell proliferation experiment (cell proliferation assay)
(1) get 96 porocyte culture plates, in every hole, add 0.1ml containing 2 × 10
4the culture fluid (containing the DMEM culture fluid of 10% calf serum) of target cell, at 37 DEG C of 5% CO
2saturation vapour CO2 gas incubator in cultivate and within 2～3 hours, allow cell note wall.
(2) add 30ng/ml TF antibody MAB-5G9 with DMEM culture fluid, and add Tamoxifen 1 × 10
-6mol/L, 3 repeating holes.Another three repeating holes only add Tamoxifen, do not add TF antibody.3 negative control holes, every hole adds 0.1mlDMEM culture fluid.At 37 DEG C of 5% CO
2saturation vapour CO2 gas incubator in cultivate 72 hours.
(3) suck culture fluid, with once (as being suspension cell, should suck centrifugal culture plate before supernatant) of PBS washing.
(4) every hole adds 0.1ml PBS and 10 μ l MTT dye liquors, at 37 DEG C of 5% CO
2saturation vapour CO2 gas incubator in cultivate 6 hours.
(5) every hole adds 0.1ml acidify isopropyl alcohol, on agitator, vibrates and mixes, and allows reduzate fully dissolve.Put on enzyme connection detector and measure optical density (OD) value, detect wavelength 570nm, reference wavelength 630nm.With OD value, to the mapping of diluted sample degree, standard of comparison curve and testing sample curve can be tried to achieve the content of cytokine in testing sample.Select 490nm wavelength, measure each hole absorbance value on enzyme linked immunological monitor, record result, taking the time as abscissa, light absorption value is that vertical coordinate is drawn cell growth curve.
2) cellular infiltration experiment (cell migration assay) is by 5 × 10
4cell is put into the upper chamber of BOYDEN cell, and lower chamber adds 30ng/ml TF antibody MAB-5G9 with DMEM culture fluid, and adds Tamoxifen 1 × 10
-6mol/L, 3 repeating holes.Another three repeating holes only add Tamoxifen, do not add TF antibody.3 negative control holes, every hole adds PBS.After 24 hours.Remove upper strata, lower confluent monolayer cells is fixed poststaining, under scene, carries out cell counting for ten random visuals field.
Experimental result shows, TF inhibitor can effectively promote the expression of estrogen receptor in different tumor cells.And in breast cancer cell for estrogen receptor negative, it is obvious that Tamoxifen invades profit inhibitory action for the propagation of tumor cell.
Specific embodiments two, experiment in body
By MCF-7 MDA-MB231 and MCF-7 at 37 degree, 5%CO
2cultivate in the DMEM of 10%FBS and 1%LNN culture fluid, gather in the crops with EDTA at exponential phase, after MCF-7 cell transfecting wTF and TF Δ CT, corresponding cell strain 5 × 10
6individual cell/ML, the amount of 100 μ l is implanted in mice butter oil pad.
6 mice/groups, are divided into totally 7 groups of matched group and experimental grouies, are respectively injection MAB-5G9 antibody 0.01mg/KG, 0.1mg/KG, 1mg-/KG, 10mg/KG; And tamoxifen 1 × 10
-6mol/L; Injection tamoxifen separately; PBS is as negative reference in injection.At the 0th day, tumor cell injection is arrived to the butter oil pad of mice.Started injection of antibodies treatment and endocrine medicine in passages through which vital energy circulates at the 3rd day, during studying, monitor weekly and record gross tumor volume, once in a week according to body weight administration, until the 30th day or tumor reach the volume of 2,000mm2.With clamp with three-dimensional measurement tumor and give V=(L × W × T)/2 and calculate the volume of tumor.Cervical vertebra dislocation method is put to death mice afterwards.According to the tumor growth trend statistical data under different situations.
Claims (10)
1. the antagonist of tissue factor improves curative effect effect for playing for mammary cancer endocrine medicine.
2. in claim 1, the tissue factor antagonist of indication can be the human monoclonal antibodies of Mus source, Ren Yuan and inserted type or their fragment.Described antibody possesses and prevents that tissue factor from passing through the characteristic of the signal conduction that its cell intracellular domain produces.
3. according in claim 2, this antibody is a kind of Fab, Fab ', fragment or the derivant of F (ab ') 2.
4. according in claim 2, this antibody possesses and prevents that tissue factor from passing through the characteristic of the signal conduction that its cell intracellular domain produces.
This antibody can with breast carcinoma hormone Drug combination, include but not limited to the Drug combinations such as tamoxifen (tamoxifen), toremifene (fareston), fulvestrant (fulvestrant), raloxifene.
6. this antibody plays facilitation for the expression of the estrogen receptor in breast cancer cell.
7. this antibody and breast carcinoma hormone Drug combination, all produces effect for estrogen receptor positive or negative patient with breast cancer.
8. according to claim 5, this antibody includes but not limited to MDA-MB-231 for breast cancer cell.
9. tissue factor antagonist can be used for anti-tumor medicine, refers in particular to the adjuvant therapy medicaments into mammary cancer endocrine medicine.
10. according to claim 9, this antibody is for taking estrogen receptor as targeting, or taking retardance or reduce estrogen and play optimum assosting effect as the mammary cancer endocrine medicine of object.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829531A (en) * | 2003-05-30 | 2006-09-06 | 森托科尔公司 | Method of inhibiting tumor growth with anti-tissue factor antibodies |
| CN102317317A (en) * | 2009-03-19 | 2012-01-11 | 伯拉考成像股份公司 | Reagents for the atherosclerotic coronary plaque and uses thereof |
-
2013
- 2013-03-18 CN CN201310084936.2A patent/CN104056267A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829531A (en) * | 2003-05-30 | 2006-09-06 | 森托科尔公司 | Method of inhibiting tumor growth with anti-tissue factor antibodies |
| CN102317317A (en) * | 2009-03-19 | 2012-01-11 | 伯拉考成像股份公司 | Reagents for the atherosclerotic coronary plaque and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| COLLIER等: "Influence of Exogenous Tissue Factor on Estrogen Receptor α Expression in Breast Cancer Cells: Involvement of β1 -Integrin, PAR2, and Mitogen-Activated Protein Kinase Activation", 《MOLECULAR CANCER RESEARCH》 * |
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Application publication date: 20140924 |