KR101217565B1 - Composition for Enhancing Anti-Cancer Effect or Inhibiting Metastasis of Cancer Comprising Extract of Abalone Viscera - Google Patents

Abstract

The present invention relates to an anticancer or cancer metastasis inhibiting composition comprising an abalone viscera extract as an active ingredient. The composition of the present invention comprising the abalone viscera extract as an active ingredient, anti-cancer or cancer as a whole in vivo by enhancing the Th1 immune response (ie, cellular immune response) and cytotoxic T cells, ie cancer cell killing ability by increasing CD8 activity Greatly improves metastasis inhibition effect. In addition, the present invention provides basic data as a medicine and food of abalone viscera extract having an immune enhancing function.

Description

Composition for Enhancing Anti-Cancer Effect or Inhibiting Metastasis of Cancer Comprising Extract of Abalone Viscera}

The present invention relates to an anticancer enhancement or cancer metastasis inhibiting composition comprising an abalone viscera extract as an active ingredient.

Overture drinks with "Abalone research on the processing of vitamin B ₁ and vitamin B ₂ Lots of calcium and phosphorus, etc., and the mineral-rich healthy food is a basic research on some component analysis D performed two euros bars, rollover There is a study on the lawsuit of 'but there is still a lack of scientific evidence on the functionality. Dried abalone is known to be effective in dissolving gallstones and improving liver detoxification with taurine, lowering cholesterol, improving heart function, and restoring vision, but developing processed foods and functional food materials using abalone in Korea. Very little research has been done.

Abalone is rich in protein and vitamins, so it is effective in skin care, nourishing tonic and postpartum cooking, and in particular, it is rich in taurine, which is effective in protecting liver, fatigue and myocardial infarction. In terms of taste and taste, it is incomparable with other seafood.

The efficacy of Korean abalone, which has been recognized for its excellence since ancient times, has been described as “Efficacy of abalone for lightening the body and brightening the eyes” in the Oriental medicine book, Myungbyeong Myungbyeong and Kyuhap. . In the Joseon Dynasty, Seokjeong Jeong Yakjeon introduces abalone in the name of blowfish, saying, `` Red meat is sweet and can be eaten raw or cooked, but the best way is to make pho. The intestines can be eaten, soaked in salted seafood, and boiled. In the spring and summer, there is a poison, and when it comes into contact with it, the flesh boils and the affected area bursts. In general, shellfish are known to be effective in restoring tired nerves, and abalone is known to be effective in relieving dull optic nerve fatigue. The abalone shell is also used in Korean medicine, so it is useful for conjunctivitis and cataracts. It is used to relieve the symptoms of burning or chest swelling, strengthens the liver function, and boils abalone when the body is weak. It is known to be good for urine, jaundice and cystitis.

Abalone has excellent nutritional value, even as feces, which can be used to treat diabetes and high blood pressure. Abalone is rich in protein, rich in the amino acids that make up it, and also rich in minerals and vitamins such as phosphorus, iron and iodine. For this reason, abalone has been treated as a high-grade seafood since ancient times, and has excellent efficacy in skin beauty, nourishing tonic, postpartum cooking, and weak constitution, and has been widely used for edible as well as medicinal purposes.

A lack of iodine and thyroid hormones can lead to obesity due to a slowing of metabolism or the accumulation of fat in the blood vessels. In addition, thyroid hormones are involved in the physical, mental and sexual growth of growing children. Lack of iodine can lead to significant hypothyroidism, resulting in rough skin and thinning hair. Therefore, eating iodine with foods like seaweed is a safe way. Abalone is known to increase the strength of the liver because it has a high content of iodine, pain in the head, ringing, and dry tongue and throat. It is said that orphan feeding abalone when mother's milk does not come out within 7 days of old, has a great effect, because there are many minerals in abalone. In addition, abalone is a high protein ingredient, so high absorption in the body is good food for pregnant women, obesity, liver cirrhosis. Therefore, it is also used as a medicine for liver function recovery and pulmonary tuberculosis. Abalone is rich in sulfur-containing amino acids such as methionine and cysteine, which is good for restoring and restoring fatigue after illness.

Cancer treatment can be roughly divided into methods of directly killing cancer cells such as surgery, administration of anticancer drugs, and irradiation, and methods of activating and treating in vivo immune functions that remove cancer cells, such as immunotherapy. Conventionally, the former method of direct killing is used, but the latter uses the latter immunotherapy and combination therapy. In immunotherapy, as the mechanism of action of immune function is known recently, many attempts have been made to use immunomodulators. The immunomodulatory substance enhances the body's defense against disease factors by non-specifically stimulating the immune cells in vivo to enhance the biological immune function. Such non-specific immunomodulators include dead bacteria, chemical syntheses (synthetic nucleic acid derivatives or glycosides), and biologics (cytokines or hormones). Is being performed.

As described above, efforts have been continuously made to treat cancer through activation of immune function, and there is a need for a natural anticancer agent having no side effects.

For example, US Patent Application Publication No. 2008-0107760 discloses an anticancer composition comprising a bamboo extract, and US Patent Application Publication No. 2008-0019996 discloses an anticancer composition comprising a Trametes zonata mushroom extract as an active ingredient. Doing.

In addition, US Patent Application Publication No. 2007-0160691 describes an anticancer composition comprising a cinnamon extract, US Patent Application Publication No. 2004-0116394 describes an anticancer comprising a plant extract containing betulinic acid (betulinic acid) The composition is disclosed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety and the level of the technical field to which the present invention belongs and the present invention.

The present inventors tried to identify the physiological activity of Abalone viscera extract. As a result, the present inventors have found that Abalone viscera extract not only enhances the activity of T cells involved in gene expression related to cytokine, which is an immune mediator and cancer cell death, but also the expression of genes involved in cancer cell growth and metastasis. The present invention was completed by identifying the amount of anticancer or cancer metastasis effect in vivo by suppressing the amount.

Therefore, an object of the present invention to provide a composition for inhibiting cancer or cancer metastasis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, there is provided an anticancer or cancer metastasis inhibiting composition comprising an abalone viscera extract as an active ingredient.

The present invention is based on antitumor or cancer metastasis suppression activation of Abalone viscera extract. Abalone viscera extract used as an active ingredient in the composition of the present invention can be obtained using a variety of abalone. According to a preferred embodiment of the present invention, the abalone used in the present invention is Haliotis discus hannai , Abalone ( Holiotis gigantea ), Haliotis discus , Haliotis discus ( Haliotis sieboldi ) or Haliotis diversicolor super-texta ), more preferably true abalone, horse abalone, black abalone or sibolt abalone, even more preferably true abalone or horse abalone, even more preferably true abalone, and most preferably This is the abalone abalone that is inhabited or farmed in Jindo-gun, Jeollanam-do, Korea.

The composition of the present invention includes the abalone viscera extract used as an active ingredient. The term “extract” as used herein referring to abalone viscera is an abalone viscera workpiece formulated (eg, powdered) for administration to an animal of the abalone viscera itself as well as the extraction result obtained by treating the extract solvent in the abalone viscera. Also has a meaning to include.

If the abalone viscera extract used in the composition of the present invention is obtained by treating the abalone viscera with an extracting solvent, various extracting solvents may be used. Preferably, (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (e.g. methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol and normal-butanol, etc.), (c) Mixed solvent of the lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) 1,3-butylene glycol, (h) hexane, (i) diethyl ether, or (j) Butyl acetate may be treated with abalone intestine to obtain an extract.

As used herein, the term "extract" has a meaning commonly used as a crude extract in the art as described above, but broadly includes a fraction additionally fractionating the extract. That is, the abalone viscera extract includes not only those obtained by using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the abalone viscera extract of the present invention.

Abalone viscera extract used in the present invention may be prepared in a powder state by an additional process such as vacuum distillation and freeze drying or spray drying.

Alternatively, abalone viscera extract may be used abalone viscera processing formulated (eg, powdered) to administer the abalone viscera itself to the animal.

The composition of the present invention is administered to a living body to have an anti-cancer or cancer metastasis inhibiting action. As used herein, the term "anticancer" means inhibiting or killing the growth of cancer cells or cancer tissues in vivo. For example, stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, rectal cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, endometrial cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, leukemia , but not limited to, non-Hodgkin lymphadenopathy, parathyroid cancer and ureter cancer cells or tissues.

According to a preferred embodiment of the present invention, the composition of the present invention exhibits very excellent efficacy, in particular for treating, preventing or inhibiting cancer metastasis by breast and lung cancer.

According to a preferred embodiment of the present invention, the composition of the present invention enhances the Th1 immune response and cancer cell killing ability. As used herein, the term "Th1 immune response" refers to a response that induces a cellular immune response.

The composition of the present invention promotes the expression of various cytokines involved in the Th1 immune response. For example, the compositions of the present invention enhance the expression of interleukin-2 (IL-2), interleukin-17 (IL-17), interferon gamma (IFN-γ) or tumor necrosis factor-α (TNF-α). .

The composition of the present invention promotes the expression of various genes involved in cancer cell killing ability. For example, the composition of the present invention enhances granzyme and Fas Ligand gene expression. More specifically, granzyme is an exogenous serine protease secreted into granules from cytotoxic T cells and natural killer cells, and the pars ligand is a type II membrane transmembrane protein belonging to a tumor necrosis factor (TNF). Binding to receptors induces apoptosis and plays an important role in controlling immune response and tumor development.

As used herein, the term “cytokine” is a generic term for proteins released by cell populations that act on other cells as intercellular mediators. Examples of such cytokines are lymphokine, monocaine and typical polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone: parathyroid hormone; Thyroxine; insulin; Proinsulin; Rilacin; Prolylacin; Glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); Liver growth factor; Fibroblast growth factor; Prolactin; Placental lactogen; Tumor necrosis factor-α and β; Inhibitors of Müller; Mouse gonadotropin-associated peptide; Inhivin; Actibin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factors such as NGF-β; Platelet-growth factor; Converting growth factors (TGF) such as TGF-α and TGF-β; Insulin-like growth factor-I and II; Erythropoietin (EPO); Osteoinduction factors; Interferons such as interferon-α, -β and -γ; Colony stimulating factor (CSF) such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF); Interleukins such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 (IL); Tumor necrosis factors such as TNF-α and TNF-β; And other polypeptides including LIF and kit ligands (KL). As used herein, cytokines include biologically active equivalents of cytokines of proteins and native sequences from natural sources or from recombinant cell culture.

According to a preferred embodiment of the present invention, the composition of the present invention exhibits an excellent effect of promoting the proliferation of T cells.

As used herein, the term “cancer metastasis” refers to a phenomenon in which a primary tumor in vivo spreads to other non-neighboring tissues or cells, and also generates a new completely different cancer having characteristics of neighboring tissues. It means to cause.

The composition of the present invention not only inhibits the expression of the cox-2 (Cyclooxygenase-2) gene involved in cancer cell growth and metastasis in vivo, but also affects the growth and metastasis of cancer cells among subgenes regulated by the cox-2 gene. By inhibiting expression of important VEGF (Vascular endothelial growth factor) , FGF (Fibroblast growth factor) , EGF (Epidermal growth factor) and MMP-13 (matrix metallopeptidase 13 or collagenase 3) genes, thereby enhancing anticancer effects and / or inhibiting cancer metastasis Very good effect on activity.

According to a preferred embodiment of the present invention, the composition of the present invention exhibits anti-cancer effect enhancement or cancer metastasis inhibiting activity by inhibiting the expression level of one or more genes from the group consisting of cox-2 , VEGF , FGF and EGF genes.

The composition of the present invention may be provided in a pharmaceutical composition or a food composition.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

When the composition of the present invention is prepared as a food composition, as an active ingredient, as well as abalone viscera extract, it contains components that are commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors Includes the first. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides an anticancer composition using the abalone viscera extract.

(Ii) The composition of the present invention comprising the abalone viscera extract as an active ingredient greatly improves the antitumor or cancer metastasis suppression effect in vivo by enhancing the Th1 immune response (ie, cellular immune response) and cancer cell killing ability.

(Iii) The present invention provides basic data as a medicine and food of abalone viscera extract having anticancer efficacy.

1 is a graph showing the weight change of mice after oral administration once daily for 36 days 1 mg per mouse PBS or abalone viscera extract. The control group is a group administered with PBS, and the sample is a group administered with abalone viscera extract.
FIG. 2 is an image of tumor masses implanted subcutaneously after oral administration of 1 mg per day of PBS or abalone viscera extract mice for 36 days. The control group is a group administered with PBS, and the sample is a group administered with abalone viscera extract.
Figure 3 is a graph measuring the size of tumor mass using Vernier caliper every two days after cancer induction (square square x length x 0.52).
Figure 4 is the final 36 days, the resulting rock mass is separated from each group by weight measurement graph.
Figures 5a-5b is the result of comparing the spleen separated from the mouse of each group with the naked eye on the 36th day image and the weight is a specific result graph.
Figure 6a-6b is a result of measuring the proliferation change of T cells using a radioisotope scintillator after 72 hours stimulation with T3 cells and CD28 antibody to the T cells of mice orally administered PBS or abalone viscera extract, respectively. (FIG. 6A shows CD4 T cells; FIG. 6B shows CD8 T cells). The control group is PBS administration group, Abalone is the group to which the abalone viscera extract was administered. W / O are cells stimulated with CD3 and CD8 antibodies, and CD3 / CD8 are cells stimulated with CD3 and CD8 antibodies.
7 is a result of measuring the degree of cell lysis of CD8 T cells by using a scintillator by co-culture with EL4 cells (mouse T lymphoma cells) and respective indicated ratios in order to confirm the cell lysis capacity of the isolated CD8 T cells. (1: 1 = control: sample; 1: 3 = control: sample). The control group is PBS administration group, Abalone is the group to which the abalone viscera extract was administered.
Figure 8 shows the interferon-gamma (IFN-γ), granzyme (granzyme B: GzmB), granzyme (granzyme c: GzmC) of CD4 T cells isolated from the group orally administered PBS or abalone viscera extract, respectively ), Fas ligand (FasL), Tumor Necrosis Factor-α (TNF-α), Interleukin-4 (IL-4) and Interleukin-17: IL -17) expression is compared between the groups. The control group is a group administered with PBS, and the sample is a group administered with abalone viscera extract.
9A-9B show TNF-α (Tumor Necrosis Factor-α), granzyme (GzmB), and granzyme (GzmC) of CD8 T cells isolated from the group orally administered PBS or abalone viscera extract, respectively. ), The expression of the Fas ligand (Fas ligand: FasL) by the quantitative RT-PCR method using SYBR green (SYBR green). The control group is the PBS administration group, the sample is administered abalone viscera extract, the control + IL-2 is administered PBS and IL-2, and the sample + IL-2 is administered abalone gut extract and IL-2 .
FIG. 10 shows the results of images of normal lung tissue, PBS-administered group lungs, and abdominal visceral-administered group lungs and histopathological samples through H & E staining.
11a-11c measure the changes in the expression level and protein of cox-2 using mRNA and protein isolated from primary cancer cell tissues of PBS group and abdominal viscera group, and the VEGF , FGF and EGF genes related to cancer cell growth. It is the result of comparing the expression amount of.
Figure 12a-12c is a measure of gene expression changes and protein changes of cox-2 using mRNA and protein isolated from lung tissue in which cancer cell metastasis occurred in the PBS-administered group and abalone viscera-administered group, VEGF , The expression levels of the FGF and MMP-13 genes were compared.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental material

Abalone and mouse

The abalone abalone ( Haliotis discus hannai) was used as a sample of cultured abalone produced in Jindo-gun, Jeollanam-do, and the abalone viscera was extracted from the abalone and then lyophilized to convert it into a powder form. Mice for the study used Balb c / J mice (SLC, Japan).

Experimental Method

Animal Experiment Time Schedule

The 6-week-old mice were divided into two groups, and the experimental group orally administered abalone visceral extract 1 mg per mouse once a day for 36 days using a syringe, and the control group was orally administered PBS in the same manner. At this time, the weight change of the two groups of mice was monitored before administration. After 36 days of oral administration, T cells were isolated from the spleen and intestinal lymph nodes of each group of mice in the same manner in each group of mice. In order to measure the activity of T cells, experiments were performed to measure T cell proliferation, cytokine and target gene expression.

Cancer cell line transplantation (xenotransplation) in mice

The abalone visceral extract was orally administered once daily for 36 days (1 mg per mouse in the case of the control group, orally administered with the same volume of PBS) to measure the weight change of the experimental animals during the administration period. After oral administration for the first 10 days, cancer models were induced by subcutaneous injection of 4T1 metastatic breast cancer cell lines (4T1p24, ATCC, USA) on both days. At this time, 10 ⁶ cells per mouse were mixed and injected into 100 µl volume of PBS.

Isolation of T Cells

Two groups of mice were orally administered with PBS or abdominal intestine for 36 days, and spleen and intestinal lymph nodes were extracted from each mouse. The lymph nodes thus harvested were made to single cell level using collagenase V (Sigma, USA).

CD4 ⁺ T cells were isolated by separating magnetic beads and spleen from mice in each group to investigate the mechanism of suppression of cancer cell formation in the immune system by the administration of abalone viscera extract. Miltenyi, Germany, Cat No. 130 -049-201) and a column (columns; Miltenyi Biotech, Germany, Cat No. 130-042-401). CD8 ⁺ T cells were isolated using isolation magnetic beads (Miltenyi, Germany, Cat No. 130-049-201).

To do this, a separate cells ^{(1 X 10 5) 1 ㎍} / ㎖ CD3 antibody concentration of the (BD Pharmigen Cat No.553058) or CD3 antibody and 1 ㎍ / ㎖ concentration of CD28 antibody (BD Pharmigen Cat No.553294) 60 sigan After stimulation, the cell proliferation was measured using a scintillator for 12 hours after adding the radioisotope 0.5 μCi [H ³ ] -thymidine (NEN Life science, Boston, MA, USA). It was.

In addition, after treatment with CD3 antibody and CD28 antibody to CD8 ⁺ T cells isolated in the same manner as the above experiment [H ³ ] -thymidine was added to measure the cell proliferation.

CD8 located in mouse spleen and intestinal lymph nodes ⁺  Cancer Cell Fracture Capacity Analysis of T Cells

To confirm the cancer cell disruption ability of T cells isolated from orally administered abalone extract, 0.5 μCi of [H ³ ] -thymidine was added to EL4 cells (mouse T lymphoma cells, ATCC, USA) as target cells. Cell culture was carried out for 12 hours. Next, EL8 cells cultured by adding CD8 ⁺ T cells and [H ³ ] -thymidine, which were separated from each group, were co-cultured at 1: 1 or 1: 3 ratio, respectively, to determine the degree of cancer cell destruction of CD8 ⁺ T cells. It measured using the scintillator.

Measurement of Cytokines and Target Gene Expression

Spleen and lymph nodes were isolated from PBS and abdominal viscera groups, and mRNA was isolated from CD4 ⁺ and CD8 ⁺ T cells using Trizol reagent (Molecular Research Center). 1 μg of the isolated mRNA was synthesized using oligo (dT) primer (Promega) and Improm-II Reverse Transcription System (Promega).

In order to analyze the target gene expression level related to specific cytokines, cell disruption and killing ability using the synthesized cDNA, SYBR green (Takara, Japan) was used for quantitative real-time PCR. Analyzed. Quantitative real-time PCR was incubated at 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. 30 sec, 62 ° C. 30 sec, and 72 ° C. 30 sec. The sequence of the primers used is as follows: IFN-γ (forward primer 5'-GAGCCAGATTATCTCTTTCTACC-3 'and reverse primer 5'-GTTGTTGACCTCAAACTTGG-3'); TNF-α (forward primer 5′-CATCTTCTCAAAATTCGAGTGACAA-3 ′ and reverse primer 5′-TGGGAGTAGACAAGGTACAACCC-3 ′); GzmB (forward primer 5'-CCACTCTCGACCCTACATGG-3 'and reverse primer 5'-GGCCCCCAAAGTGACATTTATT-3'); GzmC (forward primer 5'-GCAGAGGAGATAATCGGAGGC-3 'and reverse primer 5'-GCACGAATTTGTCTCGAACCA-3'); FasL (forward primer 5'-TCCGTGAGTTCACCAACCAAA-3 'and reverse primer 5'-GGGGGTTCCCTGTTAAATGGG-3'); IL-2 (forward primer 5′-CCT GAG CAG GAT GGA GAA TTA CA-3 ′ and reverse primer 5′-TCC AGA ACA TGC CGC AGA G-3 ′); IL-17 (forward primer 5'-TTCATCTGTGTCTCTGATGCTG-3 'and reverse primer 5'-TTGACCTTCACATTCTGGAGG-3').

Histopathology

Mouse lung tissue was separated from the PBS administration group and the abdominal intestine administration group, cut into pieces, and fixed in 4% formaldehyde. After infiltrating paraffin to form embedded tissue, the sections were floated to a thickness of 5 μm and attached to glass slides, and tissue staining was performed using hematoxylin and eosin.

Measurement of Cox-2 and Target Gene Expression

Primary cancer cells and metastasized secondary cancer cells (lungs) were isolated from PBS administration group and abdominal visceral administration group, homogenized using homogenizer, and mRNA was isolated using Trizol reagent (Molecular Research center). It was. 1 μg of isolated mRNA was synthesized using oligo (dT) primer (Promega) and Improm-II reverse transcription system (Promega). In order to analyze the expression level of cox-2 gene and target genes related to cancer growth and metastasis using the synthesized cDNA, SYBR Green (Takara, Japan) was used for quantitative real-time PCR. Analyzed. Quantitative real-time PCR was incubated at 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. 30 sec, 62 ° C. 30 sec, and 72 ° C. 30 sec. The sequences of the primers used are shown in Table 1 below.

Table 1

Cell Extracted Protein Preparation and Immunoblot

Isolate primary cancer cells and lung tissues, which are secondary metastatic cancer cell tissues, and homogenize them using homogenizers, and then centrifuge and digest with RIPA buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40). Enzyme inhibitor (Calbiochem) was mixed and stored on ice for 10 minutes. To analyze Cox-2 protein, electrophoresis was performed on 10% SDS-PAGE gels using protein size markers as samples prepared above. After electrophoresis, the protein separated on the nitrocellulose membrane was transferred, and 5% non-fat milk was prepared in TBST (Tris Buffered Saline Tween 20) buffer to prevent nonspecific binding. The Cox-2 antibody (Cayman chemical) was reacted overnight at 4 ° C., and then a secondary antibody was prepared. After the reaction, the presence of the protein was confirmed using an ECL solution. In order to confirm the same amount of protein electrophoresis, the protein-transported nitrocellulose membrane was detected using the β-tubulin antibody (Abcam) in the same manner as above.

Experiment result

Changes in Mouse Weight According to Oral Administration of Abalone Intestine Extract

Abalone and abalone viscera are currently used as foods, but foods can also cause abdominal pain and upset stomach in general when excessive doses are taken. Oral administration once a day for 36 days at 1 mg per mouse to measure the weight change of the experimental animals appearing during the period. Prior to this measurement, the abalone viscera extract was orally administered for the first 10 days, and on day 10, a 4T1 metastatic breast cancer cell line was injected subcutaneously in both experimental groups to induce a cancer model.

As a result, it was confirmed that there was no difference in weight between the group administered the abalone viscera extract and the control group administered with PBS. Through this, the 1 mg abalone viscera extract used in this study was orally administered to the mouse for 36 days (1 dose / day). It was confirmed that the toxicity is not shown at (1).

Changes in the Size of Tumor Transplanted in Mice Following Oral Administration of Abalone Visceral Extracts

As a result of visual comparison of the size of tumors implanted in each group on the last 36 days, it was confirmed that cancer formation was suppressed in terms of size in the abalone extract administration group (FIG. 2).

Tumor size was measured using Vernier caliper (width x length x 0.52) every two days after cancer induction. It was found that the size of cancer was suppressed in the oral administration group of abalone viscera extract compared to the control group (Fig. 3). As a result of weighing the tumor mass generated on the last 36 days from each group, it was observed that the weight of the tumor mass was also reduced in the abalone oral administration group compared to the control group (FIG. 4).

On the 36th day, visual comparison of the spleens from the mice of each group showed that the control group had a larger spleen size due to cancer metastasis, whereas the abdominal visceral oral administration group suppressed the enlargement of the spleen despite cancer metastasis. It could be observed (Figs. 5a-5b).

A Study on the Change of T Cell Proliferation in Abalone Intestine Extracts

The activity of T cells could be evaluated by the degree of proliferation by which the cells themselves proliferate and the degree of secretion of immune mediators secreted by the cells. Therefore, in this experiment, the change in the degree of proliferation of T cells that may appear upon ingestion of abalone viscera extract was confirmed. In general, immune cells begin to proliferate only when given an external stimulus, such as invading pathogens, and the extent to which they respond to the external stimulus depends on how proliferates the same stimulus. Therefore, in order to give the same stimulation as the external stimulation, the change of T cell proliferation was measured using radioisotope after stimulation using CD3 antibody and CD28 antibody, which are specifically antibodies capable of stimulating T cells. .

As can be seen in Figures 6a-6b, little differentiation of T cells in the absence of antibody stimulation. However, when stimulated with CD3 antibody and CD28 antibody, T cells of mice treated with Abalone viscera extract showed about 30% increased cell proliferation compared to T cells of control group. Through this, intake of abalone viscera extract was found to increase the degree of T cell proliferation.

CD8 ⁺  Cancer cell death and disruption of T cells

Excellent cancer cells when mice orally administered abalone viscera extract were stimulated with CD3 antibody and CD8 antibody on CD8 ⁺ T cells and co-cultured with the addition of 0.5 μCi [H ³ ] -thymidine and co-cultured EL4 cells It can be seen that the crushing ability. In other words, the oral administration of abalone viscera extract in CD8 ⁺ T cells of spleen and intestinal lymph nodes of mice showed superior anticancer efficacy compared to the control (Fig. 7).

Expression of gene targets involved in cytokine or cancer cell disruption and cancer cell death of T cells

Spleen and lymph nodes were isolated from PBS and abdominal visceral groups to separate mRNAs from CD4 ⁺ and CD8 ⁺ T cells, respectively, and then the expression of genes related to cytokine and cancer cell death of various functions were analyzed in real time. time PCR) was used to compare with T cells of the control group. In addition, in the case of CD8 ⁺ T cells, IL-2 is known to act on cell disruption ability, and compared with both the isolated cells themselves and the treated groups with IL-2.

As a result, Interferon-gamma (IFN-γ), granzyme B (granzyme B: Gzm B), and granzyme C for CD4 ⁺ T cells located in mouse spleen and intestinal lymph nodes orally administered abalone viscera extract. granzyme C: Gzm C, Fas ligand: FasL, Tumor Necrosis Factor-α (TNF-α), Interleukin-4 (IL-4), and Interleukin-17 (Interleukin-17: IL-17) expression was found to be significantly increased compared to the control (Fig. 8).

In addition, TNF-α, Granzyme B, Granzyme C, and Fas ligand in CD8 ⁺ T cells treated with oral abdominal viscera extract or oral abalone viscera extract and IL-2. Was significantly higher than the control (Fig. 9a), interferon-γ also showed the same results (Fig. 9b).

Study on the Inhibition of Metastasis in Abalone Intestine Extracts

To compare the degree of metastasis of cancer cells, the tissues of the lungs were first observed by eye. As can be seen in Figure 10, compared with the lungs of the normal rat lungs of the group administered with PBS it was observed that the formation of secondary cancer masses by metastasis. On the other hand, the lungs of the abdominal viscera were not significantly different from the lungs of normal rats. For more accurate comparisons, lung histology was performed using histopathological methods. Compared with normal lung tissue, lung tissue of PBS group was found to be infiltrated with cancer cells indistinguishable from specific alveolar structure. On the other hand, the lung tissue of the abalone administration group was confirmed that there is no significant difference compared to the histology of normal lung tissue. Through this, intake of abalone viscera extract was found to inhibit the metastatic capacity of cancer cells.

Mechanism of Primary Cancer Cell Growth Inhibition by Inhibition of Cox-2 Expression by Abalone Visceral Extract

Cox-2, known as one of the oncogenes, is involved in the growth and development of cancer cells. For tumor growth inhibition seen in the group administered internal upset to determine if the connection with the expression of cox-2, the mRNA expression level of cox-2 was measured in the primary cancer cells isolated from each group. Compared to the PBS group, the expression level was decreased by 20% in the abalone intestine group. Even in the case of measuring the expression level of cox-2 at the protein level, it was confirmed that the abdominal visceral administration group significantly reduced the cox-2 expression level compared to the PBS administration group. In addition, the target genes related to the growth of primary cancer cells, VEGF , FGF and EGF was confirmed that the decrease in the abdominal visceral administration group. This suggests that the intake of abalone viscera extract inhibits growth by reducing the expression of cox-2 and several genes, which are important for cancer cell growth.

Inhibitory Mechanism of Cancer Cell Metastasis by Inhibiting Cox-2 Expression by Abalone Viscera Extracts

Overexpression of cox-2 is involved in metastatic capacity as well as in the growth of cancer cells. In order to determine whether the inhibition of cancer cell metastasis to the lung shown in the abdominal visceral administration group was associated with cox-2 expression, the mRNA expression level of cox-2 was measured from lung tissues isolated from each group. Compared with the PBS-administered group, the abdominal visceral-administered group showed a decrease in expression level of 90% or more. Even when the cox-2 expression level was measured at the protein level, it was confirmed that the abdominal visceral administration group significantly reduced the cox-2 expression level compared to the PBS administration group. In addition, it was confirmed that the expression of target genes VEGF , FGF and MMP-13 related to the metastatic capacity of secondary cancer cells that metastasized to the lung decreased in the abdominal visceral group. This suggests that ingestion of abalone viscera extract inhibits growth by reducing the expression levels of cox-2 and several genes that are important for cancer cell metastasis.

conclusion

In conclusion, continuous intake of abalone viscera extract was found to have anticancer efficacy through functional enhancement of T cells in the human immune system. The present invention is the first data to scientifically prove the anticancer efficacy of abalone viscera extract.

Also,

Review

To date, abalone has been studied in terms of its pharmacologic function, but there are no studies on anticancer efficacy.

The present invention carried out an evaluation study on the effect of intake of abalone viscera extract on anticancer efficacy in order to prepare more empirical and scientific evidence on the function of abalone viscera extract. To evaluate the toxicity of the abalone viscera extract used in this experiment, mice were orally administered various abalone viscera extracts, and even at the highest concentration of 1 mg / ml, any weight loss was observed during the 36-day oral administration period. No weight gain, as well as a higher level of weight gain compared to the control. This confirmed that the abalone viscera extract used in this study was not toxic and indirectly confirmed high nutritional function.

Evaluation of the effect of abalone viscera extract on T cell function The conclusions of the study suggest that sustained ingestion of abalone viscera extract increases T cell function and ultimately improves anticancer efficacy. The first evaluation of the effect of intake of abalone viscera extract on the proliferation of T cells showed that sustained intake of abalone viscera extract increased the proliferation of T cells to a significant level compared to the control group. there was. Secondly, the results of gene expression related to cytokine and cancer cell disruption of T cells showed that Abalone viscera extract improved anticancer efficacy.

In addition, it was confirmed that the abalone viscera extract can inhibit the growth and metastasis of cancer cells through the effect of the expression of cox-2 , a cancer gene. In other words, the first pathological analysis showed that ingestion of abalone viscera inhibited cancer cell metastasis to the lung. Secondly, the growth inhibition mechanism of primary cancer cells revealed that Abalone viscera extract inhibited the growth of cancer cells by inhibiting the expression of cox-2 , VEGF , FGF and EGF genes. Third, the metastatic inhibition mechanism of cancer cells was confirmed to inhibit the metastasis of cancer cells by inhibiting the expression of cox-2 , VEGF , FGF, and MMP-13 in lung tissues from which abalone visceral extracts were metastasized.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

A composition for inhibiting cancer metastasis comprising an abalone viscera extract as an active ingredient, the composition enhances Th1 immune response and cancer cell killing ability of CD8 ⁺ T cells, and cox-2 , VEGF , FGF, EGF and MMP A composition for inhibiting cancer metastasis, wherein the amount of expression of one or more genes is suppressed from the group consisting of -13 genes.
The method of claim 1, wherein the abalone is Haliotis discus hannai , horse abalone ( Holiotis gigantea ), black abalone ( Haliotis discus ), Haliotis sieboldi or Haliotis diversicolor super-texta characterized in that A composition for inhibiting cancer metastasis.
The composition for inhibiting cancer metastasis according to claim 2, wherein the abalone is abalone.
delete The composition for inhibiting cancer metastasis according to claim 1, wherein the composition promotes proliferation of T cells.
delete A food composition comprising an abalone viscera extract as an active ingredient, the food composition enhances the Th1 immune response and cancer cell killing ability of CD8 ⁺ T cells, cox-2 , VEGF , FGF, EGF and MMP-13 Food composition for preventing or alleviating cancer metastasis, characterized in that to suppress the expression of one or more genes from the group consisting of genes.
The composition for inhibiting cancer metastasis according to claim 1, wherein the composition is a pharmaceutical composition.

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