KR20120044805A - Suppression of cancer metastasis via upregulation of task-3 potassium channel - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing recombinant expression vector containing a gene encoding TASK-3 proteins is provided to effectively suppress cancer metastasis. CONSTITUTION: A pharmaceutical composition for suppressing cancer metastasis contains TASK-3 protein, a gene encoding the protein, or expression vector for gene therapy as an active ingredient and further contains pharmaceutically acceptable protein. The TASK-3 protein has an amino acid sequence of sequence number 21. The gene encoding TASK-3 contains a base sequence of sequence number 22. The recombinant expression vector contains the gene. A composition additionally contains an anticancer agent.

Description

Inhibition of cancer metastasis via upregulation of TASK-3 potassium channel

The present invention relates to inhibition of cancer metastasis by increasing expression of TASK-3 potassium channel, and specifically, TASK-3 protein, a gene encoding TASK-3 protein, or a recombinant expression comprising the gene as a target molecule for cancer metastasis inhibition. Pharmaceutical composition for inhibiting cancer metastasis, comprising a vector as an active ingredient, a method for inhibiting cancer metastasis using the same; Cancer metastasis prediction kit; A method for selecting a cancer metastasis risk group using the kit; And screening methods for cancer metastasis inhibitors.

Cancer is a disease in which cells multiply indefinitely and interfere with normal cell function. Abnormal cells, whose proliferation and suppression are not controlled, proliferate uncontrolled and excessively proliferate and invade surrounding tissues or organs, leading to destruction of normal tissues. The site of cancer is not limited, and the cause of cancer is still not known exactly. To date, it is known that mutations occur in genes of normal cells. Aberrant gene expression is closely related to unregulated cell growth and dedifferentiation, a common feature of all cancers. Numerous cancer studies have shown that the genomes of individuals with certain cancers are generally called tumor suppressor genes and normally reduce the expression of recessive genes that function to inhibit indiscriminate cell division and overgrowth. Oncogenes with the ability to modify are overexpressed. Therefore, searching for genes specifically overexpressed or suppressed in cancer cells compared to normal cells based on the relationship between gene expression and the onset or progression of cancer can develop a target for diagnosis or treatment of cancer.

In addition, one of the most important biological characteristics of cancer is that it can cause metastasis, which is considered to be the biggest obstacle in treating cancer. In fact, about 60% of all solid tumor patients already have cancers that have been clinically metastasized at the time of primary tumor diagnosis, and it is well known that the major cause of death in most cancer patients is cancer metastasis. Most cancer metastases are multiple and systemic, and it is difficult to determine whether they exist or not, and current therapeutic methods for cancer are not satisfactory. However, cancer metastasis is an inefficient process in which only a small portion of the cancer cells that make up the primary tumor have successfully completed the various stages of metastasis and become metastatic cancer. Therefore, if we understand the metastasis process and develop a method that can effectively inhibit this by discovering clinical and biologically useful therapeutic targets, we can effectively control death from cancer metastasis.

Accordingly, various studies have been conducted in relation to cancer occurrence and cancer metastasis, and as part of this research, studies on potassium channels, which are molecules that play an important role in cancer progression, are being actively conducted.

Potassium channels are protein complexes present in cell membranes that play an important role in many physiological processes due to their influence on cell membrane potential, and regulate the membrane potential by controlling the flow of potassium through the cell membrane. They are also important molecules in many types of cancer progression And voltage-dependent switch (Kv), calcium-activated (K _Ca ), ATP-sensitive (K _ATP ), double-pore domain (K _2P ) channels, and the like. These channels are expressed in various cancer cells from prostate, colon, lung, breast and tissue and have been reported to be associated with cancerous and malignant tumor growth. Potassium channels play a very important role in cell proliferation, apoptosis and differentiation. Some potassium channel modulators have anticancer effects by directly inhibiting cancer growth or by enhancing the efficacy of typical anticancer agents in combination therapy. In the process of turning normal epithelial cells into cancer cells, a series of genetic variations occur that can affect the expression of potassium channels or cause changes in channel activity. Therefore, by controlling the expression level and activity of these abnormal ion channels it will be possible to suppress the proliferation and metastasis of cancer cells. While the effects of Kv channels on cancer cell proliferation and tumorigenesis have been actively studied, few studies have been conducted on the relationship between K _2P channels and cancer.

Meanwhile, K _2P Channels are known as the main pathway of extracellular K ⁺ currents, which affect resting membrane potential and play an important role in the excitability of nerve or muscle cells. K _2P TASK channel among channels is K _2P It has all the characteristics of the channel and is known to be very sensitive to pH due to the antagonism of K ⁺ current flow due to acidosis of cells. To date, the TASK channel has been reported for TASK-1, TASK-2, TASK-3, TASK-4 and TASK-5. Of these, TASK-1 and TASK-3 channels with at least 50% amino acid identity form the largest homologous relationship. K _2P Dimerization of the channel forms a functional potassium channel having a total of four pore units, for example heterozygous of TASK-1 and TASK-3, in addition to homodimerization of two TASK-1 or two TASK-3 proteins Dimerization is possible.

K _2P It has been reported that various potassium channels, including channels, are amplified and overexpressed in various carcinoma tissues of humans, such as breast cancer, lung cancer, colon cancer and metastatic prostate cancer. In addition, point mutations on TASK-3 have been shown to alter tumor function and eliminate tumor formation. Thus, TASK-3 antagonists that reduce cancer development and growth have been proposed as new anticancer agents. However, despite the great physiological importance of TASK channels, little is known about the pharmacological regulation of these channels, and in particular, the association between cancer metastasis and potassium channels (K _2P ) has not been reported yet.

Therefore, the present inventors made a thorough study to determine the association between cancer metastasis and potassium channel TASK-3 expression level, K _2P TASK-3 expression, one of the channels, increases at both mRNA and protein levels in non-invasive cancer cells, and overexpression of TASK-3 inhibits cancer cell migration and invasion, while silencing thereof increases cancer cell migration. The present invention was completed by revealing that it is possible to inhibit cancer metastasis by regulating expression of TASK-3.

An object of the present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing TASK-3 protein, a gene encoding TASK-3 protein, or a gene therapy expression vector containing the gene as an active ingredient as a target molecule for inhibiting cancer metastasis. To provide.

Another object of the present invention is to provide a method for inhibiting cancer cell migration and invasion, comprising administering the pharmaceutical composition for inhibiting cancer metastasis to a subject in need thereof.

It is another object of the present invention to provide a method for screening a cancer metastasis inhibitor, comprising analyzing a change in the level of TASK-3 expression by treatment of a test substance.

Another object of the present invention is to provide a cancer metastasis predictive kit comprising an antibody specific for a TASK-3 protein or a primer pair specific for a gene encoding the protein.

Still another object of the present invention is to provide a method for screening cancer metastasis risk groups including analyzing TASK-3 expression levels from cancer tissue samples.

Another object of the present invention to provide a pharmaceutical composition for inhibiting cancer metastasis containing a PKC inhibitor that increases the expression of TASK-3 as an active ingredient, and a method for inhibiting the movement and invasion of cancer cells using the same.

In order to achieve the above object, the present invention contains a TASK-3 protein, a gene encoding the protein or a gene therapy expression vector containing the gene as an active ingredient as a target molecule for inhibiting cancer metastasis, and is pharmaceutically acceptable. It provides a pharmaceutical composition for inhibiting cancer metastasis comprising a possible carrier.

As used herein, the term "cancer metastasis" refers to the proliferation of cancer cells by leaving the primary organs and moving to other organs. The spread of cancer to other parts of the body is largely divided into primary cancers in which cancerous tissues grow and directly invade surrounding organs and distant metastases along blood vessels or lymphatic vessels to other organs in the distance.

As used herein, the term "TASK-3" is designated as KCNK9 or K _2P 9.1 as a subclass of the dual-pore domain K ⁺ channel of the various potassium channels. This channel is inhibited at acidic pH and activated at alkaline pH. Carcinoma in which the migration and invasion of cancer cells can be inhibited by the expression control of TASK-3 in the present invention is not particularly limited as long as it can cause metastasis, preferably breast cancer, lung cancer, colon cancer, prostate cancer, etc. .

In the present invention, in order to determine the role of the dual-pore domain K ⁺ (K _2P ) channel in cancer metastasis, the association of K _2P channel gene / protein level and cell migration is invasive cancer cells MCF-7 cells and non-invasive cancer cells Confirmed in MDA-MB-231. Specifically, in a preferred embodiment of the present invention, the expression of TASK-3 in non-invasive MCF-7 breast cancer cells and invasive MDA-MB-231 breast cancer cells was determined by RT-RCR, real-time PCR, Western blot and immunohistochemical staining. Comparison was made through (FIG. 1). As a result, it was confirmed that mRNA and protein levels of TASK-3 were higher in MCF-7 cells than in MDA-MB-231 cells. In addition, the effects of TASK-3 expression on the migration of breast cancer cells by 2-D and 3-D mobility analysis showed that overexpression of TASK-3 in both cells reduced cell migration and invasion, while TASK Silencing of -3 was found to increase the migration of these cells (Figure 2). As a result of examining the correlation between PKC activation and TASK-3 expression, it was confirmed that the expression of TASK-3 was inhibited by PVOC activator, poboliristate acetate (PMA), thereby increasing cell mobility (FIG. 3). . However, reduction of TASK-3 expression induced by PMA was recovered by treatment with rottlerin, a PKCδ inhibitor. The results indicate that TASK-3 expression level is associated with cancer cell migration and invasion and that TASK-3 expression level can be regulated through PKC activation. Therefore, the results suggest that metastasis of cancer cells can be regulated through the regulation of TASK-3 expression.

The present invention is the first to identify that TASK-3 expression levels, which can be regulated by PKC activation, are associated with migration invasion of human cancer cells. To date, the role of TASK-3 has been studied in relation to cell proliferation and apoptosis in cancer cells by regulation of channel activity and not by regulation of expression levels. The TASK-3 gene has been reported to be overexpressed in a number of human carcinomas including breast cancer, colon cancer, lung cancer, gastrointestinal cancer, melanoma cancer, and prostate cancer, but the association between its expression level and cancer cell metastasis has not been reported. none. The present invention reveals that TASK-3 expression levels are associated with cell migration and invasion, which are critical processes of cancer metastasis, and suggest the use of TASK-3 as a new target molecule for cancer treatment through cancer metastasis inhibition.

Accordingly, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing TASK-3 protein, a gene encoding the protein, or an expression vector for gene therapy comprising the gene as an active ingredient.

The present invention also provides a method for inhibiting cancer cell migration and invasion, comprising administering the pharmaceutical composition for inhibiting cancer metastasis to a subject in need thereof.

As used herein, the term "subject" refers to a mammalian animal having a disease in which cancer metastasis can be inhibited by administering the pharmaceutical composition of the present invention, preferably human, monkey, dog, goat, pig or rat.

In the present invention, TASK-3 protein refers to a TASK-3 protein of SEQ ID NO: 22 or a protein having a physiological activity substantially equivalent thereto. Proteins having substantially equivalent physiological activity include TASK-3 protein of SEQ ID NO: 22, and its functional equivalents and functional derivatives.

The "functional equivalent" includes an amino acid sequence variant in which part or all of the amino acid of SEQ ID NO: 22 is substituted or a part of the amino acid is deleted or added, and has substantially the same physiological activity as TASK-3 protein of SEQ ID NO: 22 Say.

The "functional derivative" is a protein that has been modified to increase or decrease the physicochemical properties of the TASK-3 protein, which means that it has a physiological activity substantially equivalent to that of the TASK-3 protein of SEQ ID NO: 22.

TASK-3 gene in the present invention is characterized in that it comprises a nucleotide sequence encoding the TASK-3 protein or a functional equivalent thereof, the nucleotide sequence includes all DNA, cDNA and RNA sequences. Preferably, the TASK-3 gene is represented by the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 22, most preferably represented by the nucleotide sequence of SEQ ID NO: 21.

In the present invention, TASK-3 protein may be prepared using DNA recombination technology, and methods known in the art may be used for this purpose. Specifically, the TASK-3 protein may be prepared by a genetic engineering method of recombinantly injecting bacteria, yeasts, plant cell lines, and animal cell lines using the TASK-3 gene.

The expression vector for gene therapy included in the pharmaceutical composition of the present invention refers to a recombinant expression vector into which the TASK-3 gene is inserted, but is not limited thereto, and preferably includes plasmids, viruses or other media known in the art. it means. In a preferred embodiment of the present invention, the TASK-3 gene was inserted into the pcDNA3.1 vector to express the TASK-3 gene and a recombinant expression vector TASK-3 / pcDNA3.1 was prepared.

In the present invention, the term "gene therapy" is intended to treat various genetic diseases and cancers caused by gene abnormalities, by directly injecting a gene associated with the disease into the patient's body and injecting the gene into cells. Refers to a method of normalizing its function by expression in. Therefore, the expression vector for gene therapy in the present invention refers to an expression vector capable of inhibiting the movement and invasion of cancer cells by directly expressing TASK-3 gene in the body.

The expression vector according to the present invention can be introduced into cells using methods known in the art. For example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells It can be introduced into cells by the method of.

In one embodiment of the present invention, the expression vector was prepared by inserting the TASK-3 gene of SEQ ID NO: 1 into the pcDNA3.1 vector, and named it 'TASK-3 / pcDNA3.1'. The prepared expression vector was transfected into MCF-7 or MDA-MB-231 cells to confirm that TASK-3 protein was successfully expressed from the expression vector.

TASK-3 protein produced by the expression vector according to the present invention is characterized by inhibiting the movement and invasion of cancer cells can effectively inhibit cancer metastasis. Therefore, the expression vector or TASK-3 protein can be used as an active ingredient of the pharmaceutical composition for inhibiting cancer metastasis.

In addition, according to the present invention, it was confirmed that the expression of TASK-3 was inhibited by the treatment of the PKC activator, and that the inhibition of TASK-3 expression was recovered by the treatment of the PKC inhibitor. It is to suggest that can be achieved through the control of.

Accordingly, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis, which contains a PKC inhibitor that induces expression of TASK-3 as an active ingredient.

The present invention also provides a method for inhibiting the migration and invasion of cancer cells, comprising administering the pharmaceutical composition to a subject in need thereof.

The term “protein kinase C (PKC)” in the present invention is the superfamily of lipid-activated Ser / Thr kinases associated with multiple signal transduction pathways. Thirteen PKC-isoforms have been identified that are classified according to their regulatory action by intracellular signaling molecules such as diacylglycerol, phospholipids and calcium. Protein kinase C is a ubiquitous phospholipid-dependent enzyme, generally associated with cell proliferation, differentiation, metabolism and apoptosis, and is known to play a number of roles in radiation-induced cellular responses involving apoptosis. By regulating the activation of PCK, it was found that the expression of TASK-3, which plays an important role in inhibiting cancer cell migration and invasion, can be regulated.

The term "PKC inhibitor" in the present invention refers to a protein that targets, decreases or inhibits not only protein kinase C but also its isoenzyme. In the present invention, a PKC inhibitor refers to a substance that inhibits PKC activity to induce overexpression of TAKS-3 or restore TASK-3 expression reduced by a PKC activator such as pobol myristate acetate.

PKC inhibitors suitable for the present invention can be used any substance known in the art to reduce or inhibit the activity of PCK, such as 1-H-pyrrolo-2,5-dione, 3- [1- [3- (dimethylamino) propyl] -1H-indol-3-yl] -4- (1H-indol-3-yl); Bisindoleylmaleimide IX; Spin high; Staurosporin and its derivatives; Tyrphostin 51; Hypericin; UCN-01; Sapphine; BAY 43-9006; Bryostatin 1; Perifosine; Llmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521; LY333531 / LY379196; Rottlerin and the like are included, but are not limited to these.

Cancer metastasis inhibiting pharmaceutical composition according to the present invention and a method of inhibiting the movement and invasion of cancer cells using the same can be effectively inhibited cancer metastasis, but not limited to these brain tumor (Brain Tumor) Low-grade astrocytoma, High-grade astrocytoma, Pituitary adenoma, Meningioma, CNS lymphoma, Oligodendrogliryn, Craniophaloma ), Ependymoma, Brain stem tumor, Head & Neck Tumor, Larygeal cancer, Oropharyngeal cancer, Nasal cavity / PNS tumor, Nasopharyngeal tumor, Salivary gland tumor, Hypopharyngeal cancer, Thyroid cancer, Oral cavity tumor, Chest Tumor, Small cell lung cancer Non-small cell Lung cancer (NSCLC), thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, breast cancer, male breast cancer, abdomen-pelvis tumor, stomach cancer cancer, liver cancer, gallbladder cancer, biliary tract tumor, pancreatic cancer, small intestinal tumor, large intestinal tumor, anal cancer (Anal cancer), Bladder cancer, Renal cell carcinoma, Prostatic cancer, Cervical cancer, Endometrial cancer, Ovarian cancer, Uterine sarcoma (Uterine sarcoma), Skin Cancer, and the like. Preferably, the cancer is breast cancer, prostate cancer, colon cancer and lung cancer.

Since the pharmaceutical composition of the present invention specifically inhibits the migration and invasion of cancer cells, effectively inhibits cancer metastasis, when used in combination with other anticancer agents known to inhibit the growth and proliferation of cancer cells, both anti-cancer and cancer metastasis inhibitory effects are observed. Achievements can lead to significant progress in chemotherapy.

Anticancer agents that can be administered in combination with the pharmaceutical composition of the present invention include DNA alkylating agent (DNA alkylating agent), methyllothamine (mechloethamine), chlorambucil, phenylalanine (phenylalanine), mustard (mustard), cyclo Phosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin (streptozotocin), busulfan, thiotepa, Cisplatin and carboplatin; Anti-cancer antibiotics include dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin (idaunorubicin), idarubicin, mitoxantrone, and pyrimamycin (plicamycin), mitomycin and C Bleomycin; And plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan And iridotecan (iridotecan) etc. can be illustrated, but it is not limited to these.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient described above for administration. As used herein, the term "acceptable carrier, excipient or diluent" refers to a carrier, excipient or diluent that does not irritate the organism and does not impair the biological activity and properties of the active ingredient administered together. The carrier, excipient and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. have. Specifically, when formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like.

In addition to the simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral administration include, but are not limited to, suspensions, solvents, emulsions, syrups, and the like, and various excipients, such as wetting agents, sweeteners, and fragrances, in addition to commonly used simple diluents such as water and liquid paraffin. , Preservatives and the like can be added. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and duration, it may be appropriately selected by those skilled in the art.

The pharmaceutical compositions of the present invention may also be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modulators for effective cancer metastasis inhibition.

The present invention also provides a cancer metastasis predictive kit comprising a TASK-3 specific antibody or a primer pair specific to a gene encoding the protein.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to the TASK-3 protein of the present invention, which is encoded by the marker gene by cloning each gene into an expression vector according to a conventional method. Proteins can be obtained and prepared from conventional proteins by conventional methods. The antibody suitable for the present invention is preferably a monoclonal antibody, more preferably a humanized monoclonal antibody.

As used herein, the term “primer” refers to a nucleic acid sequence having a short free 3′-terminal hydroxyl group, which may form a base pair that interacts with a complementary template, Short nucleic acid sequence as a starting point for copying a template. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffers and temperatures. In the present invention, by performing PCR amplification using the sense and antisense primers of the polynucleotide encoding the TASK-3 protein, it is possible to analyze the expression level of the TASK-3 protein through the generation of a desired product. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art. Primer pairs suitable for the present invention can be used without limitation as long as it can specifically recognize and amplify the gene encoding TASK-3, but is not particularly limited to, the base sequence described in SEQ ID NOs: 5 to 8 It is preferable to have.

As used herein, the term “kit” refers to a tool capable of predicting cancer metastasis by confirming mRNA expression level or protein expression level of TASK-3 gene, which is a target molecule. The cancer metastasis predictive kit of the present invention includes at least one other component composition suitable for analytical methods as well as an antibody that recognizes a primer, a probe or optionally a marker for measuring TASK-3 expression level as a target molecule for cancer metastasis diagnosis. , Solutions, or devices may be included.

The present invention also provides a method for selecting a cancer metastasis risk group using the cancer metastasis predictive kit. Screening method according to the invention

1) preparing a sample for detecting TASK-3 protein or a gene encoding the same from a cancer tissue sample;

2) detecting TASK-3 protein or a gene encoding the same using a cancer metastasis predictive kit from the detection sample; And

3) comparing the level of the detected TASK-3 protein or the gene encoding the same with the level of normal tissue to determine the possibility of cancer metastasis.

The detection sample in step 1) is not limited thereto, but may be a peeled sample, a crushed solution, or total RNA obtained from a cancer tissue sample.

Step 2) is a TASK-3 protein or a gene encoding the same from a cancer tissue sample using a TASK-3 specific antibody or a primer pair specific to the gene encoding the protein included in the cancer metastasis predictive kit of the present invention. It is a step of detecting. At this stage, the detection of the TASK-3 protein or the gene encoding it can be performed by Northern blot, Western blot, immunohistochemical staining, in situ hybridization, reverse transcriptase. The reaction may be performed using a reverse transcriptase-polymer chain reaction (RT-PCR), real-time quantitative RT-PCR (enzyme-linked immunosorbent assay), or the like.

Step 3) determines the possibility of cancer metastasis. When the level of the TASK-3 protein detected in step 2) or the gene encoding the same is lower than that of normal tissue, cancer metastasis is determined to be high and normal tissue is determined. If it is higher than the level of dark cancer is determined to be low.

The invention also provides a method for screening cancer metastasis inhibitors by targeting TASK-3 protein.

The screening method according to the present invention

1) treating a test substance to a cell comprising a TASK-3 protein or a gene encoding the same; And

2) analyzing the expression level of the intracellular TASK-3 protein or the gene encoding the same before and after treatment with the test substance, and selecting the test substance having increased therein.

In step 1), the sample may be cancer cells separated from tumor tissues of cancer patients or body fluids such as blood and lymph fluid. The sample is treated with a test substance to determine whether it is a cancer metastasis inhibitor.

The term "test substance" in the present invention is a substance capable of increasing the expression level of TASK-3, and includes without limitation oligonucleotides, proteins, compounds, and the like.

In step 2), the expression level of TASK-3 protein or gene encoding it was expressed by Northern blot, Western blot, immunohistochemical staining, in situ hybridization method, reverse transcriptase polymerization (RT-PCR), real-time quantitative RT-PCR, ELISA It detects using methods, such as these. A test substance that significantly increases the expression level of a TASK-3 protein or a gene encoding it is selected as a cancer metastasis inhibitor by comparing the detected expression level with that before treatment.

As described above, the present invention confirms that cancer metastasis can be effectively inhibited by inhibiting the migration and invasion of cancer cells through an increase in the level of TASK-3 expression. Therefore, TASK-3 of the present invention can be usefully used as a target molecule for inhibiting cancer metastasis.

1 compares the expression of double-pore domain potassium channels in human breast cancer MCF-7 cells and MDA-MB-231 cells. (A) RT-PCR amplification of calcium-activated potassium channel (BK _Ca ) mRNA in MCF-7 cells and MDA-MB-231 cells. The RT reaction under the absence of reverse transcriptase BK _Ca No genomic DNA was found. The middle lane is a 1 kb DNA marker. Right Arrow BK _Ca The sequence of PCR reactions was analyzed, indicating the expected location of the bands. MRNA expressions of (B) and (C) TASK-1, TASK-3, TREK-1, TREK-2 and TRAAK were measured by semi-quantitative PCR and real-time PCR, respectively. (D) and (E) are Western blot and immunohistochemical analysis of TASK-1, TASK-3, TREK-1, TREK-2 and TRAAK, respectively. -Labeled with rabbit IgG antibody. Nuclei were stained with PI and observed with an optical laser microscope. Scale bars represent 20 μm and IF represents immunofluorescence. Each bar represents the mean ± SD of 3 to 5 independent experiments. Asterisks each indicate a significant difference from the corresponding control (p <0.05). mRNA and protein expression levels were normalized to GAPDH and β-actin, respectively.
2 shows the role of TASK-3 in the migration of human breast cancer cells. (A) Phase difference images of wounds after 0, 9 and 24 hours after wounding of MCF-7 cells and MDA-MB-231 cells. The bar graph shows the migration capacity of MCF-7 cells and MDA-MB-231 cells. (B) Representative microscopic images of moving cells. MCF-7 cells and MDA-MB-231 cells were seeded in a transwell culture insert and incubated at 37 ° C. for 6 hours. (C) and (D) the effect of TASK-3 on the migration of MDA-MB-231 cells is shown. Decreased mobility in MDA-MB-231 cells overexpressed TASK-3 and increased mobility in cells knocked down to TASK-3 siRNA in 2-D (C) and 3- Confirmed by D (D) mobility analysis. Scale bars represent 500 μm. (E) Overexpression of TASK-3 blocks MCF-7 cell migration. All migrated cells were counted and the results are expressed in% of migrated cells compared to the initial seeded cell number. (F) Invasion assay of MDA-MB-231 cells overexpressed or knocked down TASK-3. Invaded cells were stained at the membrane bottom. All invasive cells were counted and the results are expressed as% of cells invaded compared to the number of cells initially seeded. Each bar represents the mean ± SD of 4-5 independent experiments. Asterisks each indicate a significant difference from the corresponding control (p <0.05).
Figure 3 shows the decrease in TASK-3 expression by PKC activation in MDA-MB-231 cells. (A) shows the effect of TASK-3 channel blocker on cell proliferation of MDA-MB-231 cells. (B) shows the effect of TASK-3 channel blocker on cell migration of MDA-MB-231 cells. 15,000 cells per well were inoculated into transwell culture inserts and incubated with channel blockers at 37 ° C. for 6 hours. Bupi stands for bupivacaine. (C) TASK-3 expression down regulated by PMA was measured by Western blot. a. Treatment with PMA (500 nM) inhibited TASK-3 expression. b. Pretreated with bisindole maleimide I (1 uM) and rottlerin (1 uM) for 1 hour prior to PKC activation. After stimulating cells with 10 nM PMA for 20 hours, PKC activation and TASK-3 expression were measured. The same amount (30 μg) of total protein was loaded into each lane and the molecular weight was indicated on the right side of the blot. (D) Cell migration was promoted by PMA in MDA-MB-231 cells. Each bar represents the mean ± SD of 3 to 5 independent experiments. Asterisks each indicate a significant difference from the corresponding control (p <0.05).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

All compounds were purchased from Sigma. Stock solutions of PMA (phorbol myristate acetate; 12.5 mM, Calbiochem, Darmstadt, Germany), BSM I (bisindolymaleimide I; 2.42 mM, Calbiochem) and rottlerin (5 mM) were prepared using DMSO. Bupivacaine (100 mM), ruthenium red (20 mM) and ZnCl 2 (100 mM) were dissolved in distilled water. The solutions were diluted in culture medium to the concentration used. When using DMSO as a solvent, a solution containing the same concentration of DMSO was used as a control. The final DMSO concentrations of PMA, BSM I and Lotlane are ˜0.1% v / v.

Human breast cancer cell lines, MCF-7 cells and MDA-MB-231 cells, were obtained from Korea Cell Line Bank _, 5% FBS _at 37 ° C, 95% air, 5% CO ₂ , 100 U / ml penicillin, 100 μg / ml streptomycin This was incubated in RPMI 1640 medium containing. The medium was changed to a new medium every two days.

Experimental Example  1: transfection

Recombinant expression vector TASK-3 / pcDNA3.1 was prepared by cloning the human TASK-3 gene (GenBank ^™ accession number NM 016601) and subcloning into pcDNA 3.1 vector. MCF-7 or MDA-MB-231 cells were seeded in each well of a 24-well plate at a concentration of 1 × 10 ⁵ cells and incubated in RPMI medium containing 5% FBS for 24 hours. MCF-7 cells and MDA-MB-231 cells were transfected with the recombinant expression vector TASK-3 / pcDNA3.1 using a Magnettofection ^™ system. 1 μg of DNA was mixed in 1 μl of polyMAG (Chemicell GmbH, Berlin, Germany) and incubated at room temperature for 20 minutes. The mixture was added to each well containing 500 μl serum free medium . The well plate was placed in a MagnetoFACTOR plate 24 device at room temperature for 20 minutes, replaced with fresh medium, and incubated at 37 ° C., 95% air 5% CO ₂ . Expression of the transgene was observed after two days.

Experimental Example  2 : Cell viability  analysis

Cell viability was determined colorimetrically using MTT (3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide) reagent. Cells in the logarithmic phase were seeded in 96 well plates at a density of 1 × 10 ⁴ cells / well. After 24 hours of treatment with the chemicals, 10 μl of 0.05 mg / ml MTT solution was added to each well and incubated for 4 hours. The supernatant was aspirated off and the formazan crystals in each well were dissolved in 100 μl of DMSO at 37 ° C. for 30 minutes. Absorbance values of each well were measured using a microplate reader.

Experimental Example  3: Reverse transcription -Polymerase ( RT - PCR ) analysis

The first-stranded cDNA was synthesized from total RNA of MCF-7 cells and MDA-MB-231 cells using oligo dT (SuperScript ^™ First-Strand Synthesis System for RT-PCR, Invitrogen Carlsbad, Calif., USA). First-strand cDNA was used as template for PCR amplification, each K _2P PCR was performed using a channel specific primer and Taq polymerase (Takara, Otsu, Japan). Each K _2P in Table 1 below The DNA sequence of the primer used to detect the expression of the channel is shown. PCR conditions were: initial denaturation at 94 ° C. for 4 minutes, 30 cycles at 94 ° C., 30 seconds at 58 ° C., 60 seconds at 72 ° C., and 30 cycles of amplification, and finally 10 minutes at 72 ° C. Was performed. The DNA fragments obtained by RT-PCR were sequenced using ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, CA, USA).

Experimental Example  4: real time PCR  analysis

K _2P confirmed by RT-PCR using FastStart DNA Master SYBR Green I (Roche Applied Science, Mannheim, Germany) using LightCycler System (LightCycler 2.0 instrument, Roche) Expression of the channels was quantified. Each K _2P The mRNA expression level of the channel was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers for real time PCR are shown in Table 1 above. PCR conditions are as follows. Initial denaturation at 95 ° C. for 10 minutes; 50 cycles of amplification for 7 seconds at 95 ° C., 7 seconds at 56 ° C. and 10 seconds at 72 ° C .; A melting cycle of 0 seconds at 95 ° C and 60 seconds at 65 ° C, including a step cycle starting at 65 ° C and up to 95 ° C at a transition rate of 0.1 ° C / sec; 30 seconds cooling cycle at 40 ° C. Melt-curve analysis was performed to confirm homogeneity of each product, and the product was electrophoresed on 1.2% (w / v) agarose gel.

Experimental Example  5: Weston Blotting Analysis

MCF-7 and MDA-MB-231 cells were treated with 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.5% NP-40, 1% Triton X-100, 1% Protein extraction solution containing deoxycholate, 0.1% SDS, 1 mM EDTA, 1 μM leupetin, 1 μM pepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 μM aprotinin (PRO-PREP ^™ , iNtRON Biotechnology Inc, Seongnam, Korea). The homogenization mixture was then incubated for 60 minutes in ice under intermittent vortex. The obtained extract was fractionated by centrifugation (16,609X g , Micro 17TR, Hanil, Korea) for 20 minutes at 13,000 rpm at 4 ℃. The supernatant protein thus obtained was separated using 10% SDS-polyacrylamide gel and then transferred to PVDF membrane via semi-dry transfer (Bio-Rad, CA, SUA). After blocking the membrane with 5% skim milk powder, K _2P Incubated with channel polyclonal primary antibody (1: 200 dilution), β-actin and α-tubulin monoclonal antibody (1: 10000 dilution). Primary antibodies include Chemicon (Billerica, MA, USA; TASK-3 and TREK-1), Abcam (Cambridge, UK; TASK-1 and TRAAK), Alomone Labs (Jerusalem, Israel; TREK-2), and Cell Signaling (Danvers) , MA, USA; Phospho-PKCδ) and then incubated with peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1: 10000 dilution). Immune-positive bands were observed using an ECL kit (ECL Plus kit, ELPIS, Taejon, Korea).

Experimental Example  6: immunostaining of cancer cells

MCF-7 cells and MDA-MB-231 cells incubated in poly-L-lysine coated round coverslides were fixed for 30 minutes with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS). The samples were then washed and incubated for 2 hours with gentle shaking in a blocking solution containing 1% normal serum and 0.1% Triton X-100. Cells are incubated with polyclonal antibodies against TASK-1, TASK-3, TREK-1, TREK-2 and TRAAK proteins, diluted with PBS containing 81% gelatin, 0.05% NaN3 and 1% BSA and overnight at 4 ° C. Incubated. After washing three times with PBS, cells were incubated with FITC-conjugated anti-rabbit IgG secondary antibody (1: 400) diluted 1: 400 in PBS for 1 hour and 30 minutes in the dark. The cells were treated with propidium iodide (PI) to stain the nuclei. Negative controls (NC) are cells that omit primary antibody binding, and positive controls (PCs) are cells transfected with plasmid DNA. The competition control was preadsorbed to 2 fold excess antigenic peptide. No staining of green fluorescence was observed in negative control or competition control cells, only PI staining. Stained cells were observed with an optical-laser microscope (Olympus, Tokyo, Japan).

Experimental Example  7: Mobility Analysis

For wound-healing (2-D) mobility analysis, cells were placed in a 24-well plate at 2 × 10 ^5. Inoculated at a density of cells / well. Cells were scraped off using sterile pipette tips, replaced with fresh medium and treated with mitomycin (8 μg / ml) for 2 hours to inhibit cell proliferation. After incubation for 0, 9 and 24 hours, micrographs were taken. Cells were obtained and each photomicrograph was obtained after incubation for the indicated period. Relative cell migration distances were determined by measurement of the wound depth of the tomography on a microscope (Axiovert 40C, Carlzeiss MicroImaging, Germany) and then subtracted the initial value (wound depth at the first 0 hour). Each data was obtained from three wounds in each well, expressed as a percentage of wound distance at a given time.

Transwell (3-D) mobility assays were performed using the CytoSelect ^™ Cell Migration Assay Kit (Cell Biolabs, Inc., CA, USA), including polycarbonate membrane inserts. Briefly, 300 μl of cell suspension at a concentration of 1 × 10 ⁵ cells / ml in serum-free medium was added to the upper chamber of each insert. The lower chamber contained medium containing 10% FBS to allow cell migration to the bottom surface of the transwell culture insert. Cells were incubated at 37 ° C. for 6 hours. Cells that did not migrate on the inner side of the transwell culture insert were removed by stealing with a cotton swab. Cells migrated to the bottom surface were stained with cell-staining solution (0.5% methylene blue) for 10 minutes. After photographing the cells under the microscope, the number of cells moved was counted. The stained inserts were washed, dissolved in extract solution (Triton X-100) and incubated for 10 minutes. Each sample was transferred to a 96 well microtight plate and the absorbance was measured at 560 nm using a plate reader (Infinite F200, Tecan, Switzerland).

Experimental Example  8: invasive analysis

Invasiveness of MCF-7 and MDA-MB-231 cells was measured using quantitative CytoSelect ^™ 96 well cell invasion assay (Cell Biolabs). Specifically, the invasion assay used an invasion chamber consisting of cell culture inserts placed in tissue culture wells. This insert has an 8 μm pore size polycarbonate membrane coated with a single layer of dryer bottom membrane substrate solution. MCF-7 cells and MDA-MB-231 cells suspended in a serum-free medium at a density of 2.3 × 10 ⁵ cells / ml were inoculated into the culture inserts, respectively. The cells were allowed to invade the basement membrane layer for 12 hours. Cells invaded through the membrane were stained with cell-staining solution (0.5% crystal violet), and non-invasive cells were removed from the upper surface of the membrane and observed under a microscope.

Experimental Example  9: siRNA Human using TASK -3 channels of gene Silencing

MCF-7 and MDAMB-231 cells were treated with DharmaFECT 3 transfection reagent, 100 nM of NC (ON-TARGET Non-targeting Pool, Thermo Pflugers Archiv-European Journal of Scientific Dharmacon, Lafayette, CO, USA) and PC ( transfection with human TASK-3 ON-TARGETplus SMARTpool siRNA (Dharmacon) in siGLO Lamin A / C siRNAs, Dharmacon) or serum-free medium. The target sequences of TASK-3 are AGGCAGAACGUGCGGACUC (SEQ ID NO: 23), AGGAGAAACUCAAAGCCGA (SEQ ID NO: 24), CAGAACUCCUUCAGCGCCA (SEQ ID NO: 25), and GCCUUUAGCUUUAUGUAUA (SEQ ID NO: 26). After 6 hours incubation, cells were changed to fresh medium and grown for another day. The effect of gene silencing was determined by RT-PCR and Western blot analysis. In cells transfected with 100 nM siRNA, TASK-3 mRNA and protein were reduced by 90% after 2 days of transfection. Positive controls were used for visual confirmation of transfection efficiency using fluorescently-labeled siRNA.

Experimental Example  10; Quantitative PCR  And Weston Blot  Data Analysis

Images of agarose gels and Western blots were captured with a luminescence image analyzer LAS-4000 (Fujifilm corp, Tokyo, Japan). Bands obtained from RT-PCR and Western blot analysis were quantified using Sigma Gel image analysis software (version 1.0, Jandel Scientific, San Rafael, CA, USA) or Quantity One software (version 4.6.3). Light Cycler Software 4.0 (Roche, Mannheim, Germany) and Fluoview (FV1000, Ver. 1.5, Olympus) were used to obtain real time PCR data and immunofluorescence analysis data, respectively. Relative mRNA and protein levels were calculated based on the amount of GAPDH and β-actin.

Example  One: MCF With -7 cells MDA - MB231  Comparison of Expression Patterns of Dual-pore Domain Potassium Channels in Breast Cancer Cells

First, mRNA expression levels of calcium-activated potassium channels between MCF-7 cells and MDA-MB-231 cells were compared by quantitative RT-PCR. At this time, a highly conductive calcium-activated potassium channel (BKca) expressed in MCF-7 cells was used as a positive control. As a result, as shown in Figure 1A, it was confirmed that the mRNA expression level of the BKca channel in all of these cells is similar.

Subsequently, mRNA and protein expression levels of K _2P channels (TASK-1, TASK-3, TREK-1, TREK-2, and TRAAK) in MCF-7 cells and MDA-MB-231 cells were measured by quantitative RT-PCR and Western blot. Comparisons were made using the analysis. As shown in FIG. 1B, quantitative RT-PCR analysis showed that the mRNA expression levels of TASK-3, TREK-1 and TREK-2 in MDA-MB-231 cells were significantly lower than those in MCF-7 cells. (P <0.05, n = 5). MRNA levels of TASK-1 and TRAAK were insignificant in both MCF-7 and MDA-MB-231 cells. TASK-3 and TREK-2 expression showed a positive correlation between quantitative PCR (B) and real time PCR (C) (FIG. 1C). In the K _2P channels examined, only TASK-3 showed significantly down-regulated protein expression by ˜20% in MDA-MB-231 cells (p <0.05, n = 4) (FIG. 1D), which was intracellular. Does not necessarily mean that mRNA is translated into protein.

Immunostaining results indicate that all channel proteins tested were expressed in all regions of the cell. The immunofluorescence intensity of TASK-3 expressed in MDA-MB-231 cells was lower than in MCF-7 cells, which is correlated with the results of quantitative PCR and Western blot analysis (FIG. 1E). However, unlike PCR and Western blot analysis, immunohistochemical analysis indicated that TRAAK was very strongly expressed in MCF-7 cells. In addition, the immune strength of TASK-3, TREK-1, TREK-2 and TRAAK was higher in MCF-7 cells than in MDA-MB-231 cells, whereas TASK-1 expression level was higher in MCF than in MDA-MB-231 cells. Low at -7 cells. The results strongly suggest that TASK-3 is expressed differently in MCF-7 cells and MDA-MB-231 cells. Therefore, the subsequent experiments were performed focusing on the TASK-3 channel.

Example  2: TASK -3 inhibition of cell migration by overexpression

The cell migration capacity of MDA-MB-231 cells is one of the well known features. In the present invention, the following experiment was conducted to investigate whether the difference in the TASK-3 expression level between MCF-7 cells and MDA-MB-231 cells to adjust the characteristics of these cells.

First, the mobility of MCF-7 cells and MDA-MB-231 cells was examined by 2-D and 3-D migration assays. MCF-7 and MDA-MB-231 cells were scraped off with sterile pipette tips and harvested at 0, 9 and 24 hours. As a result, the mobility was 8.4 ± 4.4% and 13.4 ± 6.6% at 9 and 24 hours in MCF-7 cells and 59.2 ± 10.0% and 86.8 at 9 and 24 hours in MDA-MB-231 cells, respectively. Mobility of ± 2.8% was shown (FIG. 2A). As a result of evaluating this mobility by 3-D mobility analysis, the mobility of MDAMB-231 cells was higher than that of MCF-7 cells (FIG. 2B).

To investigate whether TASK-3 expression modulates cell mobility, empty vectors, TASK-3, NC siRNA, and TASK-3 siRNA are transiently applied to MCF-7 and MDA-MB-231 cells. Transfection. As a result, cell migration was reduced by overexpression of TASK-3 in MDA-MB-231 cells, whereas cell migration and invasion was observed in cells transfected with empty vectors or NC siRNAs, similar to non-transfected control cells ( 2C). In the 3-D mobility assay, TASK-3 overexpression reduced the migration of MDA-MB-231 cells by 38%, while TASK-3 siRNA showed 35% increased cell migration compared to the control (FIG. 2D). As a result of 2-D and 3-D mobility analysis, it was confirmed that there is a positive correlation with the inhibitory effect of TASK-3 on cell migration. The other K _2P channels (TREK-1, TREK-2 and TRAAK) had no effect on cell migration of MDA-MB-231 cells. In addition, overexpression of TASK-3 in MCF-7 cells significantly reduced cell migration (p <0.05, n = 5) (FIG. 2E) and knock-down of TASK-3 by siRNA. Showed a tendency to improve mobility, but there was no significant difference (p> 0.05, n = 5).

Invasiveness of MDA-MB-231 cells was also reduced by TASK-3 overexpression (4.5 ± 1.2% in cells transfected with the empty vector versus 0.8 ± 0.1% in cells transfected with TASK-3), while Increased by knock-down (4.4 ± 0.5% in NC siRNA versus 8.9 ± 0.3% in TASK-3 siRNA (FIG. 2F).

The results demonstrate that TASK-3 expression levels can modulate cell migration and malignancy in MDA-MB-231 cells.

Example  3: PKC  By activation TASK -3 adjustment of expression

The following experiments were conducted to investigate whether modulation of TASK-3 channel activity regulates proliferation and migration of MDA-MB-231 cells.

The cell viability assay was performed by adding 10 μM bupivacaine, 10 μM RR and 10 μM ZnCl ₂ to the culture medium containing the cells as TASK-3 blockers. While it did not have a significant effect on the proliferation of 231 cells (Fig. 3A), it was confirmed that the cell mobility is enhanced (Fig. 3B). Bupivacaine and RR increased cell migration by 59% and 156%, respectively. ZnCl ₂ had no effect on the migration of MDA-MB-231 cells. In this regard, the present inventors conducted experiments focusing on TASK-3 expression again. It has been reported that inhibition of PKC inhibits migration of MDA-MB-231 cells, where PKC is a regulator of gene expression, in particular PKCδ is the only subtype of PKC shown to be associated with cytoskeleton associated with cell migration. to be. Based on these findings, we investigated the association between PMA activation and TASK-3 expression levels.

As a result, as shown in FIG. 3, PMA treatment (10-500 nM), which is a PKC activator, decreased TASK-3 expression in MCF-7 cells and MDA-MB-231 cells, whereas rotelin (inhibitor of PKCδ) rottlerin (1 μM) treatment maintained TASK-3 expression level without reduction. However, treatment with the PKC inhibitor BSM I (bisindolymaleimide I: 1 μM) did not show this effect. The results indicate that PKCδ may be involved in the regulation of TASK-3 expression levels.

To investigate whether PKC activation is actually involved in the mobility of MDA-MB-231 cells, PMA, BSM I and rottlerin were treated in the upper chamber of the transwell. As a result, cell migration was significantly increased by 18% in 10 nM PMA-treated MDA-MB-231 cells, whereas PSM-induced PMA induced MMA-MB-231 cells when BSM I and PMA were treated together. The increase in cell migration was reversed. In addition, treatment with PMA and rottlerin reduced the increase of PMA-induced cell migration in MDA-MB-231 cells, indicating that PKCδ is associated with the migration and proliferation of MDA-MB-231 cells. To indicate. In addition, cell migration was reduced to a lower level than the PMA untreated control. The results indicate that PKC activation induces inhibition of TASK-3 expression and promotes cell migration with low TASK-3 expression.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Suppression of cancer metastasis via upregulation of TASK-3          potassium channel <130> PA100622KR <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-1) <400> 1 cgtgtgcacc ttcacctacc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-1) <400> 2 cctcaccaag gtgttgatgc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-1) <400> 3 gtcgtgctgc gcctcaagcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-1) <400> 4 gcccgtagcc gatggtggtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-3) <400> 5 ctacgtggcc tttagcttta 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-3) <400> 6 gtcggtaaag ctgtgtaacc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-3) <400> 7 ggctccttct actttgcgat 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer (TASK-3) <400> 8 cggcgtagaa catgcaga 18 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-1) <400> 9 ggtggttgtc ctctatctga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-1) <400> 10 aagaaggaac ttcccaaatc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-1) <400> 11 gcttgcttac tttgctgctg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-1) <400> 12 agcgtgtgct ctgaactctc 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-2) <400> 13 gtcattacga ccatagggta tgg 23 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-2) <400> 14 tcaccaaagc ccaccgtggt cag 23 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-2) <400> 15 agccagaaga ataccatcgc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TREK-2) <400> 16 caagagcatg ctggatcaac 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TRAAK) <400> 17 acccagaaac caactcgacc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TRAAK) <400> 18 agcttgctcc agtcctccta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (TRAAK) <400> 19 gacccagaaa ccaactcgac 20 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer (TRAAK) <400> 20 catagccgat ggtggtgat 19 <210> 21 <211> 374 <212> PRT <213> Homo sapiens <400> 21 Met Lys Arg Gln Asn Val Arg Thr Leu Ser Leu Ile Val Cys Thr Phe   1 5 10 15 Thr Tyr Leu Leu Val Gly Ala Ala Val Phe Asp Ala Leu Glu Ser Asp              20 25 30 His Glu Met Arg Glu Glu Glu Lys Leu Lys Ala Glu Glu Ile Arg Ile          35 40 45 Lys Gly Lys Tyr Asn Ile Ser Ser Glu Asp Tyr Arg Gln Leu Glu Leu      50 55 60 Val Ile Leu Gln Ser Glu Pro His Arg Ala Gly Val Gln Trp Lys Phe  65 70 75 80 Ala Gly Ser Phe Tyr Phe Ala Ile Thr Val Ile Thr Thr Ile Gly Tyr                  85 90 95 Gly His Ala Ala Pro Gly Thr Asp Ala Gly Lys Ala Phe Cys Met Phe             100 105 110 Tyr Ala Val Leu Gly Ile Pro Leu Thr Leu Val Met Phe Gln Ser Leu         115 120 125 Gly Glu Arg Met Asn Thr Phe Val Arg Tyr Leu Leu Lys Arg Ile Lys     130 135 140 Lys Cys Cys Gly Met Arg Asn Thr Asp Val Ser Met Glu Asn Met Val 145 150 155 160 Thr Val Gly Phe Phe Ser Cys Met Gly Thr Leu Cys Ile Gly Ala Ala                 165 170 175 Ala Phe Ser Gln Cys Glu Glu Trp Ser Phe Phe His Ala Tyr Tyr Tyr             180 185 190 Cys Phe Ile Thr Leu Thr Thr Ile Gly Phe Gly Asp Tyr Val Ala Leu         195 200 205 Gln Thr Lys Gly Ala Leu Gln Lys Lys Pro Leu Tyr Val Ala Phe Ser     210 215 220 Phe Met Tyr Ile Leu Val Gly Leu Thr Val Ile Gly Ala Phe Leu Asn 225 230 235 240 Leu Val Val Leu Arg Phe Leu Thr Met Asn Ser Glu Asp Glu Arg Arg                 245 250 255 Asp Ala Glu Glu Arg Ala Ser Leu Ala Gly Asn Arg Asn Ser Met Val             260 265 270 Ile His Ile Pro Glu Glu Pro Arg Pro Ser Arg Pro Arg Tyr Lys Ala         275 280 285 Asp Val Pro Asp Leu Gln Ser Val Cys Ser Cys Thr Cys Tyr Arg Ser     290 295 300 Gln Asp Tyr Gly Gly Arg Ser Val Ala Pro Gln Asn Ser Phe Ser Ala 305 310 315 320 Lys Leu Ala Pro His Tyr Phe His Ser Ile Ser Tyr Lys Ile Glu Glu                 325 330 335 Ile Ser Pro Ser Thr Leu Lys Asn Ser Leu Phe Pro Ser Pro Ile Ser             340 345 350 Ser Ile Ser Pro Gly Leu His Ser Phe Thr Asp His Gln Arg Leu Met         355 360 365 Lys Arg Arg Lys Ser Val     370 <210> 22 <211> 1303 <212> DNA <213> Homo sapiens <400> 22 gtgggacgcg cgcggctgtg agcctgcggg acatgccccc cgcgccggct ccttgctggc 60 ggccatgaag aggcagaacg tgcggactct gtccctcatc gtctgcacct tcacctacct 120 gctggtgggc gccgccgtgt tcgacgccct cgagtcggac cacgagatgc gcgaggagga 180 gaaactcaaa gccgaggaga tccggatcaa ggggaagtac aacatcagca gcgaggacta 240 ccggcagctg gagctggtga tcctgcagtc ggaaccgcac cgcgccggcg tccagtggaa 300 attcgccggc tccttctact ttgcgatcac ggtcatcacc accataggtt atgggcacgc 360 tgcacctggc accgatgcgg gcaaggcctt ctgcatgttc tacgccgtgc tgggcatccc 420 gctgacactg gtcatgttcc agagcctggg cgagcgcatg aacaccttcg tgcgctacct 480 gctgaagcgc attaagaagt gctgtggcat gcgcaacact gacgtgtcta tggagaacat 540 ggtgactgtg ggcttcttct cctgcatggg gacgctgtgc atcggggcgg ccgccttctc 600 ccagtgtgag gagtggagct tcttccacgc ctactactac tgcttcatca cgttgactac 660 cattgggttc ggggactacg tggccctgca gaccaagggt gccctgcaga agaagccgct 720 ctacgtggcc tttagcttta tgtatatcct ggtggggctg acggtcatcg gggccttcct 780 caacctggtc gtcctcaggt tcttgaccat gaacagtgag gatgagcggc gggatgctga 840 agagagggca tccctcgccg gaaaccgcaa cagcatggtc attcacatcc ctgaggagcc 900 gcggcccagc cggcccaggt acaaggcgga cgtcccggac ctgcagtctg tgtgctcctg 960 cacctgctac cgctcgcagg actatggcgg ccgctcggtg gcaccgcaga actccttcag 1020 cgccaagctt gccccccact acttccactc catctcttac aagatcgagg agatctcacc 1080 aagcacatta aaaaacagcc tcttcccatc gcctattagc tccatctctc ctgggttaca 1140 cagctttacc gaccaccaga ggctgatgaa acgccggaag tccgtttagg ggaactaact 1200 gcacattcaa gagaggcgtc cgtggatgct gggtctcact gccaaagccg aacacggctt 1260 cgggatttct gccttctcaa gtggacctct gctgtgctgg gcg 1303 <210> 23 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TASK-3 target <400> 23 aggcagaacg ugcggacuc 19 <210> 24 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TASK-3 target <400> 24 aggagaaacu caaagccga 19 <210> 25 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TASK-3 target <400> 25 cagaacuccu ucagcgcca 19 <210> 26 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> TASK-3 target <400> 26 gccuuuagcu uuauguaua 19

Claims (13)

A pharmaceutical composition for inhibiting cancer metastasis, comprising a TASK-3 protein, a gene encoding a TASK-3 protein, or an expression vector for gene therapy comprising the gene as an active ingredient, and comprising a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 1, wherein the TASK-3 protein has an amino acid sequence as set forth in SEQ ID NO: 21. The pharmaceutical composition of claim 1, wherein the gene encoding the TASK-3 protein has a nucleotide sequence set forth in SEQ ID NO: 22. The pharmaceutical composition of claim 1, wherein the recombinant expression vector comprises a gene encoding TASK-3 protein having a nucleotide sequence set forth in SEQ ID NO: 21. The pharmaceutical composition for inhibiting cancer metastasis of claim 1, wherein the composition further comprises an anticancer agent. The method according to claim 5, wherein the anticancer agent is a DNA alkylating agent (DNA alkylating agent) methacryamine (mechloethamine), chlorambucil (chlorambucil), phenylalanine (phenylalanine), mustard (mustard), cyclophosphamide (cyclophosphamide) ), Ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin (streptozotocin), busulfan, thiotepa, cisplatin and Carboplatin; Anti-cancer antibiotics include dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin (idaunorubicin), idarubicin, mitoxantrone, and pyrimamycin (plicamycin), mitomycin and C Bleomycin; And plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan And iridotecan (iridotecan) is selected from the group consisting of, the pharmaceutical composition for inhibiting cancer metastasis. A cancer metastasis predictive kit comprising an antibody specific for a TASK-3 protein or a primer pair specific for a gene encoding the protein. 8. The cancer metastasis predictive kit according to claim 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody. According to claim 7, wherein the primer pair has a base sequence described in SEQ ID NO: 5 to 8, cancer metastasis prediction kit. 1) preparing a sample for detecting TASK-3 protein or a gene encoding the same from a cancer tissue sample;
2) detecting a TASK-3 protein or a gene encoding the same using a cancer metastasis predictive kit according to claim 8 from the detection sample; And
3) comparing the detected level of TASK-3 protein or gene encoding the same with the level of normal tissue to determine the possibility of cancer metastasis. 1) treating a test substance to a cell comprising a TASK-3 protein or a gene encoding the same; And
2) A method for screening a cancer metastasis inhibitor comprising the step of selecting the test substance having increased by analyzing the expression level of the intracellular TASK-3 protein or the gene encoding the same before and after the test substance treatment. A pharmaceutical composition for inhibiting cancer metastasis, comprising as an active ingredient a PKC inhibitor that increases the expression of TASK-3 and comprising a pharmaceutically acceptable carrier. The method of claim 12, wherein the PKC inhibitor is 1-H-pyrrolo-2,5-dione, 3- [1- [3- (dimethylamino) propyl] -1H-indol-3-yl] -4- (1H-indol-3-yl); Bisindoleylmaleimide IX; Spin high; Staurosporin and its derivatives; Tyrphostin 51; Hypericin; UCN-01; Sapphine; BAY 43-9006; Bryostatin 1; Perifosine; Llmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521; LY333531 / LY379196; Rottlerin (Rottlerin) is selected from the group consisting of, inhibiting cancer metastasis pharmaceutical composition.

Images