阅读说明：本技术 Met和axl双靶点抑制剂在制备防治胃癌的药物中的用途 (Application of MET and AXL double-target inhibitor in preparation of medicine for preventing and treating gastric cancer ) 是由 魏霞蔚 朱晨静 魏于全 于 2019-11-15 设计创作，主要内容包括：本发明涉及MET和AXL双靶点抑制剂在制备防治胃癌的药物中的用途,属于医药领域。本发明提供了MET和AXL双靶点抑制剂在制备防治胃癌的药物中的用途。通过利用MET与AXL高表达细胞株MKN45,以及MET与AXL中等表达细胞株SNU719进行体外实验,并建立裸鼠移植瘤模型,本发明证实了MET和AXL双靶点抑制剂对胃癌能够发挥显著的治疗作用,为临床防治胃癌提供的新的策略。(The invention relates to application of a MET and AXL double-target inhibitor in preparation of a medicine for preventing and treating gastric cancer, belonging to the field of medicines. The invention provides application of a MET and AXL double-target inhibitor in preparation of a medicine for preventing and treating gastric cancer. The invention proves that the MET and AXL double-target inhibitor can play a remarkable treatment role on gastric cancer and provides a new strategy for clinically preventing and treating the gastric cancer by utilizing a MET and AXL high-expression cell strain MKN45 and a MET and AXL medium-expression cell strain SNU719 to perform in-vitro experiments and establishing a nude mouse transplantation tumor model.)

Use of a MET and AXL dual-target inhibitor in the preparation of a medicament for the prevention and treatment of gastric cancer.

2. Use according to claim 1, characterized in that: the gastric cancer over-expresses MET and AXL.

3. Use according to claim 1 or 2, characterized in that: the gastric cancer is gastric adenocarcinoma.

4. Use according to claim 1, characterized in that: the medicine can inhibit gastric cancer metastasis.

5. Use according to claim 1, characterized in that: the drug inhibits the generation of microangioses in gastric cancer tissues.

6. Use according to claim 1, characterized in that: the medicine can inhibit gastric cancer cell proliferation and promote gastric cancer cell apoptosis.

7. Use according to claim 1, characterized in that: the medicine can inhibit the generation of M2 type macrophage in gastric cancer tissue.

8. Use according to claim 1, characterized in that: the MET and AXL dual-target inhibitor is LY2801653 or a pharmaceutically acceptable salt thereof.

9. Use according to any one of claims 1 to 8, characterized in that: the medicament is a preparation prepared by taking a MET and AXL double-target inhibitor as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

10. Use according to claim 9, characterized in that: the preparation is an oral preparation or an injection preparation.

Technical Field

The invention relates to application of a MET and AXL double-target inhibitor in preparation of a medicine for preventing and treating gastric cancer, belonging to the field of medicines.

Background

Gastric cancer is one of the most common malignancies. Although the incidence of gastric cancer worldwide is gradually reduced in recent years, gastric cancer is still one of the high-incidence tumors in China, and 29.9 new gastric cancer cases exist in every 10 ten thousand per year on average, which causes huge social burden. The five-year survival rate of early stage gastric cancer can be greater than 95%, but since most patients often neglect the symptoms of early stage gastric cancer, they have already entered the late stage at the time of diagnosis, missing the optimal surgical period. The treatment means of the advanced gastric cancer is more complex, new auxiliary radiotherapy and chemotherapy, molecular targeted therapy, immunotherapy and the like need to be combined, and the median survival time is less than one year. The standard chemotherapy regimen for gastric cancer is based on cisplatin in combination with other cytotoxic drugs such as taxanes, fluorouracil, anthracyclines, VP-16, etc. However, these regimens often do not improve the efficiency of chemotherapy fundamentally due to drug resistance, and patients often die due to tumor recurrence and metastasis.

In recent years, molecular targeted therapy has become a focus of cancer research. Some inhibitors targeting EGFR, VEGF, cyclin-dependent kinases (CDKs), Matrix Metalloproteinases (MMPs) show anti-tumor effects in many malignancies, such as the use of trastuzumab and lapatinib against HER-2 in HER-2 positive breast cancer, the use of the EGFR inhibitors erlotinib and gefitinib in non-small cell lung cancer, and many other molecularly targeted drugs including Gastrointestinal Stromal Tumors (GIST), hematologic Tumors, colorectal cancer and renal cancer as therapeutic approaches.

MET, also called tyrosine protein kinase MET (C-MET), is a protein product encoded by the MET proto-oncogene, the heterodimer has tyrosine kinase activity and the ligand is Hepatocyte Growth Factor (HGF). Binding of hepatocyte growth factor to MET results in MET phosphorylation and activation of downstream cascade signaling pathways that mediate cell proliferation, migration, invasion, survival and branching morphogenesis. Fluorescence In Situ Hybridization (FISH) analysis showed MET gene amplification in about 8% of tumor patients, and PCR analysis showed an increase in MET gene copy number in about 20% of patients.

AXL (also known as UFO, Ark, Tyro7) is a member of the TAM family, encoded by the AXL gene located on human chromosome 19q13.1, and also belongs to the large family of receptor tyrosine kinases. TAMs have 3 members: TYRO-3, AXL and MER, each of which is composed of an extracellular region, a transmembrane region and an intracellular region, and has a common ligand growth repression specific Protein 6 (GrowArrestspecific Protein 6, Gas6) and Protein S (Protein S). Gas6 can bind to all three members of the TAM family, which have an affinity for Gas6 of AXL > Tyro3> Mer. In Non-Small cell lung Cancer (NSCLC), activation of AXL causes acquired resistance of NSCLC cell lines to first generation Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), while inhibition of AXL can preserve the sensitivity of cell lines and tumor grafts to erlotinib (one of EGFR-TKI). AXL also plays an important role in stem cell phenotype and is associated with the expression of stem cell markers such as CD44, ALDH1, and the like. For example, the expression of AXL is positively regulated by EZH2 in brain glioma cells, EZH2 has important significance for maintaining stem cell morphology, and the knockout of AXL can simulate the inhibition of EZH 2.

So far, reports of using the MET/AXL double-target inhibitor for preparing a gastric cancer prevention and treatment medicine are not found.

Disclosure of Invention

The invention aims to provide application of a MET and AXL double-target inhibitor in preparation of a medicine for preventing and treating gastric cancer.

The invention provides application of a MET and AXL double-target inhibitor in preparation of a medicine for preventing and treating gastric cancer.

Further, the gastric cancer overexpresses MET and AXL.

Further, the gastric cancer is gastric adenocarcinoma. In the embodiment of the invention, 7 human gastric cancer cell lines are specifically detected: SNU719, MGC803, SNU16, MKN28, MKN45, AZ521 and GT39, all gastric adenocarcinoma cells.

Further, the drug inhibits gastric cancer metastasis.

Further, the drug inhibits microangiogenesis in gastric cancer tissues.

Furthermore, the medicine inhibits the proliferation of gastric cancer cells and promotes the apoptosis of the gastric cancer cells.

Further, the drug inhibits the production of macrophage M2 in gastric cancer tissues.

Further, the MET and AXL dual-target inhibitor is LY2801653 or a pharmaceutically acceptable salt thereof. LY2801653 is a micromolecule tyrosine kinase inhibitor which aims at MET and AXL double targets, and its CAS No. 1206799-15-6 has the following chemical structure:

furthermore, the medicament is a preparation prepared by taking a MET and AXL double-target inhibitor as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Further, the preparation is an oral preparation or an injection preparation.

The invention utilizes MET and AXL high expression cell strain MKN45 and MET and AXL medium expression cell strain SNU719 to carry out in vitro experiments, and establishes a nude mouse transplantation tumor model, thereby proving that the MET and AXL double-target inhibitor can play a remarkable treatment role on gastric cancer and providing a new strategy for clinically preventing and treating gastric cancer.

Drawings

FIG. 1 is a graph showing the results of detecting the expression of MET and AXL genes of 7 human gastric cancer cell lines in example 1 by a real-time fluorescent quantitative PCR method;

FIG. 2 is a graph showing the results of Western blotting for detecting the expression of MET and AXL proteins of 7 human gastric cancer cell lines in example 1;

FIG. 3 is a representation of the high/low expression of MET and AXL profiles selected in example 1;

FIG. 4 is a graph showing the survival rate of the gastric cancer patient in example 1;

FIG. 5 is a graph showing the effect of LY2801653 on gastric cancer cell proliferation measured by the CCK8 method in example 2;

FIG. 6 is a graph showing the results of Western blotting of the changes in the levels of MKN45 apoptosis-related proteins after treatment with LY2801653 in example 2;

FIG. 7 is a graph showing the results of measuring the change in the number of apoptotic cells 24 hours after treating MKN45 cells with LY2801653 by flow cytometry in example 2;

FIG. 8 is a graph showing the results of flow cytometry detecting the change in the number of apoptotic cells after LY2801653 treated SNU719 cells for 72h in example 2;

FIG. 9 is a graph of apoptosis of MKN45 cells detected by TUNEL in example 2;

FIG. 10 is a graph of the induction of MKN45 cell cycle arrest by LY2801653 in example 2;

FIG. 11 is a graph of the results of flow cytometry analysis of SNU719 cells treated with LY2801653 and solvent in example 2;

FIG. 12 is a graph showing the results of Western blotting for detecting the changes in the levels of cycle-associated proteins Cyclin A2 and Cyclin D1 after action of LY2801653 on MKN45 cells in example 2;

FIG. 13 is a graph of the results of a scratch test conducted on SNU719 cells treated with LY2801653 and solvent in example 2;

FIG. 14 is a graph showing the results of Western blotting detecting changes in the level of protein associated with epithelial-mesenchymal transition after treatment of SNU719 cells with LY2801653 in example 2;

FIG. 15 is a graph showing the results of Western blotting detecting changes in the levels of proteins associated with the downstream pathway 24 hours after treating MKN45 cells with LY2801653 in example 2;

FIG. 16 is a graph showing the results of Western blotting for detecting the changes in the levels of proteins associated with the downstream pathway 72 hours after the SNU719 cell line treated with LY2801653 in example 2;

FIG. 17 is a graph of the in vivo inhibition of MKN45 cell growth by LY2801653 in example 3;

FIG. 18 is a photograph of subcutaneous tumors of the control group and the experimental group in example 3;

FIG. 19 is a graph showing the weight statistics of all subcutaneous nodules in groups of nude mice in example 3;

FIG. 20 is a graph showing the average body weight changes of control and experimental nude mice in example 3;

FIG. 21 is a pathological section staining pattern of the important organs of nude mice in HE staining test control group and LY2801653 drug-treated group in example 3;

FIG. 22 is a staining chart of HE pathological sections of liver, spleen and kidney organs in example 3;

FIG. 23 is a graph showing the statistics of the indexes of the control group and the experimental group in example 3, which reflect liver function, kidney function, and heart function;

FIG. 24 is a graph showing HE staining patterns of tumor graft morphology in the experimental group and the control group in example 3;

FIG. 25 is an immunohistochemical graph of LY2801653 inhibiting phosphorylation of tumor cells MET and AXL in the nude mouse transplant tumor model of cell line MKN45 in example 3;

FIG. 26 is a graph showing Ki67 immunohistochemistry results for subcutaneous tumors of control and experimental groups in example 3;

FIG. 27 is a graph showing the results of TUNEL assay for apoptosis in subcutaneous tumors in control and experimental groups in example 3;

FIG. 28 is a graph showing the results of microangiogenesis of subcutaneous tumors in control and experimental groups detected by CD31 immunohistochemical staining in example 3;

FIG. 29 is a graph of the in vivo inhibition of growth of cell line SNU719 by LY2801653 in example 3;

FIG. 30 is a graph showing the body weight changes of the control and experimental nude mice in example 3;

FIG. 31 is a graph of LY2801653 inhibiting in vivo growth of tumor cell SNU719 in example 3;

FIG. 32 is a graph showing the results of CD31 immunofluorescent staining for microangiogenesis in subcutaneous tumors in control and experimental groups in example 3;

FIG. 33 is a graph showing the results of analyzing the proportion of M2-type macrophages in the subcutaneous tumors of the experimental and control nude mice by flow analysis in example 3;

FIG. 34 is a graph showing the expression of macrophage-associated gene of nude mouse subcutaneous tumor M2 by qRT-PCR in example 3.

Detailed Description

The invention uses Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, qRT-PCR) and Western immunoblotting (Western Blot, WB) to detect the total MET and AXL genes and protein expression of seven human gastric cancer cell lines; clinical data from gastric cancer patients were collected and a tissue chip containing 90 patients with gastric cancer was analyzed to investigate the relationship between MET and AXL and patient prognosis. A Cell proliferation/toxicity assay Kit (Cell Counting Kit-8, CCK-8) was used to observe the Cell proliferation status after the action of LY2801653 drugs. Flow cytometry examined the effect of drugs on apoptosis and cell cycle, WB reflected changes in apoptosis, cycle and MET and AXL downstream pathway-associated protein levels. In addition, a human gastric cancer transplantation tumor model of a MET and AXL high expression cell strain MKN45 and a moderate expression cell strain SNU719 is constructed in female Balb/c nude mice with the weight of 18-20g and the week 6-8, and the in vivo anti-tumor effect of LY2801653 is observed. Taking a nude mouse transplanted tumor as a histopathological section, and staining hematoxylin-eosin (H & E) to observe the morphological and pathological changes of the tumor after administration; the expression changes of MET and AXL activated forms (namely, phosphorylated MET and AXL protein) and a cell proliferation marker Ki67 after the action of the immunohistochemical determination medicine; changes in microvessel density (CD31), apoptosis (TUNEL) were measured by immunofluorescence and flow cytometry for changes in microvessel density (CD31), apoptosis (TUNEL) in control and experimental groups, and macrophage markers in the tumor microenvironment were detected by flow cytometry.

The above experiment, using LY2801653 as a typical test drug, revealed the anti-tumor effect of MET/AXL dual-target inhibitors in gastric cancer. LY2801653 can inhibit proliferation of MET and AXL high expression cell strain in vitro, promote tumor cell apoptosis, induce cell cycle arrest, inhibit phosphorylation of MET and AXL and related proteins such as AKT, ERK, STAT3 in downstream pathway, and prevent cell migration and EMT.

In a nude mouse transplantation tumor model of human gastric cancer, LY2801653 at a low dose can inhibit the in vivo growth of MKN45 cells with high expression of MET and AXL, and SNU719 cell grafts with medium expression of MET and AXL can also be inhibited after increasing the drug dose. For the tumor cells with high MET and AXL expression, LY2801653 has the functions of directly inhibiting the proliferation of the tumor cells and promoting the apoptosis of the tumor cells; for MET and AXL moderately expressed tumor cells, LY2801653 may act on the tumor microenvironment, reducing the production of M2 type macrophages and microvessels, thus playing an in vivo anti-tumor role. The invention provides evidence of MET and AXL as new targets for treating gastric cancer through the experiments.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.