KR101051135B1 - Apoptosis inducer composition comprising elapin and preparation method thereof - Google Patents

Abstract

The present invention relates to a composition comprising elafin, specifically, apoptosis inducer composition of skin cancer or kidney cancer cell comprising elapine as an active ingredient, prevention of the cancer comprising elapine Or a health functional food composition for preventing and improving cancer by inducing apoptosis of skin cancer or kidney cancer cells, comprising a pharmaceutical composition for treatment, elapine, and a food supplement acceptable food additive, and (a ) Preparing a vector comprising the elapin gene; (b) transforming or transfecting the vector with a host cell; (c) a method for producing the apoptosis inducer composition, pharmaceutical composition or health functional food composition comprising recovering elapine produced from the transformant or transfectant.

Ellafin, anticancer, apoptosis, skin cancer, melanoma, kidney cancer

Description

Apoptosis inducer composition comprising elapine and a method of manufacturing the same {A COMPOSITION FOR INDUCING APOPTOSIS COMPRISING ELAFIN AND A METHOD OF PREPARING THE SAME}

The present invention relates to a composition comprising elafin, specifically, apoptosis inducer composition of skin cancer or kidney cancer cell comprising elapine as an active ingredient, prevention of the cancer comprising elapine Or pharmaceutical compositions for treatment, and methods of making the same.

Cancer is one of the most deaths in the world, with about 7.6 million people dying from cancer in 2007, and it is predicted that the age of cancer will gradually decrease, and the incidence of cancer will increase as life expectancy increases. According to the American Cancer Society (ACS) data, more than 12 million new cases of cancer were diagnosed worldwide in 2007 alone, with 7.6 million deaths, killing about 20,000 people every day.

Melanoma, a type of skin cancer, is a cancer caused by melanocyte abnormality, and the US National Cancer Institute's SEER report shows a six-fold increase in incidence compared to 1950. More than 2,000 people were diagnosed with melanoma and 8,420 died of the disease (Cancer Facts & Figures 2008, ACS 2008). Among the cancers occurring in the United States, melanoma ranks sixth in men and seventh in women and is the most common cancer among women in their twenties (Cancer Facts & Figures 2008, ACS 2008). In addition, of the three major skin cancers known as skin cancer, malignant melanoma has the highest risk of death from metastasis (Schalick et al. (1998) Blackwell Science, Inc. Malden, MA ISO-348; Jemal et al. (2001) J. Nat. Cancer Inst. 93: 678-683; Jemal et al. (2002) Ca: A Cancer Journal for Clinicians 52: 23-47).

Melanoma can be cured more than 90% by surgical treatment in the early stage, but difficult to cure due to resistance to chemotherapy when metastasis occurs. In addition, melanoma is known to have a better metastasis compared to other cancers, and is poor in response to treatment after metastasis. The average survival time for melanoma patients with metastases is 6-10 months and only 5% or less survive for more than 5 years. At the end of the disease the patient's prognosis shows a very poor life expectancy of 6 to 10 months (Jemal et al. (2002) Ca: A Cancer Journal for Clinicians 52: 23-47; Soengas et al. (2003) Oncogene 22: 3138-3151). At present, despite numerous clinical trials examining the efficacy of a variety of therapies ranging from surgical operations to immuno-, radio- and chemotherapy, no effective long-term therapy exists for patients at advanced stages of the cancer (Soengas et al. (2003) Oncogene 22: 3138-3151; Ballo et al. (2003) Surgical Clinics North Am 83: 323-342; Hersey, P. (2003) Int Med J 33: 33-43). There are currently dacarbazine and high doses of interleukin-2, which have been approved by the US FDA for the treatment of melanoma, but still exhibit only low efficacy. In order to overcome this, various therapeutic agents are in clinical trials, but no drug has yet been found to exhibit breakthrough therapeutic effects. Therefore, the development of therapeutic agents using new molecular mechanisms is required for effective melanoma treatment.

Accordingly, the present inventors have made an effort to confirm that the composition containing the elapin protein is effective for the prevention and treatment of skin cancer or kidney cancer disease, in particular for the prevention or treatment of melanoma, and completed the present invention.

An object of the present invention is to provide an apoptosis inducer composition of skin cancer or kidney cancer cells comprising elafin as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of skin cancer or kidney cancer comprising elapine.

Another object of the present invention to provide a health functional food composition for preventing and improving the cancer by inducing apoptosis of skin cancer or kidney cancer cells, including elapine and food acceptable food additives will be.

Still another object of the present invention is to prepare a vector comprising an elapin gene; (b) transforming or transfecting the vector with a host cell; (c) to provide a method for producing the apoptosis inducer composition, pharmaceutical composition or health functional food composition comprising the step of recovering the elapine produced from the transformant or transfectant.

Therefore, as one embodiment, the present invention relates to an apoptosis inducer composition of skin cancer or kidney cancer cells comprising elafin as an active ingredient.

As used herein, the term "elafin" is one of protease inhibitors, which are enzymes that inhibit the breakdown of proteins by protease. Such protease inhibitors include PMSF (phenylmethylsulfonyl fluoride), Aprotinin, Leupeptin, Na3VO4 (Sodium orthovanadate), NaF (sodium Fluoride), and the like. Elastin of the present invention is a protein known as a serine protease inhibitor among these protease inhibitors.

The elapine gene is located on the human chromosome 20q11.2-13.1 and is about 1.7 kb in size and is divided into three exons. The preproelafin produced here is a protein consisting of 117 amino acids, including 25 signal peptides, 34 pro-sequences, and 57 amino acids at the C-terminus (Saheki). T et al. (1992) Biochem Biophys Res Commun; 185: 240-5). Elastin can be induced even when excessive proliferation of epithelial cells such as psoriasis, wound healing, and cell culture, and is abundantly expressed in keratinocytes. These elapins can also be separated from the stratum corneum of human skin, acting as a strong inhibitor against elastase (Wiedow O, J. (1990) Biol Chem 265: 14791-5), and elastin from neutrophils Also inhibits Protease 3 (Wiedow OJ, (1991) Biochem Biophys Res Commun; 174: 6-10).

As used herein, the term "apoptosis (apoptosis)" is one of the methods by which cells are controlled and killed by genes, and is distinguished from necrosis of cells. Apoptosis removes abnormal cells and takes an active killing process, unlike necrosis. Apoptosis of cells by gene expression in multicellular organisms is an essential process for development and maintenance of homeostasis. In this process, cellular gene fragmentation and cell contraction occur, and fragmented cell genes are wrapped in the cytoplasm and are removed by phagocytic activity in the form of small cysts. Thus, apoptosis, unlike cell necrosis, does not cause an inflammatory response and thus plays an important role in normal development.

One of the typical features of tumors is that division and proliferation are not controlled at an appropriate level, but they progress excessively. One of the mechanisms controlling this is apoptosis, and the gene involved in this process is p53. In many higher animals, including humans, the p53 protein, which acts as a tumor suppressor, is responsible for inhibiting abnormal proliferation of cells or inhibiting or eliminating the proliferation of damaged cells. Therefore, p53 protein is associated with important factors in cells such as ATM / ATR, Chk2, mdm2, p21 and Bax, and these factors maintain a constant interaction with p53 protein, which is called p53 signaling. In addition, p53 protein is known to be associated with tumor development, inhibition and aging, and drug cytotoxicity. The p53 protein is phosphorylated by various compounds and viruses that cause nucleic acid damage and stabilizes in the cytoplasm, thereby quantitatively increasing, and the stabilized p53 protein migrates to the cell nucleus. In the cell nucleus, the p53 protein acts as a transcription factor that specifically binds to the promoter region of the target gene, causing transcription and protein production of the target protein, resulting in cell cycle pauses to repair gene damage or permanent removal of damaged cells. Causes poptosis. The composition comprising the elapin of the present invention induces apoptosis of abnormal or skin cancer or kidney cancer cells, preferably can induce apoptosis in melanoma cells. Also preferably the composition will induce apoptosis by increasing the activity of p53.

As used herein, the term "cancer" refers to a disease associated with cell death control, and refers to a disease caused by excessive proliferation of cells when a normal apoptotic balance is broken. These abnormally overproliferating cells sometimes invade surrounding tissues and organs to form masses and destroy or modify the normal structure of the body, which is called cancer. In general, the term "tumor" refers to a mass abnormally grown by autonomous overgrowth of body tissues, and may be classified into a benign tumor and a malignant tumor. Malignant tumors grow much faster than benign tumors and invade surrounding tissues, resulting in metastasis, which is life threatening. These malignant tumors are commonly called 'cancer', and the types of cancers are cerebral spinal cord tumor, head and neck cancer, lung cancer, breast cancer, thymic tumor, esophageal cancer, cancer, colon cancer, liver cancer, pancreatic cancer, biliary cancer, kidney cancer and bladder cancer. , Prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma and skin cancer, but for the purposes of the present invention include elapine The composition to induce apoptosis of skin cancer or kidney cancer cells, and is preferably useful for inducing apoptosis of malignant melanoma.

Pharmaceutical compositions comprising elapin of the present invention are useful for preventing or treating skin cancer or kidney cancer as described above, in particular the cancer may be melanoma.

Skin cancer refers to all malignant tumors occurring on the skin, including malignant melanoma, sarcoma, lymphoma, and other skin malignancies. Skin cancer is also similar to other cancers, it is common for the cells of the area to proliferate rapidly, invading surrounding tissues, and causing metastases to other areas. The frequency of such skin cancers varies according to race or region, and the frequency is high, but it is easy to identify with the naked eye. Skin cancers can be classified into primary metastases that develop from primary cancer and metastases from other organs, including primary cell carcinoma, squamous cell carcinoma, and other malignant melanoma and Kaposi's sarcoma. , Paget's disease and mycosis fungoides. Treatment methods for skin cancer include surgical resection, radiation, cryotherapy, systemic or local chemotherapy, laser treatment, or immunotherapy, but the treatment method for skin cancer using the composition containing elapin of the present invention Nothing is known to date.

Kidney cancer is a term used to describe a carcinoma of the kidney, and may be classified into two types according to the age of onset: kidney cancer in infants and kidney cancer in adults. Representative kidney cancers in infants include Wilms tumor, and representative kidney cancers in adults include Grawitz tumors. Other cancers that occur in the pyelone are urothelial carcinoma. Wilms' tumor occurs from 1 to 4 years and is considered one of the teratomas. Gravitz tumors, on the other hand, are the most frequent of adult malignant kidney tumors, accounting for about 85-95% of adult kidney cancers. It is reported to occur between 60 and 70 years of age and is reported to occur mainly in men. Diagnosis of kidney cancer is performed by radiological methods such as ultrasonography, pyelogram, renal artery, etc., and most of the treatments are performed by nephrectomy as soon as possible. By using a composition containing a protein as in the present invention, There is no known method for effectively treating kidney cancer. In a preferred embodiment of the present invention by administering elapine to the kidney cancer cell line to observe the rate of cell growth inhibition of the kidney cancer cell line, it was confirmed that elapine can be used as a treatment for kidney cancer.

Melanoma is a tumor caused by the malignation of melanocytes that produce melanin pigment, which can occur anywhere in the melanocytes, but most frequently occurs on the skin. Malignant melanoma has 1) malignant melanoma melanoma, 2) superficial diffuse melanoma, 3) distal melanoma melanoma, and 4) nodular melanoma, which has the highest metastasis rate among skin cancers. It has been reported to metastasize to any organ, including leaf nodes, bones, lungs, liver, spleen, and central nervous system. Compositions comprising elapin of the present invention are not limited to apoptosis of certain forms of malignant melanoma, and can induce apoptosis of all malignant melanoma associated with malignant melanocytes.

As another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of skin cancer or kidney cancer comprising elapine.

As described above, the composition comprising the elapin of the present invention is useful for preventing or treating skin cancer or kidney cancer, and preferably may be effective for preventing or treating malignant melanoma.

As used herein, the term "prophylaxis or treatment" refers to any action that inhibits or delays the onset of all diseases associated with skin or kidney cancer disease by using a pharmaceutical composition comprising elapine, in particular 'treatment' "I mean all the actions of improving or beneficially modifying skin cancer or kidney cancer disease using the composition. Preferably, the pharmaceutical composition comprising the elapin of the present invention can be used to effectively treat malignant melanoma by inhibiting the growth of skin cancer or kidney cancer, more preferably melanoma cells.

The present invention provides an anticancer composition for skin cancer or kidney cancer using the elapine as an active ingredient, and the anticancer composition may be more preferably a composition for treating melanoma, and the composition for treating melanoma is required. According to the present invention may further include a chemotherapeutic agent for treating melanoma.

As used herein, the term "therapeutic chemotherapeutic agent" refers to a chemical that inhibits the activity or reproduction of pathogenic microorganisms with little toxicity to the human body. The chemotherapeutic agent should be harmless to the human body or have little effect on it, exert a powerful effect on the subject such as microorganisms, penetrate to the target site when administered by the proper route of administration, have sufficient time to act, It should be easy to obtain by culture or synthesis. The elapin composition of the present invention may be used alone or in combination with one or more chemotherapeutic agents as needed. When used in combination, the chemotherapeutic agent that can be included in the pharmaceutical composition comprising the elapin of the present invention can be used without limitation as long as it satisfies the requirements of the chemotherapeutic agent, and according to conventional methods in the art, chemotherapy The kind of agent can be selected suitably, and can be used.

In addition, the pharmaceutical composition for treating skin cancer or kidney cancer comprising the elapin of the present invention may further include a pharmaceutically acceptable carrier. The composition of the present invention according to the purpose of use, oral formulations, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external forms such as sterile injectable solutions, ointments, etc. according to conventional methods, Carriers, excipients and diluents that may be included in such compositions 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, mineral oil and the like. .

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, or the like. Mix and formulate. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be used. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Bases for injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

Oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, transdermal of the composition of the present invention can be administered using a variety of methods, the dosage may vary depending on the age, sex, and weight of the patient It can be easily determined by those skilled in the art.

In addition, since the dosage of the composition may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, etc., the scope of the present invention is not limited by the dosage.

As another aspect, the present invention relates to a composition for dietary supplements for preventing and improving skin cancer or kidney cancer by inducing apoptosis of cancer cells, comprising elapine and food acceptable food additives. will be.

As used herein, the term "food supplement" means a component that can be added to food supplements, and the food supplement is added to prepare a health functional food of each formulation. It may be appropriately selected and used by those skilled in the art. Examples of food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, The pH adjusting agent, stabilizer, preservatives, glycerin, alcohol, carbonated beverages used in carbonated beverages and the like may be included, but the types of food supplements of the present invention is not limited by the above examples.

As used herein, the term "health functional food" refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, 'functional' means to obtain a useful effect for health purposes such as nutrient control or physiological action on the structure and function of the human body. The present invention is a health functional food containing elapine as an active ingredient can be prepared by a method commonly used in the art, in the preparation can be prepared by adding the raw materials and ingredients commonly added in the art. . In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a health functional food.

In another embodiment, the present invention provides a method for preparing a vector comprising: (a) preparing a vector comprising an elapin gene; (b) transforming or transfecting the vector with a host cell; (c) a method for producing the apoptosis inducer composition, pharmaceutical composition or health functional food composition comprising recovering elapine produced from the transformant or transfectant.

As used herein, the term "vector" refers to a gene construct, which is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert. Preferably, the present invention can prepare a recombinant vector comprising the elapine gene, by transforming or transfecting the prepared recombinant vector into a host cell, it is possible to obtain the elapine of the present invention do. In a preferred embodiment of the present invention, the elaffin protein was recovered by introducing the elapine gene into pYG601-mycHis (pLFG258), and transforming (or transfecting) the vector into CHO cells to culture the elapine protein. .

The recombinant vector of the present invention can be obtained by linking (inserting) the gene of the present invention into an appropriate vector. The vector into which the gene of the present invention is to be inserted is not particularly limited as long as it can be replicated in the host. For example, plasmid DNA, phage DNA and the like can be used. Specific examples of plasmid DNA include commercial plasmids such as pCDNA3.1 + (Invitrogen). Other examples of plasmids used in the present invention include E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50). There is). Specific examples of phage DNA include λ-phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP). In addition, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses can also be used.

In addition, a fusion plasmid (eg, pJG4-5) to which a gene expression activating protein (eg, B42) is linked may be used as a vector of the present invention, and the fusion plasmid may be GST, GFP, or His-tag. , Myc-tag, etc., but the fusion plasmid of the present invention is not limited by the above examples.

In order to insert the gene of the present invention into a vector, a method of cutting the purified DNA with an appropriate restriction enzyme and inserting the restriction or cloning site of the appropriate vector DNA can be used.

The gene of the present invention must be operably linked to a vector. To this end, the vector of the present invention may be used in addition to the promoter and the gene of the present invention, a cis element such as an enhancer, a splicing signal, a poly A addition signal, and a selection marker. (selection marker), a ribosome binding sequence (ribosome binding sequence, SD sequence) and the like can be further included. As an example of the selection marker, chloramphenicol resistance gene, ampicillin resistance gene, dihydrofolate reductase, neomycin resistance gene, etc. may be used, but the additional components that are operatively linked by the above examples are limited. no.

As used herein, the term "transformation" means that DNA is introduced into a host so that the DNA can be reproduced as a factor of a chromosome or by completion of chromosome integration. It means a phenomenon causing change. As the transformation method of the present invention, any transformation method may be used, and may be easily performed according to conventional methods in the art. In general, the transformation method uses the CaCl ₂ precipitation method, the CaCl ₂ method, a Hanahan method that increases efficiency by using a reducing material called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, plasma fusion method, silicon carbide Agitation with fibers, agro bacteria mediated transformation, transformation with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation.

As used herein, the term "transfection" refers to a phenomenon in which a desired nucleic acid is introduced into a host cell of a virus and is expressed in the host cell by chemically separating and purifying only the nucleic acid from the virus. , A compound word formed from transformation and infection. By separating and purifying DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from virus particles and introducing them into the original host cell and inducing viral infection, nucleic acid penetrates the cell and expresses its genetic information. Same as transformation, but also distinguished from transformation in that viral particles are formed. Such transfection may be carried out using plant viruses, animal viruses, bacterial viruses (bacteriophage), and host cells may be apes apes COS-7 or VERO cells, CHO cells (chinese hamster ovary cells), mouse L cells, Murine GH3, PC12 or NG108-15 cells, human FL, HEK293, HeLa or Jurkat cells and the like can be used, but the above examples do not limit the transfection method of the present invention.

Methods for transforming or transfecting a vector comprising the elapin gene of the present invention are not limited to the above examples, and transformation or transfection methods commonly used in the art may be used without limitation. Preferably, the present invention uses lipofectamine to introduce a vector containing elapine into the host cell.

The transformant or transfectant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the gene of interest is expressed. The host is not particularly limited as long as the host is allowed to express the gene of the present invention. Specific examples of hosts that can be used in the present invention include Escherichia bacteria, such as E. coli; Bacillus bacteria, such as Bacillus subtilis; Pseudomonas bacteria, such as Pseudomonas putida; Yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe; Animal cells and insect cells.

When a bacterium such as Escherichia coli is used as the host, the recombinant vector of the present invention is preferably capable of autonomous replication in the host, as well as a promoter, ribosomal binding sequence, the gene and transcription termination sequence of the present invention. It consists of). Specific examples of E. coli strains that can be used in the present invention are BL21 (DE3), JM109 and HB101, specific examples of Bacillus subtilis strains are WB700 and LKS87. As the promoter of the present invention, any promoter can be used so long as the gene of the present invention is expressed in a host such as E. coli. E. coli or phage-derived promoters such as, for example, trp promoter, lac promoter, PL promoter or PR promoter; E. coli infected phage-derived promoters such as the T7 promoter can be used. Artificially modified promoters such as the tac promoter can also be used. Preferably, the present invention encodes a human elapine gene to prepare a recombinant vector, and insert it into CHO cells to induce expression.

In order to facilitate the purification of the protein of interest to be recovered in the present invention, other sequences may be further included as needed in the preparation of the plasmid vector. The additionally included sequence may be a tag sequence for protein purification, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine Quiagen, USA), and most preferably 6x His (hexa histidine), but the examples do not limit the type of sequence required for purification of the target protein. In the case of a fusion protein expressed by a vector containing the fusion sequence, it may be purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of this enzyme, can be used, and when 6x His is used, a Ni-NTA His-binding resin column (Novagen, USA) can be used for desired protein. Can be easily recovered.

In addition, the method for preparing a composition comprising the elapine may further include a method further comprising (d) purifying the produced elapine. Such a protein purification method may be preferably performed using the protein purification tag, and the protein containing the purification tag may be easily purified using affinity chromatography.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for carrying out the present invention by way of example, but the scope of the present invention is not limited to these examples.

Using the elapin of the present invention, it is possible to effectively treat the cancer disease by inducing apoptosis of skin cancer or kidney cancer cells. In particular, induction of apoptosis on malignant melanoma cells can be effectively used in the development of anticancer drugs for skin cancer treatment.

Example 1 Cloning of full-length cDNA (Elafin) corresponding to differentially expressed mRNA species

Full length DNA with elapine was obtained by oligo-pulling method. Gene specific oligomers were designed based on the elaffin sequence, and the oligomers were labeled with biotin and hybridized with 2 μg single chain plasmid DNA (cDNA recombinants) from the human placenta archive using the method of Sambruck et al. (hybridization). Hybridized cDNA was isolated using streptavidin bound beads and extracted by heat. The longest cDNA was obtained by transforming the extracted cDNA into double-stranded plasmid DNA and transforming E. coli cells (DH10B). Then, positive selection was performed using PCR using gene specific primers, and DNA sequencing was performed through cDNA clones.

The nucleic acid sequence of full-length human cDNA corresponding to differentially regulated mRNA was detected with elapin. cDNA contains 351 base pairs. Northern blotting was performed to determine the size of the mRNA transcript corresponding to elapin. Northern blots were performed including total RNAs from various human tissues (Human 12-Lane MTN Blot, Clontech, Palo Alto, CA), and EST containing 40469_at sequence was radiolabeled by a random primer method. And used as a probe in the blot. The blots were hybridized at 42 ° C. in 50% formamide, 5 × SSPE, 0.1% SDS, 5 × Denhart's solution and 0.2 mg / ml herring sperm DNA and washed with 0.2 × SSC containing 0.1% SDS at room temperature. Northern blot showed a single transcript of this gene of approximately 0.35 Kb in size.

Example 2. Production of Cell Lines Transfected with Ellapine

The coding region of elapine is the forward primer (SfiIF: 5'-CTATGGCCCAGCCGGCCGCTGTCACGGGAGTTCCT-3 ') including the SfiI site and the reverse primer (SfiIB: 5'-CATAGGCCCCACCGGCCGACTGGGGAACG AAACAG) Amplification using -3 '). PCR was performed using TaqPlus precision DNA polymerase (Stratagene, CA) according to the product instructions. The PCR amplification cycle performed a total of 27 cycles for 2 minutes of initial denature at 94 ° C., 30 seconds at 94 ° C., 30 seconds at 50 ° C., and 1 minute at 72 ° C., followed by a final extension of 10 minutes at 72 ° C. The product was obtained. The PCR product was cloned into the SfiI / SfiI site of the mammalian expression vector pYG601-mycHis (Invitrogen). Cloning was performed to confirm the original structure of the replicated plasmid (pLFG258). The plasmid was stably transfected into subconfluent CHO cells with pLFG258 or pcDNA3.1-mycHis vector using LipofectaminePLUS reagent (Invitrogen) according to the manufacturer's instructions. After 24 hours, the cells were cultured in Ham's F12 (Invitrogen) containing 10% FBS and 400 μg / ml G418 (Sigma) for selection of transfected cells. The selection medium was exchanged once every two days and selected based on the cloning ring for identification of positive clones after 10-12 days. The culture was expanded to examine the expression of elapin. The cells were treated with 50 mM HEPES (pH 7.2), 150 mM NaCl, 25 mM beta-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 1% NP-40, 1 mM sodium orthovanadium oxide, 0.1 mM PMSF, and protease inhibitors. It was dissolved in lysis buffer containing a tease inhibitor cocktail (Leupeptin, Peptstatin, Aprotinin and antipain 5 μL / ml). The culture was concentrated to 3k digested Microcon (Amicon) in a volume of 15 μl. Protein was reduced by incubation at 95 ° C. for 5 min in 4 × SDS loading buffer. Polypeptides were digested at 10 mA / gel on a 12% SDS-PAGE gel and electrophoresis was performed on 0.45 μm immobilon P-transfer menmbrane (Millipore) for 2 hours at 200 mA / gel at 4 ° C. Was transfected. The membrane was blocked for 1 hour in TBST buffer (25 mM Tris, pH 7.5; 125 mM NaCl and 0.1% (v / v) Tween-20) containing 5% (w / v) nonfat milk. Blots were incubated with anti-His HRP antibody (Santa Cruz) for one day to attach probes. The immunoactive material can be visualized by the enhanced chemiluminescence method (Slpis Biotech) according to the manufacturer's instructions. As shown in Figure 2, the elapin protein was detected only in the culture supernatant, not the cell extract, and from these results confirmed the expectation that the elapine encodes secreted protein.

Example 3. Purification of Ellapine Protein

A CHO-DHFR (-) cell line capable of expressing a large amount of elapine protein was established, and the protein was expressed while growing in CHO culture in a tissue culture flask. Various methods including affinity chromatography and ion exchange chromatography may be used to purify the protein of the present invention purely, but in this embodiment, the expressed protein is the first protein that can recognize and bind to the Fc portion of the antibody. Affinity chromatography was performed using an A column. The culture solution containing the expressed antibody was once passed through a filter having a 0.45 μm membrane, and then loaded onto an equilibrated Protein A column by washing with 20 mM sodium phosphate buffer (pH 7.0). Unbound proteins were washed out by dropping 20 mM sodium phosphate buffer (pH 7.0) onto a Protein A column. The monoclonal antibody was then eluted with 100 mM sodium citrate buffer (pH 3) and the pH of the elution aliquot was adjusted to 7-7.5 with 1M Tris buffer (pH 9.0). Elution fractionated proteins were combined to form a protein pool and subjected to buffer exchange and concentration with 20 mM sodium phosphate buffer (pH 7.0) using a spin column with a 3K MWCO standard membrane for application to cation exchange chromatography. Was performed. The sample was added dropwise to cation exchange chromatography pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.0), the unbound material was washed with the buffer and 20 mM sodium phosphate, 1M NaCl (pH 7.0) solution was added to the target. The eluting antibody was eluted. As a result, the monoclonal antibody was purified in a state in which endotoxin was minimized.

Example 4 Apoptosis Induction Effect by Elastin

In order to analyze the distribution rate and apoptosis incidence of elaffin-treated cancer cells by cell cycle was performed using Annexin V apoptosis detection kit (BD pharmingen). Melanocytes A375, A2058, SKMEL5, WM2664 and normal cell lines melanocytes 1 * 10 ⁶ were treated with elapine at a concentration of 100 ng / ml and incubated at 37 ° C., 5% CO ₂ for 48 hours, then recovered. Collected cells were washed twice with PBS. The cells were mixed in 100 μl binding buffer, 5 μl V-FITC and 5 μl propidium iodide were added and allowed to react at room temperature for 25 minutes. 400 μL of the binding buffer was added and analyzed by flow cytometry.

As a result, as shown in FIG. 3A, the percentage of apoptosis-induced cells in the melanoma cell line at 100 ng / ml was 13.78% for A375, 21.2% for A2058, 24.805% for SKMEL5, and WM2664. 27.2%. However, melanocytes, normal cells, showed an apoptosis rate of only 3%. These results indicate that elapine induces apoptosis in melanoma cell lines at a concentration of 100 ng / ml, but not apoptosis in melanocytes, which are normal cells.

Example 5 Cell Distribution Induced by Ellapine

Propidium iodide was used to analyze the distribution ratio and apoptosis incidence of elaffin-treated cancer cells. Cancer cells treated with elapine at a concentration of 0-100 ng / ml were incubated at 37 ° C. and 5% CO ₂ for 48 hours, then recovered, and then collected and washed twice with PBS and 70% ethanol. Fixed at 4 ° C. for 4 hours. The fixed cells were washed with PBS and reacted for 30 minutes at 4 ° C. with PBS containing 250 μg / ml RNaseA and 25 μg / ml propidium iodine. Stained DNA content analysis was analyzed by FACS. During 40 hours of incubation, the cyclic changes of cells and the distribution of cells induced by apoptosis were measured. As a result, as shown in Table 1 and Figure 2 in the normal melanocytes, there was no difference compared to the control group not treated with elapine, but in the melanoma cell line compared with the control group not treated with elapine, the group treated with elapine In G1 / G0 was increased, it was confirmed that the S group decreases. These results indicate that treatment with melanoma in melanoma cell lines not only inhibits cell cycle regulation, but also induces apoptosis.

Example 6 Apoptosis Inducing Activity of Ellapine through Tunnel Assay

Tunnel analysis was performed to confirm DNA fragmentation, a characteristic of apoptosis. That is, by labeling the fragmented DNA of apoptosis-induced cells with TdT (Terminal Deoxynucleotidyl Transferase) enzyme, the fluorescein-12-dUTP (fluorescein-12-dUTP) is labeled on the DNA 3'-OH terminal. Measured.

Melanoma cell lines A375, A2058, SKMEL5, WM2664 and Colo829 and melanocytes were plated at 1 * 10 ⁴ cells / well, and elapine was treated at 100ng / ml each for 72 hours at 37 ° C and 5% CO ₂ . After incubation, washed twice with PBS, and the cells were fixed at 4 ° C. for 25 minutes with PBS to which 5 ml of 4% paraformaldehyde was added. After washing with 5 ml of PBS for 5 minutes, 70% ethanol was added and fixed at -20 ° C for at least 4 hours. The reaction was performed at room temperature for 5 minutes with PBS added with 0.2% Triton X-100. After washing three times for 5 minutes with 5 ml of PBS, 100 μl of equilibration buffer was added and suspended well, and reacted at room temperature for 5 minutes to remove the solution. 50 μl of TdT incubation buffer (45 μl equilibration buffer, 5 μl nucleotide mix, 1 μl TdT enzyme) was suspended in the cells, and then blocked by light. The reaction was carried out for a time. After the reaction was completed, 2XSSC was added and reacted for 15 minutes, and then washed three times for 5 minutes with 5 ml of PBS, followed by 0.5 ml of propidium iodine solution containing 250 µg DNase-free RNase A (5 µg propidium iodine, 1 ml). PBS) and DAPI were mixed well and allowed to react for 30 minutes at room temperature while blocking the light. After washing three times for 5 minutes with 5 ml of distilled water and sealed, it was observed under a fluorescence microscope. As a result, A375 was labeled with 18% fluorescence, 15% with A2058, 23% with SKMEL5, 23% with WM2664, and less than 5% with melanocytes as controls. As a result, it was confirmed that elapin induced apoptosis much more than the control as in FIG. 3B. The above results support the results of Examples 3, 4 and 7, as well as clearly confirming the apoptosis-inducing activity of cancer cells by elapine.

Example 7 Cell Growth Inhibition Activity by Elastin

Cell growth assay was performed using normal cells, melanocytes and various types of melanoma cancer cells A375, A2058, SKMEL5, WM2664 and colo829 to observe the cell growth inhibitory activity by elapine. In addition, cell growth analysis was performed using ACHN, a kidney cancer cell, to observe cell growth inhibitory activity by elapine. In a 24-well plate, 300 μl of medium in which cancer cells were adjusted to 1.5 × 10 ⁴ cells / well and 0-100 ng / ml were added thereto, and then cultured at 37 ° C. in a CO ₂ incubator for 0-10 days. Cells were incubated in a 37 ° C. CO ₂ incubator and exchanged with elapine added medium every 2 days. After 10 days the cells were harvested and stained in 0.2% Tryphan Blue solution to compare the number of viable cells with the control. As a result, as shown in Figure 1A, the cell growth inhibition rate of elapine is 20% cell growth inhibition rate in melanocytes, as shown in Table 2 of the cell growth inhibition rate of the control group, whereas the melanoma cell line 60-95% inhibition rate Showed. In addition, as shown in FIG. 1D, the cell growth inhibition rate of the kidney cancer cell line ACHN by elapin was 69%. This is the result of elapin showing cell growth inhibition in melanoma cell line and kidney cancer cells.

Cell line A375 A2085 SKMEL5 WM2664 Colo829 Melanocyte % Inhibition 60 80 65 90 95 20

In addition, cell growth assay was performed using anti-elapin antibody and control antibody to determine whether cell growth inhibitory activity occurred only in melanoma cell line. A group treated with elapine (100 ng / ml) only for 1.5x10 ⁴ cells / well cancer cells in a 24 well plate, and a group treated with elapine (100 ng / ml) and anti-elapine antibody (20 ug / ml) , 300 medium treated with the group treated with elapine (100 ng / ml) and control IgG (20 ug / ml), 37, and incubated for 2 days in a CO ₂ incubator. Cells were incubated in a 37 CO ₂ incubator, then recovered and stained in 0.2% Tryphan Blue solution to compare the number of viable cells with controls. As a result, as shown in FIG. 1B, in the group treated with elapin as compared to the control group, 60-70% of the cell activity was inhibited, but in the group treated with the anti-elapin antibody, the cell inhibitory activity was about 10-20%. In the group treated with the control antibody, it was confirmed that the cell activity was suppressed to 65-75% of the cell activity, and when the elapine was blocked with the anti-elapine antibody, less cell growth inhibition occurred. As a result, it was confirmed that the elapin showed inhibition of cellular activity of the melanoma cell line.

Example 8. Spheroid Analysis

A375, a melanoma cell line, was placed in agar (agar, DIFCO) coated plate with 25,000 / ml of 200 µl in a 96-well plate and incubated for 48 hours at 37 ° C and 5% CO ₂ . When the spheroid was obtained, the spheroid was obtained using a p1000 pipette. Obtaining spheroid cells were transplanted into 12 well plates coated with 2% Rat tail collagen. Thereafter, 100 ng / ml of elapine was treated for 48 hours, and then stained with calcein-AM and etiBr for 1 hour. At this time, the living cells are stained with calcein-AM (calcein-AM) to have a green fluorescence, and the dead cells are stained with etiBal (EtBr) to have a red fluorescence. This method is a two-dimensional observation in the usual dish (dish), but a more realistic three-dimensional method to analyze the proliferation, death and penetration of cells using the cell line. Compared to the control group not treated with elapine by the method described above, it was confirmed that many cells showing red fluorescence were stained with etiBal (EtBr) in the elapine-treated group (FIG. 3C).

Example 9 Production of Dox Inducible Cell Lines Overexpressing Ellapine

In order to produce the Dox inducible cell lines were used Tet-On ^® Advanced inducible gene expression systems ^{(Tet-On ® Advanced Inducible Gene} Expression System). Two vector systems were used to produce Dox inducible cell lines. In order to prepare an elaffin packaging cell line, first, a pTet-on plasmid (Clontech) capable of expressing a tetratracycline regulatory element (Clontech) through a gene transfer method by Lipofectin (Invitrogen) Melanoma cell line (A2058) was transfected (transfection). After transfection, the cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 400 μg / ml G418 to select only cell lines transfected with pTet-on plasmid.

The coding region of elapine is preproElafin consisting of 25 signal sequences, 34 pro-sequences, and 57 amino acids at the C-terminus. Therefore, each clone was constructed to make three different kinds of sequences.

gene Primer sequence preproelafin 5'- actacgaattcatgagggccagc-3 5'-atccagtcgaccagaaggaactcttt-3 ' proelafin 5'- actacgaattcatggctgtcacg-3 ' 5'-atccagtcgaccagaaggaactcttt-3 ' matureelafin 5'- actacgaattcatggtttcag-3 ' 5'-atccagtcgaccagaaggaactcttt-3 '

Each sequence was amplified using a forward primer containing an EcoRI site and a reverse primer containing a SalI site. PCR was performed using TaqPlus precision DNA polymerase (Stratagene, CA) according to the product instructions. PCR amplification was obtained by performing an initial denaturation at 94 ° C. for 2 minutes, 30 seconds at 94 ° C., 30 seconds at 50 ° C., and 1 minute at 72 ° C. and a final extension of 10 minutes at 72 ° C. The PCR product replicated the EcoRI and SalI sites of the mammalian expression vector pTre-tight (Clontech). Replicated plasmids (pGR288, 289, 290) were cloned to confirm the original structure. A linear hygromycin marker expressing the packaging construct pTre-tight and the hygromycin resistance gene prepared in the above example was added to a cell line containing the selected tetracycline regulatory element. Cotransfection was carried out in the same manner as above. At this time, the plasmid pTre-tight and the linear hygromycin marker were all used by Invitrogen. Using the above prepared sequence as a backbone, the hygromycin resistance gene fused with the elapine (propre-, pro-, mature-) gene of each cavity was prepared at the EcoRI / SalI restriction enzyme site. Transfected cells were selected by culturing in DMEM medium containing 200 μg / ml of hygromycin. The selection medium was changed every other day and after 10-12 days the cloning ring was used to identify positive clones. The culture was expanded to examine the expression of elapin. The cells were treated with 50 mM HEPES (pH 7.2), 150 mM NaCl, 25 mM beta-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 1% NP-40, 1 mM sodium orthovanadium oxide, 0.1 mM PMSF, and protease inhibitors. It was dissolved in lysis buffer containing a tease inhibitor cocktail (Leupeptin, Peptstatin, Aprotinin and antipain 5 μL / ml). The culture was concentrated to 3k cut Amicones made up to a volume of 3 ml. Protein was reduced by incubating for 5 min at 95 ° C. in 4 × SDS loading buffer. The polypeptide was digested at 10 mA / gel on a 12% SDS-PAGE gel and subjected to electrophoresis on 0.45 μm immobilon P-transfer menmbrane (Millipore) for 2 hours at 200 mA / gel at 4 ° C. It was transfected. The membrane was blocked for 1 hour in TBST buffer (25 mM Tris, pH 7.5; 125 mM NaCl and 0.1% (v / v) Tween-20) containing 5% (w / v) nonfat milk. The blot was reacted with the first anti-Elafin antibody (Santa Cruz), and the second reaction with the anti-His HRP antibody (Santa Cruz) for 30 minutes followed by ECL (amersham). It was confirmed by labeling on X-ray (amersham). As a result, as shown in Fig. 4A, the overexpressed elapine was confirmed by Western blot, while in the control group not treated with doxycycline (Doxycycline), the expression was low, but it was confirmed that the overexpressed in the cell line in which doxycycline was added.

Example 10 Cell Growth Inhibition, Cell Change Observation and Tunnel Analysis of Dox Induced Cell Lines Overexpressed Ellapine

Cell growth assays were performed using A2058, a selected melanoma cell line overexpressed with elafin (prepro-elafin, pro-elafin). Cancer cells were treated in a 24-well plate at a concentration of 1000 ng / ml of doxycycline in a medium adjusted to 1.5 × 10 ⁴ cells / well and incubated at 37 ° C. in a CO ₂ incubator for 0-4 days. . Cells were incubated at 37 ° C. in a CO ₂ incubator and exchanged with elapine added medium every two days. After 4 days, the cells were harvested and stained in a 0.2% Tryphan Blue solution to compare the number of viable cells with controls. As a result, cell proliferation was inhibited by 45% on day 2 and by 60% on day 4, compared to the control group, compared to the control cell line transformed with elapin overexpression. Pro-elafin was inhibited by 40% at 2 days and 58% at 4 days. It was confirmed that overexpression of elapine showed the same aspect as the protein treatment, showing cell growth inhibition by elapine (FIG. 4B).

In addition, changes in cell morphology were observed with or without apoptotic cell appearance when doxycycline was treated in the same cell line. Experiments were carried out under the same conditions as cell growth inhibition. Shape observation was performed using a Nikon microscope, and as a result, when compared with the vector control, it was confirmed that the nucleus of the apoptotic cells was condensed and the cells became smaller. As a result, it was confirmed that apoptosis by elapin was induced (FIG. 4C).

In addition, tunnel analysis was also performed to identify cell segments. The experimental method of Example 6 was followed and analyzed. As a result, the fluorescent substance was not seen in the vector control, and the fragmented DNA of the cell could be confirmed in the cell line overexpressing the elapine (FIG. 4D). This proves that elapin induces apoptosis.

Example 11. Activity analysis of p53 and its subgenes by elapin

To investigate the degree of p53 activity induced by elapine, 13 copies of the p53 binding site derived from the ribosomal gene cluster upstream of the polyoma virus minimal promoter were lucifered. PG13-luciferase vector located in front of the luciferase gene (provided by Prof. Hong-Gil Yoon of Seoul National University; El-Deiry W, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B. Nat Genet. 1:45 -49 (1992); El-Deiry W, Tohino T, Velculescu VE, Levy DB, Parsons R, Trent JR, Lin D, Mercer WE, Kinzler KW, Vogelstein B. Cell. 75: 817-825 (1993)). Luciferase assay was used. PG13-luciferase, p21-luciferase, p21-luciferase, Bax-luciferase (Bax-luciferase) on melanoma cell lines A375, A2058, SKMEL5, WM2664 and Colo829 and normal cell lines melanosite ), NOXA-luciferase, NOXA mut-luciferase and NOXA del-luciferase, respectively, as lipofectamin (Invitrogen). After 4 hours after transformation, the medium containing fetal bovine serum (FBS) was stabilized, and then the medium was removed, and the cells were dissociated after treatment with elapine at a concentration of 100 ng / ml for 48 hours. After centrifugation of the dissociated cell solution at 1000rpm, only the supernatant was collected, and a certain amount of firefly luciferase and renilla luciferase were reacted, and then the amount of luciferase emission was measured to determine p53, p21, Bax, The degree of NOXA activity was quantified. As a result, when compared with the control as shown in Figure 6B, it was confirmed that the difference was 9.2 times for A375, 7 times for A2058, 6.8 times for SKMEL5, 6 times for WM2664, and 13 times for Colo829. Similar results were obtained in p21, Bax and Noxa, respectively, confirming that elapin induces apoptosis through p53.

Example 12 Analysis of Activity Levels in the Treatment of Inhibitors of p53 by Elafin

P53 siRNA (cel signaling) and PFT-a (Biomol) were used to determine the degree of inhibition of p53 activity by elapin. PG13-luciferase, p21-luciferase, Bax-luciferase, NOXA-luciferase, NOXA mut-luciferase and NOXA del in melanoma cell lines A375, A2058, SKMEL5 and WM2664 and normal cell lines melanocytes P53 siRNAs were transfected with lipofectamin (Invitrogen) as shown in the luciferase vector, and then stabilized by adding medium containing fetal bovine serum (FBS) 4 hours later. The cells were dissociated after 48 hours of treatment with the pin at a concentration of 100 ng / ml. PFT-α, an inhibitor of p53 activity, was treated with elapine at a concentration of 20 uM for 48 hours. After centrifugation of the dissociated cell solution at 13000 rpm, only the supernatant was collected, and a certain amount of firefly luciferase was reacted with Renilla luciferase, and then the amount of luciferase emission was measured to determine the p53, p21, Bax, The degree of activity was quantified. As a result, as compared with the control siRNA-treated control group as shown in Figure 7, it was observed that the p53 siRNA-treated A375, A2058, SKMEL5 and WM2664 the p53 activity is inhibited in the elapine-treated group, Figure As in 8, A375 and WM2664 in the group treated with the p53 inhibitor PFT-α confirmed that p53 activity decreased when the elapine treatment, it was confirmed that the elapine regulates the p53 activity.

Example 13. Apoptosis Induction by Caspase-3, 8, and 9 Activity Assays

After lysis buffer was added and mixed well, it was left at room temperature for 10 minutes. Take these cell lysates and transfer them to the fluorescence measurement plate, and add 100 µl of analytical buffer and 10 µl of Kephase-3, Kephase-8 and Kephase-9 substrates, and mix and react for 1 hour at 37 ° C. Fluorescence activity was measured by the emission at 400 nm and the absorption at 505 nm. The results are expressed as the ratio of the control group and the experimental group. Kespase-3 showed a 2-4 fold difference in activity compared to the control group, while Kespaz-9 showed a 2 ~ 3 fold difference in activity, while Kespaz-8 did not show any difference from the control group.

The melanoma cell lines A375, A2058, SKMEL5, WM2664 and Colo829 and normal melanocytes were laid at 1x10 ⁶ / well, treated with elapine at a concentration of 100 ng / ml, and the cells were recovered 48 hours later. Sediment Dissolve in 50 μl of cold lysis buffer. This was left on ice for 10 minutes, and then the cell lysate was centrifuged at 4 ° C and 12,000 rpm for 3 minutes to transfer the supernatant to a new tube. 2 μl of the inhibitor was mixed in the sample to be treated with the kease phase-3, the kease phase-8 (peptron), and the ke phase 9 (Clon tech) inhibitor, and allowed to react at room temperature for 5 minutes. 50 μl of 2 × reaction buffer with freshly added 10 mM DTT was added. 2 μl of substrate was added to each reaction solution, and the reaction was carried out at 37 ° C. for 30 minutes.

Kess Phase temperament Inhibitor Cez Phase-3 2.5mM Ac-DEVD-AMC 2.5mM Ac-DEVD-CHO Kephas-8 2.5mM Ac-IETD-AMC 2.5mM Ac-IETD-CHO Kephas-9 2.5mM Ac-LEHD-AMC 2.5mM Ac-LEHD-CHO

The reaction sample was measured using a fluorescence spectrometer (LS50B) using a 360-nm emission filter and a 460-nm emission filter. As a result, as shown in Figure 5 represented by the ratio of the control group and the experimental group. Kespase-3 showed a 2-4 fold difference in activity compared to the control group, while Kespaz-9 showed a 2 ~ 3 fold difference in activity, while Kespaz-8 did not show any difference from the control group.

Example 14. Western Blot

Since apoptosis of melanoma cells is observed when melanoma is treated with melanoma cell lines, in order to investigate the effect of elapin on the expression of p53 and its genes that are involved in the regulation of apoptosis, A375 Melanoma cell lines and melanocytes, A2058, SKMEL5, WM2664 and Colo829, were treated with 1 × 10 ⁶ / well, treated with elapine at a concentration of 100 ng / ml, and then recovered cells after 48 hours, lysate. ) And 20 μg / ml of protein was mixed and heated with SDS-PAGE loading buffer to denature the protein. Subsequently, the cell debris was separated by centrifugation, the supernatant was taken, and 60 µl of these was electrophoresed at 100 volts in 10% SDS-PAGE for 3 hours. The gel is then transferred to the transfer membrane and transferred. The membrane was blocked for 1 hour in TBST buffer (25 mM Tris, pH 7.5; 125 mM NaCl and 0.1% (v / v) Tween-20) containing 5% (w / v) fat-free milk. Primary antibody was diluted 1: 1000 in 5% BSA solution. The blots include primary anti-p53 (cell signaling), anti-p21 (cell signaling), anti-Bax (cell signaling), anti-PUMA (cell signaling), anti-NOXA (cell signaling), and anti-actin (action) ) Was reacted with Santa Cruz, and the secondary was reacted with heath HRP antibody (anti-IgG HRP antibody; Santa Cruz) for 30 minutes and then reacted with ECL (amersham) to label X-ray (amersham). . In the results of FIG. 6A, there was no change in the melanocytes treated group in melanocytes, but the melanoma cell line increased the expression of the protein compared to the group not treated with elapin.

Example 15 Cancer Cell Growth Inhibition Experiments of Ellapine in a Mouse Model

5 × 10 ⁶ SKMEL5 cells were implanted subcutaneously in 6-7 week old nude mice Balb / c nu / nu. From day 10, the monoclonal antibody was injected into the tumor at a concentration of 4 mg / kg twice a week for 3 weeks. Tumor volume was measured twice a week using a caliper. On the final day of the experiment nude mice were sacrificed using CO ₂ and then tumors were isolated and weighed. In addition, tunnel analysis was performed to determine if apoptosis was induced. 9A and 9B confirmed that the tumor size was reduced by 40%, and in the tunnel analysis, the results of FIG. 9C confirmed that the apoptotic cells increased by 30% compared to the control group.

1 shows the growth inhibition of melanoma cell lines (A375, A2058, SKMEL5, WM2664 and Colo829) and kidney cancer cell lines (ACHN) by elapine.

2 shows cell cycle changes of melanoma cell lines (A375, A2058, SKMEL5, WM2664 and Colo829) and melanocytes by elapine

Figure 3 shows the double staining, TUNEL analysis and spheroid analysis of melanoma cell lines (A375, A2058, SKMEL5, WM2664 and Colo829) and melanocytes by Annexin V / PI by elapine.

Figure 4 shows elapin expression, growth inhibition and apoptosis induction in elapine overexpressing cell lines.

5 shows the caspase activity by elapine.

Figure 6 shows an increase in the expression of p53 and p53 subgenes by elapin, and shows an increase in the activity of p53 by the reporter assay according to the p53 dependent pathway.

Figure 7 shows the activity of p53 by elapine by siRNA treatment, comparing the difference with the control.

Figure 8 shows the activity of p53 by elapine in comparison with the control treated p53 activity inhibitors.

Figure 9 shows the therapeutic effect on the growth of SKMEL5 melanoma cell line xenografts in mice by elapin.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A COMPOSITION FOR INDUCING APOPTOSIS COMPRISING ELAFIN AND A          METHOD THE SAME <130> PA080543 / KR <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer for elafin comprising SfiI <400> 1 ctatggccca gccggccgct gtcacgggag ttcct 35 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> backward primer for elafin comprising SfiI <400> 2 cataggcccc accggccgac tggggaacga aacag 35 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for preproelafin <400> 3 actacgaatt catgagggcc agc 23 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> backward primer for preproelafin <400> 4 atccagtcga ccagaaggaa ctcttt 26 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for proelafin <400> 5 actacgaatt catggctgtc acg 23 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> backward primer for proelafin <400> 6 atccagtcga ccagaaggaa ctcttt 26 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mature elafin <400> 7 actacgaatt catggtttca g 21 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> backward primer for mature elafin <400> 8 atccagtcga ccagaaggaa ctcttt 26  

Claims (13)

Apoptosis inducer composition of skin cancer cells comprising elafin as an active ingredient. The apoptosis inducer composition of claim 1, wherein the skin cancer cells are malignant melanoma cells. The apoptosis inducer composition of claim 1, wherein the elapin increases p53 activity to induce apoptosis. Pharmaceutical compositions for the prevention or treatment of skin cancer or kidney cancer comprising elapine. The pharmaceutical composition of claim 4, wherein the skin cancer is malignant melanoma. The pharmaceutical composition of claim 5 further comprising a chemotherapeutic agent for treating melanoma. The pharmaceutical composition of claim 4, wherein the composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 4, wherein the prevention or treatment of skin cancer induces apoptosis of cells. The pharmaceutical composition according to claim 4, wherein the treatment of skin cancer induces apoptosis by increasing the activity of p53. delete delete delete delete

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