KR20180085387A - Composition for the prevention or treatment of TRAIL resistant cancer - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of TRAIL-resistant cancer containing galbanic acid and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as effective components. The composition of the present invention has a remarkable synergistic effect on TRAIL-resistant cancer cell death due to the combination of two effective components, and is effective for preventing or treating TRAIL-resistant cancer.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention or treatment of TRAIL resistant cancer,

The present invention relates to a composition for preventing or treating TRAIL-resistant cancer.

Cancer is one of the world's most deadly diseases, with the cancer age gradually decreasing, while the average life expectancy is increasing, and cancer incidence is expected to increase further. According to the 2013 statistics provided by the National Cancer Center, the number of cancer patients registered in the National Cancer Statistics Department in 2010 is 202,053, which is estimated to reach about 270,000 by 2015 .

TRAIL (TRAIL receptor 1) / DR5 (TRAIL receptor 2), a receptor for TRAIL, is a member of the cytokine protein belonging to the TNF (Tumor Necrosis Factor) superfamily. Is a protein that induces apoptosis cell death in cancer cells.

TRAIL-induced apoptosis, specifically, when TRAIL binds to DR4 / DR5, the three receptors form a trimer, and a complex called a death-inducing signaling complex (DISC) , And then activating caspase-8 / caspase-3 sequentially to induce apoptosis in cancer cells.

These TRAILs are known to be cytotoxic only to cancer cells without significant effect on normal cells, and are known to be very useful proteins for the development of anticancer drugs. TRAIL-mediated apoptosis cell death in various types of cancer cells Is known.

However, some cancer cell lines have been shown to be resistant to TRAIL proteins because TRAIL receptors are expressed at low levels on the cell surface, and resistance to these specific cancer cell lines has been reported to have limitations in the use of TRAIL.

Administration of more than two drugs to treat a given condition may be considered, but it can cause a number of potential problems. In vivo interactions between the two drugs are complex and when two drugs are introduced into the body, each drug can affect the absorption, distribution and elimination of the other, thus potentially altering the effect of the other have. For example, one drug is likely to induce inhibition, activation, and the like of the production of enzymes involved in the metabolic pathway of the other drug. Thus, when two drugs are administered to treat the same condition, there is a need to select a drug that can supplement the therapeutic activity of the other drug in the human subject, respectively. However, there is a very limited research and development for a combination drug for the combination administration of such a drug, particularly a combination drug for achieving the effect of the drug by controlling the tolerance of a carcinoma having resistance to a specific drug.

Under these circumstances, new therapeutic methods for the treatment of TRAIL-resistant cancers are being actively researched and developed, and the need for research and development related to the combined therapy of drugs is urgently required.

Apr 10, 2012 KR10-2014-0051555, 2014.05.02

The present invention provides a pharmaceutical composition for preventing or treating TRAIL-resistant cancer, which comprises galvanic acid and a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating TRAIL resistant cancer, which comprises galbanic acid and a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein as an active ingredient.

The inventors of the present invention found that the combination of galbanic acid and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) protein was effective in preventing and treating TRAIL-resistant cancer, And the present invention was completed.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating TRAIL-resistant cancer, which comprises galvanic acid and a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein as an active ingredient.

The composition of the present invention is a synergistic effect of a combination of galbanic acid and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) protein to produce galbanic acid or TRAIL (tumor necrosis factor-related apoptosis-inducing ligand ) Protein, it has a remarkable effect on the prevention or treatment of TRAIL-resistant cancer, and is particularly effective in the TRAIL-resistant cancer cell death.

In the present invention, Galbanic acid is a compound having the structure of the following formula (I).

(I)

Galbanic acid can be isolated and extracted from plants such as avian plants according to methods known in the art, and used or synthesized.

In the present invention, "TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) protein" means a member of cytokine protein belonging to the Tumor Necrosis Factor (TNF) superfamily, Can be obtained from GenBank of NCBI, which is a known database. Preferably, the TRAIL protein has the amino acid sequence of SEQ ID NO: 1.

Such TRAIL protein has been attracting attention as a next-generation anticancer drug. However, it is a protein that does not show toxicity in normal cells but selectively kills cancer cells. However, there is a problem that TRAIL treatment effect is low due to current resistance. Thus, the newly identified combination of galvanic acid and TRAIL-resistant cancer has the effect of effectively killing cancer cells.

In the present invention, TRAIL-resistant cancer means a cancer in which prevention, improvement or treatment of cancer is not effectively performed by treatment with TRAIL protein. More specifically, TRAIL protein receptor is expressed on the cancer cell surface at a low level, In addition to all the types of cancer, in which TRAIL protein-induced cell apoptosis mechanism is not effective, because of low sensitivity to TRAIL protein from the beginning, anticancer effect by treatment with TRAIL protein was initially observed, but repeated TRAIL protein treatment And the cancer has gradually been reduced in its anti-cancer effect against the TRAIL protein or has lost its effect.

The TRAIL-resistant cancer includes all types of cancer, without any particular limitation, as long as the cancer is resistant to TRAIL. Non-limiting examples of the TRAIL-resistant cancer include lung cancer, stomach cancer, liver cancer, breast cancer, arsenic lung cancer, prostate cancer, colon cancer, renal cancer, skin cancer, glioma cancer, thyroid cancer, blood cancer and the like. More preferably, it may be TRAIL-resistant arsenic-pulmonary cancer.

The term "prevention" as used in the present invention means any action to inhibit or delay the onset of cancer by administering the pharmaceutical composition of the present invention to a subject.

The term "treatment" as used in the present invention means all the actions that cause the symptom of cancer to be improved or benefited by administering the pharmaceutical composition of the present invention to a subject.

The pharmaceutical composition of the present invention activates the pro-apoptotic protein. For example, the pro-apoptotic proteins of PARP and procaspase-9 are significantly reduced by treatment with galactic acid and TRAIL, and the expression of cleaved caspase 8 and cleaved PARP is markedly increased.

The pharmaceutical composition of the present invention inhibits the expression of Survival protein. For example, the expression of bcl-2 and bcl-xl is greatly reduced by the treatment with galvanic acid and TRAIL.

In addition, sub-G1 distribution cells are greatly increased to induce apoptosis induction.

The composition of the present invention may contain 0.01 to 50% by weight of galvanic acid and 0.01 to 50% by weight of TRAIL based on the total weight of the composition. It may also contain 1, 2, 3 or 4 times more of the individual doses of galvanic acid and TRAIL, or may contain 1/2, 1/3, 1/4 times.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate Starch glycolate, starch glycolate, starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, sodium carboxymethylcellulose, sodium carboxymethylcellulose, sodium carboxymethylcellulose, Calcium stearate, white sugar, dextrose, sorbitol and talc may be used.

The pharmaceutically acceptable additives according to the present invention are preferably contained in an amount of 0.1 to 90 parts by weight based on 100 parts by weight of the pharmaceutical composition, but are not limited thereto.

That is, the pharmaceutical composition of the present invention can be administered in various forms of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, Or may be formulated using excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration may include sterile 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. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally in accordance with the intended method, and can be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, It is preferable to select the injection method. The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. Kg body weight, preferably 10 to 1000 占 퐂 / kg, and TRAIL is 1 to 1000 占 퐂 / kg body weight, preferably 10 to 100 占 퐂 / kg. In the case of administration, it can be administered 1 to 6 times a day.

The present invention provides a health functional food composition for preventing or ameliorating TRAIL resistant cancer, which comprises galbanic acid and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as an active ingredient.

As used herein, the term "improvement" means any action that at least reduces the degree of symptom associated with the condition being treated.

When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method.

The kind of the food is not particularly limited, and includes food in a conventional sense. Non-limiting examples of the food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, , A drink, an alcoholic beverage, and a vitamin complex.

When the health functional food composition of the present invention is a beverage composition, various flavoring agents, natural carbohydrates, and the like may be contained as additional components such as ordinary beverages. Non-limiting examples of such natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweetening agents such as dextrin, cyclodextrin; Synthetic sweetening agents such as saccharin and aspartame, and the like. The proportion of the additional component added may be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit drink or vegetable drink. These components may be used independently or in combination of two or more. The ratios of these additives can also be suitably selected by those skilled in the art.

The composition comprising galvanic acid and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) protein according to the present invention as an active ingredient has a synergistic effect on TRAIL-resistant cancer cell death by the combination of two active ingredients It is effective for prevention or treatment of TRAIL resistant cancer.

FIG. 1 shows the results of TRAIL-resistant cancer cell death upon administration of galbanic acid and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) protein.
FIG. 2 shows the results of examining changes in apoptosis-related protein expression by the combination of galbanic acid and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).
FIG. 3 shows the result of confirming the increase in the population of Sub-G1 period according to administration of galbanic acid and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).
FIG. 4 shows the results of confirming an increase in cell death upon administration of galbanic acid and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

< Example  1> TRAIL  Resistance Cancer cell  Confirm culture and cell viability

H460 cells and TRAIL-resistant H460 (H460 / R) cells (provided by Professor Nongjin Kyung) were cultured in RPMI1640 medium (10% FBS) at 37 ° C and 5% CO ₂ . TRAIL-resistant H460 cells were cultured in the presence of TRAIL and treated with TRAIL.

The TRAIL-resistant H460 (H460 / R) cells and the H460 cells were treated with the TRAIL protein (Sigma, USA) and the galvanic acid (GBA) having the structure of the formula I alone or in combination for 24 hours And cell viability was measured by MTT assay. The results are shown in Fig.

Fig. 1 (A) shows a single treatment of galvanic acid, and Fig. 1 (B) shows a result of single treatment of TRAIL. As shown in FIGS. 1A and 1B, the treatment of TRAIL protein and galactic acid alone did not show any effect on the cell death of TRAIL-resistant H460 (H460 / R) cells.

However, the combined treatment of TRAIL protein and galactic acid significantly reduced the cell survival rate of TRAIL resistant H460 (H460 / R) cells in a dose dependent manner.

This demonstrates synergistic effects for the prevention or treatment of TRAIL-resistant cancer by the combined administration of the two drugs.

< Example  2> Western Blat  through pro - apoptotic  And survival  Identification of protein expression changes

Western blot analysis confirmed the expression of pro-apoptotic and survival proteins. After culturing TRAIL-resistant H460 (H460 / R) cells with Galbanic acid and / or TRAIL for 24 hours, the cells were washed with PBS. The washed cells were resuspended in lysis solution (20 mM sucrose, 1 mM EDTA, 20 μM Tris-CL, pH 7.2, 1 mM DTT, 10 mM KCL, 1.5 mM MgCl ₂ , 5 μg / mL pepstatin A, 10 μg / / mL &lt; / RTI &gt; aprotinin). After centrifugation, the supernatant was collected and the protein concentration was measured. 30 μg aliquots were separated on SDS-polyacrylamide gel using a bio-rad protein assay kit and treated with each of the indicated primary and secondary antibodies And analyzed with an ECL detection system. The proteins to be detected were PARP, Cleaved PARP, pro caspase 9, cleaved caspase 8, bcl-2 and bcl-xl, and the analysis results are shown in FIG.

As shown in FIG. 2, it was confirmed that both of the cleaved PARP and cleaved caspase 8 were increased while the content of PARP, procaspase-9, Bcl-2 and Bcl-xL was greatly decreased by the treatment with the galvanic acid and TRAIL.

The above results show that the combination treatment of galvanic acid and TRAIL significantly activates apoptosis and reduces the expression of Survival protein in comparison with treatment with each of the galvanic acid and TRAIL, Confirm synergy effect.

< Example  3> Flow cell  Through analysis Sub -G1 cell identification

To confirm the occurrence of apoptosis, cells of Sub-G1 phase were identified through cell cycle analysis.

TRAIL-resistant H460 (H460 / R) cells were cultured, washed once with PBS, fixed with cold 70% ethanol, and stored at 4 ° C. Suspended in a cold PI solution containing 100 μg / mL RNase A, 50 μg / mL PI, 0.1% (w / v) sodium citrate and 0.1% (v / v) NP-40 and further incubated for 30 minutes . Flow cytometer: FACS was used to analyze sub-G1 of cells.

The results are shown in Fig.

FIG. 3A is a graph showing the effect of galactic acid and TRAIL on the sub-G1 population in TRAIL-resistant H460 (H460 / R) cells by cell cycle analysis, and FIG. 3B shows the histogram thereof.

As shown in FIG. 3A, it was confirmed that the treatment with the combination of galvanic acid and TRAIL significantly increased the number of sub-G1 cells to 36.55%.

From the above results, the synergistic effect of cancer cell death by the combination treatment of Galbanic acid and TRAIL was confirmed.

< Example  4> TUNEL  Confirmation of induction of cell death by assay

The increase of cell death by combination treatment of galbanic acid and TRAIL was confirmed by TUNEL assay. A terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay was performed using the Sigma method. Cells were fixed with 3.7% formaldehyde for 25 minutes in a Lab-Tek II chamber slide (Thermo Scientific, Roachester, NY, USA) and treated with 0.1% Triton X-100 for 5 minutes to increase permeability. The TUNEL reaction mixture was then treated for 60 minutes in a 37 ° C in a humidified atmosphere and DNA fragments were measured using the DeadEnd ™ Fluorometric TUNEL System.

The results are shown in Fig.

As shown in Fig. 4, it was confirmed that TUNEL fluorescence increased by treatment with galvanic acid and TRAIL.

From the above results, it was confirmed that the treatment with the combination of galvanic acid and TRAIL of the present invention had a synergistic effect on the TRAIL resistant cancer cell line, and showed a remarkable effect on TRAIL resistant cancer cell death.

Claims (7)

A pharmaceutical composition for preventing or treating TRAIL-resistant cancer comprising galvanic acid and a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein as an active ingredient. The method of claim 1, wherein the TRAIL resistant cancer is at least one cancer selected from the group consisting of lung cancer, stomach cancer, liver cancer, arsenic lung cancer, breast cancer, prostate cancer, colon cancer, kidney cancer, skin cancer, A pharmaceutical composition for preventing or treating TRAIL resistant cancer. 3. The pharmaceutical composition according to claim 2, wherein the TRAIL resistant cancer is arsenic lung cancer. 2. The pharmaceutical composition according to claim 1, wherein the galvanic acid is separated from the bottom. 2. The pharmaceutical composition according to claim 1, wherein the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein comprises the amino acid sequence of SEQ ID NO: 1. The pharmaceutical composition for preventing or treating TRAIL resistant cancer according to claim 1, wherein said TRAIL resistant cancer is prevented or treated by promoting TRAIL resistant cancer cell death. The present invention relates to a health functional food composition for preventing or ameliorating TRAIL-resistant cancer, which comprises galbanic acid and TRAIL as an active ingredient.

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