KR101821498B1 - Pharmaceutical composition for preventing or treating cancer diesease comprising broussochalcone A - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer diseases comprising broussochalcone A as an active ingredient, and it was confirmed that the broussochalcone A inhibits the activity of NR4A1 in cancer cells overexpressing NR4A1 to effectively inhibit cancer cell growth and induce apoptosis, and thus the composition comprising the broussochalcone A as the active ingredient can be provided as a pharmaceutical composition for preventing or treating cancer diseases and health food.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating cancer,

The present invention relates to a composition for preventing or treating cancer diseases containing brucocarcon A as an active ingredient.

The pancreas is an organ that produces enzymes that help digesting insulin and the body to help turn glucose into energy in the body. Pancreatic cancer is a malignant growth of the pancreas, mainly occurring in cells of the pancreatic duct, and less than 3% is a fatal cancer disease with a 5-year survival rate.

Early diagnosis of pancreatic cancer is very difficult, and when it is diagnosed, it is often impossible to perform radical resection because there are many conditions that have already metastasized to the surrounding major organs. Unlike gastric, duodenal, lung, and breast cancers, which are widely used for various types of cancer drugs, there are few known anticancer drugs that are effective against pancreatic cancer. Currently, only a limited number of cancer drugs are used.

The most common treatment modality for advanced pancreatic cancer is the treatment with gemcitabine, an intravenous 2'-deoxycytidine nucleoside analogue that can induce apoptosis of human pancreatic cancer cells and inhibit tumor growth and progression However, since the survival of patients treated with the above treatment is less than 21 months, an effective treatment strategy for treating pancreatic cancer is required.

NR4A1 has been identified as an orphan nuclear receptor because its endogenous ligand has not yet been identified and is first known as a gene that is activated by nerve growth factor in PC12 cells. Thymic cell-induced thalassemia induced by T-cell receptor plays a key role in apoptosis and functions as a transcriptional regulator and plays an important role in animal growth, development, differentiation and maintenance of homeostasis.

In particular, NR4A1 is overexpressed in many types of tumor tissues including pancreatic cancer, and has been shown to play an important role in growth and survival of cancer cells through various pathways. Thus, NR4A1, which is a nuclear transcription factor, is inhibited and pancreatic cancer cells are killed Can be a new strategy for the treatment of pancreatic cancer.

Korean Laid-Open Patent No. 2014-0147163 (disclosed on December 30, 2014)

The present invention provides a pharmaceutical composition for preventing or treating cancer diseases, which contains brucite cone A as an active ingredient, as a novel strategy for the treatment of cancer diseases which inhibits the activity of nuclear transcription factor NR4A1 and induces the death of cancer cells.

The present invention provides a pharmaceutical composition for preventing or treating cancer diseases, which comprises brucocarcon A as an active ingredient.

In addition, the present invention provides a health food for improving cancer diseases, which contains brucocarcon A as an active ingredient.

According to the present invention, it has been confirmed that brucocarcon A inhibits the activity of NR4A1 in cancer cells overexpressing NR4A1, effectively inhibiting cancer cell growth and inducing apoptosis, A pharmaceutical composition for preventing or treating disease, and a health food.

FIG. 1 shows Western blot results of confirming NR4A1 expression levels in pancreatic cancer cell lines Capan-2, Panc-1 and MiaPaCa-2, AGS human gastric cancer cell AGS, human colon cancer cells HCT116 and human lung cancer cell line H460.
FIG. 2 shows the results of confirming luciferase activity to confirm the NR4A1 activity inhibitory effect of Brusochon Ace.
FIG. 3 is a graph showing the results of the Brassensis assay of concentrations of 0, 5, 10, 20 and 30 μM in pancreatic cancer cell lines Capan-2, Panc-1 and MiaPaCa-2 and AGS human gastric cancer cell AGS, human colon cancer cell HCT116, human lung cancer cell line H460 Cone A treatment and cell viability of each cancer cell.
FIG. 4 is a graph showing the effect of brucocarcon A concentration of 0, 10, 20 and 30 μM on Capan-2 and MiaPaCa-2 cells in order to examine the effect of inducing apoptosis-related protein expression of Brusochon A This is the confirmed Western blot result.

The present invention can provide a pharmaceutical composition for preventing or treating cancer diseases containing brucocarcon A as an active ingredient.

The cancer can be selected from the group consisting of pancreatic cancer, stomach cancer, colon cancer, lung cancer, breast cancer, liver cancer and brain cancer.

The brucocarcinoma cone inhibits NR4A1 activation, thereby inhibiting cancer cell growth and inducing apoptosis.

According to one embodiment of the present invention, in order to confirm whether the inhibition of the nuclear transcription factor NR4A1 activation is related to the inhibition of cancer cell growth, basically NR4A1, including three kinds of pancreatic cancer human cell lines Capan-2, MiaPaCa-2 and Panc- The inhibitory effect of H460, AGS, and HCT116 on cell proliferation was confirmed by MTT assays.

As a result, as shown in FIG. 3, basal NR4A1-expressing 5-cell cancer cells showed a dose-dependent increase in the cancer cell proliferation inhibitory effect by Brusochon A (10-30 μM), while the Capan-2 cell line exhibited a less NR4A1 expression The inhibitory effect of Brucella cone A (10 ~ 30 μM) on cancer cell proliferation was relatively low.

Reporter gene assays were performed on the MiaPaCa-2 and Panc-1 pancreatic cancer cell lines, which were found to have a high NR4A1 activity, in order to confirm whether the results of the previous experiment were influenced by Brusapachia cone A on nuclear transcription factor NR4A1-dependent receptor gene activity As shown in FIG. 2A, brucite cone A (10-30 .mu.M) was shown to inhibit the nuclear transcription factor Gal4-NR4A1 luciferase activity of NR4A1 overexpressed MiaPaCa-2 and Panc-1 cells in a concentration-dependent manner.

In order to investigate the correlation between the induction of apoptosis induced by brucite cone A and its regulatory protein expression, Capan-2 cell line, which is basically expressed with NR4A1, and MiaPaCa-2 cell line with high expression, As a result of comparing the expression of apoptosis-related proteins, the expression of the pro-apoptosis protein, the cleaved caspase-8, cleaved pap (c-PARP) in the MiaPaCa-2 cell line, Respectively.

From the above results, it was confirmed that Brusociticone A inhibits the activity of NR4A1 in cancer cells overexpressing NR4A1, effectively inhibiting cancer cell growth and inducing apoptosis.

The pharmaceutical composition may contain 0.1 to 90 parts by weight of brucocarcon A per 100 parts by weight of the total pharmaceutical composition.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer diseases containing the bruczacin A as an active ingredient can be administered orally or parenterally in the form of injections, granules, powders, tablets, pills, capsules, suppositories, Any one of the formulations selected from the group consisting of suspensions, emulsions, drops, and liquid preparations can be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating a cancerous disease containing brucocarcon A as an active ingredient may be prepared by mixing the appropriate carriers, excipients, disintegrants, sweeteners, , At least one additive selected from the group consisting of lubricants, lubricants, flavors, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.

The preferred dose of the brucocarpone A may vary depending on the condition and body weight of the subject, the type and degree of disease, the type of drug, the route of administration and the period of time, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

In addition, the present invention can provide a health food for improving cancer diseases containing brucocarcon A as an active ingredient.

The health food is used together with other food or food additives other than the brucocarpone A, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the above-mentioned health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range for health and hygiene purposes or for long-term intake for health control purposes, It is clear that the component can be used in an amount of more than the above range since there is no problem in terms of safety.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example  1> Cell culture

All the cell lines used were sold from the Korean Cell Line Bank (KCLB, Seoul, Korea). AGC human gastric cancer cells, HCT116 human colorectal cancer cells and H460 human lung cancer cell lines including Capan-2 human pancreatic cancer cell line were cultured in RPMI1640 (Welgene, Daegu, Korea) medium supplemented with 10% fetal bovine serum (Welgene, Daegu, Panc-1 and MiaPaCa-2 cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium high glucose; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (Welgene, Daegu, Korea) supplemented with penicillin / streptomycin , Daegu, Korea) and 1% antibiotics (penicillin / streptomycin) (Welgene, Daegu, Korea) were used for incubation in a 5% CO ₂ incubator at 37 ° C. Cells were treated with trypsin / EDTA at the end of the logarithmic growth phase, and the cells were detached and centrifuged at 1,000 rpm for 3 minutes. Cells were collected, subcultured and subcultured.

< Experimental Example  2 > transfection &lt; Luciferase  Verify Active

Broussochalcone A was purchased from Toronto Research Chemicals (Ohtario, Canada) and used.

The MiaPaCa-2, Panc-1 and Capan-2 human pancreatic cancer cell line 2.5 × 10 ⁴ cells / well seeded 48-well plate after plate culture manufacturer OPTI-MEM according to the instructions of (Invitrogen, Carlsbad, CA, USA ), Gal4-DBD (DNA Binding Domain) -NR4A1-chimera construct, Gal4-RE (Response Elements) -luciferase reporter construct and pCMV-β-galactosidase reporter plasmid were mixed together using a mixture of plasmid and Lipofectamine 2000 And transiently transfected.

The transformed cells were treated with Brusociticone A at a concentration of 5 to 30 μM for 18 hours and then luciferase activity was measured using a luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions Respectively.

< Experimental Example  3> Cell growth analysis

3- (3,4-dimethyl-thiazolyl-2) -2,5-diphenyl tetrazolium, which is a modification of the previously reported method (Green, LC et al .. J Immunol Methods, 70, 257-268) bromide (MTT) analysis was performed.

First, Capan-2, MiaPaCa-2, and Panc-1 human pancreatic cancer cell lines and H460, AGS, and HCT116 human solid tumor cell lines were plated at 250 μl per well of 48-well plates and seeded at a density of 3.0 × 10 ⁴ cells / Lt; / RTI &gt; for 24 hours. Brucella cone A was treated at a concentration of 5 to 30 μM for 24 hours, and an MTT reagent (Amresco, Solon Ind., Columbus, Ohio, USA) at a concentration of 2.5 mg / And cultured for 4 hours. After the completion of the incubation, the supernatant was removed and 200 μl of DMSO was added to each well to dissolve the resulting Formazin crystals and the absorbance at 550 nm was measured.

Cytotoxicity was expressed as a percentage of the absorbance of the control in the absorbance of the sample.

< Experimental Example  4> Protein analysis (Western blot assay)

For Western blot analysis, dividing the respective cell lines in 6-well plates at 3 × 10 ⁵ cells / well density, and cultured for 24 hours at 37 ℃, 5% CO2 incubator. The cultured cells were treated with Brusocitic cone and cultured for 24 hours, and then cells were lysed using RIPA buffer.

The protein was separated from the cell lysate by electrophoresis, transferred to PVDF membrane, blocked with 5% defatted milk powder, reacted with primary antibody for 18 hours at 4 ° C, and incubated with Tris-Buffered Saline Tween 20) solution. Subsequently, the secondary antibody was reacted for 30 minutes to 2 hours and then washed three times with TST.

The washed membrane was mixed with the ECL detection kit color development reagents I and II in a ratio of 1: 1, and the mixture was applied to the membrane and exposed to an X-ray film (CP-G plus, Agfa Healthcare Ltd, New Orleans, And then apoptosis-related protein expression was measured on the film.

< Example  1> Cancer cell line NR4A1  Confirmation of expression

The expression level of NR4A1 in 6 kinds of human cancer cells was confirmed by Western blotting.

As a result, it was confirmed that NR4A1 was overexpressed in five kinds of cells except Capan-2 as shown in Fig.

< Example  2> Bruschet cone  Of A NR4A1  Check the activity modulating effect

Reporter gene assays were performed on the MiaPaCa-2 and Panc-1 pancreatic cancer cell lines previously identified as having a high NR4A1 activity in order to confirm the effect of brucite cone A on nuclear transcription factor NR4A1-dependent receptor gene activity.

First, Gal4-NR4A1-chimeric construct and Gal4-luc construct were transiently transfected into MiaPaCa-2 and Panc-1 pancreatic cancer cell lines, and the cells were treated with 10 ~ 30 μM Brusotacon cone.

As a result, as shown in FIG. 2A, it was confirmed that brucite cone A (10-30 μM) inhibited the nuclear transcription factor Gal4-NR4A1 luciferase activity of MiaPaCa-2 and Panc-1 cell lines overexpressing NR4A1 in a concentration-dependent manner . On the other hand, the Capan-2 cell line, which basically showed less expression of NR4A1, was found to have a remarkably low activity of Gal4-NR4A1 luciferase and also showed no inhibitory effect by Brusotaconic A ( P <0.05).

On the other hand, in order to examine the effect of Brusociticone A on the activity of NBRE gene as a binding site promoter of NR4A1, MiaPaCa-2 and Panc-1 pancreatic cancer cell lines were transfected with NBRE-luc construct and NR4A1 gene, Brucella cone A at a concentration of 30 [mu] M was treated.

As a result, it was confirmed that the NBRE promoter activity in the MiaPaCa-2 and Panc-1 cell lines was markedly decreased by Brusociticone A as shown in FIG. 2B.

< Example  3> Bruschet cone  Confirmation of cancer cell death effect of A

To confirm whether the inhibition of nuclear transcription factor NR4A1 activation is related to the inhibition of cancer cell growth, H460, AGS, and HCT116 cells, which are basically NR4A1-expressing, including three kinds of pancreatic cancer human cell lines Capan-2, MiaPaCa-2 and Panc- Were used to confirm the cancer cell proliferation inhibitory effect through MTT assays.

As a result, as shown in FIG. 3, the inhibitory effect of Brusochon-kon A (10-30 μM) on the cancer cell proliferation was increased in a concentration-dependent manner in the 5-cell cancer cell in which NR4A1 was highly expressed, whereas the Capan-2 cell line, The inhibitory effect of Brucella cone A (10 ~ 30 μM) on cancer cell proliferation was relatively low.

< Example  4> Bruschet cone  Cell death by A Mechanism  Confirm

Apoptosis-related protein expression was examined to determine the correlation between induction of apoptosis by brucite cone A and its regulatory protein expression. Protein expression was compared after treatment with Brusochrone cone at different concentrations in Capan-2 cell line and MiaPaCa-2 cell line, which were basically expressed with low NR4A1 expression.

As a result, as shown in Fig. 4, the pro-apoptotic protein divisible caspase-8, cleaved pap (c-PARP) expression was relatively higher in the MiaPaCa-2 cell line than the Capan-2 cell line.

Meanwhile, the brucite cone A according to the present invention can be formulated into various forms according to the purpose. Hereinafter, some formulation methods in which brucocarpone A according to the present invention is contained as an active ingredient are exemplified, and the present invention is not limited thereto.

Formulation Example 1 Prescription Example of Pharmaceutical Composition

&Lt; Formulation Example 1-1 > Preparation of injection

10 mg of brucite cone A, 3.0 mg of sodium metabisulfite, 0.8 mg of methylparaben, 0.1 mg of propylparaben and an appropriate amount of sterilized distilled water for injection were mixed and made into a final volume of 2 ml by a conventional method, Ampicillin and sterilized to prepare an injection.

&Lt; Formulation Example 1-2 > Preparation of tablets

10 mg of brucite cone A, 100 mg of lactose, 100 mg of starch and an appropriate amount of magnesium stearate were mixed and tableted according to a conventional tablet preparation method.

&Lt; Formulation Example 1-3 > Preparation of capsules

10 mg of brucite cone A, 50 mg of lactose, 50 mg of starch, 2 mg of talc, and an appropriate amount of magnesium stearate were mixed and filled in gelatin capsules according to a conventional capsule preparation method to prepare capsules.

&Lt; Formulation Example 2 > Preparation of health food

Brucella cone A 0.5 mg Vitamin A acetate 70, Vitamin E 1.0 0.13 mg Vitamin B 2 0.15 mg Vitamin B 6 0.5 mg Vitamin B 12 0.2 ㎍ Vitamin C 10 mg , Fibrates (10 비 of biotin, 1.7 ㎎ of nicotinic acid amide, 50 ㎍ of folic acid, 0.5 mg of calcium pantothenate) and an appropriate amount of mineral mixture (1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 55 mg of calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed to prepare a granule, and a health food was prepared according to a conventional method.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

The present invention relates to a reagent composition for inhibiting NR4A1 activity, which contains as an active ingredient con A (Broussochalcone A) and inhibits intracellular NR4A1 activity in vitro. delete delete delete delete delete

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