JP2020531811A - Gastric cancer diagnosis using GKN1 protein in blood - Google Patents

Abstract

The present invention relates to a gastric cancer diagnostic composition containing a preparation for measuring GKN1 protein (Gastrokine 1 Protein) level, or a gastric cancer diagnostic kit containing the composition, and also contains GKN1 protein (Gastrokine 1 Protein) as an active ingredient. It relates to a composition for preventing or treating gastric cancer, which is included as.

Description

The present invention relates to a composition for diagnosing, preventing or treating gastric cancer containing GKN1 protein (Gastrokine 1 Protein) as an active ingredient.

Gastric cancer is a cancer that occurs frequently in South Korea, China, Japan, etc. worldwide, and the incidence is low in Western Europe such as the United States and Europe, but in the case of South Korea, gastric cancer has the highest cancer incidence. Mortality rate is second only to lung cancer. Looking at the classification of gastric cancer, 95% of the total is adenocarcinoma that occurs in the adenocarcinoma of the gastric wall mucosa, and there are also lymphoma that develops in the lymphatic system and gastrointestinal stromal tumor that develops in the stromal tissue.

Currently, there are three main treatment methods for treating cancer: surgical treatment, drug therapy, and radiation therapy, but drug therapy is less painful to treat and is suitable for surgical treatment and radiation treatment. In comparison, the recurrence of cancer is relatively small even after treatment, so expectations are high for this. Therefore, many anti-cancer agents have been developed and used to respond to this, but most of these anti-cancer agents selectively kill cells that actively divide in consideration of overgrowth of cancer, thereby causing anticancer cancer. It shows the effect. Such an anticancer drug has a serious side effect of killing both epidemic cells and normal cells such as hair root cells, which are cells that generally divide actively in the human body, and thus has a problem that it cannot be used for a long period of time. I have. Further, as a method for treating gastric cancer, methods such as lymph node dissection, endoscopic mucosal resection, and laparoscopic gastrectomy are used, but endoscopic mucosal resection is performed intramucosally. For early gastric cancer, gastrectomy is painful with simple endoscopic surgery as a method of injecting physiological saline around the gastric mucosa with cancer lesions to inflate the lesion site and then excise the mucosa with lesions. However, it has the advantage that it can be used only when the possibility of lymph node metastasis is low in early gastric cancer limited to mucous membranes.

Recently, research has been conducted worldwide to discover therapeutic targets through functional studies of genes related to cancer generation and treatment, and to utilize these for the development of diagnostic and therapeutic agents. With the activation of genome research, human gene DNA chips or proteome analysis research is actively carried out, and a large number of genes related to cancer are excavated, so that a database related to a list of many genes has been constructed, but many However, it is uncertain whether the specific intracellular biological function and the association of cancer with these genes have not been studied yet, and in fact, along with the association or diagnosis of cancer and its utilization as a target gene, further cancer There are considerable difficulties in finding genes that can effectively treat. Therefore, it is necessary to discover new genes in addition to the cancer-related genes that have been clarified so far.

An object of the present invention is to provide a composition for diagnosing gastric cancer, which comprises a preparation for measuring GKN1 protein (Gastrokine 1 Protein) level.

Another object of the present invention is to provide a gastric cancer diagnostic kit containing the gastric cancer diagnostic composition.

Another object of the present invention is to (a) measure the GKN1 protein level from a biological sample isolated from a patient and (b) compare the GKN1 protein level with a reference value obtained from a control group sample. The purpose is to provide a method for providing information for diagnosing or predicting the prognosis of gastric cancer, which is characterized by inclusion.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating gastric cancer containing GKN1 protein (Gastrokine 1 Protein) as an active ingredient.

Yet another object of the present invention relates to a method for preventing or treating gastric cancer, which comprises the step of administering GKN1 protein (Gastrokine 1 Protein).

To achieve the above object, the present invention provides a diagnostic composition for gastric cancer, which comprises a preparation for measuring GKN1 protein (Gastrokine 1 Protein) level.

In one example of the present invention, the preparation for measuring the GKN1 protein level can be an antibody that specifically binds to the protein.

In one embodiment of the invention, the GKN1 protein can be present in blood, plasma or serum exosomes.

The present invention also provides a gastric cancer diagnostic kit containing a gastric cancer diagnostic composition.

In one embodiment of the invention, the kit can be an ELISA kit or a protein chip kit.

The present invention also includes (a) measuring the GKN1 protein level from a biological sample isolated from the patient and (b) comparing the GKN1 protein level with a reference value obtained from the control group sample. To provide a method for providing information for diagnosing or predicting the prognosis of gastric cancer.

In one embodiment of the invention, the GKN1 protein can further include a step of heat treatment at 60-80 ° C. for 5-15 minutes, preferably heat treatment at 75-85 ° C. for 8-12 minutes. More preferably, it can be heat treated at 70 ° C. for 10 minutes.

In one embodiment of the present invention, a step of determining gastric cancer when the GKN1 protein level is lower than the reference value can be further included.

In one embodiment of the present invention, a step of determining that when the GKN1 protein level is lower than the reference value, the possibility of developing gastric cancer is high or the prognosis is not good can be further included.

In one embodiment of the invention, the biological sample can be blood, plasma or serum, preferably serum.

In one embodiment of the present invention, the measurements are: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay (sandwich assay), Western blotting, immunoprecipitation, immunohistochemical staining. (Immunoassaystic styling), flow cytometry, fluorescence activated cell classification (FACS), enzyme-linked immunosorbent assay and antigen-antibody aggregation method using any one or more of the methods selected from the group. Can be done.

The present invention also provides a pharmaceutical composition for preventing or treating gastric cancer, which comprises GKN1 protein (Gastrokine 1 Protein) as an active ingredient.

In one embodiment of the invention, the GKN1 protein can be an exosome secreted by a cell expressing the GKN1 protein.

In one embodiment of the present invention, the GKN1 protein can be separated from the culture upper layer fluid of cells expressing the GKN1 protein.

In one example of the present invention, the GKN1 protein can be separated from the culture upper layer fluid of cells into which the recombinant GKN1 (rGKN1: recombinant GKN1) protein has been introduced.

In one example of the invention, the recombinant GKN1 protein can be treated at a concentration of 1-10 ng / ml, preferably at a concentration of 3-7 ng / ml, even more preferably. Can be processed at a concentration of 5 ng / ml, but is not limited thereto.

In one example of the invention, the GKN1 protein can be treated at a concentration of 1-20 ng / ml, preferably at a concentration of 5-15 ng / ml, and more preferably. It can be processed at a concentration of 10 ng / ml, but is not limited to this.

In one embodiment of the invention, the GKN1 protein can be internalized in cancer cells by clathrin.

In one embodiment of the present invention, the GKN1 protein can suppress cell growth of cancer cells and induce cell death of cancer cells.

The present invention also provides a method for preventing or treating gastric cancer, which comprises the step of administering GKN1 protein (Gastrokine 1 Protein).

The method of treating cancer of the present invention comprises administering the GKN1 protein to an individual in a therapeutically effective amount. The specific therapeutically effective amount for a particular individual includes the type and extent of the reaction to be achieved and, in some cases, whether other formulations are used, the specific composition, the age, weight of the individual, general health. Various factors including status, gender and diet, time of administration, route of administration and rate of secretion of composition, duration of treatment, drugs used with or co-use with specific composition and similar factors well known in the pharmaceutical field. It is preferable to apply differently depending on. Therefore, it is preferable to determine the effective amount of the composition suitable for the purpose of the present invention in consideration of the above-mentioned matters.

The individual is applicable to any mammal, which includes livestock such as cattle, pigs, sheep, horses, dogs, and cats as well as humans and primates.

The GKN1 protein according to the present invention contains the GKN1 protein of the exosome as a biomarker useful for predicting or diagnosing gastric cancer, and when the total GKN1 protein level in the sample is lower than that of a normal person, in gastric cancer. If there is, it is possible to diagnose gastric cancer or predict the prognosis early and conveniently.

In addition, the GKN1 protein (Gastrokine 1 Protein) according to the present invention is secreted as an exosome in cells expressing the GKN1 protein, separated from the culture upper layer fluid of the cells expressing the GKN1 protein, and is endogenous to the cancer cells by Krathrin. It can be usefully used for the prevention or treatment of gastric cancer by being converted to suppress the cell proliferation of cancer cells and induce the cell death of cancer cells.

As a result of identifying the GKN1 binding protein, (a) relates to a list of exosome proteins showing binding to the recombinant GKN1 protein on a protein microarray, and (b) immunoprecipitates the binding force of GKN1 to COMT and YWHAZ proteins. It shows the result confirmed by the method and Western blotting. HFE-145 cells, exosomes isolated from AGS and MKN1 cells treated / untreated with recombinant GKN1 (rGKN1) protein, exosome-removed medium (soble) and GKN1 in whole cell lysate (WCL), It shows the result of Western blotting for TSG-101 and CD81. GKN1 concentration after treating the whole culture solution (Total media), exosome fraction (Exosome), and exosome-free medium (Soluble) in HFE-145 cells with a dilution buffer solution in the ELISA kit for measuring the concentration of GKN1 protein. It shows the result of measuring. It shows the result of performing the enzyme digestion assay (trypsin digestion assay) by the treatment / untreatment of trypsin and Triton X-100 on the exosome derived from HFE-145 cells. It shows the result of confirming the isolated exosome after treating the recombinant GKN1 protein into AGS cells. It shows the result of confirming by Western blotting that the expression of Clathrin protein is suppressed in AGS and MKN1 cells treated with siClathrin. It shows the result of investigating the localization of exosomes containing GKN1 protein in AGS and MKN1 cells treated with siClathrin. It shows the result of confirming the expression level of GKN1 and CD81 by Western blotting in AGS and MKN1 cells treated with siClathrin. The concentration of GKN1 protein in the whole medium (Media), the exosome (Exosome), and the medium from which the exosome was removed (exosome depleted media) when the HFE-145 cells were not heat-treated (Non-heating) or heat-treated (Heating). It shows the result of analysis. Whole serum (Whole Serum) or exosome with or without heat treatment (Non-heating) heat treatment (Heating) with serum of 10 healthy subjects (Normal serum) and 10 gastric cancer patients (cancer serum) ) And the results of analysis of the concentration of GKN1 protein in the serum from which exosomes have been removed (W / O Essome) are shown. It shows the result of having analyzed the degree of association of age and sex by serum GKN1 protein concentration from 100 healthy subjects. The results of analysis of GKN1 protein concentration in healthy subjects, those with atrophic gastritis (Atrophy), those with atrophic gastritis with intestinal metaplasia (IM), and patients with gastric cancer. (P <0.0001). Results of analysis of serum GKN1 concentration in 100 healthy subjects (Normal serum), 150 gastric cancer patients (GC serum), 45 liver cancer patients (LC serum) and 50 large-scale cancer patients (CC serum) Is shown. According to serum GKN1 level in patients with gastric cancer for those with normal gastric mucosa (normal), those with atrophic gastritis (Atrophy) and those with atrophic gastritis with intestinal metaplasia (IM) It shows the result of ROC curve analysis. It shows the ROC curve analysis result as to whether the gastric cancer patient can be classified into the liver cancer and the large sickle cancer patient by the serum GKN1 concentration. GKN1 in healthy subjects, those with atrophic gastritis (Attrophy), those with atrophic gastritis with intestinal metaplasia (IM), and patients with gastric cancer who have early gastric cancer and advanced gastric cancer It shows the result of analyzing the protein concentration (P <0.0001). ROC curve for GKN1 level in healthy subjects, those with atrophic gastritis (Atrophy), those with atrophic gastritis with intestinal metaplasia (IM), and patients with early gastric cancer among patients with early gastric cancer It shows the analysis result. It shows the result of an immunofluorescence assay to confirm whether or not exosomes derived from HFE-145 cells were attached to the cell membranes of Jurkat T cells, THP-1 protein spheres, and U937 macrophages. It shows the result of analysis whether or not the exosome GKN1 and CD81 protein derived from HFE-145 cells are present in the cell membrane, cytoplasm or nucleus of Jurkat T cells, THP-1 protein spheres, and U937 macrophages. It shows the result of analysis whether or not the exosome GKN1 derived from HFE-145 cells affects the cell viability of Jurkat T cells, THP-1 protein spheres, and U937 macrophages. It shows the result of analysis whether or not the exosome GKN1 derived from HFE-145 cells affects the function of THP-1 protein sphere and U937 macrophage. After treating MKN1 (recombinant GKN1 treated / untreated), AGS (recombinant GKN1 treated / untreated) and exosomes derived from HFE-145 cells into HFE-145, AGS and MKN1 cells, respectively, cell viability through MTT assay. It shows the result of confirming. After treating exosomes derived from MKN1 (recombinant GKN1 treated / untreated), AGS (recombinant GKN1 treated / untreated) and HFE-145 cells into HFE-145, AGS and MKN1 cells, cell proliferation ability through BrdU Corporation Assay. It shows the result of confirming. It shows the result of confirming the cell viability and cell proliferation ability with time through MTT and BrdU Corporation assay after treating an exosome (Exosome carrying gastrokine 1 Protein) containing recombinant GKN1 (rGKN1) or GKN1 protein. The results of cell cycle analysis after treatment (rGKN1_exosome) / non-treatment (w / oGKN1_exosome) of recombinant GKN1 on AGS and MKN1 cells are shown. HFE-145, AGS (recombinant GKN1 treated / untreated) and MKN1 (recombinant GKN1 treated / untreated) cells p53, p21, CDK4, Cyclin D, Cdc25c, p-Cdc2, Cyclin B, BAX, BCL2, Caspase It shows the result of confirming the expression change of -3 (including cleared) and Caspase-8 (including cleared) by western blotting. It shows the result of confirming the degree of cell mortality through a cell mortality assay after treating exosomes containing GKN1 protein derived from cells treated / untreated with recombinant GKN1 into AGS and MKN1 cells.

The term "diagnosis" in the present invention means to confirm the pathological state, and for the purpose of the present invention, the above diagnosis is to confirm the GKN1 protein expression level of exosomes and confirm the progress of gastric cancer. means.

The term "antibody" in the present invention means a specific protein molecule indicated for an antigenic site as a term known in the art. For the purposes of the present invention, an antibody means an antibody that specifically binds to the GKN1 protein, which is a marker of the present invention. This also includes a partial peptide that can be made from the above proteins, and the partial peptide of the present invention contains at least 7 amino acids, preferably 9 amino acids, and more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or a part thereof as long as it has antigen-binding property is included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes a special antibody such as a humanized antibody. Such antibodies against the GKN1 protein of the present invention include all antibodies that can be produced by methods known in the art.

The gastric cancer diagnostic kit of the present invention can contain an antibody that specifically binds to the GKN1 protein of the exosome, and the kit for measuring the protein level is an ELISA kit or protein chip used for "measurement of protein expression level". Can be a kit.

The presence or absence of protein expression using the above antibody is measured by forming an antigen-antibody complex between the GKN1 protein and its antibody, and quantitatively by measuring the amount of the complex formed by various methods. Can be detected.

The measurement by the above-mentioned antigen-antibody complex includes enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay (sandwich assay), Western blotting, immunoprecipitation, and immunohistochemical staining. It is performed using any one or more methods selected from the group consisting of blotting), flow cytometry, fluorescence-activated cell assay (FACS), assay substrate color development method and antigen-antibody aggregation method. However, it is not limited to this.

The term "biological sample" in the present invention specifically means a sample containing the GKN1 protein in an exosome and capable of detecting the total GKN1 protein expression level, and includes, for example, blood, plasma or serum. However, it preferably means serum.

The present invention can include the step of obtaining a biological sample isolated from the patient and then heat treating at 60-80 ° C. for 5-15 minutes. After the above heat treatment, the expression level of GKN1 protein in the total sample or the exosome is measured, and if the GKN1 protein level of the exosome is lower than the reference value compared with the reference value obtained from the control group sample, it is judged to be gastric cancer. By doing so, it is possible to predict or diagnose gastric cancer.

The present invention relates to a pharmaceutical composition for preventing or treating gastric cancer, which comprises GKN1 protein (Gastrokine 1 Protein) as an active ingredient.

The term "prevention" of the present invention means any act of suppressing or delaying the onset of gastric cancer by administration of the pharmaceutical composition according to the present invention.

The term "treatment" of the present invention means all actions that improve or change the disease caused by gastric cancer by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the invention can include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are usually used in preparation and include saline solution, sterilized solution, Ringer solution, buffered saline solution, cyclodextrin, dextrin solution, maltodextrin solution, glycerol, ethanol, liposomes and the like. , But not limited to this, other conventional additives such as antioxidants, buffers and the like can be further included as needed. Injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets with the addition of diluents, dispersants, surfactants, binders, lubricants, etc. Can be formulated as. Suitable pharmaceutically acceptable carriers and formulations should be preferably formulated with each component using the methods disclosed in the Remington's Pharmaceutical Science, Mac Publishing Company, Easton PA. Can be done. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but can be formulated as an injection, an inhalant, an external preparation for skin, or the like.

The pharmaceutical composition of the present invention can be orally or parenterally administered (for example, applied intravenously, subcutaneously, skin, nasal cavity, respiratory tract) according to a method of interest, and the dose is the patient's condition and weight, disease. It depends on the degree, drug form, route of administration and time of administration, but can be appropriately selected by those skilled in the art.

The compositions according to the invention are administered in pharmaceutically effective amounts. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is a patient's disease. Determined by the type, severity, activity of the drug, sensitivity to the drug, duration of administration, route of administration and rate of excretion, duration of treatment, factors including the drugs used at the same time and other factors well known in the medical field. be able to. The composition according to the invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple times. Can be done. It is important to consider all of the above factors and administer an amount that produces the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the invention can be varied depending on the age, sex and body weight of the patient, generally 0.001-150 mg / kg body weight, preferably 0.01-100 mg daily or. It can be administered every other day or divided into 1 to 3 times a day. However, since the dose can be increased or decreased depending on the route of administration, the severity of obesity, sex, body weight, age, etc., the above-mentioned dose does not limit the scope of the present invention by any method.

Hereinafter, the present invention will be described in more detail through examples. These examples are for more specific explanation of the present invention, and the scope of the present invention is not limited to these examples.

Example 1. experimental method

1.1. Production of recombinant vector into which GKN1 was introduced In order to produce the recombinant GKN1 protein, a recombinant vector was produced as follows. First, human GKN1 cDNA was amplified using a PCR method, and a pCMV6-AN-FC-hGKN1 recombinant vector was produced using a restriction enzyme site on a pCMV6-AN-FC (Origene ID PS10055) vector. More specifically, the pCMV6-AN-FC vector and GKN1 cDNA were treated with restriction enzymes of SgfI and MluI, and then the pCMV6-AN-FC and hGKN1 cDNA were ligated to introduce the GKN1 gene into the vector. After that, after transforming into a large bacterium and taking a colony generated on the agar plate, this was cultured to extract DNA, and then a sequence was performed to confirm whether the GKNI gene was correctly introduced. Then, the recombinant GKN1 protein was purified by using protein A affinity chromatography, and then the GKN1 recombinant protein was confirmed by using Western blotting. Hereinafter, the sequence of the GKNI gene is shown as SEQ ID NO: 1, and the amino acid sequence of the GKN1 protein encoded therein is shown as SEQ ID NO: 2.

1.2. Cell culture AGS and MKN1 gastric cancer cell lines that do not express GKN1 (Gastrokine 1) protein and HFE-145 immortalized gastric epithelial cell line that express GKN1 (immortalized gastric epithel The cells were cultured in RPMI-1640 medium containing activated FBS (fetal bovine cellum) at 37 ° C. and 5% CO ₂ . In addition, Jurkat T cells, THP-1 mononuclear cells and U937 macrophages were also cultured under the above conditions.

1.3. Identification of GKN1-binding proteins through HuProtTM microarrays Human Protein Microarrays (CDI Labs, USA) containing more than 19,000 total-length recombinant human proteins were used to identify GKN1-binding proteins. .. Briefly, protein microarrays were treated with Blocking Buffer (5% BSA in PBS with 0.05% tween-20) for 2 hours, followed by 1 μg of biotinylated GKN1 at 4 ° C. for 8 hours. .. After that, 1 μg of streptavidin-fluorescence (Alexa-Fluor 635 nm) was treated on the microarray. The results were confirmed by GenePix 4100A Microarray Laser Scanner (Molecular Devices, USA).

1.4. Exosome Isolation
To investigate the presence of GKN1 protein in exosomes, AGS and MKN1 cells were treated with recombinant GKN1 protein (rGKN1, ANRT, Daejeon, Korea). Exosomes were isolated from the upper fluid of HFE-145, AGS and MKN1 cells. Briefly, cells of passage 3-8 are cultured in serum-free medium, added with 10% FBS and 1% penicillin / streptomycin for 48 hours prior to collection of medium and cultured at 37 ° C. and 5% CO ₂ . Was done. The conditioned medium was centrifuged at 2000 g for 10 minutes at 4 ° C. to remove cell debris and then passed through a 0.22 μm filter. The filtered top layer was transferred to a new Glass tube and placed on ice. After that, 2 ml of the upper layer solution was mixed with 0.75 ml of A / B / C solution (101 Bio Company, CA, USA) in a new glass tube, vortexed strongly for 30 seconds, and left at 4 ° C. for 30 minutes. The mixture is separated into two layers, the upper layer is removed, the lower layer is transferred to a microcentrifuge tube and centrifuged at 5,000 g for 3 minutes, and the middle layer is transferred to a new microcentrifuge tube and centrifuged at 5,000 g for 3 minutes. After that, the lid was opened and air-dried at room temperature for 10 minutes. A total of 4 times the volume of 1X PBS was added to the tube and pipette strongly. Tubes were treated with a horizontal shaker at high speed for 15 minutes and then centrifuged at 5,000 g for 5 minutes. The upper layer liquid was carefully transferred to PureExo Company (101Bio company, CA, USA) and then centrifuged at 1,000 g for 5 minutes. The transit fluid contains a separated exosome fraction suspended in PBS.

1.5. Exosome Labeling
Exosomes were labeled with PKH26 (Sigma, St. Louis, USA) according to the manufacturer's protocol (slightly modified). Briefly, the exosome pellets were suspended in 1 ml Diluent C and separately 1 ml Diluent C was mixed with 4 μl PKH26 to make a Stein Solution. The exosome suspension was mixed with the stain solution and reacted for 4 minutes, and the labeling reaction was stopped with the addition of the same amount of 1% BSA (bovine serum albumin). Labeled exosomes were isolated by the manufacturer's protocol using the Total Exosome Isosome Isolation Kit (Invitrogen). Briefly, 0.5 volume of Isolation Reagent was added to and mixed with the labeled exosomes. Labeled exosomes were allowed to settle overnight at 4 ° C. and sequentially centrifuged at 4 ° C. for 1 hour at 10,000 g. After that, the upper layer liquid was discarded, and each pellet was suspended in 100 μl of PBS.

1.6. Expression of GKN1 protein in exosomes Expression of GKN1 in HFE-145, AGS and MKN1 cell cell lysates and exosomes was analyzed by Western blotting. Equal amounts of cell lysates and exosomes were separated from 12.5% SDS-PAGE and transferred to Hybond-PVDF (polyvinylidene fluoride) transfer membranes (Amersham). After blocking with 0.5% skim milk, the membrane is reacted with a primary antibody that is anti-GKN1 (Abcam, MA, USA), anti-TSG101 and anti-CD81 antibodies (Santa Cruz Biotechnology, TX, USA). , HRP (horseradish peroxidase) -conjuged secondary antibodies. The protein band was detected on the Westensure ECL substrate (LI-COR Biosciences, NE, USA) and the band was visualized on LAS 4000 (Fuji Film, Japan).

1.7. Co-immunoprecipitation
Exosomes derived from AGS and MKN1 cells treated with recombinant GKN1 (rGKN1) were washed with PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM as described above. Was dissolved in PBS (pH 7.2) containing NaF, 1.0 mM NaVO4 and 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA) at 4 ° C. (J. Biol. Chem. 2010; 285: 20547-57). As mentioned above, the same amount of protein aliquot (1.0 mg) is a Protein A / G to which 2.0 μg of antibodies against GKN1 (Abcam, Cambridge, UK), COMT and YWHAZ (Genetex, CA, USA) are bound. -Immune-precipitated with agarose (Santa Cruz Biotechnology) (EBioMedine.2016; 9: 97-109).

1.8. Trypsin digestion assay
To confirm the presence of GKN1 protein in exosomes, as described above, exosomes isolated from HFE-145 cells (35 μg protein in 7 μl PBS) were 1% Triton X- at 4 ° C. for 30 minutes. The lipid membrane was degraded by 100 (Blood. 2009; 113: 1957-66). Exosomes were cultured under the same conditions without Triton X-100. The exosome suspension was degraded with trypsin at 37 ° C. at two enzyme concentrations (15 and 0.01 μg / mL). Triton X-100 treatment had little effect on trypsin activity (data not show). After treatment with trypsin, the samples were immediately mixed in the same amount of Laemmli buffer, boiled for 3 minutes and then gel loaded.

1.9. Measurement of cell survival and death In the case of cell viability analysis, after treating each cell with exosomes derived from HFE-145 cells and 5 ng recombinant GKN1 (rGKN1) -treated / untreated AGS and MKN1 cells. MTT assays were performed on HFE-145, AGS and MKN1 cells at 24, 48 and 72 hours. Absorbance was measured at 540 nm using a spectrophotometer and cell viability was expressed relative to untreated cells.

In the case of cell proliferation assay, each cell is treated with exosomes derived from HFE-145, AGS and MKN1 cells treated with 5 ng recombinant GKN1 (rGKN1), and then BrdU Cell Proliferation Assay Kit (Millipore, Billilica, MA, USA). ) Was performed at 24, 48 and 72 hours by the BrdU Corporation Assay according to the manufacturer's protocol. Absorbance was measured at 450 nm using a spectrophotometer and cell proliferation was expressed relative to untreated cells. To compare the effects of exosome GKN1 protein and recombinant GKN1 (rGKN1) protein on cell viability and cell proliferation, exosome or recombinant GKN1 (rGKN1) protein (10 ng / ml) containing GKN1 protein (10 ng / ml) After the treatment, the cell viability and cell proliferation ability of AGS and MKN1 cells were analyzed.

For the cell killing assay, the Annexin V-binding Assay was performed according to the manufacturer's instructions. Annexin V binds to cells with phosphatidylserine on the cell membrane, and PI (propidium iodide) stains the DNA of cells with weakened cell membranes. AGS and MKN1 cells were treated with exosomes containing GKN1 protein, washed twice with cold PBS and suspended in 100 μl binding buffer. In each case, 100 μl of the upper layer solution is mixed with 400 μl of the blocking solution, 5 μl Annexin V-FITC (1 μg / ml) and 5 μl PI (2 μg / ml) are added to the mixed solution, and the cells are reacted in a dark room for about 15 minutes. Was analyzed by FACS (fluorescein-activated cell planter, BD Biosciences, San Jose, CA, USA). Only fluorescent-positive cells that were not PI-stained were considered dead cells.

1.10. Cell Cycle Measurement In the case of cell cycle analysis, AGS and MKN1 cells are treated with exosomes isolated from AGS cells treated / untreated with 5 ng recombinant GKN1 (rGKN1), and then the cells are collected and darkened before analysis. The cells were stained with PI (propidium iodide) for 45 minutes. The percentage of cells in each phase during the cell cycle was determined using FACSCalibur Flow Cytometer with CellQuest 3.0 software (BD Biosciences, Heidelberg, Germany). The experiment was repeated twice.

1.11. Expression of Cell Cycle Regulators Derived from HFE-145 cells and recombinant GKN1 (rGKN1) treated AGS and MKN1 cells to determine if the exosome GKN1 protein is involved in the regulation of cell cycle and cell death. 48 hours after treatment with the exosomes, changes in the expression of p53, p21, CDK4, cyclin D1, Cdc25, p-Cdc2, BAX, BCL2, Caspase-3 and 8 proteins were analyzed in HFE-145, AGS and MKN1 cells. It was. Cell lysates were separated from a 10% polyacrylamide gel and transferred to a Hybrid PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). After blocking, the membranes were sequentially labeled with antibodies against G0 / G1 phase proteins. Protein bands were confirmed using ECL (enhanced chemiluminescence reagents, Amersham Pharmacia Biotech, Piscataway, NJ, USA) solutions.

1.12. Study of immunofluorescence Mouse anti-FITC conjugated GKN1 (Genetex), anti-clathrin (Abcam) and Texas-red conjugated goat anti-mouse IgG (invitrogen) used for immune reaction send. The specificity of the reaction was tested on the Non-immune Mouse Serum (invitrogen).

1.13. Serum sample The study was conducted in a population of 150 patients with gastric cancer who will undergo gastrectomy and 100 healthy volunteers. In addition, 45 patients with hepatocellular carcinoma and 50 patients with colorectal cancer were used as controls, respectively. Whole blood was collected from 245 preoperative cancer patients and 100 healthy volunteers, sampled, and then the sera were separated and immediately stored at -80 ° C. Frozen serum is thawed in ice and then diluted with 15 μg / ml in serum total protein concentration in phosphate buffered solution (PBS) to normalize the difference in total protein concentration in individual serum. And used. Samples were treated or untreated at 70 ° C. to investigate whether the GKN1 protein was present as an exosome protein in HFE-145 cell cultures and human serum. Concentrations of GKN1 protein in whole cultures, whole serum, exosome-removed samples and exosome fractions were calculated using an ELISA kit (USCN, Wuhan, China) according to the manufacturer's instructions. The announced consent form was provided by the Declaration of Helsinki and received written consent form from all subjects. The present invention has been approved by the Instituteal Review Board of The Catholic University of Korea, College of Medicine (MC16SISI0132). None of the cancer patients had evidence of hereditary cancer.

1.14. Statistical analysis ROC (receiver operating characteristic) curve analysis was used to evaluate the diagnostic usefulness of markers based on the bifurcation classification method. The ROC curve is a plot of true-positive fraction vs. false-positive fraction that evaluates to all possible limits. In addition, Student's t-test was used to analyze the effect of GKN1 on cell viability and cell proliferation. All experiments were repeated more than once to prove reproducibility. Data are average ± S. from two independent experiments. D. It was expressed as (standard deviation), and if the P value was less than 0.05, it was considered to be statistically significant.

Example 2. Identification of GKN1-binding protein

We performed a protein microarray assay to identify proteins that bind to GKN1. As a result, 27 exosome proteins having normal or strong binding force to recombinant GKN1 (rGKN1) were found (Fig. 1a). To confirm the binding force of GKN1 to exosome protein, we extracted the exosome protein from HFE-145 cells and AGS and MKN1 cells treated with recombinant GKN1 (rGKN1) and performed joint-immunoprecipitation analysis. The interaction between GKN1 and COMT and the interaction between GKN1 and YWHAZ in the exosome protein was demonstrated (Fig. 1b).

Example 3. Detection of GKN1 protein in exosomes derived from gastric cell lines

Due to the fact that the recombinant GKN1 (rGKN1) protein has a binding force to the exosome protein, the present inventors have investigated whether GKN1 is naturally secreted and internalized by the exosome protein. An experiment was conducted in which exosomes were separated from the culture upper layer fluid of HFE-145, AGS and MKN1 cells to confirm the presence or absence of GKN1. As a result, it was confirmed that the exosomes derived from HFE-145 cells contained the GKN1 protein, but the exosomes derived from the AGS and MKN1 gastric cancer cell lines did not express the GKN1 protein (Fig. 2a). The exosome marker TSG101 was confirmed in the isolated exosomes and cell lysates, but CD81 was confirmed to be present only in exosomes, and CD81 was subsequently used as an exosome marker in experiments (Fig. 2a). Moreover, when the recombinant GKN1 (rGKN1) protein was treated into AGS and MKN1 cells, it was confirmed that most of the GKN1 protein was present in exosomes (Fig. 2a). Consistent with this, the concentrations of GKN1 protein in whole culture medium, exosome fraction and exosome-free medium in HFE-145 cells were 1.13 ± 0.04 and 1.08 ± 0.07, respectively. It was 0.05 ± 0.03 ng / ml (Fig. 2b).

Next, a Trypsin Digestion Assay with / without trypsin and Triton X-100 was performed to confirm the location of the GKN1 protein in the exosome, followed by Western blotting (Blood. 2009; 113: 1957-66). When the exosomes were untreated with Triton X-100 and treated with trypsin only, GKN1 did not undergo trypsin-dependent enzymatic degradation. However, when Triton X-100 and trypsin were all treated, GKN1 was completely degraded (Fig. 2c). From the above results, it was confirmed that the GKN1 protein exists inside the exosome.

Example 4. Clathrin-dependent internalization of exosomes containing GKN1 protein

We conducted an experiment to confirm whether Clathrin was associated with the internalization of the GKN1 protein in exosomes. An immunofluorescence assay and Western to confirm the presence or absence of GKN1 protein expression in AGS and MKN1 cells after treatment of exosomes containing siClathrin and GKN1 protein with siClatrin, which is a siRNA, to knock down Cathrin. Blotting was performed.

First, when PKH26-positive exosomes containing the GKN1 protein were treated into AGS and MKN1 cells, the exosomes were clearly internalized in the cytoplasm of AGS and MKN1 (FIGS. 3a and 3c). However, when Clathrin was knocked down in AGS and MKN1 cells, the presence of PKH26-positive exosomes in the cell membrane and cytoplasm was definitely suppressed (FIGS. 3b and 3c). It was also confirmed that silencing of Clathrin by siClathrin markedly suppressed GKN1 protein expression in the cell membrane and cytoplasm (Fig. 3d).

Therefore, it was confirmed that Clathrin plays an important role in adhering and internalizing exosomes containing the GKN1 protein to the mucosal epithelial cells.

Example 5. Diagnosis of gastric cancer by serum GKN1 protein concentration

To confirm whether GKN1 protein is present as exosome protein in medium and human serum, HFE-145 cell culture medium and human serum were treated at 70 ° C. for 10 minutes, and then the concentration of GKN1 protein was measured by ELISA. Was measured. As a result, when the HFE-145 cells were not heat-treated (Non-heating), the GKN1 protein was not confirmed in the whole medium, the medium from which the exosomes and exosomes were removed, while the heat treatment was performed at 70 ° C. for 10 minutes (heating). ) Is a whole medium, a medium from which exosomes and exosomes have been removed (solve), and the concentrations of GKN1 protein are 1.13 ± 0.06, 1.08 ± 0.1 and 0.05 ± 0.04 ng / ml, respectively. It was confirmed (Fig. 4a). Consistent with this, GKN1 protein was not identified in unheat-treated whole serum, exosomes and exosome-depleted sera (Fig. 4b). Among the 10 healthy subjects, the expression concentrations of GKN1 protein in the heat-treated whole serum and exosomes were 6.53 ± 0.58 ng / ml and 6.24 ± 0.55 ng / ml, respectively (Fig.). 4b). In addition, in 10 gastric cancer patients, the expression concentrations of GKN1 protein in the total serum and exosomes were 3.58 ± 0.54 ng / ml and 3.28 ± 0.82 ng / ml, respectively (Fig. 4b). This means that the majority of serum GKN1 protein is present in exosomes. Based on the above results, the present inventors obtained whole sera in 100 healthy subjects and 245 patients with gastric cancer, large-scale cancer and liver cancer, heat-treated at 70 ° C. for 10 minutes, and then analyzed the GKN1 concentration. ..

As shown in FIG. 4c, serum GKN1 protein levels tended to be age-related in normal healthy individuals, but were not statistically significant (P = 0.338). In addition, no difference was observed between men and women at the serum GKN1 level (P = 0.074). Interestingly, those with atrophic gastritis and those with atrophic gastritis with intestinal metaplasia (IM) showed lower serum GKN1 levels than subjects without gastritis, but had gastric cancer. It was significantly higher than the patient (Fig. 4d).

GKN1 concentrations in the total sera of 100 healthy subjects and 150 gastric cancer patients were 6.35 ± 0.82 ng / ml and 3.50 ± 0.57 ng / ml, respectively (FIG. 4e). , It was confirmed that the serum GKN1 protein level in gastric cancer patients was significantly lower than that in healthy subjects (P <0.0001). In addition, serum GKN1 concentrations in patients with liver cancer and large-scale cancer were 6.07 ± 0.92 and 6.26 ± 0.95 ng / ml, respectively, similar to the levels of healthy subjects and compared to gastric cancer patients. It was a remarkably high level (Fig. 4e). In particular, by confirming that the area values of the ROC curve are 1.0, 1.0, and 0.9964, respectively, healthy subjects, those with atrophic gastritis, and intestinal metaplasia (intestinal) depending on the concentration of serum GKN1 protein. It was confirmed that it is possible to distinguish between patients with atrophic gastritis associated with metaplasia (IM) and patients with gastric cancer (Fig. 4f). In addition, in order to confirm that the present inventors can distinguish gastric cancer patients from liver cancer and large-scale cancer patients by the serum GKN1 level, ROC curve values were measured, and the ROC curves were 0.9938 and 0.9987, respectively. It was confirmed that gastric cancer can be predicted and diagnosed based on the GKN1 protein concentration in the whole serum (Fig. 4 g).

Gastric cancer is classified into early gastric cancer and advanced gastric cancer according to the degree of gastric wall infiltration of cancer cells. In this study, the GKN1 concentration in the serum of patients with early gastric cancer was 3.75 ± 0.47 ng / ml, which was higher than the serum GKN1 concentration of 3.38 ± 0.58 ng / ml in patients with advanced gastric cancer (P <0). .001) (Fig. 4h), which was lower than the serum GKN1 concentration in persons with atrophic gastritis with intestinal metaplasia (IM) (P <0.001) (Fig. 4h). By confirming that the area values of the ROC curves are all 1.0, the concentration of serum GKN1 protein indicates that healthy subjects, those with atrophic gastritis, and atrophic metaplasia with intestinal metaplasia (IM). It was confirmed that those with gastritis and those with early gastric cancer can be distinguished (Fig. 4i).

From the above results, the present inventors confirmed that the exosome GKN1 protein in human serum can be used as a useful biomarker for the diagnosis of gastric cancer.

Example 6. Cytotoxicity of GKN1 protein in exosomes to blood cells

Based on the evidence that GKN1 protein produced and secreted by gastric epithelial cells and having a tumorigenesis inhibitory function was confirmed in serum, the present inventors have exosomes containing GKN1 protein in blood lymphocytes and mononuclear cells. And whether it affects the cell viability and function of macrophages was investigated. Interestingly, immunofluorescence assay results showed that exosomes containing the GKN1 protein from HFE-145 cells were attached to the cell membranes of THP-1 protein spheres and U937 macrophages, but not to Jurkat T cells (Fig. 5a). , Western blotting confirmed that the exosome GKN1 protein was present only in the cell membrane portion of THP-1 protein spheres and U937 macrophages (Fig. 5b). In addition, the exosome GKN1 protein had no effect on the cell viability of THP-1 protein spheres and U937 macrophages and the secretion of cytokines including IL-6 (FIGS. 5c and 5d). From the above results, the present inventors confirmed that the exosome GKN1 protein is not cytotoxic to lymphocytes, mononuclear cells and macrophages.

Example 7. Measurement of cell proliferation and cell death

The present inventors performed MTT assay and BrdU Incorporation Assay to confirm the effect of GKN1 protein on exosomes on cell viability and proliferation. Exosomes derived from HFE-145, AGS (recombinant GKN1 treated / untreated) and MKN1 (recombinant GKN1 treated / untreated) cells were treated into HFE-145, AGS and MKN1 cells, respectively, for cell viability and cell proliferation. I experimented with Noh.

As a result, when GKN1-negative exosomes derived from AGS and MKN1 cells (group in which recombinant GKN1 was not treated) were treated, the cell viability was definitely increased, but they were derived from HFE-145, AGS and MKN1 cells. It was confirmed that exosomes containing the GKN1 protein (group treated with recombinant GKN1) significantly suppressed cell viability and cell proliferation ability in AGS and MKN1 cells (FIGS. 6a and 6b).

Next, the present inventors conducted an experiment to compare the cell viability of AGS and MKN1 cells with time after treating the exosome GKN1 protein and the recombinant GKN1 (rGKN1) protein. As a result, it was confirmed that when the exosome GKN1 protein and the recombinant GKN1 (rGKN1) protein were treated in the above cells, the cell viability and cell proliferation ability were suppressed in a time-dependent manner, and in particular, the exosome GKN1 protein was treated. In this case, it was confirmed that the cell viability and cell proliferative ability were sharply reduced in the cells treated with the recombinant GKN1 (rGKN1) protein (Fig. 6c).

In addition, the present inventors conducted an experiment to analyze the cell cycle after treating recombinant GKN1 (rGKN1) protein into AGS and MKN1 cells (rGKN1_exosome) / untreated (w / oGKN1_exosome). As a result, when AGS and MKN1 cells were treated with recombinant GKN1 (rGKN1) and then treated with exosomes (GKN-positive exosomes) containing the isolated GKN1 protein, the cell group in both AGS and MKN1 cells was in S phase. It was confirmed that there was a marked decrease and that the G1 and G2 / M phase cell groups increased (Fig. 6d).

In addition, the present inventors conducted an experiment to confirm the expression or absence of a cell cycle regulator affecting G1 or G2 / M phase arrest by the GKN1 protein of exosomes. As a result, the exosome GKN1 protein increased the expression of positive cell cycle regulators such as p53 and p21 and decreased the expression of positive cell cycle regulators such as cdk4 and cyclin D due to the G1 phase arrest. This was confirmed (Fig. 6e). It was also confirmed that the exosome GKN1 protein down-regulated the expression of p-cdc2, cdc25c and cyclin B proteins in HFE-145, AGS and MKN1 cells for G2 / M phase arrest (FIG. 6e).

Next, we performed a cell mortality assay after treating AGS and MKN1 cells with exosomes containing the GKN1 protein derived from cells treated / untreated with recombinant GKN1. Cell killing was measured by cells stained with FITC-labeled Annexin V, but cell killing in AGS and MKN1 cells by administration of exosomes containing GKN1 protein derived from recombinant GKN1 (rGKN1) treated AGS cells. Was confirmed to have increased remarkably (Fig. 6f). The results of Western blotting confirmed that the expression levels of cleared caspase-3 and -8 were definitely increased, while the expression of BAX and BCL2 was not changed (Fig. 6e).

Therefore, the exosome GKN1 protein is internalized in gastric cancer cells and can interact with cytoplasmic proteins to suppress cell proliferation, induce cell death and treat gastric cancer, and blood cells are not cytotoxic. It was confirmed.

Claims (20)

A composition for diagnosing gastric cancer, which comprises a preparation for measuring GKN1 protein (Gastrokine 1 Protein) level. The composition according to claim 1, wherein the preparation for measuring the GKN1 protein level is an antibody that specifically binds to the protein. The composition according to claim 1, wherein the GKN1 protein is present in blood, plasma or serum exosomes. A gastric cancer diagnostic kit comprising the composition according to claim 1. The kit according to claim 4, wherein the kit is an ELISA kit or a protein chip kit. (A) The step of measuring the GKN1 protein level from the biological sample isolated from the patient, and
(B) A method for providing information for diagnosing or predicting the prognosis of gastric cancer, which comprises a step of comparing the GKN1 protein level with a reference value obtained from a control group sample. The method of claim 6, wherein the GKN1 protein further comprises a step of heat treating at 60-80 ° C. for 5-15 minutes. The method according to claim 6, further comprising a step of determining gastric cancer when the GKN1 protein level is lower than the reference value. The method according to claim 6, further comprising a step of determining that when the GKN1 protein level is lower than the reference value, the possibility of developing gastric cancer is high or the prognosis is not good. The method of claim 6, wherein the biological sample is blood, plasma or serum. The measurement includes enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay (sandwich assay), Western blotting, immunoprecipitation, immunohistochemical staining, and flow cytometry. 6. The method according to claim 6, wherein any one or more methods selected from the group consisting of a fluorescence activated cell classification method (FACS), an enzyme-linked immunosorbent assay and an antigen-antibody aggregation method are used. The method described. A pharmaceutical composition for the prevention or treatment of gastric cancer containing GKN1 protein (Gastrokine 1 Protein) as an active ingredient. The composition according to claim 12, wherein the GKN1 protein is derived from the exosome of a cell expressing the GKN1 protein. The composition according to claim 12, wherein the GKN1 protein is separated from the culture upper layer solution of cells expressing the GKN1 protein. The composition according to claim 12, wherein the GKN1 protein is separated from the culture upper layer solution of the cells into which the recombinant GKN1 (rGKN1: recombinant GKN1) protein has been introduced. The composition according to claim 15, wherein the recombinant GKN1 protein is treated at a concentration of 1 to 10 ng / ml. The composition according to claim 12, wherein the GKN1 protein is treated at a concentration of 1 to 20 ng / ml. The composition according to claim 12, wherein the GKN1 protein is internalized in cancer cells by clathrin. The composition according to claim 12, wherein the GKN1 protein suppresses cell growth of cancer cells and induces cell death of cancer cells. A method for preventing or treating gastric cancer, which comprises the step of administering GKN1 protein (Gastrokine 1 Protein).