KR102150239B1 - Anticarcinogenic composition comprising an inhibitor of DRG2 as an active ingredient - Google Patents

Abstract

The present invention relates to an anticancer composition comprising a DRG2 inhibitor as an active ingredient, wherein the expression inhibitor of the DRG2 (Developmentally regulated GTP-binding protein 2) gene or the DRG2 protein activity inhibitor according to the present invention inhibits cell migration, Since it inhibits Rac 1 activity and can effectively inhibit metastasis of cancer cells by a mechanism that inhibits the expression of VE-cadherin protein, an inhibitor of the expression of the developmentally regulated GTP-binding protein 2 (DRG2) gene or an inhibitor of the activity of the DRG2 protein It will be useful as an anticancer composition that can block cancer metastasis.

Description

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

The present invention relates to an anticancer composition comprising a DRG2 inhibitor as an active ingredient, and more specifically, to a pharmaceutical composition comprising a DRG2 inhibitor capable of preventing or treating cancer diseases by inhibiting metastasis of cancer as an active ingredient. .

Cancer is one of the major diseases that pose a risk to human life. In Korea, cancer and cancer are the number one cause of death in the past several years, and it is reported that cancer is the second cause of death after cardiovascular disease in the United States. Therefore, although a number of studies are being conducted to conquer cancer, millions of people die of cancer every year to this day, and the resulting economic loss is also astronomical.

Cancer is known as a genetic disease caused by mutations in genes such as oncogenes and tumor suppressor genes, and is particularly a disease that has a cause at the cellular level. Currently, surgical treatment, drug therapy, radiation therapy, and immunotherapy are used as treatments for cancer, but suppression and recurrence of malignant tumors are still not resolved.

One of the most important biological properties of cancer is that it can cause metastasis, which is considered the biggest obstacle to cancer treatment. In fact, about 60% of all solid tumor patients already have cancer that has metastasized clinically at the time of primary tumor diagnosis, and it is well known that cancer metastasis is an important cause of death for most cancer patients.

That is, the cause of death of cancer patients is mainly due to metastasis of cancer cells rather than due to early tumors. The metastasis of these cancer cells is that metastatic cancer cells are first detached from the initial tumor and invasion into the extracellular matrix (ECM) such as the interstitial stroma and the basal membrane (BM) of normal tissues, thereby invading the blood vessels. After entering the or lymphatic vessels, they are attached to the capillary walls of other target tissues, leached from the capillaries through the matrix and basement membrane of cells, and proliferate in new tissues, forming a secondary tumor (metastatic cancer).

Until now, expectations for the development of a substance having such a cancer metastasis inhibitory action are very high, but in fact, there are very few examples of the development of a substance for the purpose of suppressing cancer metastasis. Sulfated polysaccharides, N-diazoacetylglycine derivatives, noiraminase and fibronectin degrading enzyme (FNS) have been reported to have such inhibitory effects, but there is no report of their practical use yet, and they themselves have inhibitory effects on cancer metastasis. There is no report. Therefore, there is an urgent need to develop a therapeutic agent that can effectively inhibit metastasis of cancer.

Accordingly, the present inventors have conducted research with interest in a novel substance capable of effectively inhibiting cancer metastasis, and as a result, it has been confirmed that the metastasis of cancer cells is significantly inhibited when DRG2 expression is suppressed, thereby completing the present invention.

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 anticancer composition that can effectively inhibit cancer metastasis.

In addition, another object of the present invention is to provide a novel method for screening for cancer metastasis inhibitors.

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 anticancer composition comprising an expression inhibitor of DRG2 (Developmentally regulated GTP-binding protein 2) gene or an activity inhibitor of 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 DRG2 (Developmentally regulated GTP-binding protein 2) gene expression inhibitor is from the group consisting of an antisense nucleotide for the gene represented by SEQ ID NO: 1, siRNA sh RNA, and ribozyme. It may be any one selected.

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

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

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

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

In one embodiment of the present invention, the cancer is lung cancer, gastric cancer, colon cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle 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 ureter 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 a test substance on a DRG2 protein-expressing cell line; Measuring the expression level of the DRG2 protein in the cell line; And when the expression level of the DRG2 protein is decreased compared to the control cell line not treated with the test substance, determining the test substance as an anticancer 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) may be measured by any one selected from the group consisting of.

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

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

In addition, the present invention comprises the steps of measuring the expression level of the DRG2 protein in the sample derived from the subject; And it provides a method of providing information necessary for cancer diagnosis, comprising the step of determining a subject having an increased expression level of the DRG2 protein compared to the control group as an individual at risk of developing cancer.

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

1 is an experimental result of inducing DRG2 overexpression in Jurkat T cells (A), and observing cell growth (B), cell cycle (C) and cell death (D) accordingly.
2 is a result of observation of 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 level of Rac1 activation (A), the level of mRNA expression of VE-cadherin (B), and the level of protein expression of VE-cadherin (C, D) in B16F10/scRNA and B16F10/shDRG2 cell lines.
4 is an experimental result of observing the mouse survival rate (A), the number of lung metastases (B), and the degree of lung metastasis (C) after treatment with B16F10/scRNA or B16F10/shDRG2 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, any subject who participated in a clinical study trial showing no clinical manifestation of any disease, or a subject who participated in an epidemiological study or a subject used as a control group is included. In an 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. The source of the tissue or cell sample may be a fresh, frozen and/or preserved organ or tissue sample or solid tissue from a biopsy or aspirate; Blood or any blood component; It may be a cell at any point in the subject's pregnancy or development. The tissue sample can also be a primary or cultured cell or cell line.

Optionally, a tissue or cell sample is obtained from a primary or metastatic tumor. The tissue sample may contain compounds that do not mix with the tissue due to their nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. For the purposes of the present invention, a "section" of a tissue sample means a portion or piece of a tissue sample, for example a thin slice of tissue or cells cut from a tissue sample. If it is understood that the present invention encompasses a method in which the same section of a tissue sample is analyzed at both the morphological and molecular level or both protein and nucleic acid, then multiple sections of a tissue sample are taken and applied to the analysis according to the invention. It is understood that it can be done.

The term “growth” of a cell refers to the increase in the number of cells in the body of a multicellular organism, usually by dividing a cell and increasing the homogeneity. When the number of cells proliferates (amplifies) and reaches a certain period, the trait changes. (Differentiation) is usually controlled at the same time as the number of cells is increased in the body, and when the cytoplasm in the cell is emerging, it is often distinguished by growth, but biologically, the number of cells is different. In view of the increase, the period in which differentiation does not occur during the development of multicellular organisms is justified as proliferation or growth, and the two terms are used interchangeably in the present invention.

“Cancer”, “tumor” or “malignant” generally refers to or describes a physiological condition in a mammal characterized by 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 treated as a representative.

"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, polymer compounds such as proteins, carbohydrates, lipids, and composites of various compounds. For example, a'lymph node metastasis inhibitor' may include a substance that inhibits, blocks or reduces lymph node metastasis in a tumor.

"Prevention" refers to inhibiting the occurrence of a disease or disease in an animal that has not been diagnosed as having a disease or disease, but is prone to the disease or disease.

"Treatment" means (a) inhibition of the disease or development of the disease; (b) alleviation of the disease or disease; And (c) means the elimination of a disease or disorder. Accordingly, the term therapeutically effective amount as used herein means an amount sufficient to achieve the aforementioned pharmacological effect.

All technical terms used in the present invention, unless otherwise defined, are used in the same sense as those of ordinary skill in the art generally understand in the related field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention. The contents of all publications referred to herein by reference 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 containing an inhibitor of the expression or activity of DRG2 gene or protein as an active ingredient.

DRG2 is a type of G protein, and has all five regions of G1 to G5, which are required for GTP binding. The size is similar to the trimeric G protein, but the amino acid sequence is different from the existing G protein subfamily, so it is classified into a new subfamily. DRG is known to have DRG1 and DRG2, and is present in almost all types of cells from bacteria to humans. On the other hand, it was found that all DRG proteins found in humans from bacteria are evolutionarily conserved, and the amino acid sequence of human DRG2 is 98% of mouse and bovine DRG2, 95% of Xenopus DRG2, and zebrafish DRG2 and 91. % Homology

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

The cancer is lung cancer, gastric cancer, colon cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle 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 cancer, chronic or acute leukemia, lymphocytic lymphoma , Bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, preferably one or more solid cancers selected from the group consisting of, breast cancer or black It is more preferable that it is a species, but is not limited thereto.

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

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

The shRNA is a small hairpin structure RNA that inhibits gene expression by interfering with RNA. The shRNA is introduced into cancer cells using various vectors, but according to a preferred example according to the present invention, the pLK0.1 vector is used. It could be introduced into cancer cells. At this time, the shRNA sequence is not particularly limited, but is preferably SEQ ID NO: 3.

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

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

The aptamer is a single-stranded DNA or RNA molecule, and has high affinity for a specific chemical or biological molecule by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). It can be obtained by separating an oligomer that binds to and has a selectivity. Aptamers can specifically bind to a target and modulate the activity of the target, such as blocking the ability of the target to function through binding.

The antibody specifically and directly binds to DRG2 and can effectively inhibit the activity of DRG2. It is preferable to use a polyclonal antibody or a monoclonal antibody as the antibody that specifically binds to DRG2. Antibodies that specifically bind to DRG2 may be prepared by known methods known to those skilled in the art, and commercially known DRG2 antibodies may be purchased and used. The antibody can be prepared by injecting the immunogen DRG2 protein into an external host according to a conventional method 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 an adjuvant to increase antigenicity. Antibodies can be isolated by collecting blood from an external host on a regular basis and collecting serum showing the shaped titer and specificity for the antigen.

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

The present inventors experimented on the anticancer effect of DRG2 based on the experimental results (see FIG. 1) that DRG2 is closely related to cell growth, cell cycle and cell death. To this end, as a result of transfecting the MCF-7 cell line, which is a breast cancer cell line, with shRNA capable of inhibiting the expression of DRG2 and observing cell migration, 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 Fig. 3).

Furthermore, in the mouse animal model injected with the melanoma cell line that inhibited DRG2 expression, 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 (see FIG. 4).

Therefore, it was found that inhibition of the expression or activity of DRG2 exhibits an effect of inhibiting the proliferation of cancer cells and inhibiting cancer metastasis. Therefore, the DRG2 expression or activity inhibitor may be usefully utilized as an active ingredient in a pharmaceutical composition for cancer treatment or cancer metastasis inhibition.

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

The pharmaceutical composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and if necessary, antioxidants , Buffers, bacteriostatic agents, and other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to form injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets, and can act specifically 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 according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. have.

The nucleotides or nucleic acids used in the present invention may be prepared for oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal administration, or the like.

Preferably, nucleic acids or vectors are used in injectable form. Thus, it can be mixed with any pharmaceutically acceptable vehicle for injectable compositions, especially for direct injection into the area to be treated. The pharmaceutical composition of the present invention is in particular according to the addition of an isotonic sterile solution or drying in particular sterile water or an appropriate physiological saline solution.

It may include a lyophilized composition that allows the composition of an injectable solution. Direct injection of the nucleic acid into the patient's tumor is advantageous because 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 the gene, the vector, the mode of administration used, the disease in question or alternatively the duration of treatment required. In addition, the range varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage is about 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, and is preferably administered once to several times a day.

Preferably, nucleic acids or vectors are used in injectable form. Thus, it can be mixed with any pharmaceutically acceptable vehicle for injectable compositions, especially for direct injection into the area to be treated. The pharmaceutical composition of the present invention is in particular according to the addition of an isotonic sterile solution or drying in particular sterile water or an appropriate physiological saline solution.

It may include a lyophilized composition that allows the composition of an injectable solution. Direct injection of the nucleic acid into the patient's tumor is advantageous because 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 the gene, the vector, the mode of administration used, the disease in question or alternatively the duration of treatment required. In addition, the range varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage is about 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg, and is preferably administered once to several times a day.

In addition, the present invention provides a method for treating cancer or 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 vary depending on 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 at the time of clinical administration, and when administered parenterally, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or chest It can be administered by intramuscular injection, and can be used in the form of a general pharmaceutical formulation.

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

Specifically, the method comprises the steps of treating a test substance to a cell line expressing the DRG2 protein; Measuring the expression level of the DRG2 protein in the cell line; And selecting a test substance whose expression 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 expression level of the protein is immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western Blotting, and flow cytometry ( FACS) is preferably measured by any one selected from the group consisting of, but any method of measuring the amount of a transcript or protein encoded therefrom can be used.

Another method includes treating the DRG2 protein with a test substance; Measuring the degree of activity of the DRG2 protein; And selecting a test substance whose 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 degree of activity of the protein is preferably measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme 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 the DRG2 protein. Specifically, the method comprises the steps of measuring the expression level of the DRG2 protein in a sample derived from the subject; And determining the subject having an increased expression level of the DRG2 protein compared to the control group as an individual at risk of developing cancer, but is not limited thereto.

In the above method, the DRG2 preferably has an amino acid sequence represented by 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>

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

As disclosed in Prior Document 1, it is known that overexpression of DRG2 has an effect of inhibiting the growth of T cells. In order to more clearly confirm the effect of DRG2 on the cell cycle of T cells, the present inventors analyzed the cell growth curve of the human T cell line Jurkat cell line due to overexpression of DRG2 and the cell cycle using a flow cytometer.

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

In order to confirm the effect of DRG2 on the cell cycle of T cells, the present inventors first tried to clone the human DRG2 gene into the pLNCX2 vector. To this end, the human DRG2 gene was amplified through RT-PCR using the following primers after RNA was extracted from the HT1080 cell line.

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 retrovirus, and the Jurkat cell line was infected with the pLNCX2-DRG2 retrovirus, and then the manufacturer Jurkat-LNCX2-DRG2 cell line stably expressing DRG2 was established according to the method (Clontech). On the other hand, the control Jurkat-LNCX2 was prepared by infecting the Jurkat cell line with the pLNCX2 retrovirus. The Jurkat cell line was supplemented with 10% FBS, 100 μg/ml of streptomycin, and 100 U/ml of penicillin in RPMI 1640 medium, and then cultured in a humidified incubator maintaining 37° C. and 5% CO ₂ .

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

1-2. Cell growth curve according to DRG2 expression

In order to confirm the cell growth of Jurkat-LNCX2 and Jurkat-LNCX2-DRG2 prepared in Example 1-1, the cell growth curve was measured at intervals of 2 days. 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 FIG. 1B, it was confirmed that the growth in DRG2 overexpressed T cells was significantly reduced compared to the control.

1-3. Cell cycle changes according to DRG2 expression

It can be seen that the growth of T cells is inhibited when DRG2 is overexpressed through Example 1-2, and the present inventors further confirmed how DRG2 expression affects the cell cycle of T cells.

To this end, the 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) at room temperature, and then resuspended in PBS at 2×10 ⁶ cells/ml. For propidium iodide staining, cells were fixed overnight at -20°C using pure ethanol, followed by staining according to the manufacturer's method (Molecular Probes). Thereafter, the cells were washed twice with PBS, resuspended in FACS buffer (PBS, 0.2% BSA and 1% sodium azide), and 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 the DRG2 overexpressed T cells stopped in the G2/M phase.

1-4. Observation of apoptosis according to DRG2 expression

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

Annexin V staining was performed according to the manufacturer's method (Roche Molecular Biochemicals) using an 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 a binding buffer containing Annexin V and propidium iodide (0.01 M HEPES, pH 7.4, After suspending in 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 apoptosis was 52.13%, Jurkat-LNCX2-DRG2 apoptosis was 17.92%, it was confirmed that the apoptosis significantly decreased.

The above results suggest that DRG2 is an important protein regulating cell cycle and cell death. Accordingly, the present inventors assumed that the DRG2 protein could play an important role in the regulation of cancer cells, which occurs due to abnormal division of cells due to abnormal cell cycle regulation, and further experiments were conducted on this.

<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 determine whether DRG2 exerts an effect of inhibiting cell migration in addition to its role in regulating cell cycle and cell death.

2-1. Establishment of shRNA-treated MCF-7 cell line

In order to confirm the effect of DRG2 on cell migration, the present inventors transfected with shRNA (small hairpin RNA) for DRG2 to stably suppress the expression of DRG2 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 in which human DRG2 shRNA (hereinafter referred to as'shDRG2') was cloned, into MCF-7 cells. A cell line was prepared (hereinafter, the cell line is referred to as'MCF-7/shDRG2'). Meanwhile, as a control for this, an MCF-7 cell line into which the shRNA control vector was introduced was also produced (hereinafter referred to as'MCR-7/scRNA').

shRNA sequence Base 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, only the MCF-7/shDRG2 cell line effectively inhibited the expression of DRG2. Appeared.

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

A wound healing assay was performed to confirm the effect of DRG2 expression inhibition on cell migration. To this end, wild-type MCF-7 cell line, MCF-7/shRNA-control cell line, and MCF-7/shDRG2 cell line were added to a 6-well plate in a medium containing 10% FBS for 12 hours at a concentration of 5×10 ⁵ cells/well. After incubation and attachment, a line was drawn on the plate using a 200 µl pipet tip. Thereafter, the cells were washed twice with PBS, replaced with a medium without serum, and cultured for an additional 24 hours, and the degree of cell migration was observed under a microscope.

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

Through the results as described above, the present inventors found that DRG2 plays an important role in cell migration, and by knock-down of DRG2 in breast cancer cell lines, it is possible to inhibit cell migration. Therefore, inhibition of DRG2 prevents cancer cell metastasis. It was found that it exerts a suppressive 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 combined with GDP, and is known to play an important role in the development of various cancers such as cell cycle, cell migration and melanoma, small cell lung cancer. have. Therefore, the present inventors tried to confirm whether the effect of inhibiting cell migration by inhibiting DRG2 expression is related to Rac 1 activity.

To this end, a cell line stably inhibiting DRG2 expression was produced by transfecting the B16F10 cell line, a melanoma cell line, with shDRG2 (hereinafter referred to as'B16F10/shDRG2'). On the other hand, as a control for this, a cell line into which the shRNA control vector was introduced was also produced (hereinafter referred to as'B16F10/scRNA').

In each of the above cell lines, the degree of activation of Rac1 according to the inhibition of DRG2 expression was confirmed by Western blotting, and it was confirmed that the amount of Rac1-GTP was significantly reduced in B16F10/shDRG2, which suppressed the expression of DRG2, compared to the control group. It was found that the cell migration inhibitory effect was exhibited by inhibiting the activity of. Therefore, it was determined that the effect of inhibiting the expression of DRG2 on inhibiting cancer cell metastasis has a close relationship with Rac1 activation (see FIG. 3A).

<Example 4>

Effect of DRG2 expression inhibition on VE-cadherin expression

Meanwhile, in order for cancer cells to pass through endothelial cells in blood vessels during cancer cell metastasis, the binding between endothelial cells must be weakened. Vascular endothelial cells are bound to each other using VE-cadherin, and when VE-cadherin is expressed in cancer cells, they weaken VE-cadherin binding between vascular endothelial cells, allowing cancer cells to easily pass through the vessel wall. Conventionally, Rac1 has been reported to affect the 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 inhibition of DRG2 expression may also affect the expression of VE-cadherind.

First, in the wild-type B16F10 cell line, the B16F10/scDRG2 cell line, and the B16F10/shDRG2 cell line prepared in Example 3, the amounts of mRNA and protein 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 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 cDNA as a template using IQ ^TM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Quantification was derived by the efficiency-corrected Cq method. Relative expression levels were normalized using GAPDH.

Primer name Base 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 was confirmed that the amount of VE-cadherin remained constant regardless of whether DRG2 was inhibited, as can be seen in FIGS. 3B and C.

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

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

As a result, as can be seen in Figure 3C, it was found that the expression of the protein of VE-cadherin was also suppressed 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 expression pattern of VE-cadherin protein in the B16F10/scRNA cell line and the B16F10/shDRG2 cell line with a fluorescence microscope in order to confirm the effect of DRG2 expression on the expression of VE-cadherin protein 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 for 5 minutes each. Thereafter, 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 3 times with PBS, 2 μg/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 can be seen in Figure 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 found that the inhibition of DRG expression can inhibit metastasis of cancer 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, the present inventors have found that inhibition of DRG2 expression inhibits the migration of cancer cells through regulation of Rac1 activity, and inhibits the ability of cancer cells to pass through endothelial cells by regulating the protein expression of VE-cadherin. The present inventors tried to confirm whether the mechanism identified from the results of the in vitro experiment as described above actually exhibits 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 a breeding environment maintained at a temperature of 23°C, humidity 55%, illumination 300-500 lux, and a 12-hour contrast cycle. Breed in. For feed, a solid feed for experimental animals (Purina Rodent Laboratory Chow 5001, Purina Mills, USA) of Purina Co., Ltd. was purchased and supplied freely, and tap water was freely available for drinking. After 2 weeks of acclimatization, the mice were randomly classified into two groups of 10 mice each. B16F10/scRNA or B16F10/shDRG2 cell line per mouse of each group was suspended in PBS, and 5×10 ⁵ was injected intravenously into the tail.

Subsequently, the survival rate of mice over time was investigated and the number of nodules metastasized from the lung tissue was counted. As a result, the survival rate of mice 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 melanomas metastasized to the lung tissue was significantly low (see FIGS. 4B and 4C).

Through the above experimental results, the present inventors found that DRG2 is a protein that plays an important role in the occurrence and metastasis of cancer. When DRG2 is suppressed, it is possible to effectively inhibit cancer metastasis such as breast cancer and melanoma by inhibiting the migration of cancer cells. And it was found.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to 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 from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed 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)

A pharmaceutical composition for inhibiting cancer metastasis comprising an antisense nucleotide specific for DRG2 (Developmentally regulated GTP-binding protein 2) gene, siRNA, sh RNA or ribozyme, or an aptamer or antibody specific for DRG2 protein as an active ingredient As, the composition is characterized in that it has an activity to inhibit metastasis of cancer cells, the cancer is breast cancer or melanoma, and the composition inhibits the activity of Rac1 or inhibits the expression of VE-cadherin protein in cancer cells A pharmaceutical composition for inhibiting metastasis of cancer, characterized in that it inhibits the metastasis of. The method of 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. Treating the test material to the DRG2 protein-expressing cell line;
Measuring the expression level of the DRG2 protein in the cell line; And
When the expression level of the DRG2 protein is decreased compared to the control cell line not treated with the test material, the cancer is breast cancer by a screening method for a substance for inhibiting cancer metastasis, comprising determining the test substance as a substance for inhibiting cancer metastasis. Or a method for screening a substance for inhibiting cancer metastasis, which is melanoma. The method of 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 method for screening a substance for inhibiting cancer metastasis, characterized in that measuring by any one selected from the group, wherein the cancer is breast cancer or melanoma. Treating the test substance to the DRG2 protein;
Measuring the degree of activity of the DRG2 protein; And
When the level of activity of the DRG2 protein is reduced compared to the control cell line not treated with the test material, the cancer is breast cancer by a screening method for a substance for inhibiting cancer metastasis, comprising determining the test substance as a substance for inhibiting cancer metastasis. Or a method for screening a substance for inhibiting cancer metastasis, which is melanoma. The method of claim 5,
The degree of activity of the DRG2 protein is measured by any one selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme immunoassay (ELISA), mass spectrometry, and protein chip. Cancer is a method for screening a substance for inhibiting metastasis of breast cancer or melanoma. Measuring the expression level of the DRG2 protein in the sample derived from the subject; And
A method of providing information necessary for cancer metastasis, comprising the step of determining a subject having an increased expression level of DRG2 protein than a control group as an individual at risk of cancer metastasis, wherein the cancer is breast cancer or melanoma. How to provide.

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