Composition for inhibiting cancer metastasis comprising seongsanamide B as effective component

Abstract

The present invention relates to a composition for inhibiting cancer metastasis containing seongsanamide B as an active ingredient. Seongsanamide B, the active ingredient of the present invention, effectively inhibited the migration and invasion of lung cancer cells enhanced by radiation irradiation even at concentrations that did not exhibit cytotoxicity. Furthermore, in a metastasis animal model established by administering irradiated cells, the effect of inhibiting lung cancer cell metastasis at concentrations that did not exhibit cytotoxicity was significant. Therefore, the composition of the present invention can enhance the therapeutic effect against cancer by inhibiting the metastasis of cancer cells increased by radiation irradiation without side effects caused by the toxicity of existing synthetic agents, while enhancing the effect of radiation therapy for anticancer purposes without cytotoxicity. Accordingly, the composition of the present invention containing seongsanamide B as an active ingredient can be usefully utilized as a cancer metastasis inhibitor or as a material for health functional foods for inhibiting cancer metastasis.

Description

Technology Field

[0001] The present invention relates to a composition for inhibiting cancer metastasis containing saccharamide B as an active ingredient. Background Technology

[0002] Lung cancer is the second most common cancer in both sexes, accounting for 15% of all cancers. According to a 2011 report by the American Cancer Society, over 220,000 cases of lung cancer are diagnosed annually, with approximately 70% resulting in death, accounting for 27% of all cancer deaths. Among lung cancers, non-small cell lung cancer (NSCLC) is a type of epithelial carcinoma that refers to all epithelial lung cancers that are not small lung cancer, accounting for approximately 85% to 90% of all lung cancers. NSCLC is relatively less sensitive to chemotherapy compared to small lung cancer, and the stages of the cancer are classified based on the TNM classification system: the size of the tumor, the extent of cancer spread to regional lymph nodes, and the presence or absence of metastasis. In the treatment of non-small cell lung cancer (NSCLC), early-stage, non-metastatic NSCLC is generally treated with surgery accompanied by ancillary chemotherapy involving platinum-containing cisplatin, as the cancer has very low sensitivity to chemotherapy and radiation. On the other hand, for metastatic NSCLC that has progressed beyond the early stages, various chemotherapy and radiation therapies are used. Symptoms of NSCLC include a persistent cough, chest pain, weight loss, nail damage, joint pain, and shortness of breath; however, because NSCLC generally progresses slowly, symptoms are rarely present in the early stages. Consequently, early detection and treatment of NSCLC are difficult, and the disease is highly likely to be discovered only after it has metastasized throughout the body, including to the bones, liver, small intestine, and brain.Therefore, despite the high incidence and mortality rates, no drug or treatment method has yet been developed to overcome non-small cell lung cancer, and the need for such a method is emerging.

[0003] One of the most important biological characteristics of cancer is its ability to metastasize, which is considered the biggest obstacle to treating cancer. In fact, about 60% of all patients with solid tumors already have clinically metastasized cancer at the time of diagnosis of the primary tumor, and it is already well known that cancer metastasis is a major cause of death for most cancer patients.

[0004] Neovascularization is a phenomenon that accompanies the invasion of local tissues by cancer during the process of metastasis; tumor angiogenesis factors are involved in this process, and because the blood vessels newly formed by the tumor are often defective, they are easily invaded by cancer cells. Furthermore, the processes of cancer invasion and metastasis require the expression of numerous cancer cell surface receptors, such as laminin receptors necessary for adhesion to the tissue stroma and basement membrane; numerous enzymes necessary for dissolving the stroma of normal tissues, such as collagenase type IV, plasminogen activator, and cathepsin D; growth factors; autocrine motility factors; and oncogenes.

[0005] To date, while there are high expectations for the development of substances possessing such cancer metastasis-inhibiting effects, there are actually very few examples of substances developed specifically for this purpose. Although sulfated polysaccharides, N-diazoacetylglycine derivatives, neuraminidase, and fibronectinase (FNS) have been reported to possess such inhibitory effects, there are no reports of their practical application yet, nor are there any reports that they themselves possess cancer metastasis-inhibiting effects. Therefore, developing a method to effectively inhibit cancer metastasis would enable the development of a useful therapeutic method capable of effectively controlling mortality caused by cancer metastasis.

[0006] Regarding prior art related to cancer metastasis, Korean Registered Patent No. 1818187 discloses a "pharmaceutical composition for inhibiting lung cancer metastasis containing fibulin-3 protein as an active ingredient," and Korean Registered Patent No. 2575465 discloses a "composition for inhibiting cancer metastasis and / or enhancing cancer treatment effects containing docetaxel and calkitoxin thiolamide as active ingredients." However, there has not yet been any disclosure regarding the "composition for inhibiting cancer metastasis containing saccharamide B as an active ingredient" of the present invention. The problem to be solved

[0007] The present invention was derived from the above-mentioned requirements. The present invention provides a composition for inhibiting cancer metastasis containing seongsanamide B as an active ingredient. More specifically, the invention was completed by confirming that the active ingredient, seongsanamide B, inhibits the migration and invasion of lung cancer cells increased by radiation irradiation, and effectively inhibits the metastasis of irradiated lung cancer cells in an animal model even at a concentration that is non-cytotoxic. means of solving the problem

[0008] To solve the above problem, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis containing seongsanamide B represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for inhibiting cancer metastasis containing seongsanamide B represented by Chemical Formula 1 or a food-grade acceptable salt thereof as an active ingredient.

[0010] In addition, the present invention provides a veterinary composition for inhibiting cancer metastasis containing seongsanamide B represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] In addition, the present invention provides a feed additive for inhibiting cancer metastasis containing seongsanamide B represented by Chemical Formula 1 or a food-grade acceptable salt thereof as an active ingredient. Effects of the invention

[0012] The present invention relates to a composition for inhibiting cancer metastasis containing seongsanamide B as an active ingredient. Seongsanamide B, the active ingredient of the present invention, effectively inhibited the migration and invasion of lung cancer cells enhanced by radiation irradiation even at concentrations that did not exhibit cytotoxicity. Furthermore, in a metastasis animal model established by administering irradiated cells, the effect of inhibiting the metastasis of lung cancer cells at concentrations that did not exhibit cytotoxicity was significant. Therefore, the composition of the present invention can enhance the therapeutic effect against cancer in radiation therapy for anticancer purposes by inhibiting the metastasis of lung cancer cells increased by radiation irradiation without side effects caused by the toxicity of existing synthetic agents, while enhancing the effect of radiation therapy without cytotoxicity. Brief explanation of the drawing

[0013] Figure 1 shows the results of confirming the cytotoxicity of seongsanamide B on A549 and H460 cells. Figure 2 shows the results of confirming the degree of cell migration and infiltration of A549 and H460 cells. A and B are photographs confirming the degree of cell migration and the quantification results depending on whether A549 and H460 cells were irradiated and treated with sylsanamide B, and C and D are photographs confirming the degree of cell invasion and the quantification results depending on whether A549 and H460 cells were irradiated and treated with sylsanamide B. Figure 3 shows the results of confirming the changes in protein expression levels of signaling factors and IL-6 in A549 and H460 cells depending on irradiation and treatment with sylsanamide B. A represents the signaling factor (β-catenin, p-STAT3, Src, Bcl-X L B is the result of confirming changes in the expression of , SOD2) proteins through Western blot experiments, B is the result of confirming changes in IL-6 protein expression through ELISA experiments, and C is the result of confirming changes in STAT3 and p-STAT3 protein expression in the nuclear fraction through Western blot experiments. β-actin and Lamin B1 were used as loading controls. Figure 4 shows the results of Western blot experiments confirming changes in the expression of E-cadherin and vimentin, epithelial-mesenchymal transition (EMT) related proteins, in A549 and H460 cells depending on irradiation and treatment with sylsanamide B. β-actin was used as a loading control. Figure 5 shows the results of confirming the degree of tumor formation according to the dosage of sylsanamide B in an animal model. A is a schematic diagram of an experiment to confirm the degree of tumor formation according to the dosage of sylsanamide B in an animal model, B is a graph showing the change in body weight of the animal model according to the dosage of sylsanamide B, and C is a graph quantifying the change in tumor size according to the dosage of sylsanamide B. Figure 6 shows the results confirming the metastasis inhibitory effect of sylsanamide B on radiation-induced metastasis. A is a workflow diagram for the experiment to confirm the inhibitory effect of sylsanamide B on radiation-induced metastasis; B is a graph showing the change in body weight of an animal model following the injection of irradiated cells and the administration of sylsanamide B; C is a photograph of the lungs of an animal model following the injection of irradiated cells and the administration of sylsanamide B; D is the result of quantifying the number of nodules formed in the lungs of an animal model following the injection of irradiated cells and the administration of sylsanamide B; E is the result of H&E staining of the lung tissue; and F is Bcl-X in the lung tissue L This is the result of confirming changes in the expression of , SOD2, p-Src, Src, E-cadherin, and vimentin proteins. Specific details for implementing the invention

[0014] The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis containing seongsanamide B represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015]

[0016] The above salt includes conventional acid addition salts, for example, salts derived from inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, and bromic acid, and salts derived from organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, citric acid, maleic acid, malonic acid, malic acid, tartaric acid, gluconic acid, lactic acid, gestic acid, fumaric acid, lactobionic acid, salicylic acid, phthalic acid, emvonic acid, aspartic acid, glutamic acid, and acetylsalicylic acid. Additionally, the above salt includes salts derived from amino acids such as glycine, alanine, valine, isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine, and proline. In addition, the above salt includes salts of sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0017] The above cancer metastasis may be induced by radiation, but is not limited thereto.

[0018] The above cancer metastasis may be metastasis of lung cancer cells, preferably metastasis of non-small cell lung cancer cells, but is not limited thereto.

[0019] The above-mentioned lung cancer cells may be radiation-resistant lung cancer cells, but are not limited thereto. The radiation-resistant lung cancer cells of the present invention refer to cells whose ability to metastasize or invade is increased by radiation irradiation.

[0020] In one embodiment of the present invention, the composition may inhibit the invasion or metastasis of cancer cells induced by radiation, but is not limited thereto.

[0021] In this invention, the term "invasion" refers to a symptom in which cancer cells proliferate while infiltrating and destroying surrounding tissues near where they first originated, and "metastasis" refers to a phenomenon in which cancer cells infiltrate blood vessels and lymph nodes, circulate along the blood and lymph, and then settle and proliferate in other tissues.

[0022] The above composition can enhance the effect of radiation therapy against cancer by inhibiting cancer cell infiltration or metastasis, which are side effects that occur during radiation therapy, but is not limited thereto.

[0023] The term 'radiation therapy enhancement' in this invention refers to maintaining the destructive effect of radiation therapy on cancer cells while suppressing the occurrence of metastatic or recurrent cancer and minimizing side effects.

[0024] The composition of the present invention may be administered simultaneously with radiation therapy, or before or after radiation therapy. As long as the effects of radiation therapy and the composition of the present invention are within a range where they can interact with each other, radiation therapy and the administration of the composition of the present invention may be performed with a time gap between them.

[0025] The composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient, and may be in various oral or parenteral formulations. When formulating, it is prepared using commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid formulations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid formulations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, emulsions, syrups, and aerosols; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cocoa dough, laurin dough, and glycerogelatin may be used as bases for suppositories. For parenteral administration, it is preferable to select a method of topical application or injection via the abdominal cavity, rectum, vein, muscle, subcutaneous, intrauterine dura mater, or cerebrovascular system.

[0026] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective amount may be determined according to factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above-mentioned factors into account, and this can be easily determined by a person skilled in the art.

[0027] The dosage of the composition of the present invention varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modulators.

[0028] In addition, the present invention relates to a health functional food composition for inhibiting cancer metastasis containing seongsanamide B represented by the above chemical formula 1 or a food-grade acceptable salt thereof as an active ingredient.

[0029] In this specification, the term "acceptable salt" refers to any salt of a compound of the present invention that does not exhibit toxicity or is not unsuitable for food or pharmaceutical use while maintaining the biological properties of the compound of the present invention. Such salts may be derived from, and include, various organic and inorganic counterions known in the art. These salts include organic or inorganic acids, such as hydrochloric acid, hydrobromide, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvate, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, chrysanthenic acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, Camponic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.Acid addition salt formed with [2]-oct-2-en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, cyclohexylsulfamic acid, quinic acid, or muconic acid, etc.; Or, it may include salts formed when an acidic proton present in the parent compound is substituted with a metal ion (e.g., alkali metal ion, alkaline earth metal ion, or aluminum ion), an alkali metal or alkaline earth metal hydroxide (e.g., sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxide), or ammonia, or when an organic base, such as an aliphatic, alicyclic, or aromatic organic amine, such as ammonia, methylamine, dimethylamine, diethylamine, picolin, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucarmine, piperazine, tris(hydroxymethyl)-aminomethane, or tetramethylammonium hydroxide, is coordinately bonded to it.

[0030] The health functional food composition for inhibiting cancer metastasis of the present invention is preferably manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages, but is not limited thereto.

[0031] When the health functional food composition of the present invention is used as a food additive, the saturamide B may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to its purpose of use (prevention or improvement). Generally, when manufacturing food or beverages, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw material. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there are no issues regarding safety, the active ingredient may be used in an amount greater than the above range.

[0032] There are no special restrictions on the types of the above foods. Examples of foods to which the above saturamide B can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0033] When the composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as taumarin and stevia extract, or synthetic sweeteners such as saccharin or aspartame may be used. The proportion of the above natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention. The composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

[0034] In addition, the present invention relates to a veterinary composition for inhibiting cancer metastasis containing seongsanamide B represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0035] The veterinary composition containing saccharamide B of the present invention may further include suitable excipients and diluents according to conventional methods. Examples of excipients and diluents that may be included in the veterinary composition containing saccharamide B 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, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

[0036] The veterinary composition containing saccharamide B according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powder, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

[0037] The veterinary composition according to the present invention may be administered once or several times a day, depending on the age, sex, and weight of the animal, and the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc. Accordingly, the above dosage does not limit the scope of the present invention in any way.

[0038] In addition, the present invention relates to a feed additive for inhibiting cancer metastasis containing seongsanamide B represented by Chemical Formula 1 or a food-grade acceptable salt thereof as an active ingredient.

[0039] The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act. In the present invention, the term "feed" may refer to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest. The types of said feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0040] The present invention will be described in detail below by way of embodiments. However, the following embodiments are merely illustrative of the present invention, and the scope of the present invention is not limited to the following embodiments.

[0041] Materials and Methods

[0042] 1. Seongsanamide B

[0043] Seongsanamid B used in the present invention is Bacillus saphensis ( Bacilus safensis) was isolated from the KCTC 12796BP culture medium, and in detail, the procedure was carried out according to the reference (Geum Jin Kim et al., Org Lett. 2018 Dec 7;20(23):7539-7543). HPLC was performed using a Gilson HPLC system (321 pump and UV / VIS-155 detector) equipped with a Hector-M C18 column (22.5×250 mm). Nuclear magnetic resonance spectra were recorded with dimethyl sulfoxide-d6 using an AVANCE 400 MHz spectrometer (1H, 400 MHz and 13C, 100 MHz, Bruker, Billerica, MA, USA). Ultraviolet and electronic circular dichroism spectra were acquired at the Research Center for Natural Products and Medical Materials using Cary5000 (Agilent, Santa Clara, CA, USA) and J-810 (Jasco, Tokyo, Japan) spectrophotometers, respectively. 1 Sanamide B, whose structure was confirmed through H NMR analysis, was used in the following examples.

[0044] 2. Cell culture, gamma irradiation, and dendrobin treatment

[0045] A549 and H460 non-small cell lung cancer cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Gibco Invitrogen Corp, Grand Island, NY, USA) containing 10% FBS (fetal bovine serum, Hyclone) under conditions of 5% (v / v) CO2 and a temperature of 37°C.

[0046] For irradiation, 5×10⁶ A549 and H460 cells were placed in a 60-pipe dish. 5 Inoculate at a cell density, and after culturing until the cells fill 70–80%, 137γ-gamma rays (A549: 10 Gy, H460: 2 Gy) were irradiated using a Cs gamma ray source (Atomic Energy of Canada, Mississauga, ON, Canada). The irradiated cells were treated with seongsanamide B of the present invention for 24 or 48 hours.

[0047] 3. Measurement of cell viability

[0048] A549 and H460 cells were seeded into 96-well culture dishes (A549: 1×10⁶ 4 Cell / well, H460: 2×10 4 Cells / well) were treated with sylsanamide B at various concentrations (3.12, 6.25, 12.5, 25, 50, 100, 200, and 400 μM) for 48 hours. Subsequently, 10 μL of EZ-Cyto cell viability assay reagent (DoGenBio, SEOUL, Korea) was added to each well, and the plates were incubated for 4 hours. Then, the effect of sylsanamide B on cell viability was confirmed by measuring the absorbance at a wavelength of 450 nm using a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

[0049] 4. Wound healing assay

[0050] To confirm the inhibitory ability of soxanamide B on radiation-induced cancer cell migration, A549 and H460 cells were seeded into 96-well culture dishes (A549: 2.5×10⁶ 5 Cell / Well, H460: 5×10 5 Cells / well), cultured for 24 hours. Wounds of similar thickness were made, and after γ-gamma irradiation (A549: 10 Gy, H460: 2 Gy), 50 μM sylsanamide B was administered and cultured for 48 hours. Cells were Moticam 3 +Cell migration was analyzed by imaging at 0 hours and 24 hours of culture using a CKX31 microscope (Olympus, Tokyo, Japan) connected to a digital camera (Motic, Hong Kong).

[0051] Relative wound width was calculated using the following formula: Wound width (%) = (Wound width after 48 hours / Initial wound width) × 100.

[0052] 5. Invasion assay

[0053] Since cancer cells must invade the blood vessel walls and enter the bloodstream to metastasize, invasion assays can measure not only invasion but also the risk of metastasis. Cell invasion was evaluated using the Transwell assay. Gamma-gamma-irradiated cells (A549: 10 Gy, H460: 2 Gy) were inoculated into wells on the upper surface of Matrigel-coated 6.5 mm Transwell chambers (Corning, Glendale, AZ) in serum-free RPMI-1640 medium (A549: 1×10⁶ 4 Cell, H460: 2×10 4 Cells / well). Saturamide B was added. After 16 hours, the infiltrated cells were fixed with 3.8% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes and stained with 0.23% crystal violet (Sigma-Aldrich) for 30 minutes. The infiltrated cells were imaged and counted using a CKX31 microscope.

[0054] 6. Nuclear fractionation

[0055] Nuclear fractionation was performed using the NE-PER Nuclear and Cytoplasm Extraction Reagent Kit (Thermo Fisher Scientific). Briefly, harvested cells were washed with PBS (Thermo Fisher Scientific) and suspended in cold CER I in a protease-containing inhibitor cocktail (Sigma-Aldrich). After 10 minutes on ice, cold CER II was added. The lysate was separated from the supernatant and pelleted using centrifugation (4°C, 5 min, ~16,000×g). The pellet was suspended in cold NER for 40 minutes using a vortex mixer, and the nuclear extract (supernatant) was separated using centrifugation (4°C, 10 min, ~16,000×g).

[0056] 7. Animal testing

[0057] BALB / c mice (5-6 week old females; Orient Bio Co., Ltd., Gyeonggi-do) were acclimatized for one week in a climate-controlled room (22 ± 2℃, relative humidity 55% ± 5%, 12-hour light-dark cycle). All animal protection and ethics protocols and experiments were conducted with the approval of the Animal Protection and Use Committee of Jeonbuk National University Hospital (Approval No. JBUH-IACUC-2020-7, Jeonju-si, Korea, JBUH-IACUC-2021-10-1).

[0058] 8. Radiation-induced metastasis animal model

[0059] A549 cells cultured for 24 hours after irradiating 0, 2, and 5 Gy of gamma rays through the tail vein of 5-6 week old female BALB / c mice (Orient Bio, Korea) (1×10⁶ per mouse) 6 Dog cells were injected. After 5 weeks, the mice were euthanized, the lungs were removed, images were taken, and the number of metastatic tumors was counted. Histological analysis or Western blot analysis was performed on the tumor tissues.

[0060] 9. A549 xenograft model

[0061] 5-6 week old female BALB / c mouse flank (1×10 6Inject cells (100 µl) subcutaneously, and the tumor is 100-200 mm 3 Upon reaching [date], the mice were randomly divided into four groups. Mice were intraperitoneally injected daily with phosphate amide B (1, 2.5, or 5 mg / kg) or DPBS (dulbecco's phosphate-buffered saline, vehicle). Tumors were measured twice a week for 27 days using a digital caliper, and tumor volume was calculated using Equation 1 below.

[0062] [Equation 1]

[0063] V(average tumor volume) = (width) 2 × length) / 2

[0064] 10. Analysis of metastasis in animal models with metastasis induced by radiation

[0065] 100 mm of A549 cells 2 Cells were cultured in dishes until 70–80% full, then re-cultured for 24 hours after irradiating with 2 Gy of gamma rays using a TrueBeam device (Varian Medical Systems, Palo Alto, CA, USA). Gamma-irradiated or non-gamma-irradiated A549 cells were administered intraperitoneally daily via the mouse tail vein for 7 weeks. Six-week-old female BALB / c mice were divided into four groups: 0 Gy / DPBS (n=5), sylsanamide B 0 Gy / 5 mg / kg (n=7), 2 Gy / DPBS (n=5), and sylsanamide B 2 Gy / 5 mg / kg (n=7). Body weight was measured once a week for 7 weeks. After euthanizing the mice with ketamine, the lungs were excised and imaged, and the number of metastatic tumors was counted. Tumor tissues were subjected to histological and Western blot analysis.

[0066] 11. Histological analysis

[0067] Lung tissue was fixed with 10% formalin and embedded in paraffin, and after staining with hematoxylin and eosin (H&E), metastatic lung nodules were imaged and analyzed using a Motic EasyScan One Digital Slide Scanner (Motic, Kowloon, Hong Kong).

[0068] 12. Western blotting

[0069] Lysates of A549 and H460 cells, respectively, and mouse lung tissue were lysed using the T-PER™ Tissue Protein Extraction Reagent Kit (Thermo Fisher Scientific) containing cell lysis buffer and a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Equal amounts of cell lysates (15 µg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Thermo Fisher Scientific). After blocking with 5% skim milk, β-catenin (#8480), p-STAT3 (#9145), STAT3 (#9139), and Bcl-X L The primary antibodies against (#2762) and p-Sr (#12432) were reacted overnight, and the secondary antibody containing horseradish peroxidase was reacted, and the antibody signal was detected using an iBright 1500 Imaging System (Invitrogen, Waltham, MA, USA).

[0070] 13. Enzyme-linked immunosorbent assay (ELISA)

[0071] The expression level of IL-6 protein in cell lysates was measured using the Quantikine ELISA kit (LSBio, Seattle, WA, USA) according to the manufacturer's protocol.

[0072] 14. Statistical Analysis

[0073] The results were expressed as the mean ± SD of three independent experiments, and statistical significance was determined using Student's t-test or one-way ANOVA in Tukey post-hoc comparison using the GraphPad Prism statistical program (GraphPad Software, San Diego, CA, USA). p If the value was less than 0.05, it was considered statistically significant.

[0074] Example 1. Toxicity test of saccharamide B on non-small cell lung cancer cells

[0075] A549 and H460 cells were treated with various concentrations (3.12, 6.25, 12.5, 25, 50, 100, 200, 400 μM) of sylsanamide B and cultured for 48 hours.

[0076] As a result, as disclosed in Fig. 1, the saccharamide B of the present invention did not exhibit cytotoxicity at a concentration of less than 50 μM.

[0077] Example 2. Confirmation of the inhibitory effect of spheramide B on cancer cell metastasis and invasion by radiation.

[0078] 1) Wound healing assay

[0079] To confirm the ability of spheramide B to inhibit metastasis and invasiveness, 50 μM of spheramide B was administered to A549 and H460 cells irradiated with gamma rays (γ-irradiation), and a wound healing analysis was performed.

[0080] As a result, as disclosed in Figures 2A and 2B, it was confirmed that cell migration increased by radiation was significantly reduced by treatment with sangsanamide B. In addition, it was confirmed that sangsanamide B also had the effect of reducing the migration of A549 and H460 cells that were not irradiated.

[0081] 2) Invasion assay

[0082] An invasion assay was performed to confirm the ability of synthamide B to inhibit invasion and metastasis. For the invasion assay, Matrigel, known to create an environment similar to the extracellular matrix, was used.

[0083] As a result, as disclosed in Figures 2C and 2D, it was confirmed that the infiltration ability of cells increased by radiation was significantly reduced by treatment with sangsanamide B. In addition, it was confirmed that sangsanamide B also had the effect of reducing the infiltration ability of A549 and H460 cells that were not irradiated.

[0084] Example 3. Analysis of changes in expression levels of proteins that activate cancer cell migration and invasion

[0085] β-catenin, IL-6, STAT3, Bcl-X L It is known that SOD2 and Src are involved in activating the migration and invasion of irradiated cancer cells. Therefore, the effect of sangsanamide B on their expression was analyzed.

[0086] As a result of confirming the effect of synthamide B on the protein expression levels of signaling molecules induced by radiation, β-catenin, p-STAT3, and Bcl-X in A549 and H460 cells induced by radiation, as disclosed in Figures 3A and 3B L The expression levels of p-Src and SOD2, and the production of IL-6 increased. When irradiated cells were treated with spheramide B, the increased Bcl-X L SOD2 and p-Src expression were reduced, but the expression levels of β-catenin, IL-6, and p-STAT3 showed little change due to treatment with saccharamide B.

[0087] In addition, sangsanamide B is STAT3-Bcl-X induced by radiation L It inhibited signal transduction. Therefore, synthamide B blocked the activated STAT3 potential, thereby inhibiting Bcl-XL To determine whether it reduces expression, the expression level of p-STAT3 in cells that underwent nuclear fractionation was measured using Western blot. As a result, radiation irradiation increased the expression of p-STAT3 in the nuclear fraction, and p-STAT3 expression was inhibited after treatment with 50 μM of sangsanamide B (Fig. 3C). These results indicate that sangsanamide B can inhibit radiation-induced migration and invasion of cancer cells by blocking the p-STAT3 nuclear potential in non-small cell lung cancer cells.

[0088] Example 4. Analysis of changes in EMT-related protein expression induced by radiation exposure

[0089] The process of epithelial-mesenchymal transition (EMT) is activated by various intrinsic and extrinsic signaling factors, including STAT3, which regulates migration and invasion. Radiation induces cell migration and invasion by regulating STAT3 phosphorylation in various cancer cells and promotes EMT through the radiation-mediated Src signaling pathway. Therefore, to evaluate the effect of sangsanamide B on radiation-induced EMT activity in A549 and H460 cells, the expression levels of the EMT markers E-cadherin and vimentin were measured by Western blot after radiation.

[0090] As a result, as disclosed in Figure 4, the expression levels of E-cadherin and vimentin were reduced and increased in irradiated A549 and H460 cells, but it was confirmed that when treated with the saccharamide B of the present invention, the expression levels of E-cadherin increased and the expression levels of vimentin decreased and recovered.

[0091] Example 5. Effects of senoamide B in the A549 xenograft model

[0092] Inside the body ( in vivoIn order to determine the effect of senoside amide B on cancer growth, A549 cells irradiated with 2 Gy were subcutaneously injected into the flanks of 5-6 week old female BALB / c mice to induce tumor formation, and the tumors were approximately 100-200 mm 3 When the volume grew to [a certain level], sylsanamide B (1, 2.5, or 5 mg / kg / day) or DPBS (vehicle) was injected intraperitoneally daily for 2 weeks (Fig. 5A).

[0093] As a result, as disclosed in Fig. 5B, no difference in body weight was observed between the experimental groups of mice, and it was confirmed that senoside amide B did not exhibit cytotoxicity up to a concentration of 5 mg / kg (Fig. 5C).

[0094] Therefore, in order to confirm the cancer metastasis inhibitory effect of sylsanamide B at a concentration that does not exhibit cytotoxicity in the present invention, it was decided to treat a cancer metastasis animal model using irradiated cells with a dose of 5 mg / kg of sylsanamide B that does not exhibit cytotoxicity.

[0095] Example 6. Metastasis-inhibiting effect of spheramide B in an animal model of cancer metastasis using irradiated cells

[0096] Inside the body ( in vivo In order to confirm the cancer metastasis inhibitory effect of sylsanamide B, an experiment was conducted as disclosed in Fig. 6A using a radiation dose (2 Gy) to induce cancer metastasis and an optimal sylsanamide B dose (5 mg / kg) that does not exhibit cytotoxicity.

[0097] As a result, as disclosed in Fig. 6B, there was no difference in mouse body weight between the experimental groups, but as disclosed in Figs. 6C to 6E, metastatic lung nodules significantly increased in the mouse group injected with irradiated A549 cells and were confirmed to be effectively suppressed by treatment with sylsanamide B. Sylsanamide B also showed an effect of suppressing metastatic lung nodules in the mouse group injected with non-irradiated A549 cells, and the effect of suppressing metastatic lung nodules that increased after irradiation was even more pronounced.

[0098] To determine whether sylsanamide B inhibits the expression of radiation-induced signaling pathways in vivo, Western blot analysis was performed, and as disclosed in Fig. 6F, Bcl-X by radiation L While the expression levels of , SOD2, p-Src and vimentin were increased, the expression of the present invention was inhibited by treatment with saccharamide B.

[0099] Through this, sangsanamide B is Bcl-X L It was confirmed that in vivo metastasis induced by radiation is suppressed by inhibiting EMT through the Src signaling pathway.

Claims

Claim 1 A pharmaceutical composition for inhibiting lung cancer metastasis containing seongsanamide B represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Chemical Formula 1] Claim 2 A pharmaceutical composition for inhibiting lung cancer metastasis according to claim 1, characterized in that the lung cancer metastasis is induced by radiation. Claim 3 delete Claim 4 A pharmaceutical composition for inhibiting lung cancer metastasis, characterized in that, in claim 1, the lung cancer is radiation-resistant lung cancer. Claim 5 A pharmaceutical composition for inhibiting lung cancer metastasis according to claim 1, characterized in that the composition inhibits the invasion or metastasis of lung cancer cells induced by radiation. Claim 6 A pharmaceutical composition for inhibiting lung cancer metastasis according to claim 1, characterized in that the composition enhances the radiation therapy effect against lung cancer. Claim 7 A pharmaceutical composition for inhibiting lung cancer metastasis according to claim 1, characterized by further comprising, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent. Claim 8 A pharmaceutical composition for inhibiting lung cancer metastasis according to claim 1, characterized in that the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. Claim 9 A health functional food composition for inhibiting lung cancer metastasis containing seongsanamide B represented by the following chemical formula 1 or a food-grade acceptable salt thereof as an active ingredient. [Chemical Formula 1] Claim 10 A health functional food composition for inhibiting lung cancer metastasis according to claim 9, characterized in that the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages. Claim 11 A veterinary composition for inhibiting lung cancer metastasis containing seongsanamide B represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Chemical Formula 1] Claim 12 A feed additive for inhibiting lung cancer metastasis containing seongs anamide B represented by the following chemical formula 1 or a food-acceptable salt thereof as an active ingredient. [Chemical Formula 1]

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