KR20200076660A - Anticarcinogenic composition comprising an inhibitor of DRG2 as an active ingredient - Google Patents

Abstract

The present invention relates to an anticancer composition including a DRG2 inhibitor as an active ingredient. According to the present invention, the inhibitor against expression of developmentally regulated GTP-binding protein 2 (DRG2) genes or the DRG2 activity inhibitor can effectively inhibit transition of cancer cells through a mechanism of inhibiting cell migration and suppressing Rac1 activity and expression of VE-cadherin protein. Therefore, the inhibitor against expression of developmentally regulated GTP-binding protein 2 (DRG2) genes or the DRG2 activity inhibitor can be used advantageously for an anticancer composition capable of interrupting metastasis.

Description

{Anticarcinogenic composition comprising an inhibitor of DRG2 as an active ingredient}

The present invention relates to an anti-cancer composition comprising a DRG2 inhibitor as an active ingredient, and more particularly, to a pharmaceutical composition comprising a DRG2 inhibitor as an active ingredient that can prevent or treat cancer disease by inhibiting metastasis of cancer. .

Cancer is one of the major risks to human life. In Korea, cancer has been the number one cause of death in the past few years, and in the United States, cancer has been reported to be the second leading cause of death following cardiovascular disease. Therefore, although numerous studies are underway to conquer cancer, millions of people die of cancer each year to this day, and the economic loss resulting from this is also astronomical.

Cancer is known as a genetic disease caused by mutations in genes such as oncogenes and tumor suppressor genes, and is a disease particularly caused at the cell level. Surgical surgery, drug therapy, radiation therapy, and immunotherapy are currently used as treatments for cancer, but the suppression and recurrence of malignant tumors are still unresolved.

One of the most important biological properties of cancer is that it can cause metastasis, which is considered the biggest obstacle to treating cancer. In fact, about 60% of all patients with solid tumors already have microscopically metastasized cancer at the time of primary tumor diagnosis, and it is well known that cancer metastasis is a major cause of death in most cancer patients.

That is, the cause of death in cancer patients is mainly due to metastasis of cancer cells, rather than due to initial tumors. Metastasis of these cancer cells is first caused by metastatic cancer cells detached from the initial tumor and invasion of extracellular matrix (ECM) such as interstitial stroma and basal membrane (BM) of normal tissues, thereby invasion of blood vessels. After entering into the lymphatic vessel, it attaches to the capillary blood vessel wall of other target tissues, leaches from the capillaries through the cell's matrix and basement membrane, and proliferates in new tissue to form secondary tumors (metastatic cancer).

Until now, expectations for the development of a substance having an inhibitory effect on cancer metastasis are very high, but there are very few examples of the development of a substance aimed at suppressing cancer metastasis. It has been reported that sulfated polysaccharides, N-diazoacetylglycine derivatives, noiraminitases, and fibronectin degrading enzymes (FNS) have such inhibitory action, but there are no reports of their practical use, and they themselves have cancer metastasis inhibitory effects. There is no report. Therefore, there is an urgent need to develop a therapeutic agent that can effectively suppress cancer metastasis.

Accordingly, the present inventors completed the present invention by confirming that metastasis of cancer cells is significantly inhibited when DRG2 expression is suppressed as a result of conducting research with interest in a novel substance capable of effectively inhibiting cancer metastasis.

1.Overexpression of DRG2 suppresses the growth of Jurkat T cells but does not induce apoptosis. Arch Biochem Biophys. 2004 Feb 15;422(2):137-44.

Accordingly, an object of the present invention is to provide a novel anti-cancer composition that can effectively suppress cancer metastasis.

Another object of the present invention is to provide a novel method for screening for metastasis inhibitors of cancer.

Furthermore, another object of the present invention is to provide a novel method for diagnosing cancer in an individual.

In order to achieve the above object of the present invention, the present invention provides an anti-cancer composition comprising an inhibitor of expression of a DRG2 (Developmentally regulated GTP-binding protein 2) gene or an inhibitor of activity of a DRG2 protein as an active ingredient.

In one embodiment of the present invention, the DRG2 (Developmentally regulated GTP-binding protein 2) gene may be composed of the nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the expression inhibitor of the developmentally regulated GTP-binding protein 2 (DRG2) gene is from a group consisting of antisense nucleotides, siRNA sh RNA and ribozymes for the gene represented by SEQ ID NO: 1. It may be any one selected.

In one embodiment of the present invention, the activity inhibitor of the DRG2 protein is any one selected from the group consisting of compounds, peptides, peptide mimetics, substrate mimetics, aptamers and antibodies that complementarily bind to the DRG2 protein. It can be one.

In one embodiment of the present invention, the composition may have an activity that inhibits metastasis of cancer cells.

In one embodiment of the present invention, the composition may be to inhibit the metastasis of cancer cells by inhibiting the activity of Rac1.

In one embodiment of the present invention, the composition may be to inhibit the metastasis of cancer cells by inhibiting the VE-cadherin protein expression of the cancer cells.

In one embodiment of the present invention, the cancer is lung cancer, stomach cancer, colon cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anus near rectum cancer, colon cancer , Breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate One or more selected from the group consisting of cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urinary tract cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma It may be a solid cancer.

In addition, the present invention comprises the steps of treating the test substance in the DRG2 protein expressing cell line; Measuring the expression level of DRG2 protein in the cell line; And when the expression level of the DRG2 protein is reduced compared to a control cell line that is not treated with the test substance, determining the test substance as an anti-cancer substance.

In one embodiment of the present invention, the expression level of the DRG2 protein is immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, western blotting ) And flow cytometry (FACS).

In addition, the present invention comprises the steps of treating the test substance to the DRG2 protein; Measuring the activity level of the DRG2 protein; And when the activity level of the DRG2 protein is reduced compared to a control cell line that is not treated with the test substance, determining the test substance as an anti-cancer substance.

In one embodiment of the present invention, the activity level of the DRG2 protein may be measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzymatic immunoassay (ELISA), mass spectrometry and protein chip.

In addition, the present invention comprises the steps of measuring the expression level of DRG2 protein in a sample derived from a subject; And determining a subject having an increased expression level of DRG2 protein than a control group as an individual at risk of developing cancer.

DRG2 (Developmentally regulated GTP-binding protein 2) gene expression inhibitor or DRG2 protein activity inhibitor according to the present invention inhibits cell migration, inhibits Rac 1 activity, and inhibits VE-cadherin protein expression Since the metastasis of cancer cells can be effectively suppressed, an inhibitor of the expression of the DRG2 (Developmentally regulated GTP-binding protein 2) gene or an inhibitor of the activity of the DRG2 protein will be useful as an anticancer composition that can block the metastasis of cancer.

1 is an experimental result of inducing DRG2 overexpression in Jurkat T cells (A), and thus observing cell growth (B), cell cycle (C), and cell death (D).
2 is a result of observing DRG2 expression inhibition (B) and cell migration inhibition (C) in the MCF-7 cell line transfected with shDRG2.
3 is an experimental result of observing the Rac1 activation level (A), VE-cadherin mRNA expression level (B), and VE-cadherin protein expression level (C, D) in the B16F10/scRNA and B16F10/shDRG2 cell lines.
4 is an experimental result of observing mouse survival rate (A), number of lung metastasized tumors (B), and degree of lung metastasis (C) after B16F10/scRNA or B16F10/shDRG2 treatment in a mouse animal model.

Definitions of terms used in the present invention are as follows.

“Subject” or “patient” means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. In addition, the subject includes any subject who participated in a clinical research trial that did not show any disease clinical manifestations, or a subject who participated in epidemiological studies or a control group. In one embodiment of the present invention, a mouse was targeted.

"Tissue or cell sample" means a collection of similar cells obtained from a subject or patient's tissue. Sources of tissue or cell samples may include fresh, frozen and/or preserved organ or tissue samples or solid tissue from biopsies or aspirates; Blood or any blood component; It can be a cell at any time in the subject's pregnancy or development. The tissue sample can also be primary or cultured cells or cell lines.

Optionally, tissue or cell samples are obtained from primary or metastatic tumors. Tissue samples may include compounds that do not mix with tissue due to their properties, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. For the purposes of the present invention, “slice” of a tissue sample refers to one portion or piece of a tissue sample, for example a thin slice of tissue or cell cut from a tissue sample. If the present invention understands that the same fragments of tissue samples include methods that are analyzed at both morphological and molecular levels or for both proteins and nucleic acids, then multiple sections of tissue samples are taken and applied to the analysis according to the invention It is understood that it can be done.

The term "growth" of cells refers to an increase in the number of cells in the body of a multicellular organism as the cells are divided and the homogeneous ones expand. When the cell number multiplies (amplifies) and reaches a certain time, the trait changes. It is usually controlled at the same time as it is (differentiated), and the cells are increased in the body, and when the cytoplasm is newly formed in the cell, it is often distinguished by growth. In view of the increase, it is justified to consider proliferation or growth as the period when the differentiation does not occur in the generation phase of a multicellular organism, and the two terms are used interchangeably in the present invention.

“Cancer”, “tumor” or “malignant” generally refers to or describes the physiological condition of a mammal with characteristics of unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, melanoma, and sarcoma. In the present invention, melanoma is typically treated.

“Inhibitor or inhibitor” means a substance that inhibits, blocks or reduces the activity or expression of the substance. The mechanism of action of the inhibitor is not particularly limited. Examples include organic or inorganic compounds, polymers such as proteins, carbohydrates, lipids, and composites of various compounds. For example,'lymph node metastasis inhibitors' may include substances that inhibit, block or reduce lymph node metastasis of a tumor.

"Prevention" means that the disease or occurrence of a disease is suppressed in an animal that has never been diagnosed as having a disease or disease, but is prone to such disease or disease.

“Treatment” means (a) inhibition of a disease or development of disease; (b) disease or relief of disease; And (c) disease or elimination of disease. Thus, the term therapeutically effective amount in this specification means an amount sufficient to achieve the above pharmacological effect.

All technical terms used in the present invention, unless defined otherwise, are used in the sense as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although a preferred method or sample is described herein, similar or equivalent ones are included in the scope of the present invention. The contents of all publications described by reference herein are incorporated into the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for treating cancer or inhibiting cancer metastasis, which contains an inhibitor of expression or activity of a DRG2 gene or protein as an active ingredient.

DRG2 is a type of G protein and has all five regions of G1 to G5 required for GTP binding. The size is similar to that of the trimeric G protein, but the amino acid sequence is different from the existing G protein subfamily and is classified as a new subfamily. DRG is known to have DRG1 and DRG2, and is present in almost all kinds of cells, from bacteria to humans. On the other hand, all DRG proteins found in humans from bacteria have been found to be highly conserved evolutionarily, and human DRG2 amino acid sequences are DRG2 and 98% in mice and cows, DRG2 and 95% in Xenopus, and DRG2 and 91 in zebrafish. % Homology

In the present invention, the DRG2 gene has a nucleic acid sequence represented by SEQ ID NO: 1, and the DRG2 protein preferably has an amino acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

The cancer is lung cancer, stomach cancer, colorectal cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, rectal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma , Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma , Bladder cancer, kidney or urinary tract cancer, kidney cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, preferably one or more solid cancers, breast cancer or black It is more preferably a species, but is not limited thereto.

The DRG2 protein expression inhibitor is preferably one or more selected from the group consisting of antisense nucleotides, small hairpin RNA (shRNA), small interfering RNA (siRNA), and ribozymes (combined with mRNA) of the DRG2 gene. In addition, the DRG2 protein activity inhibitor is preferably one selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that complementarily bind to the DRG2 protein, but is not limited thereto. .

The antisense nucleotide binds (hybridizes) to the complementary sequence of DNA, immature-mRNA or mature mRNA, as defined by Watson-Crick base pairing, thereby interfering with the flow of genetic information as a protein in DNA. Since antisense nucleotides are long chains of monomer units, they can be easily synthesized for target RNA sequences.

The shRNA is a small hairpin structure RNA that interferes with RNA to suppress gene expression. The shRNA can be introduced into cancer cells using various vectors, but according to a preferred embodiment of the present invention, the pLK0.1 vector is used. It could be introduced into cancer cells. In this case, the shRNA sequence is not particularly limited, but is preferably SEQ ID NO: 3.

The siRNA comprises, but is not limited to, a 15 to 30 mer sense sequence selected from the base sequence of the mRNA of the gene encoding the human DRG2 protein and an antisense sequence that repeatedly binds to the sense sequence. .

The peptide minetics (Peptide Minetics) is to inhibit the activity of the protein by inhibiting a specific domain of the DRG2 protein. Peptide mimetics can be peptides or non-peptides, and can be composed of amino acids bound by non-peptide bonds, such as psi bonds. In addition, "conformationally constrained" peptides, cyclic mimetics, cyclics comprising at least one exocyclic domain, binding moiety (binding amino acid) and active site It can be mimetics. Peptide mimetics can be novel small molecules that are structured similar to the secondary structure properties of the DRG2 protein and can mimic the inhibitory properties of large molecules, such as antibodies or water-soluble receptors, and can act with the same effects as natural antagonists.

The aptamer is a single chain DNA or RNA molecule, and has a high affinity for a specific chemical or biological molecule by an evolutionary method using an oligonucleotide library called systemic evolution of ligands by exponential enrichment (SELEX). It can be obtained by separating the oligomers that bind with and selectivity. Aptamers can specifically bind to targets and modulate the activity of the target, for example, binding can block the ability of the target to function.

The antibody can specifically and directly bind to DRG2 and effectively inhibit the activity of DRG2. It is preferable to use a polyclonal antibody or a monoclonal antibody as the antibody specifically binding to DRG2. The antibody specifically binding to DRG2 may be prepared by a known method known to those skilled in the art, and a commercially known DRG2 antibody may be purchased and used. The antibody can be prepared by injecting a DRG2 protein that is an immunogen into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. Immunogens are injected by intramuscular, intraperitoneal or subcutaneous injection, and can generally be administered with adjuvants to increase antigenicity. Antibodies can be isolated by collecting blood from the external host regularly and collecting serum showing specific titer and specificity for antigen.

The composition may have an inhibitory activity of cancer proliferation, migration, infiltration or metastasis.

The present inventors tested the anti-cancer effect of DRG2 on the basis of the experimental results (see FIG. 1) that DRG2 is closely related to cell growth, cell cycle, and cell death. To this end, the MCF-7 cell line, a breast cancer cell line, was transfected with shRNA capable of inhibiting the expression of DRG2 and observed cell migration. As a result, it was confirmed that cell migration was significantly reduced in the cell line that suppressed DRG2 expression (FIG. 2 ). Reference).

On the other hand, as a result of inhibiting DRG2 expression in the melanoma cell line B16F10 cell line, it was confirmed that the activity of Rac1, which is known to play an important role in cell migration, is inhibited and the expression of VE-cadherin protein, which is important for cancer cell metastasis, is inhibited. There was (see Figure 3).

Furthermore, in the mouse animal model in which the melanoma cell line in which DRG2 expression was suppressed was injected, it was confirmed that the mouse survival rate was significantly increased and the lung metastasis of the melanoma cell line was significantly decreased compared to the control group (see FIG. 4 ).

Therefore, it was found that suppression of DRG2 expression or activity inhibits cancer cell proliferation and suppresses cancer metastasis. Therefore, the DRG2 expression or activity inhibitor may be usefully used as an active ingredient in a cancer treatment or a pharmaceutical composition for inhibiting cancer metastasis.

The pharmaceutical composition containing the inhibitor of expression or activity of the DRG2 protein of the present invention as an active ingredient, it is preferable to include the active ingredient in an amount of 0.0001 to 50% by weight based on the total weight of the composition, but is not limited thereto.

The pharmaceutical composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. Pharmaceutically acceptable carriers can be used by mixing one or more of these components: saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and if necessary, antioxidants Other buffering additives, such as buffers and bacteriostatic agents, may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and specifically act on target organs. Thus, a target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated in accordance with each disease or ingredient by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. have.

The nucleotide or nucleic acid used in the present invention can be prepared for administration such as oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, and transdermal.

Preferably, nucleic acids or vectors are used in injectable form. Accordingly, particularly to the area to be treated, it can be mixed with any pharmaceutically acceptable medium for injectable compositions for direct infusion. The pharmaceutical composition of the present invention is particularly suitable for isotonic sterile solutions or drying, particularly with the addition of sterile water or appropriate physiological saline.

And lyophilized compositions that allow for the composition of injectable solutions. Direct injection of the nucleic acid into the patient's tumor is advantageous as it allows treatment efficiency to be focused on the infected tissue. The dosage of the nucleic acid used can be adjusted by various parameters, in particular genes, vectors, the mode of administration used, the disease in question, or alternatively the required duration of treatment. In addition, the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage is about 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, and is preferably administered once or several times a day.

Preferably, nucleic acids or vectors are used in injectable form. Accordingly, particularly to the area to be treated, it can be mixed with any pharmaceutically acceptable medium for injectable compositions for direct infusion. The pharmaceutical composition of the present invention is particularly suitable for isotonic sterile solutions or drying, particularly with the addition of sterile water or appropriate physiological saline.

And lyophilized compositions that allow for the composition of injectable solutions. Direct injection of the nucleic acid into the patient's tumor is advantageous as it allows treatment efficiency to be focused on the infected tissue. The dosage of the nucleic acid used can be adjusted by various parameters, in particular genes, vectors, the mode of administration used, the disease in question, or alternatively the required duration of treatment. In addition, the range varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage is about 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, and is preferably administered once or several times a day.

In addition, the present invention provides a method for treating cancer or a method for inhibiting cancer metastasis, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to an individual suffering from cancer.

The pharmaceutically effective amount is 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, but is not limited thereto. The dosage may be changed according to the weight, age, sex, health status, diet, administration period, administration method, elimination rate, disease severity, etc. of a specific patient.

The pharmaceutical composition can be administered orally or parenterally during clinical administration, and when administered parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, intracranial injection or chest It can be administered by intravenous injection and can be used in the form of generic pharmaceutical formulations.

In addition, the present invention provides a method for screening a candidate substance for cancer treatment or cancer metastasis suppression using DRG2 protein.

Specifically, the method comprises the steps of treating the test substance in the cell line expressing the DRG2 protein; Measuring the expression level of DRG2 protein in the cell line; And it is preferable that the expression level of the DRG2 protein is reduced compared to the control group not treated with the test material, but it is preferable to include, but is not limited to.

In the above method, the expression level of the protein is immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, western blotting and flow cytometry ( It is preferable to measure any one selected from the group consisting of FACS), but any method of measuring the amount of transcripts or proteins encoded therefrom known to those skilled in the art can be used.

Another method comprises the steps of treating the DRG2 protein with a test substance; Measuring the activity level of the DRG2 protein; And selecting a test substance in which the activity level of the DRG2 protein is reduced compared to a control group not treated with the test substance, but is not limited thereto.

In the above method, the activity level of the protein is preferably measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzymatic immunoassay (ELISA), mass spectrometry and protein chip, but is not limited thereto.

In addition, the present invention provides a method for monitoring or diagnosing cancer using DRG2 protein. Specifically, the method includes measuring the expression level of DRG2 protein in a sample derived from a subject; And determining that the subject having an increased expression level of DRG2 protein is greater than the control group as an individual at risk of developing cancer.

In the above method, the DRG2 preferably has an amino acid sequence set forth in SEQ ID NO: 2, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples specifically illustrate the present invention, and the contents of the present invention are not limited by the above examples.

<Example 1>

Checking the effect of DRG2 overexpression on the cell cycle of T cells

As disclosed in the prior document 1, it is known that overexpression of DRG2 has an effect of inhibiting the growth of T cells. The present inventors analyzed the cell cycle using the flow cytometry and the cell growth curve of the Jurkat cell line, which is a human T cell line due to overexpression of DRG2, in order to confirm the effect of DRG2 on the cell cycle of T cells.

1-1. DRG2 cloning and DRG2 overexpression cell line establishment

The present inventors first tried to clone the human DRG2 gene into the pLNCX2 vector to confirm the effect of DRG2 on the cell cycle of T cells. For this, the human DRG2 gene was extracted from the HT1080 cell line, and then amplified through RT-PCR using the following primers.

Forward primer: 5'-CTCGAGCTGCTGCTACCATGGGGATCTTA-3'

Reverse primer: 5'-GTCGACTTACTTCTTCACGATCTGGATGA-3'

The amplified DNA was cloned into the pLNCX2 vector, and the cloned pLNCX2-DRG2 was transfected with the RetroPact PT67 cell line according to the manufacturer's method (Clontech) to obtain a retro virus, and the Jurkat cell line was infected with the pLNCX2-DRG2 retro virus, and then the manufacturer According to the method (Clontech), a Jurkat-LNCX2-DRG2 cell line stably expressing DRG2 was established. On the other hand, the control group Jurkat-LNCX2 was prepared by infecting the pLNCX2 retrovirus on the Jurkat cell line. The Jurkat cell line was supplemented with RPMI 1640 medium in 10% FBS, 100 μg/ml streptomycin, and 100 U/ml penicillin, and then cultured in a wet incubator maintaining 37° C. and 5% CO ₂ .

As a result, as can be seen in Figure 1A, it was confirmed that the DRG2 is overexpressed specifically in the Jurkat-LNCX2-DRG2 cell line.

1-2. Cell growth curve according to DRG2 expression

To confirm the cell growth of Jurkat-LNCX2 and Jurkat-LNCX2-DRG2 prepared by Example 1-1, the cell growth curve was measured at 2-day intervals. The cell growth curve was measured by measuring the cell volume and counting the number of cells using a hematocytometer.

As a result, as can be seen in Figure 1B, it was confirmed that the growth in T cells overexpressing DRG2 is significantly reduced compared to the control group.

1-3. Cell cycle changes according to DRG2 expression

Through the above Examples 1-2, it can be confirmed that the growth of T cells is inhibited when DRG2 is overexpressed, and the present inventors additionally confirmed how DRG2 expression affects the cell cycle of T cells.

For this, Jurkat-LNCX2 and Jurkat-LNCX2-DRG2 cells prepared in Example 1 were collected and PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na ₂ HPO ₄ 7H ₂ O, 1.4 mM KH ₂ PO ₄ , pH 7.2), washed twice at room temperature, and resuspended in PBS at 2×10 ⁶ cells/ml. For staining propidium iodide, the cells were fixed overnight at -20°C using pure ethanol, and then stained according to the manufacturer's method (Molecular Probes). Then, the cells were washed twice with PBS, resuspended in FACS buffer (PBS, 0.2% BSA and 1% sodium azide), and then analyzed using a FACS flow cytometer (Becton Dickinson, Inc., San Jose, CA). .

As a result, as can be seen in Figure 1C, it was confirmed that the cell cycle of T cells overexpressing DRG2 was stopped at the G2/M phase.

1-4. Apoptosis observation according to DRG2 expression

The present inventors confirmed that DRG2 affects the cell cycle, and Annexin V staining was performed to further confirm the effect of overexpression of DRG2 on apoptosis.

Annexin V staining was performed according to the manufacturer's method (Roche Molecular Biochemicals) using the Annexin-V-FLUOS staining kit. That is, the cells were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 7H2O, 1.4 mM KH2PO4, pH 7.2), and binding buffer (0.01 M HEPES, pH 7.4, containing Annexin V and propidium iodide). After suspending with 0.14 M NaCl, 2.5 mM CaCl2), the fluorescence intensity of the cells was analyzed using a FACS flow cytometer (Becton Dickinson).

As a result, as can be seen in Figure 1D, Jurkat-LNCX2 cell death was 52.13%, but Jurkat-LNCX2-DRG2 cell death was 17.92%, confirming that the cell death was significantly reduced.

These results suggest that DRG2 is an important protein that regulates cell cycle and cell death. Therefore, the present inventors assumed that the DRG2 protein may play an important role in the regulation of cancer cells caused by abnormal division of cells due to abnormality in cell cycle regulation, and further experiments will be conducted.

<Example 2>

Effects of DRG2 on cell migration

Cell migration is known to play an important role in the invasion and metastasis of cancer cells as well as various physiological responses. Therefore, the present inventors tried to confirm whether DRG2 exerts an effect of inhibiting cell migration in addition to the role of regulating cell cycle and cell death.

2-1. Established MCF-7 cell line treated with shRNA

In order to confirm the effect of DRG2 on cell migration, the present inventors transfected small hairpin RNA (shRNA) against DRG2 so that the expression of DRG2 is stably suppressed in the human breast cancer cell line MCF-7 cell line. That is, the expression of DRG2 was stably suppressed by transfecting pLK0.1-shDRG2 (Sigma, TRCN0000047195), a pLK0.1 vector cloned with the shRNA of human DRG2 (hereinafter referred to as'shDRG2') into MCF-7 cells. Cell lines were prepared (hereinafter, referred to as'MCF-7/shDRG2'). Meanwhile, an MCF-7 cell line into which a shRNA control vector was introduced was also prepared as a control for this (hereinafter referred to as'MCR-7/scRNA').

shRNA sequence Sequence Sequence number shDRG2 5'-CCGGGCCCTGCCTGTATGTTTATAACTCGAGTTATAAACATACAGGCAGGGCTTTTTG-3' 3 shRNA control 5'-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3' 4

The effect of inhibiting DRG2 expression in the MCF-7/shDRG2 and MCF-7/shRNA-control cell lines was confirmed through Western blotting, and as shown in FIG. 2A, the expression of DRG2 is effectively suppressed only in the MCF-7/shDRG2 cell line. Appeared.

2-2. Observation of the effect of DRG2 expression inhibition on cell migration through Wound Healing Assay

To confirm the effect of DRG2 expression inhibition on cell migration, a wound healing assay was performed. To this end, the wild-type MCF-7 cell line, MCF-7/shRNA-control cell line, and MCF-7/shDRG2 cell line were cultured in a 6-well plate using a medium in which 10% FBS was added at a concentration of 5×10 ⁵ cells/well for 12 hours. After incubating and attaching for a while, a line was drawn on the plate using a 200 μl pipet tip. Thereafter, the cells were washed twice with PBS, then replaced with serum-free medium, and further cultured for 24 hours, and the degree of cell migration was observed under a microscope.

As a result, as shown in FIG. 2, it was found that cell migration was significantly reduced in the MCF-7/shDRG2 cell line compared to the wild-type MCF-7 cell line and the MCF-7/scRNA cell line.

Through the above results, the present inventors can play a major role in the movement of DRG2 cells, and can inhibit the movement of cells by knocking down (Gnock-down) DRG2 in a breast cancer cell line, thus suppressing DRG2 inhibits cancer cell metastasis. It was found that it exhibited an inhibitory effect.

<Example 3>

Effect of DRG2 expression inhibition on Rac1 activity

Rac1 is a type of Rho family small GTPase, which is activated by binding to GTP by various signals in an inactive form associated with GDP, and is known to play an important role in cell cycle, cell migration, and various cancers such as melanoma and small cell lung cancer. have. Therefore, the present inventors tried to confirm whether the effect of cell migration inhibition by DRG2 expression inhibition is related to Rac 1 activity.

To this end, shDRG2 was transfected into the melanoma cell line B16F10 cell line to stably suppress DRG2 expression (hereinafter referred to as'B16F10/shDRG2'). Meanwhile, a cell line into which a shRNA control vector was introduced was also prepared as a control for this (hereinafter referred to as'B16F10/scRNA').

The degree of activation of Rac1 according to the inhibition of DRG2 expression in each cell line was confirmed by Western blotting, and it was confirmed that the amount of Rac1-GTP in B16F10/shDRG2, which suppressed the expression of DRG2, was significantly reduced compared to the control group. By inhibiting the activity of it was found to exert a cell migration inhibitory effect. Therefore, it was determined that the effect of suppressing DRG2 expression suppressing cancer cell metastasis is closely related to Rac1 activation (see FIG. 3A ).

<Example 4>

Effect of DRG2 expression inhibition on VE-cadherin expression

Meanwhile, in the process of metastasis of cancer cells, in order for cancer cells to pass through endothelial cells of blood vessels, the binding between endothelial cells must be weakened. Vascular endothelial cells are mutually bound using VE-cadherin. When VE-cadherin is expressed in cancer cells, they weaken VE-cadherin binding between vascular endothelial cells, so that cancer cells can easily pass through the blood vessel wall. It has been previously reported that Rac1 affects cell surface expression of VE-cadherin. Therefore, the present inventors conducted experiments on this due to the possibility that the inhibitory effect of Rac1 activation by suppressing DRG2 expression may also affect the expression of VE-cadherind.

First, in the wild-type B16F10 cell line, the B16F10/scDRG2 cell line prepared in Example 3, and the B16F10/shDRG2 cell line, mRNA and protein amounts of VE-cadherin were observed by RT-PCR and Western blotting, respectively.

4-1. VE-cadherin mRNA analysis using RT-PCR

Total RNA from each cell line was isolated with Welprep ^TM reagent (Jeil Biotechservices Inc., Daegu, Korea), and the reverse transcription reaction was performed using ImProm II ^TM reverse transcription reagent (Promega, Madison, WI, USA) according to the manufacturer's method. RT-PCR was performed with IQ5 (Bio-Rad) using IQ ^TM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) as a cDNA template. Quantification was derived by the efficiency-corrected Cq method. The relative expression level was normalized using GAPDH.

Primer name Sequence GAPDH forward 5'-agtccatgccatcactgccacc-3' GAPDH reverse 5'-ccagtgagcttcccgttcagc -3' VE-cadherin forward 5'-AACTTCCCCTTCTTCACCC-3' VE-cadherin reverse 5'-AAAGGCTGCTGGAAAATG-3'

As a result, it can be seen from FIGS. 3B and C that the amount of VE-cadherin remains constant regardless of whether DRG2 is inhibited.

4-2. VE-cadherin protein analysis using Western blotting

In addition, to analyze the VE-cadherin protein expression level from each cell line, each cell line was captured and centrifuged to collect only cells. Each cell line was lysed using RIPA buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.4), 1% NP-40, 0.1% sodium deoxycholate, 1 mM EDTA) containing protease inhibitor (Sigma), and the protein After separation with 5X reduction sample buffer, electrophoresis was performed by SDS-PAGE, and the protein was transferred to the PVDF membrane. After blocking the PVDF membrane for 1 hour at room temperature using a blocking solution (5% skim milk), mouse anti-DRG2 antibody (1:2000, Sigma) and mouse anti-VE-cadherin antibody (1:2000, Santacruz) for 2 hours at room temperature. After washing the PVDF membrane with TBS-T buffer three times for 10 minutes, the PVDF membrane was reacted for 1 hour at room temperature with an anti-mouse HRP conjugated (1:4000, Sigma) secondary antibody.

As a result, as shown in FIG. 3C, it was found that the protein expression of VE-cadherin was also inhibited in the B16F10 cell line in which DRG2 expression was suppressed.

4-3. Analysis of VE-cadherin protein expression using fluorescence microscopy

The present inventors observed the VE-cadherin protein expression pattern in a fluorescence microscope in a B16F10/scRNA cell line and a B16F10/shDRG2 cell line to confirm the effect of DRG2 expression on VE-cadherin protein expression on cells.

To this end, DAPI staining and FITC staining were used. First, the medium was removed from the cell line, washed three times with PBS, and then the cells were fixed with a 3.7% formaldehyde solution for 20 minutes, and then the fixed cells were again washed with PBS. Washed 3 times every 5 minutes. After this, 0.2% Triton X-100 was treated for 5 minutes, washed with PBS, and then treated with FITC-conjucated VE-cadherin (1:500, Santacruz) antibody. After washing with PBS three times, 2µg/ml DAPI solution was added and maintained in dark condition for 5 minutes. The stained cells were observed using a fluorescent lamp of a phase contrast microscope (Leica DC100, Wetzlar, Germany).

As a result, as shown in FIG. 3D, it was found that the expression of VE-cadherin was also suppressed in the B16F10 cell line in which DRG2 expression was suppressed. Through this, the present inventors have found that DRG expression inhibition can inhibit cancer metastasis by inhibiting the ability of cancer cells to pass through vascular endothelial cells.

<Example 5>

Effect of DRG2 expression inhibition on melanoma metastasis

Through the above examples, it was found that the present inventors inhibited the expression of DRG2 and inhibited the movement of cancer cells through the regulation of Rac1 activity, and the protein expression of VE-cadherin, thereby inhibiting the ability of cancer cells to pass through vascular endothelial cells. The present inventors tried to confirm whether the mechanism identified from the above in vitro experiment results actually exerts an effect of inhibiting cancer metastasis in vivo.

To confirm this, a 5 week old C57BL/6J mouse (male) was purchased from a central laboratory animal (Korea), and the breeding ground was maintained at a temperature of 23℃, humidity of 55%, illuminance of 300-500 lux, and a 12-hour breeding cycle. Was bred in. The feed was purchased freely from Purina Rodent Laboratory Chow 5001 (Purina Mills, USA) and supplied freely for drinking water. After 2 weeks of purification, the mice were randomly divided into two groups of 10 mice each. The B16F10/scRNA or B16F10/shDRG2 cell line per mouse in each group was suspended in PBS and intravenously injected into the tail by 5×10 ⁵ .

Then, as a result of investigating the mouse survival rate over time and counting the number of nodules metastasized from lung tissue, the mouse survival rate was significantly increased in the melanoma cell line in which DRG2 expression was suppressed compared to the control group in which DRG2 expression was not suppressed (FIG. 4A), it was observed that the number of melanoma metastasized to the lung tissue was significantly lower (see FIGS. 4B and 4C).

Through the above experimental results, the present inventors, DRG2 is a protein that plays an important role in cancer development and metastasis. When DRG2 is inhibited, cancer cell migration can be suppressed to effectively inhibit cancer metastasis such as breast cancer and melanoma. And it was found.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

<110> University of Ulsan Foundation For Industry Cooperation <120> Anticarcinogenic composition comprising an inhibitor of DRG2 as an active ingredient <130> PN2006-229 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 364 <212> PRT <213> Artificial Sequence <220> <223> human drg2 amino acid seq. <400> 1 Met Gly Ile Leu Glu Lys Ile Ser Glu Ile Glu Lys Glu Ile Ala Arg 1 5 10 15 Thr Gln Lys Asn Lys Ala Thr Glu Tyr His Leu Gly Leu Leu Lys Ala 20 25 30 Lys Leu Ala Lys Tyr Arg Ala Gln Leu Leu Glu Pro Ser Lys Ser Ala 35 40 45 Ser Ser Lys Gly Glu Gly Phe Asp Val Met Lys Ser Gly Asp Ala Arg 50 55 60 Val Ala Leu Ile Gly Phe Pro Ser Val Gly Lys Ser Thr Phe Leu Ser 65 70 75 80 Leu Met Thr Ser Thr Ala Ser Glu Ala Ala Ser Tyr Glu Phe Thr Thr 85 90 95 Leu Thr Cys Ile Pro Gly Val Ile Glu Tyr Lys Gly Ala Asn Ile Gln 100 105 110 Leu Leu Asp Leu Pro Gly Ile Ile Glu Gly Ala Ala Gln Gly Lys Gly 115 120 125 Arg Gly Arg Gln Val Ile Ala Val Ala Arg Thr Ala Asp Val Ile Ile 130 135 140 Met Met Leu Asp Ala Thr Lys Gly Glu Val Gln Arg Ser Leu Leu Glu 145 150 155 160 Lys Glu Leu Glu Ser Val Gly Ile Arg Leu Asn Lys His Lys Pro Asn 165 170 175 Ile Tyr Phe Lys Pro Lys Lys Gly Gly Gly Ile Ser Phe Asn Ser Thr 180 185 190 Val Thr Leu Thr Gln Cys Ser Glu Lys Leu Val Gln Leu Ile Leu His 195 200 205 Glu Tyr Lys Ile Phe Asn Ala Glu Val Leu Phe Arg Glu Asp Cys Ser 210 215 220 Pro Asp Glu Phe Ile Asp Val Ile Val Gly Asn Arg Val Tyr Met Pro 225 230 235 240 Cys Leu Tyr Val Tyr Asn Lys Ile Asp Gln Ile Ser Met Glu Glu Val 245 250 255 Asp Arg Leu Ala Arg Lys Pro Asn Ser Val Val Ile Ser Cys Gly Met 260 265 270 Lys Leu Asn Leu Asp Tyr Leu Leu Glu Met Leu Trp Glu Tyr Leu Ala 275 280 285 Leu Thr Cys Ile Tyr Thr Lys Lys Arg Gly Gln Arg Pro Asp Phe Thr 290 295 300 Asp Ala Ile Ile Leu Arg Lys Gly Ala Ser Val Glu His Val Cys His 305 310 315 320 Arg Ile His Arg Ser Leu Ala Ser Gln Phe Lys Tyr Ala Leu Val Trp 325 330 335 Gly Thr Ser Thr Lys Tyr Ser Pro Gln Arg Val Gly Leu Thr His Thr 340 345 350 Met Glu His Glu Asp Val Ile Gln Ile Val Lys Lys 355 360 <210> 2 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> human drg2 polynucleotide seq. <400> 2 atggggatct tagagaagat ctcggagatc gagaaggaga tcgctcggac acagaagaac 60 aaggccactg agtatcatct gggcctgctg aaagctaagc tcgccaagta tcgggcccag 120 ctcctggaac cgtccaaatc ggcctcgtcc aaaggagagg gctttgatgt catgaagtcg 180 ggtgatgccc gtgtggcgct gattggattt ccctctgtgg gtaagtccac attcttgagt 240 ctgatgacct ccacggccag cgaggcagcg tcctatgagt tcaccactct gacgtgtatt 300 cctggggtca ttgaatacaa aggtgccaac atccagctcc tggaccttcc tggaatcatt 360 gaaggcgcag cccaaggaaa aggccgtggc cggcaggtga tcgctgtggc gcgcacggct 420 gacgtcatca tcatgatgct ggatgccacc aagggagagg tgcagaggtc tctgctggag 480 aaggagctgg agtctgtggg catccgcctc aacaagcaca agcctaacat ctacttcaag 540 cccaagaaag gtggtggcat ctcctttaac tcgacagtca cgctgaccca gtgctcggaa 600 aagctggtgc agctcatcct gcacgaatac aagatcttca atgcagaagt gcttttccga 660 gaagactgct ccccggacga gttcatcgat gtgatcgtgg gcaaccgggt gtacatgccc 720 tgcctgtatg tttataacaa aatcgaccag atctccatgg aagaggtgga ccgcctggcc 780 cgaaaaccca acagtgtggt catcagctgc ggcatgaagc tgaacctgga ctatctgctg 840 gagatgcttt gggagtactt ggccctgacc tgcatctaca ccaagaagag aggacagagg 900 ccagacttca cagacgccat cattctccgg aaaggggcct cagtggagca cgtgtgccac 960 cgcatccacc ggtcactcgc cagccagttc aagtacgccc tggtgtgggg caccagcacc 1020 aagtacagtc cgcagcgggt gggcctgacc cacaccatgg agcatgagga cgtcatccag 1080 atcgtgaaga agtaa 1095 <210> 3 <211> 58 <212> RNA <213> Artificial Sequence <220> <223> shDRG2 polynucleotide seq. <400> 3 ccgggccctg cctgtatgtt tataactcga gttataaaca tacaggcagg gctttttg 58 <210> 4 <211> 57 <212> RNA <213> Artificial Sequence <220> <223> shRNA control polynucleotide seq. <400> 4 ccggcaacaa gatgaagagc accaactcga gttggtgctc ttcatcttgt tgttttt 57

Claims (7)

DRG2 (Developmentally regulated GTP-binding protein 2) gene-specific antisense nucleotide, siRNA, sh RNA or ribozyme (ribozyme), or DRG2 protein specific aptamer or antibody pharmaceutical composition for inhibiting cancer metastasis as an active ingredient As, the composition is characterized in that it has the activity of inhibiting the metastasis of cancer cells, the cancer is breast cancer or melanoma, the composition inhibits the activity of Rac1 or inhibits the expression of VE-cadherin protein in cancer cells to cancer cells Pharmaceutical composition for inhibiting cancer metastasis, characterized in that to inhibit the metastasis of. According to claim 1,
The DRG2 (Developmentally regulated GTP-binding protein 2) gene is a pharmaceutical composition for inhibiting cancer metastasis, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. Processing the test substance in the DRG2 protein expressing cell line;
Measuring the expression level of DRG2 protein in the cell line; And
When the expression level of the DRG2 protein is reduced compared to a control cell line that has not been treated with the test substance, determining the test substance as a substance for inhibiting cancer metastasis. Or a method for screening a substance for inhibiting cancer metastasis that is melanoma. According to claim 3,
The expression level of the DRG2 protein is composed of immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western Blotting and flow cytometry (FACS). A screening method of a substance for inhibiting cancer metastasis, characterized in that it is measured by any one selected from the group, wherein the cancer is a breast cancer or melanoma. Treating the test substance with DRG2 protein;
Measuring the activity level of the DRG2 protein; And
When the level of activity of the DRG2 protein is reduced compared to a control cell line that is not treated with the test substance, comprising the step of determining the test substance as a substance for inhibiting cancer metastasis. Or a method for screening a substance for inhibiting cancer metastasis that is melanoma. The method of claim 5,
The activity level of the DRG2 protein is measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzymatic immunoassay (ELISA), mass spectrometry, and protein chip. A method for screening a substance for inhibiting cancer metastasis, which is cancer or melanoma. Measuring the expression level of DRG2 protein in a sample derived from the subject; And
A method of providing information necessary for cancer metastasis, comprising determining a subject having an increased expression level of DRG2 protein than a control group as an individual at risk of cancer metastasis. How to provide.

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