KR20230056505A - Composition for preventing, improving or treating radiation-resistant cancer comprising durumamide A - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing durumamide A for the prevention or treatment of radiation-resistant cancer. The pharmaceutical composition containing durumamide A as an active ingredient according to one embodiment of the present invention inhibits the expression of ACSL4 and FOXM1 protein in radiation-resistant cancer, and inhibits DNA damage response, invasion ability, and migration ability, thereby being able to be usefully used for the prevention or treatment of radiation-resistant cancer by inducing cell death in radiation-resistant cancer.

Description

Composition for preventing, improving or treating radiation-resistant cancer comprising durumamide A

The present invention relates to a composition comprising durumamide A.

For cancer treatment, surgery, chemotherapy and radiation therapy are mainly performed. Among them, surgery and radiation therapy are local therapies that are effective only in the ablation area or irradiated area, and anticancer drug therapy is a systemic therapy that is effective throughout the body. Most cancers are diseases that develop locally and metastasize throughout the body, and local therapy is primarily used for most cancer treatments.

On the other hand, the effectiveness of radiation therapy among local therapies varies depending on the nature of the cancer, the patient, and the treatment method used in combination with radiation. In general, cancers for which radiation therapy is widely performed include head and neck cancer, laryngeal cancer, cervical cancer, pharynx cancer, lung cancer, brain cancer, breast cancer, colon cancer, and the like. However, although these carcinomas are the main targets of radiation therapy, there are many cases where the response is not good during radiation therapy. In addition, even if the initial response to radiation therapy is excellent, recurrence is frequent, and it is known as a problem of radiation therapy that radiation-resistant cancer occurs due to radiation therapy. Therefore, in radiation therapy, a more effective cancer treatment strategy is needed.

Cancer cells are known to change cellular components to protect themselves from chronic radiation damage. For example, proliferation regulatory proteins such as epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K)/Akt, and RAS are known to be activated or overexpressed in radiation-resistant tumors. Status/expression of p53 or Bcl-2/-xl plays an important role in cell cycle and apoptosis, is associated with tumor radioresistance, and is associated with peroxiredoxin-II and manganese superoxide dismutase (MnSOD) Cellular antioxidant proteins such as are known to be involved in oxidative stress resistance in cancer cells. In addition, by changing the expression pattern of P- glycoprotein and proteins related to multi-drug resistance by radiation treatment, radiation resistance and anticancer drug resistance may occur acquired.

Factors related to radiation resistance reported so far include cyclooxygenase-2 (Terakado N, et al., Oral Oncol 2004; 40:383-9), EGFR (Milas L, et al., Int. J Radiat Oncol Biol Phys 2004;58(3);966-71), cyclin D1 (Milas L, et al., Int J Radiat Oncol Biol Phys 2002;52;514-21), insulin-like There are growth factor 1 receptor (IGFBP-1 receptor), MnSOD, and survivin, but the mechanisms related to radiation resistance of cancer cells are not yet clearly understood. Accordingly, there is a need for research to develop materials for the treatment of radiation-resistant cancer.

On the other hand, Actinomycetes are microorganisms belonging to Gram-positive bacteria that are widely distributed in nature and play an important role in biotechnology because they produce beneficial substances such as amino acids, vitamins, enzymes, anticancer substances, and antibiotics. In addition, 130 to 140 kinds of useful compounds produced by actinomycetes are used as human medicines, and furthermore, some 15 to 20 kinds of compounds are widely used as plant protection factors, insecticides, food additives, and pharmaceuticals.

The present inventors have completed the present invention by confirming whether durumamide A, which has been identified as an effective substance of Corynebacterium durum , a type of actinomycete, is effective against radiation-resistant cancer.

Republic of Korea Patent No. 10-0768476

An object of the present invention is to provide a pharmaceutical composition for preventing or treating radiation-resistant cancer comprising durumamide A as an active ingredient.

In addition, an object of the present invention is to provide a food composition for preventing or improving radiation-resistant cancer containing durumamide A.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating radiation-resistant cancer comprising durumamide A as an active ingredient.

The structure of durumamide A is shown in Formula 1 below.

[Formula 1]

According to one embodiment, the durumamide A may be derived from Corynebacterium durum or synthesized using tryptamine. For example, the durumamide A may be obtained from Corynebacterium durum by the following method.

According to one embodiment, the durumamide A is obtained by culturing Corynebacterium durum to obtain a culture; extracting the culture with acetone to obtain an acetone extract; obtaining fractions by fractionating the acetone extract with an organic solvent; and performing chromatography on the fraction to obtain durumamide A.

According to one embodiment, the durumamide A may be synthesized from tryptamine. Preparing a mixed solution by mixing the durumamide A with 3-indoleglyoxylyl chloride and 4-dimethylaminopyridine; and preparing a reaction solution by adding tryptamine to the mixed solution, or preparing a lysate by dissolving tryptamine in DMSO; And it can be synthesized by a method comprising the step of refluxing the lysate to obtain a reflux extract.

As used herein, the term "active ingredient" means a component that exhibits the desired activity alone or that can exhibit activity in combination with a carrier that is not active itself.

As used herein, "radiation-resistant cancer" means a state in which the effect of radiation is less than that of general cancer, and there is little cell damage or reduction in proliferation when exposed to radiation. The radiation may be electromagnetic radiation including X-rays, ultraviolet light, alpha particles, or gamma rays.

According to one embodiment, the radiation-resistant cancer is not particularly limited as long as it is radiation-resistant cancer, and is, for example, selected from the group consisting of head and neck cancer, laryngeal cancer, pharynx cancer, lung cancer, brain cancer, colon cancer, breast cancer, and pancreatic cancer. It may be any type of cancer that is affected by cancer, and may have acquired radiation resistance during or after treatment, or it may be essential radiation-resistant cancer.

As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of radiation-resistant cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms of radiation-resistant cancer by administration of the pharmaceutical composition according to the present invention.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described durumamide A.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. can be administered.

A suitable dosage of the pharmaceutical composition of the present invention may be determined by factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. .

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container.

According to one embodiment, the composition may inhibit the expression of acyl-CoA synthetase long chain family member 4 (ACSL4) in radiation-resistant cancer.

The ACSL4 gene is a gene encoding long chain fatty acid CoA ligase 4, and its association with resistance to radiation therapy has been reported.

ACSL4 is a parent factor of FOXM1 in radiation-resistant breast cancer cells, and FOXM1 is a transcription factor that regulates the expression of various genes required for DNA damage repair, proliferation, and survival of cancer cells.

The expression may mean mRNA expression or protein expression.

The ACSL4 is one of the isomers of ACSL consisting of ACSL1, 3, 4, and 5.

In the present specification, it was confirmed that only the expression of ACSL4 among the ACSL isomers increased in cancer showing radiation resistance, and the expression of ACSL1, ACSL3, and ACSL5 decreased, and the survival rate of cancer cells was improved by suppressing the expression of ACSL4 in radiation-resistant cancer cells. found to be significantly reduced.

Another aspect of the present invention provides a food composition for preventing or improving radiation-resistant cancer containing durumamide A.

Descriptions common to the foregoing here are omitted in order to avoid excessive complexity.

As used herein, the term "improvement" refers to all actions that at least reduce the parameters related to the condition to be treated, for example, the severity of symptoms. In this case, the food composition may be used simultaneously with or separately from a drug for treatment before or after the onset of the disease for the prevention or improvement of radiation-resistant cancer.

The food composition according to the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, chewing gum, ice cream, vitamin complexes, and health supplements. .

In addition to durumamide A, the food composition of the present invention may include ingredients commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made into drinks and beverages, in addition to durumamide A of the present invention, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included. can

A pharmaceutical composition comprising durumamide A as an active ingredient according to an embodiment of the present invention inhibits the expression of ACSL4 and FOXM1 protein in radiation-resistant cancer, and inhibits DNA damage response, invasion ability and migration ability, thereby inhibiting radiation-resistant cancer. It can be usefully used for prevention or treatment of radiation-resistant cancer by inducing apoptosis of resistant cancer.

Figure 1 shows the process of constructing radiation-resistant breast cancer cells, SR and MR, from normal breast cancer cells SK-BR-3 and MCF-7, respectively.
Figure 2 shows the results of comparing the tissue viability of SR and SK-BR-3 (a), and the tissue viability of MR and MCF-7 (b) to irradiation.
FIG. 3 shows results showing that radiation-resistant cancer cells (SR) have anticancer drug resistance to HER2-targeting drug trastuzumab (a), EGFR/HER2 dual-targeting drug lapatinib (b), and general anticancer drug doxorubicin (c).
Figure 4 shows the result of confirming that the expression of the ACSL4 gene is increased in SR (a) and MR (b), which are radiation-resistant cancer cells, through cDNA microarray analysis.
5 shows the results confirming that the expression of ACSL4 mRNA is increased in radiation-resistant cancer cells SR and MR.
Figure 6 shows the results confirming that the expression of ACSL4 protein (a) and FOXM1 protein (b) increased in radiation-resistant cancer cells, SR and MR.
7 shows the results confirming that only the expression of ACSL4 gene among ACSL isomers increased in radiation-resistant cancer cells SR (a) and MR (b), and the expression of ACSL4 protein among ACSL isomers in radiation-resistant cancer cells SR (c) and MR (d). The result of confirming that only the expression is increased is shown.
8 shows the results confirming that Triacsin-C, an ACSL1, 3, and 4 inhibitor, exhibits a high apoptotic effect selectively in radiation-resistant cancer cells SR (a) and MR (b).
9a and 9b show results confirming that durumamide A selectively exhibits a high apoptotic effect in radiation-resistant cancer cells SR (a) and MR (b).
9c and 9d show results confirming that durumamide A effectively suppresses the increased expression of ACSL4 protein in radiation-resistant cancer cells SR (c) and MR (d).
9e and 9f show results confirming that durumamide A effectively suppresses FOXM1 protein expression in radiation-resistant cancer cells SR(e) and MR(f).
9g and 9h show results confirming that durumamide A does not inhibit the expression of moesin protein (g) and MAP4K4 protein (h) in radiation-resistant cancer cell MR.
9i and 9j show results confirming that the DNA damage response is effectively inhibited by durumamide A in radiation-resistant cancer cells SR(i) and MR(j).
9k and 9l show results confirming that apoptosis is effectively increased by durumamide A in radiation-resistant cancer cells SR(k) and MR(l).
9m and 9n show results confirming that durumamide A effectively suppresses the high invasiveness of radiation-resistant cancer cells SR(m) and MR(n).
9o and 9p show results confirming that durumamide A effectively inhibits the high migration capabilities of SR(o) and MR(p) radiation-resistant cancer cells.
10 shows the mechanism of the selective anticancer effect of durumamide A on radiation-resistant breast cancer cells.
11 shows results showing that PANC-1 pancreatic cancer cells are essential radiation-resistant cells.
12 shows the results confirming that the expression of ACSL4 protein is increased in essential radiation-resistant pancreatic cancer cells, PANC-1.
13 shows the results confirming that durumamide A selectively exhibits a high apoptotic effect on essential radiation-resistant pancreatic cancer cells, PANC-1.
14 shows results confirming that the increased expression of ACSL4 protein in essential radiation-resistant pancreatic cancer cells, PANC-1, was effectively inhibited by durumamide A.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Durumamide A was prepared by the method described in Preparation Examples 1 to 3 for use in Example 9 below.

Preparation Example 1. Corynebacterium durum ( Corynebacterium durum ) Separation of durumamide A from acetone extract

1-1. Corynebacterium durum culture

An oral microbial strain, Corynebacterium durum, was cultured in a brain heart infusion (BHI) medium (9 × 1 L) for 4 weeks at 37 ° C. to obtain a culture, and the porous resin XAD-7-HP (20 g / L) ) was administered and shaken at room temperature for 2 hours.

1-2. acetone extraction

The resin to which the organic compounds were adsorbed was filtered and an acetone extract was obtained with 99.5% acetone (14.7 g).

1-3. fractionation with organic solvents

The acetone extract was fractionated with chloroform (270 mL × 3) and demineralized distilled water (270 mL), and organic substances (1.6 g) dissolved in the organic solvent layer were mixed with n -nucleic acid (15 mL × 3) to remove fat components. Fractionation was made again with acetonitrile (15 mL) to give fractions.

1-4. Chromatography performed and purification of durumamide A

Chromatography was performed using C ₁₈ HPLC (4.6 × 250 mm, 1.0 mL/min) from the fraction dissolved in the acetonitrile layer, and then 5% acetonitrile/water containing 0.1% formic acid was used as a mobile phase. After elution for 15 minutes, gradient elution was increased to 100% in 15 minutes to give durumamide A ( t _R 17.2 min).

Preparation Example 2. Synthesis of durumamide A 1

2-1. Preparation of a mixed solution of 3-indoleglyoxylyl chloride and 4-dimethylaminopyridine (DMAP)

A mixed solution was prepared by dissolving 3-indoleglyoxylyl chloride (777 mg, 1.2 equiv) and 4-dimethylaminopyridine (457 mg, 1.2 equiv) in dry pyridine (64 mL).

2-2. Addition of tryptamine

Tryptamine (500 mg, 1.0 equiv) was administered to the mixture and reacted. The reaction mixture was stirred at room temperature for 22 hours and fractionated with dichloromethane (100 mL) and demineralized distilled water (100 mL × 3) to obtain fractions.

2-3. Chromatography performed and durumamide A purification

From the material (902 mg) dissolved in an organic solvent, n -nucleic acid/ethyl acetate (800 mL each of 7:3, 5:5, 3: 7), followed by stepwise elution with pure ethyl acetate (800 mL) to purify durumamide A (618 mg, 60%).

Preparation Example 3. Synthesis of durumamide A 2

3-1. Tryptamine lysate preparation

Lysates were prepared by dissolving tryptamine (1.9 g) in 67% DMSO/water (360 mL).

3-2. Reflux extract preparation

The lysate was refluxed for 20 hours while stirring at 100° C. using an oil bath to obtain a reflux extract.

3-3. Preparation of chloroform extract

The reflux extract was extracted with chloroform (350 mL × 3) to prepare a chloroform extract (1.1 g).

Preparation Example 4. Material preparation - Preparation of radiation-resistant cancer cells

4-1. Induction of radiation tolerance in cancer cells

SK-BR-3 (human breast cancer cells, a HER2-overexpressing subtype) and MCF-7 (human breast cancer cells, a hormone receptor-positive subtype) were used as normal breast cancer cells.

Normal breast cancer cells of each subtype were subcultured by repeatedly irradiating 4 to 5 Gy of radiation every week (FIG. 1). In order to determine the presence or absence of radiation resistance of radiation-resistant human breast cancer cells derived from each of the normal cancer cells (SK-BR-3 and MCF-7), a colony forming assay was performed to determine whether resistance was obtained. Each of the radiation-resistant human breast cancer cells established from the normal breast cancer cells SK-BR-3 and MCF-7 were named SR and MR.

4-2. Confirmation of acquisition of radiation tolerance of SR and MR

4-2-1. Experiment method - colony forming ability assay

A colony forming assay was performed to confirm the acquisition of radiation resistance of SR and MR. Tissue survival fractions of normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR) were compared and evaluated.

300 to 900 cells were suspended in DMEM (Dulbecco's Modified Eagle's Medium) (10% FBS DMEM) containing 10% FBS (fetal bovine serum), and then plated in a 6-well plate. After culturing for 24 hours, irradiation with 0, 2, and 4Gy, respectively, was carried out and cultured for 10 to 30 days in an incubator maintained at 37° C. and 5% CO ₂ . The formed colonies were fixed with 10% formalin for 15 minutes, stained with 0.01% crystal violet, and then counted to calculate tissue viability.

4-2-2. Experiment result

Referring to Figures 2a and 2b, tissue viability after irradiation of SR and MR derived from normal cancer cells (SK-BR-3 and MCF-7) is higher than that of normal cancer cells (SK-BR-3 and MCF-7) appear. Through this, it was confirmed that both SR and MR acquired resistance to radiation.

Preparation Example 5. Confirmation of whether radiation-resistant cancer cells have acquired resistance to target therapeutics or general anticancer drugs

5-1. Experiment method - MTT assay

In order to investigate the anticancer effect of trastuzumab, a HER2-targeting treatment used as a breast cancer treatment, lapatinib, an EGFR/HER2 dual-targeting treatment, and doxorubicin, a general anticancer drug, on radiation-resistant cancer cells, each anticancer drug was tested. After treating normal cancer cells (SK-BR-3) and radiation-resistant cancer cells (SR) for 72 hours, MTT assay was performed.

Normal cancer cells (SK-BR-3) (5,000 cells/well) and radiation-resistant cancer cells (SR) (2,000 cells/well) were suspended in 10% FBS DMEM medium, seeded in a 96-well plate, and incubated for 24 hours. cultured. Subsequently, various concentrations of trastuzumab (3, 6, 12.5, 25, 50 μg/mL), lapatinib (0.01, 0.05, 0.1, 0.5, 1, 5 μM) or doxorubicin (0.05, 0.1, 0.2, 0.4, 0.6 μM) were administered. μM) and then cultured for 3 days. MTT reagent was added to the cultured cells, incubated for 4 hours, and the absorbance value was measured at 570 nm to compare and analyze the viability of normal cancer cells and radiation-resistant cancer cells.

5-2. Experiment result

Referring to FIGS. 3A and 3B , cell viability by trastuzumab and lapatinib was significantly higher in radiation-resistant cancer (SR) than in normal cancer cells (SK-BR-3). Referring to Figure 3c, the cell viability by doxorubicin was also higher in radiation resistant cancer (SR) than in normal cancer cells (SK-BR-3).

These results indicate that radiation-resistant cancer cells (SR) acquired not only radiation resistance but also resistance to targeted therapies (trastuzumab and lapatinib) and general anticancer drugs (doxorubicin).

Preparation Example 6. Confirmation of increased expression of the ACSL4 gene in radiation-resistant cancer cells

6-1. Experimental method - cDNA microarray analysis and realtime polymerase chain reaction (realtime PCR)

6-1-1. cDNA microarray analysis

In order to analyze gene expression changes according to the acquisition of radiation tolerance in radiation-resistant cancer cells (SR and MR), cDNA microarray was performed using GeneChip® Human Gene 2.0 ST array (Affymetrix, USA). Total RNA was extracted from each cell using the easy-BLUE total RNA extraction kit (iNtRON Biotechnology, Korea), and then cDNA microarray analysis was requested (DNP Biotech Co., Ltd., Korea) for ACSL4 Gene expression changes were comparatively analyzed.

6-1-2. real-time polymerase chain reaction

To compare and analyze the expression of ACSL4 mRNA in normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR), real-time polymerase chain reaction was performed. Total RNA was extracted in the same manner as described in the c-DNA microarray analysis of Preparation Example 6-1-1, and then cDNA was prepared using reverse-transcriptase. Using the prepared cDNA as a template, ACSL4 primers [Forward 5'-GGGAAGAAGGACAGCCTTGGG-3' (SEQ ID NO: 1), Reverse 5'-TCGGCCCTGGTCTCACAGAA-3' (SEQ ID NO: 2)] and 18s rRNA primer [Forward 5'-GTAACCCGTTGAACCCCATT-3 ' (SEQ ID NO: 3), Reverse 5'-CCATCCAATCGGTAGTAGCG-3' (SEQ ID NO: 4)] using a denaturation step (95 ° C, 10 seconds), annealing step (60 ° C, 20 seconds) and an extension step (72 ° C, ACSL4 in normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR) after real-time polymerase chain reaction was performed by performing 45 cycles with 20 seconds as one cycle. Gene expression was analyzed comparatively.

6-2. Experiment result

6-2-1. cDNA microarray analysis results

As a result of analyzing gene expression changes in normal cancer cells (SK-BR-3) and radiation-resistant cancer cells (SR) through cDNA microarray analysis, there were changes in the expression of about 2,000 genes.

Referring to Figures 4a and 4b, compared to normal cancer cells (SK-BR-3 and MCF-7), the expression of the ACSL4 gene increased more than 5 times in SR, which is a radiation-resistant cancer cell, and increased more than 12 times in MR.

6-2-2. Real-time polymerase chain reaction results

Referring to FIG. 5 , it was shown that the expression of the ACSL4 gene was increased in radiation-resistant cancer cells (SR and MR) compared to normal cancer cells (SK-BR-3 and MCF-7).

Preparation Example 7. Confirmation of increased expression of ACSL4 protein in radiation-resistant cancer cells

7-1. Experimental method - western blot assay

In order to analyze the expression of ACSL4 protein in normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR), Western blotting was performed using the ACSL4 antibody (Santa Cruz Biotechnology, Inc., #SC-271800). A blot was performed. RIPA lysis buffer (radio-immuno precipitation assay lysis buffer), which is a mixture of protease inhibitor and phosphatase inhibitor at a ratio of 1:100, was added to the cells and reacted at room temperature for 5 minutes. Then, the supernatant obtained by centrifugation at 13,000 rpm for 10 minutes was used as a protein sample.

For each protein sample, protein was quantified with BSA (bovine serum albumin), and then the same amount of protein was separated by 8% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis), transferred to a PVDF (polyvinylidene fluoride) membrane, and ACSL4 antibody ( antibody) was used to analyze expression changes.

β-actin was used as a protein loading control. HRP-conjugated anti-mouse IgG (Thermo Fisher Scientific, goat anti-Rabbit lgG (H+L) secondary antibody, HRP) was used, and the protein band was developed with ECL solution (enhanced chemiluminescence solution), followed by LAS-4000 (Fujifilm, Japan).

7-2. Experiment result

Referring to FIG. 6a , it was confirmed that the ACSL4 protein was expressed in a large amount in radiation-resistant cancer cells (SR and MR), compared to not being expressed in normal cancer cells (SK-BR-3 and MCF-7).

Preparation Example 8. Check whether the expression of the ACSL4 gene alone increased among the 5 ACSL isomers in radiation-resistant cancer cells

8-1. Experiment method

Expression of ACSL1, 3, 4, 5, and 6 genes using GeneChip® Human Gene 2.0 ST array (Affymetrix, USA) in the same manner as described above in the c-DNA microarray analysis of Preparation Example 6-1-1 Changes were comparatively analyzed.

8-2. Experiment result

Referring to Figures 7a and 7b, the gene expression of ACSL1, 3, 5 and 6 in radiation-resistant cancer cells (SR and MR) was decreased or lowered (less than 3-fold) in normal cancer cells (SK-BR-3 and MCF-7). ), ACSL4 increased by 5.5 times in SR and 12.8 times in MR than in normal cancer cells (SK-BR-3 and MCF-7).

Preparation Example 9. Check whether the expression of the ACSL4 protein alone among the 5 ACSL isomers increased in radiation-resistant cancer cells

9-1. Experimental Method - Western Blot

In order to analyze the expression of ACSL1, 3, 4 and 5 proteins in normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR), ACSL4 antibody as well as ACSL1 antibody (Cell Signaling, # 9189), the ACSL3 antibody (Santa Cruz Biotechnology, # SC-166374) and the ACSL5 antibody (Santa Cruz Biotechnology, # SC-365478), except that the expression change of the isomer was analyzed as described above in Preparation Example 7-1. Western blotting was performed in the same manner as described above.

9-2. Experiment result

Referring to FIGS. 7c and 7d , it was confirmed that among the four isoforms ACSL1, 3, 4, and 5, the expression of only the ACSL4 protein specifically increased in radiation-resistant cancer cells (SR and MR).

Preparation Example 10. ACSL1, 3 and 4 inhibitors (Triacsin-C) confirmed the selective apoptosis effect on radiation-resistant cancer cells

10-1. Experiment method - MTT assay

In order to analyze the survival rate of radiation-resistant cancer cells by ACSL4 inhibition, triacsin-C (triacsin-C), which is known to inhibit ACSL1, 3 and 4, was treated with trastuzumab, lapatinib, or doxorubicin and then cultured for 3 days. C) normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR) at various concentrations, except that they were cultured for 24 hours, the same as described above in Preparation Example 5-1. MTT assay was performed in the same way as in the previous study.

10-2. Experiment result

Referring to FIGS. 8a and 8b , it was confirmed that triaxin-C exhibits a high apoptotic effect selectively in radiation-resistant cancer cells (SR and MR) in which only the expression of ACSL4 among ACSL isomers increases. Through this, it can be seen that ACSL4 is a factor that plays an important role in the growth and survival of radiation-resistant cancer cells.

Preparation Example 11. Confirmation of increased expression of FOXM1 (forkhead box M1) protein in radiation-resistant cancer cells

11-1. Experimental Method - Western Blot

To analyze the expression of FOXM1 protein in normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR), FOXM1 antibody (Santa Cruz Biotechnology, #SC-376621) was used instead of ACSL4 antibody. Western blotting was performed in the same manner as described in Preparation Example 7-1, except that expression changes were analyzed.

11-2. Experiment result

Referring to FIG. 6B , it was confirmed that the FOXM1 protein was not expressed in normal cancer cells (SK-BR-3 and MCF-7), but was highly expressed in radiation-resistant cancer cells (SR and MR).

Example 1. Confirmation of selective anticancer effect of durumamide A through targeting of ASCL4 in radiation-resistant cancer cells - Confirmation of cell death effect

1-1. Experiment method - SRB assay (Surfohodamine assay)

The cytotoxicity of durumamide A against normal cancer cells (SK-BR-3 and MCF-7) and radiation-resistant cancer cells (SR and MR) was measured by SRB assay.

Normal cancer cells (5,000 cells/well) and radiation-resistant cancer cells (2,000 cells/well) were seeded in a 96-well plate and cultured in 10% FBS DMEM medium for 24 hours. Then, the cells were treated with 0-10 μM of durumamide A and cultured for 48 hours, then the supernatant was removed, and a 10% (w/v) TCA (trichloroacetic acid) solution (100 μL) was added and 1 hour at 4°C. Cells were fixed while incubated. The plate was washed 4 times using slowly flowing tap water, left at room temperature to dry all water, and then 100 μL of 0.4% (w/v) SRB solution was added and reacted at room temperature for 30 minutes. After removing excess SRB not bound to cell proteins using a 1% (v/v) acetic acid solution, the plate was left at room temperature to dry completely. 200 μL of 10 mM Tris base solution (pH 10.5) was put into each well to dissolve SRB bound to cell protein, and then the absorbance value was measured at 510 nm to calculate the cell viability for comparative analysis.

1-2. Experiment result

As shown in FIGS. 9a and 9b, it was confirmed that durumamide A exhibited a more significant apoptotic effect in radiation-resistant cancer cells (SR and MR) than normal cancer cells (SK-BR-3 and MCF-7). Through this, it was confirmed that durumamide A has an inhibitory effect on the growth and survival of radiation-resistant cancer cells.

Example 2. Analysis of the inhibitory effect of durumamide A on the increased expression of ACSL4 protein in radiation-resistant cancer cells

2-1. Experimental Method - Western Blot

Western blotting was performed using an ACSL4 antibody (Santa Cruz Biotechnology, # SC-271800) to analyze changes in ACSL4 protein expression in radiation-resistant cancer cells treated with durumamide A. SR and MR cells, which are radiation-resistant cancer cells, were treated with 0.5, 1, 2, 4, and 8 μM of durumamide A for 24 hours, and then protein samples were prepared in the same manner as described above in Preparation Example 7-1, and tested. used

2-2. Experiment result

As shown in FIGS. 9c and 9d, it was confirmed that the increased expression of ACSL4 protein in SR and MR cells was effectively inhibited depending on the concentration of durumamide A.

Example 3. Analysis of the inhibitory effect of durumamide A on the increased FOXM1 protein expression in radiation-resistant cancer

3-1. Experiment method

Western blotting was performed using FOXM1 antibody (Santa Cruz Biotechnology, #SC-376621) to analyze changes in ACSL4 subfactor protein expression in radiation-resistant cancer cells treated with durumamide A. SR and MR cells, which are radiation-resistant cancer cells, were treated with 0.5, 1, 2, 4, and 8 μM of durumamide A for 24 hours, and then protein samples were prepared in the same manner as described above in Preparation Example 7-1 for the experiment. used

3-2. Experiment result

As a result of confirming the change in FOXM1 expression by durumamide A, as shown in FIGS. 9e and 9f, it was confirmed that the expression of FOXM1 protein was effectively inhibited depending on the concentration of durumamide A.

Example 4. Other radiation resistance factors such as moesin and MAP4K4 are not inhibited by durumamide A

4-1. Experimental Method - Western Blot

Western blotting was performed using moesin antibody (Cell Signaling, #3150) and MAP4K4 antibody (Abcam, ab155583) to analyze changes in the expression of moesin and MAP4K4 proteins in radiation-resistant cancer cells treated with durumamide A. After treating radiation-resistant cancer cells (SR and MR) with durumamide A for 24 hours, protein samples were prepared in the same manner as described in Preparation Example 7-1 and used in the experiment.

4-2. Experiment result

9g and 9h, it was shown that durumamide A did not inhibit the expression of moesin and MAP4K4.

Example 5. Analysis of the inhibitory effect of durumamide A on DNA damage response in radiation-resistant cancer cells

5-1. Experimental Method - Western Blot

p-H2AX antibody (Cell Signaling, #9718), p-p38 antibody (Cell Signaling, #4511) and RAD51 antibody (Santa Cruz Biotechnology, sc-8349) were used for Western blotting. SR and MR cells, which are radiation-resistant cancer cells, were treated with 0.5, 1, 2, 4, and 8 μM of durumamide A for 24 hours, and then protein samples were prepared in the same manner as described above in Preparation Example 7-1, and tested. used

5-2. Experiment result

9i and 9j, the expression of p-H2AX and p-p38 proteins, which are DNA damage markers, increased in SR and MR cells depending on the concentration of durumamide A, whereas the expression of RAD51, a DNA damage repair protein, effectively confirmed to be reduced.

Example 6. In radiation-resistant cancer cells Analysis of apoptosis induction effect by durumamide A

6-1. Experimental Method - Western Blot

To analyze the expression changes of cleaved caspase 3 and cleaved PAR, which are apoptosis marker proteins, in radiation-resistant cancer cells treated with durumamide A, caspase 3 antibody (Cell Signaling, #9662) and PARP antibody (Cell Signaling, # 9542) was used to perform Western blotting. SR and MR cells, which are radiation-resistant cancer cells, were treated with 0.5, 1, 2, 4, and 8 μM of durumamide A for 24 hours, and then protein samples were prepared in the same manner as described above in Preparation Example 7-1, and tested. used

6-2. Experiment result

Referring to FIGS. 9K and 9L , it was confirmed that the expression of the apoptosis marker cleaved caspase 3 and its substrate, the cleaved form of PARP, increased in a concentration-dependent manner of durumamide A in SR and MR cells.

Example 7. Analysis of the effect of inhibiting the invasiveness of radiation-resistant cancer cells by durumamide A

7-1. Experimental method - invasion assay

The effect of durumamide A on the metastatic activity of radiation-resistant cancer cells was confirmed through invasion assay. First, after coating Matrigel on a transwell chamber equipped with a polycarbonate filter with 8 μm pores, the same amount of SK-BR-3 and SR cells (10,000 cells/well) or the same amount of MCF-7 or MR cells (30,000 cells/well) were seeded and cultured for 6 hours (MR cells) to 24 hours (SR cells). A medium containing 10% FBS was applied to the lower part of the chamber, and a medium containing durumamide A (1 or 4 μM) and 2% FBS was applied to the upper part of the chamber, incubated for 24 hours, and then filtered using the H&E staining method. The passing cells were observed using a microscope.

7-2. Experiment result

Referring to FIGS. 9m and 9n , it was confirmed that the invasiveness of radiation-resistant cancer cells (SR, MR) was improved compared to normal cancer cells (SK-BR-3, MCF-7), and this was effectively inhibited by durumamide A.

Example 8. Analysis of the effect of inhibiting migration of radiation-resistant cancer cells by durumamide A

8-1. Experimental method - wound healing assay

The effect of durumamide A on the migration ability of radiation-resistant cancer cells was confirmed through a wound healing assay. SK-BR-3 cells (750,000 cells/well), SR cells (500,000 cells/well), MCF-7 cells (700,000 cells/well) or MR cells (600,000 cells/well) in a collagen-coated 6-well plate After dispensing, the cells were cultured so that the cell onfluency was 95% or more. After making a wound using a yellow tip for a pipette, treatment with durumamide A (1 or 4 μM), and observing the migration distance of cells after 0 or 24 hours using a microscope.

8-2. Experiment result

Referring to FIGS. 9O and 9P , it was confirmed that the migration ability of radiation-resistant cancer cells (SR and MR) was increased compared to normal cancer cells (SK-BR-3 and MCF-7), which was effectively inhibited by durumamide A.

Summarizing the contents of Examples 1 to 8, durumamide A effectively inhibits the ACSL4-FOXM1 pathway in radiation-resistant breast cancer cells, SR and MR, thereby inhibiting the intracellular DNA damage response, inhibiting cell proliferation, and inducing apoptosis, It can be seen that it effectively suppresses the metastatic activity of radiation-resistant breast cancer cells and exhibits a selective anticancer effect on radiation-resistant breast cancer cells (FIG. 10).

Example 9. Confirmation of essential radiation resistance of Capan-1 and PANC-1 human pancreatic cancer cells

9-1. Experiment method

As normal pancreatic cancer cells, Capan-1 and PANC-1 human pancreatic cancer cells were used. Each cell was irradiated with 2Gy, 4Gy, 8Gy, and 16Gy and then cultured for 72 hours. The cultured cells were fixed with 10% trichloroacetic acid and stained with SRB for 30 minutes. After washing the excess dye with 1% acetic acid, 10 mM Tris base solution was added to dissolve the protein-bound dye, and the absorbance value was measured at 510 nm to compare the viability of Capan-1 and PANC-1 pancreatic cancer cells.

9-2. Experiment result

Referring to FIG. 11 , the viability of PANC-1 pancreatic cancer cells was significantly higher than that of Capan-1 pancreatic cancer cells. These results show that PANC-1 pancreatic cancer cells are essential radiation-resistant cells that are difficult to treat with radiation.

Example 10 Confirmation of increased expression of ACSL4, known as a radiation resistance factor, in essential radiation-resistant PANC-1 pancreatic cancer cells

10-1. Experimental Method - Western Blot

In order to analyze the expression of the ACSL4 protein, Western blotting was performed in the same manner as described in Preparation Example 7-1.

10-2. Experiment result

Referring to FIG. 12 , it was confirmed that the expression of the ACSL4 protein was significantly increased in PANC-1 pancreatic cancer cells, which are essential radiation-resistant pancreatic cancer cells, whereas it did not appear in Capan-1.

Example 11. Confirmation of selective apoptosis effect of durumamide A on essential radiation-resistant PANC-1 pancreatic cancer cells

11-1. Experimental method - SRB analysis

In order to analyze the survival rate of radiation-resistant cancer cells by inhibition of ACSL4, ACSL1 and 3 were used in the same manner as described in Example 1 except that Capan-1 was used as normal cancer cells and PANC-1 was used as radiation-resistant cancer cells. After treating pancreatic cancer cells with various concentrations of durumamide A, which is known to inhibit , and 4 , cultured for 72 hours, SRB analysis was performed to compare and analyze cell viability.

11-2. Experiment result

Referring to FIG. 13 , it was found that durumamide A exhibited a selectively high apoptotic effect in PANC-1, an essential radiation-resistant pancreatic cancer cell with higher expression of ACSL4 than in Capan-1, a normal pancreatic cancer cell.

Example 12. Durumamide for increased ACSL4 protein expression in essential radiation-resistant pancreatic cancer cells, PANC-1 Analysis of the inhibitory effect of A

12-1. Experimental Method - Western Blot

Western blotting was performed using an ACSL4 antibody (Santa Cruz Biotechnology, #SC-271800) to analyze changes in ACSL4 protein expression in PANC-1 cancer cells treated with durumamide A. Cells were treated with 10, 20, or 40 μM of durumamide A for 24 hours, and protein samples were prepared and used in experiments.

12-2. Experiment result

Referring to FIG. 14, it was confirmed that the increased expression of ACSL4 protein in PANC-1 cells was effectively inhibited depending on the concentration of durumamide A.

Summarizing the results of Examples 9 to 12, durumamide A selectively exhibits a high cytotoxic effect by effectively inhibiting ACSL4, a radiation resistance factor, in PANC-1, an essential radiation-resistant pancreatic cancer cell, showing essential radiation resistance. It indicates that it can be used as a treatment for pancreatic cancer.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

<110> Dongguk University Gyeongju Campus Industry-Academy Cooperation Foundation <120> Composition for preventing, improving or treating radiation-resistant cancer comprising durumamide A <130> BPN211036 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> artificial sequence <220> <223> ACSL4 forward primer <400> 1 gggaagaagg acagccttgg g 21 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> ACSL4 reverse primer <400> 2 tcggccctgg tctcacagaa 20 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> 18s rRNA forward primer <400> 3 gtaacccgtt gaaccccatt 20 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> 18s rRNA reverse primer <400> 4 ccatccaatc ggtagtagcg 20

Claims (10)

A pharmaceutical composition for preventing or treating radiation-resistant cancer comprising durumamide A as an active ingredient.
The method according to claim 1, wherein the durumamide A is derived from Corynebacterium durum ,
A pharmaceutical composition for preventing or treating radiation-resistant cancer.
The method according to claim 1, wherein the durumamide A is synthesized from tryptamine,
A pharmaceutical composition for preventing or treating radiation-resistant cancer.
The method according to claim 1, wherein the radiation-resistant cancer is any one selected from the group consisting of head and neck cancer, laryngeal cancer, pharynx cancer, lung cancer, brain cancer, colon cancer, breast cancer, and pancreatic cancer,
A pharmaceutical composition for preventing or treating radiation-resistant cancer.
The method according to claim 1, wherein the pharmaceutical composition inhibits the expression of ACSL4 in radiation-resistant cancer,
A pharmaceutical composition for preventing or treating radiation-resistant cancer.
A food composition for preventing or improving radiation-resistant cancer containing durumamide A.
The method according to claim 6, wherein the durumamide A is derived from Corynebacterium durum,
A food composition for preventing or improving radiation-resistant cancer.
The method according to claim 6, wherein the durumamide A is synthesized from tryptamine,
A food composition for preventing or improving radiation-resistant cancer.
The method according to claim 6, wherein the radiation-resistant cancer is any one selected from the group consisting of head and neck cancer, laryngeal cancer, pharynx cancer, lung cancer, brain cancer, colon cancer, breast cancer, and pancreatic cancer,
A food composition for preventing or improving radiation-resistant cancer.
The method according to claim 6, wherein the food composition inhibits the expression of ACSL4 in radiation-resistant cancer,
A food composition for preventing or improving radiation-resistant cancer.

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