KR101975640B1 - Pharmaceutical composition for preventing or treating cell damage from irradiation or anticancer drug - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cell damage caused by radiation irradiation comprising a COH (coniferyl alcohol) compound or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutical composition for preventing or treating cells To a method of preventing or treating damage. The pharmaceutical composition according to the present invention is excellent in preventing or treating cell damage caused by irradiation or an anticancer agent. In particular, when a cell damage is caused by radiation or an anticancer agent, the expression of Heat Shock Factor 1 (HSF1), Heat Shock Protein 27 (HSP27) and Heat Shock Protein 70 (HSP70) Can be prevented or treated.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating cell damage caused by irradiation or an anticancer drug,

The present invention relates to a pharmaceutical composition for preventing or treating cytotoxicity caused by radiation or an anticancer agent. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating cell damage by irradiation with radiation or an anticancer agent comprising a COH (coniferyl alcohol) compound or a pharmaceutically acceptable salt thereof as an active ingredient The present invention also relates to a method for preventing or treating cell damage caused by radiation or an anticancer drug using the above pharmaceutical composition.

Heat shock factor 1 (HSF1) is a major regulator of heat shock protein (HSP) gene expression in stressed conditions such as inflammation, ischemia, oxidative stress, starvation and viral infection. Under stressful conditions, HSF1 is released from the chaperone complex and induces the expression of heat shock proteins such as HSP25, HSP27, HSP70, HSP72 and HSP90. Increased expression of heat shock protein (HSP) has been reported to inhibit cell damage and promote cell regeneration. Particularly, the activity of the HSF1 is abruptly lowered in an environment in which radiation is irradiated, which leads to a problem that the induction of the expression of the heat shock protein is inhibited. Accordingly, researches are underway to develop compounds that induce the expression of heat shock proteins while maintaining the induction activity of HSF1 in spite of various stresses, particularly, irradiation with radiation.

For example, there has been developed a technique for a composition having an inducing activity of HSF1 and a heat shock protein (Korean Patent Registration No. 10-1507221), but the heat shock protein inducing activity of the derivative of the compound as an active ingredient contained in the composition, The effect of preventing or treating cell damage by radiation or an anticancer agent is not known at all.

Accordingly, the present inventors have made intensive researches to develop a drug that can more effectively prevent or treat damage to cells caused by radiation or an anticancer drug. As a result, it has been found that COH (Coniferyl alcohol) The present inventors have confirmed that the pharmaceutical composition of the present invention exhibits a therapeutic effect and is more excellent in stability, and completed the present invention.

The main object of the present invention is to provide a pharmaceutical composition for preventing or treating cytotoxicity by radiation or an anticancer agent comprising a compound of the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating cell damage caused by irradiation or an anticancer agent comprising the step of administering the above pharmaceutical composition to an individual other than a human.

The inventors of the present invention conducted various studies to develop a pharmaceutical composition for preventing or treating cytotoxicity caused by irradiation or an anticancer agent, and found that when a COH (coniferyl alcohol) compound as a derivative of a CA (coniferyl aldehyde) The present invention has been found to be effective in preventing or treating cell damage, and having excellent stability.

Specifically, the present inventors have conducted studies in anticipation of the fact that derivatives of CA compounds have an effect of preventing or treating cytotoxicity caused by irradiation or an anticancer agent, while other derivatives are effective in preventing or treating cell damage by radiation irradiation or an anticancer agent However, the COH compound showed the effect of preventing or treating the cell damage caused by the irradiation or the anticancer drug. Furthermore, it was confirmed that the compound was more stable than CA because of low cytotoxicity.

Of the derivatives of the CA compounds, the fact that COH compounds exhibit the effect of preventing or treating cell damage by irradiation or an anticancer agent has not been known at all and has been first identified by the present inventors.

As an embodiment for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating cytotoxicity caused by irradiation with radiation or an anticancer agent comprising a compound (COH) of the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient Lt; / RTI >

[Chemistry]

The compound of Chemical Formula 1 corresponds to a derivative of CA (Coniferyl aldehyde) compound. The term 'CA' of the present invention is a compound which induces the expression of HSF1, HSP27 and HSP70 while having a vinyl structure. ≪ RTI ID = 0.0 > 1 < / RTI >

The compound of formula (1) may be named as a COH (coniferyl alcohol) compound as a CA compound derivative. The term 'COH compound' of the present invention is structurally similar in that it has a vinyl structure with the CA compound, Unlike CA, it contains a hydroxyl moiety, specifically a structure substituted by a hydroxyl moiety in place of the aldehyde moiety of CA.

In the present invention, the compound of Formula 1 may increase the expression of Heat Shock Factor 1 (HSF1), Heat Shock Protein 27 (HSP27) and Heat Shock Protein 70 (HSP70).

As used herein, the term " thermal shock protein inducing activity " means activating a regulatory factor (HSF1) that induces the expression of a heat shock protein and activating the expression of heat shock protein or inducing an increase in expression of heat shock protein (HSP) . Heat shock factor 1 (HSF1) is a transcription factor that regulates the heat-shock response of a cell, and the heat shock protein is a protein whose synthesis is induced when the cell, tissue or individual is at a temperature 5 to 10 캜 above the physiological temperature It is a protein.

Exposure of cells to various environmental disorders, including heat shock, alcohol, heavy metals and oxidative disorders, typically results in the accumulation of many chaperones known as heat shock proteins (HSPs). Induction of HSP protects cells against early impairment impairment and enhances maintenance recovery and induction of impaired resistance status. In addition, any HSP can act as a key molecular chaperone under normal disorder-free conditions by regulating the correct folding, degradation, integration and function of growth lists of important cellular proteins.

They are classified according to molecular weight, for example, HSP25, HSP27, HSP70, HSP72 and HSP90. Some diseases of humans can be obtained according to the results of incorrectly folded proteins. Since HSP is involved in preventing protein denaturation in cells or regenerating denatured proteins, it can be useful for the treatment of inflammatory diseases of the organs such as tissue damage.

Therefore, by inducing the expression of HSP, it is possible to treat diseases related to the regeneration of cells by irradiation with radiation or an anticancer drug.

In one embodiment of the present invention, the effect on expression of HSF1 protein and its transcription target, HSP27 and HSP70 was measured, and thus it was found that the compound of formula 1 has an inducing activity of HSF1, HSP27 and HSP70.

That is, the compound of Formula 1 of the present invention can regulate the expression of HSF1 and induce the expression of HSP27 and / or HSP70. In particular, the compound of formula (1) of the present invention can induce the expression of HSF1, HSP27 and HSP70, and thus has excellent HSP inducing ability. In particular, the compound of Formula 1 induces the expression of HSF1, HSP27, and HSP70, and thus has an excellent effect of preventing or treating cell damage caused by irradiation or an anticancer agent.

The term " radiation " of the present invention means an electromagnetic wave having a short particle wave flow and a short wavelength. The radiation may include alpha rays, beta rays, gamma rays, protons, neutrons, and heavy fluxes of the nucleus. Although there is no particular limitation, the radiation that causes cell damage by irradiation in the present invention may be a specific example And may be ionized radiation (IR).

The term " anticancer agent " of the present invention may include any composition or medicament showing a preventive or therapeutic effect on cancer. The cancer can be included in any of the cancerous tumors without any particular limitation as long as it is classified as a malignant tumor in the body. Specific examples of the cancer include colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, (Hodgkin's disease), esophageal cancer, small bowel cancer, endometrial carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Cancer of the kidney, renal cell carcinoma, renal pelvic carcinoma, central nervous system carcinoma, renal pelvic carcinoma, renal pelvic carcinoma, renal pelvic carcinoma, renal pelvic carcinoma, pancreatic cancer, And central nervous system (CNS) tumors.

The term " cell damage " of the present invention may be collectively referred to as a case where a cell is killed by ionizing radiation or an anti-cancer agent, which is an embodiment of radiation, or a case where a deformed structure, form, have. The term " prophylactic " in the present invention means all the actions of inhibiting or delaying the cell damage by radiation or anticancer drugs upon administration of the composition of the present invention. &Quot; Treatment " Or all of the actions that can be beneficially altered.

The pharmaceutical composition for preventing or treating cytotoxicity by radiation or an anticancer agent according to the present invention may be prepared by mixing a suitable carrier usually used in the preparation of a pharmaceutical composition (specific examples of which may include non-naturally occurring carriers) , A foam, or a diluent.

In addition, although the pharmaceutical composition is not particularly limited, it may contain a pharmaceutically acceptable salt for the compound of the formula (1) as an active ingredient. As an example of the pharmaceutically acceptable salt, any of those for increasing the solubility of the compound of Chemical Formula 1 may be applicable. In one preferred embodiment, the acid addition salt may be applicable.

On the other hand, the pharmaceutically acceptable salts should not be toxic to humans and adversely affect the biological activity and physicochemical properties of the parent compound. The free acids that can be used in the preparation of the pharmaceutically acceptable salts can also be divided into inorganic acids and organic acids. As the inorganic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid and the like can be used. The organic acid may be selected from the group consisting of acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, citric acid, gluconic acid, tartaric acid, Benzoic acid, embonic acid, aspartic acid, glutamic acid and the like can be used. Organic bases that can be used to prepare organic base addition salts include tris (hydroxymethyl) methylamine, dicyclohexylamine, and the like. Amino acids that can be used in the production of amino acid addition bases are natural amino acids such as alanine and glycine. The compounds of formula (I) according to the present invention may include not only pharmaceutically acceptable salts, but also all hydrates and solvates. The hydrate or solvate may be obtained by dissolving the compound of formula 1 in a solvent which can be mixed with water such as methanol, ethanol, acetone, or 1,4-dioxane, adding a free acid or a free base, and then crystallizing or recrystallizing . In such cases, solvates (especially hydrates) may be formed. Accordingly, the compounds of formula (I) of the present invention may also include stoichiometric solvates, including hydrates, in addition to various amounts of water-containing compounds that can be prepared by methods such as lyophilization.

The compound of Formula 1 or a pharmaceutically acceptable salt thereof is not particularly limited, but may be specifically contained in an amount of 1-80 parts by weight based on 100 parts by weight of the total composition, more specifically, 20-60 parts by weight By weight. When the compound of Formula 1 or a pharmaceutically acceptable salt thereof is contained in an amount of less than 1 part by weight, it is difficult to sufficiently exhibit the effect of preventing or treating cell damage due to irradiation of the present invention. When the amount of the compound represented by the formula (1) or the pharmaceutically acceptable salt thereof is more than 80 parts by weight, the content of the remaining materials for producing a pharmaceutical application product can be limited, It can not correspond to the embodiment of Fig.

On the other hand, the method of administering the pharmaceutical composition is not particularly limited, but it may be specifically injected into the artery or vein, subcutaneously, rectally, nasally, or any other parenteral route. More specifically, Arterial or intravenous, orally, or directly into muscle cells.

Also, the dosage level selected from the composition will depend on the activity of the compound, the route of administration, the severity of the condition being treated, and the condition and previous history of the patient being treated. It is, however, within the knowledge of the art to gradually increase the dose until the desired effect is achieved, starting with the dose of the compound at a level lower than that required for achieving the desired therapeutic effect, , Body shape, and body weight. The composition may be further processed before being formulated into a pharmaceutically acceptable pharmaceutical formulation, and may in particular be comminuted or ground into smaller particles. Also, the composition will vary depending on the condition and the patient being treated, but it can be determined non-ingeniously.

In addition, the pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, May have one formulation, and may be various forms of oral or parenteral administration. 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 formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention will depend on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, and the appropriate total daily dose may be determined by treatment, Generally, an amount of 0.001 to 1000 mg / kg, specifically, 0.05 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, can be administered once a day to several times a day.

According to another aspect of the present invention, there is provided a method for preventing or treating cell damage caused by radiation or an anticancer agent comprising the step of administering the pharmaceutical composition to a subject other than a human.

In the present invention, the term " individual " means an animal other than a human, in which cell damage may occur or may occur due to radiation or an anticancer agent. By administering the pharmaceutical composition of the present invention to a suspect cell, It can be treated efficiently.

The term " administration " in the present invention means introduction of the pharmaceutical composition of the present invention to a subject suspected of impaired cytotoxicity by irradiation or an anticancer agent by any suitable method, and the administration route may be either oral or parenteral May be administered via various routes of administration. Specifically, it can be administered by a transdermal administration method such as topical application, but is not limited thereto.

The pharmaceutical composition according to the present invention is excellent in preventing or treating cell damage caused by irradiation or an anticancer agent. In particular, when a cell damage is caused by radiation or an anticancer agent, the expression of Heat Shock Factor 1 (HSF1), Heat Shock Protein 27 (HSP27) and Heat Shock Protein 70 (HSP70) Can be prevented or treated.

FIG. 1 is a graph showing the HSF1 activity by the promoter activity of CA and its derivatives, wherein (a) shows the structure of the derivatives including CA and COH, (b) shows the activity of promoter In the case of the present invention.
Fig. 2 shows the effect of treating CAs and COHs with cells treated with taxol, cisplatin or gamma ray to protect cells. Fig. 2 (a) shows cells treated with Taxol, cisplatin or gamma- (B) is an electrophoresis photograph and a graph showing cytotoxicity and cytotoxicity of gamma-ray with HSF1 - / - and HSF1 + / + cells, and (c) (D) is a graph showing cytotoxicity and cytotoxicity by cisplatin with HSF1 - / - and HSF1 + / + cells, and (d) cytotoxicity by taxol with HSF1 - / - and HSF1 + And electrophoresis photographs and graphs showing the results of cell death.
FIG. 3 is a photograph showing Western blotting results showing that only COH among derivatives of CA increases the expression of HSPs protein.
FIG. 4 shows electrophoresis images and graphs showing that COH increases expression of HSF1, HSP70, and HSP27 in a concentration or time-dependent manner, (a) showing concentration-dependent increase in expression, and (b) Electrophoresis photographs and graphs showing that the expression is increased in a time-dependent manner.
FIG. 5 is a graph showing that COH increases the expression level of HSF1, HSP27, HPS70 protein or mRNA, wherein (a) is an electrophoresis photograph and graph comparing relative protein expression levels, (b) And electrophoresis photographs and graphs comparing relative gene expression changes.
FIG. 6 is a graph showing that COH promotes the promoter activity, cytotoxicity and phosphorylation of HSF 1, wherein (a) shows that COH increases the promoter activity of HSP27 and HSP70, and (b) (C) is an electrophoresis image showing that COH phosphorylates HSF1 at Ser326. FIG.
FIG. 7 is a chart and graph showing the results of in vivo experiments showing that the radiological disease-related characteristics of the COH-treated mouse group are reduced compared to those of the group not treated with COH.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example  1: Promoter Issei  through HSF1  Measurement of expression activity

In Example 1, the expression activity of HSF1 of each compound was measured in order to identify a compound showing the inducing activity of HSF1 among the compounds.

(Coniferyl aldehyde), CIN (cinnamaldehyde), CNOH (cinnamylalcohol), Gu (guaiacol) and beta-PV (beta-propiovanilone) were used for Sigma Aldrich ). RCA and RCOH were synthesized. All compounds were dissolved in DMSO and diluted in cell culture medium. The synthesis procedure of RCA (4-hydroxy-3-methoxyphenylpropanal, BPA) and RCOH (4-hydroxy-3-methoxyphenylpropanol, BPOH) is as shown in the following reaction formula.

[Reaction Scheme]

Compound RCA : Rf 0.41 (ethyl acetate / n-hexane = 1: 2); (T, J = 7.6 Hz, 2H), 2.79 (t, J = 7.6 Hz, 2H), 3.77 , 2.0 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 7.56 (s, 1H), 9.72 (t, J = 1.2 Hz, 1); 13 C-NMR (DMSO-d 6, 100 MHz) 27.4, 45.1, 55.5, 111.4, 114.9, 120.2, 131.3, 144.3, 147.0, 201.7 ppm.

Compound RCOH : Rf 0.16 (ethyl acetate / n-hexane = 1: 2); J = 8.0 Hz, 2H), 2.64 (t, J = 8.0 Hz, 2H), 3.68 (t, J = 6.4 Hz, 2H), 3.87 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 1.6 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H); 13 C-NMR (CDCl 3, 100 MHz) 31.2, 34.6, 55.5, 60.2, 112.5, 115.2, 120.3, 132.9, 144.3, 147.3 ppm.

On the other hand, the promoter activity of HSP25 or HSP70 was evaluated using a luciferase reporter device which stably expresses HSP25 or HSP70. Human lung cancer NCI-H460 cells were plated at 1 × 10 ⁵ cells in 60 mm dishes. Twenty-four hours after the culture, the compounds were treated with the cells, and after 12 hours of treatment, the cells were harvested and luciferase activity was measured. Luciferase activity was analyzed from cell lysates using the Luciferase Assay Systems (Promega). And the results normalized to? -Galactosidase expression (Promega). All results were found in three independent experiments. The promoter of various compounds (CA, COH, CNA, CNOH, Gu, beta-PV, BPA (RCA), and BPOH (RCOH), Figure 1a) was also used to investigate HSF1 and radiation- RTI ID = 0.0 > HSF1 < / RTI > Stable Pgl4-hsp70 H460 cells were plated at 1 × 10 ⁵ cells in 12 well plates. After 24 hours of incubation, the compounds were treated with the compounds and after 5 hours of treatment the cells were harvested and the luciferase activity was measured. Luciferase activity was analyzed from cell lysates using luciferase kit and detected by a luminometer.

As a result, in the case of RCA or RCOH, the promoter activity was increased when CA or COH was treated at 10 μM or 30 μM (FIG. 1B). Thus, it was confirmed that COH increases the promoter activity of HSF1 among CA derivatives.

Example  2: Results from treatment with anticancer agent or irradiation (IR, irradiation)

Example 2 is intended to confirm the effect of treatment of CA and COH on cells despite anticancer drugs or irradiation.

For this purpose, the waste L-132 cells, a human embryo is American Type Culture Collection was purchased from (ATCC, Manassas, VA, USA), 10% this, in 37 ℃, humidity, 5% CO ₂ incubator FBS (fetal bovine serums) and 1 % Penicillin streptomycin (GIPCO BRL) supplemented with RPMI. The HSF1 null out mouse embryonic fibroblast (HSF1 + / + and HSF1 - / - MEF) Ivor Benjamin (University of Utah, Salt Lake City, UT). The cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with heat-inactivated 10% FBS (Life Technologies), 0.1 mmol / L amino acid, glutamine, HEPES and antibiotics in a 37 ° C, 5% humidity CO ₂ incubator. The thus cultured L-132 cells were exposed to gamma rays operated by a 137Cs gamma-ray source (Elan 3000, Atomic Energy of Canada, Mississauga, Canada) at 10 Gy. For taxol treatment, cells in a 600 mm Petri dish were treated with taxol (0.5 μM) for 24 and 48 hours. For cisplatin treatment, cells in a 600 mm Petri dish were treated with cisplatin (20 μM) for 24 and 48 hours. Gamma rays were also treated at 10 Gy.

To confirm the induction of apoptosis and cell survival, cut-type PARP and cut-off caspase-3 or cell death using FACS were measured, respectively. Taxol, cisplatin or gamma rays were treated in L-132 cells for 24 hours or 48 hours, and CA and COH were pretreated with cells for 1 hour before their treatment. Pretreatment with CA or COH (3 μM) inhibited the production of truncated PARP and truncated caspases by taxol, cisplatin or gamma irradiation in L132 cells. Taxol, cisplatin, and gamma rays increased the cytotoxicity of L-132 cells, but significantly inhibited cytotoxicity by taxol, cisplatin, or gamma irradiation when CA or COH was pretreated for 1 hour at 3 μM. From these results, it was confirmed that pretreatment of CA or COH protects cells against chemotherapeutic agents or gamma rays (Fig. 2A).

The cytoprotective effect of CA or COH depends on HSF1. HSF1 - / - and HSF1 + / + cells were tested to confirm this. Pretreatment of CA or COH reduced cytotoxicity and cell death induced by taxol, cisplatin or gamma irradiation in HSF1 + / + cells. However, in HSF1 - / - cells, CA or COH did not inhibit cytotoxicity and cell death induced by taxol, cisplatin or gamma ray (IR) (FIGS. 2B-2D). In particular, FIG. 2b shows that it does not inhibit cytotoxicity and cell death induced by gamma ray (IR) in HSF1 - / - cells, and FIG. 2c shows cytotoxicity and cell death induced by cisplatin in HSF1 - / - And FIG. 2d confirms that it can not inhibit cytotoxicity and cell death induced by taxol in HSF1 - / - cells. Therefore, these results indicate that cytotoxicity or cytotoxicity by gamma-ray (IR) irradiation can be suppressed by the activity of HSF1, and that HSF1 activity can be induced by COH compounds.

On the other hand, the following statistical significance, including FIG. 2 in the second embodiment, was analyzed by Student's t-test.

Example  3: Immunoblotting ( Immunoblotting )

For the experiments of Example 3, the monoclonal antibody HSF1 was purchased from Abcam (diluted 1: 1000, Cambridge, Mass., USA). Anti-HSP70, HPS27, and β-actin were purchased from Santa Cruz Biotechnology (diluted 1: 1000, Santa Cruz, CA, USA). Anti-truncated PARP, or truncated caspase-3 was purchased from Cell Signaling Technology (diluted 1: 1000, Danvers, MA, USA).

On the other hand, in Example 3, total RNA was extracted using TRIzol (Invitrogen). cDNA was synthesized using ReverTra Ace RT-PCR kit (Toyobo, Osaka, Japan). Transcription levels of HSF1, HSP70, HSP27 and gapdh were measured by RT-PCR (GenDEPOT, Barker, TX, USA). gapdh was used as an internal regulatory gene. Relative gene expression changes were normalized to gapdh mRNA levels using the NIH ImageJ program.

L-132 cells were also plated in 2x10 ⁵ mm cell culture dishes and grown to 30% fused. Equal amounts of cells were dissolved in cell lysis buffer. And cell lysates were centrifuged at 13,000 rpm for 30 minutes at a temperature of 4 ° C. Protein concentrations were determined by Bradford (Biorad, USA) assay. BSA (bovine serum albumin) was used as a standard curve. Standard optical density and each sample was prepared by a 595 nm microplate reader. The prepared samples were mixed with the sample buffer and boiled for 5 minutes at 100 ° C. Proteins were separated by electrophoresis on 8% SDS-PAGE gel (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The separated protein bands migrated to the nitrocellulose membrane. After blocking with skim milk in PBS (Phosphate Buffered Saline), the membrane was incubated with monoclonal antibody against HSF 1 for 18 hours at 4 ° C. It was again washed and incubated again with horseradish peroxidase, a chemiluminescent enhancing visualization. The membrane after this process was detected by Bio-med.

On the other hand, after treatment of the compounds with 5 [mu] M or 10 [mu] M for 12 hours, the expression levels of HSPs were observed by Western blotting. As a result, expression of HSF1 and HSPs was increased in the case of CA and COH. However, the expression of the protein was not increased even at 10 μM in the RCA or RCOH having no vinyl structure and the remaining derivative described in Example 1 (FIG. 3). Through these results It was confirmed that COH is a derivative which increases the expression of the protein among the derivatives of CA.

FIGS. 4A and 4B are graphs showing the results of Western blotting of cell lysates collected from cells treated with COH or CA at 12 hours after L132 cells were treated with COH or CA, showing that COH increases the expression of HSP27 and HSP70 in a concentration- , Showing that COH increases the expression of HSF1, HSP70, and HSP27 in a concentration (Fig. 4A) or time (Fig. 4B) -like manner, similar to CA. The graph shows the quantification of the banding of the western blotting using a densitometer. Celastrol was used as a positive control.

5A and 5B are graphs showing the cell lysis by Western blotting or RT-PCR after collecting the cells 12 hours after treatment with 3 μM of COH or CA to L132 cells. Specifically, in FIG. 5A, HSP27, and HPS70 protein in the same manner as the CA. In FIG. 5B, the expression level of HSF1, HSP27, and HPS70 mRNA is increased similarly to that of CAH. The graph shows the quantification of the banding of Western blotting or RT-PCR using a densitometer. Celastrol was used as a positive control.

In addition, MTT assay was carried out. Specifically, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT: Amersham Pharmacia Biotech, Little Chalfont, UK) . After L132 cells were cultured, the culture medium was removed and replaced with 100 μl of MTT reagent (5 mg / ml) in PBS. The plate was incubated at 37 DEG C for 4 hours. 100 [mu] l DMSO was added to dissolve the formazan crystals. The multi-well plate was shaken for 15 minutes and signals were detected using ELISA at a wavelength of 540 nm. The results of the MTT Assay were expressed as the number of cells converted from total cell number to adsorption using a calibration curve. Measurements of the calibration curve were prepared by distributing UMR 106 cells in 96-well plates the number of times assigned for 24 hours before adding the MTT solution.

Both CA and COH were found to increase the promoter activity of HSP27 and HSP70 in L132 cells (Fig. 6A). On the other hand, L132 cells show a decrease in cell viability by treatment with Celastrol, a positive control.

6B is an MTT Assay method, and FIG. 6C is a Western blotting experiment. As can be seen from FIG. 6B, neither CA nor COH showed cytotoxicity even at a concentration of 20 μM. In particular , according to FIG. 6B, the cytotoxicity of COH was decreased at a concentration of 3 μM compared with that of CA, and the cytotoxicity was remarkably reduced at a concentration of 20 μM . Through these results, it was confirmed that the COH will be more reliable cytotoxicity lower than CA. 6C, it was confirmed that COH phosphorylates HSF1 in Ser326 similarly to CA. This suggests that COH increases the half-life of HSF1 by inhibiting protein degradation of HSF1 similarly to CA.

Example  4: Measurement of survival days

Example 4 is an experiment to measure the effect of CA or COH treatment on the survival days of animals when irradiated with gamma rays.

Female ICR mice (5-6 weeks old, 25-30 g) were purchased from Central Lab Animal, Inc. These mice were irradiated with 7 Gy gamma rays throughout the body. They were then raised in normal laboratory conditions and checked twice a day until the end of the experiment. CA and COH stock solutions were prepared by dissolving in DMSO. It was diluted with PBS to obtain the desired concentration to be injected into the mice. Fresh CA solution was prepared prior to injection into the mouse. Then, the mice were injected into the mice to measure survival days.

Previous studies have shown that CA increases survival days despite exposure to radiation for the measurement of survival days. In addition, it is an experiment to determine whether COH is associated with radiation protection in animal systems. The group exposed to radiation (IR) without any treatment began to show signs of radiation disease, reduced food or water intake, appeared lethargic ten days later, and died within 22 days. However, once CA or COH was treated before radiation treatment, or 3 times treatment with CA or COH at 5 mg / kg or 10 mg / kg after radiation treatment, the average survival days increased. And the features of radiation disease were reduced compared to the group of mice irradiated alone (FIG. 7).

From the above results, it was confirmed that the COH compound increases the expression of HSF1, HSP27, and HSP70 and thus has the effect of preventing or treating cell damage by irradiation, and it is confirmed that the COH compound is more stable than CA.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (4)

A pharmaceutical composition for preventing or treating cytotoxicity caused by radiation or an anticancer agent, wherein the anticancer agent is taxol or cisplatin, wherein the anticancer agent is a compound of the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

.
The method according to claim 1,
Wherein the compound of Formula 1 increases the promoter activity of Heat Shock Factor 1 (HSF1).
The method according to claim 1,
The compound of Chemical Formula 1 may increase the expression of a protein selected from the group consisting of Heat Shock Factor 1 (HSF1), Heat Shock Protein 27 (HSP27), Heat Shock Protein 70 (HSP70) ≪ / RTI >
A method for treating a cell injury caused by radiation or an anticancer drug, which comprises administering the pharmaceutical composition of any one of claims 1 to 3 to a subject other than a human, wherein the chemotherapeutic agent is taxol or cisplatin .

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