CN1899616A - New use of non-receptor tyrosine kinase c-Ab1 specific inhibitor - Google Patents
New use of non-receptor tyrosine kinase c-Ab1 specific inhibitor Download PDFInfo
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Abstract
The present invention discloses the new use of non-receptor tyrosine kinase c-Abl specific inhibitor in preparing antitumor medicine or auxiliary tumor treating medicine. Experiments proves that non-receptor tyrosine kinase c-Abl specific inhibitor can inhibit the activity of c-Abl kinase to lead the denaturation aggregation and lysosome degradation of Gal3 and to lower the Gal3 protein level in the tumor cell obviously, so as to make tumor cell die in autophagic apoptosis. The antitumor medicine with the non-receptor tyrosine kinase c-Abl specific inhibitor as the active component has the following advantages: obvious curative effect, high safety, convenient oral taking and low cost. The present invention opens one new way for preventing and treating tumor.
Description
Technical field
The present invention relates to the new purposes of non-receptor tyrosine kinase c-Ab 1 specific inhibitor, particularly relate to the application of non-receptor tyrosine kinase c-Ab 1 specific inhibitor in the preparation antitumor drug.
Background technology
The individualized treatment that carries out tumor according to the molecule mechanism of patient's genetic background and morbidity is the developing direction of oncotherapy.Galectin 3 (Gal3) is that the beta galactose glycosides is conjugated protein, has multiple biological function, participates in the sticking of tumor cell, propagation, differentiation, angiogenesis, deterioration and transfer as a kind of cancer associated protein.Clinical research shows, in the histiocyte of many types, when cell generation vicious transformation, the expression of Gal3 raises the cancer of organs such as colon, thyroid, central nervous system, pancreas, bladder, kidney, stomach (for example), and the rise of Gal3 expression can make the grade malignancy of tumor and transfer invade the also enhancing thereupon of profit ability in the tumor cell.Therefore think that Gal3 is a kind of cancer associated protein, plays an important role in carcinogenesis and tumor progression.The anti-apoptosis activity of Gal3 shows and can suppress the inductive apoptosis of medicine and thereby the anchorage dependence apoptosis makes cell survival.Result of study shows that tumor cell can be secreted Gal3, but excretory Gal3 by combining the apoptosis of inducing T cell with the T cell surface receptor, thereby play the effect of immune evasion, make tumor cell in development process, can escape catching and killing of T cell like this.At present, Gal3 is as an index of immunocytochemistry Clinical detection, and the good pernicious diagnosis and the cancer prognosis diagnosis of tumor cell all had certain value.Therefore, aspect polytype cancer diagnosis and treatment, Gal3 has extraordinary application prospect as a new target.
Protein tyrosine kinase is the protein that a class has tyrosine kinase activity, can be divided into two kinds of receptor type and non-receptor types, they can catalysis phosphate on the ATP transfer on the tyrosine residue of many key proteins, make it that phosphorylation take place.Protein tyrosine kinase has occupied crucial status in intracellular signal transduction pathway, regulating a series of physiology processes such as growth, differentiation, death of cell.
ABL family in the non-receptor protein tyrosine kinase comprises two member: Abl and Ara.The Abl gene is positioned at No. 9 chromosomes long-armed (9q34.1), the Abl albumen of coding 145kD.C-Abl albumen has tyrosine kinase activity, contain SH3, SH2, PTK, DNA in conjunction with (Blume-Jensen P such as territory, actin binding structural domains, HunterT.Oncogenic kinase signalling.Nature, 2001,411 (6835): 355-65).
Non-receptor tyrosine kinase c-Ab 1 specific inhibitor can make ubiquitous Abelson tyrosine kinase (Abl) inactivation.
Summary of the invention
The new purposes that the purpose of this invention is to provide non-receptor tyrosine kinase c-Ab 1 specific inhibitor, i.e. application in preparation antitumor drug or adjuvant drug for tumor therapy thing.
The selection of the described non-receptor tyrosine kinase c-Ab 1 specific inhibitor of application that application of the present invention is is active component with the non-receptor tyrosine kinase c-Ab 1 specific inhibitor in preparation antitumor drug or adjuvant drug for tumor therapy thing is diversified, as STI571, Sunitinib, PKC412, vandetanib, Sorafenib, erlotinib, gefitinib or dasatinib etc.
For improving tumor killing effect, in antitumor drug for preparing with non-receptor tyrosine kinase c-Ab 1 specific inhibitor or adjuvant drug for tumor therapy thing, also can add chemotherapeutics such as cisplatin.
Studies show that, Galectin 3 wide expression in tumor cell, thereby but non-receptor tyrosine kinase c-Ab 1 specific inhibitor has broad-spectrum function of tumor, comprise breast carcinoma, carcinoma of prostate, cervical cancer, adenocarcinoma of lung, hepatocarcinoma, colon cancer, thyroid carcinoma, cancer of pancreas, neuroendocrine tumor, bladder cancer, renal carcinoma and gastric cancer etc.
When needing, in application of the present invention, can also add one or more acceptable accessories, described adjuvant comprises diluent, excipient, filler, binding agent, wetting agent, absorption enhancer, surfactant, lubricant, stabilizing agent of pharmaceutical field routine etc., also can add flavouring agent, sweeting agent and pigment etc. in case of necessity.
Application of the present invention can also be made multiple medicament forms such as capsule, tablet, powder, granule, injection except that making oral liquid.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
The oral consumption of the adult of this medicine is generally 400mg/kg/d, can use by one or many, and be 20 days-30 days the course of treatment.
The invention provides a kind of new purposes of non-receptor tyrosine kinase c-Ab 1 specific inhibitor, i.e. application in preparation antitumor drug or adjuvant drug for tumor therapy thing.Experimental results show that non-receptor tyrosine kinase c-Ab 1 specific inhibitor can cause Gal3 that degeneration takes place by the kinase activity that suppresses c-Abl and assemble and degrade by lysosome, cause that the Gal3 protein level significantly reduces in the tumor cell, thereby make tumor cell generation autophagy sexual cell apoptosis and death.Simultaneously, non-receptor tyrosine kinase c-Ab 1 specific inhibitor also can make tumor cell very responsive to the kinds of tumors medicine, the inducer of apoptosis of low concentration can make cell all dead in short time, thereby non-receptor tyrosine kinase c-Ab 1 specific inhibitor can strengthen the therapeutic effect of existing cell toxicant based chemotherapy medicine, and can toxic and side effects be alleviated greatly by reducing the using dosage of chemotherapeutics.Be that active component prepares antitumor drug and has the following advantages with the non-receptor tyrosine kinase c-Ab 1 specific inhibitor: 1) evident in efficacy, tumour inhibiting rate can reach 79.34%; 2) safe; 3) main route of administration is oral, and medication is convenient; 4) this inhibitor is cheap, thereby lower with the cost of its preparation antitumor drug, thereby can alleviate patient's financial burden.The present invention provides a new way for the control of tumor, having a extensive future of medicine and pharmacology field.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is immunoprecipitation and the immunoblotting testing result of non-receptor tyrosine kinase c-Ab 1 to the phosphorylation of Gal3 specificity
Fig. 2 is that non-receptor tyrosine kinase c-Ab 1 specific inhibitor STI571 is to inhibiting immunoprecipitation of the special phosphorylation of c-Abl and immunoblotting testing result
Fig. 3 detects the result of Gal3 albumen distribution situation in the MCF-7 cell that 10uM STI571 handles for immunostaining
Fig. 4 detects the result that the Gal3 protein content changes in 5uM STI571,0.5uM inducer of apoptosis STS individual processing and both common MCF-7 cells of handling for immunostaining
Fig. 5 detects the result that the Gal3 protein content changes in 5uM STI571,0.5uM inducer of apoptosis STS individual processing and both common MCF-7 cells of handling for immunoblotting
Fig. 6 is the TUNEL method testing result through 10uM STI571,0.5uM STS individual processing and both common MCF-7 apoptosis situations of handling
Fig. 7 is the Annexin-V-FLUOS method testing result through 10uM STI571,0.5uM STS individual processing and both common MCF-7 apoptosis situations of handling
The tumour inhibiting rate statistical result that Fig. 8 handles for different pharmaceutical
The specific embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
One, the structure of Flag-Gal3 expression vector
1, the structure of recombinant vector pcDNA 3-Flag
The DNA sequence of coding Flag label (is contained 8 amino acid whose peptide sections: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) be cloned between the restricted enzyme Kpn I and BamH I restriction enzyme site of carrier pcDNA3 (Invitrogen company) multiple clone site, obtain containing the recombinant vector of Flag label coding sequence, called after pcDNA 3-Flag.
2, the pcr amplification of Gal3 gene
With the Gal3 gene (GenBank number: BC001120) be template, at primer P1 (forward primer):
5 '-C
GGATCCATGGCAGACAATTTTTCGCTCCA-3 ' (leukorrhagia line base is a restricted enzyme BamHI recognition site) and P2 (downstream primer): 5 '-C
GGAATTCPcr amplification Gal3 gene under the guiding of TTATATCATGGTATATGAAGCA-3 ' (leukorrhagia line base is a restricted enzyme EcoRI recognition site), and add restricted enzyme BamHI and EcoRI recognition site respectively at the two ends of genetic fragment, after reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, the result has obtained the dna fragmentation of 753bp, conform to expected results, reclaim and this purpose fragment of purification, order-checking, sequencing result shows and has obtained the correct Gal3 genetic fragment of sequence.
3, the acquisition of pcDNA 3-Flag-Gal3 expression vector
Two, the structure of Myc-c-Abl, Myc-c-Abl (K290R) expression vector
1, the structure of Myc-c-Abl expression vector
1) pcr amplification of c-Abl gene
With wild type c-Abl gene (GenBank number: NM_005157) be template, at primer Myc-Abl-1 (forward primer):
(leukorrhagia line base is a restricted enzyme EcoRI recognition site) and Myc-Abl-2 (downstream primer):
Pcr amplification c-Abl gene under the guiding of (leukorrhagia line base is a restricted enzyme BglI recognition site), and add restricted enzyme EcoRI and BglI recognition site respectively at the two ends of genetic fragment, after reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, the result has obtained the dna fragmentation of 3393bp, conforms to expected results, reclaims and this purpose fragment of purification, order-checking, sequencing result shows and has obtained the correct c-Abl genetic fragment of sequence.
2) acquisition of Myc-c-Abl expression vector
Step 1) clone's c-Abl genetic fragment is carried out double digestion with restricted enzyme EcoRI and BglI, with endonuclease bamhi be connected through the carrier pMyc-CMV of same enzyme double digestion (Clotech company), to connect product Calcium Chloride Method transformed into escherichia coli DH5 α competent cell, the screening positive transformant, the upgrading grain, carrying out enzyme action with restricted enzyme EcoRI and BglI identifies, obtain 3800bp and the segmental positive clone of 3393bp through enzyme action, again the positive colony plasmid is done further evaluation with the method for PCR, the primer is Myc-Abl-1 and Myc-Abl-2, the PCR product is checked order, testing result shows that the c-Abl genetic fragment inserted between the EcoRI and BglI restriction enzyme site of pMyc-CMV, and sequence is correct, with this recombinant expression carrier called after Myc-c-Abl.The Abl gene can be expressed the fusion rotein that has the Myc label under the effect of CMV promoter.
2, the structure of Myc-c-Abl (K290R) expression vector
1) pcr amplification of Myc-c-Abl (K290R) mutation gene
With wild type c-Abl gene (GenBank number: NM_005157) be template, at primer Myc-Abl-1:
(leukorrhagia line base is a restricted enzyme EcoRI recognition site) and K290R-II:5 '-GGTGTCCTCCTTCAAGGT
CCT5 ' the terminal sequence (called after M1) of pcr amplification amplification c-Abl under the guiding of CACGGCCACCGTCAGGC-3 ' (leukorrhagia line base is the mutational site), again at primer Myc-Abl-2:
(leukorrhagia line base is a restricted enzyme BglI recognition site) and K290R-I:5 '-GCCGTACGGTGGCCGTG
AGG3 ' the terminal sequence (called after M2) of pcr amplification c-Abl under the guiding of ACCTTGAAGGAGGACACC-3 ' (leukorrhagia line base is the mutational site), after reaction finishes, above-mentioned pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, the result has obtained the M1 fragment of 888bp and the M2 fragment of 2405bp, conform to expected results, reclaim and two purpose fragments of purification, then, get each 1 μ l of M1 and M2, mix, as template, the complete sequence of pcr amplification c-Abl mutant gene under the guiding of primer Myc-Abl-1 and Myc-Abl-2 is after reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, the result has obtained the dna fragmentation of 3393bp, conforms to expected results, reclaims and this purpose fragment of purification.
2) acquisition of Myc-c-Abl (K290R) expression vector
Step 1) clone's c-Abl mutation gene fragment is carried out double digestion with restricted enzyme EcoRI and BglI, endonuclease bamhi is connected with carrier pMyc-CMV through the same enzyme double digestion, to connect product Calcium Chloride Method transformed into escherichia coli DH5 α competent cell, the screening positive transformant, the upgrading grain, carrying out enzyme action with restricted enzyme EcoRI and BglI identifies, obtain 3800bp and the segmental positive clone of 3893bp through enzyme action, again the positive colony plasmid is done further evaluation with the method for PCR, the primer is Myc-Abl-1 and Myc-Abl-2, the PCR product is checked order, testing result shows that c-Abl mutation gene fragment inserted between the EcoRI and BglI restriction enzyme site of pMyc-CMV, and sequence is correct, with this recombinant expression carrier called after Myc-c-Abl (K290R).The Abl mutation gene can be expressed the fusion rotein that has the Myc label under the effect of CMV promoter.
Three, detect the specificity phosphorylation of non-receptor tyrosine kinase c-Ab 1 to Gal3
C-Abl is as nonreceptor tyrosine kinase, and is main by making the life process of protein tyrosine phosphatase adjusting cell.Now use the method validation Gal3 of immunoprecipitation and immunoblotting (Western blot) can be by the special phosphorylation of c-Abl, and avoid the phosphorylation of another kind of cell endogenous nonreceptor tyrosine kinase Arg to disturb, concrete grammar may further comprise the steps:
1, cotransfection
PcDNA 3-Flag-Gal3 is born two l cell MEF (the Abl/Arg double knockout mouse embryo fibroblast cells that knock out of expression vector pCMV-Myc-c-Abl (K290R) cotransfection Abl, Arg of dominant mutant respectively with pCMV-Myc-c-Abl, pCMV-Myc empty carrier, c-Abl, the DKO cell, phenotype be Abl-/-Arg-/-, Koleske AJ, Essential roles for the Abl and Arg tyrosine Kinasesin neurulation, Neuron 1998 Dec; 21 (6): 1259-72).
2, with anti--Flag antibody carry out immunoprecipitation (Immunoprecipitation, IP)
Cracking transfectional cell after transfection 36-48 hour carries out immunoprecipitation with anti--Flag antibody (M5, Sigma company), the all operations of this process should strict carry out at ice bath or cryogenic conditions, concrete grammar is: collecting cell, centrifugal 3 minutes of 1000g abandons most culture medium.With 1 * PBS washed cell of 5mL ice pre-cooling, centrifugal 2 minutes of 1000g abandons supernatant, and same procedure is given a baby a bath on the third day after its birth inferior.Every ware cell of Φ 100mm need add the single detergent lysate of 600 μ l (50mmol/LTrisCl, 150mmol/L NaCl, 0.02% sodium azide, 1%NP-40 and protease inhibitor (Roche, Inc) 1/25mL), every ware cell of Φ 60mm adds the single detergent lysate of 200 μ l.With cell and lysate mixing, ice bath 5-10 minute, centrifugal 10 minutes of 16000g.The careful lysis supernatant of drawing adds 10-15 μ l and resists-the antibody linked sepharose 4B (Sigma) of Flag, hatches 2 hours 4 ℃ of rotations, carries out immunoprecipitation.After 2 hours, centrifugal 30 seconds of 1000g, careful supernatant discarded with 250 μ l lysates washing three times, exhausts the IP product as far as possible with washing liquid.In the IP product, add 40 μ l, 1 * sds gel sample-loading buffer, 100 ℃ of water-baths 5 minutes, centrifugal 2 minutes of 10000g.Getting centrifugal supernatant and 5 μ l protein dyes Marker in advance (Invitrogen Inc) carries out the SDS-PAGE gel electrophoresis, and voltage is set at 80-100 volt.
3, change film
After electrophoresis finishes, albumen is transferred to nitrocellulose filter (NC film), concrete grammar is: with two filter paper (Bio-Rad that 2.5mm is thick, Inc.) and a nitrocellulose filter be cut into the gel size, at 1 * transfering buffering liquid (39mmol/L glycine, 48mmol/L Tris, 0.037% SDS and 20% methanol) the middle immersion 30 minutes.Subsequently, (Bio-Rad places on Inc.), catches up with the bubble in the most interlayer as far as possible at half-dried transfer instrument according to the order of filter paper-gel-nitrocellulose filter-filter paper from top to bottom.Change membrane voltage 15V, after 2-2.5 hour the NC film is taken out, add the 5mL confining liquid and (contain 1 * PBST) of 5% defatted milk powder, be placed on the horizontal shaking table that shakes gently the room temperature incubation 1 hour.After the sealing, wash film three times, each 5-10 minute with 1 * PBST.
4, immuning hybridization
With the anti-phosphorylated tyrosine antibody of the PBST preparation specific non-marked of target protein that contains 0.2 ‰ sodium azides as one anti-(anti-P-Tyr, 4G10, Upstate Biotechnology, Inc.); With PBST preparation horseradish peroxidase (HRP) the labelling sheep anti-mouse antibody (HRP-mouse Ig G) that contains 5% defatted milk powder and 0.2 ‰ sodium azides as two anti-(Amersham Pharmacia Biotech, Inc).NC film and after step 3 sealing was resisted at the room temperature incubation after 1 hour, wash film three times, each 5-10 minute with 1 * PBST; Add ELIAS secondary antibody subsequently, after 1 hour, wash film three times at the room temperature incubation equally, each 5-10 minute with 1 * PBST.Above-mentioned incubation process is all carried out on horizontal shaking table.
5, colour developing
Carry out chemiluminescence reaction at last, method is: will contain horseradish peroxidase substrate 3, colour developing liquid WESTERN LIGHTNING (the PerkinElmer Life Sciences of 3 '-diaminobenzidine, Inc.) drop in equably on the NC film, react the liquid that to develop the color after 1 minute and blot, with X-ray sheet expose (X-ray sheet and developer solution, fixative solution are all available from Kodak company).
The result is ("+" expression transfection as shown in Figure 1, "-" expression untransfected), compare with pCMV-Myc empty carrier transfection results, the expression product of Flag-Gal3 transfectional cell can be by the expression product phosphorylation of Myc-c-Abl, but the expression product of Myc-c-Abl (K290R) transfectional cell can not be by the expression product phosphorylation of Myc-c-Abl, reason is that the K290 site of Abl is the key amino acid of ATP-binding site in the c-Abl kinase domain, make c-Abl forfeiture tyrosine kinase activity after being sported arginine, above-mentioned testing result shows that Gal3 can be specifically by the c-Abl phosphorylation.
Four, detect the inhibitory action of non-receptor tyrosine kinase c-Ab 1 specific inhibitor STI571 to the c-Abl phosphorylation
Detect the inhibitory action of non-receptor tyrosine kinase c-Ab 1 specific inhibitor STI571 to the c-Abl phosphorylation, detection method is: with Flag-Gal3 plasmid transfection 293 cells, with 10 μ mol/L STI571 transfectional cell was handled 12 hours after 24 hours, with the transfectional cell with equivalent DMSO (solvent of STI571) processing is contrast, cell lysis uses the method identical with step 3 to carry out immunoprecipitation and immunoblotting detects.
(through STI571 handle as shown in Figure 1 by "+" expression for the result, "-" expression is handled without STI571), the expression product Gal3 of Flag-Gal3 can be by tyrosine phosphorylation in the transfectional cell that equivalent DMSO handles, and still the tyrosine phosphorylation of the Gal3 that expresses in the transfectional cell that 10 μ mol/L STI571 handle is suppressed.This result proves that Gal3 can be specific by cell endogenous c-Abl phosphorylation, and STI571 is inhibited to the phosphorylation of c-Abl simultaneously.
One, detects the proteic distribution situation of Gal3 in the MCF-7 cell that 10uM STI571 handles
Detect the proteic distribution situation of Gal3 in the MCF-7 cell that STI571 handles with immunostaining, concrete grammar is: human breast cancer cell MCF-7 is inoculated into 2 culture dishs (Glass Bottom Culture Dishes, available from Instrumental Analysis test center of Military Medical Science Institute) in, inoculum concentration 10
2Individual/ware, carry out following processing behind the cell attachment 24h: first group of cell do not handled, second group of cell handled 18h with 10uM STI571, then two groups of cells are carried out mitochondrion probe (Invitrogen company) labelling: change the DMEM culture fluid in the Tissue Culture Dish (Gibco company) into the preheating good DMEM culture fluid that adds 0.2uM mito-probe solution, in 37 ℃ of incubators, hatch 15min, clean with fresh DMEM culture fluid pair cell then, decide cell with the DMEM that contains 3.7% paraformaldehyde is liquid-solid, in 37 ℃ of incubators, hatch 15min, it is inferior to give a baby a bath on the third day after its birth with PBS, each 5min.Add 0.1%TritonX-100, incubated at room 5min, reuse PBS give a baby a bath on the third day after its birth inferior, each 5min.Then with 1% BSA sealing 1h, reuse PBS gives a baby a bath on the third day after its birth time, each 5min.Gal3 protein staining and lysosome dyeing one are anti-for being respectively galectin-3 (H-160) (santa cruzbiotechnology.inc) antibody and LAMP-2 antibody (santa cruz biotechnology.inc), dilute in 1: 200 ratio during use, incubated at room 1h, it is inferior to give a baby a bath on the third day after its birth with PBS, each 5min.It is FITC-rabbit (company of China fir Golden Bridge in Beijing) that Gal3 protein staining two resists, and dilutes in 1: 200 ratio during use, and incubated at room 1h, reuse PBS give a baby a bath on the third day after its birth inferior, each 5min.Carry out nucleus dyeing at last, the working concentration of nuclear dyestuff Hochest33342 (available from ancient cooking vessel state biotech company) is 1ug/mL, incubated at room 30min, and it is inferior to give a baby a bath on the third day after its birth with PBS, each 5min.Put under the fluorescence microscope and observe.
The immunostaining of the MCF-7 cell of handling without STI571 the results are shown in Figure the figure A in 3, and redness is a mitochondrion, and green is Gal3, and blueness is a nucleus, and Gal3 is distributed in full cell, and with mitochondrion common location is arranged; The immunostaining of the MCF-7 cell behind 10uM STI571 processing 18h the results are shown in Figure figure B and the figure C in 3, and Gal3 albumen generation point-like is assembled, and this moment, proteic aggregation of Gal3 and mitochondrion were not located altogether, but located altogether with lysosome, promptly entered lysosome.Above-mentioned testing result shows that non-receptor tyrosine kinase c-Ab 1 specific inhibitor STI571 can cause Gal3 that degeneration takes place by the kinase activity that suppresses c-Abl and assemble and degrade by lysosome.
Two, detect the variation of Gal3 protein content in 5uM STI571,0.5uM inducer of apoptosis STS individual processing and both common MCF-7 cells of handling
1, immunostaining detects
With the variation of immunostaining detection Gal3 protein content in 5uM STI571,0.5uM inducer of apoptosis STS individual processing and both common MCF-7 cells of handling, experiment is divided into four groups: first group of cell do not handled; Second group of cell handled 18h through 5uM STI571; The 3rd group of cell handled 150min through 0.5uM inducer of apoptosis STS (strurosporine), and the 4th group of cell adds 0.5uM STS and handle 150min again after 5uM STI571 handles 18h.Immuning dyeing method is identical with step 1.
The result is (the picture left above: unprocessed cell as shown in Figure 4, top right plot: handle the 150min cell through 0.5uM inducer of apoptosis STS, lower-left figure: handle the 18h cell through 5uM STI571, bottom-right graph: after 5uM STI571 handles 18h, add 0.5uM STS and handle the 150min cell), redness is a mitochondrion, and green is Gal3, and blueness is a nucleus, when cell was handled with inducer of apoptosis STS separately, significant change did not take place in Gal3 in the cell; When handling with STI571, Gal3 assembles in the cell; When handling cell jointly with STI and STS, the Gal3 protein content obviously reduces.
2, immunoblotting detects
In MCF-7 cell inoculation to 4 a 100mm Tissue Culture Dish, the processing identical with above-mentioned immunostaining experiment is divided into four groups, do further to detect with the variation of Gal3 protein content in the immunoblotting Western blot method pair cell, Gal3 detects an anti-galectin-3 (B-2) (santa cruz biotechnology.inc) of being, two anti-are HRP-mouse Ig G (santa cruz biotechnology.inc), with β-actin as confidential reference items (one anti-be β-actin monoclonal antibody (santa cruz biotechnology.inc), two anti-are HRP-mouse Ig G (santa cruzbiotechnology.inc), and above antibody is all available from joining bio tech ltd in the friendship.
Result ("+" expression is added, and "-" expression is not added) as shown in Figure 5 detects under the equilibrated situation of β-actin, and after handling with STI571, Gal3 content descends in the cell; Handling back Gal3 albumen jointly with STS and STI571 almost all disappears; And when using STS to handle separately, Gal3 content does not almost change in the cell.Testing result conforms to the immunostaining result of step 1.
Above-mentioned experimental result shows, non-receptor tyrosine kinase c-Ab 1 specific inhibitor STI571 degrades the proteic kinases dependency of Gal3 loss of stability by the inhibition to the c-Abl kinase activity, lost the function of Gal3, thereby can suppress the sticking of tumor cell, propagation, differentiation, angiogenesis, deterioration and transfer as the cancer associated protein.
Experiment is divided into four groups: first group of MCF-7 cell do not handled; Second group of MCF-7 cell handled 18h through 10uM STI571; The 3rd group of MCF-7 cell handled 4-5h through 0.5uM inducer of apoptosis STS, and the 4th group of MCF-7 cell adds 0.5uM STS and handle 4-5h again after 10uM STI571 handles 18h.
One, the TUNEL method detects the apoptosis situation through 10uM STI571,0.5uM STS individual processing and both common MCF-7 cells of handling
Detect the apoptosis situation of above-mentioned four groups of MCF-7 cells through different disposal with TUNEL-FLUOS test kit (available from Roche company) and reference reagent box description, concrete grammar is: with 4% paraformaldehyde of the new preparation fixing 30min of pair cell at room temperature, and PBS develop a film back and blocker (H
2O
2Methanol solution) at room temperature hatch 30min, PBS develops a film, and the Triton X-100 of adding 0.1% is hatched 2min in ice bath.PBS washes twice, and Dropwise 50 μ l TUNEL reaction mixture is hatched 60min for 37 ℃ in wet box, and PBS gives a baby a bath on the third day after its birth inferior, carries out dyeing of Gal3 protein immunization and nucleus dyeing then, and immuning dyeing method is seen embodiment 2, and the dyeing back is observed under fluorescence microscope.
(redness is a Gal3 albumen to the result as shown in Figure 6, green is the apoptosis situation that FITC marker enzyme traget antibody shows, blueness is a nucleus), shown in Figure 1 as among Fig. 6, normal MCF-7 cell detection is less than apoptosis, be not have green fluorescence in the nucleus, but can see that red Gal3 albumen is distributed in full kytoplasm; Shown in Figure 2 as among Fig. 6, cell has apoptosis after 10uM STI571 handles 18h, can see that green fluorescence is arranged in the nucleus, and red Gal3 albumen is point-like and assembles; Shown in Figure 3 as among Fig. 6, cell also has apoptosis after 0.5uM STS handles 4-5h, can see that green fluorescence is arranged in the nucleus, and red Gal3 albumen still is distributed in full kytoplasm; Shown in Figure 4 as among Fig. 6, cell adds after 10uM STI571 handles 18h after 0.5uM STS handles 4-5h again, can see that very strong green fluorescence expresses in nucleus, and the nucleus shrinkage that diminishes, this moment red Gal3 protein expression extremely a little less than, apoptosis is remarkable.
Thereby showing non-receptor tyrosine kinase c-Ab 1 specific inhibitor STI571, above-mentioned testing result can make Gal3 protein degradation inducing tumor cell generation apoptosis.
Two, the Annexin-V-FLUOS method detects the apoptosis situation through 10uM STI571,0.5uM STS individual processing and both common MCF-7 cells of handling
Reuse Annexin-V-FLUOS test kit (available from Roche company) and reference reagent box description detect the apoptosis situation of above-mentioned four groups of MCF-7 cells through different disposal, concrete grammar is: the centrifugal 5min collecting cell of 500g, use the PBS re-suspended cell, wash once, cell is resuspended among the binding buffer (test kit carries) of 200 μ l, adds 10 μ l Annexin-V-FITC and 5 μ l PI, mixings gently, lucifuge incubated at room 15min detects with the fluidic cell detector.
The result is (the picture left above: normal MCF-7 cell as shown in Figure 7, top right plot: the MCF-7 cell of handling 4-5h through 0.5uM inducer of apoptosis STS, lower-left figure: the MCF-7 cell of handling 18h through 10uM STI571, bottom-right graph: after 10uM STI571 handles 18h, add the MCF-7 cell that 0.5uM STS handles 4-5h again), all can make MCF-7 cell generation apoptosis to a certain degree with STI571 and STS individual processing, apoptosis is remarkable when handling jointly with STI571 and STS, but further proves non-receptor tyrosine kinase c-Ab 1 specific inhibitor STI571 inducing tumor cell generation apoptosis.
With BABL/C nude mice (male, 18-20g is available from Military Medical Science Institute's Experimental Animal Center) is laboratory animal.The take the logarithm MCF-7 cell of trophophase, after the trypsinization by every 1 * 10
6Cell number inoculation nude mice, after 5 days nude mice is divided into 7 groups at random: blank group, control drug cyclophosphamide (CTX) group, cisplatin group, STI571 group, STI571 add cisplatin combined medication group, Sutent group, Sutent and add cisplatin combined medication group, 6 every group, administration in the following manner:
Matched group: do not give any Drug therapy after the inoculation;
The CTX group: every nude mice using dosage 100mg/kg is administered once the administration of set time every day every other day;
Cisplatin (CDDP) group: every nude mice using dosage 7mg/kg, lumbar injection is 1 time weekly, in start injection next day of inoculation transplanted tumor, administration every other week;
STI group: every nude mice oral STI571 50mg/kg every day, the administration of set time every day;
STI571 adds cisplatin combined medication group: STI571 and cisplatin are used simultaneously, and method is with medication is identical separately.
Sutent group: every nude mice oral Sutent 12.5mg/kg every day
Sutent adds cisplatin combined medication group: Sutent and cisplatin are used simultaneously, and method is with medication is identical separately.
IR=(1-treatment group tumor weight/matched group tumor is heavy) * 100%
The inhibition effect that different pharmaceutical is handled the nude mice tumor sees Table 1, wherein the block diagram of tumour inhibiting rate statistical result as shown in Figure 8, compare with control drug cyclophosphamide administration group, though the inhibition rate of tumor growth of drug combination group is low, but the nude mice of drug combination group does not see any unusual, body weight is also similar with the nude mice of normal saline group, after the execution, dissecting nude mice finds, the obvious atrophy of nude mice spleen of cyclophosphamide group, weight also obviously is lighter than normal saline group and drug combination group, each internal organs no abnormality seen of drug combination group; In addition, the enlargement of cyclophosphamide administration group group nude mice liver, color and luster is paler, and is covered with the neoplasm metastasis kitchen range, and normal saline group and the equal color and luster of drug combination group group liver are ruddy, have only 2-3 neoplasm metastasis kitchen range.The above results shows that STI571 adds cisplatin combined medication and Sutent and adds cisplatin combined medication group the MCF-7 growth of tumor is had significant inhibitory effect, and can effectively suppress the transfer of tumor cell, and toxic and side effects is faint.
The inhibition effect of table 1 different pharmaceutical processed group tumor
| Group | Dosage (mg/kg) | Number of animals | The weight of animals (g) | Heavy (the g) ± SD of tumor | Tumour inhibiting rate % |
| All the time | All the time | ||||
| Matched group (normal saline) | 25mL/kg | 6 6 | 20.36 23.79 | 1.2173±0.2764 | |
| CTX | 100 | 6 6 | 20.52 19.98 | 0.22±0.052** | 81.93 |
| CDDP | 7 | 6 6 | 20.07 21.69 | 0.561±0.100** | 53.87 |
| STI | 50 | 6 6 | 20.60 21.67 | 0.4757±0.1939** | 60.92 |
| STI+CDDP | 7+50 | 6 6 | 20.03 21.36 | 0.2514±0.2424** | 79.34 |
| Sutent | 12.5 | 6 6 | 20.68 21.55 | 0.4579±0.1124** | 62.38 |
| Sutent+CDDP | 7+12.5 | 6 6 | 20.46 21.23 | 0.2432±0.1222** | 80.02 |
Compare with the normal saline group: * P＜0.05, * * P＜0.01.
Claims (3)
1, the application of non-receptor tyrosine kinase c-Ab 1 specific inhibitor in preparation antitumor drug or adjuvant drug for tumor therapy thing.
2, application according to claim 1 is characterized in that: described tumor is for expressing the tumor of Galectin 3.
3, application according to claim 1 and 2 is characterized in that: described non-receptor tyrosine kinase c-Ab 1 specific inhibitor is STI571, Sunitinib, PKC412, vandetanib, Sorafenib, erlotinib, gefitinib or dasatinib.
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