KR101988118B1 - Anti-cancer composition including GKN1 protein - Google Patents

Abstract

The present invention relates to a composition for the prophylaxis or treatment of gastric cancer, which comprises GKN1 protein (Exosomal Gastrokine-1 protein) as an active ingredient, wherein the GKN1 protein-containing exosome is secreted from cells expressing GKN1 protein, Is isolated from the culture supernatant of cells expressing the cancer cell, and is internalized as cancer cells by Clathrin to inhibit cell proliferation of cancer cells and induce apoptosis of cancer cells, thereby being useful for prevention or treatment of gastric cancer.

Description

Anti-cancer composition including GKN1 protein < RTI ID = 0.0 >

The present invention relates to a composition for the prophylaxis or treatment of gastric cancer containing GKN1 protein (Exosomal Gastrokine-1 protein) as an active ingredient.

Gastric cancer is a common cancer worldwide in Korea, China, and Japan. In the United States and Europe, the incidence rate is low. In Korea, cancer incidence is the first and stomach cancer is the second most common cancer. Gastric cancer is classified as an adenocarcinoma arising in the glandular cells of the mucous membrane of the stomach, and 95% of the gastric cancers are gastrointestinal stromal tumors arising from lymphoma and epilepsy.

Currently, three major treatment modalities for cancer are surgical treatment, pharmacotherapy, and radiotherapy. Pharmacotherapy is associated with less pain and is associated with recurrence of cancer after treatment compared to surgical or radiotherapy. This is relatively small, so expectations are gathering. Therefore, many anticancer drugs have been developed and used to cope with them. Most of these anticancer drugs exhibit anticancer effects by selectively killing cells that are actively dividing in consideration of abnormal growth of cancer. Such anticancer drugs have serious problems such as immune cells and hair follicle cells, which are actively dividing cells in the human body, and they are accompanied with serious side effects, so that they can not be used for a long time. Endoscopic mucosal resection has been used for the treatment of gastric cancer, including lymphadenectomy, endoscopic mucosal resection, and laparoscopic gastrectomy. Endoscopic mucosal resection has been used for the treatment of early gastric cancer in the stomach, This method is effective in avoiding the pain of gastrectomy due to the simple endoscopic procedure. However, if the possibility of lymph node metastasis is limited among mucosal-limited early gastric cancer, There is a limitation that it can be used.

In recent years, research on the function of genes related to the generation and treatment of cancer has been conducted worldwide, and researches have been conducted to find therapeutic targets and to use them for diagnosis and therapeutic drug development. With the activation of the genome research, studies on human gene DNA chip or proteome analysis have been actively conducted, and a large number of genes related to cancer have been discovered. Therefore, a list of many genes and related databases have been established, but most of these genes Biological function and cancer relatedness have not yet been studied or are uncertain. Therefore, there is considerable difficulty in finding genes that can effectively treat cancer, in addition to actual cancer-related or diagnosis and utilization as target genes. Therefore, in addition to the cancer-related genes identified so far, it is necessary to discover new genes.

Korean Patent No. 10-1215069

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of gastric cancer comprising GKN1 protein (Exosomal Gastrokine-1 protein) as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating gastric cancer, which comprises GKN1 protein (Exosomal Gastrokine-1 protein) as an active ingredient.

In one embodiment of the present invention, the GKN1 protein may be an exosome secreted from a cell expressing GKN1 protein.

In one embodiment of the present invention, the GKN1 protein may be isolated from the culture supernatant of cells expressing the GKN1 protein.

In one embodiment of the present invention, the GKN1 protein may be isolated from the culture supernatant of cells transfected with recombinant GKN1 (rGKN1: recombinant GKN1) protein.

In one embodiment of the present invention, the recombinant GKN1 protein may be treated at a concentration of 1 to 10 ng / ml, preferably at a concentration of 3 to 7 ng / ml, more preferably May be treated at a concentration of 5 ng / ml, but is not limited thereto.

 In one embodiment of the present invention, the GKN1 protein may be treated at a concentration of 1 to 20 ng / ml, preferably at a concentration of 5 to 15 ng / ml, 10 ng / ml. ≪ / RTI >

In one embodiment of the present invention, the GKN1 protein may be internalized into cancer cells by Clathrin.

In one embodiment of the present invention, the GKN1 protein may inhibit cell proliferation of cancer cells and induce apoptosis of cancer cells.

The GKN1 protein (Exosomal Gastrokine-1 protein) according to the present invention is secreted into the exosome in the GKN1 protein-expressing cell and isolated from the culture supernatant of the GKN1 protein-expressing cell, and is isolated by the Clathrin It can be used for prevention or treatment of gastric cancer by inhibiting cell proliferation of cancer cells and inducing apoptosis of cancer cells.

FIG. 1 shows the result of identification of GKN1 binding protein. (A) is a list of exosomal proteins showing binding to recombinant GKN1 protein in a protein microarray, (b) shows the binding ability of GKN1 to COMT and YWHAZ protein Immunoprecipitation method and Western blotting.
FIG. 2A shows the results of immunohistochemical staining for HFE-145 cells, exosomes isolated from AGS and MKN1 cells treated / untreated with recombinant GKN1 (rGKNl) protein, exosome-depleted and whole cell lysates (WCL) The results of Western blotting on GKN1, TSG-1 and CD81 are shown in FIG.
FIG. 2B is a graph showing the effect of the total medium, exosome and exosome-removed medium (Soluble) in HFE-145 cells after diluting buffer solution in the ELISA kit for measuring the concentration of GKN1 protein, The results are shown in FIG.
FIG. 2C shows the result of trypsin digestion assay by treatment / non-treatment of trypsin and Triton X-100 on exosome derived from HFE-145 cells.
FIG. 3A shows the results of treatment of HFE-145, AGS and MKN1 cells with MKN1 (recombinant GKN1 treated / untreated), AGS (recombinant GKN1 treated / untreated) and HFE- The results are shown in Fig.
FIG. 3B shows the results of treatment of HFE-145, AGS and MKN1 cells with MKN1 (recombinant GKN1 treated / untreated), AGS (recombinant GKN1 treated / untreated) and HFE-145 cell derived exosomes, followed by BrdU Incorporation Assay The results are shown in Fig.
FIG. 3c shows the results of confirming cell viability and cell proliferation activity over time through MTT and BrdU Incorporation assay after treating exosome carrying Gastrokine-1 protein containing recombinant GKN1 (rGKN1) or GKN1 protein .
FIG. 3D shows the results of cell cycle analysis after treatment (rGKN1_exosome) / non-treatment (w / o GKN1_exosome) of recombinant GKN1 with AGS and MKN1 cells.
Figure 3e shows the expression of p53, p21, CDK4, CyclinD, Cdc25c, p-Cdc2, Cyclin B, BAX, BCL2 in HFE-145, AGS (recombinant GKNl treated / untreated) and MKNl (recombinant GKNl treated / , Caspase-3 (including cleaved) and Caspase-8 (including cleaved) by Western blotting.
FIG. 3f shows the result of treating AGS and MKN1 cells with exosomes containing GKN1 protein derived from treated / non-treated cells and confirming apoptosis through cell death assay.
FIG. 4A shows the result of isolating exosome after treating recombinant GKN1 protein with AGS cells.
FIG. 4B shows Western blotting results of inhibiting the expression of Clathrin protein in AGS and MKN1 cells treated with siClathrin.
Figure 4c shows the results of localization of exosomes containing GKN1 protein in AGS and MKNl cells treated with siClathrin.
FIG. 4d shows the results of Western blotting of the expression levels of GKN1 and CD81 in siClathrin-treated AGS and MKN1 cells.

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of gastric cancer containing exosomal GKN1 protein (Exosomal Gastrokine-1 protein) as an active ingredient.

The term "prevention" of the present invention means all actions that inhibit or delay 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 the actions of improving or alleviating symptoms by gastric cancer by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical compositions according to the present invention may comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. In addition, it can be formulated into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders, lubricants and the like. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, subcutaneously, nasally, or intracavitally) depending on the intended method, and the dosage may vary depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal 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 present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.001 to 150 mg, preferably 0.01 to 100 mg, One to three doses may be administered. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for further illustrating 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

A recombinant vector was prepared as follows to prepare recombinant GKN1 protein. First, the human GKN1 cDNA was amplified by the PCR method and a recombinant vector pCMV6-AN-FC-hGKN1 was prepared using a restriction enzyme site in pCMV6-AN-FC (Origene ID PS100055) vector. More specifically, pCMV6-AN-FC vector and GKN1 cDNA were treated with restriction enzymes SgfI and MluI, and pCMV6-AN-FC and hGKN1 cDNA were ligated to introduce the GKN1 gene into the vector. Thereafter, E. coli was transformed to colonies formed on agar plates, followed by culturing and extracting DNA, followed by sequencing to confirm whether the GKNI gene was correctly introduced. Then, recombinant GKN1 protein was purified using protein A affinity chromatography, and the recombinant GKN1 protein was identified by Western blotting. Thereafter, the sequence of the GKNI gene is shown in SEQ ID NO: 1, and the amino acid sequence of the GKN1 protein encoded thereby is represented by 2.

1.2. Cell culture

AGS and MKN1 gastric cancer cell lines that do not express the GKN1 (Gastrokine-1) protein and HFE-145 immortalized gastric epithelial cells that express GKN1 were cultured in 10% heat-inactivated FBS fetal bovine serum) in RPMI-1640 medium at 37 ° C and 5% CO ₂ .

1.3. Identification of GKN1 binding protein through HuProt (TM) microarray

Human Proteome Microarray (CDI Labs, USA) containing over 19,000 full-length recombinant human proteins was used to identify GKN1 binding proteins. Briefly, the protein microarray was treated with 1 μg of biotinylated GKN1 at 4 ° C for 8 h after 2 h of blocking buffer (5% BSA in PBS with 0.05% tween-20). The microarray was then treated with 1 μg of streptavidin-fluorescence (Alexa-Fluor 635 nm). Results were confirmed with a 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 supernatants of HFE-145, AGS and MKN1 cells. Briefly, cells from passage 3 to 8 were cultured in serum-free medium and cultured at 37 ° C, 5% CO 2 with 10% FBS and 1% penicillin / streptomycin for 48 hours before collecting the medium. The conditioned media was centrifuged at 2000 g for 10 min at 4 ° C to remove cell debris and then passed through a 0.22 μm filter. The filtered supernatant was transferred to a new glass tube and placed on ice. Then, 2 ml of supernatant was mixed with 0.75 ml of A / B / C solution (101Bio company, CA, USA) in a new glass tube, vortexed vigorously for 30 seconds and left at 4 ° C for 30 minutes. The mixture was separated into two layers and the upper layer was removed. The lower layer was transferred to a microcentrifuge tube and centrifuged at 5000 g for 3 minutes. The intermediate layer was transferred to a new microcentrifuge tube and centrifuged at 5000 g for 3 minutes. Lt; / RTI > for 10 minutes. A total volume of 4X 1X PBS was added to the tube and strongly pipetted. Tubes were treated at high speed for 15 minutes in a horizontal shaker and centrifuged at 5000 g for 5 minutes. The supernatant was carefully transferred onto a PureExo Column (101Bio company, CA, USA) and centrifuged at 1000 g for 5 min. The passage liquid contains a separate exosomal fraction suspended in PBS.

1.5. Exosome Labeling

Exosomes were labeled with PKH26 (Sigma, St. Louis, USA) according to the manufacturer's protocol (slight modification). Briefly, the exosomal pellet was suspended in 1 ml of Diluent C, and 1 ml of Diluent C was mixed with 4 μl of PKH26 to make a Stain Solution. The exosomal suspension was mixed with the stain solution for 4 minutes, and the labeling reaction was stopped when the same amount of 1% BSA (bovine serum albumin) was added. Labeled exosomes were isolated using the Total Exosome Isolation Kit (Invitrogen) according to the manufacturer's protocol. Briefly, a 0.5 volume of Isolation Reagent was added to the labeled exosomes and mixed. Labeled exosomes were kept at 4 ° C overnight and subsequently centrifuged at 10,000 g for 1 hour at 4 ° C. Thereafter, the supernatant was discarded and each pellet was suspended in 100 μl of PBS.

1.6. Expression of GKN1 protein in exosomes

Expression of GKN1 in cell lysate and exosomes of HFE-145, AGS and MKN1 cells was analyzed by Western blotting. Equivalent cell lysates and exosomes were separated by 12.5% SDS-PAGE and transferred to Hybond-PVDF (polyvinylidene difluoride) transfer membranes (Amersham). After blocking with 0.5% skim milk, the membranes were reacted with primary anti-GKN1 (Abcam, MA, USA), anti-TSG101 and anti-CD81 antibodies (Santa Cruz Biotechnology, TX, USA) peroxidase-conjugated secondary antibodies. Protein bands were detected on Westernsure ECL substrate (LI-COR Biosciences, NE, USA) and bands were visualized on LAS 4000 (Fuji Film, Japan).

1.7. Co-immunoprecipitation (co-immunoprecipitation)

AGS and MKN1 cell-derived exosomes treated with recombinant GKN1 (rGKNl) were washed with PBS and incubated with 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1.0 mM NaVO4 (J. Biol. Chem. 2010; 285: 20547-57) with PBS (pH 7.2) containing 1% protease inhibitor cocktail (Sigma, St. Louis, Mo., USA). As described previously, the same amount of protein aliquat (1.0 mg) was added to 2.0 μg of Protein A / G-protein coupled antibody to GKN1 (Abcam, Cambridge, UK), COMT and YWHAZ (Genetex, CA, USA) agarose (Santa Cruz Biotechnology) (EBioMedicine. 2016; 9: 97-109).

1.8. Trypsin digestion assay

Exosomes isolated from HFE-145 cells (35 μg protein in 7 μl of PBS) as previously described were incubated with 1% Triton X-100 for 30 min at 4 ° C. to find the presence of GKN1 protein in the exosome Lipid membrane was degraded (Blood. 2009; 113: 1957-66). Exosomes were incubated under the same conditions without Triton X-100. The exosomal suspension was digested with trypsin at 37 ° C with two enzyme concentrations (15 and 0.01 μg / mL). Triton X-100 treatment had little effect on trypsin activity (data not shown). After the trypsin treatment, the samples were immediately mixed with an equal volume of Laemmli buffer and boiled for 3 minutes before gel loading.

1.9. Measurement of cell viability and death

For cell survival analysis, each cell was treated with exosomes derived from AGS and MKN1 cells treated / untreated with HFE-145 cells and 5 ng recombinant GKNl (rGKNl), and then treated at 24, 48 and 72 hours with HFE -145, MTT assays were performed on AGS and MKN1 cells. Absorbance was measured using a spectrophotometer at 540 nm and cell viability was expressed relative to non-treated cells.

For cell proliferation assays, each cell was treated with exosomes derived from HFE-145, AGS and MKN1 cells treated with 5 ng recombinant GKN1 (rGKNl), and then stained with BrdU Cell Proliferation Assay Kit (Millipore, Billerica, MA, USA) at 24, 48, and 72 hours according to the manufacturer's protocol. Absorbance was measured using a spectrophotometer at 450 nm and the cell proliferation rate was expressed relative to non-treated cells. Exogenous GKN1 protein (10 ng / ml) or recombinant GKN1 (rGKN1) protein (10 ng / ml) was used to compare cell survival and cell proliferation of exogenous GKN1 protein and recombinant GKN1 ml), and cell viability and cell proliferative capacity of AGS and MKN1 cells were analyzed.

For the cell death assay, 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 DNA on 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 of binding buffer. In each case, 100 μl of the supernatant was mixed with 400 μl of blocking solution and 5 μl of annexin V-FITC (1 μg / ml) and 5 μl of PI (2 μg / ml) Cells were analyzed by FACS (fluorescence-activated cell sorter, BD Biosciences, San Jose, Calif., USA). Only PI-untreated fluorescence-positive cells were considered dead cells.

1.10. Cell cycle measurement

For cell cycle analysis, exosomes isolated from AGS cells treated with 5 ng recombinant GKNl (rGKNl) (rGKNl) were treated with AGS and MKNl cells and then harvested and analyzed for PI propidium iodide). The percentage of cells in each phase of the cell cycle was determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software (BD Biosciences, Heidelberg, Germany). The experiment was repeated twice.

1.11. Expression of cell cycle regulators

To determine whether exosomal GKN1 protein is involved in the regulation of cell cycle and apoptosis, exosomes derived from AGS and MKN1 cells treated with HFE-145 cells and recombinant GKN1 (rGKN1) were treated and cultured for 48 hours Expression of p53, p21, CDK4, cyclin D1, Cdc25, p-Cdc2, BAX, BCL2, Caspase-3 and 8 proteins in HFE-145, AGS and MKN1 cells was analyzed. Cell lysates were separated on a 10% polyacrylamide gel and transferred to a Hybond PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). After blocking, the membrane was sequentially labeled with antibodies to G0 / G1 phase proteins. Protein bands were identified using ECL (enhanced chemiluminescence reagents, Amersham Pharmacia Biotech, Piscataway, NJ, USA).

1.12. Immunofluorescence studies

Mouse anti-FITC conjugated GKN1 (Genetex), anti-clathrin (Abcam) and Texas-red conjugated goat anti-mouse IgG (invitrogen) secondary antibodies were used for the immune response. The specificity of the reaction was tested with Non-immune Mouse Serum (Invitrogen).

1.13. Statistical analysis

Student's t-test was used to analyze the effect of GKN1 on cell viability and cell proliferation. All experiments were repeated two or more times to demonstrate reproducibility. Data were expressed as mean ± SD (standard deviation) from two independent experiments, and a P value of less than 0.05 was considered statistically significant.

Example 2. Identification of GKN1 binding protein

The present inventors performed a protein microarray assay to identify the protein binding to GKN1. As a result, 27 exosome proteins with normal to strong binding ability to recombinant GKN1 (rGKN1) were found (Fig. 1A). To confirm the binding ability of GKN1 to exo-somatic protein, the present inventors extracted exo-somatic protein from AGS and MKN1 cells treated with HFE-145 cells and recombinant GKN1 (rGKN1), and co- The interaction between GKN1 and COMT in the protein and the interaction between GKN1 and YWHAZ was demonstrated (Fig. 1B).

Example 3 Detection of GKN1 Protein from Exocytosis from Stomach Cell Lines

From the fact that the recombinant GKN1 (rGKN1) protein has binding capacity to exosomal proteins, the inventors of the present invention found that the culture supernatant of HFE-145, AGS and MKN1 cells in order to investigate whether GKN1 is naturally secreted and internalized into exosome protein To confirm the presence or absence of GKN1. As a result, it was confirmed that GKN1 protein was contained in exosome derived from HFE-145 cell, but GKN1 protein was not expressed in exosome derived from AGS and MKN1 gastric cancer cell line (Fig. 2a). TSG101, an exosomal marker, was identified in isolated exosomes and cell lysates, but CD81 was only present in exosomes and CD81 was used as an exosomal marker in further experiments (FIG. 2a). In addition, when recombinant GKN1 (rGKN1) protein was treated on AGS and MKN1 cells, most of the GKN1 protein was present in the exosome (Fig. 2a). Consistent with this, the concentrations of GKN1 protein in the whole culture medium, exosome fraction and exosome-removed medium in HFE-145 cells were 1.13 ± 0.04, 1.08 ± 0.07 and 0.05 ± 0.03 ng / ml, respectively (Figure 2b) .

Next, trypsin digestion assays by treatment / non-treatment of trypsin and Triton X-100 were performed to confirm the presence of GKN1 protein in exosomes, and then Western blotting was performed (Blood. 2009; 113 : 1957-66). GKN1 did not undergo trypsin-dependent enzymatic degradation when Triton X-100 was untreated and only trypsin was treated with exosomes. However, when both Triton X-100 and trypsin were treated, GKN1 was completely degraded (FIG. 2C). From the above results, it was confirmed that GKN1 protein is present in the exosome.

Example 4. Measurement of cell proliferation and apoptosis

The present inventors performed MTT assay and BrdU Incorporation Assay to confirm cell survival and proliferative effect of exosome with GKN1 protein. Exosomes derived from HFE-145, AGS (recombinant GKN1 treated / untreated) and MKN1 (recombinant GKN1 treated / untreated) cells were treated with HFE-145, AGS and MKN1 cells, respectively, and cell viability and cell proliferation Respectively.

As a result, when the GKN1-negative exosome (recombinant GKN1 untreated group) derived from AGS and MKN1 cells was treated, the cell survival rate was significantly increased, but the GKN1 protein derived from HFE-145, AGS and MKN1 cells (Group treated with recombinant GKN1) significantly inhibited cell viability and cell proliferation in AGS and MKN1 cells (FIGS. 3A and 3B).

Next, the present inventors conducted experiments comparing the cell survival rate of AGS and MKN1 cells over time after treatment of GKN1 protein and recombinant GKN1 (rGKN1) protein of exosomes. As a result, it was confirmed that treatment of exocase GKN1 protein and recombinant GKN1 (rGKN1) protein in the cells suppressed cell survival rate and cell proliferation in a time-dependent manner. In particular, when GKN1 protein of exosomes was treated, recombinant GKN1 (rGKNl) protein-treated cells (Fig. 3 (c)).

In addition, the inventors performed an experiment to analyze cell cycle after treatment of recombinant GKN1 (rGKN1) protein with AGS and MKN1 cells (rGKN1_exosome) / non-treatment (w / o GKN1_exosome). As a result, when treated with recombinant GKN1 (rGKN1) in AGS and MKN1 cells and treated with exogenous GKN1 protein-containing exosomes (GKN-positive exosome), the cells in S phase in AGS and MKN1 cells were significantly , And the G1 and G2 / M phase cells were increased (Fig. 3d).

In addition, the present inventors conducted an experiment to confirm expression of a cell cycle regulator that affects G1 or G2 / M phase arrest by GKN1 protein of exosome. As a result, it was confirmed that GKN1 protein of exosomes increased expression of negative cell cycle regulators such as p53 and p21 for G1 phase arrest and decreased expression of positive cell cycle regulators such as cdk4 and cyclin D (FIG. 3E ). In addition, for G2 / M arrest, exosomal GKN1 protein down-regulated the expression of p-cdc2, cdc25c and cyclin B proteins in HFE-145, AGS and MKN1 cells (Fig.

Next, the present inventors treated AGS and MKN1 cells with exosomes containing GKN1 protein derived from recombinant GKN1 treated / non-treated cells, and then performed cell death assays. Cell death was measured by cells stained with FITC-labeled Annexin V. Exosome treatment with GKN1 protein derived from recombinant GKN1 (rGKN1) -treated AGS cells markedly increased cell death in AGS and MKN1 cells (Fig. 3F). Western blotting revealed that the expression level of cleaved caspase-3 and -8 was significantly increased, while that of BAX and BCL2 was not changed (Fig. 3e).

Therefore, it was confirmed that GKN1 protein of exosomes is internalized by gastric cancer cells and interacts with cytoplasmic proteins, thereby inhibiting cell proliferation and inducing apoptosis, thereby treating gastric cancer.

Example 5. Clathrin-dependent internalization of exosomes containing GKN1 protein

The present inventors conducted an experiment to confirm whether Clathrin was involved in the internalization of GKN1 protein of exosomes. In order to knock down Clathrin, siRNA, siClathrin, was used. Exosomes containing siClathrin and GKN1 proteins were treated with AGS and MKN1 cells. To confirm the expression of GKN1 protein in these cells, immunofluorescence assay Blotting was performed.

First, the exosomes were clearly internalized in the cytoplasm of AGS and MKNl when PKH26-positive exosomes containing GKN1 protein were treated on AGS and MKN1 cells (FIGS. 4A and 4C). However, the knockdown of clathrin in AGS and MKN1 cells significantly inhibited the presence of PKH26-positive exosome in the cell membrane and cytoplasm (FIGS. 4B-4C). In addition, it was confirmed that the expression of GKN1 protein in the cell membrane and cytoplasm was significantly inhibited by the clathrin synthase caused by siClathrin (FIG. 4d).

Therefore, Clathrin plays an important role in attaching and internalizing exocose containing GKN1 protein to stomach cells.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Anti-cancer composition including GKN1 protein <130> PN1705-172 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 600 <212> DNA <213> Artificial Sequence <220> <223> cDNA sequence of GKN1 <400> 1 atgcttgcct actcctctgt ccactgcttt cgtgaagaca agatgaagtt cacaattgtc 60 tttgctggac ttcttggagt ctttctagct cctgccctag ctaactataa tatcaacgtc 120 aatgatgaca acaacaatgc tggaagtggg cagcagtcag tgagtgtcaa caatgaacac 180 aatgtggcca atgttgacaa taacaacgga tgggactcct ggaattccat ctgggattat 240 ggaaatggct ttgctgcaac cagactcttt caaaagaaga catgcattgt gcacaaaatg 300 aacaaggaag tcatgccctc cattcaatcc cttgatgcac tggtcaagga aaagaagctt 360 cagggtaagg gaccaggagg accacctccc aagggcctga tgtactcagt caacccaaac 420 aaagtcgatg acctgagcaa gttcggaaaa aacattgcaa acatgtgtcg tgggattcca 480 acatacatgg ctgaggagat gcaagaggca agcctgtttt tttactcagg aacgtgctac 540 acgaccagtg tactatggat tgtggacatt tccttctgtg gagacacggt ggagaactaa 600                                                                          600 <210> 2 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of GKN1 <400> 2 Met Leu Ala Tyr Ser Ser Val His Cys Phe Arg Glu Asp Lys Met Lys   1 5 10 15 Phe Thr Ile Val Phe Ala Gly Leu Leu Gly Val Phe Leu Ala Pro Ala              20 25 30 Leu Ala Asn Tyr Asn Ile Asn Val Asn Asp Asp Asn Asn Asn Ala Gly          35 40 45 Ser Gly Gln Gln Ser Val Ser Val Asn Asn Glu His Asn Val Ala Asn      50 55 60 Val Asp Asn Asn Asn Gly Trp Asp Ser Trp Asn Ser Ile Trp Asp Tyr  65 70 75 80 Gly Asn Gly Phe Ala Ala Thr Arg Leu Phe Gln Lys Lys Thr Cys Ile                  85 90 95 Val His Lys Met Asn Lys Glu Val Met Pro Ser Ile Gln Ser Leu Asp             100 105 110 Ala Leu Val Lys Glu Lys Lys Leu Gln Gly Lys Gly Pro Gly Gly Pro         115 120 125 Pro Pro Lys Gly Leu Met Tyr Ser Val Asn Pro Asn Lys Val Asp Asp     130 135 140 Leu Ser Lys Phe Gly Lys Asn Ile Ala Asn Met Cys Arg Gly Ile Pro 145 150 155 160 Thr Tyr Met Ala Glu Glu Met Gln Glu Ala Ser Leu Phe Phe Tyr Ser                 165 170 175 Gly Thr Cys Tyr Thr Thr Ser Val Leu Trp Ile Val Asp Ile Ser Phe             180 185 190 Cys Gly Asp Thr Val Glu Asn         195

Claims (8)

A pharmaceutical composition for the prophylaxis or treatment of gastric cancer, comprising an exosome having the GKN1 protein (Exosomal Gastrokine-1 protein) as an active ingredient,
Wherein the exosome in which the GKN1 protein is present is isolated from a culture supernatant of a cell expressing the GKN1 protein. delete delete The method according to claim 1,
Wherein the exosome in which the GKN1 protein is implanted is isolated from the culture supernatant of cells after expressing GKN1 protein by introducing recombinant GKN1 (rGKN1: recombinant GKN1) protein into cells that do not express GKN1 protein. 5. The method of claim 4,
Wherein the recombinant GKN1 protein is treated at a concentration of 1 to 10 ng / ml. The method according to claim 1,
Wherein the exosome in which the GKN1 protein is present is treated at a concentration of 1 to 20 ng / ml. The method according to claim 1,
Wherein the exosome in which the GKN1 protein is implanted is internalized by cancer cells by Clathrin. The method according to claim 1,
Wherein the exosome having the GKN1 protein inhibits cell proliferation of cancer cells and induces apoptosis of cancer cells.

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