KR101535895B1 - Pharmaceutical composition for preventing and treating cancer comprising CopA3 peptide as an active ingredient - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention and treatment of cancer diseases, which comprises the coprinos peptide CopA3 as an active ingredient, and more particularly to a pharmaceutical composition for preventing and treating cancer diseases, The present invention relates to a pharmaceutical composition for preventing and treating cancer diseases, which comprises lysine peptide CopA3 as an active ingredient. The coprecipine peptide CopA3 of the present invention has an anticancer activity and is effective for use as a pharmaceutical composition for prevention and treatment of cancer diseases because of its excellent cell proliferation inhibitory effect in a gastric cancer cell line.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating cancer diseases, which comprises Copreis peptide CopA3 as an active ingredient,

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention and treatment of cancer diseases, which comprises the coprinos peptide CopA3 as an active ingredient, and more particularly to a pharmaceutical composition for preventing and treating cancer diseases, The present invention relates to a pharmaceutical composition for preventing and treating cancer diseases, which comprises lysine peptide CopA3 as an active ingredient.

Gastric cancer is the fourth most common disease in the world and the second leading cause of cancer-related mortality. Because gastric cancer is not asymptomatic externally, most cases are missed due to the late detection period. This is the reason why the survival rate of stomach cancer over 5 years is 20% and the survival rate is relatively low. Although gastric cancer is the most common type of surgery, recurrence rate is high and some chemotherapy regimens are also known to have high side effects. Therefore, a more effective gastric cancer treatment method is needed.

Insects possess biological defense systems that can effectively combat the infestation of external microorganisms and viruses in order to survive in a variety of environments. In other words, unlike the acquired immune factors of higher animals, it has an immune system that effectively protects itself by secreting innate immune factors including cellular immune and humoral immune responses such as invertebrates. The innate immune response of insects includes the phagocytosis of protozoa including bacteria, fungi, and nematodes, nodule formation and encapsulation, and in the humoral immune response, various types of proteins and peptides are involved in the invasion of pathogens, They are synthesized in cells and secreted into the blood, which is used as a powerful defense against pathogens.

Peptides secreted through innate immunity have the advantage that resistance to microorganisms is less likely to induce resistance due to different mechanisms and selectivity than conventional antibiotics in question. Therefore, these insect-derived peptides can be used as natural antibiotics of the next generation, and thus are highly likely to be industrially applicable not only for human diseases such as cancer but also for prevention and treatment of diseases caused by various pathogenic bacteria and crops. In addition, peptide synthesis procedures are simplified, synthesis time and unit cost are reduced, and more patients are expected to be effectively treated. Many studies are being conducted for clinical application because of excellent antimicrobial activity and safety for human cells. In addition, it can be developed not only as an antifungal agent, an antimicrobial agent, an anticancer agent, but also a drug, a vaccine, and a diagnostic reagent whose physiological function is controlled by utilizing such peptide engineering technique.

 Accordingly, the present inventors have paid attention to the anticancer ability of insect-derived peptides and have studied the possibility of being an anticancer therapeutic agent for gastric cancer which shows a recent high incidence. As a result, it was confirmed that coplysin CopA3 has excellent anticancer activity against gastric cancer cells. Completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing and treating cancer diseases, which comprises a coprecipine peptide CopA3 as an active ingredient.

In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition for preventing and treating cancer diseases, which comprises a coprin peptide CopA3 as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases, which comprises as an active ingredient a coplysin peptide CopA3 (LLCIALRKK: SEQ ID NO: 1).

The coplysin peptide CopA3 of the present invention may have the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1.

Corp. has high antimicrobial activity with respect to the mechanism of action of the lysine peptide CopA3 is harmful bacteria on crops and human health, especially high antibacterial effect on the resistant strains, especially harmless intestinal beneficial microorganisms and bacteria that cause acute pseudomembranous colitis (Clostridium difficile ).

The pharmaceutical composition for treating cancer diseases of the present invention is a pharmaceutical composition characterized by the treatment of gastric cancer disease, though the kind of cancer disease is not particularly limited.

In addition, the pharmaceutical composition for treating cancer diseases of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI > Preferably, the pharmaceutical composition of the present invention can be transdermally administered. Refers to administering the pharmaceutical composition of the present invention to cells or skin to allow the active ingredient contained in the composition of the present invention to be delivered into the skin. For example, the pharmaceutical composition of the present invention can be administered in a scanning type formulation, pricking the skin lightly with a 30 gauge needle, or directly applying it to the skin.

The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective amount of the peptides of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the preferred total dose of the peptides of the invention may be from about 0.01 μg to 1,000 mg, and most preferably from 0.1 μg to 100 mg, per kg body weight of the patient per day. However, the dose of the peptide of the present invention is not limited to the effective route of administration to the patient in consideration of various factors such as the route of administration and the number of treatments of the pharmaceutical composition, as well as the patient's age, body weight, health condition, sex, severity of disease, It will be understood by those skilled in the art that the peptide of the present invention can be used to determine an appropriate effective dose according to the specific use as an immunological enhancer. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

Therefore, the coprecipine peptide CopA3 of the present invention has an anticancer activity and is effective for use as a pharmaceutical composition for prevention and treatment of cancer diseases because of its excellent cell proliferation inhibitory effect in gastric cancer cell lines.

Fig. 1 shows the effect of inhibiting gastric cancer cells by treatment with coplysin CopA3 peptide.
Fig. 2 shows a fragmentation phenomenon of gastric cancer cells (SNU 668) by treatment with coplysin CopA3 peptide. (A: DNA laddering, B: TUNEL assay)
FIG. 3 shows the changes of AIF (apoptosis inducing factor), PARP, and Endo G (endonuclease G) protein by the coplysin CopA3 peptide treatment.
Lane 1 (0 uM), Lane 2 (12.5 uM) Lane 3 (25 uM)
A: AIF protein change, B: PARP protein change, C: Endo G protein change

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

Chemical synthesis of antimicrobial peptides

The peptides used in the experiments were synthesized in AnyGen (Gwang-ju, Korea) at a purity of 98% or higher and dissolved in 0.01% acetic acid.

< Example  2>

Cell culture of gastric cancer cell culture

Human-derived cell lines used in the study were tested using the gastric cancer cell lines SNU216, SNU484, SNU638, and SNU668. The cells were cultured in DMEM containing antibiotics (100 units / ㎖ penicillin, 100 ㎍ / ㎖ streptomycin) and 10% fetal bovine serum (FBS) in a 5% CO ₂ incubator at 37 ℃. The reagents required for culture were purchased from Gibco Respectively.

< Example  3>

Verification of cell viability by peptide treatment

100 μl of 2 × 10 ⁵ cells / ml cells were dispensed into 96-well culture dishes, and the peptides were treated for 24 hours depending on concentrations of 0, 5, 10, 25, 50 and 75 μM. Then, 10 μl of CellTiter 96 AQ _ueous One Solution Reagent (MTT) (Promega, USA) was added and reacted for 3 to 4 hours. Inhibition of cell growth was confirmed by absorbance measurement at 450 nm wavelength using a multi detector (Beckman DTX8800).

As a result, 4 kinds of inhibition effect on gastric cancer cell growth was small at 5 and 10 uM of CopA3, but when treated with CopA3 at a high concentration (25 uM to 75 uM), the cells were sequentially inhibited the growth of gastric cancer cells. In particular, SNU638 and SNU668 were superior in inhibiting cell proliferation in cancer cells.

< Example  4>

Genomic DNA  Fragmentation Observation

The cells were treated with 12.5, 25 and 50 μM of CopA3 for 24 hours or the buffered cells were reacted with 100 μl of lysis buffer (10 mM Tris-HCl buffer pH 7.4, 10 mM EDTA and 0.5% Triton X-100) And centrifuged at 15,000 rpm for 5 minutes at 4 ° C. 50 μg / ml RNase and 200 μg / ml proteinase K were added to the supernatant and reacted at 55 ° C for 1 hour. 20 μl 5 M NaCl and 120 μl isopropanol were added and kept at -20 ° C overnight. The mixture was centrifuged at 15,000 rpm for 15 minutes at 4 ° C, dried to take up the precipitate, and dissolved in 20 μl of tertiary distilled water. The obtained gDNA was electrophoresed in ethidium bromide (EtBr) -added 2.0% agarose gel for 1 hour at 50 V, and fragmentation pattern of genomic DNA by CopA3 treatment through UV permeance (Core Bio i-MAX TM Gel Image Analysis System) Respectively.

As a result, DNA fragmentation was observed when SNU668 cancer cells were treated with 25 uM CopA3 or more (FIG. 2A).

< Example  5>

Fission observation

Fission was observed using DeadEnd ^TM Fluorometric TUNEL System (Promega, USA). The cells were treated with 12.5, 25, and 50 μM of CopA3 for 24 h, or the buffered cells were fixed for 20 min on ice with 1% paraformaldehyde in phosphate-buffered saline (Bio-Rad, USA). After increasing the permeability of the cells with 0.1% Triton X-100 for 15 minutes, 80 μl Equilibration buffer was added at room temperature and stabilized for 5 minutes. The reaction was carried out in a dark environment at 37 ° C for 60 minutes with the rTdT incubation buffer containing green fluorescent substance (45 μl Equilibration Buffer, 5 μl of nucleotide mix, 1 μl of rTdT Enzyme), and stained with DAPI solution. The stained nuclei were observed with a fluorescence microscope (Nikon Eclipse E600 Epifluorescence Microscope).

DNA fragments were re-confirmed using the TUNEL assay, and it was confirmed that DNA fragmentation was increased depending on the concentration of CopA3 (FIG. 2B). Therefore, it is presumed that CopA3 induces apoptosis of gastric cancer cells.

< Example  6>

Apoptosis  Related protein Expression  Confirm

Cells treated with 25 or 50 uM CopA3 for 24 h or buffer were incubated with 30 μl of NP40 buffer (50 mM Tris-Cl pH 8.0, 1% NP-40 or Triton X-100, 150 mM NaCl, Protease inhibitor cocktail) After lysis, protein concentration was measured using Protein assay (Bio-rad). Each cell extract was subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) and the proteins present in the gel were transferred to a nitrocellulose membrane (BIO-RAD) at 250 mA for 2 hours. In order to eliminate the nonspecific reaction, an unnecessary portion of the membrane was blocked with 5% skim milk solution. Then, the primary antibody was reacted overnight, and HRP-conjugated anti-rabbit antibody (Promega, USA) and anti-mouse antibody (Promega, USA) were reacted with the secondary antibody for 2 hours to 3 hours. The cells were developed with the following ECL system (Amersham, USA) and confirmed by FluorChem (Alpha Innotech).

As a result of protein expression, it was confirmed that the expression of AIF and Endo G in SNU638 and SNU668 of gastric cancer cells, which were superior in inhibiting cell growth by CopA3, was higher than that of untreated samples (Figs. 3A, 3C). In the stomach cancer cells of SNU 668, cleaved PARP proteins were also observed in the samples treated with 50 uM CopA3 compared to the control and 25 uM samples (Fig. 3B). Based on the above results, it was confirmed that apoptosis is progressing in SNU638 and SNU668 cells.

As described above, the coprecipine peptide CopA3 of the present invention has anticancer activity and is excellent in the inhibition of cell proliferation in gastric cancer cell line, so that it can be used as a pharmaceutical composition for prevention and treatment of cancer diseases, and thus it is highly likely to be used industrially.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION <120> Pharmaceutical composition for preventing and treating cancer          comprising CopA3 peptide as an active ingredient <130> NP12-0043 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 9 <212> PRT <213> CopA3 <400> 1 Leu Leu Cys Ile Ala Leu Arg Lys Lys   1 5

Claims (7)

1. A pharmaceutical composition for the prevention and treatment of gastric cancer disease, which comprises as an active ingredient Coprein peptide CopA3 having the amino acid sequence represented by SEQ ID NO: 1.
delete delete delete delete 1. A composition for inducing apoptosis in gastric cancer, comprising an effective amount of a coplysin peptide CopA3 having an amino acid sequence represented by SEQ ID NO: 1.
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