KR102238927B1 - Peptide controlling migration of NAG-1 protein in cell and uses thereof - Google Patents

Abstract

The present invention relates to a peptide that modulates the movement of NAG-1 protein in a cell and its use, wherein the peptide of the present invention increases the accumulation of NAG-1 protein in the cytoplasm and thus is useful for anticancer, antiobesity, and anti-inflammatory treatment. -1 It can maximize the action of protein.

Description

Peptide controlling migration of NAG-1 protein in cell and uses thereof

The present invention relates to a peptide that modulates the movement of NAG-1 protein in cells and uses thereof.

Among proteins, it is termed "moonlighting protein" when it exhibits various functions according to its intracellular location. In addition to the conventional cell research, it is called NAG-1 (or NAG-1/GDF15), which is an additional functional protein. ), a new mechanism for how proteins enter the nucleus and how they exit the cell, it is necessary to study the physiological function of each cell's location through this movement pathway.

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) showed 15-29% identity to the 7-cysteine region (domain) that characterizes the TGF-β superfamily cytokine in sequence analysis. As belonging cytokines, macrophage inhibitory cytokine 1 (MIC-1), growth differentiation factor 15 (GDF15), prostate-derived factor (PDF), placental bone Also known as placental bone morphogenetic protein (PLAB) and placental transforming growth factor-β (PTGF-β). NAG-1 protein It is known that it has many biologically active functions like other TGF-β proteins, and is known to affect the progression and prevention of adult diseases such as cancer, inflammation or obesity. NAG-1 is synthesized as pro-NAG-1 consisting of 308 amino acids in the cell, and is secreted outside the cell by using vesicles, at which time the C-terminal 112 amino acids form a coalescence by a specific disulfide bond. The formed C-terminal dimer and pro-NAG-1 dimer have various functions outside the cell, but the exact mechanism of action shows contradictory results in several papers, and it is believed that they have different effects depending on the expression in the cell and the presence or absence of a receptor. . Recently, since NAG-1, a secreted protein, is expressed in the nucleus and is used for various mechanisms of action such as regulating transcription, studies on various functions of NAG-1 are required.

According to the present invention, after NAG-1 is made in the cytoplasm, it moves to the nucleus, and then shows a dynamic migration pattern in which it moves out of the cell again. It was found that the function of NAG-1 varies depending on the location in each cell. By studying nuclear receptor regulatory mechanisms such as CRM1 (chromosome region maintenance 1), which is an important nuclear protein for cell migration, and the migration pathway of NAG-1, it will contribute to studies such as cancer, inflammation, and obesity.

On the other hand, Korean Patent No. 1998029 discloses'Antibody specifically binding to MIC-1 protein and its use', and Korean Patent Publication No. 2010-0114259 discloses '6,7-dihydroxy-2, A composition for inhibiting proliferation of cancer cells containing 4-dimethoxy phenanthrene is disclosed. However, there has been no disclosure of the'peptide that regulates the movement of NAG-1 protein in cells and its use' of the present invention.

The present invention was derived from the above requirements, and the present inventors used a nuclear localization signal (NLS) peptide produced by mimicking the nuclear localization signal of NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) protein. When treated with cancer cells, it was observed that the migration of NAG-1 protein to the nucleus was inhibited, and the loss of mitochondrial membrane potential of colon cancer cells was confirmed to induce apoptosis of colon cancer cells, thereby confirming that the present invention Was completed.

In order to solve the above problems, the present invention is a nuclear localization signal (NLS) peptide that inhibits the migration of the NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) protein to the nucleus, consisting of the amino acid sequence of SEQ ID NO: 1. Provides.

In addition, the present invention provides a polynucleotide encoding the NLS peptide.

In addition, the present invention provides a recombinant vector comprising the polynucleotide.

In addition, the present invention provides a method for producing an NLS peptide comprising the step of overexpressing a polynucleotide encoding an NLS peptide by transforming a host cell with the recombinant vector.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, inflammatory disease or obesity containing the NLS peptide as an active ingredient.

The present invention provides an NLS peptide that inhibits the migration of NAG-1 protein to the nucleus, thereby increasing the accumulation of NAG-1 protein in the cytoplasm to maximize the action of NAG-1 protein useful for anticancer, antiobesity, and anti-inflammatory treatment. I can.

1 shows the amino acid sequence of an NLS peptide prepared by mimicking the nuclear localization signal of a nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) protein. A represents an NLS peptide with improved acetylation at the N-terminus and amination stability at the C-terminus, and B represents an NLS peptide tagged with FITC (fluorescein isothiocyanate) instead of acetylation at the N-terminus. Show.
2 is a photograph taken with a fluorescence microscope after transfection of the NLS peptide into HCT116 cells using a PULSin delivery reagent. A is a negative control treated with only HEPES buffer; B is a positive control treated with HEPES buffer, R-phycoerythrin (R-PE) and PULSin; C is an experimental group treated with HEPES buffer, NLS-FITC peptide and PULSin; D is an experimental group treated with HEPES buffer and NLS-FITC peptide without PULSin. Orange fluorescence indicates R-phycoerythrin (R-PE), and green fluorescence indicates FITC tagged to NLS peptide.
Figure 3 is a picture taken with a confocal microscope after transfection of the NLS peptide into HCT116 cells using a PULSin delivery reagent, fixation with 4% paraformaldehyde, and staining the nucleus with DAPI. The left is a negative control group treated with only HEPES buffer, and the right is an experimental group treated with HEPES buffer, NLS-FITC peptide and PULSin. Blue fluorescence indicates DAPI stained nuclei, and green fluorescence indicates FITC tagged to NLS peptide.
Figure 4 is a picture taken in a time lapse of the NLS-FITC peptide entering the cell using the CellInsight CX7 LZR High-Content Screening (HCS) Platform to confirm the temporal or spatial activity of living cells. . The left is a picture after 1 hour and 30 minutes after treatment with NLS-FITC peptide and PULSin, and the right is a picture after 3 hours and 30 minutes.
FIG. 5 shows HCT116 cells (HCT116-NAG-1-GFP) overexpressing GFP (Green fluorescence protein) conjugated NAG-1 were transfected using an NLS peptide and a PULSin delivery reagent and cultured for a certain period of time, CellInsight It is a picture (A) confirming the GFP fluorescence intensity expressed in the cell nucleus and cytoplasm using the CX7 LZR HCS Platform, and a graph (B) confirming the distribution ratio of the nucleus and cytoplasm of NAG-1. In (B), *** means that the decrease in the nuclear/cytoplasmic distribution ratio of NAG-1 protein is statistically significant when NLS peptide is treated compared to the group not treated with NLS peptide (Control), and the p value is 0.001. Means less than, ** means that the decrease in the nuclear/cytoplasmic distribution ratio of the NAG-1 protein is statistically significant when treated with the NLS peptide compared to the group not treated with the NLS peptide (Control), and the p value is 0.01 Means less than, and NS means that there is no statistically significant difference.
6 is a GFP-conjugated HCT116 cells (HCT116-NAG-1-GFP) overexpressing NAG-1 were transfected using an NLS peptide and a PULSin delivery reagent and cultured for 6 hours, followed by MitoTracker ^TM Orange CM TM Ros A photograph taken after staining the mitochondria of viable cells using (A) and a graph (B) showing the mitochondrial membrane potential by measuring the fluorescence intensity of ^{MitoTracker TM Orange CM TM Ros.} In (A), blue fluorescence indicates DAPI staining, green fluorescence indicates GFP tagged to NAG-1, orange fluorescence indicates MitoTracker ^TM Orange CM TM Ros staining, and in (B) trans-chalcone indicates It was used as a positive control to reduce mitochondrial membrane potential, and *** indicates that the loss of mitochondrial membrane potential was statistically significant when NLS peptide was treated compared to the group not treated with NLS peptide (Control); Compared to the group treated with trans-chalcone alone (trans-chalcone), when treated with NLS peptide and treated with trans-chalcone, mitochondrial membrane potential loss was statistically significant, and p value was less than 0.001.

In order to achieve the object of the present invention, the present invention is a nuclear localization signal (NLS) that inhibits the migration of the NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) protein to the nucleus, consisting of the amino acid sequence of SEQ ID NO: 1 ) Peptides are provided.

The scope of the NLS peptide according to the present invention may consist of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a polynucleotide encoding the NLS peptide. The polynucleotide of the present invention may be DNA or RNA encoding the NLS peptide. DNA includes cDNA, genomic DNA or artificial synthetic DNA, which may be single-stranded or double-stranded.

Preferably, the polynucleotide of the present invention may include a nucleotide sequence represented by SEQ ID NO: 2. In addition, homologs of the nucleotide sequence are included within the scope of the present invention. Specifically, the polynucleotide is a base sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 2 It may include. The "% of sequence homology" for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including). The nucleotide sequence of SEQ ID NO: 2 of the present invention is a polynucleotide encoding a full-length NLS peptide.

In addition, the present invention provides a recombinant vector comprising the polynucleotide.

The term "vector" is used to refer to a DNA fragment(s), a nucleic acid molecule, which is delivered into a cell. Vectors replicate DNA and can be reproduced independently in host cells. The term “carrier” is often used interchangeably with “vector”. The term "expression vector" refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a particular host organism. In general, any plasmid and vector can be used as long as they can replicate and stabilize in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element. Translational regulatory elements that can be used in eukaryotic cells, enhancers, ribosome binding sites, termination signals, polyadenylation signals, and promoters are known in the art. The expression vector can be constructed by a method well known in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. In addition, the expression vector may include a ribosome binding portion and a transcription terminator as a translation initiation site.

The vectors of the present invention can typically be constructed as vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pLλ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.), It is common to include a ribosome binding site and a transcription/translation termination sequence for initiation of translation. When E. coli is used as a host cell, the promoter and operator site of the tryptophan biosynthetic pathway of E. coli, and the left-facing promoter (pLλ promoter) of the phage (λ) may be used as the regulatory site.

Meanwhile, vectors that can be used in the present invention include plasmids that are often used in the art (eg pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (eg, λgt4·λB). , λ-Charon, λΔz1 and M13, etc.) or a virus (eg, SV40, etc.). On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter) or a promoter derived from a mammalian virus (eg, adeno Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may contain an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin. And resistance genes for tetracycline.

Host cells capable of stably cloning and expressing the vector of the present invention can be used any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, Bacillus strains such as E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis , Bacillus thuringiensis , Salmonella typhimurium , Serratia marcescens , and Enterobacteriaceae and strains such as various Pseudomonas species.

In addition, when transforming the vector of the present invention into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells can be used.

The method of transporting the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)). ), one method (Hanahan, D., J. Mol. Biol., 166:557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)) and Gene Bombadment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) The vector can be injected into the host cell by, for example.

In addition, the present invention provides a method for producing an NLS peptide comprising the step of overexpressing a polynucleotide encoding an NLS peptide by transforming a host cell with the recombinant vector.

In the method for producing the peptide of the present invention, transformed host cells are cultured in a medium suitable for production of a recombinant protein (peptide) using a known technique. For example, host cells can be cultured by small-scale or large-scale fermentation, shake flask culture in laboratory or industrial fermentors conducted under conditions that permit expression and/or isolation of suitable media and proteins. Cultivation takes place in a suitable medium containing carbon, nitrogen sources and inorganic salts using known techniques. Suitable media can be obtained commercially or can be prepared according to the ingredients and composition ratios described in publications such as, for example, catalogs of the American Type Culture Collection (ATCC).

In the above production method, the expressed protein (peptide) can be recovered using a protein separation method known in the art. For example, the protein may be separated from the nutrient medium by conventional methods including centrifugation, filtration, extraction, spray drying, evaporation or precipitation, but is not limited thereto. Furthermore, proteins can be obtained through a variety of known methods including chromatography (e.g. ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractionation (e.g. ammonium sulfate precipitation), SDS-PAGE or extraction. Can be purified.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, inflammatory disease or obesity containing the NLS peptide as an active ingredient.

In the pharmaceutical composition according to the present invention, the NLS peptide may consist of the amino acid sequence of SEQ ID NO: 1.

In the pharmaceutical composition of the present invention, the cancer is laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, breast cancer, cervical cancer, ovarian cancer, kidney cancer, gallbladder cancer, oral cancer, colon cancer, It may be any one selected from the group consisting of liver cancer, bladder cancer, and colon cancer, preferably colon cancer, but is not limited thereto.

The NLS peptide of the present invention can inhibit the migration of NAG-1 protein into the nucleus in cancer cells, reduce mitochondrial membrane potential of cancer cells, and induce death of cancer cells.

In addition, the inflammatory diseases are allergy, dermatitis, atopic, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, Any one selected from the group consisting of psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder joint inflammation, tendinitis, tendonitis, tendonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases. May be, but is not limited thereto.

In addition to the NLS peptide, the pharmaceutical composition of the present invention may further include a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage form of the pharmaceutical composition of the present invention may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate combination.

The pharmaceutical composition according to the present invention may be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and injections according to a conventional method. . Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in the fermented product. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

Hereinafter, it will be described in detail by examples of the present invention. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

1. Cell preparation

Autoclave and PBS-washed coverslips were laid on the bottom of a 6-well plate, and then colon cancer cell line HCT116 (6×10 ⁵ cells/well) was seeded in each well. 3 ml of RPMI 1640 medium containing sugar FBS (fetal bovine serum) was dispensed.

2. Reagent mix formulation

As a negative control, only 200 μl of 20 mM HEPES buffer was used, 200 μl of 20 mM HEPES buffer, and 4 μg and 16 μl of fluorescent protein R-phycoerythrin (R-PE) were used as a positive control. Was used, which was treated with the PULSin ^TM delivery reagent (Polyplus-transfection), and the NLS peptide of the present invention (amino acid sequence) prepared by mimicking the nuclear localization signal (NLS) of the NAG-1 protein and 200 μl of 20 mM HEPES buffer as an experimental group. : CRLHTVRA, SEQ ID NO: 1) and 16 µl of PULSin treated and 20 mM HEPES buffer 200 µl and 4 µg NLS peptide treated without PULSin treatment were used.

3. Peptide Transfection

After diluting 4 µg of NLS peptide in 200 µl of 20 mM HEPES buffer, it was slowly vortexed and spun down briefly. The PULSin delivery reagent was vortexed for 5 seconds and then spun down before use. The 16 µl of the PULSin delivery reagent was added to the prepared HEPES buffer and NLS peptide mixture, vortexed, spun down briefly, and incubated at room temperature for 15 minutes to form a complex. The wells inoculated with HCT116 were washed with serum-free medium or 1×PBS (phosphate buffer saline). Thereafter, 2.8 ml of serum-free culture medium per well was dispensed, and 200 µl of NLS peptide and PULSin complex were added and mixed, and then slowly homogenized, followed by incubation at 37° C. for 4 hours. After the cultivation was completed, the medium was removed and replaced with 1×PBS.

4. Cell fixation

After the peptide transfection process, 1×PBS was removed, and cells were fixed by treating each well with 4% paraformaldehyde.

5. DAPI staining and encapsulation

100 µl of DAPI (4',6-Diamidino-2-Phenylindole) working solution was dropped on a coverslip, and the plate was wrapped with silver paper, incubated for 15 minutes at room temperature, and washed 3-4 times with 1X PBS. Thereafter, the coverslip was taken out from the well, dried well, and a drop of a mounting medium was added to the slide glass, and then covered with a coverslip.

Example 1. Confirmation of influx of NLS peptide into cells

FITC (fluorescein isothiocyanate) tagged NLS-FITC peptide produced by mimicking the nuclear localization signal of the NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) protein in the present invention (Fig. 1) Was then transfected into HCT116 cells and observed with a fluorescence microscope. As a result, when the NLS-FITC peptide was treated with the PULSin delivery reagent as disclosed in FIG. 2, green fluorescence was clearly observed in the cells. In addition, the NLS-FITC peptide was transfected into HCT116 cells using a PULSin delivery reagent, fixed with 4% paraformaldehyde, stained with DAPI, and washed with PBS, and photographed with a confocal microscope. Green fluorescence could be confirmed both around the nucleus and inside the nucleus (Fig. 3).

On the other hand, in order to check the temporal or spatial activity of living cells, the results of taking a time lapse of the NLS-FITC peptide entering the cells using the CellInsight CX7 LZR High-Content Screening (HCS) Platform. As disclosed in 4, it was possible to confirm in real time that the NLS-FITC peptide enters the cell.

Example 2. Confirmation of inhibition of migration of NAG-1 to cell nucleus by NLS peptide treatment

A single colony (HCT116-NAG-1-GFP-#1) was isolated from HCT116 cells (HCT116-NAG-1-GFP) overexpressing NAG-1 bound to GFP (Green fluorescence protein) using G148, a selection marker. I did. The isolated HCT116-NAG-1-GFP-#1 cells were transfected with NLS peptides using a PULSin delivery reagent and incubated for 6 hours, 12 hours and 18 hours, and then CellInsight CX7 LZR High-Content Screening (HCS) ) By measuring the GFP fluorescence intensity expressed in the nucleus and cytoplasm through the platform, the distribution ratio of the nucleus and cytoplasm was confirmed. As a result, as shown in FIG. 5, the nuclear/cytoplasmic distribution ratio of NAG-1 was significantly reduced compared to the control group by treatment with the NLS peptide in the measurement after 6 hours and 12 hours after the NLS peptide was transfected. I could confirm that. In other words, it was found that NAG-1 did not enter the nucleus and stayed in the cytoplasm increased by treatment with the NLS peptide.

Example 3. Effect of loss of mitochondrial membrane potential of colon cancer cells according to NLS peptide treatment

NLS peptide in HCT116-NAG-1-GFP-#1 cells isolated from HCT116 cells (HCT116-NAG-1-GFP) overexpressing NAG-1 bound to GFP (Green fluorescence protein) using G148, a selection marker And PULSin were used for transfection and incubated for 6 hours. ^{Cells were stained using MitoTracker TM} Orange CM TM Ros, which is an orange fluorescent dye that stains the mitochondria of living cells, which accumulates according to the membrane potential, and fixed with 4% paraformaldehyde, followed by counterstaining with DAPI. ^{Thereafter, the fluorescence intensity of MitoTracker TM} Orange CM TM Ros was measured using the CellInsight CX7 LZR High-Content Screening (HCS) Platform to confirm the mitochondrial membrane potential of the cell. As a result, a lower mitochondrial membrane potential was confirmed in cells treated with the NLS peptide compared to the control (Contorl), and cells treated with trans-chalcone to reduce mitochondrial membrane potential were placed as positive controls, and the NLS peptide and When compared with the cells treated with trans-chalcone, a pattern similar to the result was shown (FIG. 6). That is, it was confirmed that the NLS peptide of the present invention can induce apoptosis of colon cancer cells by causing a loss of mitochondrial membrane potential of colon cancer cells.

<110> Seoul National University R&DB Foundation <120> Peptide controlling migration of NAG-1 protein in cell and uses thereof <130> PN19274 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 1 Cys Arg Leu His Thr Val Arg Ala 1 5 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic peptide <400> 2 tgccgtctgc acacggtccg cgcg 24

Claims (6)

delete delete delete delete delete Prevention of colon cancer containing NLS (nuclear localization signal) peptide that inhibits the migration of NAG-1 (nonsteroidal anti-inflammatory drug-activated gene-1) protein to the nucleus, consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient Or a pharmaceutical composition for treatment.

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