CN1899616A - New use of non-receptor tyrosine kinase c-Ab1 specific inhibitor - Google Patents

Abstract

The invention discloses a new application of a non-receptor tyrosine kinase c-Abl specific inhibitor, that is, the application in the preparation of antitumor drugs or auxiliary drugs for tumor treatment. Experiments have proved that non-receptor tyrosine kinase c-Abl-specific inhibitors can cause Gal3 to undergo denaturation and aggregation by inhibiting the kinase activity of c-Abl and degrade it through lysosomes, resulting in a significant decrease in the level of Gal3 protein in tumor cells, thereby making tumors Cells die by autophagic apoptosis. The preparation of antitumor drugs with non-receptor tyrosine kinase c-Abl specific inhibitors as active ingredients has the following advantages: 1) significant curative effect; 2) high safety; 3) the main route of administration is oral administration, which is convenient for administration; 4) The inhibitor is cheap, so the cost of preparing antitumor drugs is relatively low. The invention provides a new approach for the prevention and treatment of tumors, and has broad application prospects in the field of medicine.

Description

New uses of non-receptor tyrosine kinase c-Abl specific inhibitors

technical field

The invention relates to a new application of a non-receptor tyrosine kinase c-Abl specific inhibitor, in particular to the application of the non-receptor tyrosine kinase c-Abl specific inhibitor in the preparation of antitumor drugs.

Background technique

Individualized treatment of tumors based on the patient's genetic background and molecular mechanism of disease is the development direction of cancer treatment. Galectin 3 (Gal3) is a β-galactoside-binding protein with multiple biological functions. As a cancer-related protein, it participates in the adhesion, proliferation, differentiation, angiogenesis, progression and metastasis of tumor cells. Clinical studies have shown that in many types of tissue cells, when the cells undergo malignant transformation, the expression of Gal3 is up-regulated (such as in cancers of the colon, thyroid, central nervous system, pancreas, bladder, kidney, stomach, etc.), and, Up-regulation of Gal3 expression level in tumor cells will increase the degree of malignancy and the ability of metastasis and invasion of tumors. Therefore, Gal3 is considered to be a cancer-associated protein, which plays an important role in carcinogenesis and tumor progression. The anti-apoptotic activity of Gal3 is manifested in the ability to inhibit drug-induced apoptosis and anchorage-dependent apoptosis so that cells survive. The research results show that tumor cells can secrete Gal3, and the secreted Gal3 can induce the apoptosis of T cells by binding to T cell surface receptors, thereby playing the role of immune evasion, so that tumor cells can evade T cells during the development process. kill. At present, Gal3, as an indicator of immunocytochemical clinical detection, has certain value in the diagnosis of benign and malignant tumor cells and the prognosis of cancer. Therefore, Gal3 has a very promising application prospect as a new target in the diagnosis and treatment of various types of cancer.

Protein tyrosine kinases are a class of proteins with tyrosine kinase activity, which can be divided into receptor type and non-receptor type. They can catalyze the transfer of phosphate groups on ATP to tyrosine residues in many important proteins. to phosphorylate it. Protein tyrosine kinase plays a very important role in the intracellular signal transduction pathway, regulating a series of physiological and chemical processes such as cell growth, differentiation, and death.

The ABL family of non-receptor protein tyrosine kinases includes two members: Abl and Ara. The Abl gene is located on the long arm of chromosome 9 (9q34.1), encoding a 145kD Abl protein. c-Abl protein has tyrosine kinase activity and contains SH3, SH2, PTK, DNA binding domain, actin binding domain, etc. (Blume-Jensen P, Hunter T. Oncogenic kinase signaling. Nature, 2001, 411 (6835): 355-65).

Non-receptor tyrosine kinase c-Abl specific inhibitors inactivate the ubiquitous Abelson tyrosine kinase (Abl).

Contents of the invention

The purpose of the present invention is to provide a new application of the non-receptor tyrosine kinase c-Abl specific inhibitor, that is, the application in the preparation of antitumor drugs or auxiliary drugs for tumor treatment.

The application of the present invention is the use of non-receptor tyrosine kinase c-Abl specific inhibitor as an active ingredient in the preparation of anti-tumor drugs or tumor treatment auxiliary drugs. The non-receptor tyrosine kinase c-Abl The choice of specific inhibitors is varied, such as STI571, Sunitinib, PKC412, vandetanib, Sorafenib, erlotinib, gefitinib or dasatinib, etc.

In order to improve the anti-tumor effect, chemotherapy drugs such as cisplatin can also be added to anti-tumor drugs or auxiliary drugs for tumor treatment prepared by using non-receptor tyrosine kinase c-Abl specific inhibitors.

Studies have shown that Galectin 3 is widely expressed in tumor cells, so the non-receptor tyrosine kinase c-Abl specific inhibitor has a broad-spectrum anti-tumor effect, including breast cancer, prostate cancer, cervical cancer, lung adenocarcinoma, liver cancer , Colon cancer, thyroid cancer, pancreatic cancer, neuroendocrine tumors, bladder cancer, kidney cancer and gastric cancer.

When needed, one or more pharmaceutically acceptable adjuvants can also be added in the application of the present invention, and the adjuvants include conventional diluents, excipients, fillers, binders, wetting agents in the pharmaceutical field , absorption enhancers, surfactants, lubricants, stabilizers, etc., and flavoring agents, sweeteners, and pigments can also be added if necessary.

The application of the present invention can also be made into various pharmaceutical forms such as capsules, tablets, powders, granules, and injections, in addition to being made into oral liquids. The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy.

The adult oral dose of the drug is generally 400mg/kg/d, which can be used once or multiple times, and the course of treatment is 20-30 days.

The invention provides a new application of a non-receptor tyrosine kinase c-Abl specific inhibitor, that is, the application in the preparation of antitumor drugs or auxiliary drugs for tumor treatment. Experiments have proved that non-receptor tyrosine kinase c-Abl-specific inhibitors can cause Gal3 to denature and aggregate and degrade through lysosomes by inhibiting the kinase activity of c-Abl, resulting in a significant decrease in Gal3 protein levels in tumor cells, thereby making tumors Cells die by autophagic apoptosis. At the same time, non-receptor tyrosine kinase c-Abl-specific inhibitors can also make tumor cells extremely sensitive to a variety of tumor therapeutic drugs. The specific inhibitor of body tyrosine kinase c-Abl can enhance the therapeutic effect of existing cytotoxic chemotherapeutic drugs, and can greatly reduce the toxic and side effects by reducing the dosage of chemotherapeutic drugs. The preparation of antitumor drugs with non-receptor tyrosine kinase c-Abl specific inhibitors as active ingredients has the following advantages: 1) significant curative effect, tumor inhibition rate can reach 79.34%; 2) high safety; 3) main route of administration It is taken orally and is convenient to use; 4) The inhibitor is cheap, so the cost of preparing antitumor drugs is relatively low, thereby reducing the economic burden of patients. The invention provides a new approach for the prevention and treatment of tumors, and has broad application prospects in the field of medicine.

The present invention will be described in further detail below in conjunction with specific embodiments.

Description of drawings

Figure 1 shows the results of immunoprecipitation and western blot detection of the specific phosphorylation of Gal3 by the non-receptor tyrosine kinase c-Abl

Figure 2 shows the results of immunoprecipitation and western blot detection of the inhibitory effect of non-receptor tyrosine kinase c-Abl specific inhibitor STI571 on c-Abl specific phosphorylation

Figure 3 is the result of immunostaining detection of Gal3 protein distribution in MCF-7 cells treated with 10uM STI571

Figure 4 shows the results of immunostaining detection of Gal3 protein changes in MCF-7 cells treated with 5uM STI571, 0.5uM apoptosis inducer STS alone or both

Figure 5 shows the results of Western blot detection of Gal3 protein changes in MCF-7 cells treated with 5uM STI571, 0.5uM apoptosis inducer STS alone or both

Figure 6 shows the TUNEL detection results of the apoptosis of MCF-7 cells treated with 10uM STI571, 0.5uM STS alone or both.

Figure 7 shows the Annexin-V-FLUOS detection results of the apoptosis of MCF-7 cells treated with 10uM STI571, 0.5uM STS alone or both

Figure 8 is the statistical result of tumor inhibition rate treated with different drugs

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1. Detection of Gal3 specific phosphorylation by non-receptor tyrosine kinase c-Abl and inhibition of c-Abl phosphorylation by non-receptor tyrosine kinase c-Abl specific inhibitor STI571

1. Construction of Flag-Gal3 expression vector

1. Construction of recombinant vector pcDNA 3-Flag

The DNA sequence encoding the Flag tag (peptide segment containing 8 amino acids: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) is cloned into the restriction endonuclease of the multiple cloning site of the vector pcDNA3 (Invitrogen Company) Between Kpn I and BamH I restriction sites, a recombinant vector containing the Flag tag coding sequence was obtained, which was named pcDNA 3-Flag.

2. PCR amplification of Gal3 gene

Using the Gal3 gene (GenBank number: BC001120) as a template, in primer P1 (upstream primer):

5'-C GGATCCA TGGCAGACAATTTTTCGCTCCA-3' (the underlined base is the restriction endonuclease BamHI recognition site) and P2 (downstream primer): 5'-C GGAATTC TTATATCATGGTATATGAAGCA-3' (the underlined base is the restriction endonuclease Under the guidance of PCR amplification Gal3 gene, and the restriction endonuclease BamHI and EcoRI recognition sites were added at the two ends of the gene fragment respectively, after the reaction was finished, the PCR amplification product was carried out on 1% agar As a result of sugar gel electrophoresis detection, a 753bp DNA fragment was obtained, which was consistent with the expected result. The target fragment was recovered and purified, and sequenced. The sequencing result showed that the Gal3 gene fragment with the correct sequence was obtained.

3. Acquisition of pcDNA 3-Flag-Gal3 expression vector

The Gal3 gene fragment cloned in step 2 was double-digested with restriction endonucleases BamHI and EcoRI, and the cleavage fragment was ligated with the vector pcDNA 3-Flag constructed in step 1 after double-digestion with the same enzymes, and the ligated product was digested with Transform Escherichia coli DH5α competent cells with calcium chloride method, screen positive transformants, extract plasmids, and use restriction endonucleases BamHI and EcoRI to carry out enzyme digestion and identification. The 5400bp and 753bp fragments obtained by enzyme digestion are positive clones, and then positive The cloned plasmid was further identified by PCR, the primers used were P1 and P2, and the PCR product was sequenced. The test results showed that the Gal3 gene fragment had been inserted between the BamHI and EcoRI restriction sites of pcDNA 3-Flag, and the sequence was correct. The recombinant expression vector was named Flag-Gal3. Gal3 gene can express the fusion protein with Flag tag under the action of CMV promoter.

Two, the construction of Myc-c-Abl, Myc-c-Abl (K290R) expression vector

1. Construction of Myc-c-Abl expression vector

1) PCR amplification of c-Abl gene

(带下划线碱基为限制性内切酶BglI识别位点)的引导下PCR扩增c-Abl基因，并在基因片段的两端分别添加限制性内切酶EcoRI和BglI识别位点，反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，结果获得了3393bp的DNA片段，与预期结果相符，回收并纯化该目的片段，测序，测序结果表明获得了序列正确的c-Abl基因片段。Using the wild-type c-Abl gene (GenBank number: NM_005157) as a template, in the primer Myc-Abl-1 (upstream primer): (The underlined base is the restriction endonuclease EcoRI recognition site) and Myc-Abl-2 (downstream primer):

(The underlined base is the restriction endonuclease BglI recognition site), PCR amplifies the c-Abl gene under the guidance, and respectively adds the restriction endonuclease EcoRI and the BglI recognition site at the two ends of the gene fragment, and the reaction ends Finally, the PCR amplification product was detected by 1% agarose gel electrophoresis. As a result, a DNA fragment of 3393bp was obtained, which was consistent with the expected result. The target fragment was recovered and purified, and sequenced. The sequenced result showed that the c-Abl with the correct sequence was obtained. Gene fragment.

2) Acquisition of Myc-c-Abl expression vector

The c-Abl gene fragment cloned in step 1) was double-digested with restriction endonucleases EcoRI and BglI, and the digested fragment was connected with the vector pMyc-CMV (Clotech Company) double-digested with the same enzymes, and the ligated The product was transformed into Escherichia coli DH5α competent cells by the calcium chloride method, positive transformants were screened, plasmids were extracted, and restriction endonucleases EcoRI and BglI were used for enzyme digestion and identification, and the 3800bp and 3393bp fragments obtained by enzyme digestion were positive clones, and then The positive cloned plasmid was further identified by PCR method, the primers used were Myc-Abl-1 and Myc-Abl-2, and the PCR product was sequenced, and the test results showed that the c-Abl gene fragment had been inserted into EcoRI and BglI of pMyc-CMV Between the restriction sites and the correct sequence, the recombinant expression vector was named Myc-c-Abl. Abl gene can express fusion protein with Myc tag under the action of CMV promoter.

2. Construction of Myc-c-Abl (K290R) expression vector

1) PCR amplification of Myc-c-Abl (K290R) point mutation gene

(带下划线碱基为限制性内切酶EcoRI识别位点)和K290R-II：5’-GGTGTCCTCCTTCAAGGT CCTCACGGCCACCGTCAGGC-3’(带下划线碱基为突变位点)的引导下PCR扩增扩增c-Abl的5’端序列(命名为M1)，再在引物Myc-Abl-2：

(带下划线碱基为限制性内切酶BglI识别位点)和K290R-I：5’-GCCGTACGGTGGCCGTG AGGACCTTGAAGGAGGACACC-3’(带下划线碱基为突变位点)的引导下PCR扩增c-Abl的3’端序列(命名为M2)，反应结束后，对上述PCR扩增产物进行1％琼脂糖凝胶电泳检测，结果获得了888bp的M1片段和2405bp的M2片段，与预期结果相符，回收并纯化两个目的片段，然后，取M1和M2各1μl，混合，以此为模板，在引物Myc-Abl-1和Myc-Abl-2的引导下PCR扩增c-Abl突变基因的全序列，反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，结果获得了3393bp的DNA片段，与预期结果相符，回收并纯化该目的片段。Using the wild-type c-Abl gene (GenBank number: NM_005157) as a template, in the primer Myc-Abl-1:

(The underlined base is the restriction endonuclease EcoRI recognition site) and K290R-II: 5'-GGTGTCCTCCTTCAAGGT CCT CACGGCCACCGTCAGGC-3' (the underlined base is the mutation site) under the guidance of PCR amplification c- The 5' end sequence of Abl (named as M1), and then in the primer Myc-Abl-2:

(The underlined base is the restriction endonuclease BglI recognition site) and K290R-I: 5'-GCCGTACGGTGGCCGTG AGG ACCTTGAAGGAGGACACC-3' (the underlined base is the mutation site) for PCR amplification of c-Abl 3' end sequence (named as M2), after the reaction, the above-mentioned PCR amplification products were detected by 1% agarose gel electrophoresis, as a result, the M1 fragment of 888bp and the M2 fragment of 2405bp were obtained, which were consistent with the expected results, recovered and Purify the two target fragments, then take 1 μl of each of M1 and M2, mix them, and use this as a template to amplify the full sequence of the c-Abl mutant gene by PCR under the guidance of primers Myc-Abl-1 and Myc-Abl-2, After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and a DNA fragment of 3393bp was obtained, which was consistent with the expected result, and the target fragment was recovered and purified.

2) Acquisition of Myc-c-Abl (K290R) expression vector

The c-Abl point mutation gene fragment cloned in step 1) was double-digested with restriction endonucleases EcoRI and BglI, and the digested fragment was connected with the vector pMyc-CMV double-digested with the same enzymes, and the ligated product was digested with Transform Escherichia coli DH5α competent cells with calcium chloride method, screen positive transformants, extract plasmids, and use restriction endonucleases EcoRI and BglI for enzyme digestion and identification. The fragments of 3800bp and 3893bp obtained by enzyme digestion are positive clones, and then positive The cloned plasmid was further identified by PCR. The primers used were Myc-Abl-1 and Myc-Abl-2. The PCR products were sequenced. The test results showed that the c-Abl point mutation gene fragment had been inserted into EcoRI and BglI of pMyc-CMV Between the restriction sites and the correct sequence, the recombinant expression vector was named Myc-c-Abl(K290R). The Abl point mutation gene can express the fusion protein with Myc tag under the action of CMV promoter.

3. Detection of specific phosphorylation of Gal3 by non-receptor tyrosine kinase c-Abl

As a non-receptor tyrosine kinase, c-Abl mainly regulates the life process of cells by phosphorylating protein tyrosine. Immunoprecipitation and Western blot methods are now used to verify that Gal3 can be specifically phosphorylated by c-Abl, and avoid phosphorylation interference of another endogenous non-receptor tyrosine kinase Arg in cells. The specific methods include the following step:

1. Co-transfection

pcDNA 3-Flag-Gal3 and pCMV-Myc-c-Abl, pCMV-Myc empty vector, c-Abl negative dominant mutant expression vector pCMV-Myc-c-Abl (K290R) were co-transfected with Abl, Arg Double knockout mouse fibroblast MEF (Abl/Arg double knockout mouse embryo fibroblast cells, DKO cells, phenotype is Abl-/-Arg-/-, Koleske AJ, Essential roles for the Abl and Arg tyrosine Kinasesin neurulation, Neuron 1998 Dec;21(6):1259-72).

2. Immunoprecipitation (IP) with anti-Flag antibody

Lyse the transfected cells 36-48 hours after transfection, and perform immunoprecipitation with anti-Flag antibody (M5, Sigma Company). All operations in this process should be strictly performed in ice bath or low temperature conditions. The specific method is: collect cells, 1000g Centrifuge for 3 minutes and discard the medium. Wash the cells with 5 mL ice-cold 1×PBS, centrifuge at 1000g for 2 minutes, discard the supernatant, and wash three times in the same way. Add 600 μl single detergent lysate (50mmol/LTris·Cl, 150mmol/L NaCl, 0.02% sodium azide, 1% NP-40 and protease inhibitor (Roche, Inc) 1 tablet/25mL to each dish of Φ100mm cells ), add 200 μl single detergent lysate to each dish of Φ60mm cells. Mix the cells with the lysate, place on ice for 5-10 minutes, and centrifuge at 16000g for 10 minutes. The cell lysed supernatant was carefully pipetted, and 10-15 μl of anti-Flag antibody cross-linked agarose beads (Sigma) was added, and incubated at 4° C. for 2 hours with rotation for immunoprecipitation. After 2 hours, centrifuge at 1000 g for 30 seconds, carefully discard the supernatant, wash the IP product with 250 μl lysate three times, and suck up the washing solution as much as possible. Add 40 μl of 1×SDS gel loading buffer to the IP product, bathe in water at 100°C for 5 minutes, and centrifuge at 10,000g for 2 minutes. The centrifuged supernatant and 5 μl protein prestained Marker (Invitrogen, Inc) were taken for SDS-PAGE gel electrophoresis, and the voltage was set at 80-100 volts.

3. Transfer film

After electrophoresis, the protein was transferred to a nitrocellulose membrane (NC membrane). The specific method was: two 2.5mm thick filter papers (Bio-Rad, Inc.) and one nitrocellulose membrane were cut to the size of the gel, Soak in 1× transfer buffer (39mmol/L glycine, 48mmol/L Tris, 0.037% SDS and 20% methanol) for 30 minutes. Then, place it on a semi-dry transfer apparatus (Bio-Rad, Inc.) in the order of filter paper-gel-nitrocellulose membrane-filter paper from top to bottom, and try to drive out the air bubbles in the interlayer as much as possible. The membrane transfer voltage was 15V, and after 2-2.5 hours, the NC membrane was taken out, 5 mL of blocking solution (1×PBST containing 5% skim milk powder) was added, and incubated on a gently shaking horizontal shaker for 1 hour at room temperature. After blocking, wash the membrane three times with 1×PBST, 5-10 minutes each time.

4. Immunohybridization

Use PBST containing 0.2‰ sodium azide to prepare a target protein-specific non-labeled anti-phosphotyrosine antibody as a primary antibody (anti-P-Tyr, 4G10, Upstate Biotechnology, Inc.); use 5% skimmed milk powder and Horseradish peroxidase (HRP)-labeled goat anti-mouse antibody (HRP-mouse IgG) was prepared in PBST with 0.2‰ sodium azide as the secondary antibody (Amersham Pharmacia Biotech, Inc). After incubating the NC membrane blocked in step 3 with the primary antibody for 1 hour at room temperature, wash the membrane three times with 1×PBST for 5-10 minutes each time; then add the enzyme-labeled secondary antibody, and also incubate at room temperature for 1 hour, Wash the membrane three times with 1×PBST, 5-10 minutes each time. The above incubation process was carried out on a horizontal shaker.

5. Color rendering

Finally, the chemiluminescent reaction was carried out by dropping the chromogenic solution WESTERN LIGHTNING (PerkinElmer Life Sciences, Inc.) containing the horseradish peroxidase substrate 3,3'-diaminobenzidine evenly on the NC membrane, After reacting for 1 minute, the developing solution was blotted dry, and exposed with X-ray film (X-ray film, developing solution, and fixer were all purchased from Kodak Company).

The results are shown in Figure 1 ("+" means transfection, "-" means no transfection), compared with pCMV-Myc empty vector transfection results, the expression product of Flag-Gal3 transfected cells can be detected by Myc-c- The expression product of Abl is phosphorylated, but the expression product of Myc-c-Abl (K290R) transfected cells cannot be phosphorylated by the expression product of Myc-c-Abl, because the K290 site of Abl is in the kinase domain of c-Abl The key amino acid of the ATP binding site was mutated to arginine to make c-Abl lose the tyrosine kinase activity. The above detection results indicated that Gal3 could be specifically phosphorylated by c-Abl.

4. Detection of the inhibitory effect of non-receptor tyrosine kinase c-Abl specific inhibitor STI571 on c-Abl phosphorylation

To detect the inhibitory effect of non-receptor tyrosine kinase c-Abl specific inhibitor STI571 on c-Abl phosphorylation, the detection method is: transfect 293 cells with Flag-Gal3 plasmid, and use 10 μmol/L STI571 to The transfected cells were treated for 12 hours, and the transfected cells treated with an equal amount of DMSO (the solvent of STI571) were used as a control, and the cells were lysed, and immunoprecipitation and immunoblotting were performed by the same method as Step 3.

The results are shown in Figure 1 ("+" means treated with STI571, "-" means not treated with STI571), the expression product Gal3 of Flag-Gal3 in the transfected cells treated with equal amount of DMSO can be phosphorylated by tyrosine, but Tyrosine phosphorylation of Gal3 expressed in transfected cells treated with 10 μmol/L STI571 was inhibited. This result proves that Gal3 can be specifically phosphorylated by endogenous c-Abl, and STI571 can inhibit the phosphorylation of c-Abl.

Example 2, detection of changes in Gal3 protein in MCF-7 cells treated with STI571

1. Detection of the distribution of Gal3 protein in MCF-7 cells treated with 10uM STI571

The distribution of Gal3 protein in MCF-7 cells treated with STI571 was detected by immunostaining method. The specific method was: inoculate human breast cancer cell MCF-7 into 2 culture dishes (Glass Bottom Culture Dishes, purchased from the Academy of Military Medical Sciences Instrument Analysis and Testing Center), the inoculum amount was 10 ² cells/dish, and the following treatments were performed after the cells adhered to the wall for 24 hours: the first group of cells was not treated, the second group of cells was treated with 10uM STI571 for 18 hours, and then the mitochondrial probe was performed on the two groups of cells (Invitrogen Company) labeling: replace the DMEM culture solution (Gibco Company) in the cell culture dish with the preheated DMEM culture solution to which 0.2uM mito-probe solution has been added, incubate in a 37°C incubator for 15min, and then use fresh The cells were washed with DMEM medium, fixed with DMEM solution containing 3.7% paraformaldehyde, incubated in a 37°C incubator for 15 minutes, and washed three times with PBS, 5 minutes each time. Add 0.1% TritonX-100, incubate at room temperature for 5 minutes, and then wash with PBS three times, 5 minutes each time. Then block with 1% BSA for 1 h, and wash with PBS three times, 5 min each time. The primary antibodies for Gal3 protein staining and lysosome staining were galectin-3 (H-160) (santa cruzbiotechnology.inc) antibody and LAMP-2 antibody (santa cruz biotechnology.inc), respectively, and were diluted at a ratio of 1:200 when used , incubated at room temperature for 1 h, washed three times with PBS, 5 min each time. The secondary antibody for Gal3 protein staining was FITC-rabbit (Beijing Zhongshan Jinqiao Company), which was diluted at a ratio of 1:200 when used, incubated at room temperature for 1 h, and then washed three times with PBS, 5 min each time. Finally, nuclear staining was carried out. The nuclear dye Hochest33342 (purchased from Dingguo Biotechnology Co., Ltd.) was used at a concentration of 1 ug/mL, incubated at room temperature for 30 minutes, and washed three times with PBS for 5 minutes each time. Observe under a fluorescence microscope.

The results of immunostaining of MCF-7 cells not treated with STI571 are shown in panel A in Figure 3. Red is mitochondria, green is Gal3, and blue is nucleus. Gal3 is distributed in the whole cell and co-localized with mitochondria; after 10uM STI571 The results of immunostaining of MCF-7 cells treated for 18 hours are shown in panels B and C in Figure 3. Gal3 protein aggregates in a punctate form. At this time, Gal3 protein aggregates do not co-localize with mitochondria, but co-localize with lysosomes. Localization, that is, into the lysosome. The above detection results indicated that the non-receptor tyrosine kinase c-Abl specific inhibitor STI571 can cause Gal3 denatured aggregation and degradation through lysosome by inhibiting the kinase activity of c-Abl.

2. Detection of changes in the amount of Gal3 protein in MCF-7 cells treated with 5uM STI571, 0.5uM apoptosis inducer STS alone or both

1. Immunostaining detection

The changes of Gal3 protein in MCF-7 cells treated with 5uM STI571 and 0.5uM apoptosis inducer STS alone or co-treated with the immunostaining method were divided into four groups: the first group cells were not treated; The cells in the group were treated with 5uM STI571 for 18h; the cells in the third group were treated with 0.5uM apoptosis inducer STS (strurosporine) for 150min; The immunostaining method is the same as step 1.

The results are shown in Figure 4 (upper left panel: untreated cells, upper right panel: cells treated with 0.5uM apoptosis inducer STS for 150min, lower left panel: cells treated with 5uM STI571 for 18h, lower right panel: cells treated with 5uM STI571 for 18h Add 0.5uM STS to treat cells for 150min), red is mitochondria, green is Gal3, blue is nucleus, when cells are treated with apoptosis inducer STS alone, there is no significant change in Gal3 in cells; when treated with STI571, Gal3 in cells Aggregation occurred; when cells were co-treated with STI and STS, the amount of Gal3 protein was significantly reduced.

2. Western blot detection

MCF-7 cells were inoculated into four 100mm cell culture dishes, and the same treatment as the above immunostaining experiment was divided into four groups, and the changes of the Gal3 protein amount in the cells were further detected by Western blot method, and the primary antibody was detected by Gal3 galectin-3 (B-2) (santa cruz biotechnology.inc), the secondary antibody was HRP-mouse IgG (santa cruz biotechnology.inc), and β-actin was used as an internal reference (the primary antibody was β-actin monoclonal antibody ( santa cruz biotechnology.inc), the secondary antibody was HRP-mouse IgG (santa cruzbiotechnology.inc), and the above antibodies were purchased from Youyi Zhonglian Biotechnology Co., Ltd.

The results are shown in Figure 5 ("+" means addition, "-" means no addition) detected under the condition of β-actin balance, after treatment with STI571, the content of Gal3 in cells decreased; after co-treatment with STS and STI571, Gal3 The protein almost completely disappeared; and when STS was used alone, the Gal3 content in the cells was almost unchanged. The detection results were consistent with the immunostaining results in step 1.

The above experimental results show that the non-receptor tyrosine kinase c-Abl specific inhibitor STI571 degrades the kinase-dependent stability of the Gal3 protein by inhibiting the c-Abl kinase activity, and loses Gal3 as a cancer-related protein function, which can inhibit the adhesion, proliferation, differentiation, angiogenesis, deterioration and metastasis of tumor cells.

Example 3, detection of the apoptosis of MCF-7 cells treated with 10uM STI571, 0.5uM STS alone or co-treated with both

The experiment was divided into four groups: the first group of MCF-7 cells were not treated; the second group of MCF-7 cells were treated with 10uM STI571 for 18h; the third group of MCF-7 cells were treated with 0.5uM apoptosis inducer STS for 4-5h, Four groups of MCF-7 cells were treated with 10uM STI571 for 18h, then added 0.5uM STS for another 4-5h.

1. TUNEL method to detect the apoptosis of MCF-7 cells treated with 10uM STI571, 0.5uM STS alone or both

Use the TUNEL-FLUOS kit (purchased from Roche Company) and refer to the kit instruction manual to detect the apoptosis of the above four groups of MCF-7 cells with different treatments. The specific method is: use newly prepared 4% paraformaldehyde at room temperature Cells were fixed for 30 min, washed with PBS and incubated with blocking agent (H ₂ O ₂ methanol solution) for 30 min at room temperature, washed with PBS, added with 0.1% Triton X-100 and incubated in ice bath for 2 min. Wash twice with PBS, add 50 μl of TUNEL reaction mixture dropwise, incubate at 37°C for 60 min in a wet box, wash with PBS three times, then carry out Gal3 protein immunostaining and cell nucleus staining, immunostaining method see Example 2, after staining, under a fluorescent microscope observe.

The results are shown in Figure 6 (red is the Gal3 protein, green is the cell apoptosis shown by the FITC-labeled enzyme-labeled antibody, and blue is the nucleus), as shown in Figure 1 in Figure 6, normal MCF-7 cells cannot be detected. When cell apoptosis occurs, that is, there is no green fluorescence in the nucleus, but the red Gal3 protein can be seen distributed in the whole cytoplasm; There is green fluorescence in the nucleus, and the red Gal3 protein aggregates in dots; as shown in Figure 3 in Figure 6, the cells also undergo apoptosis after being treated with 0.5uM STS for 4-5 hours, and there is green fluorescence in the nucleus, The red Gal3 protein is still distributed in the whole cytoplasm; as shown in Figure 4 in Figure 6, after the cells were treated with 10uM STI571 for 18 hours and then added with 0.5uM STS for another 4-5 hours, strong green fluorescence can be seen in the nucleus expression, and the nucleus becomes smaller and shrunken, at this time the expression of the red Gal3 protein is extremely weak, and the cell apoptosis is significant.

The above detection results show that non-receptor tyrosine kinase c-Abl specific inhibitor STI571 can degrade Gal3 protein and induce tumor cell apoptosis.

2. Annexin-V-FLUOS method to detect the apoptosis of MCF-7 cells treated with 10uM STI571, 0.5uM STS alone or both

Then use the Annexin-V-FLUOS kit (purchased from Roche Company) and refer to the kit instructions to detect the apoptosis of the above four groups of MCF-7 cells with different treatments. The specific method is: collect the cells by centrifugation at 500g for 5min, and re- Suspend the cells, wash once, resuspend the cells in 200 μl of binding buffer (included with the kit), add 10 μl Annexin-V-FITC and 5 μl PI, mix gently, incubate at room temperature in the dark for 15 minutes, and detect with flow cytometry Instrument detection.

The results are shown in Figure 7 (upper left: normal MCF-7 cells, upper right: MCF-7 cells treated with 0.5uM apoptosis inducer STS for 4-5h, lower left: MCF-7 treated with 10uM STI571 for 18h Cells, lower right panel: MCF-7 cells treated with 10uM STI571 for 18h and then treated with 0.5uM STS for 4-5h), treatment with STI571 and STS alone can induce apoptosis of MCF-7 cells to a certain extent, when used When STI571 and STS were co-treated, cell apoptosis was significant, which further proved that non-receptor tyrosine kinase c-Abl specific inhibitor STI571 could induce tumor cell apoptosis.

Example 4. Detection of the inhibition of STI571 on the growth of transplanted tumors in nude mice

BABL/C nude mice (male, 18-20 g, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences) were used as experimental animals. MCF-7 cells in the logarithmic growth phase were taken, and after trypsinization, nude mice were inoculated according to the number of 1× ¹⁰⁶ cells per mouse. After 5 days, the nude mice were randomly divided into 7 groups: blank control group, control drug cyclophosphamide (CTX ) group, cisplatin group, STI571 group, STI571 plus cisplatin combination group, Sutent group, Sutent plus cisplatin combination group, 6 rats in each group, administered in the following manner:

Control group: no drug treatment was given after vaccination;

CTX group: each nude mouse was given a dose of 100 mg/kg, administered once every other day, at a fixed time every day;

Cisplatin (CDDP) group: each nude mouse was given a dose of 7mg/kg, intraperitoneally injected once a week, and the injection was started on the next day after the transplanted tumor was inoculated, and administered every other week;

STI group: Each nude mouse was orally administered STI571 50mg/kg per day, at a fixed time every day;

STI571 plus cisplatin combination group: STI571 and cisplatin are applied at the same time, and the method is the same as single administration.

Sutent group: each nude mouse orally administered Sutent 12.5mg/kg per day

Sutent plus cisplatin combined drug group: Sutent and cisplatin are applied at the same time, and the method is the same as single drug.

After 4 weeks of administration, the nude mice were killed by neck dislocation, the tumor mass was dissected, and the tumor weight was weighed. The tumor growth inhibition rate was calculated according to the following formula:

IR=(1-Tumor weight in treatment group/Tumor weight in control group)×100%

The inhibitory effects of different drug treatments on tumors in nude mice are shown in Table 1, and the histogram of the statistical results of the tumor inhibition rate is shown in Figure 8. Compared with the control drug cyclophosphamide administration group, the tumor growth inhibition rate of the combined drug group is although However, no abnormality was found in the nude mice in the combined drug group, and their body weight was similar to that of the normal saline group. After the nude mice were sacrificed, the dissected nude mice found that the spleen of the nude mice in the cyclophosphamide group was obviously atrophied, and the weight was also significantly lighter than that of the physiological saline group. In the saline group and the combined drug group, no abnormalities were found in the organs of the combined drug group; in addition, the liver of the nude mice in the cyclophosphamide-administered group was enlarged, pale in color, and covered with tumor metastases. The livers in the group were all ruddy, with only 2-3 tumor metastases. The above results indicated that the combination of STI571 and cisplatin and the combination of Sutent and cisplatin had a significant inhibitory effect on the growth of MCF-7 tumors, and could effectively inhibit the metastasis of tumor cells with weak side effects.

Table 1 The inhibitory effects of different drug treatment groups on tumors group Dose (mg/kg) number of animals Animal weight (g) Tumor weight (g)±SD Inhibition rate% Always Always Control 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

Compared with normal saline group: *P<0.05, **P<0.01.

Claims (3)

1. The application of non-receptor tyrosine kinase c-Abl specific inhibitor in the preparation of anti-tumor drugs or auxiliary drugs for tumor treatment. 2. The application according to claim 1, characterized in that: the tumor expresses Galectin 3. 3. The application according to claim 1 or 2, characterized in that the non-receptor tyrosine kinase c-Abl specific inhibitor is STI571, Sunitinib, PKC412, vandetanib, Sorafenib, erlotinib, gefitinib or dasatinib.

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