KR20170111274A - Pharmaceutical Composition for the prevention or treatment of EBV associated gastric cancer comprising quercetin - Google Patents

Abstract

The present invention relates to a process for the preparation of 2- (3,4-dihydrophenyl) -3,5,7-trihydroxy-4H-chromen- The present invention relates to a pharmaceutical composition for the prevention and treatment of gastric cancer, comprising quercetin, quercetin or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the quercetin of the present invention is Epstein-Barr virus Quercetin induces the proliferation of EBV in the Epstein-Barr virus (EBV) -positive gastric cancer cell line, thereby killing the infected cells and effectively inhibiting horizontal infection of the EBV offspring virus dissolved in the cells outside the normal cells, Since the anticancer effect is superior to that of isoleucillithinin known to have viral activity and anti-EBV-positive gastric cancer activity, the quercetin of the present invention inhibits the growth of EBV-induced tumor in vivo, Drugs for treatment and prevention of diseases May be usefully used as an active ingredient of a pharmaceutical composition.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating Epstein-Barr virus-associated gastric cancer, which comprises quercetin as an active ingredient,

The present invention relates to a pharmaceutical composition for the prevention and treatment of Epstein-Barr virus (EBV) -related gastric cancer comprising quercetine as an active ingredient.

Epstein-Barr virus (EBV) is a human gamma-1 herpesvirus, an opportunistic infectious life-threatening virus that infects more than 90% of the world's population. During the incubation period, the virus is mostly bound to chromatin and is expressed in multicopy episomes in the nuclei of various tumor cells such as B cells, T cells, NK cells and epithelial cells ). ≪ / RTI > It may be a cause of various malignancies during the latency period and may be associated with various diseases such as Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disease, malignant lymphoma (NK / T -cell lymphoma, nasopharyngeal carcinoma, and gastric carcinoma.

As a reported cause of gastric cancer, about 10% of gastric cancer patients are diagnosed as having EBV-related disease. In the case of EBV-positive stomach cancer, almost all gastric cancer cells are infected with EBV, and more than 50,000 cases of EBV-associated gastric cancer are reported worldwide each year. Thus, stomach cancer associated with EBV is considered to be the most common of the EBV-induced tumors.

Recently, researchers working on stomach cancer have divided gastric cancer into four subtypes: EBV-positive gastric cancer (tumor positive for Epstein, -Barr virus, microsatellite unstable tumor, tumors with genomically stable tumors, and tumors with chromosomal instability. Among the four subtypes, EBV-positive stomach cancer is characterized by repeated PIK3CA mutations, prominent DNA hypermethylation, and amplification of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2) Which is a feature of the present invention. Thus, EBV-positive stomach cancer is characterized by DNA hypermethylation in the genome, so developing a drug or composition that induces DNA demethylation is an important factor in finding new EBV-positive stomach cancer therapies and diagnostic methods It is expected to be available.

Quercetin is a compound known as a flavonoid component contained in licorice extract. The IUPAC name is 2- (3,4-dihydroxyphenyl) -3,5,7-trihydroxy-4H-chromene (3,4-dihydroxyphenyl) -3,5,7-trihydroxy-4H-chromen-4-one). In fact, quercetin belongs as a class of flanonol, but it is a kind of flavonoid with a 3-hydroxy-2-phenylchromen-4-one (3-hydroxyflavone backbone) (Committee of Pharmacognosy Publication. (2013). Pharmacognosy. Pharmacognosy. (Seoul Korea: Dong Myoung), pp. 102-105).

In various studies, it has been reported that quercetin has various biological activity effects such as antiviral activity, anti-asthmatic activity, anticancer activity, anti-inflammatory activity, and monoamine-oxidase inhibitor. Quercetin is capable of damaging oncoviral replication and is specifically reported to inhibit the secretion of HBsAg and HBeAg in cells infected with hepatitis B virus (HBV). In addition, quercetin has an effective inhibitory effect on HCV replication among most flavonoids and can be used in combination with interferon-a as an anti-HCV therapeutic agent. In addition, quercetin and its analogues have strong inhibitory activity against HIV-1 reverse transcriptase, and studies are under way to use it as a therapeutic agent for HIV.

There are also reports related to the use of quercetin as an anticancer drug. It has been reported that this is due to the antioxidant or anti-inflammatory activity of quercetin, and also has the activity of inhibiting the growth of cancer cells and inducing apoptosis (Dajas F. Life or death: neuroprotective and anticancer effects of quercetin. Journal of ethnopharmacology. 2012; 143: 383-396). However, no specific evidence that quercetin has a protective effect on human cancer has been studied, and in particular, whether gastric cancer induced by EBV has quercetin exhibited anticancer activity has not been studied.

Therefore, the present inventors have made efforts to develop an effective therapeutic agent for gastric cancer induced by Epstein-Barr virus (EBV) infection. As a result, quercetin isolated from licorice extract induces the proliferation of EBV, And effectively suppresses the horizontal infection of EBV progeny viruses dissolved outside the cells into normal cells as well as effectively suppresses the growth of gastric cancer-induced EBV infection in vivo, -EBV-positive gastric cancer, the quercetin of the present invention can be effectively used as an active ingredient of a pharmaceutical composition for the prevention and treatment of gastric cancer, .

Thus, the inventors of the present invention completed the present invention by confirming the apoptosis-suppressing effect of quercetin on EBV-associated gastric cancer cells, the EBV infection-inhibiting effect, and the in vivo gastric cancer growth inhibition effect.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of gastric cancer.

Another object of the present invention is to provide a health functional food for preventing and improving gastric cancer.

It is another object of the present invention to provide a method for measuring the level of gene or protein expression for providing information for diagnosis of gastric cancer.

To achieve the above object, the present invention provides a process for preparing 2- (3,4-dihydrophenyl) -3,5,7-trihydroxy-4H-chromen- -3,5,7-trihydroxy-4H-chromen-4-one; Quercetin, quercetin) or a pharmaceutically acceptable salt thereof as an active ingredient.

According to a preferred embodiment of the present invention, the quercetin may be isolated from licorice extract or its fractions.

According to another preferred embodiment of the present invention, the gastric cancer may be gastric cancer caused by Epstein-Barr virus (EBV) infection.

The present invention also includes a health functional food for prevention and improvement of gastric cancer, comprising quercetin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also relates to

i) treating and culturing the sample derived from the subject with quercetin;

ii) analyzing the characteristics of the sample cultured in step i); And

iii) comparing the result of the analysis in step ii) with the untreated control group to determine the level of gene or protein expression to provide information for gastric cancer diagnosis.

According to a preferred embodiment of the present invention, the quercetin of step i) may be treated at a concentration of 32 to 250 [mu] M.

According to another preferred embodiment of the present invention, the characteristics of step ii) may be characterized by any one or more of the following a) to d):

a) decreased expression of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A) protein;

b) decreased methylation of the BCL7A gene;

c) estrogen, heat shock response, upregulation of MAPK / ERK and MAPK / JNK signaling pathways; And

d) Downregulation of the glucocorticoid, heavy metal stress, interferon gamma, liver X receptor and TGF-beta signaling pathways.

According to another preferred embodiment of the present invention, the gastric cancer may be gastric cancer caused by EBV infection.

Accordingly, the present invention provides a process for preparing 2- (3,4-dihydrophenyl) -3,5,7-trihydroxy-4H-chromen- -trihydroxy-4H-chromen-4-one; quercetin, quercetin) or a pharmaceutically acceptable salt thereof as an active ingredient.

The quercetin of the present invention induces the proliferation of EBV by solubilizing quercetin in Epstein-Barr virus (EBV) -positive gastric cancer cell line, killing the infected cells, and allowing the EBV progeny virus, which is dissolved outside the cells, And significantly inhibits the growth of tumors induced by EBV infection in vivo, which has superior anticancer effects than iscloquidizine, which is known to have antiviral activity and anti-EBV-positive gastric cancer activity The quercetin of the present invention is effective as an active ingredient of a pharmaceutical composition for prevention and treatment of gastric cancer caused by EBV infection.

In particular, EBV-positive gastric cancer induced by EBV infection is characterized by repeated PIK3CA mutations, prominent DNA hypermethylation, and amplification of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2) Since it has characteristics distinguishing it from gastric cancer, quercetin of the present invention is more effective because it can be specifically used for diagnosis and treatment of EBV-positive gastric cancer.

FIG. 1 shows cytotoxicity of quercetin or isocyanuric acid to the SNU719 cell line, which is an Epstein-Barr virus (EBV) -related gastric cancer cell line.
FIG. 2 is a chart showing the apoptosis of quercetin or isoricillitinin in an EBV-related gastric cancer cell line.
FIG. 3 is a graph showing changes in DNA methylation and demethylation patterns by quercetin or isocyanuric triinine in an EBV-related gastric cancer cell line.
FIG. 4 is a graph showing changes in expression level of an intracellular signal transduction pathway by quercetin or isoriculiidinin in an EBV-related gastric cancer cell line.
FIG. 5 is a graph showing a change in the transcription pattern of the EBV gene caused by quercetin or isocyanuricidin in an EBV-related gastric cancer cell line.
FIG. 6 is a graph showing the effect of quercetin or isocyanuric triinine on the protein production of EBV in EBV-related gastric cancer cell lines.
FIG. 7 is a diagram for confirming whether the frequency of use of the promoter of EBV gene is changed by quercetin or isoleucyllithinine in an EBV-related gastric cancer cell line.
8 is a graph showing the proliferation of EBV progeny virus in an EBV-related gastric cancer cell line by quercetin or isoriculiidin.
FIG. 9 is a graph showing the inhibitory effect of quercetin or isoricillitidin on infection of cell-cell EBV horizontal infection.
FIG. 10 is a chart for confirming the growth inhibitory effect of quercetin on tumors formed by EBV-related cell line transplantation in vivo .

Hereinafter, the present invention will be described in more detail.

As described above, although EBV infection has been reported as a cause of carcinogenesis of stomach cancer, there has been no report on the treatment of gastric cancer caused by EBV infection.

The quercetin of the present invention induces the proliferation of EBV by solubilizing quercetin in Epstein-Barr virus (EBV) -positive gastric cancer cell line, killing the infected cells, and allowing the EBV progeny virus, which is dissolved outside the cells, And effectively inhibits the growth of tumors induced by EBV infection in vivo. Thus, it is effective as an active ingredient of a pharmaceutical composition for the prevention and treatment of gastric cancer caused by EBV infection.

Accordingly, the present invention provides a process for the preparation of 2- (3,4-dihydrophenyl) -3,5,7-trihydroxy-4H-chromen- 7-trihydroxy-4H-chromen-4-one; Quercetin, quercetin) or a pharmaceutically acceptable salt thereof as an active ingredient.

The "quercetin" of the present invention is preferably, but not limited to, a structure of the following formula (1):

[Chemical Formula 1]

.

.

The "quercetin" of the present invention is preferably isolated from, but not limited to, an extract of licorice ( Glycyrrhiza uralensis ) or a fraction thereof.

The "gastric cancer" of the present invention is preferably gastric cancer induced by Epstein-Barr virus (EBV) infection.

In a specific example of the present invention, the inventors of the present invention found that, in order to determine whether quercetin exhibits an anticancer effect on EBV-induced gastric cancer, quercetin is added to SNU719 cells, which are gastric cancer cell lines containing an EBV genome as an episome As a result of confirming the CD ₅₀ concentration, when the drug was treated for 48 hours, the CD ₅₀ value of quercetin was 62 μM and the cell viability was decreased (FIG. 1). In order to compare the effect of quercetin, it was confirmed that the CD ₅₀ value of isoliquiritigenin used as a control group in the present invention was 45 μM (FIG. 1).

Further, as a result of confirming cell death by quercetin in an EBV-related gastric cancer cell line, it was confirmed that both quercetin and isoriculiidinin induce apoptosis of SNU719 cells (Fig. 2A), and treatment with quercetin inhibited EBV- The expression of PARP is completely inhibited by the treatment of quercetin in EBV-positive cells, which is an essential condition for the cell death involving quercetin, and quercetin is inhibited by apoptosis Indicating high cytotoxicity, which was induced by JNK pathway and EBV infection (Fig. 2B-2H).

As a result of confirming whether the transcription pattern of EBV gene was changed in quercetin or isoriculiidin-treated SNU719 cells, the quercetin-treated EBV-soluble gene was up-regulated to a high level, whereas the eosinly quercitidin was EBV The expression of the latent gene was upregulated to a high level, confirming that the isoriqulitizine increased the latent replication cycle of EBV, while the quercetin enhanced the soluble replication cycle of EBV (Fig. 5).

In addition, since quercetin regulates the transcription of EBV gene in SNU719 cells, the expression of the related protein is regulated. As a result, quercetin induces soluble replication of EBV in gastric cancer cells induced by EBV infection, (Fig. 6) plays a role in maintaining latency replication of EBV.

In addition, as a result of confirming the change of the frequency of promoter use of EBV gene by quercetin in EBV-related gastric cancer cell line, quercetin increased the use of F promoter for EBV-soluble replication as well as EBV gene transcription and related protein expression, Quiritizin was found to increase the frequency of use of the Q promoter for EBV latency replication (Fig. 7).

In addition, since most of EBV genes were confirmed to be regulated by quercetin or isocyanuricidin, quercetin was found to increase the proliferation of EBV progeny virus. As a result, quercetin induced EBV-soluble replication, resulting in EBV offspring virus in SNU719 cells (Fig. 8). On the other hand, it was confirmed that the sorbitol quercitidinin did not induce EBV-soluble replication and did not express the EBV offspring virus outside the cells (Fig. 8).

In addition, since it was confirmed that quercetin causes release of EBV to the outside of cells through induction of soluble replication in EBV-positive cells, the effect of quercetin on the EBV-negative peripheral cells infected with dissolved progeny EBV virus was confirmed. As a result, (Fig. 9), whereas the isoliquiritinin did not affect the EBV-positive cells to horizontal infection with EBV-negative cells.

In addition, in order to confirm whether quercetin can exert a therapeutic effect on EBV-induced gastric cancer in vivo, SNU719 cells were transplanted in immunodeficient mice to induce solid tumors and quercetin was orally administered. As a result, It was confirmed that the growth was inhibited (Fig. 10).

Therefore, the quercetin of the present invention is effective in quercetin in the EBV-associated gastric cancer cell line to induce the proliferation of EBV solubility, to kill the infected cells, effectively inhibit horizontal infection of the EBV progeny virus dissolved in the cells outside the normal cells, EBV-infected tumors, which exhibit superior anticancer effects over the known antiretroviral activity and anti-EBV-positive gastric cancer activity. Thus, the quercetin of the present invention can inhibit EBV Can be used as an active ingredient of a pharmaceutical composition for prevention and treatment of gastric cancer caused by infection.

In particular, EBV-positive gastric cancer induced by EBV infection is characterized by repeated PIK3CA mutations, prominent DNA hypermethylation, and amplification of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2) Because of its distinctive features from stomach cancer, the quercetin of the present invention is more effective because it can be used specifically for the treatment of EBV-positive gastric cancer.

The quercetin of the present invention can be used in the form of a pharmaceutically acceptable salt, and the acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention may be prepared by dissolving quercetin in an excess amount of an aqueous acid solution and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile Can be manufactured. Heating the same amount of quercetin and an acid or alcohol in water, and then evaporating and drying the mixture, or by filtering the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

In addition, the quercetin of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method. For example, quercetin can be dissolved in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile or the like and added with an excessive amount of an organic acid, Followed by precipitation or crystallization. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

When the composition of the present invention is used as a medicine, the pharmaceutical composition containing quercetin or a pharmaceutically acceptable salt thereof as an active ingredient may be formulated into various oral or parenteral dosage forms at the time of clinical administration, , But is not limited thereto.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing quercetin or a pharmaceutically acceptable salt thereof as an active ingredient of the present invention can be administered parenterally, and parenteral administration can be carried out by injecting subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection . In this case, in order to formulate the formulation for parenteral administration, a pharmaceutical composition containing quercetin or a pharmaceutically acceptable salt thereof as an active ingredient is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, and the ampule or vial Unit dosage form. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the composition of the present invention to the human body may be varied depending on the age, body weight, sex, dosage form, health condition, and disease severity of the patient. When the patient is 60 kg body weight, And may be 0.01 to 500 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

The present invention also provides a health functional food for preventing and improving gastric cancer, comprising quercetin or a pharmaceutically acceptable salt thereof as an active ingredient.

The "quercetin" of the present invention is preferably, but not limited to, a structure of the following formula (1):

[Chemical Formula 1]

.

.

The "quercetin" of the present invention is preferably isolated from, but not limited to, an extract of licorice ( Glycyrrhiza uralensis ) or a fraction thereof.

The "gastric cancer" of the present invention is preferably gastric cancer induced by Epstein-Barr virus (EBV) infection.

The quercetin of the present invention induces the proliferation of EBV by solubilization of quercetin in EBV-associated gastric cancer cell lines, thereby killing the infected cells, effectively inhibiting horizontal infection of EBV offspring virus dissolved in the cells outside the normal cells, and inhibiting EBV infection Induced quadricepsin significantly inhibits the growth of tumors, which exhibits anticancer effects superior to the conventional antiviral activity and anti-EBV-positive gastric cancer activity known to have anti-EBV-positive gastric cancer activity, Can be used as an active ingredient of a health functional food for prevention and improvement of gastric cancer induced.

In particular, EBV-positive gastric cancer induced by EBV infection is characterized by repeated PIK3CA mutations, prominent DNA hypermethylation, and amplification of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2) Since it has characteristics distinguishing it from gastric cancer, quercetin of the present invention is more effective because it can be specifically used for diagnosis and treatment of EBV-positive gastric cancer.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The quercetin of the present invention or a pharmaceutically acceptable salt thereof may be added directly to the food or may be used together with other food or food ingredients and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients except that it contains the compound of the present invention as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the quercetin of the present invention or a pharmaceutically acceptable salt thereof may be in the form of a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), a synthetic flavoring agent and a natural flavoring agent, a coloring agent and a thickening agent (cheese, Acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the quercetin or pharmaceutically acceptable salt thereof of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the quercetin or pharmaceutically acceptable salt thereof of the present invention.

In addition,

i) treating and culturing the sample derived from the subject with quercetin;

ii) analyzing the characteristics of the sample cultured in step i); And

iii) comparing the result of the analysis in the step ii) with a non-treatment control, to provide a method for measuring the level of gene or protein expression for providing information for gastric cancer diagnosis.

The "quercetin" of step i) of the present invention is preferably treated at a concentration of 32 to 250 μM, more preferably at a concentration of 40 to 125 μM, more specifically at a concentration of 62 μM Most preferably, but not limited thereto.

The step ii) "characteristics" of the present invention is preferably, but not limited to, any one of the following a) to d).

a) decreased expression of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A) protein;

b) decreased methylation of the BCL7A gene;

c) estrogen, heat shock response, upregulation of MAPK / ERK and MAPK / JNK signaling pathways; And

d) Downregulation of the glucocorticoid, heavy metal stress, interferon gamma, liver X receptor and TGF-beta signaling pathways.

The "gastric cancer" of the present invention is preferably gastric cancer caused by EBV infection.

In another embodiment of the present invention, the inventors of the present invention found that expression of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A), which are methyltransferases, were detected in the EBV-related gastric cancer cell line by the quercetin- (Fig. 3A), and the methylation level of BCL7A, a SNU719 cell tumor suppressor gene, was decreased by quercetin (Fig. 3B).

In addition, the present inventors have examined the level of intracellular signal transduction pathway by quercetin in an EBV-related gastric cancer cell line. As a result, in the quercetin-treated SNU719 cells, four estrogen, heat shock response, MAPK / ERK, The MAPK / JNK pathway is up-regulated and the pathway of glucocorticoid, heavy metal stress, interferon gamma, liver X receptor and TGF-beta pathway is upregulated (FIG. 4).

Therefore, the quercetin of the present invention reduces the expression of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A) in the EBV-associated gastric cancer cell line, decreases the methylation level of the BCL7A gene, , Which can be useful in diagnosing stomach cancer induced by EBV infection in gastric cancer patients.

In particular, EBV-positive gastric cancer induced by EBV infection is characterized by repeated PIK3CA mutations, prominent DNA hypermethylation, and amplification of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2) Since it has characteristics distinguishing it from gastric cancer, quercetin of the present invention is more effective because it can be specifically used for diagnosis of EBV-positive gastric cancer.

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 examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Epstein-Barr virus ( Epstein - Barr  virus; EBV ) Cytotoxicity of quercetin to related gastric cancer cell lines

Order with respect to the stomach cancer caused by EBV to ensure that quercetin is showing an anti-cancer effect, and it contained the EBV genome in the epi bit (episome) and confirm the CD ₅₀ levels of quercetin on the gastric cancer cell lines SNU719 cells. Isoliquiritigenin, which has a structure similar to quercetin, was used as a control for comparison with the antitumor activity of quercetin.

<1-1> Culture of SNU719 cell line and preparation of sample

SUN719 cells were cultured in RPMI1640 (Welgene, Korea) medium containing 10% fetal bovine serum (FBS, Welgene), antibiotic / antifungal agent (Gibco, Grand Island, NY, USA) and glutamax Respectively. The culture was incubated in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 95% humidity.

Quercetin and isolequittinin were purchased from Sigma-Aldrich (Catalog Nos. I3766 and Q4951). Quercetin and isocyanuric acid were dissolved in 200 mM DMSO solution, and impurities were removed with a 0.22 ㎛ filter and stored at -20 캜.

&Lt; 1-2 > CD of quercetin on SNU719 cells ₅₀  Verify the value

The cytotoxicity of quercetin or isocyanuric acid to SNU719 cells was assayed using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan).

Specifically, 100 μl of the suspension was inoculated so that the cultured SNU719 cells had a cell number of 1 × 10 ⁴ cells per well. Then, 0, 4, 8, 16, 32, 62, 125, 250, 500 or 1000 μM Quercetin or isocyanuric acid was added at various concentrations. Immediately after addition, 24 hours and 48 hours after addition, 10 μl of CCK-8 solution was added to each well and incubated for 3 hours. Then, the absorbance of each cell sample was measured using an ELISA reader. All other procedures were performed according to the protocol provided by the manufacturer, and the average value was used after three experiments of the same experiment.

As a result, the more the concentration of quercetin and with yisori Curie T increases as shown in Figure 1, it was confirmed that the cell death is SNU719, and calculated the concentration of CD ₅₀ represents the cytotoxic for 50% of cells. When the drug was treated for 48 hours, the CD ₅₀ value of quercetin was 62 μM (FIG. 1A), and the CD50 value of isocyanuric acid was 45 μM (FIG. 1B). In addition, CD ₅₀ value of quercetin when treated 24 hours 125 μM, and (Fig. 1C), yisori CD ₅₀ value of the Curie T with is to check that the 57 μM (Fig. 1D), the longer the processing time of the drug CD ₅₀ value . &Lt; / RTI &gt;

<1-3> Identification of the decrease in the survival rate of SNU719 cells by quercetin treatment

In order to confirm the cell survival curve over time, SNU719 cells treated with 62 μM quercetin or 45 μM isocyanuric triinine for 48 hours were obtained and the number of cells was counted by trypan blue staining. The number of viable cells and apoptotic cells was counted by cell count, and the cell viability (%) was determined by calculating the ratio of viable cells to total cells. DMSO was used for untreated control.

As a result, as shown in FIG. 1E and FIG. 1F, when quercetin was treated, it was found that the cell survival rate was slightly lower than that when compared with DMSO or isocyanuritizin. In all cases, cell survival rate after 48 hours was 85 % Or more (Figs. 1E and 1F).

Confirmation of cell death by quercetin in EBV-related gastric cancer cell line

<2-1> Determination of the cell death of EBV-positive gastric cancer cell line by quercetin

Since quercetin exhibits cytotoxicity against the EBV-related gastric cancer cell line SNU719 cell line, annexin V-FITC apoptosis detection assay is performed to confirm the manner in which SNU719 cell line is killed by quercetin Respectively.

Specifically, the embodiment and inoculated with <1-1> and SNU719 cells cultured in a 6 ㎝ plate at a concentration of 1 × 10 ^6/5 ㎖, and cultured by treatment of 62 μM quercetin. After 48 hours, the cells were stained using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA), and the cells were stained for 1 hour with the initial cell death of stained SNU719 cells apoptosis, necrosis / late apoptosis, and cell membrane integrity were analyzed using a FACSAria III cell sorter. As a control for comparison, 45 μM isocyanuric triinine was treated, and the same method was performed by treating with DMSO as a non-treated control group.

As a result, as shown in FIG. 2A, quercetin and isoriculiidinin induced apucin death of SNU719 cells, and in particular, they increased initial apoptosis by about 175% as compared with those treated with DMSO, and late apoptosis / And increased by about 215% (FIG. 2A). On the other hand, since the cell membrane integrity of SNU719 cells in which apoptosis occurred did not decrease (Fig. 2A), it was confirmed that the apoptosis by quercetin did not increase the destruction of cell membrane integrity.

<2-2> Identification of changes in cell death related factors by quercetin

Quercetin induces apoptosis in SNU719 cells. Therefore, in order to confirm the phenomenon manifested in cells, Western blot analysis of the degree of change of apoptosis related protein was carried out. As a protein related to apoptosis, it can maintain the cell viability and cleavage degree of PRAP protein used as a marker for confirming the degree of apoptosis and induce apoptosis in the JNK signaling pathway The degree of phosphorylation of the known JNK was confirmed.

Specifically, the SNU719 cells cultured in Example <1-1> were treated with quercetin or isocyanuric triinine for a total of 48 hours, while trypsin was used at 0 hours, 24 hours and 48 hours after the treatment &Lt; / RTI &gt; The obtained cells were counted by 10 × 10 ⁶ cells, and then 100 μl of an eporter lysis buffer (Promega) containing 1 μl of a proteinase inhibitor and 10 μl of phenylmethylsulfonyl fluoride USA, USA). The lysed cells were further pulsed with an ultrasonic pulverizer (Bioruptor sonicator) at intervals of 30 seconds for 5 minutes to prepare a cell lysate sample. When storage was required, cell lysates were lyophilized in liquid nitrogen and stored at -80 ° C.

25 μl of the prepared cell lysate was separated from 7% sodium dodecyl sulfate polyacrylamide electrophoresis gel, and then washed with 1: 1000 diluted anti-PARP antibody (Cell Signaling Technology, USA Western blot analysis was performed using anti-SAPK / JNK antibody (Cell Signaling Technology) or anti-phospho-SAPK / JNK antibody (Cell Signaling Technology) as the primary antibody. Horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (Amersham Biosciences) or horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (Amersham Biosciences, Switzerland) was used as the secondary antibody. conjugated goat anti-rat IgG antibody (Bethyl Laboratories, USA) was used.

As a result, it was confirmed that some of the PARP proteins were cleaved (Fig. 2B), as shown in Figs. 2B and 2C, than the treatment with quercitinin in the quercetin treatment. In addition, the treatment of quercetin also increased the production of phosphorylated-SAPK / JNK, which was higher than that of isocyanuric acid (Fig. 2C).

<2-3> Confirmation of the degree of cell death by inhibition of JNK phosphorylase

In an EBV-positive gastric cancer cell line, SNU719 cells, quercetin increased PARP protein modification and phosphorylation of JNK protein. To confirm this specifically, SP600125, an anthrapyrazolone inhibitor of JNK phosphorylase, was treated with quercetin.

Specifically, quercetin or isocyanuric triinine was treated in the same manner as in Example <2-2>, and 10 μM of SP600125 was treated with JNK phosphorylation inhibitor as an inhibitor and cultured for 48 hours. Then, western blot analysis was performed in the same manner as in Example <2-2> to confirm the modification of PRAP protein and the degree of phosphorylation of JNK protein. DMSO was used as an untreated control group and TPA / NaB was used as a positive control.

As a result, phosphorylation of c-Jun was inhibited when SP600125 was treated with DMSO, isoleucyltidine, or quercetin, except in the case of TPA / NaB used as a positive group, as shown in Figs. 2D and 2E (Fig. 2D). It was also confirmed that the treatment of SP600125 was dependent on quercetin-induced apoptosis and was at a higher level than the effect of isocyanuric acid (Fig. 2E).

<2-4> Cell death by quercetin in EBV-infected gastric cancer cell line

In order to confirm whether or not the quercetin-induced cell death by the quercetin was specifically detected in EBV-infected cells, anti-RARP antibody and anti-phosphorylated JNK antibody were used in EBV-negative gastric cancer cell line, AGS cell line, Bleed analysis was performed.

Specifically, AGS cells were cultured in RPMI1640 medium containing 10% FBS (Omega, Canada), antibiotics / antifungal (Gibco, USA) and glutamax (Gibco) Carbon dioxide and 95% humidity were maintained in a CO2 incubator. AGS cells (AGS-wt EBV) infected with wild-type EBV and recombinant EBV-infected AGS cells (AGS-EBV-GFP) conjugated with GFP were donated by KRIBB and Catholic University of Korea I used what I received. AGS cells infected with GFP-conjugated recombinant EBV were screened at 400 μg / ml G418.

The cultured AGS cells, AGS-wt EBV cells and AGS-EBV-GFP cells were subjected to Western blot analysis in the same manner as in Example <2-2> to confirm the modification of PRAP protein and the degree of phosphorylation of JNK protein.

As a result, as shown in FIGS. 2F to 2H, phosphorylation-JNK was significantly decreased in EBV-positive AGS cells compared with EBV-negative AGS cells, and JNK expression was significantly elevated (FIGS. 2F, 2G, 2H). However, it was confirmed that the phosphorylation of JNK in EBV-positive AGS cells was induced by treatment with quercetin and isorqualitizine when compared with EBV-negative AGS cells (Fig. 2F, 2G, 2H).

That is, the quercetin treatment significantly increased the phosphorylated JNK in EBV-positive cells as compared to the isocyanuricidin, and the expression of PARP was completely inhibited by the quercetin treatment in EBV-positive cells, and the quercetin treatment in EBV- The expression of the protein was not changed.

When these results are compared, EBV infection is an essential condition for quercetin-induced apoptosis, and quercetin is highly cytotoxic by apoptosis, which is induced by JNK pathway and EBV infection.

EBV  Related gastric cancer cell line On quercetin  DNA methylation by Demethylation  Identify changes in aspects

According to previous studies, gastric cancer can be classified into four types according to the etiology of the disease. EBV-positive gastric cancer induced EBV infection is characterized by repeated PIK3CA mutations, prominent DNA hypermethylation and JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2), and have been reported to have features distinct from other types of gastric cancer. Therefore, it is confirmed that confirming the effect of quercetin on DNA methylation or demethylation in EBV-positive gastric cancer can be used as an important factor.

<3-1> SNU719  In a cell On quercetin  The level of expression of intracellular DNA methylase

In order to confirm whether quercetin or isocyanuric acid influences the methylation of cellular genes, the expression of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A) were confirmed.

Specifically, in the same manner as in Example <2-2>, SNU719 cells were treated with quercetin or isocyanuric triinine, cultured for 48 hours, and Western blot analysis was performed to confirm the expression levels of DNMT1 and DNMT3A enzymes . Anti-DNMT1 antibody (Santa Cruz Biotechnology) and anti-DNMT3A antibody (Santa Cruz Biotechnology) were used as primary antibodies for Western blot. DMSO was used as a negative control, and TPA / NaB was used as a positive control.

As a result, as shown in FIG. 3A, the expression of DNMT1 and DNMT3A was significantly decreased when quercetin was treated, whereas it was found that DNKT1 and DNMT3A were not affected by the treatment with isoriculiidin 3A).

<3-2> Identification of changes in cellular gene methylation level by quercetin in SNU719 cells

In SNU719 cells, the reduction of DNMT expression by quercetin was confirmed to demethylate tumor suppressor gene and EBV locus. Since the SNU719 cell line is known to show dense methylation in the TP73, BLU, FSD1, BCL7A, MARK1, SCRN1 and NKX3.1 genes, methylation-specific PCR methylation-specific PCR (MCR) was performed to determine whether quercetin and isoriculi tizinin affect methylation in the tumor suppressor gene BCL7A.

Specifically, in the same manner as in Example <2-2>, 62 μM quercetin or 45 μM isocyanuric triinine was treated, followed by culturing and disruption to obtain a cell lysate. The obtained cell lysate was subjected to CpGenome DNA Modification Kit (Millipore, USA) according to the protocol provided by the manufacturer to convert the DNA into sodium bisulphite. Then, 5 μl of 5 × reaction mixture (NanoHelix, Korea), 5 μl of 5 × TuneUp solution (NanoHelix), 1 μl of Taq-plus polymerase (NanoHelix, 25 μl reaction sample containing 2.5 μl each of 10 μM forward primer and reverse primer was prepared. Sequences of the forward primer and the reverse primer were as shown in Table 1 below. The PCR reaction was carried out in TaKaRa PCR Tomocycler (TaKaRa, Japan) under the following conditions: 3 minutes at 95 ° C; 30 seconds at 95 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C; Then at 72 ° C for 10 min. Then, they were separated by electrophoresis on a 1.5% agarose / TBE gel. The separated resultant bands were quantitatively analyzed by densitometric analysis using ImageQuant 5.2. The intensity values of all the bands are the sum of the intensities of all the bands on the agarose gel, and the respective band intensity values are shown as relative values compared with the intensity values of the whole band. As a control, DMSO was treated as a negative control and 10 mM DAC (5-aza-2'-deoxycytidine), which is a demethylating enzyme, was treated as an internal control.

As a result, as shown in FIG. 3B, the methylation level in the CpG domain of the BCL7A gene was slightly reduced when quercetin was treated as compared with the negative control DMSO (FIG. 3B). However, the isolurquiritinin did not show a significant effect (Fig. 3B).

<3-3> Identification of the level of STAT3 pathway expression by quercetin in SNU719 cells

We confirmed the reduction of DNMT expression by quercetin in SNU719 cells and thus confirmed the effect of quercetin treatment on the expression or phosphorylation of STAT3 pathway, which is reported to be involved in the upregulation of DNMT1.

Specifically, SNU719 cells were treated with quercetin or isocyanuric triinine in the same manner as in Example <2-2>, and treated with 10 μM of SP600125 as an inhibitor of JNK phosphorylase and cultured for 48 hours. Then, western blot analysis was performed in the same manner as in Example <2-2> to confirm protein expression and phosphorylation level of STAT3 and the expression level of DNMT1 and DNMT3a proteins. Anti-DNMT1 antibody (Santa Cruz Biotechnology) and anti-DNMT3A antibody (Santa Cruz Biotechnology) were used as the primary antibodies for Western blotting, anti-STAT3 antibody (Cell Signaling Technology), anti-phospho-Stat3 antibody Biotechnology) was used. DMSO was used as a negative control, and TPA / NaB was used as a positive control.

As a result, as shown in FIGS. 3C to 3E, when quercetin did not significantly inhibit phosphorylation of STAT3 as compared with DMSO control, but it was confirmed that the quercitinin slightly increased the phosphorylation of STAT3, DNMT was downregulated in a manner independent of pathway (Figure 3C). In addition, when treated with the JNK phosphorylase inhibitor SP600125, quercetin significantly inhibited the degree of phosphorylation of STAT3, whereas it was confirmed that the isoriquritizin significantly increased the degree of phosphorylation of STAT3 (FIG. 3D). At the same time, when the SP600125 and the isolurquiritinin were simultaneously treated, the expression of DNMT3A was suppressed, whereas when the quercetin was simultaneously treated, the expression of DNMT1 and DNMT3A was completely inhibited (Fig. 3E).

These results indicate that DNMT1, which can be regulated by quercetin, is STAT3 independent and that DNMT3 is dependent on STAT3, and that the reduction of quercetin-induced demethylation is due to the presence of genes that are hypermethylated in the genomes of SNU719 cells and EBV It is shown that it plays an important role in the expression.

EBV  Related gastric cancer cell line On quercetin  Of intracellular signal transduction pathways

Since quercetin and isoleucyltidine differ in the expression patterns of DNMT1 and DNMT3A, it is determined that SNU719 cells use different signal transduction pathways, and specifically, through the modulation of a signal transduction pathway, .

Specifically, in order to use a Cignal Finder Multi-Pathway Reporter Array (Qiagen, catalog number CCA-901L), artacten was dispensed into 1-well per well on a 96-well array plate Respectively. SNU719 cells were then diluted in Opti-MEM medium, dispensed into each cell of the plate, and treated with 62 uM quercetin or 45 uM isocyanuric triinine. After 48 hours of treatment, the reverse transfection reagent and Opti-MEM medium were removed. Then, 75 μl of Dual-Glo Luciferase Reagent was added to each well and left at room temperature for 10 minutes. The measurement of the luciferase activity was then determined according to the protocol provided by the manufacturer.

As a result, as shown in FIG. 4, it was confirmed that quercetin treatment in SNU719 cells, an EBV-positive gastric cancer cell line, increased 4 pathways in 45 signaling pathways and suppressed 5 signaling pathways (Fig. 4A). Four increased pathways are estrogen, heat shock response, MAPK / ERK and MAPK / JNK pathways and five inhibited pathways are glucocorticoid, heavy metal stress, interferon Gamma (interferon gamma), liver X receptor and TGF-beta (beta) pathway. Conversely, isocyanuric acid increases the five signaling pathways of amino acid deprivation response, MAPK / ERK, MAPK / JNK, PPAR and retinoid X receptor pathways, Lt; RTI ID = 0.0 &gt; interferon &lt; / RTI &gt; regulator pathway (FIG. 4B). The signal transduction pathways regulated by quercetin or isorquiritizine are all involved in cell death and survival in the cell.

EBV  Related gastric cancer cell line On quercetin  by EBV  Identification of transcriptional changes in genes

To determine whether quercetin or isocyanuric acid has anti-viral activity against EBV transcript, RT-qPCR analysis was used to determine whether the transcription pattern of EBV gene in SNU719 cells treated with quercetin or isocyanuritizin was changed Respectively.

Specifically, SNU719 cells cultured in Example <1-1> were treated with quercetin or isocyanuric triinine for a total of 48 hours, and then cells were obtained and cultured using an RNeasy mini kit (Qiagen, USA) The intracellular RNA was extracted. Using the extracted RNA as a template, cDNA was synthesized using Superscript II Reverse Transcriptase (Invitrogen). The synthesized cDNA was diluted at a ratio of 1:50 in pure water containing no nucleic acid degrading enzyme, and the expression levels of potential genes and soluble genes of EBV were analyzed by quantitative PCR (qPCR). The sequences of the primers used for qPCR are as described in [Table 1] above. was performed using qPCR CFX96 (Bio-Rad) using iQ SYBR Green reagent (Bio-Rad).

As a result, as shown in FIG. 5, the treatment of quercetin, compared with the untreated control group, showed a lytic gene such as BNRF1, BCRF1, BLLF1, BZLF1 and BRLF1 and a latent gene such as LMP1, EBNA3A and EBNA3C lt; / RTI &gt; (Fig. 5). The treatment with isorylqualithine increased the transcription of the soluble genes of NRF1, BCRF1, BLLF1, BZLF1 and BRLF1 and the latent genes of LMP1, LMP2, EBER1, EBNA3C and EBNA1 (FIG. 5).

Compared with quercetin treatment, however, quercetin treatment resulted in higher upregulation of EBV-soluble genes such as BLLF1, BZLF1, and BRLF1, while quercitinin enhanced LMP2, The expression of latent genes such as EBER1, EBNA3A, EBNA3C and EBNA1 was upregulated to a higher level. Thus, it was confirmed that the quercitinin increases the latent replication cycle of EBV while quercetin enhances the soluble replication cycle of EBV.

Determination of the effect of quercetin or isocyanuricidin in the production of EBV protein

<6-1> Changes in EBV protein expression by quercetin in SNU719 cell line

It was confirmed that the EBV gene transcription can be regulated by quercetin in EBV-related gastric cancer cell lines. Thus, it was confirmed that quercetin can regulate the expression level at the same protein level.

Specifically, in the same manner as in Example <2-2>, SNU719 cells were treated with quercetin or isocyanuric triinine and cultured for 48 hours, followed by Western blot analysis to determine the expression levels of EBNA1, BZLF1 and LMP2A proteins Respectively. Anti-EBV EBNA1 antibody (Santa Cruz Biotechnology, USA), anti-EBV BZLF1 antibody (Santa Cruz Biotechnology) and anti-EBV LMP2A antibody (Santa Cruz Biotechnology) were used as primary antibodies for Western blot. DMSO was used as a negative control, and TPA / NaB was used as a positive control.

As a result, as shown in FIG. 6A, quercetin did not increase the expression of BZLF1 and LMP2A, as in the transcriptional tendency of the EBV gene affected by quercetin or isocyanuric triinine identified in [Example 5] (Fig. 6A). It was confirmed that the gene induces the expression of LMP2A significantly (Fig. 6A).

<6-2> EVB  Infected AGS  In the cell line On quercetin  by EBV  Identification of changes in protein expression

The expression of BZLF1 protein was confirmed by using AGS cells in order to confirm the change of EBV protein expression in quercetin treatment according to EBV infection in EBV negative cells.

Specifically, in the same manner as in Example <2-4>, AGS cells, AGS-wt EBV cells and AGS-EBV-GFP cells were treated with quercetin or isocyanuric triinine and cultured for 48 hours. Western blot analysis Were performed to confirm expression levels of EBNA1 and BZLF1 proteins.

As a result, as shown in Figs. 6B to 6D, the expression of BZLF1 was not observed in EBV-negative AGS cells, whereas in EBV-positive cells, BZLF1, which was significantly induced by quercetin or isocyanuricuridine, In the case of quercetin treatment, it was confirmed that BZLF1 was induced at a higher level than in the case of isocyanuricidin (Fig. 6B, Fig. 6C, Fig. 6D). On the other hand, EBNA1 expression in EBV-positive AGS cells was also expressed in the DMSO control group, and thus it was confirmed that it was not induced by isocyanuricidin, but it was confirmed that the expression of EBNA1 was completely inhibited by quercetin (FIGS. 6B, 6C, 6D). These results show that quercetin induces the soluble replication of EBV in gastric cancer cells induced by EBV infection and induces soluble replication of EBV in EBV-associated gastric cancer cell lines, It can be seen that it plays a role of maintaining latency replication of

EBV  Related gastric cancer cell line On quercetin  by EBV  Identification of changes in gene promoter usage frequency

It was determined whether quercetin or isori quercitidinin affects the frequency of use of EBV potential promoters such as Cp, Qp, and Fp, since quercetin and isoleucyltidine affect the expression of most EBV genes .

Specifically, the SNU719 cells were treated with quercetin or isocyanuric triinine in the same manner as in Example <2-2>, and cultured for 48 hours. Then, RNA was isolated in the same manner as in Example 5, Lt; / RTI &gt; Actin, EBV Qp, EBV Cp / Wp and EBV Fp were used as the primer sequences listed in Table 1 above. 5 μl of 5 × reaction mixture, 5 μl of 5 × TuneUp solution, 1 μl of Taq-plus polymerase and 2.5 μl of 10 μM forward / reverse primer, respectively. PCR was performed in the TaKaRa PCR Thermal Cycler under the following conditions: 3 minutes at 95 ° C; Repeating 30 seconds at 95 ° C for 10 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 10 minutes; Then at 72 ° C for 10 min. They were then separated and analyzed by 1.5% agarose / TBE gel. For use as a control, the EBV gene promoter usage was compared by treating EBV-luciferous 1-positive cells KEM1 cells and EBV type III-specific KEM3 cells in the same manner as SNU719 cells.

As a result, as shown in FIG. 7, it was confirmed that the KEM1 cells used as the control group had a high frequency of use of the Q promoter and the F promoter, and that the EBV type III-specific KEM3 cells used the C / W promoter more frequently 7A). The treatment of quercetin in SNU719 cells significantly reduced the use of the Q promoter and increased the use of the F promoter, confirming the results consistent with the effect of quercetin on EBV soluble replication (Fig. 7B). In contrast, it was found that the treatment of the isoriquiritinin completely coincided with the characteristics of the isocyanuric triinine, which makes it possible to completely suppress the use of the F promoter and maintain the EBV latency by increasing the frequency of use of the Q promoter 7B).

On quercetin  by EBV  Related gastric cancer cell line EBV  Identification of progeny virus proliferation

Since most of the EBV genes were confirmed to be regulated by quercetin or isocyanuricidin, the number of copies of EBV genome in the host cell SNU719 and in the extracellular region was checked to see if the proliferation of EBV progeny virus was increased by quercetin.

Specifically, in the same manner as in Example <2-2>, 62 μM quercetin or 45 μM isocyanuric triinine was treated, followed by culturing and disruption to obtain a cell lysate, and then the genomic DNA (gDNA) was extracted. 50 ng of extracted gDNA was subjected to qPCR and the relative amount of EBV gDNA amplified using actin, an internal control, was calculated. The number of EBV copies in the cells was calculated as the relative amount of EBV gDNA to total gDNA.

In order to calculate the number of EBV copies outside the cells, SNU719 cells were treated with quercetin or iso-nin to obtain 20 ml of a culture medium. The culture medium was filtered through a 0.45 nm syringe filter, added to a phosphate-buffered saline (PBS) containing 20% sucrose cushion, and centrifuged at 27,000 rpm for 90 minutes (ultracentrifugation; CP100WX, Hitachi). The viral precipitate was dissolved in 100 μl of FA dissolution buffer containing EDTA (1 mM, pH 8.0), HEPES-KOH (50 mM, pH 7.5) and NaCl (140 mM) and sonicated with a Bioruptor sonicator for 30 seconds The DNA was extracted by pulsing with a pulse of interval for 5 minutes. Then, the viral DNA was amplified and quantified using a primer set specific to EBV EBNA1 using the viral DNA as a template in 100 μl of RNase-free water.

As a result, as shown in Fig. 8, the intracellular EBV genome copy number was not significantly affected by quercetin or isoriculiidin (Fig. 8A). The number of extracellular EBV genome copies increased to 150% in the case of quercetin treatment, but was not significantly changed in the case of treatment with isocyanuric triinine (FIG. 8B). Through this, it was confirmed that quercetin induces EBV soluble replication to produce EBV offspring virus in SNU719 cells.

Identification of Quercetin Infection Suppressing Effect of Cell-cell Intercellular EBV Levels

It was confirmed that quercetin causes EBV release to the outside of cells through induction of soluble replication in EBV-positive cells. Therefore, the effect of quercetin on EBV-negative peripheral cells infected was confirmed.

Specifically, AGS cells were counted at 0.625 × 10 ⁶ cells per well and inoculated into a 6-well plate (Corning, USA). The culture medium of AGS was RPMI1640 (Hyclone, USA) containing 10% FBS (Hyclone), antibiotics / antifungal agent and glutamax. After 1 day of inoculation, the medium was removed and replaced with pure RPMI1640 medium. Then, LCL-EBV-GFP cells were counted at 1.25 × 10 ⁶ / ml, cultured with AGS cells, and treated with 62 μM quercetin or 58 μM isocyanuric triinine. After 72 hours of treatment, all medium was removed and the cells were washed with PBS to leave only AGS-EBV-GFP. EBV-GFP was transferred to bacto-agar (1.5%) supplemented with 2 x RPMI1640 medium containing 20% FBS (Hyclone), antibiotic / antifungal agent and glutamax. Cells were cultured for 72 hours after inoculation, and the number of cells was counted by confirming GFP in AGS cells.

As a result, as shown in Fig. 9, EBV was horizontally infected from LCL-EBV to AGS cells, but when compared with the DMSO control, the addition of quercetin effectively inhibited the migration of EBV from the LCL-EBV-GFP cells to the AGS cells (Fig. 9A). On the other hand, it was confirmed that EBV was transferred from LCL cells to AGS cells when horizontally-infected and did not show a significant difference from the DMSO control (Fig. 9B). Thus, it was confirmed that quercitinin did not affect horizontal infection of EBV-positive cells with EBV-negative cells, whereas quercitinin did not affect horizontal infections due to quercetin-induced progeny virus blockage.

In vivo In vivo )in By EBV  For gastric cancer Quercetin  Identification of tumor growth inhibitory effect

Since quercetin induced the proliferation of EBV solubility in EBV-positive gastric cancer cell lines and effectively inhibited horizontal infections of EBV offspring viruses, which have been infected and killed outside the cells, from the in vitro level, We investigated whether quercetin could exert therapeutic effects on EBV - induced gastric cancer in vivo.

Specifically, the SNU719 cells cultured in Example <1-1> were counted to 5 × 10 ⁶ cells, and then transplanted into the subcutaneous tissue of a 5-week-old immunodeficient mouse NOD-SCID mouse and cultured for 7 days to obtain SNU719 So that solid tumors were induced to develop. When solid tumors developed, quercetin was orally administered to NOD-SCID mice once a day at a dosage of 30 mg / kg. The size of solid tumors was measured 19 days after the start of administration. As a non-treated control group, drinking water was administered without quercetin, and each mouse was divided into 6 mice to determine the size of the solid tumors, and the average value was obtained.

As a result, as shown in FIG. 10, solid tumors induced from EBU-positive SNU719 cells in the subcutaneous tissue of mice showed inhibition of development from the 5th day after the oral administration of quercetin, and the size of solid tumor in the untreated control group administered with drinking water , While growth was inhibited in the quercetin-treated experimental group (Fig. 10).

Claims (10)

2- (3,4-dihydrophenyl) -3,5,7-trihydroxy-4H-chromen-4-one -chromen-4-one; Quercetin, quercetin) or a pharmaceutically acceptable salt thereof as an active ingredient.
The pharmaceutical composition for preventing and treating gastric cancer according to claim 1, wherein the quercetin is isolated from licorice extract or a fraction thereof.
The pharmaceutical composition for the prevention and treatment of gastric cancer according to claim 1, wherein the gastric cancer is gastric cancer induced by Epstein-Barr virus (EBV) infection.
A health functional food for preventing and improving gastric cancer, comprising quercetin or a pharmaceutically acceptable salt thereof as an active ingredient.
5. The health functional food for preventing and improving gastric cancer according to claim 4, wherein the quercetin is isolated from licorice extract or a fraction thereof.
The health functional food for preventing and improving gastric cancer according to claim 4, wherein the gastric cancer is gastric cancer induced by EBV infection.
i) treating and culturing the sample derived from the subject with quercetin;
ii) analyzing the characteristics of the sample cultured in step i); And
iii) comparing the result of the analysis in step ii) with the untreated control group to determine the level of gene or protein expression to provide information for gastric cancer diagnosis.
8. The method according to claim 7, wherein the quercetin of step i) is treated at a concentration of 32 to 250 [mu] M to measure the level of gene or protein expression to provide information for gastric cancer diagnosis.
The method according to claim 7, wherein the characteristic of step ii) is any one of the following a) to d): a method for measuring the level of gene or protein expression for providing information for gastric cancer diagnosis;
a) decreased expression of DNMT1 (DNA methyltransferase 1) and DNMT3A (DNA methyltransferase 3A) protein;
b) decreased methylation of the BCL7A gene;
c) estrogen, heat shock response, upregulation of MAPK / ERK and MAPK / JNK signaling pathways; And
d) Downregulation of the glucocorticoid, heavy metal stress, interferon gamma, liver X receptor and TGF-beta signaling pathways.
[Claim 7] The method according to claim 7, wherein the gastric cancer is gastric cancer induced by EBV infection.

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