KR101254244B1 - Composition for preventing or treating of irradiation-induced cell damage comprising TGF-β1 - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of cell damage by radiation exposure or irradiation containing TGF-β1 as an active ingredient, the TGF-β1 not only reduces DNA damage by radiation exposure or irradiation, As it has a protective effect against radiation exposure or irradiation, which can restore DNA, the composition containing TGF-β1 as an active ingredient prevents damage to normal cells during radiation treatment of epithelial cell-derived cancer diseases such as skin cancer and cervical cancer. It can be usefully used as a pharmaceutical or health food which can be prevented.

Description

Composition for preventing or treating of radiation-induced cell damage comprising TGF-β1}

The present invention relates to a composition for preventing or treating cell damage by radiation exposure or irradiation containing transforming growth factor-β1 (TGF-β1) as an active ingredient.

As the use of nuclear energy as an energy source increases, the necessity of taking countermeasures against radiation exposure which may occur in an accident or high dose radiation exposure is increasing. In addition, the radiation used to treat cancer damages the normal tissues around the necessity to reduce the side effects of the radiation treatment is increasing day by day.

In the case of low dose radiation, adaptive response has been introduced that can activate the cellular function of the living body to reduce damage caused by high dose radiation (Olivieri, G., Bodycote, J., Wolff, S., Adaptive response of human lymphocytes to low concentrations of radioactive thymidine. 1984: 223: 594-597). In the study of adaptive response to low-dose radiation using human lymphocytes, an adaptive response response in which cells acquire resistance by X-ray irradiation of 1.5 Gy when first treated with low concentration of 3H-thymidine Although it has been induced (Faroogi Z et. Al., Low dose radiation-induced adaptive response in bone marrow cells of mice.Mutat. Res. 1993: 302: 83-89), the characteristics of related genes have not yet been identified.

In addition, studies on high-dose radiation, which is used for therapeutic purposes or harming the human body immediately after radiation exposure due to radiological accidents, have been actively conducted, but there are almost no studies on various cellular damages to low-dose radiation. Factors involved in the adaptive response response include cell cycle related factors, cell death related factors, and DNA damage repair related factors. However, the genes involved in the adaptive response response are still unknown. not.

DNA damage is known to cause many diseases, including cancer and aging. To overcome this, a system exists that recognizes DNA damage and stops the cell cycle and repairs it. However, it has been observed that in many diseased cells the system does not operate normally. As a result, the DNA damage response after DNA damage is normal and is important for preventing and treating diseases related to DNA mutations.

DNA damage responses are complex signaling pathways that involve the organization of events in various cells that activate rapidly in response to DNA damage. These signaling pathways involve many factors that inhibit cell cycles, promote DNA damage checkpoints, and induce apoptosis when too much damage is repaired (Harper, JW and Elledge, SJ, Mol Cell, 28, 739-745, 2007; Bartek, J. and Lukas, J., Curr Opin Cell Biol, 19, 238-245, 2007).

On the other hand, TGF-β is a very important cytokine in a living body that regulates proliferative differentiation of cells, repair or regeneration after tissue failure. It is known that the disruption of the signal is associated with the development and progression of various diseases.

One well known relationship between TGF-β and disease is fibrosis of organs or tissues. Fibrosis of an organ or tissue occurs when the organ or the like is damaged by any cause, and the extracellular matrix accumulates excessively in the organ as its repair or defense mechanism. The extracellular matrix refers to a substance surrounding cells of tissues, and examples thereof include fibrous proteins such as collagen and elastin, complex saccharides such as proteoglycans, and glycoproteins such as fibronectin and laminin.

Accordingly, the present inventors have made the invention by confirming that TGF-β1 exhibits a protective effect against radiation exposure or irradiation that can not only reduce cellular damage, ie DNA damage, by radiation exposure or irradiation, but also repair damaged DNA. Completed.

An object of the present invention is to provide a composition for preventing or treating cell damage by radiation exposure or irradiation containing TGF-β1 as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing or treating cell damage by radiation exposure or irradiation containing TGF-β1 as an active ingredient.

The TGF-β1 may not only reduce cell damage, that is, DNA damage by radiation exposure or irradiation, but may also exhibit a protective effect against radiation exposure or irradiation that can repair damaged DNA.

The cell damage may be DNA damage by radiation exposure or irradiation, and in particular, damage to normal cells when radiation treatment of epithelial cell-derived cancer disease.

The epithelial cell-derived cancer diseases include skin cancer, cervical cancer, fibroadenoma, breast cancer, stomach cancer, colon cancer, ovarian cancer, head and neck cancer, lung cancer, prostate cancer, esophageal cancer, oral cancer, thyroid cancer, pancreatic cancer, biliary tract cancer, laryngeal cancer and gallbladder cancer. It may be any one selected from the group, preferably skin cancer or cervical cancer.

The skin cancer may be selected from the group consisting of squamous cell carcinoma, basal cell carcinoma, papilloma, carcinoma, spindle cell carcinoma and malignant melanoma.

The composition may include 0.01 to 50% by weight of TGF-β1 based on 100% by weight, preferably 0.05 to 10% by weight, and more preferably 0.10 to 5% by weight. If a small amount of TGF-β1 is out of the above content range, it is difficult to obtain a desired DNA damage repair effect, and an excessive amount may slow down the DNA damage repair effect compared to the dose, and may cause side effects, and is economical. Unsuccessful problems may arise.

The composition according to the invention may be provided in the form of a medicament or a functional food.

That is, the present invention provides a pharmaceutical composition for preventing or treating cell damage by radiation exposure or irradiation containing TGF-β1 as an active ingredient.

The pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin Mix the back.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. 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 base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of TGF-β1 which is an active ingredient of the pharmaceutical composition according to the present invention may vary depending on the age, sex, and weight of the patient, but is preferably 0.0001 to 100mg / kg, preferably 0.001 to 10mg / kg once or several times daily. May be administered.

In addition, the dose of TGF-β1 may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrabronchial inhalation, intrauterine dural or intracerebroventricular injection.

The present invention also provides a functional food for preventing or improving cell damage by radiation exposure or irradiation containing TGF-β1 as an active ingredient. In this case, the functional food may be provided in the form of a health functional food or a health beverage, it is preferable to use in the form of a soft or hard capsule filled with functional powders, pills, tablets or dry powdered ingredients.

The functional food is used together with other food or food additives in addition to TGF-β1, and may be appropriately 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.

An effective dose of TGF-β1 contained in the functional food may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for long term intake for health and hygiene purposes or for health control purposes. Since the active ingredient has no problem in terms of safety, it is evident that the active ingredient can be used in an amount above the above range.

There is no particular limitation on the kind of the functional food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic drinks, vitamin complexes, etc. are mentioned.

According to the present invention, TGF-β1 is first shown to not only reduce DNA damage caused by radiation exposure or irradiation, but also exhibits a protective effect against radiation exposure or irradiation that can repair damaged DNA. The composition containing as an active ingredient can be usefully used as a drug or health food that can protect the damage of normal cells in the radiotherapy of cancer diseases such as epidermal cell-derived cancer diseases such as skin cancer, cervical cancer and the like.

Figure 1 shows the results of DNA fragment analysis examining the effect of TGF-β1 on DNA damage by radiation,
Figure 2 shows the results of single cell electrophoresis analysis examining the effect of TGF-β1 on DNA damage caused by radiation,
Figure 3 shows the results of in vivo DNA end-joining assay examining the effect of TGF-β1 on damaged DNA recovery,
Figure 4 examines the effect of repairing DNA damage using immunofluorescence (Immunofluorescence),
Figure 5 shows the protective effect of TGF-β1 against DNA damage by irradiation in the experimental animal model.

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

Reference Example 1 Cell Line and Reagent Preparation

The human squamous cell carcinoma cell line A431 (epidermoid carcinoma) and human keratin-producing cell line HaCaT (keratinocyte) cells were purchased from the American type culture collection (ATCC), and the culture medium of the two cell lines was DMEM ( 10% FBS (Fetal Bovine Serum) was added to Dulbecco's Modified Eagle's Medium, and cultured at 37 ° C. and 5% CO ₂ . TGF-β1 was purchased from R & D System and used at a concentration of 0.5 ng / ml.

Example 1 Effect of TGF-β1 Treatment on DNA Damage by Radiation

DNA fragmentation assay

DNA fragment analysis was performed to determine how DNA damage by irradiation was affected by TGF-β1 treatment. That is, 8 × 10 ⁵ cells were prepared in 100 mm plate (dish) of A431 cells, which are human squamous cell carcinoma cell lines, and HaCaT cells, which are human keratin-producing cell lines, and treated with 0.5ng / ml of TGF-β1. After 24 hours, radiation was irradiated with 10 Gy and cultured again for 24 hours. The culture and cells were collected and separated using a centrifuge, and then the supernatant was removed. DNA isolation buffer (1mM PMSF) was added to release the precipitate. After placing on ice for 30 minutes, the supernatant was placed in an e-tube using a centrifuge. The same amount of phenol as the supernatant was added and mixed. The supernatant was once again separated by turning a centrifuge and mixed with the same amount of chloroform. The supernatant was once again separated using a centrifuge. Supernatant 1/10 volume of 3M sodium acetate was added and gently mixed. 1 ml of 100% alcohol was mixed and left to stand at -20 ° C for 1 hour. The supernatant was discarded by centrifugation, 1 ml of 70% alcohol was added, and the centrifuge was turned once again. The precipitate was dried in air, sterilized water was added, 1 μl of RNase was added, and the mixture was left at 37 ° C. for 30 minutes. Agarose gels were prepared and placed in an electrophoresis device to check the samples.

As a result, it was confirmed through DNA fragment analysis that the amount of fragmented DNA shown in the cells irradiated as shown in Figure 1 is reduced by treatment with TGF-β1.

2. Single cell gel electrophoresis

DNA damage was also assessed by single cell electrophoresis. That is, each cell was prepared by collecting in PBS at 1 × 10 ⁵ / ml. Cells and LM (low melting) agarose were mixed at a ratio of 1:10, aliquoted on a comet slide and hardened using a cover glass. The camelslide was added to the dissolution buffer and placed at 4 ° C. for 1 hour. The solution was placed in an alkaline solution for 30 minutes, and the slide was placed in an electrophoresis well, and the TBE buffer (Tris-Borate-EDTA buffer) was added and lowered to 20V for 20 minutes. The solution was removed and left in 70% alcohol for 5 minutes to dry the comslide. Stained with SYBR Green and analyzed using Komet 5.5.

The results are shown in FIG. 2, and it was observed that the DNA tail caused by radiation was reduced by TGF-β1 similarly to the DNA fragment analysis.

These results indicate that radiation-induced DNA damage is reduced by TGF-β1 in both cancer cells and normal cells.

Example 2 Effect of TGF-β1 Treatment on Recovery of Damaged DNA

Radiation-damaged DNA repair mechanisms include homologous recombination and non-homologous end joining (NHEJ). It is known that NHEJ is mainly responsible for DNA recovery in the G ₀ -G ₁ phase in the cell cycle, and TGF-β1 increases the G ₁ phase and slows cell death. In vivo DNA end-joining assay, one of the methods of measuring NHEJ, was used to determine the effect of TGF-β1 on DNA damage caused by radiation.

That is, the pGL2 plasmid was cut between the promoter and the luciferase cDNA with a Hind III enzyme, and the EcoR I enzyme was cut in the middle of the luciferase cDNA to form a plasmid. After 24 hours of treatment with TGF-β1, pGL2 plasmid and renilla plasmid cut with EcoR I and Hind III were transfected with fugene6 (roche) and incubated for 24 hours. Absorption was measured using a dual luciferase assay system (promega) to determine the extent of repair of damaged DNA by radiation.

The overall challenge of joining the plasmid in the form of a straight line is a measure of the recovery of pGL2 cut by Hind III, and the precipitate-joining activity of the plasmid in the form of a plasmid is a recovery of pGL2 cut by EcoRI. It is measured. As a result, as shown in Figure 3, the overallend-joining activity and preciseend-joining activity in both cell lines increased when pretreatment with TGF-β1. Through this, when DNA was damaged, it was confirmed that TGF-β1 contributes to repairing DNA damage.

Example 3 Examination of DNA Damage Recovery Using Immunofluorescence

A431 cells, a human epithelial cell carcinoma cell line, and HaCaT, a human keratin-producing cell line, were sprinkled on a 12well plate containing 7 × 10 ⁴ glass plates in a 12-well plate and cultured in a 37 ° C. 5% CO ₂ incubator for one day to fully settle the cells. treated 0.5ng / ml of β1. After 24 hours, pRFP-N1 was not cleaved with enzyme, and pGFP-N1 was not cut with enzyme and cut with enzyme. The cells were transformed into Lipofectamin2000 (Invitrogen) for 24 hours. After transferring the cover glass to the plate was fixed with 3.7% formaldehyde (in PBS) and washed three times with PBS. Nuclei were stained with a mounting solution (vectashield; H-1200) containing DAPI, and then observed with a laser scanning confocal microscope (Zeiss LSM710). At this time, the gene is not expressed in the GFP cut with Hind III, but if the cut part is recovered, the GFP is expressed, and thus the recovery degree can be known.

As a result, in the cells treated with TGF-β1 as shown in Figure 4 it was confirmed that the expression of GFP increased compared to the cells not.

Example 4 Examination of Protective Effect of TGF-β1 Against DNA Damage by Irradiation in Animal Model

In order to examine the effect of A431 cells, a human squamous cell carcinoma cell line, applied to an experimental animal model, cancer tissues were made by implanting A431 cells into the hind legs of nude mice. One hour after adding 20 µg of SB431542 (TGF-β inhibitor) to cancer tissue, 15ng of TGF-β1 was added and left for 24 hours. Radiation 8Gy was only partially irradiated to the hind limbs, and after 24 hours, cancer tissue was extracted, fixed in formalin, and stained with immunohistochemistry.

More specifically, 2 × 10 ⁶ A431 cells were transplanted into the hind legs of nude mice and waited until the cancer tissue became about 250 mm ³ in volume. 20 μg of SB431542 was added to cancer tissue using a syringe, and after 1 hour, 15 ng of TGF-β1 was added and left for 24 hours. Radiation 8Gy was only partially irradiated to the hind limbs and after 24 hours the cancer tissue was extracted and fixed in formalin. The immobilized tissue was planted in paraffin, then cut into 4 μm sections and plated on slides. After removing paraffin into xylene, immersed in 100%, 95%, 70%, 50% ethanol for 2 minutes in order to hydrate. After soaking in PBS and washing, protease K (20 μg / ml) was placed on the slide tissue and allowed to stand at room temperature for 15 minutes. After washing twice with distilled water, it was placed in 3% hydrogen peroxide (3% H ₂ O ₂ ) for 5 minutes. After washing twice with PBS, it was left in equilibration buffer (equilibration buffer) for 10 seconds. After the solution was removed, the TdT enzyme was added and left at 37 ° C. for 1 hour. The Stop / wash solution was treated for 10 minutes and washed three times with PBS. AntiDigoxigenin conjugate was added and washed three times with PBS after 30 minutes. DAB (KPL) reagent was treated to check the color development, and then washed with distilled water. Staining was performed using hematoxylin as a counterstain. After washing with running water to remove the dye was treated 50%, 70%, 95%, 100% ethanol in 2 minutes in order. After soaking in xylene twice for 10 minutes, the stained tissue was fixed using a mounting solution, and observed using an optical microscope.

As a result, as shown in Fig. 5, cell death of the irradiated cancer tissue was increased, but the pretreatment of TGF-β1 was confirmed to decrease the degree of cell death. In addition, it was confirmed that the treatment effect by TGF-β1 is inhibited by treating the TGF-β inhibitor.

Therefore, DNA damage due to irradiation can be suppressed by pretreatment of TGF-β1.

Hereinafter, an example of the preparation of a composition comprising TGF-β1 according to the present invention will be described, but the present invention is not intended to be limited thereto but only to be described in detail.

Preparation Example 1 Preparation of Powder

Powder was prepared by mixing 20 mg of TGF-β1, 100 mg of lactose and 10 mg of talc and filling into an airtight bag.

≪ Formulation Example 2 > Preparation of tablet

Tablets were prepared by mixing 20 mg of TGF-β1, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, followed by compression according to a conventional method for preparing tablets.

Preparation Example 3 Preparation of Capsule

10 mg of TGF-β1, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and the above ingredients were mixed and filled into gelatin capsules according to a conventional capsule preparation method to prepare a capsule.

Preparation Example 4 Preparation of Injection

10 mg of TGF-β1, sterile distilled water for injection, and a pH adjusting agent were mixed, and then prepared in the above-described component content per ampoule (2 ml) according to a conventional injection method.

Preparation Example 5 Preparation of Health Food

1 mg of TGF-β1, vitamin mixture (70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B 1, 0.15 mg of vitamin B 2, 0.5 mg of vitamin B 6, 0.2 μg of vitamin B 12, vitamin C 10 mg, 10 μg biotin, 1.7 mg nicotinic acid amide, 50 μg folic acid, 0.5 mg calcium pantothenate and an appropriate amount of mineral mixture (1.75 mg ferrous sulfate, 0.82 mg zinc oxide, 25.3 mg magnesium carbonate, 15 mg potassium monophosphate, diphosphate 55 mg of calcium, 90 mg of potassium citrate, 100 mg of calcium carbonate, 24.8 mg of magnesium chloride) were mixed, and then granules were prepared and health food was prepared according to a conventional method.

Preparation Example 6 Preparation of Health Beverage

1 mg of TGF-β1, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate, 1 g of taurine and purified water were added to make it total 900 ml, and the above ingredients were mixed according to a conventional healthy beverage preparation method. After stirring and heating at 85 ° C. for 1 hour, the resulting solution was collected by filtration into a sterilized 2 L container, sealed and sterilized and then stored in a refrigerator.

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 (5)

A composition for preventing or treating cell damage by radiation exposure or irradiation, containing TGF-β1 as an active ingredient and preventing damage to normal cells during radiation treatment of skin cancer. The method of claim 1, wherein the cell damage is a composition for the prevention or treatment of cell damage, characterized in that the DNA damage by radiation exposure or irradiation. delete delete The composition of claim 1, wherein the skin cancer is selected from the group consisting of squamous cell carcinoma, basal cell carcinoma, papilloma, carcinoma, spindle cell carcinoma, and malignant melanoma.

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