KR20210145922A - Composition for preventing, improving or treating cancer comprising Aplykurodin A as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating cancer comprising aplykurodin A as an active component. Aplykurodin A of the present invention strongly inhibits a Wnt/β-catenin signaling system, promotes β-catenin protein degradation, and induces apoptosis and promotes autophagy in cancer cells without showing cytotoxicity in normal cells, thereby being able to be usefully used for preventing, alleviating or treating various cancer diseases.

Description

A composition for preventing, improving or treating cancer comprising aplicurodin A as an active ingredient

The present invention relates to a composition for preventing, improving or treating cancer comprising aplicurodin A as an active ingredient.

Cancer, also called neoplasia, is generally characterized by "uncontrolled cell growth." By abnormal cell growth of cancer cells, a cell mass called a tumor is formed, which penetrates into surrounding tissues and, in severe cases, metastasizes to other organs of the body. Cancer is an intractable chronic disease that causes pain and ultimately death without being cured in many cases even with surgery, radiation, and chemotherapy.

There are various causes of cancer, but internal factors include genetic factors and immunological factors, and external factors include chemicals, radiation, viruses, and the like. It is not known exactly how normal cells are transformed into cancer cells, but the balance between genes involved in the development of cancer, that is, oncogenes and tumor suppressor genes Cancer occurs when it breaks down by the above internal or external factors.

Cancer is broadly classified into blood cancer and solid cancer. It occurs in almost any part of the body.

Among cancers, liver cancer is known as one of the most deadly cancers in the world, and in particular, it is reported that more than half a million people die from liver cancer every year in Asia and sub-Saharan Africa. Liver cancer can be broadly divided into primary liver cancer (hepatocellular carcinoma) arising from hepatocellular carcinoma and metastatic liver cancer in which cancers from other tissues have metastasized to the liver. More than 90% of liver cancers are primary liver cancer. Hepatocellular carcinoma (HCC) is the fifth most common tumor in the world, with 500,000 deaths annually (Okuda 2000). Survival rates in HCC patients have not improved over the past 20 years and have an incidence nearly equal to mortality (Marrero, Fontana et al 2005). Therefore, there is an urgent need to develop an anticancer drug for the liver cancer in the current situation.

On the other hand, among the cell signaling systems targeted for anticancer drug development, the Wnt/β-catenin signaling system is important in various processes such as development, differentiation, and cell proliferation by stimulating intracellular signaling pathways by Wnt acting as a ligand for several receptors. When Wnt does not act as a ligand, GSK3β, CK1a, Anexin, and APC form a complex to phosphorylate β-catenin, and phosphorylated β-catenin is ubiquitinated by E3 ligase to form a 26S protease. On the other hand, when Wnt binds to the frizzled receptor and the co-receptor LRP5 (lipoprotein receptor-related protein5) or LRP6, the signal is only activated After metastasis starts to occur sequentially and β-catenin is released and enters the nucleus, β-catenin binds to LEF (Lymphoid Enhancer-Binding factor) and TCF (T cell factor) proteins to activate and regulate the expression of target genes. will do At this time, it is known that the target genes whose expression is activated are Wnt/β-catenin-responsive genes such as c-myc, cyclinD1, and metalloproteinase-7, which are oncogenes related to tumorigenesis and metastasis. (Science. 1998, 281 (5382), 1509-1512.; Nature. 1999, 398 (6726), 422-426.; Oncogene, 2002, 21 (38). 5861-5867.).

In addition, it is known that when the activity of β-catenin is effectively blocked, cancer growth is effectively inhibited, and the abnormally increased degradation of β-catenin protein can be a very effective target for the development of anticancer drugs.

In the process of researching substances targeting the Wnt/β-catenin pathway, the present inventors found that aplicurodin A strongly inhibits the Wnt/β-catenin signaling system and promotes β-catenin protein degradation, The present invention was completed by confirming that it induces apoptosis and promotes autophagy in various cancer cells without showing cytotoxicity in normal cells.

KR 10-2011-0132719 A

It is an object of the present invention to provide a novel use of Aplykurodin A, which inhibits the Wnt/β-catenin signaling system to prevent, improve, or treat cancer.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising Aplykurodin A as an active ingredient.

In addition, the present invention provides a Wnt-targeted anticancer agent or a Wnt-targeted anticancer adjuvant comprising aplikurodin A as an active ingredient, which inhibits the Wnt/β-catenin signaling system of cells.

In addition, the present invention provides a health functional food composition for preventing or improving cancer comprising aplicurodin A (Aplykurodin A) as an active ingredient.

Aplicurodin A of the present invention strongly inhibits the Wnt/β-catenin signaling system, promotes β-catenin protein degradation, and induces apoptosis in various cancer cells without showing cytotoxicity in normal cells. Since it promotes autophagy, it can be usefully used for the prevention, improvement or treatment of various cancer diseases.

1 is to confirm the Wnt/β-catenin signaling pathway inhibitory activity of the aplicurodin A compound of the present invention. It is the result of the assay of lyase activity (TOPflash activity), and FIG. 1b is the result of the Wnt3a-stimulated SEAP activity analysis showing that the aplicurodin A compound suppresses the expression of β-catenin.
2 is a graph showing that the aplicurodin A compound of the present invention has no cytotoxicity to HEK293 cells.
3 is a graph showing that the aplicurodin A compound of the present invention does not have a significant effect on the expression of p53 and NF-B.
4 is a firefly luciferase activity (TOPflash activity) analysis results for comparing the Wnt/β-catenin signaling pathway inhibitory activity of the aplicurodin A compound of the present invention ( FIG. 4a ) and the small extract ( FIG. 4b ). It is a graph representing
Figure 5a is a Western blot result showing that aplicurodin A decreases the intracellular β-catenin protein level increased by Wnt3a-CM in HEK293-FL reporter cells, and Figure 5b is aplicurodin A in HEK293-FL reporter cells. Semi-quantitative RT-PCR analysis results showing that the increase in intracellular β-catenin mRNA level by Wnt3a-CM in This is a Western blot result showing that the protein level is reduced, but the effect is suppressed in the presence of MG-132, a proteasome inhibitor.
6A is a result of Firefly luciferase activity analysis showing that aplicurodin A suppresses the expression of β-catenin in HEK293-FL reporter cells even in the presence of a GSK-3β inhibitor, and FIG. 6B is the result of a GSK-3β inhibitor. Western blot results showing that aplicurodin A suppressed the expression of β-catenin in HEK293-FL reporter cells in HEK293-FL reporter cells even in the presence of aplicurodin A.
7 is a Western blot result showing that aplicurodin A reduces the level of β-catenin protein in HepG2 HCC cells, but does not affect the level of ΔN β-catenin protein.
8 is a Firefly luciferase activity (TOPflash activity) analysis result showing that aplicurodin A reduces CRT (β-catenin responsive transcription) in TOPflash-transfected SNU475 and Hep3B cells.
9 is a Western blot analysis result showing that aplicurodin A reduces CRT (β-catenin responsive transcription) in TOPflash-transfected SNU475 and Hep3B cells.
10 is a Western blot analysis result showing that aplicurodin A suppresses the expression of cyclin D1, c-myc, and Axin-2, which are sub-regulators of Wnt/β-catenin signaling, in SNU475 and Hep3B cells.
11 is a cell viability graph showing the effect of aplicurodin A on the growth of AXIN1-mutant SNU475 and Hep3B liver cancer cells.
12 is a cell viability graph showing the effect of aplicurodin A on the growth of IMR90 and WI38 normal fibroblasts.
13 shows the results of counting the number of apoptosis-induced apoptotic cells in each cell through Annexin V/PI staining after treatment with aplicurodin A in SNU475 and Hep3B cells.
14 is a result of measuring caspase 3/7 activity using Caspase-Glo 3/7 Assay (Promega) after treating SNU475 and Hep3B cells with aplicurodin A and staining with Annexin V/PI.
15 is a Western blot analysis result showing that aplicurodin A induces proteolytic cleavage of apoptosis markers pro-caspase-3 and PARP in SNU475 and Hep3B cells.
FIG. 16 is an image of the punctate distribution of GFP-LC3 observed with a confocal microscope after treatment with aplicurodin A in SNU475 and Hep3B cells.
17 shows the results of confirming the expression levels of LC3 and p62/SQSTM1 by performing western blot after treatment with aplicurodin A in SNU475 and Hep3B cells.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising aplicurodin A (Aplykurodin A) as an active ingredient.

In the present specification, "aplikurodin A (Aplykurodin A)" is a compound having the structure of Formula 1 below, and as one of the active ingredients present in Kurodai (Aplysia kurodai ), it may be isolated from Kurodai or commercially available.

[Formula 1]

The therapeutic effect of aplicurodin A on cancer has not been previously known.

The aplicurodin A may be isolated from the small extract.

As used herein, the small group (Sea hare, Aplysia kurodai ) refers to an animal belonging to the mollusk gastropod. A shellless, slow-moving marine mollusk, commonly found on rocky beaches and used as food in Japan and Korea. It is known that the small group secretes various physiologically active substances as a defense mechanism to compensate for the lack of a shell, and it is known that the physiologically active substances included in the small group exhibit various effects such as anti-inflammatory, anti-diabetic, antibacterial, and antibacterial. have.

In the present specification, the minnow extract can be prepared as it is or in the form of minced or powder, and extracted by a conventional extraction method, for example, hot water extraction or an organic solvent.

For example, the small extract of the present invention can be prepared as follows. One or more mixed solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, dichloromethane, chloroform, acetone, propylene glycol, ethyl acetate, butylene glycol and ether after washing and washing the dried small particles, preferably is dichloromethane, chloroform, acetone, ethanol or a mixed solvent thereof, more preferably, a mixed solvent of dichloromethane, acetone, and ethanol in a mixing ratio of 10-1:1-10:1-10 (w/w), more More preferably, a mixed solvent of dichloromethane and ethanol in a 5-1:1-5 (w/w) mixing ratio is mixed, and then at a temperature of 30° C. to 150° C., preferably 50° C. to 100° C. for 30 minutes to For 48 hours, preferably 1 to 12 hours, ultrasonic extraction, hot water extraction, room temperature extraction or reflux extraction, preferably reflux extraction, is repeated about 1 to 20 times, preferably 2 to 10 times, and the obtained extract is filtered , concentrated under reduced pressure, and dried to obtain the small extract of the present invention.

Also, in the present specification, the term "extract" used while referring to the extract of the small grains includes not only the crude extract obtained by treating the extract with the extract, but also the processed product of the small grain extract. For example, the small extract may be prepared in a powder state by an additional process such as vacuum distillation, freeze drying, or spray drying.

In addition, the fraction obtained by passing the extract or fraction through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. It may be isolated through various purification methods.

As an example of this separation process, aplicurodin A of the present invention is fractionated with dichloromethane to obtain a fraction, and fractionated by silica gel vacuum liquid column chromatography (VLC) or silica gel column chromatography Then, it may be separated by sequentially performing a medium-pressure liquid chromatography (MPLC) method and a silica gel column chromatography method again.

Aplicurodin A, which is an active ingredient of the composition for preventing, improving or treating cancer of the present invention, includes not only the compound of Formula 1, but also pharmaceutically acceptable salts thereof, possible solvates, hydrates, and D. It may include both semi-isomers or stereoisomers.

Aplicurodin A of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. Acid addition salts include 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, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methylbenzoate oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is prepared by a conventional method, for example, by dissolving the compound of Formula 1 in an excess aqueous acid solution, and dissolving the salt in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt.

The aplicurodin A of the present invention exhibits therapeutic efficacy against cancer, and since it is derived from a natural product, it has the advantage that it can be used stably without serious stimulation or harmful effects on normal cells.

Accordingly, aplicurodin A may be usefully used as an active ingredient in a pharmaceutical composition for preventing or treating cancer.

In the present invention, "prevention" refers to any action that inhibits or delays the onset of cancer by administering the pharmaceutical composition of the present invention to an individual, and "treatment" refers to cancer by administering the pharmaceutical composition of the present invention to an individual. It means any action that improves or benefits the symptoms of Specifically, the "treatment" inhibits the increase in the number of cancer cells, inhibits the quantitative proliferation of cancer cells, kills cancer cells, maintains the size of cancer tissue, reduces the size of cancer tissue, It may be at least one of inhibiting the development of angiogenesis in cancer tissue or inhibiting metastasis of cancer.

In the present invention, the cancer is not particularly limited, but may be liver cancer. The liver cancer may be cancer arising from or metastasized to the liver, and specifically, may be any one or more selected from hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, intrahepatic hemangiosarcoma, and intrahepatic adenocarcinoma.

Meanwhile, in the present specification, the term “consisting of an active ingredient” means including the aplicurodin A of the present invention in an amount sufficient to prevent, improve or treat cancer. The pharmaceutical composition for preventing or treating cancer of the present invention preferably contains aplicurodin A in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight based on the total weight of the composition, but is not limited thereto.

If the aplicurodin A is used in an appropriate amount within the above range, it is economical and the therapeutic effect for cancer according to the aplicurodin A is sufficiently exhibited, thereby making it more suitable for achieving the object of the present invention.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient and diluent.

In the present invention, the term "pharmaceutically acceptable" means that it is not irritating to an organism when administered, and does not inhibit the biological activity and properties of the administered compound, and is commonly used in the pharmaceutical field.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. have. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition may contain at least one active ingredient exhibiting the same or similar function in addition to aplicurodin A.

The pharmaceutical composition of the present invention may be formulated and used in various formulations suitable for oral or parenteral administration.

Non-limiting examples of the formulation for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, etc. can be heard

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose (Cellulose) or gelatin (Gelatin); excipients such as Dicalcium phosphate; disintegrating agents such as corn starch or sweet potato starch; Lubricating oils such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax can be used, and sweeteners, fragrances, syrups, etc. can also be used. can Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be additionally used.

Non-limiting examples of the parenteral preparation include injection solutions, suppositories, powders for respiratory inhalation, aerosols for spraying, ointments, powders for application, oils, creams, and the like.

Non-limiting examples of the parenteral preparation include injections, suppositories, and sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, external preparations, etc. to formulate the pharmaceutical composition of the present invention for parenteral administration. In addition, as the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. Respiratory inhalation powder, spray aerosol, ointment, application powder, oil, cream, etc. are mentioned.

In addition, more specifically, when the pharmaceutical composition of the present invention is formulated as an injection solution, the composition of the present invention is mixed with a stabilizer or buffer in water to prepare a solution or suspension, and this is an ampoule or vial. It may be formulated for unit administration. In addition, when the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like may be blended with an additive so that the water-dispersed concentrate or wet powder is dispersed.

The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the reaction to be achieved by administration of the pharmaceutical composition may vary. Including the type and extent of the subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, components of drugs or other compositions used simultaneously with or at the same time for the subject, etc. It may vary depending on several factors and similar factors well known in the pharmaceutical field, and a person skilled in the art can easily determine and prescribe an effective dosage for a desired treatment.

The dosage for a more preferable effect of the pharmaceutical composition of the present invention is preferably 0.001 mg/kg to 1,000 mg/kg per day, more preferably 0.01 mg/kg to 100 mg/kg, most preferably 0.1 mg/kg to 60 mg/kg. Administration of the pharmaceutical composition of the present invention may be administered once a day, may be administered divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any way.

The route and mode of administration of the pharmaceutical composition of the present invention may each be independent, and the method is not particularly limited, and may be applied to any route and mode of administration as long as the pharmaceutical composition can reach the desired site. can follow The pharmaceutical composition may be administered by oral administration or parenteral administration.

The parenteral administration method may be administered by, for example, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, intracerebrovascular injection or intrathoracic injection, A method of applying, spraying, or inhaling the composition to a diseased site may also be used, but is not limited thereto.

The pharmaceutical composition of the present invention may preferably be administered orally or administered by injection, and more preferably by injection.

In addition, the pharmaceutical composition of the present invention can be administered for humans or animals.

As demonstrated in the Examples of the present invention, aplicurodin A of the present invention has an effect of inhibiting the Wnt/β-catenin signaling system.

As demonstrated in the Examples of the present invention, aplicurodin A of the present invention has an effect of promoting proteasome degradation of β-catenin.

As demonstrated in the Examples of the present invention, aplicurodin A of the present invention has an effect of promoting β-catenin degradation.

As demonstrated in the Examples of the present invention, aplicurodin A of the present invention has an effect of inhibiting the proliferation of liver cancer cells.

As demonstrated in the examples of the present invention, aplicurodin A of the present invention has an effect of inducing apoptosis of liver cancer cells.

As demonstrated in the Examples of the present invention, aplicurodin A of the present invention has an effect of inducing autophagy of liver cancer cells.

When the pharmaceutical composition of the present invention is administered, cancer can be effectively prevented, improved or treated.

In addition, the present invention provides a Wnt-targeted anticancer agent comprising Aplykurodin A as an active ingredient, characterized in that it inhibits the Wnt/β-catenin signaling system of cells.

The content of aplicurodin A of the present invention is the same as described above.

In the present specification, the cells may be liver cancer cells.

The targeted anticancer agent refers to a therapeutic agent that selectively attacks only a specific target in the carcinogenesis process, and the targeted anticancer agent of the present application targets the Wnt/β-catenin signaling system involved in the carcinogenesis of various cancers, preferably liver cancer. It is a targeted anticancer drug.

In addition, the present invention provides a Wnt-targeted anticancer adjuvant comprising Aplykurodin A as an active ingredient, characterized in that it inhibits the Wnt/β-catenin signaling system of cells.

The content of aplicurodin A of the present invention is the same as described above.

In the present specification, the cells may be liver cancer cells.

The anticancer adjuvant of the present invention targets the Wnt/β-catenin signaling system, and can be used to enhance the anticancer effect of the anticancer agent and suppress or improve the side effects of the anticancer agent.

In the present invention, the term "suppression" refers to any action that reduces the side effects of anticancer drugs by administration of anticancer adjuvants. It refers to any action that improves or beneficially changes symptoms caused by cancer.

In addition, in the present invention, the "individual" means all animals, including humans, that have or can develop cancer, and by administering the anticancer adjuvant of the present invention to the subject, it is possible to effectively treat cancer while minimizing the side effects of the anticancer drug. For example, by administering the anticancer adjuvant of the present invention to a human suffering from colorectal cancer receiving an anticancer agent, it is possible to effectively treat cancer while reducing anticancer side effects.

Acceptable carriers, formulations, administration methods and dosages of the target anticancer agent or anticancer adjuvant of the present invention are as described in the pharmaceutical composition.

In addition, the present invention provides a health functional food composition for preventing or improving cancer comprising aplicurodin A (Aplykurodin A) as an active ingredient.

In the present invention, “improvement” refers to any action that at least reduces a parameter related to the condition to be treated, for example, the severity of symptoms.

When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The type of the food is not particularly limited, and includes all foods in a conventional sense. Non-limiting examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, and tea, drinks, alcoholic beverages, or vitamin complexes.

When the health functional food composition of the present invention is a beverage composition, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage. Non-limiting examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; natural sweeteners such as dextrin and cyclodextrin; and synthetic sweeteners such as saccharin and aspartame. The proportion of the additional components to be added may be appropriately determined by the selection of those skilled in the art.

The aplicurodin A of the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, the amount of the compound in the health food may be added in an amount of 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake for health and hygiene or health control, 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 above the above range.

The health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginsam and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent used in carbonated beverages, and the like. In the gourd, the health functional food composition of the present invention may contain the flesh for the production of natural fruit juice, fruit drink or vegetable drink. These components may be used independently or may be used in combination of two or more. The proportion of these additives may also be appropriately selected by those skilled in the art.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited by the following examples.

<Example>

Example: Isolation of aplicurodin A

Isolation of aplicurodin A

After the intestines were removed and washed , 35 kg (wet weight) of wet pulverized cow (Aplysia kurodai ) was refluxed and extracted three times with a dichloromethane/ethanol mixed solution (weight ratio of 1:1). Then, the extract was filtered and the solvent was removed with a reduced pressure concentrator, and the crude extract (800 g) obtained was used as a small extract.

After 800 g of the small extract was suspended in distilled water, the solvent was fractionated with dichloromethane to obtain 140 g of a dichloromethane fraction (A) and 620 g of a water-soluble fraction (B). 140 g of the obtained dichloromethane fraction (A) was mixed with n-hexane-ethyl acetate (30:1, 7:1, 4:1, 1.5:1 [v/v]); n-hexane-ethyl acetate-methanol (1:1:0.2 [v/v/v]); chloroform-methanol (3:1 [v/v]); chloroform-methanol-distilled water (1:1:0.1 [v/v/v]); And fractionation by silica gel vacuum liquid column chromatography (VLC) using methanol (100%) to obtain a total of 8 small fractions (A ~ H).

After mixing 2.7 g of the subfraction D with 5 g of the subfraction E having a similar TLC pattern to the subfraction D, acetone-methanol-distilled water (17:17:66 → 47:47:6 [v/v]) Fractionation was performed by medium-pressure liquid chromatography (MPLC) according to concentration gradient elution conditions to obtain a total of 10 small fractions (DE-1 to DE-10). At this time, MPLC was performed using a Biotage Isolera apparatus equipped with a reversed-phase C18 SNAP Cartridge KPC18-HS (340 g, Biotage AB, Uppsala, Sweden).

1.02 g of the small fraction DE-5 was applied to a silica gel column (1.5 cm × 80 cm ) by chromatography to obtain a total of 6 small fractions (DE-5-1 to DE 5-6).

337.7 mg of the small fraction DE-5-6 was purified using a silica gel column (1.0 cm×70 cm), eluted with n-hexane-ethyl acetate-acetone (5:1:0.2), and a white amorphous powder called Apple 210 mg of Licurodin A was obtained.

Identification and structural analysis of aplicurodin A

The structure of the aplicurodin A compound was confirmed using nuclear magnetic resonance spectrometer (NMR) and mass spectroscopy. The structure of the compound was ^{confirmed by measuring 1} H-NMR and ¹³ C-NMR (Bruker AVACE Ⅲ 400 MHz spectrometer, Bruker, Germany). _{Specifically, the aplicurodin A compound has a molecular formula C 20} H ₃₄ O ₃ determined from the protonation-molecular ion peak m/z 322.25 of ESI-MS data.

The ¹ H-NMR spectral data (300 MHz, methanol- d ₄ ) shows two methine oxides at δH 5.06 (1H, d, J = 6.3 Hz, H-9) and 3.86 (1H, br s, H-4). One tertiary methyl group at proton, δH 1.02 (3H, s, H-12), δH 1.02 (3H, d, J = 6.4 Hz, H-14), 0.94 (6H, d, J = 6.6 Hz, H- 19, H-20) showed three secondary methyl group signals.

The ¹³ C-NMR spectral data (300 MHz, methanol- d ₄ ) is one carboxyl group at δC 175.2 (C-1), two methine oxides at δC 82.5 (C-9), 67.4 (C-4), δC 1 tertiary methyl group at 23.4 (C-12), 3 secondary methyl groups at δC 23.2 (C-20), 22.9 (C-19) and 19.2 (C-14), and 1 quaternary carbon, 5 A total of 20 carbon signals were shown, including methine, and 7 methylene.

Through the NMR spectrum analysis (see Table 1), it was confirmed that the compound isolated in the above Example was aplicurodin A.

Position δ _H , mult. ( J in Hz) δ _C One NS 175.2 2 2.44, m; 2.14, m 38.8 3 2.35, m 34.4 4 3.86, br s 67.4 5 2.14, m; 1.96, m 29.6 6 1.78, m; 1.74, m 30.2 7 NS 44.3 8 2.14, m 44.9 9 5.06, d (6.3) 82.5 10 1.93, m; 1.66, m 34.5 11 1.94, m 48.8 12 1.02, s 23.4 13 1.57, m 36.9 14 1.02, d 6.4) 19.2 15 1.46, m; 1.13, m 37.7 16 1.46, m; 1.30, m 25.2 17 1.21, m 40.6 18 1.46, m 29.1 19 0.94, d (6.6) 22.9 20 0.94, d (6.6) 23.2

<Experiment method>

Cell culture, reporter assay, transfection and sample used

HEK293, SNU475, and Hep3B cell lines (ATCC, Manassas, VA, USA) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin. And, HEK293-FL, DEK293-SEAP reporter cells, and Wnt3a-CM were prepared by methods described in the prior art (Mol. Pharmacol. 2006, 70, 960-966; Biochem. Biophys. Res. Commun. 2088, 377, 1304-1308). ) was prepared according to FL (Firefly luciferase) and SEAP activity assays were performed using the Dual luciferase assay kit (Promega, Madison, WI, USA) and Phospha-Light assay kit (Applied Biosystems, Foster City, CA, USA), respectively, as described by the manufacturer. was performed according to

For reporter assay, HEK293-FL and HEK293-SEAP reporter cells were ^{aliquoted in duplicate at a density of 2.5 × 10 4} cells/well in 96-well plates, respectively, and 10% FBS, 120 mg/mL penicillin, 200 mg It was cultured for 24 hours in DMEM medium supplemented with /mL streptomycin. The reporter cells were further cultured with aplicurodin A in Wn3a-CM (Wnt3a-CM; DMEM medium supplemented with Wnt3a, 10% FBS, 120 mg/ml penicillin, 200 mg/ml streptomycin) for 15 hours. did FL and SEAP activities were then measured using a microplate reader (Victor V3, PerkinElmer, MA, USA).

Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

MG-132 and BIO were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Western blot and antibody

Western blot was performed according to the method described in the prior literature (Nucleic Acids Res. 1983, 11, 1475-1489). Anti-β-catenin antibodies used in Western blot were obtained from BD Transduction Laboratories (Palo Alto, CA, USA), and anti-axin2, anti-PARP, anti-caspase-3, anti-actin, and anti-lc3b antibodies was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cyclin D1, anti-c-myc, and anti-p62 antibodies were obtained from Santa Cruz Biotechnology (Dalla, TX, USA).

RNA extraction and semi-quantitative RT-PCR

RNA was isolated from HEK293-FL reporter cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA synthesis, amplification, and PCR were performed according to the method described in the prior literature (Cell Sci. 2006, 119, 4702-4709).

After the amplification reaction, 5 μl of the PCR product was stained with Dyne LoadingSTAR and electrophoresed on an agarose gel at a concentration of 2% (w/v), and then a Gel document system (InGenius, Gel doc, CT, USA) was used. to confirm.

Cell viability analysis

CellTiter-Glo Assay was performed to determine cell viability, and the method was tested according to the method described in the literature (Mol. Pharmacol. 2006, 70, 960-966).

That is, SNU475 and Hep3B cells were inoculated into 96-well plates (5000 cells), and then treated with 20 μM and 40 μM of aplicurodin A for 48 hours, respectively. Cell viability of individual samples was measured three times according to the manufacturer's instructions using the Cell Titer-Glo assay kit (Cat# G7570, Promega).

Apoptosis Assay and Caspase 3/7 Assay

After 48 hours of treatment with aplicurodin A in SNU475 and Hep3B cells, the cells were ^{stained using ApoScan TM} annexin V-FITC apoptosis detection kit (Promega) according to the manufacturer's instructions, and then Cellometer Vision image cytometer (Nexcelom Bioscience) cell death (apoptosis) analysis was performed. The caspase activity was measured using Caspase-Glo 3/7 Assay (Promega) according to the manufacturer's instructions.

Fluorescence microscopy analysis of GFP-LC3

GFP-LC3-transfected cells were incubated with aplicurodin A for 48 hours and fixed with PBS containing 4% paraformaldehyde at room temperature. The fluorescence signal was visualized and observed using a Leica 4000 confocal microscope.

statistical analysis

Student's t test (SigmaPlot) was used for comparison between the experimental group and the control group. All experiments were performed in triplicate. Statistical significance was set at p<0.05 or p<0.01, and each result was reported as mean±standard deviation (SD).

<Test Example>

Test Example 1: Evaluation of Wnt/β-catenin signaling pathway inhibitory activity of aplicurodin A

In order to determine whether the aplicurodin A compound isolated in the above example inhibits Wnt/β-catenin signaling, human Frizzled-1 (amplifies Wnt/β-catenin signaling) and HEK293-FL (human embryonic kidney cell-firefly luciferase) reporter cells stably expressing the TOPFlash reporter were used (Mol. Pharmacol. 70(3): 906-966). TOPFlash is bound to a luciferase reporter for the TCF/LEF transcription factor induced by Wnt/β-catenin. The HEK293-FL reporter cells were prepared by transforming the TOPFlash reporter and human Frizzled-1 expression plasmids into HEK293 cells.

The aplicurodin A compound was dissolved in DMSO (Sigma) in the HEK293-FL reporter cells and added at concentrations of 0, 20, and 40 μM, respectively, and then Wnt3a-CM (Wnt3a-CM; Wnt3a, 10% FBS, 120 mg) /ml penicillin, 200 mg/ml streptomycin added in DMEM medium) for 15 hours. Thereafter, FL and SEAP activities were measured using a microplate reader (Victor V3, PerkinElmer, MA, USA), and (A) Firefly luciferase activity (TOPFlash activity) was analyzed (Fig. 1a) and ( B) The results of SEAP activity analysis (FIG. 1b) are shown in FIG. The experiment was repeated three times, and the average value was shown as the result. The activity of TOPFlash was measured by normalizing to cell titer activity.

As a result, as shown in Figure 1a, as the dosage of the aplicurodin A compound increased, it was confirmed that β-catenin responsive transcription (CRT, β-catenin responsive transcription) induced by Wnt3a-CM was significantly inhibited, A decrease in TOPFlash activity was confirmed in HEK293-FL reporter cells.

In addition, using HEK293-SEAP reporter cells under the same conditions (Biochem. Biophys. Res. Commun. 377(4): 1304-1308), the effect of the aplicurodin A compound on Wnt/β-catenin signaling was confirmed. As a result, the aplicurodin A compound was shown to inhibit Wnt3a-stimulated secreted alkaline phosphatase (SEAP) activity in a concentration-dependent manner in the reporter cells ( FIG. 1B ).

On the contrary, it was specifically confirmed that the aplicurodin A compound had no significant effect on the p53 and NF-κB pathways (see FIG. 2 ).

Specifically, HCT116 cells were co-transfected with p53-FL gene and pCMV-RL plasmid to prepare HCT116-p53-FL reporter cells, and then the HCT116-p53-FL reporter cells were transfected with p53 with an aplicurodin A compound. Incubated for 15 hours in the presence or absence of doxorubicin, an activator of the pathway. The luciferase activity was measured 39 hours after the transfection, and the measured value was converted into a relative light unit (RLU) value normalized to Renilla luciferase activity, and is shown in FIG. 2A .

FIG. 2a is a graph showing whether treatment of aplicurodin A in HCT116 cells affects p53 expression.

Referring to FIG. 2a , it can be seen that although the treatment concentration of aplicurodin A in HCT116-p53-FL cells increased, it did not significantly affect the expression level of p53.

In addition, HEK293 cells were co-transfected with the NF-κB-FL gene and the pCMV-RL plasmid to prepare HEK293-NF-κB-FL reporter cells, and then the HEK293-NF-κB-FL reporter cells were transfected with aplicurodin. Compound A was incubated for 15 hours in the presence or absence of aspirin, an activator of the NF-κB pathway. The luciferase activity was measured 39 hours after the transfection, and the measured value was converted into a relative light unit (RLU) value normalized to Renilla luciferase activity, and is shown in FIG. 2B .

FIG. 2b is a graph showing whether treatment of aplicurodin A in HEK293 cells affects NF-κB expression.

Referring to FIG. 2b , it can be seen that the treatment concentration of aplicurodin A in HEK293-NF-κB-FL cells does not significantly affect the NF-κB expression level.

From these results, it was confirmed that the aplicurodin A compound is a specific antagonist of the Wnt/β-catenin signal.

Test Example 2: Cell viability analysis

It was attempted to determine whether the aplicurodin A compound of the present invention exhibits cytotoxicity against HEK293-FL reporter cells.

To this end, the HEK293-FL reporter cells were inoculated into a 96-well plate, and the aplicurodin A compound was added at concentrations of 0, 20 μM and 40 μM, respectively, and then cultured in Wnt3a-CM for 15 hours. Then, the cell viability of each sample was measured three times with the CellTiter-Glo assay kit (Promega) according to the manufacturer's instructions, and is shown in FIG. 3 .

Referring to FIG. 3 , it can be seen that the aplicurodin A compound does not exhibit cytotoxicity to the HEK293-FL reporter cells.

From the above results, it can be seen that the aplicurodin A compound of the present invention is a substance capable of effectively reducing only β-catenin responsive transcription (CRT) without affecting cell viability.

Test Example 3: Comparison of the Wnt/β-catenin signaling pathway inhibitory activity of small extract and aplicurodin A

The Wnt/β-catenin signaling pathway inhibitory activity of the aplicurodin A compound of the present invention and the small extract was compared.

Gunso extract was obtained by reflux extraction of 35 kg (wet weight) of Aplysia kurodai 3 times with a dichloromethane/ethanol mixed solution (weight ratio of 1:1).

Firefly luciferase activity was analyzed by applying the method of Test Example 1 to the aplicurodin A compound and the small extract isolated in the above example, and the results are shown in FIG. 4 .

Referring to FIG. 4 , in the case of the experimental group treated with the aplicurodin A compound of the present invention, it can be confirmed that the β-catenin response transcription (CRT) is significantly reduced compared to the experimental group treated with the small extract.

Test Example 4: Aplicurodin A β-catenin proteolytic promoting effect

In the Wnt/β-catenin signaling system, CRT (β-catenin responsive transcription) depends on the amount of intracellular β-catenin protein, and the β-catenin protein amount is regulated by proteasome degradation. In addition, since aplicurodin A inhibits CRT, Western blot and semi-quantitative RT-PCR were performed to determine whether aplicurodin A regulates β-catenin levels.

Specifically, in the presence of Wnt3a-CM, HEK293-FL reporter cells were treated with DMSO, or 20 μM or 40 μM of aplicurodin A, and cultured for 15 hours, and the cytoplasmic protein was expressed using an anti-β-catenin antibody. Western blot analysis was performed (Fig. 5a). In addition, in the presence of Wnt3a-CM, HEK293-FL reporter cells were treated with DMSO, or 20 μM or 40 μM of aplicurodin A, and then cultured for 15 hours, and gene expression of β-catenin and GAPDH was analyzed by semi-quantitative RT- It was analyzed by PCR (Fig. 5b).

Figure 5a is a Western blot result showing that aplicurodin A decreases the intracellular β-catenin protein level increased by Wnt3a-CM in HEK293-FL reporter cells, and Figure 5b is aplicurodin A in HEK293-FL reporter cells. This is the result of semi-quantitative RT-PCR analysis indicating that there is no effect on intracellular β-catenin mRNA level increased by Wnt3a-CM in

Referring to Figure 5a, it can be seen that aplicurodin A reduces the intracellular β-catenin protein level increased by Wnt3a-CM in HEK293-FL reporter cells. Meanwhile, referring to FIG. 5B , it can be seen that aplicurodin A does not affect the mRNA level of β-catenin under the same conditions.

In addition, it was attempted to determine whether the proteasome is involved in the reduction (downregulation) of β-catenin induced by aplicurodin A.

To this end, HEK293-FL reporter cells were treated with DMSO, or 20 μM or 40 μM of aplicurodin A, and then exposed to 10 μM of MG-132 for 8 hours. Thereafter, the cytoplasmic protein was analyzed by western blot using an anti-β-catenin antibody (FIG. 5c).

Figure 5c is a Western blot result showing that aplicurodin A decreases the intracellular β-catenin protein level increased by Wnt3a-CM, but suppresses the effect in the presence of the proteasome inhibitor MG-132.

Referring to FIG. 5c , it can be confirmed that the amount of β-catenin protein in the cell is decreased by aplicurodin A, but this reduction effect disappears in the presence of MG-132, a proteasome inhibitor.

These results indicate that aplicurodin A suppresses the Wnt/β-catenin signaling pathway through proteasome-dependent β-catenin degradation promotion without affecting β-catenin gene expression. That is, it can be seen that aplicurodin A inhibits the Wnt/β-catenin signaling pathway by very effectively degrading β-catenin protein and reducing the protein level of β-catenin.

Test Example 5: Aplicurodin A promotes the degradation of β-catenin by a mechanism of action independent of GSK-3β

GSK-3β induces β-catenin degradation by promoting β-catenin phosphorylation at Ser33, Ser37, and Thr41 residues. Accordingly, it was attempted to determine whether the GSK-3β is involved in aplicurodin A-induced β-catenin degradation.

Specifically, DMSO, or aplicurodin A 20 μM or 40 μM, in HEK293-FL reporter cells in the presence of 0.75 μM BIO (6-bromoindirubin-3'-oxime, which activates Wnt/β-catenin signaling) After treatment, incubated for 15 hours, and firefly luciferase activity was measured (FIG. 6a).

6A is a result of a Firefly luciferase activity assay showing that aplicurodin A suppresses the expression of β-catenin in HEK293-FL reporter cells even in the presence of a GSK-3β inhibitor.

As previously known, when HEK293-FL reporter cells are cultured with BIO, a GSK-3β inhibitor, intracellular CRT is increased (Chem. Biol. 2003, 10, 1255-1266).

However, it was confirmed that aplicurodin A still inhibited CRT under the same conditions (FIG. 6a).

In addition, in the presence of 0.75 μM BIO, HEK293-FL reporter cells were treated with DMSO, or 20 μM or 40 μM of aplicurodin A, and cultured for 15 hours, and the cytoplasmic protein was expressed using an anti-β-catenin antibody. Western blot analysis (Fig. 6b).

6B is a Western blot result showing that aplicurodin A suppresses the expression of β-catenin in HEK293-FL reporter cells even in the presence of a GSK-3β inhibitor.

Referring to FIG. 6b , it can be confirmed that aplicurodin A reduces the amount of β-catenin protein in the cell even in the presence of a GSK-3β inhibitor (BIO).

That is, it can be seen that the presence or absence of GSK-3β has no effect on aplicurodin A-induced β-catenin degradation.

From these results, it can be seen that aplicurodin A promotes the degradation of β-catenin with a mechanism of action independent of GSK-3β.

On the other hand, although the aplicurodin A has a mechanism of action independent of GSK-3β, it was attempted to determine whether β-catenin phosphorylation is required for β-catenin degradation. To this end, it was confirmed whether aplicurodin A could reduce the level of mutant β-catenin lacking the N-terminal region (ΔN β-catenin).

Specifically, HepG2 HCC cells were treated with DMSO, or aplicurodin A 20 μM or 40 μM, and cultured for 15 hours, and the cytoplasmic protein was westernized using an anti-β-catenin antibody and an anti-β-actin antibody. Blot analysis was performed ( FIG. 7 ).

7 is a Western blot result showing that aplicurodin A reduces the level of β-catenin protein in HepG2 HCC cells, but does not affect the level of ΔN β-catenin protein.

These results suggest that aplicurodin A has a mechanism of action independent of GSK-3β, but requires β-catenin phosphorylation for aplicurodin A-mediated β-catenin degradation.

Test Example 6: Aplicurodin A inhibits proliferation of AXIN1-mutant HCC cells

Since aberrant CRT activity is often observed in human hepatocellular carcinoma (HCC), we tried to determine whether aplicurodin A can downregulate CRT in SNU475 and Hep3B hepatocellular carcinoma cells. The liver cancer cells exhibit abnormally increased CRT due to an inactivating mutation of AXIN1, a key component of the degradation complex (composed of APC, GSK3β, AXIN1, and disheveled proteins).

Specifically, SNU475 and Hep3B cells co-transfected with TOPflash and pCMV-Renilla luciferase (RL) plasmids were incubated with DMSO, or aplicurodin A 20 μM or 40 μM, followed by FL activity And RL activity was measured, and the results are shown in FIG. 8 . TOPflash activity was normalized to RL activity.

8 is a Firefly luciferase activity (TOPflash activity) analysis result showing that aplicurodin A reduces CRT (β-catenin responsive transcription) in TOPflash-transfected SNU475 and Hep3B cells.

Referring to FIG. 8, when TOPflash-transfected SNU475 and Hep3B cells are cultured with aplicurodin A, respectively, it can be seen that the CRT in the SNU475 and Hep3B cells is reduced by aflacurodin A.

In addition, the TOPflash-transfected SNU475 and Hep3B cells were cultured with MSO, or 20 μM or 40 μM of aplicurodin A, and then the cytoplasmic protein was purified using anti-β-catenin and anti-β-actin antibodies. Western blot analysis was performed, and the results are shown in FIG. 9 .

9 is a Western blot analysis result showing that aplicurodin A reduces CRT (β-catenin responsive transcription) in TOPflash-transfected SNU475 and Hep3B cells.

Referring to FIG. 9 , it can be seen that aplicurodin A downregulates intracellular β-catenin levels in SNU475 and Hep3B cells.

Next, to determine whether aplicurodin A affects subgene expression of Wnt/β-catenin signaling, SNU475 and Hep3B cells with mutations in the AXIN1 gene were incubated with aplicurodin A. after, Western blot was performed on the extracts of SNU475 and Hep3B cells with anti-cyclin D1, anti-c-myc, anti-Axin2 antibody, and anti-β-actin antibody, respectively, and the results are shown in FIG. 10 .

10 is a Western blot analysis result showing that aplicurodin A suppresses the expression of cyclin D1, c-myc, and Axin-2, which are sub-regulators of Wnt/β-catenin signaling, in SNU475 and Hep3B cells.

Referring to FIG. 10, it can be confirmed that aplicurodin A suppresses the expression of cyclin D1, c-myc, and Axin-2, which are sub-regulators of Wnt/β-catenin signaling in SNU475 and Hep3B cells.

In addition, considering that aplicurodin A promotes the degradation of β-catenin, we tried to determine the effect of aplicurodin A on the growth of AXIN1-mutant liver cancer cells.

11 is a cell viability graph showing the effect of aplicurodin A on the growth of AXIN1-mutant SNU475 and Hep3B liver cancer cells.

11, aplicurodin A significantly reduced cell viability in SNU475 and Hep3B cells in a concentration-dependent manner.

From these results, it can be seen that aplicurodin A inhibits Wnt/β-catenin signaling in SNU475 and Hep3B liver cancer cells, and significantly reduces cell viability of the liver cancer cells.

In addition, under the same conditions as the cell viability experiments of SNU475 and Hep3B cells, it was attempted to determine the effect of aplicurodin A on the growth of normal fibroblasts, IMR90 and WI38 cells.

12 is a cell viability graph showing the effect of aplicurodin A on the growth of IMR90 and WI38 normal fibroblasts.

Referring to FIG. 12 , it can be confirmed that aplicurodin A does not exhibit cytotoxicity in IMR90 and WI38 normal fibroblasts.

These results suggest that aplicurodin A exhibits cytotoxicity to cancer cells such as liver cancer cells, but not cytotoxicity to normal cells.

Test Example 7: Aplicurodin A induces apoptosis in liver cancer cells

In order to elucidate the mechanism of aplicurodin A-mediated inhibition of cancer cell growth, it was attempted to determine whether aplicurodin A induces apoptosis in AXIN1-mutant liver cancer cells.

First, SNU475 and Hep3B cells were incubated with DMSO, or aplicurodin A 20 μM or 40 μM for 48 hours, followed by Annexin V-FITC/PI (propidium iodide) double staining, followed by Annexin V-FITC Apoptosis Detection The number of stained cells was measured using a Kit (BioVision, USA), and the results are shown in FIG. 13 .

13 shows the results of counting the number of apoptosis-induced apoptotic cells in each cell through Annexin V/PI staining after treatment with aplicurodin A in SNU475 and Hep3B cells.

Referring to FIG. 13, apoptotic cell counting results through Annexin V/PI staining in SNU475 and Hep3B cells treated with aplicurodin A, Annexin B-positive cells and Annexin V/PI double-positive cells were combined to determine the total number of apoptotic cells. It can be seen that the ratio significantly increased in a concentration-dependent manner.

Next, SNU475 and Hep3B cells were incubated with DMSO, or 20 μM or 40 μM of aplicurodin A for 48 hours, and then caspase3/7 activity was measured. Specifically, 48 hours after treating the cells with aplicurodin A, the cells were ^{stained using the ApoScan TM} annexin V-FITC apoptosis detection kit (Promega) according to the manufacturer's instructions, and then a Cellometer Vision image cytometer (Nexcelom Bioscience). cell death (apoptosis) analysis was performed. The caspase activity was measured using Caspase-Glo 3/7 Assay (Promega) according to the manufacturer's instructions.

14 is a result of measuring caspase 3/7 activity using Caspase-Glo 3/7 Assay (Promega) after treating SNU475 and Hep3B cells with aplicurodin A and staining with Annexin V/PI.

Referring to FIG. 14 , caspase 3/7 activity was increased in AXIN1-mutant liver cancer cells treated with aplicurodin A, and this result is consistent with the apoptosis-inducing effect of aplicurodin A.

In addition, after incubating SNU475 and Hep3B cells with DMSO, or aplicurodin A 20 μM or 40 μM for 48 hours, the respective cell extracts were treated with anti-caspase-3, anti-poly(ADP-rebose) polymerase ( PARP) and western blot analysis with anti-β-actin antibody.

15 is a Western blot analysis result showing that aplicurodin A induces proteolytic cleavage of apoptosis markers pro-caspase-3 and PARP in SNU475 and Hep3B cells.

These results suggest that aplicurodin A induces apoptosis in SNU475 and Hep3B liver cancer cells.

Test Example 8: Aplicurodin A induces autophagy in liver cancer cells

In a previous study, it was reported that autophagy is promoted by inhibition of β-catenin function in cancer cells.

Considering that aplicurodin A reduces the amount of β-catenin protein, the present inventors hypothesized that aplicurodin A would induce autophagic cell death in AXIN1-mutant liver cancer cells.

Specifically, SNU475 and Hep3B cells were incubated with DMSO, or aplicurodin A 20 μM or 40 μM for 48 hours, and then the punctate distribution of GFP-LC3 was analyzed by confocal microscopy.

Accordingly, the conversion from cytosolic microtubule-associated protein (LC3-I) in liver cancer cells SNU475 and Hep3B cells to LC3-II (autophagosome-associated light chain 3-II), an indicator of autophagy, was performed using GFP-LC3. It was observed and shown in FIG. 16 .

FIG. 16 is an image of the punctate distribution of GFP-LC3 observed with a confocal microscope after treatment with aplicurodin A in SNU475 and Hep3B cells.

As shown in FIG. 16 , when aplicurodin A was treated in SNU475 and Hep3B cells, fluorescence points were formed by GFP-LC3 conversion, thereby confirming the formation of autophagosomes.

Next, SNU475 and Hep3B cells were incubated with DMSO, or aplicurodin A 20 μM or 40 μM for 48 hours, and then extracts of each of the cells were treated with anti-LC3, p62/SQSTM1, and anti-β-actin. Western blotting was performed with the antibody, and the expression levels of LC3 and p62/SQSTM1 were confirmed and shown in FIG. 17 .

17 shows the results of confirming the expression levels of LC3 and p62/SQSTM1 by performing western blot after treatment with aplicurodin A in SNU475 and Hep3B cells.

Referring to FIG. 17 , it can be seen that aplicurodin A promotes the conversion from LC3-I to lipidated LC3-II in SNU475 and Hep3B cells, and reduces the level of p62/SQSTM1, an LC3-interacting protein.

Through the above experimental results, the mechanism of action of the Wnt/β-catenin signaling pathway inhibitory activity caused by the aplicurodin A compound of the present invention promoting the degradation of β-catenin in cancer cells is a conventional Axin1-related mechanism or GSK-3β It was confirmed that it is due to an independent mechanism different from the related mechanism.

Specifically, in an embodiment of the present invention, it was confirmed that aplicurodin A downregulates the level of β-catenin even in the presence of a GSK-3β inhibitor. In addition, it was confirmed that aplicurodin A still promotes the degradation of β-catenin even in SNU475 and Hep3B cells in which the destruction-dependent pathway is impaired due to AXIN1-mutation.

In addition, the aplicurodin A compound of the present invention inhibited the growth of AXIN1-mutant HCC cells by inducing apoptosis and autophagy.

Although the present invention has been described as the above-mentioned preferred embodiment, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also intended that the appended claims cover such modifications and variations as fall within the scope of the present invention.

Claims (11)

A pharmaceutical composition for preventing or treating cancer comprising Aplykurodin A as an active ingredient. According to claim 1,
The aplicurodin A is a pharmaceutical composition for preventing or treating cancer, characterized in that it is isolated from the small group (Aplysia kurodai). According to claim 1,
The cancer is selected from liver cancer, lung cancer, stomach cancer, breast cancer, oral cancer, cervical cancer, endometrial cancer, prostate cancer, ovarian cancer, thyroid cancer, esophageal cancer, colorectal cancer, rectal cancer, pancreatic cancer, kidney cancer and skin cancer prevention or A therapeutic pharmaceutical composition. According to claim 1,
The cancer is a pharmaceutical composition for preventing or treating cancer, characterized in that liver cancer. According to claim 1,
The aplicurodin A is a pharmaceutical composition for preventing or treating cancer, characterized in that it inhibits the Wnt/β-catenin signaling system. A Wnt-targeted anticancer agent comprising Aplykurodin A as an active ingredient, characterized in that it inhibits the Wnt/β-catenin signaling system of cells. 7. The method of claim 6,
The cell is a Wnt-targeted anticancer agent, characterized in that the liver cancer cell. A Wnt-targeted anticancer adjuvant comprising Aplykurodin A as an active ingredient, characterized in that it inhibits the Wnt/β-catenin signaling system of cells. 9. The method of claim 8,
The cell is a Wnt-targeted anticancer adjuvant, characterized in that it is a liver cancer cell. A health functional food composition for preventing or improving cancer comprising Aplykurodin A as an active ingredient. 11. The method of claim 10,
The cancer is a health functional food composition for preventing or improving cancer, characterized in that liver cancer.

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